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https://openalex.org/W4313411157	https://zenodo.org/records/7498098/files/L%20versus%20NP.pdf	English	null	L versus NP	Zenodo (CERN European Organization for Nuclear Research)	2,023	cc-by	4,459	Abstract P versus NP is considered as one of the most important open problems in computer science. This consists in knowing the answer of the following question: Is P equal to NP? It was essentially mentioned in 1955 from a letter written by John Nash to the United States National Security Agency. However, a precise statement of the P versus NP problem was introduced independently by Stephen Cook and Leonid Levin. Since that date, all efforts to find a proof for this problem have failed. Another major complexity classes are L and NL. Whether L = NL is another fundamental question that it is as important as it is unresolved. Many computer scientists believe that the problem L versus NP is easier to solve than P versus NP. However, we prove that L = NL if and only if L = NP. In this way, we conclude that a proof for a separation of the complexity classes L and NP is so hard to find as proving that L is not equal to NL. 2012 ACM Subject Classification Theory of computation →Complexity classes; Theory of compu- tation →Problems, reductions and completeness; Theory of computation →Abstract machines Keywords and phrases complexity classes, completeness, polynomial time, reduction, logarithmic space, one-way 1 Introduction In 1936, Turing developed his theoretical computational model [9]. The deterministic and nondeterministic Turing machines have become in two of the most important definitions related to this theoretical model for computation [9]. A deterministic Turing machine has only one next action for each step defined in its program or transition function [9]. A nondeterministic Turing machine could contain more than one action defined for each step of its program, where this one is no longer a function, but a relation [9].ii Let Σ be a finite alphabet with at least two elements, and let Σ∗be the set of finite strings over Σ [2]. A Turing machine M has an associated input alphabet Σ [2]. For each string w in Σ∗there is a computation associated with M on input w [2]. We say that M accepts w if this computation terminates in the accepting state, that is M(w) = “yes” [2]. Note that M fails to accept w either if this computation ends in the rejecting state, that is M(w) = “no”, or if the computation fails to terminate, or the computation ends in the halting state with some output, that is M(w) = y (when M outputs the string y on the input w) [2].i Another relevant advance in the last century has been the definition of a complexity class. A language over an alphabet is any set of strings made up of symbols from that alphabet [4]. A complexity class is a set of problems, which are represented as a language, grouped by measures such as the running time, memory, etc [4]. The language accepted by a Turing machine M, denoted L(M), has an associated alphabet Σ and is defined by: L(M) = {w ∈Σ∗: M(w) = “yes”}. L(M) = {w ∈Σ∗: M(w) = “yes”}. Moreover, L(M) is decided by M, when w /∈L(M) if and only if M(w) = “no” [4]. We denote by tM(w) the number of steps in the computation of M on input w [2]. For n ∈N we denote by TM(n) the worst case run time of M; that is: Moreover, L(M) is decided by M, when w /∈L(M) if and only if M(w) = “no” [4]. We denote by tM(w) the number of steps in the computation of M on input w [2]. L versus NP L versus NP Frank Vega ! Ï CopSonic, 1471 Route de Saint-Nauphary 82000 Montauban, France L versus NP Frank Vega ! Ï CopSonic, 1471 Route de Saint-Nauphary 82000 Montauban, France L versus NP Frank Vega ! Ï CopSonic, 1471 Route de Saint-Nauphary 82000 Montauban, France TM(n) = max{tM(w) : w ∈Σn} L1 = {w : M(w, c) = “yes” for some string c}. L1 = {w : M(w, c) = “yes” for some string c}. We measure the time of a verifier only in terms of the length of w, so a polynomial time verifier runs in polynomial time in the length of w [2]. A verifier uses additional information, represented by the symbol c, to verify that a string w is a member of L1. This information is called certificate. NP is the complexity class of languages defined by polynomial time verifiers [8]. If NP is the class of problems that have succinct certificates, then the complexity class coNP must contain those problems that have succinct disqualifications [8]. That is, a “no” instance of a problem in coNP possesses a short proof of its being a “no” instance [8]. [ ] It is fully expected that P ̸= NP [8]. Indeed, if P = NP then there are stunning practical consequences [8]. For that reason, P = NP is considered as a very unlikely event [8]. Certainly, P versus NP is one of the greatest open problems in science and a correct solution for this incognita will have a great impact not only in computer science, but for many other fields as well [3]. Whether P = NP or not is still a controversial and unsolved problem [1]. We show some results that could help us to understand better this outstanding problem. 1 Introduction For n ∈N we denote by TM(n) the worst case run time of M; that is: TM(n) = max{tM(w) : w ∈Σn} 2 1.1 The Hypothesis A function f : Σ∗→Σ∗is a polynomial time computable function if some deterministic Turing machine M, on every input w, halts in polynomial time with just f(w) on its tape [9]. Let {0, 1}∗be the infinite set of binary strings, we say that a language L1 ⊆{0, 1}∗ is polynomial time reducible to a language L2 ⊆{0, 1}∗, written L1 ≤p L2, if there is a polynomial time computable function f : {0, 1}∗→{0, 1}∗such that for all x ∈{0, 1}∗: L versus NP where Σn is the set of all strings over Σ of length n [2]. We say that M runs in polynomial time if there is a constant k such that for all n, TM(n) ≤nk + k [2]. In other words, this means the language L(M) can be decided by the Turing machine M in polynomial time. Therefore, P is the complexity class of languages that can be decided by deterministic Turing machines in polynomial time [4]. A verifier for a language L1 is a deterministic Turing machine M, where: F. Vega of which is the OR of one or more literals [4]. A Boolean formula is in 3-conjunctive normal form or 3CNF, if each clause has exactly three distinct literals [4]. For example, the Boolean formula: (x1∨⇁x1∨⇁x2) ∧(x3 ∨x2 ∨x4) ∧(⇁x1∨⇁x3∨⇁x4) is in 3CNF. The first of its three clauses is (x1∨⇁x1∨⇁x2), which contains the thre literals x1, ⇁x1, and ⇁x2. (x1∨⇁x1∨⇁x2) ∧(x3 ∨x2 ∨x4) ∧(⇁x1∨⇁x3∨⇁x4) (x1∨⇁x1∨⇁x2) ∧(x3 ∨x2 ∨x4) ∧(⇁x1∨⇁x3∨⇁x4) is in 3CNF. The first of its three clauses is (x1∨⇁x1∨⇁x2), which contains the three literals x1, ⇁x1, and ⇁x2. A logarithmic space Turing machine has a read-only input tape, a write-only output tape, and read/write work tapes [9]. The work tapes may contain at most O(log n) symbols [9]. In computational complexity theory, L is the complexity class containing those decision problems that can be decided by a deterministic logarithmic space Turing machine [8]. NL is the complexity class containing the decision problems that can be decided by a nondeterministic logarithmic space Turing machine [8]. The complexity class coNL can be defined as the set of languages such that every element inside of the language will be accepted for every possible path by a nondeterministic logarithmic space Turing machine [8]. The two-way Turing machines may move their head on the input tape into two-way (left and right directions) while the one-way Turing machines are not allowed to move the input head on the input tape to the left [7]. Hartmanis and Mahaney have investigated the classes 1L and 1NL of languages recognizable by deterministic one-way logarithmic space Turing machine and nondeterministic one-way logarithmic space Turing machine, respectively [6]. They have shown that 1L ̸= 1NL (by looking at a uniform variant of the string non-equality problem from communication complexity theory) and have defined a natural complete problem for 1NL under deterministic one-way logarithmic space reductions [6]. Furthermore, they have proven that 1NL ⊆L if and only if L = NL [6]. Another important complexity class is coNP–complete [5]. A principal coNP–complete problems is UNSAT [5]. A Boolean formula without any satisfying truth assignment is unsatisfiable. The problem UNSAT asks whether a given Boolean formula is unsatisfiable [5]. coNL is the complexity class containing the languages such that their complements belong to NL [8]. We can give a disqualification-based definition for coNL [2]. x ∈L1 if and only if f(x) ∈L2. x ∈L1 if and only if f(x) ∈L2. An important complexity class is NP–complete [5]. If L1 is a language such that L′ ≤p L1 for some L′ ∈NP–complete, then L1 is NP–hard [4]. Moreover, if L1 ∈NP, then L1 ∈ NP–complete [4]. A principal NP–complete problem is SAT [5]. An instance of SAT is a Boolean formula ϕ which is composed of: 1. Boolean variables: x1, x2, . . . , xn; 2. Boolean connectives: Any Boolean function with one or two inputs and one output, such as ∧(AND), ∨(OR), ⇁(NOT), ⇒(implication), ⇔(if and only if); 3. and parentheses. 2. Boolean connectives: Any Boolean function with one or two inputs and one output, such as ∧(AND), ∨(OR), ⇁(NOT), ⇒(implication), ⇔(if and only if); 3. and parentheses. A truth assignment for a Boolean formula ϕ is a set of values for the variables in ϕ. A satisfying truth assignment is a truth assignment that causes ϕ to be evaluated as true. A Boolean formula with a satisfying truth assignment is satisfiable. The problem SAT asks whether a given Boolean formula is satisfiable [5]. We define a CNF Boolean formula using the following terms: A literal in a Boolean formula is an occurrence of a variable or its negation [4]. A Boolean formula is in conjunctive normal form, or CNF, if it is expressed as an AND of clauses, each 3 F. Vega The disqualification- based definition of coNL assumes that a logarithmic space Turing machine has another separated read-only tape, that is the same kind of special tape called “read-once” that we use in the certificate-based definition for NL [2]. Besides, in the disqualification-based definition of coNL, we assume the disqualification string is appropriated for the instance [8]. ▶Definition 1. A language L1 is in coNL if there exists a deterministic logarithmic space Turing machine M with an additional special read-once input tape polynomial p : N →N such that for every x ∈{0, 1}∗: x ∈L1 ⇔∀appropriated u ∈{0, 1}p(\|x\|) then M(x, u) = “yes” x ∈L1 ⇔∀appropriated u ∈{0, 1}p(\|x\|) then M(x, u) = “yes” x ∈L1 ⇔∀appropriated u ∈{0, 1}p(\|x\|) then M(x, u) = “yes” x ∈L1 ⇔∀appropriated u ∈{0, 1}p(\|x\|) then M(x, u) = “yes” where by M(x, u) we denote the computation of M where x is placed on its input tape and the disqualification string u is placed on its special read-once tape, and M uses at most O(log \|x\|) space on its read/write tapes for every input x where \| . . . \| is the bit-length function. We call M as a logarithmic space disqualifier. For example, there is a well-known coNL problem that states: Given a directed graph G = (V, E) and two nodes s, t ∈V , is there no possible path from s to t? In that problem, an appropriated disqualification string u is a sequence of nodes contained in V when s is the first node and t is the last one such that this sequence of nodes is not a path: There is at least a consecutive pair of nodes in the sequence which are not connected by an edge. We state the following Hypothesis: L2 = {w : M(w, u) = y, ∀appropriated u such that y ∈L1} L2 = {w : M(w, u) = y, ∀appropriated u such that y ∈L1} when M runs in logarithmic space in the length of w, u is placed on the special read-once tape of M, and u is polynomially bounded by w. In this way, there is a coNP–complete language defined by a logarithmic space disqualifier M such that when the input is an element of the language with any of its appropriated disqualification, then M always outputs a string which belongs to a single language in 1NL. L versus NP 4 ▶Hypothesis 1. Given a nonempty language L1 ∈1NL, there is a language L2 in coNP–complete under logarithmic space reductions with a deterministic Turing machine M, where: 2 Results We define a well-known coNP–complete problem: ▶Definition 2. 3UNSAT INSTANCE: A Boolean formula ϕ in 3CNF. QUESTION: Is ϕ unsatisfiable? REMARKS: 3UNSAT ∈coNP–complete [5]. ▶Theorem 3. If the Hypothesis 1 is true for L2 = 3UNSAT, then L = NL if and only if L = NP. ▶Theorem 3. If the Hypothesis 1 is true for L2 = 3UNSAT, then L = NL if and only if L = NP. ▶Theorem 3. If the Hypothesis 1 is true for L2 = 3UNSAT, then L = NL if and only if L = NP. Proof. We can simulate the computation M(w, u) = y in the Hypothesis 1 by a nondetermin- istic logarithmic space Turing machine N such that N(w) = y, since we can read the certificate string u within the read-once tape by a work tape in a nondeterministic logar- ithmic space generation of symbols contained in u [8]. Certainly, we can simulate the reading of one symbol from the string u into the read-once tape just nondeterministically generating the same symbol in the work tapes using a logarithmic space [8]. We remove each symbol generated in the work tapes, when we try to generate the next symbol contiguous to the right on the string u. In this way, the generation will always be in logarithmic space. In addition, we can guarantee that the generation of symbols in the work tapes always produces an appropriated string u, since we can check this by symbol per symbol from the generated string u and could make a logarithm space backtracking from the generation in any case. Under the assumption that L = NL, then this nondeterministic logarithmic space Turing machine N can be simulated by a deterministic logarithmic space Turing machine M ′. Note that, the nondeterministic logarithmic space Turing machine N with multiple outputs y on w collapses to a single output y′ on w under the deterministic logarithmic space Turing machine version M ′ of N. Moreover, the language L1 ∈1NL can be decided by a deterministic logarithmic space Turing machine M ′′ since 1NL ⊆L if and only if L = NL [6]. Finally, we compose the computation as M ′′(M ′(w)) in a deterministic logarithmic space computation since L is closed under composition in deterministic logarithmic space [8]. The complexity class coNL contains those languages such that every element in the language will be accepted for every possible path by a nondeterministic logarithmic space Turing machine [8]. The Turing machine that represents the computation of M ′′(M ′(w)) will accept every element w ∈3UNSAT for every possible path by a nondeterministic logarithmic space Turing machine since we know that a deterministic computation is also a nondeterministic. Hence, we deduce that 3UNSAT ∈coNL if the Hypothesis 1 is true and L = NL. In this way, we have that 3UNSAT ∈L under the assumption of L = NL because of coNL would 5 F. Vega collapse to L. Every coNP–complete is logarithmic space reduced to 3UNSAT. Certainly, every coNP problem could be logarithmic space reduced to 3UNSAT by the Cook’s Theorem algorithm [5]. Consequently, every language L3 ∈coNP will be in L when the Hypothesis 1 is true for L2 = 3UNSAT and L = NL. Due to we know that P is closed under complement, then we obtain that L = NP [8]. The reverse implication in the other direction, it is easy to deduce since L = NL is trivially true when L = NP (it is not necessary the veracity of the Hypothesis 1 for this implication). ◀ We define a new problem: ▶Theorem 5. 0SUM ∈1NL. Proof. Given a collection of integers C, we can read its elements from left to right, verify that every element is not equal to 0, check that every element in C has the same bit-length and count the amount of elements in C to finally multiply it by 3 and compare the bit- length of this number by the single bit-length from the elements in C. In addition, we can nondeterministically pick two elements a and b from C and accept in case of a + b = 0 otherwise we reject. We can make all this computation in a nondeterministic one-way using logarithmic space. Certainly, the calculation and store of the bit-length of the elements in C could be done in logarithmic space since this is a unique value. On the one hand, we can count and store the number of elements that we read from the input and multiply it by 3 to finally compare the bit-length of this number by the stored unique bit-length from the elements of the collection, since the cardinality of C multiplied by 3 could be stored in a binary number of bit-length that is logarithmic in relation to the binary encoded length of C. On the other hand, the two elements a and b that we pick from C have a logarithmic space in relation to the encoded length of C, because of every integer in C has the same bit-length which is equal to the bit-length of the number that is the cardinality of C multiplied by 3. Indeed, we never need to read to the left on the input for the acceptance of the elements in 0SUM in a nondeterministic logarithmic space. ◀ ▶Definition 4. SUM ZERO INSTANCE: A collection of integers C such that 0 /∈C and every integer in C has the same bit-length that is equal to the bit-length of the number that is the cardinality of C multiplied by 3 (we do not take into account the symbol minus in counting the bit-length of the negative integers). QUESTION: Are there two elements a, b ∈C, such that a + b = 0? QUESTION: Are there two elements a, b ∈C, such that a + b = 0? REMARKS: We denote this problem as 0SUM. REMARKS: We denote this problem as 0SUM. ▶Theorem 6. There is a deterministic Turing machine M, where: 3UNSAT = {w : M(w, u) = y, ∀appropriated u such that y ∈0SUM} when M runs in logarithmic space in the length of w, u is placed on the special read-once tape of M, and u is polynomially bounded by w. Proof. The input could be a Boolean formula ϕ in 3CNF with n variables and m clauses such that each variable is represented by an integer between 1 and n and a positive and a negated literal correspond to the values of k and −k, respectively (similar to the DIMACS format: http://www.satcompetition.org/2009/format-benchmarks2009.html). We can create a disqualification array A which contains m positive integers between 1 and 3 which represents the position of the literals in the clauses of ϕ from left to right. We read at once 6 L versus NP L versus NP 6 L versus NP Algorithm 1 Logarithmic space disqualifier with output Algorithm 1 Logarithmic space disqualifier with output 1: /A valid instance for 3UNSAT with its disqualification/ 1: /A valid instance for 3UNSAT with its disqualification/ 1: /A valid instance for 3UNSAT with its disqualification/ 2: procedure DISQUALIFIER(ϕ, A) 3: /Initialize an index/ 4: j ←0 5: /m is the number of clauses in ϕ/ 6: /Iterate for the elements of the disqualification array A/ 7: for i ←1 to m + 1 do 8: if i = m + 1 then 9: /There exists an m + 1 element in the array/ 10: if A[i] ̸= undefined then 11: /Reject the disqualification/ 12: return “no” 13: end if 14: /Break the for loop/ 15: break 16: else if A[i] = undefined ∨A[i] < 1 ∨A[i] > 3 then 17: /Reject the disqualification/ 18: return “no” 19: else 20: j ←A[i] 21: end if 22: /f(i, j) is the literal in the position j in the clause ci/ 23: output “ , f(i, j)” 24: end for 25: end procedure 1: /A valid instance for 3UNSAT with its disqualification/ 2: procedure DISQUALIFIER(ϕ, A) 3: /Initialize an index/ 4: j ←0 5: /m is the number of clauses in ϕ/ 6: /Iterate for the elements of the disqualification array A/ 7: for i ←1 to m + 1 do 8: if i = m + 1 then 9: /There exists an m + 1 element in the array/ 10: if A[i] ̸= undefined then 11: /Reject the disqualification/ 12: return “no” 13: end if 14: /Break the for loop/ 15: break 16: else if A[i] = undefined ∨A[i] < 1 ∨A[i] > 3 then 17: /Reject the disqualification/ 18: return “no” 19: else 20: j ←A[i] 21: end if 22: /f(i, j) is the literal in the position j in the clause ci/ 23: output “ , f(i, j)” 24: end for 25: end procedure / 2: procedure DISQUALIFIER(ϕ, A) / 2: procedure DISQUALIFIER(ϕ, A) p Q /Initialize an index/ /Initialize an index/ 7 ▶Theorem 7. L = NL if and only if L = NP. ▶Theorem 7. L = NL if and only if L = NP. Proof. This is a direct consequence of Theorems 3, 5 and 6. References 1 Scott Aaronson. P ? NP. Electronic Colloquium on Computational Complexity, Report No. 4, 2017. 2 Sanjeev Arora and Boaz Barak. Computational complexity: a modern approach. Cambridge University Press, 2009. 3 Stephen A. Cook. The P versus NP Problem, April 2000. Clay Mathematics Institute. http://www.claymath.org/sites/default/files/pvsnp.pdf. Accessed 10 January 2020. 4 Thomas H. Cormen, Charles E. Leiserson, Ronald L. Rivest, and Clifford Stein. Introduction to Algorithms. The MIT Press, 3rd edition, 2009. F. Vega the elements of the array A and we reject whether this is not an appropriated disqualification: That is when the array A does not contain exactly m elements, or the array A contains a number that is not between 1 and 3. While we read the elements of the array A using the index i, we select from the ith clause in ϕ the single literal such that this one occupies the position that represents the number A[i] within the clause, that is the first, second or third place from left to right. In this way, we output the selected literals that are represented by a positive or negative integer (it is negative in case of a negated variable) just creating another instance C for 0SUM where the collection C contains those integers which are the selected literals for each clause in ϕ. We obtain that all the appropriated array A implies that: 2 Sanjeev Arora and Boaz Barak. Computational complexity: a modern approach. Cambridge University Press, 2009. 1 Scott Aaronson. P ? NP. Electronic Colloquium on Computational Complexity, Report No. 4, 2017. ϕ ∈3UNSAT ⇔(C ∈0SUM for all appropriated array A), ϕ ∈3UNSAT ⇔(C ∈0SUM for all appropriated array A), ϕ ∈3UNSAT ⇔(C ∈0SUM for all appropriated array A), because of when we obtain an instance C /∈0SUM, then this is equivalent to a satisfying truth assignment in ϕ. Furthermore, we can make this disqualification in logarithmic space such that the array A is placed on the special read-once tape, because we read at once the elements in the array A. Hence, we only need to iterate from the elements of the array A to verify whether the array is an appropriated disqualification and pick the m literals from the Boolean formula ϕ and write those integers to the output tape. We use a function f such that f evaluated in a pair (i, j) outputs the literal in the position j inside of the ith clause just filled with zeroes to the left until we reach a total of \|3 · m\| bits taking into account the bit-length of integer linked to the literal, where the bit-length of the symbol minus is ignored when we fill the negated literals.i This logarithmic space disqualification with output will be the Algorithm 1. We introduce some constraints in the Algorithm 1 in order to guarantee the theoretical procedure. For example, we assume that a value does not exist in the array A into the cell of a position i when A[i] = undefined. In addition, we immediately reject when the following comparisons: A[i] < 1 ∨A[i] > 3 hold at least into one single binary digit. Note that, in the worst case every possible literal in ϕ would have a representation by an integer between −3 · m and 3 · m with the exception of 0, where m is the cardinality of the collection C (i.e. when n = 3·m). In this way, we guarantee the output collection C is an appropriated instance of 0SUM just using the function f that maps the literals inside of the clauses to numbers with a bit-length equal to \|3 · m\| where \| . . . \| is the bit-length function. ◀ 1 Scott Aaronson. P ? NP. Electronic Colloquium on Computational Complexity, Report No. 4, 2017. 2 Sanjeev Arora and Boaz Barak. Computational complexity: a modern approach. Cambridge University Press, 2009. 3 Stephen A. Cook. The P versus NP Problem, April 2000. Clay Mathematics Institute. http://www.claymath.org/sites/default/files/pvsnp.pdf. Accessed 10 January 2020. 4 Thomas H. Cormen, Charles E. Leiserson, Ronald L. Rivest, and Clifford Stein. Introduction to Algorithms. The MIT Press, 3rd edition, 2009. Proof. This is a direct consequence of Theorems 3, 5 and 6. References L versus NP L versus NP 8 5 Michael R. Garey and David S. Johnson. Computers and Intractability: A Guide to the Theory of NP-Completeness. San Francisco: W. H. Freeman and Company, 1 edition, 1979. 6 Juris Hartmanis and Stephen R. Mahaney. Languages Simultaneously Complete for One- Way and Two-Way Log-Tape automata. SIAM Journal on Computing, 10(2):383–390, 1981. doi:10.1137/0210027. 7 Martin Kutrib, Julien Provillard, György Vaszil, and Matthias Wendlandt. Deterministic One-Way Turing Machines with Sublinear Space. Fundamenta Informaticae, 136(1-2):139–155, 2015. doi:10.3233/FI-2015-1147. 8 Christos H. Papadimitriou. Computational complexity. Addison-Wesley, 1994. 9 Michael Sipser. Introduction to the Theory of Computation, volume 2. Thomson Course Technology Boston, 2006.
https://openalex.org/W2029217034	https://europepmc.org/articles/pmc3783438?pdf=render	English	null	“Best Practice” Skills Lab Training vs. a “see one, do one” Approach in Undergraduate Medical Education: An RCT on Students’ Long-Term Ability to Perform Procedural Clinical Skills	PloS one	2,013	cc-by	10,021	Abstract This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The authors have no support or funding to report. Competing interests: The authors have declared that no competing interests exist. * E-mail: nora.celebi1@gmail.com Received February 7, 2013; Accepted August 23, 2013; Published September 25, 2013 Copyright: © 2013 Werner et al. This is an open-access article distributed under the terms of the Creative Commons Attributio unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. ner et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits ution, and reproduction in any medium, provided the original author and source are credited. Funding: The authors have no support or funding to report. Competing interests: The authors have declared that no competing interests exist. * E-mail: nora.celebi1@gmail.com Anne Herrmann-Werner1, Christoph Nikendei2, Katharina Keifenheim1, Hans Martin Bosse3, Frederike Lund4, Robert Wagner5, Nora Celebi5, Stephan Zipfel1, Peter Weyrich5 Anne Herrmann-Werner1, Christoph Nikendei2, Katharina Keifenheim1, Hans Martin B Lund4, Robert Wagner5, Nora Celebi5, Stephan Zipfel1, Peter Weyrich5 1 Department of Psychosomatic Medicine and Psychotherapy, University Hospital of Tübingen, Tübingen, Germany, 2 Department of General Internal Medicine and Psychosomatics, University Hospital of Heidelberg, Heidelberg, Germany, 3 Department of General Paediatrics, Neonatology and Child Cardiology, University Hospital of Düsseldorf, Düsseldorf, Germany, 4 Department of Anaesthesiology, University Hospital Heidelberg, Heidelberg, Germany, 5 Department of Diabetes, Endocrinology, Angiology, Nephrology and Clinical Chemistry, University Hospital of Tübingen, Tübingen, Germany Abstract Background: Benefits of skills lab training are widely accepted, but there is sparse research on its long-term effectiveness. We therefore conducted a prospective, randomised controlled-trial to investigate whether in a simulated setting students trained according to a "best practice" model (BPSL) perform two skills of different complexity (nasogastral tube insertion, NGT; intravenous cannulation, IVC) better than students trained with a traditional "see one, do one" teaching approach (TRAD), at follow-up of 3 or 6 months. Methodology and Principal Findings: 94 first-year medical students were randomly assigned to one of four groups: BPSL training or TRAD teaching with follow-up at 3 (3M) or 6 (6M) months. BPSL included structured feedback, practice on manikins, and Peyton’s "Four-Step-Approach", while TRAD was only based on the "see one - do one" principle. At follow-up, manikins were used to assess students’ performance by two independent blinded video- assessors using binary checklists and a single-item global assessment scale. BPSL students scored significantly higher immediately after training (NGT: BPSL3M 94.8%±0.2 and BPSL6M 95.4%±0.3 percentage of maximal score ± SEM; TRAD3M 86.1%±0.5 and TRAD6M 84.7%±0.4. IVC: BPSL3M 86.4%±0.5 and BPSL6M 88.0%±0.5; TRAD3M 73.2%±0.7 and TRAD6M 72.5%±0.7) and lost significantly less of their performance ability at each follow-up (NGT: BPSL3M 86.3%±0.3 and TRAD3M 70.3%±0.6; BPSL6M 89.0%±0.3 and TRAD6M 65.4%±0.6; IVC: BPSL3M 79.5% ±0.5 and TRAD3M 56.5%±0.5; BPSL6M 73.2%±0.4 and TRAD6M 51.5%±0.8). In addition, BPSL students were more often rated clinically competent at all assessment times. The superiority at assessment after training was higher for the more complex skill (IVC), whereas NGT with its lower complexity profited more with regard to long-term retention. Conclusions: This study shows that within a simulated setting BPSL is significantly more effective than TRAD for skills of different complexity assessed immediately after training and at follow-up. The advantages of BPSL training are seen especially in long-term retention. Citation: Herrmann-Werner A, Nikendei C, Keifenheim K, Bosse HM, Lund F, et al. (2013) “Best Practice” Skills Lab Training vs. a “see one, do one” Approach in Undergraduate Medical Education: An RCT on Students’ Long-Term Ability to Perform Procedural Clinical Skills. PLoS ONE 8(9): e76354. doi: 10.1371/journal.pone.0076354 Editor: Manuel João Costa, University of Minho, Portugal Received February 7, 2013; Accepted August 23, 2013; Published September 25, 2013 Copyright: © 2013 Werner et al. Introduction each other prior to performing procedural skills on real patients [2-4]. Skills lab trainings have shown to improve procedural skills in novices as well as experts [5-8]. This applies to complex surgical skills [8] as well as basic clinical skills performed by medical students [9]. Furthermore there seems to The skills lab is an established part of a medical faculties’ training programme. It offers a protected, “mistake forgiving” training environment [1] that allows students to practice procedures on manikins, with standardized patients or with September 2013 \| Volume 8 \| Issue 9 \| e76354 PLOS ONE \| www.plosone.org 1 Long-Term Effect of Skills Lab Training be evidence, that simulation-based medical education (SBME) positively influences the outcome in the clinical setting [10,11]. individual feedback, practice on manikins and Peyton’s “Four- Step-Approach” is superior to traditional bedside teaching immediately after teaching [9]. However, these findings are solely based on performance assessments immediately after the respective teaching, and research comparing long-term effects is still lacking. Knowledge about long-term retention is crucial though, as medical students often experience a time lapse between their skills lab training and actual performance on patients. This happens even more so, since the importance of early clinical teaching in the pre-clinical phase has been stressed more intensively throughout many curricula worldwide [26,27]. In light of limited resources and an already high study load there is only limited capacity for repetitive classes. Hence, there is a clear need for established methods for effective SBME providing a maximum of retention. In a systematic review, Issenberg and colleagues describe aspects that influence the effectiveness of SBME [5]. The key factor seen is educational feedback, providing a chance for reflection on procedural performance. Other elements including “deliberate practice”, “integration into curriculum” and “validity of simulators” also contribute to the significant success of SBME. However, not much is known about the long-term retention of procedural skills acquired during SBME, although practical proficiencies are known to abate over time, if they are not repeatedly practised [12]. In general, theoretical knowledge seems to be retained better than practical skills, and the performance of simpler tasks seems to be lost more slowly than complex ones [13,14]. The majority of studies looking at the long-term retention of procedural skills focus on techniques in basic and advanced cardiac life support training. Introduction In this setting, a significant decline in performance could be shown to start as early as a couple of weeks after initial training or could begin up to a year later. The most significant decline occurred between 6 and 12 months of time [15-18]. The effectiveness and retention of other skills taught in the SBME setting have been studied less, and much heterogeneity is seen with regards to performed skills, study subjects and teaching methods, rendering data interpretation difficult. Examples include competencies in laparoscopic surgery or colonoscopy by surgical residents after 3 months [13,19], a significant decay in temporary haemodialysis catheter insertion skills by nephrology fellows after 6 months [20] and a satisfactory retention of a rare but crucial procedural skill like coniotomy performed by trained anaesthetist up to one year [21]. This heterogeneity in findings makes it hard if not impossible to draw any conclusions for skills lab training in medical undergraduates. In summary, our current understanding of factors contributing to long-term retention of SBME trained skills is still limited owing to general data shortage, flaws in study design (heterogeneity in training methods, number of redundant practice, etc.) and heterogeneity in tested skills with regards to their complexity. To our knowledge, so far there has been no randomized and prospective study investigating the effect of two different teaching approaches for undergraduate medical students for skills of different complexity with regards to long-term outcomes. We therefore investigated the effects of two different teaching methods within a simulated setting on the long-term performance of undergraduate medical students: a “best practice” example of skills lab training (BPSL) incorporating structured individual feedback, practice on manikins and Peyton’s “Four-Step-Approach” vs. a traditional “see one, do one” approach (TRAD) similar to bedside teaching. As task complexity is an important variable with regards to skills retention over time [14,19], we have chosen two skills with different complexity level for investigation, namely nasogastric tube insertion, NGT, as a simpler and i.v. cannulation, IVC, as a more complex procedure. As the time lapse between training and assessment is another important variable for retention, we assessed the students’ performance at 3 and 6 months respectively, resulting in a study design comprising four independent arms. Study design We conducted a randomised controlled trial to investigate the long-term retention of “best practice” skills lab training (BPSL) versus a traditional “see one, do one” bedside teaching (TRAD) in a simulated setting of two different procedural skills (nasogastral tube insertion, NGT, and i.v. cannulation, IVC) at undergraduate medical educational level. Performances were assessed twice for each student: immediately after training and at 3 or 6 months follow-up, respectively. Introduction Our data support a “best practice” form of SBME (BPSL) to be more effective than formerly used “see one, do one” approaches (TRAD), especially for the long-term retention of trained clinical skills with higher manual complexity. g y g p y Within a SBME setting, different teaching components comprise the “best practice” skills lab training. Amongst it are feedback and repetitive practice as key factors of effective SBME [5], and instruction methods like Peyton’s “Four-Step- Approach” which seems to provide a reliable and yet quite popular teaching method [22]. In this respect, it was implemented as standard instruction for resuscitation courses of the European Resuscitation Council [23]. There is, however, conflicting evidence whether skills lab teaching following a “best practice” approach (BPSL) leads to a better performance than other established teaching methods, for example a more traditional teacher-centred “see one, do one” approach (TRAD), which is a main component of clinical bedside teaching [24]. In this form of teaching, students learn by merely watching an experienced doctor explaining and demonstrating the skill [25]. The expert acts as a role model and the first independent performance of procedural clinical skills is already with a real patient. Two recent studies could show that skills lab training following a “best practice” model with structured Sample size A power analysis was undertaken to determine the necessary sample size. An effect size according to Cohen’s d = 1.2 was expected from training assessment data obtained from our previous studies [28,29]. For this study, we aimed at a power of ≥ 0.8. PLOS ONE \| www.plosone.org September 2013 \| Volume 8 \| Issue 9 \| e76354 2 Long-Term Effect of Skills Lab Training Long-Term Effect of Skills Lab Training Skills lab setting and teachers All teaching took place within DocLab, the skills lab of the Medical Faculty of Tübingen. This was done to make the teaching conditions as comparable as possible and to control for a maximum of possible confounders. At any given time, only one method (BPSL or TRAD) was taught to avoid cross- contamination. In total, 8 teachers ran the training sessions. All teachers were experienced student tutors of our skills lab whose equality in teaching performance as compared to faculty Student sample and Randomization procedure Inclusion criteria were: first year medical student at the University of Tübingen, within the first six months of training, and at the time of investigation not within any skills lab training. Students were excluded on the basis of the following criteria: previous training as a paramedic or nurse, prior experience in intravenous cannulation, urinary catheter or nasogastric tube insertion, and/or inability to attend the teaching sessions within the given timeframes. Therefore, the whole cohort of 1st-year medical students at the University of Tübingen in 2011 (n=358) was approached for participation in the study. The 95 voluntarily participating and eligible medical students were allocated by means of blocked randomisation to one of four groups: i) “best practice” skills lab training with follow-up after 3 months (BPSL3M: n=24), ii) “best practice” skills lab training with follow-up after 6 months (BPSL6M: n=23), iii) “see one, do one” with follow-up after 3 months (TRAD3M: n=24), and iv) “see one, do one” with follow-up after 6 months (TRAD6M: n=24). For details of study design and randomisation procedure see Figure 1. Pre-assessment Before the first teaching session, we recorded socio- economic and educational background data to ensure that there were no confounders among the four groups. In addition, the students’ general learning strategies were characterised by two standardized questionnaires: The Kolb Learning Style Inventory (LSI) [30,31] and the General Self-Efficacy Scale (GSE) [32]. “Best practice” skills laboratory training The two intervention groups (BPSL3M: n=22 and BPSL6M: n=22) trained both procedural skills (NGT and IVC) in the skills laboratory using structured individual feedback, performance on manikins and Peyton’s “Four-Step-Approach” [39,40] which consists of the following four steps: 1. The teacher demonstrates the skill at his normal speed without any comments (“Demonstration”). 2. The teacher repeats the procedure, this time describing all necessary sub-steps (“Deconstruction”). 3. The student has to explain each sub-step with the teacher following the student’s instructions (“Comprehension”). 4. The student performs the complete skill on his own (“Performance”). Each student was allowed to perform step 4 once for each skill. Each session was held in a group of three students with one teacher. The “Life/form Adult Venipuncture and Injection Training Arm”, a part-task-trainer model shaped like a human arm, was used for intravenous cannulation (Nasco, Fort Atkinson, USA). The “Nursing training manikin CLA 1+8” (CLA, Coburg, Germany), a whole body model, was manikin for nasogastric tube insertion. Both skill trainings were embedded into a clinical scenario with role-play to create a more realistic training situation and to enhance the students’ involvement [4,41]. Afterwards, students received feedback about their performance. The student:teacher ratio was 3:1. Skills classification Regarding the conceptual frameworks for methods, the current study was based on Ericsson’s model of deliberate practice with feedback as the basis of our skills lab training, and Bandura’s social learning theory as basis for the traditional bedside teaching [37,38]. The complexity of the skills was determined by 10 expert interviewers who rated them on a 10-point Likert scale (0= very easy to 10 = very complex). A rating below 5 was considered a simple skill. One simple and one more complex skill were chosen. “See one, do one” teaching The two other teaching groups (TRAD3M: n=23 and TRAD6M: n=24) received training according to the well-known teaching principle of “see one, do one”, commonly part of bedside teaching, within a simulated setting. In order to control for time differences arising due to practice in BPSL training, a thorough theoretical introduction on both procedures, NGT and IVC, was given to the students. This was followed by the practical part where the teachers showed both skills on the same manikins within the same skills laboratory setting as in the intervention groups. They explained each step while they were performing it. Students were asked to watch the performance attentively and were allowed to make themselves familiar with the material used but were not allowed to practise the skills within the teaching session itself. As with the “best practice” skills lab group, the student:teacher ratio was 3:1. The Kolb Learning Style Inventory aims to define an individual’s specific learning preference. It consists of 12 items ranked on a four-point Likert scale (4 = most like me to 1= least like me) and results in one out of four learning modules: Concrete Experience (CE) - Abstract Conceptualization (AC) - Reflective Observation (RO) - Active Experimentation (AE) [30,31]. The General Self-Efficacy Scale is also ranked on a four- point Likert scale (1 = I agree to 4 = I disagree) and assesses 10 items with regards to perceived self-efficacy [32]. Both LSI and GSE have been shown to provide a reliable measurement for students’ learning motivation and self- assessment; these two parameters were considered potential confounder in addition to former education or clinical practice [33,34]. Sample size Based on our preliminary studies, power analysis showed that n=15 students were needed for each of the four groups to detect the expected effect size (α=0.05; power 0.8). Of the 358 students approached, 121 medical students volunteered to participate. After initial screening according to our criteria, 95 were eligible to be included in the study. Three students failed to attend follow-up and another video was not assessable for technical reasons, therefore the final number of students whose performance could be completely assessed was 91 (BPSL3M: n=22, BPSL6M: n=22, TRAD3M: n=23, TRAD6M: n=24). Conceptual frameworks As conceptual frameworks for the learning content we used standard up-to-date manuals, which have been used regularly in our classes as well as for our previous studies [35,36]. PLOS ONE \| www.plosone.org September 2013 \| Volume 8 \| Issue 9 \| e76354 3 Long-Term Effect of Skills Lab Training . Study design and randomisation process. BPSL = “best practice” skills lab, TRAD = “see one, do one e Structured Clinical Examination, 3M = follow-up after 3 months, 6M = follow up after 6 months * = video m 1/journal.pone.0076354.g001 Figure 1. Study design and randomisation process. BPSL = “best practice” skills lab, TRAD = “see one, do one”, OSCE = Objective Structured Clinical Examination, 3M = follow-up after 3 months, 6M = follow up after 6 months * = video material not useable. doi: 10.1371/journal.pone.0076354.g001 September 2013 \| Volume 8 \| Issue 9 \| e76354 PLOS ONE \| www.plosone.org 4 Long-Term Effect of Skills Lab Training Long-Term Effect of Skills Lab Training Statistical analysis staff has been shown previously [29]. They were randomised to one of the teaching methods. Additionally, the teachers received thorough instructions for their respective teaching session and a detailed manual including defined learning goals, a comprehensive teaching agenda and detailed information about the time available for each section of the teaching session. Intervention group teachers received a refresher in Peyton’s “Four-Step-Approach” [39,40]. All teachers were blinded to the study design and only taught one method (BPSL or TRAD) to avoid any reciprocal interference in teaching style. All data are presented as means ± standard error of the mean (SEM), if not otherwise stated. Data were tested for normal distribution using the Shapiro-Wilk-Test. Normally distributed data were compared using a Student’s t-test (assuming equal variances). A Mann-Whitney U-Test (MWU) was used for non-normally distributed parameters. For reader’s convenience, results of the MWU tests are not displayed as sum of ranks. The distribution of discontinuous group characteristics was compared by the chi-square test. Effect sizes were calculated using Cohen’s d for continuous variables and Cramer’s φ for 2-level associations. Standardized inter- rater reliability for the two video assessors was calculated based on Kappa analysis. A p-value <0.05 was considered to be statistically significant. Raw data were processed using Microsoft EXCEL (Microsoft Inc., Redmond, WA, USA). The software packages JMP (SAS Institute Inc., Cary, NC, USA) and SPSS (SPSS Inc., Chicago, IL, USA) were used for statistical analysis. Video rating For videotaping, high-resolution cameras with optical zoom were used. Afterwards, all videos were digitally processed and randomised in regard to the playing sequence so that no conclusions could be drawn from it. Two blinded video assessors evaluated students’ performance according to a binary checklist and a global rating form, both already used in our previous studies [28,29,42]. Video assessors were encouraged to fill in the boxes as soon as students showed the respective step. The single-item global rating on a six-point Likert-scale (6 = very good to 1 = unsatisfactory) referred to the overall performance and was given at the end of intravenous cannulation or nasogastric tube insertion. It was furthermore categorised into “competent students” (rated as ‘6’ and ‘5’), “borderline students” (rated as ‘4’ and ‘3’) and “incompetent students” (rated as ‘2’ and ‘1’). Assessment of trained skills After the teaching sessions, we immediately videotaped the students’ performance at two assessment stations (nasogastric tube insertion, i.v. cannulation) on the same mannequins as used in training comparable to the ones used in OSCEs (Objective Structured Clinical Examination). Each student was alone in the assessment room and had only one attempt to perform the skill learned. The other students from the corresponding teaching group waited in a different room with a supervisor present until it was their respective turn for individual assessment. The total amount of time needed was recorded for each skill at both assessments dates. Two blinded video-assessors rated the performance according to pre- defined binary and global checklists. Ethics Study participation was voluntary and all students were assured of anonymity and confidentiality. Students were informed that the purpose of the study was the comparison of different ways of teaching but they did not receive any details. The ethics committee of the University of Tübingen waived the requirement of further ethical approval based on the condition that all data were analyzed anonymously (Nr. 539/2012A and 296/2008A). However, written consent was obtained from all students. According to group randomisation, we re-invited students after either 3 or 6 months. This time, they did not receive any teaching but were asked to perform both skills again on the same manikin in the same environment as before. Once more, students were assessed alone according to the method (BPSL or TRAD). There was an assistant present to take care of any students who arrived before their allocated assessment time. Again, students were videotaped to be rated by video- assessors via the identical checklists. All students signed an agreement not to talk to their fellow students about their experienced teaching method and not to practise the skills in between assessment date one (OSCE I) and two (OSCE II, see Figure 1). Skills complexity Skills rating by experts for NGT was 1.7±1.1 and for IVC 6.5±1.1. Teaching sessions Length of teaching sessions did not differ significantly between the four different teaching groups (BPSL3m 89.6±1.0 min, BPSL6m 89.5±0.8 min, TRAD3m 89.9±0.7 min, TRAD6m 89.7±0.8 min, pANOVA=0.58). Student sample P-values p1, p2 and p3 refer to the following comparisons: p1: TRAD (3M and 6M pooled together)” and “BPSL (3M and 6M pooled together)” BPSL = “best practice” skills lab teaching, TRAD = traditional “see one, do one” teaching, 3M = assessed 3 months after training, 6M = assessed 6 months after training, ANOVA = analysis of variance doi: 10.1371/journal.pone.0076354.t001 Assessment at examination stations by binary checklists. The number of correctly performed steps for nasogastric tube insertion and i.v. cannulation identifiable on the video tapes was calculated as the percentage of maximal achievable binary checklist points (NGT: 26 points, IVC: 29 points). Immediately after teaching, students from our intervention groups trained via “best practice” skills lab teaching scored significantly higher at performance of NGT insertion (BPSL3M 94.8%±0.2, BPSL6M 95.4%±0.3) and IVC (BPSL3M 86.4%±0.5, BPSL6M 88.0%±0.5) than the comparison group receiving only teaching according to the traditional “see one, do one” approach (NGT: TRAD3M 86.1%±0.5, TRAD6M 84.7%±0.4 and IVC: TRAD3M 73.2%±0.7, TRAD6M 72.5%±0.7). According to these ratings, BPSL resulted in an effect size (Cohen’s D) of 1.32/1.36 (NGT/IVC) and 1.57/1.64 (NGT/IVC) after 3 and 6 months, respectively, compared to TRAD. Within each group, there was a significant loss of performance when tested after 3 months or 6 months respectively. The obtained percentage scores according to the binary checklists of all participants at the first (t0) and respective second assessment station (t1 after 3 or 6 months, respectively) are shown in Table 2. The corresponding percentages and p-values of skill decay according to each skill Assessment at examination stations by binary checklists. The number of correctly performed steps for nasogastric tube insertion and i.v. cannulation identifiable on the video tapes was calculated as the percentage of maximal achievable binary checklist points (NGT: 26 points, IVC: 29 points). Immediately after teaching, students from our intervention groups trained via “best practice” skills lab teaching scored significantly higher at performance of NGT insertion (BPSL3M 94.8%±0.2, BPSL6M 95.4%±0.3) and IVC (BPSL3M 86.4%±0.5, BPSL6M 88.0%±0.5) than the comparison group receiving only teaching according to the traditional “see one, do one” approach (NGT: TRAD3M 86.1%±0.5, TRAD6M 84.7%±0.4 and IVC: TRAD3M 73.2%±0.7, TRAD6M 72.5%±0.7). According to these ratings, BPSL resulted in an effect size (Cohen’s D) of 1.32/1.36 (NGT/IVC) and 1.57/1.64 (NGT/IVC) after 3 and 6 months, respectively, compared to TRAD. Student sample All participating students were in their first year. The average age of the complete study cohort was 21.4±0.5 years. A total of 26 out of 91 students were male. With two exceptions (gender distribution between BPSL3m and TRAD3m; prior practical nursing days between BPSL and TRAD at baseline), there were no significant differences between the four randomized groups regarding socio-demographic variables, former health care education, previous clinical experience or scores in the above described standardized questionnaires LSI [30,31] and GSE [32] as shown in table 1 (all p>0.06). PLOS ONE \| www.plosone.org September 2013 \| Volume 8 \| Issue 9 \| e76354 5 Long-Term Effect of Skills Lab Training Table 1. Basic socio-demographic characteristics of participants. Sociodemographic variables BPSL 3M (n=23) BPSL 6M (n=23) TRAD 3M (n=22) TRAD 6M (n=23) ANOVA p1 p2 p3 Gender [male/female] 5/18 6/17 8/14 7/16 - 0.32a 0.02 0.74 Age [years] 20.9 [19.8;22.0] 21.6 [19.5;23.8] 20.2 [19.5;21.0] 22.9 [19.9;25.8] .23 0.38b 0.75 0.13 Prior HealthCare Education [yes/no] 1 4/19 3/20 2/20 2/21 - 0.35a 0.41 0.64 Prior Study [yes/no] 2 5/18 3/20 4/18 6/17 - 0.56a 0.77 0.26 Practical Nursing [days] 22.3 [7.1;37.5] 41.6 [26.2;57.1] 45.7 [28.5;62.8] 55.4 [37.5;73.3] 0.07 0.04b 0.06 0.21 Prior Blood Sampling Procedures [yes/no] 0.7 [0;1.4] 2.2 [0.3;4.1] 2.6 [0;7.4] 0.3 [0;0.5] .35 0.35b 0.71 0.10 Prior Nasogastric Tube Procedures [yes/no] 0 0 0 0 - 1b 1 1 Prior Intravenous Cannulation Procedures [yes/no] 0 0 0 0 - 1b 1 1 Handedness [left/right] 1/22 1/22 2/20 2/21 - 0.40a 0.55 0.55 Questionnaires BPSL 3M (n=23) BPSL 6M (n=23) TRAD 3M (n=22) TRAD 6M (n=23) ANOVA p1 p2 p3 General Self-Efficacy Scale [40-10] [32] 20.0 [18.4;21.7] 21.0 [18.6;23.4] 19.3 [16.4;22.2] 20.4 [17.6;23.1] 0.76 0.88c 0.64 0.73 Kolb LSI [10-40] [30,31] Abstract_Conceptualization Concrete_Experience Active_Experimentation Reflective_Observation 32 [0;47] 25 [0;36] 35 [0;44] 29 [0;39] 31 [0;43] 21 [0;40] 32 [0;44] 28 [0;40] 34 [0;46] 22 [0;36] 34 [0;43] 31 [0;40] 32 [0;43] 23 [0;40] 33 [0;45] 28 [0;41] 0.82 0.58 0.89 0.63 0.81b 0.76b 0.72b 0.43b 0.87 0.25 0.55 0.13 0.81 0.72 1 0.77 All data are presented as means with the 95% confidence intervals provided in square brackets, except the results from the Kolb Learning Style Inventory (LSI) which are shown as medians [min; max]. 1Prior HealthCare education included: biological technical assistant, biologist, medical technical assistant, physiotherapist, social care worker, surgical technologist. Video-rating Percentages of maximal achievable points with the 95%CI provided in squared brackets on binary checklist (NGT = 26 points, IVC = 29 points). 1Cohen’s d was calculated using means and standard deviations of achieved binary checklist points. BPSL (TRAD) was considered as treatment group (control group). 2p-values were calculated using the Mann-Whitney-U test on ranks. each follow-up (3 or 6 months, respectively). Interestingly, BPSL training led to better assessment results, in particular for IVC which represents a task of higher complexity, while NGT as a lower complexity skill made its benefit from BPSL training primarily in regard to its long-term retention (see Figure 3). It should be emphasized that the resulting effect sizes attributed to BPSL can be considered quite large. This was surprising given the long time interval between the assessments (3 and 6 months, respectively) and the short intervention time (90 min each for both BPSL and TRAD teaching sessions). and time of assessment are depicted in Figure 2. BPSL students lost significantly less of their performance than their TRAD fellows at all times of assessment (see Figure 2). Assessment at examination stations by single-item global rating. The single global item “Overall ability to perform the procedure” showed similarly, that the BPSL group was rated more often clinically competent than the TRAD group for both tested skills (NGT, IVC) at all times of assessment (t0 immediately after teaching, t3M after 3 months and t6M after 6 months; see Figure 3). The categorisation into “competent”, “borderline”, and “incompetent” particularly showed the superiority of BPSL students (see table 3). NGT, as a clinical skill of lower complexity experienced a slightly greater benefit from BPSL training than IVC representing a task of higher procedural complexity. Being trained in the “best practice” skills lab group also led to a significantly shorter time needed for performance of both skills, despite the fact that both tested teaching methods occupied the identical resources in teaching time. Furthermore, BPSL students were significantly more often rated as “competent” than TRAD students. To our knowledge, the long-term retention of skills taught to medical students comparing a “best practice” model of skills lab training and a “see one, do one” approach as frequently used in clinical bedside teaching has not been investigated so far. Inter-rater reliability Standardised inter-rater reliability ranged from 0.734 to 0.870 (p<0.001) for binary checklists and 0.911 to 0.931 (p<0.001) for the single-item global rating indicating a good to very good agreement. Video-rating Most of the research contrasting simulation-based medical education (SBME) and traditional teaching methods to date has focused on residents, and was either concerned with complex surgical or intensive care procedures [43,44] or refers to cardiac life support training [18]. It is well known, that straight after training SBME with “deliberate practice” is superior to traditional clinical teaching in the acquisition of a broad variety of skills [38,45]. We could show that on top of the immediate effect, there is a long term benefit of “best practice” skills lab training with structured individual feedback, training on manikins and Peyton’s “Four-Step-Approach” improves students’ performance in a relatively simple task (nasogastric tube insertion) as well as a more complex one (intravenous cannulation). There is a relative superiority of BPSL after 3 or 6 months and a smaller loss of correctly performed steps in absolute terms. Video-rating Time needed for performance of skills. Time was measured from picking up the first item until the student announced the end of the procedure. There was a significant difference between the BPSL and the TRAD group performance time at t0, measured immediately after teaching, for both NGT (TRAD3M/6M 335±20 sec, BPSL3M/6M 294±16 sec, p<0.001) and IVC (TRAD3M/6M 657±52 sec, BPSL3M/6M 522±36 sec; p<0.001). The significant difference in favour of a lower performance time needed in the BPSL group remained stable at each respective long-term assessment for both skills (data not shown). Within each group, there was a significant loss of performance when tested after 3 months or 6 months respectively. The obtained percentage scores according to the binary checklists of all participants at the first (t0) and respective second assessment station (t1 after 3 or 6 months, respectively) are shown in Table 2. The corresponding percentages and p-values of skill decay according to each skill PLOS ONE \| www.plosone.org September 2013 \| Volume 8 \| Issue 9 \| e76354 6 Long-Term Effect of Skills Lab Training Table 2. Percentages of maximal achievable points with the 95%CI provided in squared brackets on binary checklist (NGT = 26 points, IVC = 29 points). t0 (immediately after teaching) t1 (after 3 months) TRAD 3M BPSL 3M Cohen D1 p2 TRAD 3M BPSL 3M Cohen D1 p2 NGT 86.1% [82.4;89.8] 94.8% [93.2;96.6] 1.32 <0.0001 70.3% [65.9;74.7] 86.3% [83.6;88.9] 1.59 <0.0001 IVC 73.2% [68.4;78.0] 86.4% [83.0;89.7] 1.36 <0.0001 56.5% [53.2;59.8] 79.5% [75.9;83.0] 2.87 <0.0001 t0 (immediately after teaching) t1 (after 6 months) TRAD 6M BPSL 6M Cohen D1 p2 TRAD 6M BPSL 6M Cohen D1 p2 NGT 84.7% [81.3;88.1] 95.4% [93.1;97.7] 1.57 <0.0001 65.4% [60.4;70.4] 89.0% [86.9;91.1] 2.64 <0.0001 IVC 72.5% [67.7;77.2] 88.0% [84.7;91.4] 1.64 <0.0001 51.5% [45.7;57.3] 73.2% [70.6;75.8] 2.07 <0.0001 3M = follow-up after 3 months, 6M = follow-up after 6 months. NGT = nasogastral tube insertion, IVC = intravenous cannulation. BPSL = “best practice” skills lab teaching, TRAD = traditional “see one, do one” teaching. 1Cohen’s d was calculated using means and standard deviations of achieved binary checklist points. BPSL (TRAD) was considered as treatment group (control group). 2p-values were calculated using the Mann-Whitney-U test on ranks. doi: 10.1371/journal.pone.0076354.t002 Table 2. Percentages of maximal achievable points with the 95%CI provided in squared brackets on binary checklist (NGT = 26 points, IVC = 29 points). Table 2. Discussion This study prospectively investigated the long-term retention of two different skills (nasogastric tube insertion; intravenous cannulation) taught in two different ways (“best practice” skills lab training and a “see one, do one” approach) to first year medical students. Following assessment after training, students were re-invited for a second assessment either 3 or 6 months after initial training sessions according to randomisation. Students were carefully selected according to in- and exclusion criteria and randomized to one of the four groups. There were no significant differences in socioeconomic background and other potentially influencing variables among the four different study cohorts. Students who received the “best practice” model of skills lab training showed significantly better results measured with binary checklists as well as a single-item global rating at measurement immediately after teaching (T0) and Within the “best practice” skills lab training, various factors contributed to the teaching: First, BPSL teachers acted as role models when showing the skill, helping students to observe the correct procedure. Secondly, we included Peyton’s “Four-Step- 7 PLOS ONE \| www.plosone.org September 2013 \| Volume 8 \| Issue 9 \| e76354 Long-Term Effect of Skills Lab Training oach” [22], which implies a rather unusual step, namely cting the teacher step-by-step how to perform the skills shown to be a crucial step in memory consolidation [46 Additionally BPSL students had the advantage of “learnin re 2. Loss of skills level expressed as percentage of points on the corresponding binary checklist (NGT or IVC) at re 2A) and 6 months (Figure 2B) after initial training, respectively (error bars refer to SEM). NGT = nasogastral tub tion, IVC = intravenous cannulation, BSPL = Best Practice Skills Lab Training, TRAD = Traditional “see one, do one” teaching. 1371/journal.pone.0076354.g002 Figure 2. Loss of skills level expressed as percentage of points on the corresponding binary checklist (NGT or IVC) at 3 (Figure 2A) and 6 months (Figure 2B) after initial training, respectively (error bars refer to SEM). NGT = nasogastral tube insertion, IVC = intravenous cannulation, BSPL = Best Practice Skills Lab Training, TRAD = Traditional “see one, do one” teaching. doi: 10.1371/journal.pone.0076354.g002 Figure 2. Loss of skills level expressed as percentage of points on the corresponding binary checklist (NGT or IVC) at 3 (Figure 2A) and 6 months (Figure 2B) after initial training, respectively (error bars refer to SEM). Discussion NGT t0 t1 t2 p1 (Cramer’s ϕ) p2 (Cramer’s ϕ) p3 (Cramer’s ϕ) BPSL 1 = 45 (97.8) 2 = 1 (2.2) 3 = 0 (0.0) 1 = 13 (56.5) 2 = 10 (43.5) 3 = 0 (0.0) 1 = 19 (82.6) 2 = 3 (13.0) 3 = 1 (4.4) 0.0008 (0.35) 0.0003 (0.60) <0.0001 (0.70) TRAD 1 = 33 (73.4) 2 = 12 (26.6) 3 = 0 (0.0) 1 = 2 (9.1) 2 = 12 (54.5) 3 = 8 (36.4) 1 = 3 (13.0) 2 = 10 (43.5) 3 = 10 (43.5) IVC t0 t1 t2 p1 (Cramer’s ϕ) p2 (Cramer’s ϕ) p3 (Cramer’s ϕ) BPSL 1 = 41 (89.1) 2 = 5 (10.9) 3 = 0 (0.0) 1 = 11 (47.8) 2 = 10 (43.5) 3 = 2 (8.7) 1 = 5 (21.7) 2 = 18 (78.3) 3 = 0 (0.0) <0.0001 (0.60) 0.0001 (0.63) <0.0001 (0.77) TRAD 1 = 14 (31.1) 2 = 25 (55.6) 3 = 6 (13.3) 1 = 1 (4.6) 2 = 7 (31.8) 3 = 14 (63.6) 1 = 1 (4.4) 2 = 5 (21.7) 3 = 17 (73.9) t0 = immediately after teaching, t1 = after 3 months, t2 = after 6 months. Percentages are shown in round brackets. NGT = nasogastral tube insertion, IVC = intravenous cannulation. BPSL = “best practice” skills lab teaching, TRAD = traditional “see one, do one” teaching. P-values were calculated using Chi2-test. p1: TRAD(3months and 6months pooled together) vs. BPSL(3months and 6months pooled together), p2: TRAD3months vs. BPSL3months, and p3: TRAD6months vs. BPSL6months. The effect sizes were calculated using Cramer’s phi. doi: 10.1371/journal.pone.0076354.t003 necessarily reflect long-term learning [50]. In the current study, students trained in skills labs were not only better at immediate assessment but also at the respective follow-up assessment after 3 or 6 months, respectively. As another component of the “best practice” skills lab training, BPSL students received structured individual feedback on their own performance. This is well recognised to represent a key feature of effective skills lab training [5]. Simulators alone are not enough to improve skill performance [51]. Feedback helps medical students to get a feeling for what they do and increases the likelihood of correct performance [52]. On the other hand, research in motor learning shows that feedback from instructors improves immediate performance but can be a hindrance for long-term learning [53]. Discussion In our study this was not supported, as the “best practice” skills lab teaching group had more feedback but performed better in the long run. This might be due to other components in line with the concept of self-regulated learning like review at each stage and self-monitoring as described above. Finally, by integrating the aspect of role-play into BPSL teaching a more realistic training scenario was created [4] g, g [ ] On the contrary, the alternative teaching followed the “see one – do one” principle, usually common in clinical bedside teaching. TRAD Students’ only way of learning the skill was through attentive observation as in line with the social learning theory [37]. Tutors acted as role models by demonstrating each skill to the students. However, all other components described as part of skills lab teaching were completely missing, and students never had the opportunity to practise the skill before the first assessment, as this is not part of a traditional bedside situation. This might explain their inferiority to BPSL students. Nonetheless, in general, there was a decline in the ability to perform both skills in nearly all students. Only 7 students out of the 91 (3 BPSL, 4 TRAD) showed an improvement from t0 to their respective long-term follow-up. This is likely explained by the fact that they were all amongst the bottom performers at baseline point and, thus profited by the training effect of a 2nd assessment. We added a global rating to the binary checklist in order to balance for the fact that a checklist ranks all items in the same way, potentially leading to failure because of several less important items rather than one big mistake [54]. Our good inter-rater reliability for both measurements validates our methodological approach to measure skill performance. Additionally, global ratings take into account the difference between competence shown in an assessment situation and performance shown under real life circumstances as described by Rethans et al. [55]. Furthermore, we tried to improve the validity of our skills training and testing by creating a role-play scenario as validity improves effective learning [4,56]. they could see the immediate implication as the tutor followed all instructions given by the student regardless of them being correct or not. This gives immediate feedback and allows students to monitor their progress. Discussion NGT = nasogastral tube insertion, IVC = intravenous cannulation, BSPL = Best Practice Skills Lab Training, TRAD = Traditional “see one, do one” teaching. doi: 10.1371/journal.pone.0076354.g002 Approach” [22], which implies a rather unusual step, namely instructing the teacher step-by-step how to perform the skills. This comprises the necessity to review all steps on their own and also reflect upon the procedure itself. Reflection has been shown to be a crucial step in memory consolidation [46]. Additionally, BPSL students had the advantage of “learning through teaching” by instructing someone else in step 3 of Peyton’s “Four-Step-Approach”. If students made a mistake, PLOS ONE \| www.plosone.org September 2013 \| Volume 8 \| Issue 9 \| e76354 8 Long-Term Effect of Skills Lab Training Figure 3. Single-item global rating of performance (mean + SEM) initially after training (t0) and at 3 (t3M) and 6 months (t6M) later, respectively. NGT = nasogastral tube insertion, IVC = intravenous cannulation, TRAD = traditional “see one, do one” training, BPSL = Best Practice Skills Lab Training. Significant differences (p<0.05) are marked with an asterisk. doi: 10.1371/journal.pone.0076354.g003 Figure 3. Single-item global rating of performance (mean + SEM) initially after training (t0) and at 3 (t3M) and 6 months (t6M later, respectively. NGT = nasogastral tube insertion, IVC = intravenous cannulation, TRAD = traditional “see one, do one training, BPSL = Best Practice Skills Lab Training. Significant differences (p<0.05) are marked with an asterisk. doi: 10.1371/journal.pone.0076354.g003 Figure 3. Single-item global rating of performance (mean + SEM) initially after training (t0) and at 3 (t3M) and 6 months (t6M) later, respectively. NGT = nasogastral tube insertion, IVC = intravenous cannulation, TRAD = traditional “see one, do one” training, BPSL = Best Practice Skills Lab Training. Significant differences (p<0.05) are marked with an asterisk. doi: 10.1371/journal.pone.0076354.g003 September 2013 \| Volume 8 \| Issue 9 \| e76354 PLOS ONE \| www.plosone.org 9 Long-Term Effect of Skills Lab Training they could see the immediate implication as the tutor followed Table 3. Global rating categorised into 1 = “competent students”, 2 = “borderline students” and 3 = “incompetent students”. September 2013 \| Volume 8 \| Issue 9 \| e76354 Discussion This kind of metacognitive awareness is a hallmark feature of self-regulated learning, which is an approach that actively incorporates students into the learning process [47]. Students are encouraged to show initiative and take responsibility for what and how they learn [48]. Particularly in its directed form as described by Brydges and colleagues, self-regulated learning can lead to superior long-term performance when compared to an instructor- regulated approach [49]. This could help to explain why our results do not seem to be in line with the “performance– learning paradox”, which refers to the common finding that immediate performance can be quite good but does not they could see the immediate implication as the tutor followed all instructions given by the student regardless of them being correct or not. This gives immediate feedback and allows students to monitor their progress. This kind of metacognitive awareness is a hallmark feature of self-regulated learning, which is an approach that actively incorporates students into the learning process [47]. Students are encouraged to show initiative and take responsibility for what and how they learn [48]. Particularly in its directed form as described by Brydges and colleagues, self-regulated learning can lead to superior long-term performance when compared to an instructor- regulated approach [49]. This could help to explain why our results do not seem to be in line with the “performance– learning paradox”, which refers to the common finding that immediate performance can be quite good but does not We deliberately used student tutors as teachers who are teaching in our official classes within the curriculum. They have been shown to be equally effective and accepted as staff [29,57] and student tutors are meanwhile part of most faculties’ skills lab training [58]. Additionally, all tutors participating in our study received an elaborate manual as well as a training session before teaching the students. In line with well-matched skills training, most students are being taught simple as well as more complex skills early on 10 PLOS ONE \| www.plosone.org September 2013 \| Volume 8 \| Issue 9 \| e76354 Long-Term Effect of Skills Lab Training Conclusions from the beginning of their studies. This is the first step of a process building up to the transfer of these skills to actual patient care. Following the results of our study, some recommendations for skills teaching can be made: Skills lab training comprising different teaching elements like structured individual feedback, practise on manikins and Peyton’s “Four- Step-Approach” should be an inherent part of undergraduate medical education. To optimise long-term outcome, these skills should be refreshed in at least biannual intervals for more complex skills, and annually for easier tasks. Further studies have to examine whether supervised practice outside regular curricular activities can also serve this purpose. In summary, we could show that a best practice skills lab training of intravenous cannulation and nasogastric tube insertion skills is superior to the traditional “see one, do one” approach not only immediately after training, but also at 3 or 6 months follow up. This observed superiority applies for single steps of the procedures, time needed to perform the skills, and the global clinical impression. With regards to the long-term performance, skills lab teaching seems to be particularly helpful for the reproduction of easier skills. In line with previous studies that showed superiority of skills lab training immediately after teaching and in transfer to real patients for the two skills [9,40], this study underlines the importance of skills lab training being an integral part of teaching students with respect to long-term performance. Regular fresher classes have to be provided when our students are supposed to show stable performances. We suggest a biannual interval for more complex tasks and an annual one for easier tasks. Acknowledgements We would like to thank Friederike Holderried (MD) for her constructive suggestions and organisational support, as well as Clare Blythe (Research Assistant) and Christopher Garrrett MD for proofreading the manuscript. Furthermore, we would like to thank the DocLab team Tübingen (www.doc-lab.de), and in particular all DocLab student tutors for their commitment and passion in delivering the teaching. Ethik No. 539/2012A and 296/2008A. Author Contributions Conceived and designed the experiments: AH-W CN FL NC PW. Performed the experiments: AH-W KK HMB PW CN. Analyzed the data: AH-W PW RW. Wrote the manuscript: AH- W PW CN. General supervisor of the study and was most helpful in assisting throughout: SZ. Involved in proofreading the article and gave their final approval: KK HMB RW NC FL SZ. Limitations Several limitations of our study should be mentioned. As the study was done on a voluntary basis, there is of course the possibility of a selection bias with only the very motivated students showing up. However, there were a lot more students registering in the first place that had to be excluded due to exclusion criteria or time incompatibilities. Additionally, careful randomisation of all applicants to one of our four groups was performed. Further studies should investigate whether these findings hold up when transferred to real patients. Additionally, research in this field should focus on clarification studies as suggested by Cook et al., asking for how and why skills lab training seems to be so superior to the traditional “see one, do one” training provided by bedside teaching [60]. Furthermore, we cannot exclude that some students practised the tasks on their own, although all students signed an agreement not to do so and additionally gave oral confirmation on their second testing day about it. In any case, it is very unlikely that they had a structured training in the meantime. Another limitation is the fact that all teaching took place in a simulated environment. This limits the generalizability of our findings with regards to clinical context. Additionally, students taught within the skills lab could practise each skill one time before doing the actual test in the assessment stations as opposed to bedside teaching students who had only seen someone else performing the task. However, this was part of one of the components included into the skills lab teaching, namely Peyton’s “Four-Step-Approach”. At the same time, the traditional “see one, do one” approach without this possibility to practise has been recognised as an appropriate teaching method, too [59]. Additionally, we made all other conditions like time or student to tutor ratio the same for both groups. Another limitation may be seen in that we did not undertake any assessment of students’ prior abilities. We deliberately waived this possibility during designing the study in order to minimize any training effect due to repetitive testing. 4. Nikendei C, Zeuch A, Dieckmann P, Roth C, Schäfer S et al. (2005) Role-playing for more realistic technical skills training. Med Teach 27: 122-126. doi:10.1080/01421590400019484. PubMed: 16019330. 1. Ziv A, Ben-David S, Ziv M (2005) Simulation based medical education: an opportunity to learn from errors. Med Teach 27: 193-199. doi: 10.1080/01421590500126718. PubMed: 16011941. 5. Issenberg SB, McGaghie WC, Petrusa ER, Lee Gordon D, Scalese RJ (2005) Features and uses of high-fidelity medical simulations that lead to effective learning: a BEME systematic review. Med Teach 27: 10-28. doi:10.1080/01421590500046924. PubMed: 16147767. 7. Khan K, Pattison T, Sherwood M (2011) Simulation in medical education. Med Teach 33: 1-3. doi:10.3109/0142159X.2011.530320. PubMed: 21182376. 6. Jiang G, Chen H, Wang S, Zhou Q, Li X et al. (2011) Learning curves and long-term outcome of simulation-based thoracentesis training for medical students. BMC Med Educ 11: 39. doi: 10.1186/1472-6920-11-39. PubMed: 21696584. 2. Barrows HS (1993) An overview of the uses of standardized patients for teaching and evaluating clinical skills. AAMC. Acad Med J Assoc Am Med Colleges 68: 443-451; discussion 451-443 doi: 10.1097/00001888-199306000-00002. 5. Issenberg SB, McGaghie WC, Petrusa ER, Lee Gordon D, Scalese RJ (2005) Features and uses of high-fidelity medical simulations that lead to effective learning: a BEME systematic review. Med Teach 27: 10-28. doi:10.1080/01421590500046924. PubMed: 16147767. 6. Jiang G, Chen H, Wang S, Zhou Q, Li X et al. (2011) Learning curves and long-term outcome of simulation-based thoracentesis training for medical students. BMC Med Educ 11: 39. doi: 10.1186/1472-6920-11-39. PubMed: 21696584. 7. Khan K, Pattison T, Sherwood M (2011) Simulation in medical education. Med Teach 33: 1-3. doi:10.3109/0142159X.2011.530320. PubMed: 21182376. 5. Issenberg SB, McGaghie WC, Petrusa ER, Lee Gordon D, Scalese RJ (2005) Features and uses of high-fidelity medical simulations that lead to effective learning: a BEME systematic review. Med Teach 27: 10-28. doi:10.1080/01421590500046924. PubMed: 16147767. Long-Term Effect of Skills Lab Training PubMed: 18406037. 37. Bandura A (1977) Social Learning Theory. New York: General Learning Press. 38. Ericsson KA (2004) Deliberate practice and the acquisition and maintenance of expert performance in medicine and related domains. Acad Med J Assoc Am Med Colleges 79: S70-S81. doi: 10.1097/00001888-200407001-00018. PubMed: 15383395. 15. Anderson GS, Gaetz M, Masse J (2011) First aid skill retention of first responders within the workplace. Scand J Trauma Resusc Emerg Med 19: 11. doi:10.1186/1757-7241-19-11. PubMed: 21303536. 16. Duran R, Aladağ N, Vatansever U, Küçükuğurluoğlu Y, Süt N et al. (2008) Proficiency and knowledge gained and retained by pediatric residents after neonatal resuscitation course. Pediatr Int 50: 644-647. doi:10.1111/j.1442-200X.2008.02637.x. PubMed: 19261112. 39. Peyton JWR (1998) Teaching and Learning in Medical Practice. Rickmansworth, UK: Manticore Publishing House Europe Limited. 40. Krautter M, Weyrich P, Schultz JH, Buss S, Maatouk I et al. (2011) Effects of Peyton’s Four Step Approach on objective performance measures in technical skills training - a controlled trial. Teach Learn Med (in press). 17. Ruetzler K, Roessler B, Potura L, Priemayr A, Robak O et al. (2011) Performance and skill retention of intubation by paramedics using seven different airway devices--a manikin study. Resuscitation 82: 593-597. doi:10.1016/j.resuscitation.2011.01.008. PubMed: 21353364. 41. Nikendei C, Kraus B, Lauber H, Schrauth M, Weyrich P et al. (2007) An innovative model for teaching complex clinical procedures: integration of standardised patients into ward round training for final year students. Med Teach 29: 246-252. doi:10.1080/01421590701299264. PubMed: 17701640. 18. Yang CW, Yen ZS, McGowan JE, Chen HC, Chiang WC et al. (2012) A systematic review of retention of adult advanced life support knowledge and skills in healthcare providers. Resuscitation 83: 1055-1060. doi: 10.1016/j.resuscitation.2012.02.027. PubMed: 22391016. 42. Regehr G, MacRae H, Reznick RK, Szalay D (1998) Comparing the psychometric properties of checklists and global rating scales for assessing performance on an OSCE-format examination. Acad Med 73: 993-997. doi:10.1097/00001888-199809000-00020. PubMed: 9759104. 19. Snyder CW, Vandromme MJ, Tyra SL, Hawn MT (2010) Retention of colonoscopy skills after virtual reality simulator training by independent and proctored methods. Am Surg 76: 743-746. PubMed: 20698383. 20. Ahya SN, Barsuk JH, Cohen ER, Tuazon J, McGaghie WC et al. (2012) Clinical performance and skill retention after simulation-based education for nephrology fellows. Semin Dial 25: 470-473. doi: 10.1111/j.1525-139X.2011.01018.x. PubMed: 22309946. 43. Long-Term Effect of Skills Lab Training Supe A, Prabhu R, Harris I, Downing S, Tekian A (2012) Structured training on box trainers for first year surgical residents: does it improve retention of laparoscopic skills? A randomized controlled study. J Surg Educ 69: 624-632. doi:10.1016/j.jsurg.2012.05.002. PubMed: 22910161. 21. Boet S, Borges BC, Naik VN, Siu LW, Riem N et al. (2011) Complex procedural skills are retained for a minimum of 1 yr after a single high- fidelity simulation training session. Br J Anaesth 107: 533-539. doi: 10.1093/bja/aer160. PubMed: 21659406. 44. Smith CC, Huang GC, Newman LR, Clardy PF, Feller-Kopman D et al. (2010) Simulation training and its effect on long-term resident performance in central venous catheterization. Simul Healthc 5: 146-151. doi:10.1097/SIH.0b013e3181dd9672. PubMed: 20651476. j 22. Peyton J (1998) Teaching in the theatre. In: J Peyton. Teaching and learning in medical practice. Rickmansworth, UK: Manticore Publishing House Europe, Ltd.. pp. 171-180. 45. McGaghie WC, Issenberg SB, Cohen ER, Barsuk JH, Wayne DB (2011) Does simulation-based medical education with deliberate practice yield better results than traditional clinical education? A meta- analytic comparative review of the evidence. Acad Med J Assoc Am Med Colleges 86: 706-711. doi:10.1097/ACM.0b013e318217e119. 23. Sopka S, Biermann H, Rossaint R, Knott S, Skorning M et al. (2012) Evaluation of a newly developed media-supported 4-step approach for basic life support training. Scand J Trauma Resusc Emerg Med 20: 37. doi:10.1186/1757-7241-20-S2-P37. PubMed: 22647148. Med Colleges 86: 706-711. doi:10.1097/ACM.0b013 24. Manthey D, Fitch M (2012) Stages of competency for medical procedures. Clin Teach 9: 317-319. doi:10.1111/j.1743-498X. 2012.00561.x. PubMed: 22994471. 46. Dewey J (1933) How we think: New York. Health. 47. Sandars J, Cleary TJ (2011) Self-regulation theory: applications to medical education: AMEE Guide No. 58. Med Teach 33: 875-886. doi: 10.3109/0142159X.2011.595434. PubMed: 22022899. 25. Williams GC, Lynch M, Glasgow RE (2007) Computer-assisted intervention improves patient-centered diabetes care by increasing autonomy support. Health Psychol 26: 728-734. doi: 10.1037/0278-6133.26.6.728. PubMed: 18020845. 48. Knowles M (1975) Self-Directed Learning: a Guide for Learners and Teachers. Chicago, IL: Follett Publishing. g g 49. Brydges R, Nair P, Ma I, Shanks D, Hatala R (2012) Directed self- regulated learning versus instructor-regulated learning in simulation training. Med Educ 46: 648-656. doi:10.1111/j. 1365-2923.2012.04268.x. PubMed: 22691145. 26. Schechter A, Eyal O, Zuckerman-Levin N, Amihai-Ben-Yaacov V, Weintrob N et al. (2012) A prototype of a new noninvasive device to detect nocturnal hypoglycemia in adolescents with type 1 diabetes-a pilot study. Diabetes Technol Ther 14: 683-689. doi:10.1089/dia. 2012.0002. PubMed: 22690891. Long-Term Effect of Skills Lab Training Long-Term Effect of Skills Lab Training 8. Lynagh M, Burton R, Sanson-Fisher R (2007) A systematic review of medical skills laboratory training: where to from here? Med Educ 41: 879-887. doi:10.1111/j.1365-2923.2007.02821.x. PubMed: 17696985. 29. Weyrich P, Celebi N, Schrauth M, Möltner A, Lammerding-Köppel M et al. (2009) Peer-assisted versus faculty staff-led skills laboratory training: a randomised controlled trial. Med Educ 43: 113-120. doi: 10.1111/j.1365-2923.2008.03252.x. PubMed: 19161480. j 9. Lund F, Schultz JH, Maatouk I, Krautter M, Möltner A et al. (2012) Effectiveness of IV cannulation skills laboratory training and its transfer into clinical practice: a randomized, controlled trial. PLOS ONE 7: e32831. doi:10.1371/journal.pone.0032831. PubMed: 22427895. j 30. Corbett EW (2004) The AAMC Project on the Clinical Education of Medical Students. Clinical Skills Education. 31. Colleges A (2003) Educating Doctors to; High Provide Quality Medical Care: a Vision for Medical Education in the United States, Washington, DC 10. McGaghie WC, Draycott TJ, Dunn WF, Lopez CM, Stefanidis D (2011) Evaluating the impact of simulation on translational patient outcomes. Simul Healthc 6 Suppl: S42-S47. doi:10.1097/SIH.0b013e318222fde9. PubMed: 21705966. 32. Kolb DA (1984) Experiential Learning. Englewood Cliffs, NJ: Prentice Hall. 33. DeCoux VM (1990) Kolb’s Learning Style Inventory: a review of its applications in nursing research. J Nurs Educ 29: 202-207. PubMed: 2162927. 11. Barsuk JH, McGaghie WC, Cohen ER, Balachandran JS, Wayne DB (2009) Use of simulation-based mastery learning to improve the quality of central venous catheter placement in a medical intensive care unit. J Hosp Med 4: 397-403. doi:10.1002/jhm.468. PubMed: 19753568. 34. Luszczynska A, Scholz U, Schwarzer R (2005) The general self- efficacy scale: multicultural validation studies. J Psychol 139: 439-457. doi:10.3200/JRLP.139.5.439-457. PubMed: 16285214. p j 12. Arthur W, Bennet W, Stanush PL, McNelly T (1998) Factors That Influence Skill Decay and Retention: A Quantitative Review and Analysis. Hum Perform 11: 57-101. doi:10.1207/s15327043hup1101_3. 35. Stroud M, Duncan H, Nightingale J (2003) Guidelines for enteral feeding in adult hospital patients. Gut 52 Suppl 7: vii1-vii12. PubMed: 14612488. y p _ 13. Bonrath EM, Weber BK, Fritz M, Mees ST, Wolters HH et al. (2012) Laparoscopic simulation training: Testing for skill acquisition and retention. Surgery 152: 12-20. doi:10.1016/j.surg.2011.12.036. PubMed: 22341719. 36. Ingram P, Lavery I (2007) Peripheral intravenous cannulation: safe insertion and removal technique. Nurs Stand 22: 44-48. doi:10.7748/ ns2007.10.22.5.44.c4639. PubMed: 17941430. 14. Smith KK, Gilcreast D, Pierce K (2008) Evaluation of staff’s retention of ACLS and BLS skills. Resuscitation 78: 59-65. doi:10.1016/ j.resuscitation.2008.02.007. References 1. Ziv A, Ben-David S, Ziv M (2005) Simulation based medical education: an opportunity to learn from errors. Med Teach 27: 193-199. doi: 10.1080/01421590500126718. PubMed: 16011941. 2. Barrows HS (1993) An overview of the uses of standardized patients for teaching and evaluating clinical skills. AAMC. Acad Med J Assoc Am Med Colleges 68: 443-451; discussion 451-443 doi: 10.1097/00001888-199306000-00002. 3. Bradley P, Postlethwaite K (2003) Setting up a clinical skills learning facility. Med Educ 37 Suppl 1: 6-13. doi:10.1046/j. 1365-2923.37.s1.11.x. 3. Bradley P, Postlethwaite K (2003) Setting up a clinical skills learning facility. Med Educ 37 Suppl 1: 6-13. doi:10.1046/j. 1365-2923.37.s1.11.x. 4. Nikendei C, Zeuch A, Dieckmann P, Roth C, Schäfer S et al. (2005) Role-playing for more realistic technical skills training. Med Teach 27: 122-126. doi:10.1080/01421590400019484. PubMed: 16019330. September 2013 \| Volume 8 \| Issue 9 \| e76354 11 PLOS ONE \| www.plosone.org Long-Term Effect of Skills Lab Training 50. Schmidt RA, Bjork RA (1992) New conceptualisations of practice – common principles in three paradigms suggest new concepts for training. Psychol Sci 3: 207-217. doi:10.1111/j. 1467-9280.1992.tb00029.x. 27. Pérez-Ferre N, Galindo M, Fernández MD, Velasco V, Mj, et al. (2010) A Telemedicine system based on Internet and short message service as a new approach in the follow-up of patients with gestational diabetes. Diabetes Research & Clinical Practice 87: e15-17 51. Mahmood T, Darzi A (2004) The learning curve for a colonoscopy simulator in the absence of any feedback: no feedback, no learning. Surg Endosc 18: 1224-1230. doi:10.1007/s00464-003-9143-4. PubMed: 15457382. 28. Krautter M, Weyrich P, Schultz JH, Buss SJ, Maatouk I et al. (2011) Effects of Peyton’s four-step approach on objective performance measures in technical skills training: a controlled trial. Teach Learn Med 23: 244-250. doi:10.1080/10401334.2011.586917. PubMed: 21745059. 52. Domuracki KJ, Moule CJ, Owen H, Kostandoff G, Plummer JL (2009) Learning on a simulator does transfer to clinical practice. Resuscitation PLOS ONE \| www.plosone.org 12 September 2013 \| Volume 8 \| Issue 9 \| e76354 Long-Term Effect of Skills Lab Training 80: 346-349. doi:10.1016/j.resuscitation.2008.10.036. PubMed: 19155117. controlled trial. Med Teach 29: 956-960. doi: 10.1080/01421590701601543. PubMed: 18158671. 57. Schwarzer R, [!(surname)!] (1995) Generalized Self-Efficacy Scale. In: J Weinman[!(surname)!]M Johnston. Measures in health psychology: A user’s portfolio Causal and control beliefs. Windsor, UK: NFER-Nelson. pp. 35-37. 53. Schmidt RA, Wulf G (1997) Continuous concurrent feedback degrades skill learning: implications for training and simulation. Hum Factors 39: 509-525. doi:10.1518/001872097778667979. PubMed: 9473972. 54. Moulton CA, Dubrowski A, Macrae H, Graham B, Grober E et al. (2006) Teaching surgical skills: what kind of practice makes perfect?: a randomized, controlled trial. Ann Surg 244: 400-409. PubMed: 16926566. 58. Deshazo J, Harris L, Pratt W (2010) Effective Intervention or Child’s Play? A Review of Video Games for Diabetes Education. Diabetes Technol Ther 12: 815-822. doi:10.1089/dia.2010.0030. PubMed: 20807119. 55. Rethans JJ, Norcini JJ, Barón-Maldonado M, Blackmore D, Jolly BC et al. (2002) The relationship between competence and performance: implications for assessing practice performance. Med Educ 36: 901-909. doi:10.1046/j.1365-2923.2002.01316.x. PubMed: 12390456. 59. Dent JA (2005) Bedside Teaching. In: Dent J.A. HR, editor. A Practical Guide for Medical Teachers; Edinburgh.Elsevier, editor. Churchill Livingstone. pp. 77-85 60. Cook DA, Bordage G, Schmidt HG (2008) Description, justification and clarification: a framework for classifying the purposes of research in medical education. Med Educ 42: 128-133. doi:10.1111/j. 1365-2923.2007.02974.x. PubMed: 18194162. 56. Nikendei C, Kraus B, Schrauth M, Weyrich P, Zipfel S et al. (2007) Integration of role-playing into technical skills training: a randomized PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org 13 September 2013 \| Volume 8 \| Issue 9 \| e76354
https://openalex.org/W2564141136	https://seer.ufrgs.br/ActaScientiaeVeterinariae/article/download/81077/47530	English	null	Effect of Hoof Trimming on Milk Yield in Dairy Cows with Foot Disease	Acta Scientiae Veterinariae	2,016	cc-by	4,710	ABSTRACT Backround: Milk is produced at the cost of other metabolic processes in the body and high milk yield has been associated with lameness and claw lesions. Lameness has also been associated with a decrease in milk yield. In the past, claw disorders and lameness in dairy cattle have been an increasing problem of the modern dairy industry. Hoof trimming is performed to prevent hoof lesions and improve gait by correction and maintenance of the hoof symmetry and shape. Lameness caused by hoof disorders can be treated by correct hoof trimming. The aim of this study was to test the hypothesis that one-time claw trimming affect the milk production in dairy cattle with hoof disorders on commercial dairy farms. Materials, Methods & Results: Milk yield level was examined before and after claw trimming in dairy cattle. Eighteen Holstein dairy cows were examined on a commercial dairy farm. Calving number, calving time, lactation number, lactation stage, culling date and milk yield in liters were detected from farm recording system. All cows were visual signs of claw disorders or lameness. Their hooves had not been were trimmed for several years. Trimming technique included leveling Materials, Methods & Results: Milk yield level was examined before and after claw trimming in dairy cattle. Eighteen Holstein dairy cows were examined on a commercial dairy farm. Calving number, calving time, lactation number, lactation stage, culling date and milk yield in liters were detected from farm recording system. All cows were visual signs of claw disorders or lameness. Their hooves had not been were trimmed for several years. Trimming technique included leveling the 2 claws, aiming for symmetric bulbs. The axial and abaxial walls were both intended to be parts of the bearing surface and the 2 claws were trimmed flat and balanced with each other. The caudal two-thirds of the axial sole of both claws were sloped toward the interdigital area. All of the cows checked for hoof diseases. The period of observation spanned 45 d, starting day of claw trimming. The observation period was the lactation when the claw trimming was performed. Milk yield was performed one day before and 10, 30, and 45 days after hoof trimming. Cows that were in the mid to late lactation period were selected for the study. Received: 17 November 2015 Accepted: 22 May 2016 Published: 24 June 2016 1Faculty of Veterinary Medicine, Kyrgyz Turkish Manas University (KTMU), Bishkek, Kyrgyzstan. 2Faculty of Veterinary Medicine, Selçuk University (SU), Konya, Turkey. CORRESPONDENCE: M. Kibar [muratkibartr@yahoo.com - Tel.: +996 (709) 071410]. Faculty of Veterinary Medicine, Kyrgyz Turkish Manas University (KTMU), Djal Campus, 720044, Bishkek, Kyrgyzstan. Effect of Hoof Trimming on Milk Yield in Dairy Cows with Foot Disease Murat Kibar1 & Tamer Çağlayan2 Keywords: claw trimming, cow, milk yield. Acta Scientiae Veterinariae, 2016. 44: 1370. RESEARCH ARTICLE Pub. 1370 ABSTRACT The mean days in milk for the group was 221.8 (150-272 days) and the mean number of calvings was 2.8 times (range: 2-5 times) before trimming. g ( g ) g Discussion: The shape of the lactation curve is influenced by herd factors such as management and nutrition and individual factors like genetics, parity, and disease. Discrepancies in the literature with regard to the effect of lameness and claw le- sions on milk yield are partly the result of these complex influences. Daily milk production of cows in the current study averaged 21 L/d, so milk weight represented approximately 3% of a cow’s body weight per milking. In this study cows were 2 to 5th lactation. Most hoof diseases are accrued around the time of calving. Hoof diseases is becomed visible on the bearing surface of the sole after 2 to 3 months such as white-line disease, sole ulcer, and hemorrhages. We assessed milk yield as the one time claw trimming performed, so any possible healing effect of claw trimming could have led to an underestimation of any negative effects of bad claw health on milk production. Cows with painful claw lesions eat less, are more reluctant to move, and might consequently produce less milk than cows without claw lesions. The significant associa- tions between most claw disorders and increased yield in this study do not prove direct relationships. At claw trimming, the average lactation stage in first parity was 148 DIM; 25% of the cows were before 74 DIM and 25% were later than 226 DIM. After the claw trimming between days 10-30 and 10-45 were determined differences in milk yield (P < 0.05; P < 0.01). In conclusion, this study showed that one time hoof trimming during the lactation period changed the milk yield of the dairy cows with hoof diseases. Keywords: claw trimming, cow, milk yield. RESEARCH ARTICLE Pub. 1370 ISSN 1679-9216 Received: 17 November 2015 INTRODUCTION every 20 min via a manure scraper. Their hooves had not been were trimmed for several years. The hooves were trimmed by an experienced claw trim- mer (M.K). Trimming technique included leveling the 2 claws, aiming for symmetric bulbs. The axial and abaxial walls were both intended to be parts of the bearing surface and the 2 claws were trimmed flat and balanced with each other. The caudal two-thirds of the axial sole of both claws were sloped toward the interdigital area. All of the cows checked for hoof diseases such as sole hemorrhage (SH), sole ulcer (SU), interdigital dermatitis (IDD), digital dermatitis (DD), and white line disease (WD). Incidence of these disorders was determined. Lameness were scored as sound (1), slightly uneven gait (2), lame (3), very lame (4), and extremely lame (5). Milk is produced at the cost of other metabolic processes in the body and high milk yield has been associated with lameness and claw lesions [2,16,24]. Lameness has also been associated with a decrease in milk yield [15,24]. Milk yield is decreased linearly as locomotion score increased [3,17]. Due to their economic impact [8,19], claw disorders in dairy cattle is receiving as much attention as fertility or mastitis. Decline in milk yield is start up to 2 week before lame- ness was diagnosed [26]. Decrease in milk yield after a lameness episode is persisted for 4 month [12]. In the past, claw disorders and lameness in dairy cattle have been an increasing problem of the modern dairy industry [27,28]. With increases in the incidence of hoof and leg injuries and disease, research on dairy cattle lameness has grown considerably over the last 25 years [5,10]. The period of observation spanned 45 d, start- ing day of claw trimming. The observation period was the lactation when the claw trimming was performed. To allow the cows to adapt to their trimmed claws, a second series of milk yield controlled in 10th, 30th, and 45th days after trimming. Milk yield was performed one day before and 10, 30, and 45 days after hoof trimming. Five cows were excluded because of culling. Lameness causes reduced animal welfare and is among the 3 most loss-making diseases in the dairy industry after mastitis and fertility problems [18,24]. Therefore, there is an increasing awareness of the importance of hoof trimming. INTRODUCTION Hoof trimming is performed to prevent hoof lesions and improve gait by correction and maintenance of the hoof symmetry and shape, which ensures correct weight bearing. Lameness caused by hoof disorders can be treated by correct hoof trimming [20,22]. Trimming in healthy hooves is significantly effected on milk fat and milk protein compositions [21]. Cows that were in the mid to late lactation period were selected for the study. The mean days in milk for the group was 221.8 (150-272 days) and the mean number of calvings was 2.8 times (range: 2-5 times) before trimming. Cows were fed the same diet throughout the study. The average feed composition is listed in Table 1. The objective of this study was to test the hy- pothesis that one-time claw trimming affect the milk production in dairy cattle with hoof disorders on com- mercial dairy farms. Milk yield level was examined before and after claw trimming in dairy cattle. Shapiro-Wilk W test for normality control were applied to the data. The data were analyzed by SPSS (version 15.0) utilizing General Linear Model Repeated Measures. The Paired t-test was used to analyze the results before and after hoof trimming. Table 1. The composition of ration used in the study. Table 1. The composition of ration used in the study. Ingredient Daily quantity - kg / cow Wheat hay 6 Alfalfa hay 4 Barley 4 Cottonseed meal 2 Wheat bran 1 Sugar beet pulp 15 Limestone 0.1 Salt 0.04 Vitamin-mineral premix* 0.03 Provided per 1 kg of premix: Vit. A 15000000 IU, Vit D3 3000000 IU, Vit E 30000 mg, Mn 50000 mg, Zn 50000 mg, Fe 50000 mg, Cu 10000 mg, I 800 mg, Co 150 mg, Se 150 mg. Keywords: claw trimming, cow, milk yield. Published: 24 June 2016 1 1 M. Kibar & T. Çağlayan. 2016. Effect of Hoof Trimming on Milk Yield in Dairy Cows with Foot Disease. A t S i M. Kibar & T. Çağlayan. 2016. Effect of Hoof Trimming on Milk Yield in Dairy Cows with Foot Disease. Acta Scientiae Veterinariae. 44: 1370. Acta Scientiae Veterinariae. 44: 1370. RESULTS In this study, the mean incidences of SH, SU, IDD, DD, and WD were 28.4%, 19.1%, 23.8%, 18.3% and 10.4%, respectively. Because the previous trimmings were not performed before, excessive over- growth was present on claws. At claw trimming, the average lactation stage in first parity was 148 DIM; 25% of the cows were before 74 DIM and 25% were later than 226 DIM. The lameness scores ranged from 3 (lame) to 5 (extremely lame). There were very few effects of level of milk production on lameness (P > 0.05). Lameness increased with age (P < 0.05). The effect of lactation number on these traits was apparent (P < 0.05). After the claw trimming between days 10th- 30th and 10th-45th were determined differences (P < 0.05; P < 0.01) in milk yield in Table 3. The General Linear Model results applied to the data among times differences (P = 0.031) and intercept (P = 0.000) were significant (P < 0.05; P < 0.001). There was no significantly change in milk yield between before and after claw trimming. However, the milk yields were significantly significant between 10th and 30th, and 45th days. Before hoof trimming, the average toe angle of forelimbs was 33.2 ± 4.8º (mean ± SD), and that of hind limbs was 35.7 ± 5.2º. After trimming they were 42.1 ± 3.2º and 41.4 ± 3.6º, respectively. These changes were statistically significant for both the forelimbs and hind limbs (P < 0.05). Lameness and all claw lesions occurred at a higher frequency in hind claws than in front claws and analyses were performed only in hind claws. Claw trimming, descriptive statistics on milk yield in terms of time are given in Table 2. Before and after the claw trimming, changes in milk yield by time is shown in Figure1. Tenth day milk yield after claw trimming increased by 0.88 ± 0.55 L in cows compared with before trimming (Figure 1). According to Table 4, pair wise comparison to milk yield positive, and highly significant correlation were determined (P < 0.001). Time (day) Milk yield (L) Figure 1. Changes to milk yield according to the time (Mean). Figure 1. Changes to milk yield according to the time (Mean). Table 2. Descriptive statistics on milk yield (L). MATERIALS AND METHODS Eighteen Holstein dairy cows (age = 4.5 ± 1.0 yr; weight = 692 ± 118 kg) were examined on a com- mercial dairy farm. Cows were housed in free-stalls. All animals were multiparous cow. Calving number, calving time, lactation number, lactation stage, culling date and milk yield in liters were detected from farm recording system. All cows were visual signs of claw disorders or lameness. They were housed in tie-stall barns for sepa- rate feeding all day long and therefore had no exer- cise. The walking areas were cleaned automatically Provided per 1 kg of premix: Vit. A 15000000 IU, Vit D3 3000000 IU, Vit E 30000 mg, Mn 50000 mg, Zn 50000 mg, Fe 50000 mg, Cu 10000 mg, I 800 mg, Co 150 mg, Se 150 mg. 2 bar & T. Çağlayan. 2016. Effect of Hoof Trimming on Milk Yield in Dairy Cows with Foot Disease. Acta Scient M. Kibar & T. Çağlayan. 2016. Effect of Hoof Trimming on Milk Yield in Dairy Cows with Foot Disease. A t S i M. Kibar & T. Çağlayan. 2016. Effect of Hoof Trimming on Milk Yield in Dairy Cows with Foot Disease. Acta Scientiae Veterinariae. 44: 1370. Acta Scientiae Veterinariae. 44: 1370. RESULTS Time n Min Max Mean ± SE Before 18 8.90 44.00 21.34 ± 2.21 10 days 18 10.00 46.20 22.22 ± 2.28 30 days 15 7.20 41.50 20.75 ± 2.55 45 days 13 10.80 41.20 22.45 ± 2.59 3 3 M. Kibar & T. Çağlayan. 2016. Effect of Hoof Trimming on Milk Yield in Dairy Cows with Foot Disease. Acta Scien yan. 2016. Effect of Hoof Trimming on Milk Yield in Dairy Cows with Foot Disease. Acta Scientiae Veterina M. Kibar & T. Çağlayan. 2016. Effect of Hoof Trimming on Milk Yield in Dairy Cows with Foot Disease. Acta Scientiae Veterinariae. 44: 1370. Acta Scientiae Veterinariae. 44: 1370. Table 3. Comparison on effect of hoof trimming on milk yield. Pair Time n Mean ± SE P Pair 1 Before 18 21.34 ± 2.21 0.126 10 days 18 22.22 ± 2.28 Pair 2 Before 15 21.86 ± 2.58 0.067 30 days 15 20.75 ± 2.55 Pair 3 Before 13 23.67 ± 2.62 0.181 45 days 13 22.45 ± 2.59 Pair 4 10 days 15 22.99 ± 2.66 0.009 30 days 15 20.75 ± 2.55 Pair 5 10 days 13 24.39 ± 2.86 0.023 45 days 13 22.45 ± 2.59 Pair 6 30 days 13 22.55 ± 2.58 0.879 45 days 13 22.45 ± 2.59 Paired samples statistics. Table 4. Paired samples correlations. Pair Time n Correlation P# Pair 1 Before - 10 days 18 0.971 0.000 Pair 2 Before - 30 days 15 0.976 0.000 Pair 3 Before - 45 days 13 0.945 0.000 Pair 4 10 days - 30 days 15 0.961 0.000 Pair 5 10 days - 45 days 13 0.967 0.000 Pair 6 30 days - 45 days 13 0.964 0.000 #P < 0.001. DISCUSSION Çağlayan. 2016. Effect of Hoof Trimming on Milk Yield in Dairy Cows with Foot Disease. Acta Scient M. Kibar & T. Çağlayan. 2016. Effect of Hoof Trimming on Milk Yield in Dairy Cows with Foot Disease. Acta Scientiae Veterinariae. 44: 1370. Cows with painful claw lesions eat less, are more reluctant to move, and might consequently pro- duce less milk than cows without claw lesions [9,24]. Reduction in milk yield associated with claw and limb disorders are likely to be caused by reductions in feed intake or increased energy consumption because of pain, which can also be present without visible lame- ness [11,24]. It is, however, unlikely that claw lesions that are not painful lead to decreases in milk yield. The significant associations between most claw disorders and increased yield in this study do not prove direct relationships. the toe angle was closer to these reported values, and the hoof shape was accurated. Increased milk yield after claw trimming can be the result of well being and more comfortable walk- ing and standing after trimming to correct claw shape and positive effects on different disorders. By measur- ing different blood parameters, it is concluded that cows began to eat more roughage after claw trimming [21], but in contrast to our study they did not detect higher milk yields after claw trimming. Milk yield is expected to decrease with increasing number of DIM after peak yield. Test days after claw trimming are in a later stage of lactation than test days before trimming; however, this was accounted for by adjusting milk yield according to DIM. It is indicated that a linear relationship between increasing degree of lameness and decreasing milk yield among cows in second and later lactations [15]. It is reported that milk yield decreased linearly as locomotion score increased [3,17]. It is founded that no differences in the incidence of claw and feet problems in high yield- ing vs. average-yielding cows and concluded that with adequate husbandry, management, and feeding, cows yielding >45 kg/d can be maintained without more serious problems than cows yielding 35 kg/d [1]. It is founded that milk production at the onset of foot lesions was a determining factor of the amount and pattern of milk loss, but only for cases in mid to late lactation [7]. DISCUSSION In the case of milk production and claw dis- orders in dairy cows, it seems sensible to postulate a lagged progressive path involving 3 traits. One path would describe the influence that test-day milk yield has on claw disorders, and the second path would pertain to the effect of the disorder on milk production level at the following test date. CONCLUSIONS In conclusion, this study showed that one time hoof trimming during the lactation period changed the milk yield of the dairy cows. In our opinion this change could be based on to increased comfortable walking after trimming. In this study, did not evaluated gait analyses, and hence would like to examine this control in future studies. The milk yield decreased in 10 days after trimming, but then it was increased and achieved to high level before trimming. That cows yielded more milk after claw trimming than before is an economic encouraging for routine claw trimming. Finally, hoof trimming may be priceless entity in the recovery of milk yield and suggestible in dairy cows. It is reported that mean incidences of digital dermatitis, solea ulcer, white line disease, and inter- digital hyperplasia were 13.7%, 16.5%, 9.8%, and 6.7%, respectively [19]. These values nearly identical to those found in other studies [13,25]. In this study, the mean incidences of SH, SU, IDD, DD, and WD were 28.4%, 19.1%, 23.8%, 18.3% and 10.4%, respectively, and there were substantial differences between herds. It is reported that before hoof trimming, the average toe angle of forelimbs was 32.7º, and that of hind limbs was 35.2º [21]. After trimming they were 41.5º and 40.6º, respectively. It is also reported that the same values were 44.9º in the forelimbs and 42.9º in the hind limbs at second lactation in Holstein cows [13]. Cows had not been trimmed before; they hence had long hooves and acute toe angles. But after trimming, Declaration of interest. The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. DISCUSSION trimming performed, so any possible healing effect of claw trimming could have led to an underestimation of any negative effects of bad claw health on milk produc- tion. This effect was partly accounted for by including the variable that described whether the test day was before claw trimming, 10th, 30th, and 45th days later. Milk yield is analyzed in the previous lactation to avoid direct relationships between lameness and milk yield within the same lactation [4,24]. Further research is needed to evaluate if milk yield is also related to the timing of claw trimming during the lactation. Cows with low milk yield [24] and lameness and claw lesions are more likely to be culled [23,24]. This is particularly problematic with lameness and claw lesions, because these disorders occur throughout the lactation (but usually around calving). Cows with high milk yield stay in the herd longer and have an increased chance of experiencing claw and limb disorders, thus leading to an overestimation of the association between milk yield and such disorders. The cluster effect within herd was significant for all hind claw lesions, but was most marked for hemorrhage and solea ulcers in this study. The shape of the lactation curve is influenced by herd factors such as management and nutrition and individual factors like genetics, parity, and disease [24]. Discrepancies in the literature with regard to the effect of lameness and claw lesions on milk yield are partly the result of these complex influences. Daily milk production of cows in the current study averaged 21 L/d, so milk weight represented approximately 3% of a cow’s body weight per milking. In contrast, the studies on horses and humans used relatively greater weights (15 to 20% of the subject’s body weight) car- ried on the subject’s back [14]. It is founded that high milk yield within the first third of lactation increases a cow’s risk to experience health problems [6]. In this study cows were 2 to 5th lactation. Most hoof diseases are accrued around the time of calving. Hoof diseases is becomed visible on the bearing surface of the sole after 2 to 3 months such as white-line disease, sole ulcer, and hemorrhages [24,29]. We assessed milk yield as the one time claw 4 M. Kibar & T. Çağlayan. 2016. Effect of Hoof Trimming on Milk Yield in Dairy Cows with Foot Disease. Acta Scientiae Veterinariae. 44: 1370. bar & T. REFERENCES 1 Aeberhard K., Bruckmaier R.M., Kuepfer U. & Blum J.W. 2001. Milk yield and composition, nutrition, body con- formation traits, body condition scores, fertility and diseases in high-yielding dairy cows-Part 1. Journal of Veterinary Medicine. 48(2): 97-110. 2 Alban L., Agger J.F. & Lawson L.G. 1996. Lameness in tied Danish dairy cattle: The possible influence of housing systems, management, milk yield, and prior incidents of lameness. Preventive Veterinary Medicine. 29(2): 135-149. 5 M. Kibar & T. Çağlayan. 2016. Effect of Hoof Trimming on Milk Yield in Dairy Cows with Foot Disease. Acta Scien M. Kibar & T. Çağlayan. 2016. Effect of Hoof Trimming on Milk Yield in Dairy Cows with Foot Disease. Acta Scientiae Veterinariae. 44: 1370. 3 Bach A., Dinarés M., Devant M. & Careé X. 2007. Associations between lameness and production, feeding and milk- ing attendance of Holstein cows milked with an automatic milking system. Journal of Dairy Research. 74(1): 40-46. 4 Bielfeldt J.C., Badertscher R., Tölle K.H. & Krieter J. 2005. Risk factors influencing lameness and claw disorders in dairy cows. Livestock Science. 95(3): 265-271. 5 Clarkson M.J., Downham D.Y., Faull W.B., Hughes J.W., Manson F.J., Merritt J.B., Murray R.D., Russell W.B., Sutherst J.E. & Ward W.R. 1996. Incidence and prevalence of lameness in dairy cattle. Veterinary Record. 138(23): 563-567. 6 Collard B.L., Boettcher P.J., Dekkers J.C.M., Petitclerc D. & Schaefferet L.R. 2000. Relationships between energy balance and health traits of dairy cattle in early lactation. Journal of Dairy Science. 83(11): 2683-2690. 7 Coulon J.B., Lescourret F. & Fonty A. 1996. Effect of foot lesions on milk production by dairy cows. Journal of Dairy Science. 79(1): 44-49. 8 Entig H., Kooji D., Dijkhuizen A.A., Huirne R.B.M. & Nordhuizen-Stassen E.N. 1997. Economic losses due to clinical lameness in dairy cattle. Livestock Science. 49(3): 259-267. 9 Fleischer P., Metzner M., Beyerbach M., Hoedemaker M. & Klee W. 2001. The relationship between milk yield and the incidence of some diseases in dairy cows. Journal of Dairy Science. 84(9): 2025-2035. 10 Flower F.C., Sanderson D.J. & Weary D.N. 2006. Effects of Milking on dairy cow gait. Journal of Dairy Science. 89(6): 2084-2089. 11 Fourichon C., Seegers H., Bareille N. & Beaudeau F. 2001. Effects of disease on milk production in the dairy cow: A review. Journal of Dairy Science. 84(9): 2025-2035. 12 Green L., Hedges V.J., Schucken Y.H., Blowey R.W. & Packington A.J. 2002. REFERENCES The impact of clinical lameness on milk yield of dairy cows. Journal of Dairy Science. 85(9): 2250-2256. 13 Hahn M.V., McDaniel B.T. & Wilk J.C. 1984. Genetic and environmental variation of hoof characteristics of Holstein cattle. Journal of Dairy Science. 67(12): 2986-2998. 14 Haskell M.J., Rennie L.J., Bowell V.A. & Lawrence A.B. 2006. Housing system, milk production, and zero-grazing effects on lameness and leg injury in dairy cows. Journal of Dairy Science. 89(11): 4259-4266. 15 Hernandez J.A., Garbarino E.J., Shearer J.K., Risco C.A. & Thatcher W.W. 2005. Comparison of milk yield in dairy cows with different degrees of lameness. Journal of American Veterinary Medicine Association. 227(8): 1292- 1296. 16 Hultgren J., Manske T. & Bergsten C. 2004. Associations of sole ulcer at claw trimming with reproductive perfor- mance, udder health, milk yield, and culling in Swedish dairy cattle. Preventive Veterinary Medicine. 62(4): 233-251. 17 Juarez S.T., Robinson P.H., DePeters M. & Price E. 2003. Impact of lameness on behavior and productivity of lactating Holstein cows. Applied Animal Behaviour Science. 83(1): 1-14. 18 Kossaibati M.A. & Esslemont R.J. 1997. The costs of production diseases in dairy herds in England. Veterinary Journal. 154(3): 41-51. 19 König S., Wu X.L., Gianola D., Heringstad B. & Simianer H. 2008. Exploration of relationships between claw disorders and milk yield in Holstein cows via recursive linear and threshold models. Journal of Dairy Science. 91(1): 395-406. 20 Manske T., Hultgren J. & Begsten C. 2002. The effect of claw trimming on the hoof health of Swedish dairy cattle. Preventive Veterinary Medicine. 54(2): 113-129. 21 Nishimori K., Okada K., Ikuta K., Aoki O., Sakai T. & Yasuda J. 2006. The effects of one time trimming on blood biochemical composition, milk yield, and milk composition in dairy cows. Journal of Veterinary Medicine Science. 68(3): 267-270. 22 Shearer J.K. & van Amstel S.R. 2001. Functional and corrective claw trimming. Veterinary Clinics North America Food Animal Practice. 17(1): 53-72. 23 Sogstad A.M., Fjeldaas T., Osteras O. & Forshell K.P. 2005. Prevalence of claw lesions in Norwegian dairy cattle housed in tie stalls and free stalls. Preventive Veterinary Medicine. 70(3-4): 191-209. 24 Sogstad A.M., Osteras O., Fjeldaas T. & Refsdal A.O. 2007. Bovine claw and limb disorders at claw trimming related to milk yield. Journal of Dairy Science. 90(2): 749-759. 25 Somers J.G.C.J., Frankena K., Nordhuizen-Stassen E.N. & Metz J.H.M. 2003. 27 Van der Tool P.P.J., van der Beek S.S., Metz J.H.M., Noordhuizen-Stassen E.N., Back W., Braam C.R. & Weijs W.A. 2004. The effect of preventive trimming on weight bearing and force balance on the claws of dairy cattle. Journal of Dairy Science. 87(6): 1732-1738. 28 Van der Waaij E.H., Holzhauer M., Ellen E., Kamphuis C. & de Jong G. 2005. Genetic parameters for claw dis- orders in Dutch dairy cattle and correlations with conformation traits. Journal of Dairy Science. 88(10): 3672-3678. 29 Vermunt J.J. & Greenough P.R. 1994. Predisposing factors of laminitis in cattle. British Veterinary Journal. 150(2): 151-164. 26 Rajala-Schultz P.J., Gröhn Y.T. & McCulloch C.E. 1999. Effects of milk fever, ketosis, and lameness on milk yield in dairy cows. Journal of Dairy Science. 82(2): 288-294. M. Kibar & T. Çağlayan. 2016. Effect of Hoof Trimming on Milk Yield in Dairy Cows with Foot Disease. Acta Scientiae Veterinariae. 44: 1370. 28 Van der Waaij E.H., Holzhauer M., Ellen E., Kamphuis C. & de Jong G. 2005. Genetic parameters for claw dis- orders in Dutch dairy cattle and correlations with conformation traits. Journal of Dairy Science. 88(10): 3672-3678. 29 Vermunt J.J. & Greenough P.R. 1994. Predisposing factors of laminitis in cattle. British Veterinary Journal. 150(2): 151 164 REFERENCES Prevalence of claw disorders in Dutch dairy cows exposed to several floor systems. Journal of Dairy Science. 86(6): 2082-2093. 6 M. Kibar & T. Çağlayan. 2016. Effect of Hoof Trimming on Milk Yield in Dairy Cows with Foot Disease. A t S i 27 Van der Tool P.P.J., van der Beek S.S., Metz J.H.M., Noordhuizen-Stassen E.N., Back W., Braam C.R. & Weijs W.A. 2004. The effect of preventive trimming on weight bearing and force balance on the claws of dairy cattle. Journal of Dairy Science. 87(6): 1732-1738. 28 Van der Waaij E.H., Holzhauer M., Ellen E., Kamphuis C. & de Jong G. 2005. Genetic parameters for claw dis- orders in Dutch dairy cattle and correlations with conformation traits. Journal of Dairy Science. 88(10): 3672-3678. 28 Van der Waaij E.H., Holzhauer M., Ellen E., Kamphuis C. & de Jong G. 2005. Genetic parameters for claw dis- orders in Dutch dairy cattle and correlations with conformation traits. Journal of Dairy Science. 88(10): 3672-3678. 29 Vermunt J.J. & Greenough P.R. 1994. Predisposing factors of laminitis in cattle. British Veterinary Journal. 150(2): 151-164 29 Vermunt J.J. & Greenough P.R. 1994. Predisposing factors of laminitis in cattle. British Veterinary Journal. 150(2): 151-164. 1370 1370 www.ufrgs.br/actavet 7
https://openalex.org/W4312239419	https://journals2.ums.ac.id/index.php/abditeknoyasa/article/download/201/152	Indonesian	null	Perancangan Sistem Informasi Sekolah Berbasis Website Sebagai Sarana Promosi Pada SDN Toso 02	Abdi Teknoyasa	2,021	cc-by	2,015	ABSTRAK Perkembangan teknologi informasi telah dimanfaatkan berbagai bidang, termasuk bidang pendidikan. Salah satu pemanfaatan teknologi informasi pada bidang pendidikan yaitu sebagai sarana untuk promosi kepada masyarakat. Tujuan penelitian ini adalah merancang sistem informasi sekolah berbasis website sebagai sarana promosi pada SDN Toso 02. Sistem informasi ini disusun menggunakan metode pengembangan perangkat lunak prototyping. Sistem informasi dirancang menggunakan bootstrap, javascript, dan Cascading Style Sheets (CSS). Hasil dari penelitian ini adalah dibangunnya website SDN Toso 02 yang digunakan sebagai sarana promosi kepada masyarakat luas. Website ini menampilkan beberapa informasi mengenai SDN Toso 02 diantaranya profil, galeri, prestasi, serta fasilitas sekolah. Berdasarkan hasil pengujian, sistem yang dirancang dapat berjalan sesuai dengan kebutuhan. Risa Ayu Agustina Teknik Informatika Universitas Muhammadiyah Surakarta risaayuagustina57@gmail.com Risa Ayu Agustina Teknik Informatika Universitas Muhammadiyah Surakarta risaayuagustina57@gmail.com Fischella Angieta Chelsea Teknik Informatika Universitas Muhammadiyah Surakarta fischella100599@gmail.com Dimas Aryo Anggoro* Teknik Informatika Universitas Muhammadiyah Surakarta dimas.a.anggoro@ums.ac.id KATA KUNCI: sekolah, sistem informasi, website * Corresponding author Naskah dikirim 12 November 2020 Naskah direvisi 27 Oktober 2021 Naskah diterima 30 Desember 2021 Naskah diterima 30 Desember 2021 tersebut. Berdasarkan hal tersebut, maka perancangan sistem infomasi sekolah berbasis website sebagai sarana promosi pada SDN Toso 02 perlu dilakukan. PERANCANGAN SISTEM INFORMASI SEKOLAH BERBASIS WEBSITE SEBAGAI SARANA PROMOSI PADA SDN TOSO 02 Anom Wisnu Subroto Teknik Informatika Universitas Muhammadiyah Surakarta anomwisnu13@gmail.com Anom Wisnu Subroto Teknik Informatika Universitas Muhammadiyah Surakarta anomwisnu13@gmail.com Risa Ayu Agustina Teknik Informatika Universitas Muhammadiyah Surakarta risaayuagustina57@gmail.com Fischella Angieta Chelsea Teknik Informatika Universitas Muhammadiyah Surakarta fischella100599@gmail.com Dimas Aryo Anggoro* Teknik Informatika Universitas Muhammadiyah Surakarta dimas.a.anggoro@ums.ac.id * Corresponding author Naskah dikirim 12 November 2020 Naskah direvisi 27 Oktober 2021 Naskah diterima 30 Desember 2021 Anom Wisnu Subroto Teknik Informatika Universitas Muhammadiyah Surakarta anomwisnu13@gmail.com PENDAHULUAN Teknologi informasi telah mengalami perkembangan yang pesat. Perkembangan teknologi informasi tersebut memungkinkan berbagai kegiatan dapat dilakukan dengan cepat, tepat, dan akurat. Teknologi informasi telah dimanfaatkan dalam berbagai bidang seperti perdagangan, komunikasi, pendidikan, pariwisata, dan lain sebagainya. ISSSN: 2745-701X E-ISSN: 2745-7028 PERANCANGAN SISTEM INFORMASI SEKOLAH BERBASIS WEBSITE SEBAGAI SARANA PROMOSI PADA SDN TOSO 02 ISSSN: 2745-701X E-ISSN: 2745-7028 http://journals2.ums.ac.id/index.php/abditeknoyasa/ ISSSN: 2745-701X E-ISSN: 2745-7028 http://journals2.ums.ac.id/index.php/abditeknoyasa/ Jurnal Pengabdian masyarakat teknoyasa, Volume 2, No.2 Desember 2021\| 63 METODE PENELITIAN A. Alat dan Bahan Peralatan yang digunakan dalam proses perancangan sistem, baik itu hardware maupun software dapat dilihat pada Tabel 1. Bahan yang digunakan yaitu informasi yang diperoleh dari SDN Toso 02. A. Alat dan Bahan Peralatan yang digunakan dalam proses perancangan sistem, baik itu hardware maupun software dapat dilihat pada Tabel 1. Bahan yang digunakan yaitu informasi yang diperoleh dari SDN Toso 02. A. Alat dan Bahan Peralatan yang digunakan dalam proses perancangan sistem, baik itu hardware maupun software dapat dilihat pada Tabel 1. Bahan yang digunakan yaitu informasi yang diperoleh dari SDN Toso 02. Salah satu pemanfaatan teknologi informasi pada bidang pendidikan yaitu sebagai sarana untuk promosi kepada masyarakat luas. Sekolah dapat memanfaatkan website sebagai sarana promosi yang dapat menjangkau semua lapisan masyarakat. Kegiatan promosi menggunakan website dinilai lebih efektif dan efisien dibandingkan dengan cara konvensional. Cara promosi konvensional seperti penyebaran brosur memerlukan biaya, tenaga, dan waktu yang cukup banyak. * Corresponding author Tabel 1. Peralatan yang digunakan Tabel 1. Peralatan yang digunakan B. Metode Pengumpulan Data SDN Toso 02 adalah sekolah yang terletak di Dusun Margosari, Desa Toso, Kecamatan Bandar, Kabupaten Batang. SDN Toso 02 merupakan intansi pemerintah dalam dunia pendidikan yang belum memiliki fasilitas website sebagai sarana informasi dan sarana promosi kepada masyarakat. SDN Toso 02 mempunyai harapan agar masyarakat luas dapat mengenal kegiatan belajar mengajar pada sekolah 1. Metode Wawancara Metode wawancara merupakan metode pengumpulan data atau informasi berdasarkan hasil yang diperoleh secara langsung dari pihak yang bersangkutan. Kemudian ditampung dan dijadikan sebagai data informasi. “Wawancara merupakan pertemuan dua orang untuk dengan sekolah yang nantinya akan ditampilkan di website SD Negeri Toso 02. Hardware yang dibutuhkan adalah seperangkat PC (Portable Computer) atau bisa menggunakan laptop yang tersambung dengan koneksi internet. dengan sekolah yang nantinya akan ditampilkan di website SD Negeri Toso 02. Hardware yang dibutuhkan adalah seperangkat PC (Portable Computer) atau bisa menggunakan laptop yang tersambung dengan koneksi internet. bertukar informasi dan ide melalui tanya jawab, sehingga dapat dikontruksikan makna dalam suatu topik tertentu”, Sugiono (2013:231) [1]. bertukar informasi dan ide melalui tanya jawab, sehingga dapat dikontruksikan makna dalam suatu topik tertentu”, Sugiono (2013:231) [1]. 2. Metode Observasi Menurut Supardi (2006:88), “Metode observasi merupakan metode pengumpul data yang dilakukan dengan cara mengamati dan mencatat secara sistematik gejala-gejala yang diselidiki” [2]. b. Analisis Kebutuhan Software Perangkat lunak (software) yang digunakan untuk mendukung proses website antara lain: Operating System (OS) menggunakan Windows 10 dan web browser yaitu Google Chrome. 3. Metode Dokumen Metode dokumen merupakan metode pengumpulan data berdasarkan data yang dimiliki oleh pihak yang bersangkutan maupun data dari hasil pencarian referensi baik melalui buku ataupun internet. Menurut Arikunto (2006:158), “Dokumentasi adalah mencari dan mengumpulkan data mengenai hal-hal yang berupa catatan, transkrip, buku, surat kabar, majalah, notulen, rapor, agenda dan sebagainya.” [3]. 2. Desain Tahapan ini digunakan untuk mengubah kebutuhan-kebutuhan diatas menjadi representasi kedalam bentuk sistem informasi. Desain juga harus dapat mengimplementasikan kebutuhan yang telah disebutkan pada tahap sebelumya maka proses ini juga harus didokumentasikan sebagai konfigurasi dari software. a. Use Case Diagram C. Pengembangan Sistem Metode prototyping merupakan pengembangan yang cepat dan pengujian terhadap model kerja (prototipe) dari aplikasi baru melalui proses interaksi dan berulang-ulang yang biasa digunakan ahli sistem informasi dan ahli bisnis. Disebut juga desain aplikasi cepat (Rapid Application Design/ RAD) karena menyederhanakan dan mempercepat desain sistem (O'Brien, 2005) [4]. Tabel 1. Peralatan yang digunakan Metode ini sangat baik digunakan untuk menyelesesaikan masalah kesalahpahaman antara user dan analis yang timbul akibat user tidak mampu mendefinisikan secara jelas kebutuhannya (Mulyanto, 2009) [5]. Model pengembangan prototyping ditunjukkan pada gambar 1. a. Use Case Diagram Use case diagram menggambarkan seluruh aktivitas yang dilakukan oleh sistem dari sudut pandang pengamatan luar (Sauri et al, 2015) [6]. Use case diagram ditunjukkan pada gambar 2. C. Pengembangan Sistem Metode prototyping merupakan pengembangan yang cepat dan pengujian terhadap model kerja (prototipe) dari aplikasi baru melalui proses interaksi dan berulang-ulang yang biasa digunakan ahli sistem informasi dan ahli bisnis. Disebut juga desain aplikasi cepat (Rapid Application Design/ RAD) karena menyederhanakan dan mempercepat desain sistem (O'Brien, 2005) [4]. Metode ini sangat baik digunakan untuk menyelesesaikan masalah kesalahpahaman antara user dan analis yang timbul akibat user tidak mampu mendefinisikan secara jelas kebutuhannya (Mulyanto, 2009) [5]. Model pengembangan prototyping ditunjukkan pada gambar 1. Gambar 2. Use Case Diagram (sumber: Hasil Penelitian, 2020) Gambar 1. Model Pengembangan Prototyping (sumber: Hasil Penelitian, 2020) Gambar 2. Use Case Diagram (sumber: Hasil Penelitian, 2020) Gambar 2. Use Case Diagram (sumber: Hasil Penelitian, 2020) 3. Coding Tahap coding yang dilakukan dalam perancangan sistem informasi sekolah berbasis website sebagai sarana promosi pada SDN Toso 02 menggunakan bootstrap, javascript, dan Cascading Style Sheets (CSS). 4. Uji Coba (Testing) Testing adalah aktivitas yang digunakan untuk melakukan evaluasi suatu atribut atau kemampuan dari program atau sistem dan berguna untuk menentukan apakah sistem telah memenuhi kebutuhan atau hasil yang diharapkan. Gambar 1. Model Pengembangan Prototyping (sumber: Hasil Penelitian, 2020) 1. Tahap Analis Kebutuhan (Requirements Analysis) Tahapan analisis kebutuhan ini dilakukan dengan menganalisis dari kebutuhan hardware dan software. 5. Implementasi Implementasi adalah suatu proses untuk melaksanakan kebijakan menjadi tindakan kebijakan dari politik ke dalam administrasi. Pengembangan kebijakan dalam rangka a. Analisis Kebutuhan Hardware Perangkat hardware dibutuhkan untuk melakukan input data terbaru berkaitan Gambar 6. Halaman Registrasi (sumber: Hasil Penelitian, 2020) 5. Halaman Fasilitas Halaman fasilitas, pada gambar 7, menampilkan fasilitas-fasilitas yang ada di SDN Toso 02. Gambar 7. Halaman Fasilitas (sumber: Hasil Penelitian, 2020) 6. Halaman Kesiswaan Halaman kesiswaan, pada gambar 8, menampilkan prestasi-prestasi yang diraih oleh SDN Toso 02. Gambar 8. Halaman Kesiswaan (sumber: Hasil Penelitian, 2020) Gambar 6. Halaman Registrasi (sumber: Hasil Penelitian, 2020) penyempurnaan suatu program (Harsono, 2002:67) [7]. Tahapan ini berarti sistem yang dikembangkan telah lolos uji coba dan siap diterapkan atau diimplementasikan. HASIL DAN ANALISA Bagian ini membahas dan menampilkan hasil dari perancangan sistem informasi sekolah berbasis website sebagai sarana promosi pada SDN Toso 02 yang telah selesai dirancang. Gambar 6. Halaman Registrasi (sumber: Hasil Penelitian, 2020) 1. Halaman Home Halaman home, pada gambar 3, merupakan tampilan awal dari website. Gambar 3. Halaman Home (sumber: Hasil Penelitian, 2020) 2. Halaman Profile Pada halaman profile, pada gambar 4, berisi profil sekolah, guru, sejarah, visi dan misi, serta struktur organisasi. Gambar 4. Halaman Profile (sumber: Hasil Penelitian, 2020) 1. Halaman Home Halaman home, pada gambar 3, merupakan tampilan awal dari website. 1. Halaman Home Halaman home, pada gambar 3, merupakan tampilan awal dari website. Gambar 3. Halaman Home (sumber: Hasil Penelitian, 2020) 2. Halaman Profile Pada halaman profile, pada gambar 4, berisi profil sekolah, guru, sejarah, visi dan misi, serta struktur organisasi. 5. Halaman Fasilitas Halaman fasilitas, pada gambar 7, menampilkan fasilitas-fasilitas yang ada di SDN Toso 02. Gambar 3. Halaman Home (sumber: Hasil Penelitian, 2020) 2. Halaman Profile Pada halaman profile, pada gambar 4, berisi profil sekolah, guru, sejarah, visi dan misi, serta struktur organisasi. Gambar 7. Halaman Fasilitas (sumber: Hasil Penelitian, 2020) Gambar 7. Halaman Fasilitas (sumber: Hasil Penelitian, 2020) 6. Halaman Kesiswaan Halaman kesiswaan, pada gambar 8, menampilkan prestasi-prestasi yang diraih oleh SDN Toso 02. Gambar 8. Halaman Kesiswaan (sumber: Hasil Penelitian, 2020) 6. Halaman Kesiswaan Halaman kesiswaan, pada gambar 8, menampilkan prestasi-prestasi yang diraih oleh SDN Toso 02. g Gambar 4. Halaman Profile (sumber: Hasil Penelitian, 2020) Gambar 8. Halaman Kesiswaan (sumber: Hasil Penelitian, 2020) Gambar 4. Halaman Profile (sumber: Hasil Penelitian, 2020) Gambar 4. Halaman Profile (sumber: Hasil Penelitian, 2020) Gambar 8. Halaman Kesiswaan (sumber: Hasil Penelitian, 2020) 3. Halaman Gallery Pada halaman gallery, pada gambar 5, berisi dokumentasi kegiatan belajar mengajar dan ekstrakurikuler sekolah. PERSANTUNAN Penulis mengucapkan terima kasih kepada Program Studi Informatika Universitas Muhammadiyah Surakarta karena telah memberikan kesempatan penulis untuk mendapatkan ilmu serta pengalaman di luar kampus. Penulis mengucapkan terima kasih kepada pihak sekolah SDN Toso 02 yang telah memberikan kesempatan kepada penulis untuk melakukan praktik kerja nyata di sekolah tersebut. Penulis mengucapkan terima kasih kepada seluruh pihak yang telah mmebantu dalam penyusunan jurnal penelitian. SARAN Setelah melakukan perancangan, penulis memberikan saran sebagai berikut: 1. Penelitian selanjutnya diharapkan mampu mengembangkan sistem ini menjadi lebih luas cakupannya dan lebih terintegrasi 2. Dalam perancangan website pada SDN Toso 02 masih banyak kekurangan dan perlu ditambahkan fitur-fitur yang menunjang pembelajaran dan penyebaran informasi. HASIL PENGUJIAN Tahap pengujian adalah tahap yang dilakukan untuk mengetahui tingkat kelayakan sistem, apakah sistem dapat berjalan semestinya sesuai dengan kebutuhan. Hasil pengujian sistem dapat dilihat pada Tabel 2. Tahap pengujian adalah tahap yang dilakukan untuk mengetahui tingkat kelayakan sistem, apakah sistem dapat berjalan semestinya sesuai dengan kebutuhan. Hasil pengujian sistem dapat dilihat pada Tabel 2. Tabel 2. Hasil Pengujian Sistem KESIMPULAN Tabel 2. Hasil Pengujian Sistem Gambar 5. Halaman Gallery (sumber: Hasil Penelitian, 2020) 4. Halaman Registrasi Halaman registrasi, pada gambar 6, digunakan untuk pendaftaran siswa baru. Perancangan sistem informasi sekolah berbasis website sebagai sarana promosi yang kami terapkan di SDN Perancangan sistem informasi sekolah berbasis website sebagai sarana promosi yang kami terapkan di SDN Toso 02 dirancang menggunakan metode prototyping. Sistem informasi dirancang menggunakan bootstrap, javascript, dan Cascading Style Sheets (CSS). Berdasarkan hasil pengujian, sistem yang dirancang dapat berjalan sesuai dengan kebutuhan. 66 \| Jurnal Pengabdian masyarakat teknoyasa, Volume 2, No.2 Desember 2021 DAFTAR PUSTAKA [1] Sugiyono. 2013. Metode Penelitian Kuantitatif, Kualitatif, R&D. Bandung: Penerbit Alfabeta. [1] Sugiyono. 2013. Metode Penelitian Kuantitatif, Kualitatif, R&D. Bandung: Penerbit Alfabeta. [2] Supardi, M.d. 2006. Metodologi Penelitian. Mataram : Yayasan Cerdas Press. [2] Supardi, M.d. 2006. Metodologi Penelitian. Mataram : Yayasan Cerdas Press. [3] Arikunto, S. 2006. Metodelogi Penelitian. Yogyakarta: Bina Aksara. [3] Arikunto, S. 2006. Metodelogi Penelitian. Yogyakarta: Bina Aksara. [4] O’Brein, James A. 2005. Pengantar Sistem Informasi. Jakarta: Salemba Empat. [5] A. Mulyanto. 2009. Sistem Informasi Konsep dan Aplikasi. Yogyalarta: Pustaka Pelajar. [6] S. Sauri, A. T. Haryono, I. F. Astuti, D. M. Khairina, dan D. Cahyadi, “Sistem Informasi Unit Kegiatan Mahasiswa (UKM) Sepakbola Universitas Mulawarman Berbasis Web,” J. Inform. Mulawarman, vol. 10, no. 2, pp. 46–50, 2015. [7] H. Harsono. 2002. Implementasi Kebijakan dan Politik. Bandung: PT Mutiara Sumber Widya. [7] H. Harsono. 2002. Implementasi Kebijakan dan Politik. Bandung: PT Mutiara Sumber Widya. 66 \| Jurnal Pengabdian masyarakat teknoyasa, Volume 2, No.2 Desember 2021
https://openalex.org/W4210801290	https://www.scielo.br/j/reben/a/5p5stWFspwL3tdmY8bKsM8d/?lang=en&format=pdf	English	null	Involvement of companions in patient safety in pediatric and neonatal units: scope review	Revista Brasileira de Enfermagem	2,022	cc-by	9,231	REVIEW REVIEW REVIEW ABSTRACT Thayane Gusmão Pires de OliveiraI ORCID: 0000-0003-2904-4296 Catharine Galvão DinizI ORCID: 0000-0002-0620-6435 Marina Peluci Malta CarvalhoI ORCID: 0000-0001-5567-8853 Allana dos Reis CorrêaI ORCID: 0000-0003-2208-958X Patrícia Kuerten RochaII ORCID: 0000-0002-8347-1363 Bruna Figueiredo ManzoI Objectives: to describe scientific evidence on the involvement of companions in patient safety, from their own perspective and health professionals’ perspective in neonatal and pediatric units. Methods: scoping review carried out according to The Joanna Briggs Institute’s recommendations, in eight databases, following the Preferred Reporting Items checklist for Systematic Reviews and Meta-Analyses extension for Scoping Reviews checklist, between 2011 and 2021. Results: the 13 studies included highlighted the importance of companions’ involvement in patient safety and the prevention of adverse events. However, they pointed out failures in communication and weakness in the training of professionals, which were obstacles to their involvement. The strengthening of health education, multidisciplinary rounds and educational technologies were highlighted as strategies to expand the involvement of companions. Final Considerations: this study directs elements for health professionals and managers to rethink the companions’ role in patient safety and development of collective strategies. Descriptors: Patient Participation; Family; Patient Safety; Pediatrics; Neonatology. Descriptors: Patient Participation; Family; Patient Safety; Pediatrics; Neonatology. How to cite this article: Oliveira TGP, Diniz CG, Carvalho MPM, Corrêa AR, Rocha PK, Manzo BF. Involvement of companions in patient safety in pediatric and neonatal units: scope review. Rev Bras Enferm. 2022;75(3):e20210504. https://doi.org/10.1590/0034-7167-2021-0504 Descritores: Participação do Paciente; Família; Segurança do Paciente; Pediatria; Neonatologia RESUMO Bruna Figueiredo ManzoI ORCID: 0000-0003-0064-9961 Objetivos: descrever evidências científicas sobre o envolvimento dos acompanhantes na segurança do paciente, na perspectiva desses e dos profissionais de saúde em unidades neonatais e pediátricas. Métodos: revisão de escopo realizada segundo recomendações do The Joanna Briggs Institute, em oito bases de dados, seguindo o checklist Preferred Reporting Items for Systematic Reviews and Meta-Analyses extension for Scoping Reviews, entre 2011 e 2021. Resultados: os 13 estudos incluídos evidenciaram a importância do envolvimento do acompanhante na segurança do paciente e na prevenção de eventos adversos. Entretanto, apontaram falhas na comunicação e fragilidade na formação dos profissionais, sendo esses dificultadores para o envolvimento. O fortalecimento da educação em saúde, rounds multidisciplinares e tecnologias educativas foram destacadas como estratégias para ampliar o envolvimento dos acompanhantes. Considerações Finais: esse estudo direciona elementos para que profissionais de saúde e gestores repensem a atuação do acompanhante na segurança do paciente e desenvolvam estratégias coletivas. IUniversidade Federal de Minas Gerais. Belo Horizonte, Minas Gerais, Brazil. IIUniversidade Federal de Santa Catarina. Florianópolis, Santa Catarina, Brazil. IUniversidade Federal de Minas Gerais. Belo Horizonte, Minas Gerais, Brazil. IIUniversidade Federal de Santa Catarina. Florianópolis, Santa Catarina, Brazil. 1 Rev Bras Enferm. 2022;75(3): e20210504 10 of Ethical aspects The term “patient participation” can be defined as the patient’s involvement in the decision-making process in relation to health issues(4). Based on this principle, the aim is to enable patients to know about their health status, whether encouraged to interact with professionals and participate in the decisions of their care plan. Thus, the patient and the companion are encouraged to get involved in care with their rights and singularities respected and, at the same time, collaborate in the prevention of AE(5-6). As this is a scoping review, submission to the Research Ethics Committee was not necessary. However, the reliability and reli- ability of the information contained in the selected publications was guaranteed(18). Type of study This is a scope review, which aims to identify or explain the main scientific evidence on a certain topic, highlighting existing knowledge gaps, in addition to proposing the clarification of the main concepts present in the literature(19). It is noteworthy that the research was prepared based on the guidelines of the Review Manual of the Joanna Briggs Institute(19). Regarding pediatric and neonatology units, children are more exposed to the occurrence of AE because of their specific charac- teristics. Accelerated metabolism, greater variation in body weight when compared to adults, frequent adjustment of drug doses and concentrations, immaturity in the development of organs and sys- tems, curiosity and unpredictability of movements, characteristics of child development, among other characteristics, make profes- sionals and managers pay special attention to these patients(7-9). RESUMEN Objetivos: de Objetivos: describir la evidencia científica sobre la implicación de los acompañantes en la seguridad del paciente, desde la perspectiva del susodicho y de los profesionales sanitarios de las unidades neonatales y pediátricas. Métodos: es una revisión de alcance realizada entre 2011 y 2021 en ocho bases de datos, según las recomendaciones del Instituto Joanna Briggs y siguiendo la lista de verificación Preferred Reporting Items for Systematic Reviews and Meta- Analyses extension for Scoping Reviews. Resultados: se incluyeron 13 estudios que demuestran la importancia de la implicación en la seguridad del paciente y en la prevención de eventos adversos. Sin embargo, se han detectado grietas en la comunicación y fragilidad en la formación de los profesionales, lo que dificulta dicha implicación. El fortalecimiento de la educación en salud, las rondas multidisciplinares y las tecnologías educativas se destacaron como estrategias para ampliar el envolvimiento de los acompañantes. Consideraciones Finales: Este estudio dirige elementos para que profesionales de la salud y gerentes reconsideren el papel de los acompañantes en la seguridad del paciente y desarrollo de estrategias colectivas. Descriptores: Participacón del Paciente; Família; Seguridad del Paciente; Pediatría; Neonatología. EDITOR IN CHIEF: Antonio José de Almeida Filho ASSOCIATE EDITOR: Hugo Fernandes VERSÃO ON-LINE ISSN: 1984-0446 1 Rev Bras Enferm. 2022;75(3): e20210504 10 of https://doi.org/10.1590/0034-7167-2021-0504 https://doi.org/10.1590/0034-7167-2021-0504 Involvement of companions in patient safety in pediatric and neonatal units: scope review Oliveira TGP, Diniz CG, Carvalho MPM, Corrêa AR, Rocha PK, Manzo BF. Involvement of companions in patient safety in pediatric and neonatal units: scope review Oliveira TGP, Diniz CG, Carvalho MPM, Corrêa AR, Rocha PK, Manzo BF. INTRODUCTION question arose: what does the literature present about evidence on the involvement of companions in PS, from their perspective and that of health professionals in neonatal and pediatric units? It is noteworthy that, in this study, by portraying pediatric and neonatology scenarios, parents, family members or other care- givers were characterized as companions. The discussion on initiatives to promote safety and quality in health care has been the subject of wide debate worldwide. Patient safety (PS) is understood as the reduction of the risk of unnecessary harm caused by health care through measures that offer better results(1). Thus, patient safety is linked to the adoption of strategies aimed at preventing the occurrence of preventable adverse events (AE) and, when not avoidable, minimize their ef- fects on the patient, in addition to encouraging the adoption of a safety culture, in which errors can be recognized and avoided(1). This study is justified by obtaining elements that can support discussions on effective strategies in search of greater involve- ment of the companion in PS in neonatal and pediatric units. OBJECTIVES Regarding the risk of AE in health care, the World Health Orga- nization (WHO) created the World Alliance for Patient Safety with the aim of improving the quality of health services(2). Therefore, the Ministry of Health, aiming at achieving the WHO goals, implemented the National Patient Safety Program (NPSP) aiming to contribute to safe care in the national territory(3). One of the axes of this program is the “Patient for patient safety”, since participation of patients and caregivers during the hospitalization process has been extremely important in increasing PS, as well as in reducing adverse events(2). To describe the scientific evidence on the involvement of companions in patient safety, from their and health professionals’ perspective in neonatal and pediatric units. Methodological Procedure This study was registered in the Open Science Framework Plat- form (https://osf.io/srzvw/), adopting the Preferred Reporting Items for Systematic Reviews and Meta-Analyses extension for Scoping Reviews checklist (PRISMA-ScR)(20), in order to describe the scien- tific evidence on the involvement of caregivers in patient safety. An American study developed by the Institute of Healthcare Improvement identified that, for every 100 children, 40 were victims of AE, 18 being potentially preventable(9). In Brazil, a descriptive, cross-sectional observational study found that there were a total of 73 adverse events in the Neonatal and Pediatric Intensive Care Unit, highlighting, among them, losses from the Peripherally In- serted Central Catheter (PICC), phlebitis, skin or soft tissue injury, medication errors, among others(7). In the search for PS and AE prevention, studies emphasize the importance of including com- panions in patient care, especially in pediatrics and neonatology(10-14). Encouraging the participation of companions in child care favors health education and the co-production of care between them and health professionals(15). However, the literature considers that the family’s participation in PS is permeated by many challenges, with different perceptions among the people who participate in the care circumstances, which need to be discussed in search of strategies that lead to a more effective practice(16-17). Thus, for its development, as mentioned, the recommendations published in the JBI Manual for Evidence Synthesis(19), version 2020, were followed, covering the following steps: defining the objective and research question; define inclusion criteria; define strategy for data selection and extraction; search, selection and analysis of publications in information sources; and presentation and synthesis of results. The mnemonic population, concept and context (PCC)(19), was used, where P: companions of children and newborns, C: involve- ment in patient safety and C: pediatric and neonatal hospital units, to create the guiding question: What are the available evidence on companion involvement in pediatric and neonatal patient safety? The inclusion criteria for the pre-selection of studies were: English, Portuguese or Spanish; and publications between 2011 and 2021. Duplicate and review articles, editorials, letters to the editor, abstracts and expert opinion or articles that did not meet the purpose of this review were considered as exclusion criteria. In order to provide essential information for the creation of strategies aimed at involving the companion in PS and favoring the transposition of scientific evidence on the subject exposed to care practice in neonatology and pediatrics, the following 2 Rev Bras Enferm. Data collect The next steps (extraction, creation and search of evidence) correspond to the research strategy and were reported in a comprehensive manner, in accordance with the JBI recommen- dations. The search was carried out between July 2020 and April 2021, in the Latin American and Caribbean Literature in Health Science (LILACS) database, nursing database (BDENF) Medical Literature Analysis and Retrieval System Online (MEDLINE) ac- cess via PubMed, Cochrane Library, CINAHL, SCOPUS, Web of Science and EMBASE. Appropriate descriptors were chosen for the researched databases (Medical Subject Headings - MeSH and Descriptors in Health Sciences - DeCS), as well as keywords were adopted in order to expand the textual research. Furthermore, the reference list of the articles included was used, in order to verify the articles used and retrieve the pertinent ones. Chart 1 demonstrates the strategies developed using the descriptors listed with the help of the Boolean operators AND and OR, in addition to the quantity of articles located and selected in each database. It should be noted that two more articles were included in the reference list, totaling 13 articles in the sample. The study selection was performed through careful reading of titles and abstracts, in order to verify if the studies fit in the final selection, meeting the aforementioned criteria. For the final selection, articles that presented evidence on the involvement of the companion in pediatric and neonatal patient safety were chosen. Data collection and analysis were performed by three independent reviewers and, when consensus was not possible, the evaluation of a fourth reviewer was used. The final stages of extraction and delimitation of information related to the evidence were carried out through descriptive analysis to characterize the studies. This step was performed using an instrument developed by the authors, consisting of the characterization of the publica- tion (year, publication country, title and authors), methodological characteristics (study design, scenario and sample), main results and level of evidence of the study. The classification regarding the level of evidence was based on the categorization of the Agency for Healthcare Research and Quality (AHRQ), which proposes seven levels of evidence, 1 being the highest level and 7 the lowest level of evidence(21). Data collect 3 0 Chart 1 – Search strategies and number of studies located and selected in databases, Belo Horizonte, Minas Gerais, Brazil, 2021 Database (Number of studies) Search Strategies Included study LILACS (22) (“Patient Safety” OR “Seguridad del Paciente” OR “Segurança do Paciente”) AND (“Medical Chaperones” OR “Chaperones Médicos” OR “Acompanhantes Formais em Exames Físicos” OR “Acompanhante Formal do Paciente” OR “Acompanhante Médico” OR “Acompanhante de Paciente” OR “Acompanhantes Formais de Pacientes” OR “Acompanhantes de Pacientes” OR “Apoio Familiar de Paciente” OR “caregivers” OR “cuidadores” OR “cuidador” OR “Cuidador Familiar” OR “Cuidador de Família” OR “Cuidadores Familiares” OR “Cuidadores de Família” OR “Familiar Cuidador” OR “Familiares Cuidadores” OR “Participação da Família” OR “Participação Familiar” OR “Family Participation” OR “Envolvimento da Família” OR “Envolvimento Familiar” OR “Family Involvement” OR “Engajamento da Família” OR “Engajamento Familiar” OR “Family Engagement” OR “envolvimento” OR “involvement” OR “engajamento” OR “engagement” OR “acompanhante” OR “companion”) AND (“pediatrics” OR “pediatria” OR “pediatria” OR “child” OR “niño” OR “criança” OR “crianças” OR “Paciente Pediátrico” OR “Pediatric Patient”). 1 BDENF (10) (“Patient Safety” OR “Seguridad del Paciente” OR “Segurança do Paciente”) AND (“Medical Chaperones” OR “Chaperones Médicos» OR «Acompanhantes Formais em Exames Físicos” OR “Acompanhante Formal do Paciente” OR “Acompanhante Médico” OR “Acompanhante de Paciente” OR “Acompanhantes Formais de Pacientes” OR “Acompanhantes de Pacientes” OR “Apoio Familiar de Paciente” OR “caregivers” OR “cuidadores” OR “cuidador” OR “Cuidador Familiar” OR “Cuidador de Família” OR “Cuidadores Familiares” OR “Cuidadores de Família” OR “Familiar Cuidador” OR “Familiares Cuidadores” OR “Participação da Família” OR “Participação Familiar” OR “Family Participation” OR “Envolvimento da Família” OR “Envolvimento Familiar” OR “Family Involvement” OR “Engajamento da Família” OR “Engajamento Familiar” OR “Family Engagement” OR “envolvimento” OR “involvement” OR “engajamento” OR “engagement” OR “acompanhante” OR “companion”) AND (“pediatrics” OR “pediatria” OR “pediatria” OR “child” OR “niño” OR “criança” OR “crianças” OR “Paciente Pediátrico” OR “Pediatric Patient”). 2 MEDLINE via PubMed (908) “patient safety AND medical chaperones OR caregivers OR family participation OR family involvement OR family engagement OR involvement OR engagement OR compa]nion AND pediatrics OR child OR pediatric patient ”. 6 COCHRANE (163) “patient safety AND medical chaperones OR caregivers OR family participation OR family involvement OR family engagement OR involvement OR engagement OR companion AND pediatrics OR child OR pediatric patient”. Methodological Procedure 2022;75(3): e20210504 10 of Involvement of companions in patient safety in pediatric and neonatal units: scope review Oliveira TGP, Diniz CG, Carvalho MPM, Corrêa AR, Rocha PK, Manzo BF. DISCUSSION This study contributed to investigations on the involvement of companions in PS in pediatric and neonatal units from the perspective of those and health professionals. The results obtained showed points of divergence and agreement in relation to pro- fessionals and companions regarding the perception, strategies used, facilitating and hindering aspects for the involvement of companions in patient safety. Records screened (n =2587) Full text articles excluded, with reasons (n = 143) Records excluded (n =2431) Studies included in qualitative synthesis (n =13) Inclusion Elegibility Selection Identification Records after duplicates removed (n =66) Records identified through database searching (n =2651) Additional records identified through other sources (n = 2) Full text articles assessed for eligibility (n = 156) Figure 1 – Flowchart of the review article selection process, PRISMA-ScR, Belo Horizonte, Minas Gerais, Brazil, 2021 Records identified through database searching (n =2651) Additional records identified through other sources (n = 2) Regarding the perception of the involvement of compan- ions in PS, they recognized that they can contribute to patient safety, especially in the prevention of incidents and AE(23,25,29), in addition to stressing that this participation should be mandatory(25), diverging from the professionals’ perception(33). However, professionals say that companions, as long as they are oriented and aware of their participation, can positively influence PS. They also reinforce the need for interaction, dialogue and clear communication between professionals and companions(23,33). This is a very important aspect, consid- ering that companions recognize that the lack of attention and support from professionals to the observations made by them, related to the children health and safety, contributes to increased anxiety, insecurity and even from stress, causing distancing instead of approximation(29-30). Records excluded (n =2431) Elegibility Full text articles excluded, with reasons (n = 143) Figure 1 – Flowchart of the review article selection process, PRISMA-ScR, Belo Horizonte, Minas Gerais, Brazil, 2021 Chart 2 – Characteristics of the studies included in the scoping review, Belo Horizonte, Minas Gerais, Brazil, 2021 Author Title Year Country Objective Study design Sample Scenario Results Level of evidence Biasibetti et al.(22) “Comunicação para a segurança do paciente em internações pediátricas” 2019 Brazil To analyze the perception of health professionals and companions/family members regarding the development of communication for patient safety in pediatric hospitalizations. Type of study: Qualitative. Sample: 44 health professionals and 94 companions. Scenario: Pediatric clinical-surgical inpatient units. Data collect 0 CINAHL (204) “patient safety AND medical chaperones OR caregivers OR family participation OR family involvement OR family engagement OR involvement OR engagement OR companion AND pediatrics OR child OR pediatric patient”. 0 SCOPUS (459) “patient safety AND medical chaperones OR caregivers OR family participation OR family involvement OR family engagement OR companion AND pediatric patient”. 2 WEB OF SCIENCE (15) “patient safety AND medical chaperones OR caregivers OR family participation OR family involvement OR family engagement OR involvement OR engagement OR companion AND pediatrics OR child OR pediatric patient”. 0 EMBASE (711) “patient safety AND family OR caregiver AND pediatric patient”. 0 TOTAL* 11 *Total of studies found in each database. Involvement of companions in patient safety in pediatric and neonatal units: scope review Oliveira TGP, Diniz CG, Carvalho MPM, Corrêa AR, Rocha PK, Manzo BF. Involvement of companions in patient safety in pediatric and neonatal units: scope review Oliveira TGP, Diniz CG, Carvalho MPM, Corrêa AR, Rocha PK, Manzo BF. Involvement of companions in patient safety in pediatric and neonatal units: scope review Oliveira TGP, Diniz CG, Carvalho MPM, Corrêa AR, Rocha PK, Manzo BF. The final sample consisted of a total of 13 articles, among which the oldest was published in 2014 and the most recent in 2020. Three (23.1%) were published in 2020, two (15.4%) in 2019, two (15 .4%) in 2018, three (23.1%) in 2017 and three (23.1%) in 2014. Then, a thematic analysis of the content was carried out to identify the key posts in the literature found, which were grouped into guiding axes. Finally, the results were reviewed in relation to the involvement of companions in the safety of pediatric and neonatal patients, especially with regard to facilitating and hindering aspects, as well as the strategies of involvement of the companion in patient safety. The most frequent design was qualitative, with ten (76.9%) articles, followed by quantitative, with two (15.4%), and one (7.7%) characterized as a mixed method. The studies included in the review are presented in Chart 2 with information on authors, title, year, country of publication, objective, design, sample, scenario, results and level of evidence. RESULTS A total of 2651 studies were found in the databases, 2 were identified in the reference lists. From the first analysis, 66 studies were removed for duplication, totaling 2587 for title and abstract reading. Subsequently, the three reviewers independently verified those who answered the research question or who were within the topic, with the selection of articles proper for reading the full text, with 2431 studies being excluded, resulting in articles eligible for reading. Of these, 143 were analyzed according to the inclusion criteria. Therefore, 13 articles met the inclusion criteria and were part of the final sample (Figure 1). Thus, to facilitate the presentation of the information ex- tracted from the records, the contents were grouped into four guiding axes: perception of the involvement of companions in patient safety, facilitating aspects, hindering aspects and strategies to increase the involvement of companions in the patient safety (Chart 3). DISCUSSION Participants identified problems in the academic training of health professionals, failures in institutional organization, lack of professional commitment and lack of integration between the health team and companions. As for the tools to qualify communication, the participants pointed out strategies such as the organization and compliance with standardized, computerized and bureaucratic processes, as well as the participation of all actors involved in care for the development of improvements related to pediatric patient safety through of effective communication. 6 To be continued Chart 2 – Characteristics of the studies included in the scoping review, Belo Horizonte, Minas Gerais, Brazil, 2021 4 Rev Bras Enferm. 2022;75(3): e20210504 10 of Chart 2 Author Title Year Country Objective Study design Sample Scenario Results Level of evidence Corbaly et al.(23) “Parental involvement in the preoperative surgical safety checklist is welcomed by both parents and staff” 2014 Ireland To establish how the surgical team and parents accept parental involvement during the Safe Surgery Checklist. Type of study: Qualitative. Sample: 42 parents and 42 professionals. Scenario: Mother and child hospital. Parents feel that family participation during the safe surgery checklist should be mandatory. The healthcare team recognized that parental participation is important to promote patient safety. 6 Gonçalves et al.(24) “Estratégia lúdica para promoção do engajamento de pais e acompanhantes na segurança do paciente pediátrico” 2020 Brazil To evaluate a playful strategy developed to promote the engagement of parents and companions in pediatric patient safety actions. Type of study: Qualitative. Sample: 17 parents and companions. Scenario: Pediatric inpatient unit. Participants evaluated the game as a playful, innovative, informative and educational resource regarding the process of family involvement in patient safety. Furthermore, they highlighted the change in behavior in favor of patient safety after experiencing the game. 6 Hoffman et al.(25) “Identificação de incidentes de segurança do paciente pelos acompanhantes de crianças hospitalizadas” 2019 Brazil Describe the security incidents identified by the companions of hospitalized children. Type of study: Qualitative. Sample: 40 companions. Scenario: Pediatric Inpatient Units. The incidents identified by the companions were related to falls, identification, dietary errors, medication errors, hygiene and communication failures. 6 Hoffman et al.(26) “Patient safety incidents reported by relatives of hospitalized children” 2020 Brazil To know the main safety incidents reported by family members of patients admitted to pediatric units. Type of study: Qualitative. Sample: 91 family members. Scenario: Intensive care unit Pediatrics and Pediatric Emergency Room. To be continued DISCUSSION Family members recognized incidents related to medication administration, communication failures, incorrect hand hygiene, incorrect use of Personal Protective equipment, failures in patient identification and monitoring of visits. 6 Khan et al.(27) “Families as partners in hospital error and adverse event surveillance” 2017 United States Perform comparison of adverse events records: 1)With the presence and report of the family and without the family; 2)Reported by family and physicians; 3)Reported by the family and rate established by the hospital. Type of study: Quantitative. Sample: 717 companions and 77 nursing and medical residents. Scenario: Pediatric units. 1)The rates of adverse events reported in the presence of family members were 1.1 times higher than rates without family members. 2) Families and physicians reported similar error rates (10.0 vs 12.8 per 1000 patient-days; and AEs (8.5 vs 6.2 per 1000 patient-days). 3) Family-reported error rates were 5.0 times higher than adverse events rates reported by hospital incident reports. 4 Lydon et al.(28) “Parents’ perspectives on safety in neonatal intensive care: a mixed-methods study” 2014 United States To examine parents’ perspectives regarding the safety of the patient in the Neonatal Intensive Care Unit. Type of study: Qualitative and quantitative. Sample: 46 parents responded to the questionnaires and 14 of these parents also participated in 10 interviews. Scenario: Neonatal Intensive Care. Parents showed little concern about the safety of the procedures. Therefore, participants suggested engagement strategies that address clinical treatment articulated to the domains of physical, developmental and emotional safety, which may result in safety improvements. 6 Chart 2 To be continued To be continued 5 Rev Bras Enferm. 2022;75(3): e20210504 10 of Author Title Year Country Objective Study design Sample Scenario Results Level of evidence Massa et al.(29) “Condiciones de seguridad percibidas por cuidadores familiares en atención pediátrica” 2020 Colombia To identify the perception of caregivers about the safety conditions of care in a pediatric hospital. Type of study: Quantitative. Sample: 163 caregivers. Scenario: Pediatric Units. 86% of caregivers perceived safety in the child’s hospitalization and, according to them, 60.2% of professionals explained the procedures to caregivers. Communication is clear for 70% of caregivers, 58.3% of them are alert to detect possible risks, 75.5% follow the recommendations given and 70.5% of caregivers trust the professionals. DISCUSSION 6 Peres et al.(30) “Percepção de familiares e cuidadores quanto à segurança do paciente em unidades de internação pediátrica” 2018 Brazil To recognize the perceptions of family members and companions regarding patient safety in a pediatric inpatient unit. Type of study: Qualitative. Sample: 24 companions. Scenario: Pediatric Inpatient Units. Several respondents have never heard of patient safety. Family members believe that their main role is to provide emotional support for the child, but they recognize that they can help to prevent mistakes. Adverse events related to medication, procedure and lack of communication are the most worrying of parents. 6 Rodrigues et al.(31) “Segurança do paciente em unidade neonatal: preocupações e estratégias vivenciadas por pais” 2018 Brazil To analyze how parents identify patient safety in a neonatal unit. Type of study: Qualitative. Sample: 23 family members. Scenario: Neonatal inpatient unit. The parents spoke about patient safety, mentioning their concerns regarding the flow of people, inefficient communication, problems with patient identification, the risk of infection, the risk of falling and injury due to the use of a medical device, and strategies that could prevent these incidents. 6 Rosenberg et al.(32) “Provider perspectives on partnering with parents of hospitalized children to improve safety” 2017 United States To explore the perspectives and experiences of pediatric professionals regarding family participation in pediatric patient safety. Type of study: Qualitative. Sample: 20 health professionals. Scenario: Pediatric teaching hospital. Professionals believe that parental involvement should not be mandatory, but that it helps to reduce errors. They presented, as facilitating aspects for patient safety, the clear definition of roles, the gain of trust, sympathy and effective communication. As barriers, they mentioned role conflicts, the lack of skill and time to put parents in safety, in addition to the concern with overloading the family. 6 Silva et al.(33) “Segurança da criança hospitalizada na UTI: compreendendo os eventos adversos sob a ótica do acompanhante” 2014 Brazil To describe the adverse events identified by the family member/ caregiver in a Pediatric Intensive Care Unit. Type of study: Qualitative. Sample: 13 companions. Scenario: Pediatric Intensive Care Unit. The family member/caregiver realized that many of the adverse events were related to a lack of scientific knowledge on the part of the nursing team to safely perform some procedures. Regarding the medical team, there were problems related to communication with family members/caregivers, especially the form and content of the information received. To be continued Perspective of health professionals right time, in addition to monitoring and clarifying doubts with a professional during the administration of the medication(37-38). Therefore, the importance of the companions’ participation in the prevention of medication errors is perceived, in order to effectively contribute to the quality of health care and in the PS(39). Professionals believe that involvement depends on the pro- fessional attitude of encouraging companions, as well as their desire to participate(33). The health team also argues that the lack of skills, training, time to introduce companions to safety, and the concern with the increase in work demand are limiting aspects in this process. Another reported point concerns the absence of a companion at many times and the conflict of roles that may exist between companions and professionals, which cause wear on both parties, often unnecessary, directly interfering with the quality of care and patient safety(18,33). With regard to the patient identification protocol, parents or guardians should receive information, at the time of placing the bracelets, about the importance of their use and the need for maintenance and verification of data. Thus, a study warns that only 35% of parents or guardians were informed about the importance of using an identification bracelet, which motivates the discussion about the need for companions to receive information so that they can be co-participants in the care, contributing to the safety of the neonatal care(40). The companions also mentioned the importance of recognizing risk situations for AEs, especially in relation to medication errors, patient identification(26-27,30-31) and care procedures(27,30-32). Among the incidents recognized by companions, there are delays in administration, poor communication related to therapy, mistake in drug suspension, infu- sion time and incorrect dosages, and lack of an allergy bracelet(27). Studies show that, in pediatrics, many AE could be minimized or prevented in face of a partnership and effective communication with the family(30,33-36). The companion has the right to be informed about the medications in use, being able to pay attention to some aspects, such as checking the right medication for the child, at the Regarding the incidents detected by companions related to care procedures, failures in care with probes, incorrect disposal of materials, delays in forwarding exams and surgical procedures, incorrect handling of catheters, among others, were identified(27). DISCUSSION Involvement of companions in patient safety in pediatric and neonatal units: scope review Oliveira TGP, Diniz CG, Carvalho MPM, Corrêa AR, Rocha PK, Manzo BF. Involvement of companions in patient safety in pediatric and neonatal units: scope review Oliveira TGP, Diniz CG, Carvalho MPM, Corrêa AR, Rocha PK, Manzo BF. Involvement of companions in patient safety in pediatric and neonatal units: scope review Oliveira TGP, Diniz CG, Carvalho MPM, Corrêa AR, Rocha PK, Manzo BF. Chart 3 (concluded) Chart 3 (concluded) Facilitating aspects for the involvement of companions in patient safety Perspective of companions Perspective of health professionals • Receive explanations about the rules and procedures offered by the team(30); • Having clear communication with the health team(30); • Receive information about risks, complications and procedures performed(30); • Receive support and attention from the team when identifying risks for adverse events(30); • Trust professionals(29-30); • Be aware of the responsibility of companions in monitoring the care provided to their children(29,31); • Enable companions to identify risks and adverse events(26-27,30-32). • Participation of companions in the safe surgery checklist does not change the procedure time(24); • Recognition of the importance of the participation of companions during the safe surgery checklist(24); • Family recognition as a barrier to preventing adverse events(23). Hindering aspects for the involvement of companions in patient safety Perspective of companions Perspective of health professionals • Realize that there are difficulties in reporting patient safety incidents in a form or computerized system(23); • Ineffective communication between all those involved in child care(23,27,29,32); • Lack of sufficient materials and equipment, as well as the precariousness of the hospital structure(26); • Impact of the Neonatal Intensive Care Unit environment on the bonding process between the child and companions(29); • Lack of knowledge of how to help prevent health risks(30); • Unfamiliarity of the patient/companion’s rights and duties(30); • Companions feel excluded from the child’s treatment, diagnosis and prognosis, becoming more anxious and insecure(34); • Unfamiliarity about the term patient safety(31). • Ineffective communication between everyone involved(23,33); • Feeling of incapacity on the part of professionals to deal with companions(33); • Absence or distraction of some companions(33); • Concern of professionals with overloading companions(33). DISCUSSION Strategies for the involvement of companions in patient safety Perspective of companions Perspective of health professionals • Games and playful activities that address the subject of patient safety(25); • Effective communication between everyone involved in child care(23,32); • Adoption of practices against adverse events with the participation of companions(26,29,31); • Professional qualification through continuing education and encouraging research in the area of patient safety(31,34). • Exchange of information between professionals through systematic shift change, multidisciplinary rounds, transfer of care between sectors and regular team meetings with the inclusion of companions(23); • Integration of the care team with companions(23); • Practice of double checking information with companions(23); • Creation of training and ongoing education to train the team in the process of including companions in patient care and safety(23). Facilitating aspects for the involvement of companions in patient safety Perspective of companions Perspective of health professionals • Receive explanations about the rules and procedures offered by the team(30); • Having clear communication with the health team(30); • Receive information about risks, complications and procedures performed(30); • Receive support and attention from the team when identifying risks for adverse events(30); • Trust professionals(29-30); • Be aware of the responsibility of companions in monitoring the care provided to their children(29,31); • Enable companions to identify risks and adverse events(26-27,30-32). • Participation of companions in the safe surgery checklist does not change the procedure time(24); • Recognition of the importance of the participation of companions during the safe surgery checklist(24); • Family recognition as a barrier to preventing adverse events(23). Hindering aspects for the involvement of companions in patient safety Perspective of companions Perspective of health professionals • Realize that there are difficulties in reporting patient safety incidents in a form or computerized system(23); • Ineffective communication between all those involved in child care(23,27,29,32); • Lack of sufficient materials and equipment, as well as the precariousness of the hospital structure(26); • Impact of the Neonatal Intensive Care Unit environment on the bonding process between the child and companions(29); • Lack of knowledge of how to help prevent health risks(30); • Unfamiliarity of the patient/companion’s rights and duties(30); • Companions feel excluded from the child’s treatment, diagnosis and prognosis, becoming more anxious and insecure(34); • Unfamiliarity about the term patient safety(31). DISCUSSION 6 Sousa et al.(34) ‘’A participação da família na segurança do paciente em unidades neonatais na perspectiva do enfermeiro” 2017 Brazil To understand family participation in patient safety in Neonatal Intensive Care Units from the perspective of nurses. Type of study: Qualitative. Sample: 14 nurses. Scenario: Intermediate Care and Neonatal Intensive Care Unit. Nurses recognize the importance of family participation in neonatal patient safety, as well as in the prevention of incidents. However, they highlighted the lack of preparation to deal with the family member in their daily work. They also highlighted that the welcoming and guidance of family members are important strategies for family involvement in patient safety actions. 6 Chart 2 (concluded) Professionals believe that parental involvement should not be mandatory, but that it helps to reduce errors. They presented, as facilitating aspects for patient safety, the clear definition of roles, the gain of trust, sympathy and effective communication. As barriers, they mentioned role conflicts, the lack of skill and time to put parents in safety, in addition to the concern with overloading the family. The family member/caregiver realized that many of the adverse events were related to a lack of scientific knowledge on the part of the nursing team to safely perform some procedures. Regarding the medical team, there were problems related to communication with family members/caregivers, especially the form and content of the information received. Nurses recognize the importance of family participation in neonatal patient safety, as well as in the prevention of incidents. However, they highlighted the lack of preparation to deal with the family member in their daily work. They also highlighted that the welcoming and guidance of family members are important strategies for family involvement in patient safety actions. 6 Rev Bras Enferm. 2022;75(3): e20210504 10 of To be continued Chart 3 – Analysis of the involvement of companions in patient safety, from their and health professionals’ perspective, Belo Horizonte, Minas Gerais, Brazil, 2021 Perception about the involvement of companions in patient safety Perspective of companions Perspective of health professionals • Companion as a barrier to the occurrence of adverse events(23,25,29); • Participation of companions contributes to patient safety and should be mandatory(24); • Participation of companions does not predispose to increased anxiety(24). • The involvement of companions depends on the professional’s attitude(33); • The involvement of companions should not be mandatory(33). DISCUSSION • Ineffective communication between everyone involved(23,33); • Feeling of incapacity on the part of professionals to deal with companions(33); • Absence or distraction of some companions(33); • Concern of professionals with overloading companions(33). Strategies for the involvement of companions in patient safety Perspective of companions Perspective of health professionals • Games and playful activities that address the subject of patient safety(25); • Effective communication between everyone involved in child care(23,32); • Adoption of practices against adverse events with the participation of companions(26,29,31); • Professional qualification through continuing education and encouraging research in the area of patient safety(31,34). • Exchange of information between professionals through systematic shift change, multidisciplinary rounds, transfer of care between sectors and regular team meetings with the inclusion of companions(23); • Integration of the care team with companions(23); • Practice of double checking information with companions(23); • Creation of training and ongoing education to train the team in the process of including companions in patient care and safety(23). Perspective of health professionals These incidents pointed out by the companions reveal possible flaws in the follow-up records, highlighting the importance of family involvement not only in prevention, but also in the 7 Rev Bras Enferm. 2022;75(3): e20210504 10 of 7 10 of Involvement of companions in patient safety in pediatric and neonatal units: scope review Oliveira TGP, Diniz CG, Carvalho MPM, Corrêa AR, Rocha PK, Manzo BF. Involvement of companions in patient safety in pediatric and neonatal units: scope review Oliveira TGP, Diniz CG, Carvalho MPM, Corrêa AR, Rocha PK, Manzo BF. distraction and relaxation, as the hospital environment is extremely stressful(24,41-42). evaluation process of care procedures. To enable the companion’s involvement in the child’s safety, it is necessary for professionals to improve the instrumentation of caregivers through education and in the documentation of records in medical records(39). Health education is also a relevant plan for the training of health professionals with regard to the focus of objective communication. As a form of educational strategy, members of the multidisciplinary team suggest that training be carried out within the service, with a focus on patient safety(22). This strategy is not only related to the failure of communication, but also usefully covers the correct hand hygiene, proceeding properly in the use of procedure gloves and in the use of individual and collective protective equipment, which if used, when applied, incorrectly affect the PS(25). The use of computerized methodologies has also been considered as a strat- egy to improve communication between health professionals(22). Other AEs identified by companions were risk of infec- tions(26-27,30,32), phlebitis(30), falls or risk of falling(26,27,30-31), pressure injury(30), lack of control of access to the unit(27,32), failures in the unit’s routine and team dynamics(32), errors in the diet offered to patients(26-27) and unidentified isolation beds(26). These situations reinforce the importance of involving companions as partners in the prevention of AE and, consequently, of PS(23). Thus, there is a growing concern of health and teaching institutions with the PS policy, which includes investment in bringing the companion closer to the care being provided, making them co-responsible and partners for the prevention of failures and damage, with increment of the possibilities of a safe hospitalization(40). Furthermore, an environment conducive to conflict resolution is critical to promoting safer care. The companions also pointed out that clearer communication with health professionals leads to better instruction to participate in the care, preserving possible risks to the child’s health(22). Perspective of health professionals Another relevant aspect in the findings is related to the partici- pation of the companion during the safe surgery checklist. For the professionals, the presence of companions during the checklist has been a very valuable experience, as it provides opportunities for interaction, dialogue and listening(24). The authors state that trust in professionals(29-30) and clear communication between professionals and companions are determining factors for involvement. “Clear communication” is understood as receiving explanations about the rules and procedures offered by the health team and information about risks, complications and procedures performed(30). Com- munication reveals itself as one of the main points of attention regarding the effectiveness of the companions’ involvement in PS. Another study infers that the inclusion of companions in the child’s general care is important, especially in the surveillance of patient safety(26). This surveillance expands the detection of errors committed by health professionals. Therefore, giving voice to companions and listening to reports can increase the quality and safety of care for hospitalized patients. Therefore, the inclusion of correct information dissemination within the hospital environment, as well as their amplification strategies, is extremely important, since the lack of knowledge about AE, the prevention of these errors, the lack of communication and the lack of improvements in basic patient safety techniques can put the child’s hospitalization at risk. Therefore, although studies point out multiple factors that favor the distancing of companions during the care process, it is noted that communication goes beyond all these spheres. Communication failures are identified by both professionals and companions, and it may occur within the team, as in situations of misunderstanding between professionals, high staff turnover, bureaucracy in filling out medical records and side conversations during the shift change. The lack of scientific knowledge on the part of companions can generate dependence on providing specific information in a clear and objective way(23,27,29,32-33). Thus, the strategies to promote the involvement of the companion are very much based on improving communication. Contributions to the area of nursing As a tool to qualify the communication process, especially among professionals, it is important to double check the in- formation, including the companion. This practice of checking is recommended from dispensing the drug at the pharmacy to administering it to the patient. Double verification can be a strategy that promotes communication between professionals, being proposed to avoid greater risks of AE(22). The results found favor discussions on the understanding of the companions’ participation in the promotion of PS, in addition to contributing to the planning and implementation of strategies aimed at encouraging the involvement of these companions and, consequently, offering safer care to children and newborns. The frequent exchange of information between professionals, through systematic and judicious shift change, multidisciplinary rounds, transfer of care between sectors and regular team meetings, can also increase the safety of hospitalized patients. For this practice to be incorporated and continued in care, factors such as multidis- ciplinary and the aptitude of professionals can be implemented. In addition to these, it is recommended to avoid parallel conversations, interruptions in the method, as well as early entries and exits(22). Limitations of the study It was possible to notice in some studies included in this review the lack of detailed information regarding the characteristics of the engagement strategies used and their evaluation process by the participants. Furthermore, some databases do not use controlled descriptors, which may favor missing studies. FINAL CONSIDERATIONS The study allowed unveiling how companions and health professionals in pediatrics and neonatology perceive their in- volvement in PS, the facilitating and hindering aspects and the strategies that aim for this purpose. FUNDING Another strategy refers to playful games, which are tools increas- ingly used to provide information in a simple way. These games, in addition to bringing professionals closer to companions, favor The authors did not receive any financial support for develop- ing this research, analyzing data or submitting this manuscript. 8 Rev Bras Enferm. 2022;75(3): e20210504 10 of Involvement of companions in patient safety in pediatric and neonatal units: scope review Oliveira TGP, Diniz CG, Carvalho MPM, Corrêa AR, Rocha PK, Manzo BF. REFERENCES 1. World Health Organization. Patient safety: about us [Internet]. Geneva: WHO; c2009-2016[cited 2016 Dec 02]; [about 2 screens]. Available from: http:// www.who.int/patientsafety/about/en/ 2. Agência Nacional de Vigilância Sanitária (BR). Assistência segura: uma reflexão teórica aplicada à prática agência na sanitária. Brasília, DF: Anvisa; 2017. 3. Ministério da Saúde (BR). Documento de referência para o programa nacional de segurança do paciente. Brasília, D 4. Longtin Y, Sax H, Leape LL, Sheridan SE, Donaldson L, Pittet D. Patient participation: current knowledge and applicability to patient safety. Mayo Clin Proc. 2010;85(1):53-62. https://doi.org/10.4065/mcp.2009.0248 5. Souliotis K, Agapidaki E, Peppou LE, Tzavara C, Varvaras D, Buonomo OC, et al. Assessing patient organization participation in health policy: a comparative study in France and Italy. Int J Health Policy Manag. 2018;7(1):48-58. https://doi.org/10.15171/ijhpm.2017.44 6. Heath S. Distinguishing and defining top patient engagement keywords. Patient Engagement Hit [Internet]. 2018 Aug 28; [about 3 screens]. Available from: https://patientengagementhit.com/news/distinguishing-and-defining-top-patient-engagement-keywords 7. Silva CB, Silva DG, Carvalho LL, Goulart CL, Silva, ALG, Angri D. Ocorrência de eventos adversos em unidade de terapia intensiva neopediátrica. Rev Epidemiol Control Infecc. 2017;7(4):2238-3360. http://doi.org/10.17058/reci.v7i4.7564 8. Harada MJCS, Chanes DC, Kusahara DM, Pedreira MLG. Safety in medication administration in pediatrics. Acta Paul Enferm. 2012;25(4):639- 42. https://doi.org/10.1590/S0103-21002012000400025 9. Stockwell DC, Bisarya H, Classen DC, Kirkendall ES, Landrigan CP, Lemon V, et al. A trigger tool to detect harm in pediatric inpatient settings. Pediatrics. 2015;135(6):1036-42. https://doi.org/10.1542/peds.2014-2152 10. Trier H, Valderas JM, Wensing M, Martin HM, Egebart J. Involving patients in patient safety programmes: a scoping review and consensus procedure by the LINNEAUS collaboration on patient safety in primary care. Eur J Gen Pract. 2015;21(suppl 1):56-61. https://doi.org/10.3109 /13814788.2015.1043729 11. Cavalcante AK, Cavalcante FA, Pires DC, Batista EM, Nogueira LT. Nursing perception of safety culture: integrative review. J Nurs UFPE. 2016;10(10):3890-7. https://doi.org/10.5205/1981-8963-v10i10a11457p3890-3897-2016 12. Skagerström J, Ericsson C, Nilsen P, Ekstedt M, Schildmeijer K. Patient involvement for improved patient safety: a qualitative study of nurses’ perceptions and experiences. Nurs Open. 2017;4(4):230-9. https://doi.org/10.1002/nop2.89 13. Azevedo AP, Cristino JS, Viana MF, Medeiros FP, Azevedo LS. Health education for companions of hospitalized patients. J Nurs UFPE. 2018;12(4);1168-73. https://doi.org/10.5205/1981-8963-v12i4a230649p1168-1173-2018 14. Ricciardi R, Shofer M. Nurses and patients: natural partners to advance patient safety. J Nurs Care Qual. 2019;34(1):1-3. https://doi. org/10.1097/NCQ.0000000000000377 15. Melo EMOP, Ferreira PL, Lima RAG, Mello DF. The involvement of parents in the healthcare provided to hospitalized children. Rev Latino-Am Enfermagem. 2014;22(3):432-9. https://doi.org/10.1590/0104-1169.3308.2434 16. Neves L, Gondim AA, Soares SC, Coelho DP, Pinheiro JA. Involvement of companions in patient safety in pediatric and neonatal units: scope review Oliveira TGP, Diniz CG, Carvalho MPM, Corrêa AR, Rocha PK, Manzo BF. REFERENCES Parcianello AT, Felin RB. E agora doutor, onde vou brincar?: considerações sobre a hospitalização infantil. Barbaroi. 2008;28:147-66. http:// doi.org/10.17058/barbaroi.v0i0.356 42. Fernandes CS, Martins MM, Gomes BP, Gomes JA, Gonçalves LHT. Family nursing game: developing a board game. Esc Anna Nery. 2016;20(1):33-7. https://doi.org/10.5935/1414-8145.20160005 26. Hoffman LM, Rodrigues FA, Biasibetti C, Peres MA, Vaccari A, Wegner W. Patient safety incidents reported by relatives of hospitalized children Rev Gaucha Enferm. 2020;41(esp):e20190172. https://doi.org/10.1590/1983-1447.2020.20190172 27. Khan A, Coffey M, Litterer KP, Baird JD, Furtak SL, Garcia BM. et al. Families as partners in hospital error and adverse event surveillance. JAMA Pediatr. 2017;171(4):372-81. https://doi.org/10.1001/jamapediatrics.2016.4812 28. Lyndon A, Jacobson CH, Fagan KM, Wisner K, Franck LS. Parents' perspectives on safety in neonatal intensive care: a mixed-methods study. BMJ Qual Saf. 2014;23(11):902-9. https://doi.org/10.1136/bmjqs-2014-003009 28. Lyndon A, Jacobson CH, Fagan KM, Wisner K, Franck LS. Parents' perspectives on safety in neonatal intensive care: a mixed-methods study. BMJ Qual Saf. 2014;23(11):902-9. https://doi.org/10.1136/bmjqs-2014-003009 29. Massa ER, Hooker AC, García Martínez DG. Condiciones de seguridad percibidas por cuidadores familiares en atención pediátrica. Rev Cienc Cuidad. 2019;16(3):80-92. https://doi.org/10.22463/17949831.1574 29. Massa ER, Hooker AC, García Martínez DG. Condiciones de seguridad percibidas por cuidadores familiares en atención pediátrica. Rev Cienc Cuidad. 2019;16(3):80-92. https://doi.org/10.22463/17949831.1574 30. Peres MA, Wegner W, Cantarelli-Kantorski KJ, Gerhardt LM, Magalhães AMM. Perception of family members and caregivers regarding patient safety in pediatric inpatient units. Rev Gaucha Enferm. 2018;39:e2017-0195. http://doi.org/10.1590/1983-1447.2018.2017- 0195 30. Peres MA, Wegner W, Cantarelli-Kantorski KJ, Gerhardt LM, Magalhães AMM. Perception of family members and caregivers regarding patient safety in pediatric inpatient units. Rev Gaucha Enferm. 2018;39:e2017-0195. http://doi.org/10.1590/1983-1447.2018.2017- 0195 31. Rodrigues FA, Wegner W, Kantorski KJC, Pedro ENR. Patient safety in a neonatal unit: concerns and strategies experienced by parent. Cogitare Enferm. 2018;23(2):e52166. http:// doi.org/10.5380/ce.v23i1.52166 31. Rodrigues FA, Wegner W, Kantorski KJC, Pedro ENR. Patient safety in a neonatal unit: concerns and strategies experienced by parent. Cogitare Enferm. 2018;23(2):e52166. http:// doi.org/10.5380/ce.v23i1.52166 32. Rosenberg RE, Williams E, Ramchandani N, Rosenfeld P, Silber B, Schlucter J, et al. Provider perspective on partnering with parents of hospitalized children to improve safety. Hosp Pediatr. 2018;8(6):330-7. https://doi.org/10.1542/hpeds.2017-0159 32. Rosenberg RE, Williams E, Ramchandani N, Rosenfeld P, Silber B, Schlucter J, et al. Provider perspective on partnering with parents of hospitalized children to improve safety. Hosp Pediatr. 2018;8(6):330-7. https://doi.org/10.1542/hpeds.2017-0159 33. Silva TD, Wegner W, Pedro EN. Segurança da criança hospitalizada na UTI: compreendendo os eventos adversos sob a ótica do acompanhante. Rev Eletr Enferm. 2012;14(2):337-44. http://doi.org/10.5216/ree.v14i2.12977 33. REFERENCES The impact of the hospitalization process on the caregiver of a chronic critical patient hospitalized in a semi-intensive care unit. Esc Anna Nery. 2018;22(2):e20170304. https://doi.org/10.1590/2177-9465-EAN-2017-0304 17. Brito MVN, Ribeiro DE, Lima RS, Gomes RG, Fava SMCL, Vilela SD, et al. Role of the companion in hospitalization: nursing professional’s perspective. J Nurs UFPE. 2020;14:e243005. https://doi.org/10.5205/1981-8963.2020.243005 18. Ministério da Saúde (BR). Resolução nº 466, de 12 de dezembro de 2012. Diretrizes e normas regulamentadoras de pesquisas envolvendo seres humanos [Internet]. Brasília, DF: MS; 2012. 19. Peters MDJ, Godfrey C, McInerney P, Munn Z, Tricco AC, Khalil, H. Scoping reviews: 2020. In: Aromataris E, Munn Z, editors. JBI manual for evidence synthesis. Adelaide: JBI; 2020. Chapter 11. https://doi.org/10.46658/JBIMES-20-12 20. Tricco AC, Lillie E, Zarin W, O’Brien KK, Colquhoun H, Levac D, et al. PRISMA extension for scoping reviews (PRISMA-ScR): checklist and explanation. Ann Intern Med. 2018;169(7):467-73. https://doi.org/10.7326/M18-0850 20. Tricco AC, Lillie E, Zarin W, O’Brien KK, Colquhoun H, Levac D, et al. PRISMA extension for scoping reviews (PRISMA-ScR): checklist and explanation. Ann Intern Med. 2018;169(7):467-73. https://doi.org/10.7326/M18-0850 21. Melnyk BM, Fineout-Overholt E. Evidencebased practice in nursing and healthcare: a guide to best practice. Philadelphia: Lippincot Williams & Wilkins; 2005. Making the case for evidence-based practice; p. 3-24. 21. Melnyk BM, Fineout-Overholt E. Evidencebased practice in nursing and healthcare: a guide to best practice. Philadelphia: Lippincot Williams & Wilkins; 2005. Making the case for evidence-based practice; p. 3-24. 22. Biasibetti C, Hoffmann LM, Rodrigues FA, Wegner W, Rocha PK. Communication for patient safety in pediatric hospitalizations. Rev Gaucha Enferm. 2019;40(esp):e20180337. https://doi.org/10.1590/1983-1447.2019.20180337 23. Corbally MT, Tierney E. Parental involvement in the preoperative surgical safety checklist is welcomed by both parents and staff. Int J Pediatr. 2014;2014:791490. https://doi.org/10.1155/2014/791490 24. Gonçalves KMM, Costa MTTCA, Silva DCB, Baggio ME, Corrêa AR, Manzo BF. Ludic strategy for promoting engagement of parents and caregivers in the safety of pediatric patients. Rev Gaucha Enferm. 2020;41:e20190473. https://doi.org/10.1590/1983-1447.2020.20190473 25. Hoffman MR, Wegner W, Biasibetti C, Peres MA, Gerhardt LM, Breigeiron MK. Patient safety incidents identified by the caregivers of hospitalized children. Rev Bras Enferm. 2019;72(3):707-14. http://doi.org/10.1590/0034-7167-2018-0484 9 Rev Bras Enferm. 2022;75(3): e20210504 10 of Involvement of companions in patient safety in pediatric and neonatal units: scope review Oliveira TGP, Diniz CG, Carvalho MPM, Corrêa AR, Rocha PK, Manzo BF. Involvement of companions in patient safety in pediatric and neonatal units: scope review Oliveira TGP, Diniz CG, Carvalho MPM, Corrêa AR, Rocha PK, Manzo BF. REFERENCES 26. Hoffman LM, Rodrigues FA, Biasibetti C, Peres MA, Vaccari A, Wegner W. Patient safety incidents reported by relatives of hospitalized children Rev Gaucha Enferm. 2020;41(esp):e20190172. https://doi.org/10.1590/1983-1447.2020.20190172 27. Khan A, Coffey M, Litterer KP, Baird JD, Furtak SL, Garcia BM. et al. Families as partners in hospital error and adverse event surveillance. JAMA Pediatr. 2017;171(4):372-81. https://doi.org/10.1001/jamapediatrics.2016.4812 28. Lyndon A, Jacobson CH, Fagan KM, Wisner K, Franck LS. Parents' perspectives on safety in neonatal intensive care: a mixed-methods study. BMJ Qual Saf. 2014;23(11):902-9. https://doi.org/10.1136/bmjqs-2014-003009 29. Massa ER, Hooker AC, García Martínez DG. Condiciones de seguridad percibidas por cuidadores familiares en atención pediátrica. Rev Cienc Cuidad. 2019;16(3):80-92. https://doi.org/10.22463/17949831.1574 30. Peres MA, Wegner W, Cantarelli-Kantorski KJ, Gerhardt LM, Magalhães AMM. Perception of family members and caregivers regarding patient safety in pediatric inpatient units. Rev Gaucha Enferm. 2018;39:e2017-0195. http://doi.org/10.1590/1983-1447.2018.2017- 0195 31. Rodrigues FA, Wegner W, Kantorski KJC, Pedro ENR. Patient safety in a neonatal unit: concerns and strategies experienced by parent. Cogitare Enferm. 2018;23(2):e52166. http:// doi.org/10.5380/ce.v23i1.52166 32. Rosenberg RE, Williams E, Ramchandani N, Rosenfeld P, Silber B, Schlucter J, et al. Provider perspective on partnering with parents of hospitalized children to improve safety. Hosp Pediatr. 2018;8(6):330-7. https://doi.org/10.1542/hpeds.2017-0159 33. Silva TD, Wegner W, Pedro EN. Segurança da criança hospitalizada na UTI: compreendendo os eventos adversos sob a ótica do acompanhante. Rev Eletr Enferm. 2012;14(2):337-44. http://doi.org/10.5216/ree.v14i2.12977 34. Souza FCP, Montenegro LC, Goveia VR, Corrêa AR, Rocha PK, Manzo BF. Family participation in patient safety in neonatal units from the nursing perspective. Texto Contexto Enferm. 2017;26(3):e1180016. https://doi.org/10.1590/0104-07072017001180016 35. Oyesanya TO, Bowerd B. "I'm trying to be the safety net": family protection of patients with moderate-to-severe TBI during the hospital stay. Qual Health Res. 2017;27(12):1804-15. https://doi.org/10.1177/1049732317697098 36. Leonard MS. Patient Safety and Quality Improvement: reducing risk of harm. Pediatr Rev. 2015;36(10):448-56. https://doi.org/10.1542/pir.36-10-448 37. Santos PRA, Rocha FLR, Sampaio CSJC. Actions for safety in the prescription, use and administration of medications in emergency care units. Rev Gaucha Enferm. 2019;40(esp):e20180347. https://doi.org/10.1590/1983-1447.2019.20180347 38. Franco LF, Bonelli MA, Wernet M, Barbieri MC, Dupas G. Patient safety: perception of family members of hospitalized children. Rev Bras Enferm. 2020;73(5):e20190525. https://doi.org/10.1590/0034-7167-2019-0525 39. Souza FT, Garcia MC, Rangel PPS, Rocha PK. Percepção da enfermagem sobre os fatores de risco que envolvem a segurança do paciente pediátrico. Rev Enfem. 2014;4(1):152-62. https://doi.org/10.5902/217976928781 40. Hoffmeister LV, Moura GMSS. Use of identification wristbands among patients receiving inpatient treatment in a teaching hospital. Rev Latino-Am Enfermagem. 2015;23(1):36-43. https://doi.org/10.1590/0104-1169.0144.2522 41. REFERENCES Silva TD, Wegner W, Pedro EN. Segurança da criança hospitalizada na UTI: compreendendo os eventos adversos sob a ótica do acompanhante. Rev Eletr Enferm. 2012;14(2):337-44. http://doi.org/10.5216/ree.v14i2.12977 34. Souza FCP, Montenegro LC, Goveia VR, Corrêa AR, Rocha PK, Manzo BF. Family participation in patient safety in neonatal units from the nursing perspective. Texto Contexto Enferm. 2017;26(3):e1180016. https://doi.org/10.1590/0104-07072017001180016 34. Souza FCP, Montenegro LC, Goveia VR, Corrêa AR, Rocha PK, Manzo BF. Family participation in patient safety in neonatal units from the nursing perspective. Texto Contexto Enferm. 2017;26(3):e1180016. https://doi.org/10.1590/0104-07072017001180016 35. Oyesanya TO, Bowerd B. "I'm trying to be the safety net": family protection of patients with moderate-to-severe TBI during the hospital stay. Qual Health Res. 2017;27(12):1804-15. https://doi.org/10.1177/1049732317697098 35. Oyesanya TO, Bowerd B. "I'm trying to be the safety net": family protection of patients with moderate-to-severe TBI during the hospital stay. Qual Health Res. 2017;27(12):1804-15. https://doi.org/10.1177/1049732317697098 37. Santos PRA, Rocha FLR, Sampaio CSJC. Actions for safety in the prescription, use and administration of medications in emergency care units. Rev Gaucha Enferm. 2019;40(esp):e20180347. https://doi.org/10.1590/1983-1447.2019.20180347 37. Santos PRA, Rocha FLR, Sampaio CSJC. Actions for safety in the prescription, use and administration of medications in emergency care units. Rev Gaucha Enferm. 2019;40(esp):e20180347. https://doi.org/10.1590/1983-1447.2019.20180347 38. Franco LF, Bonelli MA, Wernet M, Barbieri MC, Dupas G. Patient safety: perception of family members of hospitalized children. Rev Bras Enferm. 2020;73(5):e20190525. https://doi.org/10.1590/0034-7167-2019-0525 38. Franco LF, Bonelli MA, Wernet M, Barbieri MC, Dupas G. Patient safety: perceptio Enferm. 2020;73(5):e20190525. https://doi.org/10.1590/0034-7167-2019-0525 39. Souza FT, Garcia MC, Rangel PPS, Rocha PK. Percepção da enfermagem sobre os fatores de risco que envolvem a segurança do paciente pediátrico. Rev Enfem. 2014;4(1):152-62. https://doi.org/10.5902/217976928781 40. Hoffmeister LV, Moura GMSS. Use of identification wristbands among patients receiving inpatient treatment in a teaching hospital. Rev Latino-Am Enfermagem. 2015;23(1):36-43. https://doi.org/10.1590/0104-1169.0144.2522 41. Parcianello AT, Felin RB. E agora doutor, onde vou brincar?: considerações sobre a hospitalização infantil. Barbaroi. 2008;28:147-66. http:// doi.org/10.17058/barbaroi.v0i0.356 42. Fernandes CS, Martins MM, Gomes BP, Gomes JA, Gonçalves LHT. Family nursing game: developing a board game. Esc Anna Nery. 2016;20(1):33-7. https://doi.org/10.5935/1414-8145.20160005 42. Fernandes CS, Martins MM, Gomes BP, Gomes JA, Gonçalves LHT. Family nursing game: developing a board game. Esc Anna Nery. 2016;20(1):33-7. https://doi.org/10.5935/1414-8145.20160005 10 Rev Bras Enferm. 2022;75(3): e20210504 10 of 10 Rev Bras Enferm. 2022;75(3): e20210504 10 of
https://openalex.org/W4232476045	https://europepmc.org/articles/pmc2979823?pdf=render	Latin	null	5-Amino-7-(3-chlorophenyl)-3,7-dihydro-2<i>H</i>-thieno[3,2-<i>b</i>]pyran-6-carbonitrile 1,1-dioxide	Acta crystallographica. Section E	2,010	cc-by	3,166	organic compounds Experimental Crystal data C14H11ClN2O3S Mr = 322.76 Monoclinic, P21=c a = 9.5802 (19) A˚ b = 17.364 (4) A˚ c = 8.2521 (17) A˚ = 97.83 (3) V = 1360.0 (5) A˚ 3 Z = 4 Mo K radiation = 0.45 mm1 T = 113 K 0.20 0.18 0.12 mm Data collection Rigaku Saturn CCD area-detector diffractometer Absorption correction: multi-scan (CrystalClear; Rigaku/MSC, 2005) Tmin = 0.916, Tmax = 0.949 9115 measured reﬂections 2393 independent reﬂections 1705 reﬂections with I > 2(I) Rint = 0.096 Reﬁnement R[F 2 > 2(F 2)] = 0.053 wR(F 2) = 0.134 S = 1.08 2393 reﬂections 191 parameters H-atom parameters constrained max = 0.67 e A˚ 3 min = 0.49 e A˚ 3 Table 1 Hydrogen-bond geometry (A˚ , ). D—H A D—H H A D A D—H A N2—H2C N1i 0.88 2.20 3.060 (4) 165 N2—H2D O1ii 0.88 2.04 2.912 (4) 174 Symmetry codes: (i) x þ 1; y; z þ 1; (ii) x; y; z 1. Data collection: CrystalClear (Rigaku/MSC, 2005); cell reﬁnement: CrystalClear; data reduction: CrystalClear; program(s) used to solve Experimental Crystal data C14H11ClN2O3S Mr = 322.76 Monoclinic, P21=c a = 9.5802 (19) A˚ b = 17.364 (4) A˚ c = 8.2521 (17) A˚ = 97.83 (3) V = 1360.0 (5) A˚ 3 Z = 4 Mo K radiation = 0.45 mm1 T = 113 K 0.20 0.18 0.12 mm Data collection Rigaku Saturn CCD area-detector diffractometer Absorption correction: multi-scan (CrystalClear; Rigaku/MSC, 2005) Tmin = 0.916, Tmax = 0.949 9115 measured reﬂections 2393 independent reﬂections 1705 reﬂections with I > 2(I) Rint = 0.096 Reﬁnement R[F 2 > 2(F 2)] = 0.053 wR(F 2) = 0.134 S = 1.08 2393 reﬂections 191 parameters H-atom parameters constrained max = 0.67 e A˚ 3 min = 0.49 e A˚ 3 Acta Crystallographica Section E Structure Reports Online ISSN 1600-5368 Acta Crystallographica Section E Structure Reports Online ISSN 1600-5368 Table 1 D—H A D—H H A D A D—H A N2—H2C N1i 0.88 2.20 3.060 (4) 165 N2—H2D O1ii 0.88 2.04 2.912 (4) 174 Symmetry codes: (i) x þ 1; y; z þ 1; (ii) x; y; z 1. The title compound, C14H11ClN2O3S, with fused thiophene and pyran rings, was synthesized via the condensation of dihydrothiophen-3(2H)-one 1,1-dioxide and 2-(3-chlorobenz- ylidene)malononitrile catalysed by triethylamine in ethanol. The thiophene ring adopts an envelope conformation and the pyran ring is planar (r.m.s. deviation = 0.0067 A˚ ). The dihedral angle between the pyran and phenyl rings is 80.8 (1). The crystal packing is stabilized by intermolecular N—H N and N—H O hydrogen bonds in which the cyano N and sulphone O atoms, respectively, acting as acceptors. Data collection: CrystalClear (Rigaku/MSC, 2005); cell reﬁnement: CrystalClear; data reduction: CrystalClear; program(s) used to solve structure: SHELXS97 (Sheldrick, 2008); program(s) used to reﬁne structure: SHELXL97 (Sheldrick, 2008); molecular graphics: SHELXTL (Sheldrick, 2008); software used to prepare material for publication: SHELXTL. The authors acknowledge ﬁnancial support by the Graduate Foundation of Xuzhou Normal University (No. 09YLB030). Related literature For the use of thienopyranyl compounds, such as thieno[3,2- b]pyran derivatives, as antiviral agents, see: Friary et al. (1991) and as -2C adrenoreceptor agonists, see: Chao et al. (2009). For puckering parameters, see: Cremer & Pople (1975). Supplementary data and ﬁgures for this paper are available from the IUCr electronic archives (Reference: HG2619). 5-Amino-7-(3-chlorophenyl)-3,7-di- hydro-2H-thieno[3,2-b]pyran-6-carbo- nitrile 1,1-dioxide Data collection Rigaku Saturn CCD area-detector diffractometer Absorption correction: multi-scan (CrystalClear; Rigaku/MSC, 2005) Tmin = 0.916, Tmax = 0.949 Shi-De Shen,a* Xiao-Dong Feng,b,c Wei-Hua Yang,c Cui-Hua Wangb,c and Chang-Sheng Yaob,c aXuzhou Institute of Architectural Technology, Xuzhou 221116, People’s Republic of China, bSchool of Chemistry and Chemical Engineering, Xuzhou Normal University, Xuzhou 221116, People’s Republic of China, and cKey Laboratory of Biotechnology for Medicinal Plants, Xuzhou Normal University, Xuzhou 221116, People’s Republic of China Reﬁnement R[F 2 > 2(F 2)] = 0.053 wR(F 2) = 0.134 S = 1.08 2393 reﬂections 191 parameters H-atom parameters constrained max = 0.67 e A˚ 3 min = 0.49 e A˚ 3 191 parameters H-atom parameters constrained max = 0.67 e A˚ 3 min = 0.49 e A˚ 3 Correspondence e-mail: chshengyaonk@mail.nankai.edu.cn Received 15 December 2009; accepted 23 December 2009 Key indicators: single-crystal X-ray study; T = 113 K; mean (C–C) = 0.004 A˚; R factor = 0.053; wR factor = 0.134; data-to-parameter ratio = 12.5. Table 1 Hydrogen-bond geometry (A˚ , ). D—H A D—H H A D A D—H A N2—H2C N1i 0.88 2.20 3.060 (4) 165 N2—H2D O1ii 0.88 2.04 2.912 (4) 174 Symmetry codes: (i) x þ 1; y; z þ 1; (ii) x; y; z 1. References Chao, J. H., Zheng, J. Y. & Aslanian, R. G. (2009). WO Patent, No. 2009020578. Cremer, D. & Pople, J. A. (1975). J. Am. Chem. Soc. 97, 1354–1358. Friary, R. J., Schwerdt, J. H. & Ganguly, A. K. (1991). US patent, No. 5034531. Rigaku/MSC (2005). CrystalClear. Rigaku/MSC Inc., The Woodlands, Texas, USA. Sheldrick G M (2008) Acta Cryst A64 112 122 Chao, J. H., Zheng, J. Y. & Aslanian, R. G. (2009). WO Patent, No. 2009020578. Cremer, D. & Pople, J. A. (1975). J. Am. Chem. Soc. 97, 1354–1358. Friary, R. J., Schwerdt, J. H. & Ganguly, A. K. (1991). US patent, No. 5034531. Rigaku/MSC (2005). CrystalClear. Rigaku/MSC Inc., The Woodlands, Texas, USA. Sheldrick G M (2008) Acta Cryst A64 112 122 Sheldrick, G. M. (2008). Acta Cryst. A64, 112–122. o282 Shen et al. Acta Cryst. (2010). E66, o282 doi:10.1107/S1600536809055202 Comment Thienopyranyl compounds, such as thieno [3,2-b]pyran derivatives, can be uesed as antiviral agents (Friary et al., 1991) and α-2 C adrenoreceptor agonists (Chao et al., 2009). This led us to pay attention to the synthesis and bioactivity of these compounds. During the synthesis of thieno[3,2-b]pyran derivatives, the title compound, (I) was isolated and its structure was determined by X-ray diffraction. Here we report its crystal structure. The molecular structure of (I) is shown in Fig. 1. In the molecular structure, the thiophene ring is in envelope comforma- tion, for the deviation of C1 from the C2/C3/C7/S1 plane is 0.354 (4)Å with r.m.s. of 0.010. The pyrane ring adopts a planar conformation. Cremer & Pople puckering analysis can not be performed, for its weighted average ABS. torsion angle is 1.0°, less than 5.0°. The connection of the pyrane ring and phenyl ring C9—C14 can be described by the C5—C6—C9—C14 tor- sion angle of 78.3 (3)°. The crystal packing is stabilized by intermolecular hydrogen bonds: N2—H2C···N1, N2—H2D···O1 (Fig.2 & Table 1). Experimental The title compound was synthesized by the reaction of dihydrothiophen-3(2H)-one-1,1-dioxide (1 mmol) and 2-(3-chloro benzylidene)malononitrile (1 mmol) catalyzed by triethylamine (0.02 g) in 10 ml ethanol under reluxing until completion (monitored by TLC). Cooling the reaction mixture slowly gave single crystals suitable for X-ray diffraction. supplementary materials supplementary materials Acta Cryst. (2010). E66, o282 [ doi:10.1107/S1600536809055202 ] Refinement All H atoms were placed in calculated positions, with N–H = 0.88 and C—H = 0.95, 0.99 or 1.00 Å, and included in the final cycles of refinement using a riding model, with Uiso(H) = 1.2Ueq(parent atom). sup-1 Figures Fig. 1. The structure of (I), showing 30% probability displacement ellipsoids and the atom- numbering scheme. Fig. 2. The packing diagram of (I). Intermolecular hydrogen bonds are shown as dashed lines. Figures Fig. 1. The structure of (I), showing 30% probability displacement ellipsoids and the atom- numbering scheme. Fig. 2. The packing diagram of (I). Intermolecular hydrogen bonds are shown as dashed lines. Fig. 2. The packing diagram of (I). Intermolecular hydrogen bonds are shown as dashed lines. sup-1 supplementary materials supplementary materials 5-Amino-7-(3-chlorophenyl)-3,7-dihydro-2H-thieno[3,2-b]pyran- 6-carbonitrile 1,1-dioxide Special details Special details Geometry. All e.s.d.'s (except the e.s.d. in the dihedral angle between two l.s. planes) are estimated using the full covariance mat- rix. The cell e.s.d.'s are taken into account individually in the estimation of e.s.d.'s in distances, angles and torsion angles; correlations between e.s.d.'s in cell parameters are only used when they are defined by crystal symmetry. An approximate (isotropic) treatment of cell e.s.d.'s is used for estimating e.s.d.'s involving l.s. planes. Refinement. Refinement of F2 against ALL reflections. The weighted R-factor wR and goodness of fit S are based on F2, convention- al R-factors R are based on F, with F set to zero for negative F2. The threshold expression of F2 > σ(F2) is used only for calculating R- factors(gt) etc. and is not relevant to the choice of reflections for refinement. R-factors based on F2 are statistically about twice as large as those based on F, and R- factors based on ALL data will be even larger. 5-Amino-7-(3-chlorophenyl)-3,7-dihydro-2H-thieno[3,2-b]pyran- 6-carbonitrile 1,1-dioxide Crystal data C14H11ClN2O3S F(000) = 664 Mr = 322.76 Dx = 1.576 Mg m−3 Monoclinic, P21/c Mo Kα radiation, λ = 0.71073 Å Hall symbol: -P 2ybc Cell parameters from 4484 reflections a = 9.5802 (19) Å θ = 2.2–27.9° b = 17.364 (4) Å µ = 0.45 mm−1 c = 8.2521 (17) Å T = 113 K β = 97.83 (3)° Block, colorless V = 1360.0 (5) Å3 0.20 × 0.18 × 0.12 mm Z = 4 F(000) = 664 Dx = 1.576 Mg m−3 Mo Kα radiation, λ = 0.71073 Å Cell parameters from 4484 reflections θ = 2.2–27.9° µ = 0.45 mm−1 T = 113 K Block, colorless 0.20 × 0.18 × 0.12 mm Data collection Rigaku Saturn CCD area-detector diffractometer Radiation source: rotating anode confocal Detector resolution: 7.31 pixels mm-1 ω and φ scans Absorption correction: multi-scan CrystalClear Tmin = 0.916, Tmax = 0.949 9115 measured reflections 2393 independent reflections 1705 reflections with I > 2σ(I) Rint = 0.096 θmax = 25.0°, θmin = 2.2° h = −11→11 k = −19→20 l = −9→9 Secondary atom site location: difference Fourier map Hydrogen site location: inferred from neighbouring sites H-atom parameters constrained H-atom parameters constrained w = 1/[σ2(Fo 2) + (0.0583P)2] where P = (Fo 2 + 2Fc 2)/3 (Δ/σ)max < 0.001 Δρmax = 0.67 e Å−3 Δρmin = −0.49 e Å−3 Δρmax = 0.67 e Å−3 Δρmin = −0.49 e Å−3 Extinction correction: SHELXL, Fc=kFc[1+0.001xFc2λ3/sin(2θ)]-1/4 Fc=kFc[1+0.001xFc2λ3/sin(2θ)]-1/4 Primary atom site location: structure-invariant direct methods Extinction coefficient: 0.491 (16) Extinction coefficient: 0.491 (16) sup-2 sup-2 supplementary materials Special details Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2) Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2) x y z Uiso/Ueq S1 −0.01194 (8) 0.13574 (4) 0.99300 (11) 0.0141 (3) Cl1 0.69314 (8) 0.15345 (5) 1.25118 (11) 0.0240 (3) O1 −0.0062 (2) 0.07472 (12) 1.1133 (3) 0.0189 (6) O2 0.0250 (2) 0.21172 (12) 1.0529 (3) 0.0220 (7) O3 0.0422 (2) 0.09320 (11) 0.5466 (3) 0.0143 (6) N2 0.2045 (3) 0.05206 (14) 0.3987 (3) 0.0173 (7) H2C 0.2884 0.0361 0.3817 0.021 H2D 0.1367 0.0569 0.3159 0.021* N1 0.5321 (3) 0.03147 (15) 0.6812 (4) 0.0206 (7) C1 −0.1785 (3) 0.13702 (17) 0.8638 (4) 0.0157 (8) H1A −0.2326 0.0898 0.8802 0.019* H1B −0.2346 0.1822 0.8892 0.019* C2 −0.1464 (3) 0.14131 (16) 0.6882 (4) 0.0131 (8) H2A −0.2124 0.1085 0.6155 0.016* H2B −0.1545 0.1950 0.6474 0.016* C3 0.0022 (3) 0.11258 (17) 0.6944 (4) 0.0128 (8) C4 0.1798 (3) 0.06909 (16) 0.5505 (4) 0.0118 (7) C5 0.2722 (3) 0.06488 (17) 0.6910 (4) 0.0119 (7) C6 0.2351 (3) 0.08436 (16) 0.8608 (4) 0.0119 (7) H6 0.2458 0.0370 0.9305 0.014* C7 0.0834 (3) 0.10851 (16) 0.8370 (4) 0.0102 (7) C8 0.4147 (3) 0.04538 (16) 0.6824 (4) 0.0138 (8) C9 0.3344 (3) 0.14675 (16) 0.9401 (4) 0.0126 (8) C10 0.4501 (3) 0.12565 (18) 1.0523 (4) 0.0144 (8) H10 0.4640 0.0733 1.0839 0.017* C11 0.5450 (3) 0.18159 (19) 1.1175 (4) 0.0153 (8) C12 0.5249 (3) 0.25793 (18) 1.0762 (4) 0.0183 (8) H12 0.5893 0.2960 1.1233 0.022* C13 0.4088 (3) 0.27859 (18) 0.9644 (4) 0.0168 (8) H13 0.3941 0.3312 0.9351 0.020* C14 0.3145 (3) 0.22355 (17) 0.8953 (4) 0.0154 (8) H14 0.2365 0.2382 0.8177 0.018* sup-3 supplementary materials supplementary materials Atomic displacement parameters (Å2) U11 U22 U33 U12 U13 U23 S1 0.0099 (5) 0.0182 (5) 0.0140 (6) 0.0013 (3) 0.0011 (4) −0.0018 (3) Cl1 0.0124 (5) 0.0361 (6) 0.0215 (6) 0.0006 (3) −0.0051 (4) −0.0020 (4) O1 0.0173 (13) 0.0255 (13) 0.0136 (15) 0.0041 (9) 0.0015 (11) 0.0050 (10) O2 0.0201 (13) 0.0191 (13) 0.0280 (17) −0.0022 (9) 0.0073 (12) −0.0113 (11) O3 0.0094 (12) 0.0199 (13) 0.0136 (14) 0.0039 (9) 0.0018 (10) −0.0007 (10) N2 0.0100 (14) 0.0277 (16) 0.0137 (18) 0.0025 (12) −0.0008 (13) −0.0038 (13) N1 0.0137 (16) 0.0291 (16) 0.0185 (19) 0.0042 (12) 0.0008 (13) −0.0018 (14) C1 0.0081 (17) 0.0228 (18) 0.015 (2) 0.0021 (12) −0.0006 (15) 0.0022 (14) C2 0.0086 (17) 0.0147 (17) 0.016 (2) 0.0026 (12) 0.0004 (15) 0.0006 (13) C3 0.0128 (17) 0.0104 (16) 0.016 (2) 0.0000 (12) 0.0048 (15) 0.0000 (14) C4 0.0095 (16) 0.0117 (16) 0.015 (2) 0.0008 (12) 0.0035 (15) −0.0004 (14) C5 0.0095 (17) 0.0144 (16) 0.012 (2) 0.0026 (12) 0.0027 (15) −0.0003 (14) C6 0.0106 (17) 0.0149 (16) 0.010 (2) 0.0024 (12) −0.0004 (15) −0.0002 (13) C7 0.0081 (16) 0.0109 (15) 0.012 (2) −0.0008 (12) 0.0028 (14) −0.0003 (14) C8 0.0182 (19) 0.0133 (17) 0.010 (2) −0.0004 (13) 0.0006 (15) −0.0002 (13) C9 0.0072 (17) 0.0187 (17) 0.012 (2) 0.0004 (12) 0.0027 (15) −0.0031 (14) C10 0.0138 (18) 0.0163 (17) 0.014 (2) 0.0018 (13) 0.0032 (16) 0.0002 (14) C11 0.0075 (16) 0.0268 (19) 0.011 (2) 0.0012 (13) −0.0009 (15) −0.0019 (15) C12 0.0118 (18) 0.0242 (19) 0.020 (2) −0.0045 (13) 0.0059 (16) −0.0060 (15) C13 0.0156 (18) 0.0167 (17) 0.019 (2) −0.0010 (13) 0.0070 (16) −0.0016 (15) C14 0.0094 (16) 0.0205 (18) 0.016 (2) 0.0032 (13) 0.0017 (14) 0.0004 (15) Geometric parameters (Å, °) S1—O2 1.436 (2) C3—C7 1.322 (5) S1—O1 1.448 (2) C4—C5 1.361 (5) S1—C7 1.742 (3) C5—C8 1.418 (4) S1—C1 1.794 (3) C5—C6 1.529 (4) Cl1—C11 1.745 (3) C6—C7 1.499 (4) O3—C3 1.369 (4) C6—C9 1.529 (4) O3—C4 1.379 (4) C6—H6 1.0000 N2—C4 1.339 (4) C9—C14 1.390 (4) N2—H2C 0.8800 C9—C10 1.393 (5) N2—H2D 0.8800 C10—C11 1.388 (4) N1—C8 1.152 (4) C10—H10 0.9500 C1—C2 1.523 (5) C11—C12 1.376 (4) C1—H1A 0.9900 C12—C13 1.392 (5) C1—H1B 0.9900 C12—H12 0.9500 C2—C3 1.503 (4) C13—C14 1.383 (4) C2—H2A 0.9900 C13—H13 0.9500 C2—H2B 0.9900 C14—H14 0.9500 O2—S1—O1 116.86 (15) C8—C5—C6 116.4 (3) O2—S1—C7 111.96 (13) C7—C6—C9 113.2 (2) O1—S1—C7 109.44 (13) C7—C6—C5 106.5 (3) Atomic displacement parameters (Å2) sup-4 supplementary materials supplementary materials 110.54 (14) C9—C6—C5 109.9 (2 111.37 (14) C7—C6—H6 109.1 94.45 (15) C9—C6—H6 109.1 115.9 (3) C5—C6—H6 109.1 120.0 C3—C7—C6 125.1 (3 120.0 C3—C7—S1 109.8 (2 120.0 C6—C7—S1 125.1 (3 106.7 (2) N1—C8—C5 177.0 (4 110.4 C14—C9—C10 119.8 (3) 110.4 C14—C9—C6 120.7 (3 110.4 C10—C9—C6 119.4 (3) 110.4 C11—C10—C9 119.6 (3) 108.6 C11—C10—H10 120.2 105.3 (3) C9—C10—H10 120.2 110.7 C12—C11—C10 121.1 (3 110.7 C12—C11—Cl1 120.0 (3 110.7 C10—C11—Cl1 118.9 (2) 110.7 C11—C12—C13 119.0 (3) 108.8 C11—C12—H12 120.5 125.3 (3) C13—C12—H12 120.5 119.2 (3) C14—C13—C12 120.9 (3 115.5 (3) C14—C13—H13 119.6 127.5 (3) C12—C13—H13 119.6 109.6 (3) C13—C14—C9 119.7 (3) 123.0 (3) C13—C14—H14 120.2 119.2 (3) C9—C14—H14 120.2 124.3 (3) 97.0 (2) C9—C6—C7—S1 −60.0 (3 −131.35 (19) C5—C6—C7—S1 179.2 (2 −18.5 (2) O2—S1—C7—C3 −104.5 ( 21.1 (3) O1—S1—C7—C3 124.3 (2 −1.3 (4) C1—S1—C7—C3 9.8 (2) 178.0 (2) O2—S1—C7—C6 75.3 (3) −16.2 (4) O1—S1—C7—C6 −55.9 (3 164.4 (2) C1—S1—C7—C6 −170.4 ( 180.0 (2) C4—C5—C8—N1 149 (7) −0.4 (4) C6—C5—C8—N1 −28 (7) 4.5 (5) C7—C6—C9—C14 −40.6 (4 −175.0 (2) C5—C6—C9—C14 78.3 (3) −179.1 (3) C7—C6—C9—C10 142.6 (3 1.4 (5) C5—C6—C9—C10 −98.5 (3 −0.6 (4) C14—C9—C10—C11 −0.4 (5) 175.8 (3) C6—C9—C10—C11 176.3 (3 −123.5 (3) C9—C10—C11—C12 1.6 (5) 52.9 (3) C9—C10—C11—Cl1 −177.3 ( 2.1 (5) C10—C11—C12—C13 −1.4 (5) −177.2 (3) Cl1—C11—C12—C13 177.5 (2 −178.1 (2) C11—C12—C13—C14 0.1 (5) 2.6 (4) C12—C13—C14—C9 1.1 (5) sup-5 110.54 (14) C9—C6—C5 109.9 (2) 111.37 (14) C7—C6—H6 109.1 94.45 (15) C9—C6—H6 109.1 115.9 (3) C5—C6—H6 109.1 120.0 C3—C7—C6 125.1 (3) 120.0 C3—C7—S1 109.8 (2) 120.0 C6—C7—S1 125.1 (3) 106.7 (2) N1—C8—C5 177.0 (4) 110.4 C14—C9—C10 119.8 (3) 110.4 C14—C9—C6 120.7 (3) 110.4 C10—C9—C6 119.4 (3) 110.4 C11—C10—C9 119.6 (3) 108.6 C11—C10—H10 120.2 105.3 (3) C9—C10—H10 120.2 110.7 C12—C11—C10 121.1 (3) 110.7 C12—C11—Cl1 120.0 (3) 110.7 C10—C11—Cl1 118.9 (2) 110.7 C11—C12—C13 119.0 (3) 108.8 C11—C12—H12 120.5 125.3 (3) C13—C12—H12 120.5 119.2 (3) C14—C13—C12 120.9 (3) 115.5 (3) C14—C13—H13 119.6 127.5 (3) C12—C13—H13 119.6 109.6 (3) C13—C14—C9 119.7 (3) 123.0 (3) C13—C14—H14 120.2 119.2 (3) C9—C14—H14 120.2 124.3 (3) 97.0 (2) C9—C6—C7—S1 −60.0 (3) −131.35 (19) C5—C6—C7—S1 179.2 (2) −18.5 (2) O2—S1—C7—C3 −104.5 (2) 21.1 (3) O1—S1—C7—C3 124.3 (2) −1.3 (4) C1—S1—C7—C3 9.8 (2) 178.0 (2) O2—S1—C7—C6 75.3 (3) −16.2 (4) O1—S1—C7—C6 −55.9 (3) 164.4 (2) C1—S1—C7—C6 −170.4 (3) 180.0 (2) C4—C5—C8—N1 149 (7) −0.4 (4) C6—C5—C8—N1 −28 (7) 4.5 (5) C7—C6—C9—C14 −40.6 (4) −175.0 (2) C5—C6—C9—C14 78.3 (3) −179.1 (3) C7—C6—C9—C10 142.6 (3) 1.4 (5) C5—C6—C9—C10 −98.5 (3) −0.6 (4) C14—C9—C10—C11 −0.4 (5) 175.8 (3) C6—C9—C10—C11 176.3 (3) −123.5 (3) C9—C10—C11—C12 1.6 (5) 52.9 (3) C9—C10—C11—Cl1 −177.3 (2) 2.1 (5) C10—C11—C12—C13 −1.4 (5) −177.2 (3) Cl1—C11—C12—C13 177.5 (2) −178.1 (2) C11—C12—C13—C14 0.1 (5) 2.6 (4) C12—C13—C14—C9 1.1 (5) O2—S1—C1 110.54 (14) C9—C6—C5 109.9 (2) O1—S1—C1 111.37 (14) C7—C6—H6 109.1 C7—S1—C1 94.45 (15) C9—C6—H6 109.1 C3—O3—C4 115.9 (3) C5—C6—H6 109.1 C4—N2—H2C 120.0 C3—C7—C6 125.1 (3) C4—N2—H2D 120.0 C3—C7—S1 109.8 (2) H2C—N2—H2D 120.0 C6—C7—S1 125.1 (3) C2—C1—S1 106.7 (2) N1—C8—C5 177.0 (4) C2—C1—H1A 110.4 C14—C9—C10 119.8 (3) S1—C1—H1A 110.4 C14—C9—C6 120.7 (3) C2—C1—H1B 110.4 C10—C9—C6 119.4 (3) S1—C1—H1B 110.4 C11—C10—C9 119.6 (3) H1A—C1—H1B 108.6 C11—C10—H10 120.2 C3—C2—C1 105.3 (3) C9—C10—H10 120.2 C3—C2—H2A 110.7 C12—C11—C10 121.1 (3) C1—C2—H2A 110.7 C12—C11—Cl1 120.0 (3) C3—C2—H2B 110.7 C10—C11—Cl1 118.9 (2) C1—C2—H2B 110.7 C11—C12—C13 119.0 (3) H2A—C2—H2B 108.8 C11—C12—H12 120.5 C7—C3—O3 125.3 (3) C13—C12—H12 120.5 C7—C3—C2 119.2 (3) C14—C13—C12 120.9 (3) O3—C3—C2 115.5 (3) C14—C13—H13 119.6 N2—C4—C5 127.5 (3) C12—C13—H13 119.6 N2—C4—O3 109.6 (3) C13—C14—C9 119.7 (3) C5—C4—O3 123.0 (3) C13—C14—H14 120.2 C4—C5—C8 119.2 (3) C9—C14—H14 120.2 C4—C5—C6 124.3 (3) O2—S1—C1—C2 97.0 (2) C9—C6—C7—S1 −60.0 (3) O1—S1—C1—C2 −131.35 (19) C5—C6—C7—S1 179.2 (2) C7—S1—C1—C2 −18.5 (2) O2—S1—C7—C3 −104.5 (2) S1—C1—C2—C3 21.1 (3) O1—S1—C7—C3 124.3 (2) C4—O3—C3—C7 −1.3 (4) C1—S1—C7—C3 9.8 (2) C4—O3—C3—C2 178.0 (2) O2—S1—C7—C6 75.3 (3) C1—C2—C3—C7 −16.2 (4) O1—S1—C7—C6 −55.9 (3) C1—C2—C3—O3 164.4 (2) C1—S1—C7—C6 −170.4 (3) C3—O3—C4—N2 180.0 (2) C4—C5—C8—N1 149 (7) C3—O3—C4—C5 −0.4 (4) C6—C5—C8—N1 −28 (7) N2—C4—C5—C8 4.5 (5) C7—C6—C9—C14 −40.6 (4) O3—C4—C5—C8 −175.0 (2) C5—C6—C9—C14 78.3 (3) N2—C4—C5—C6 −179.1 (3) C7—C6—C9—C10 142.6 (3) O3—C4—C5—C6 1.4 (5) C5—C6—C9—C10 −98.5 (3) C4—C5—C6—C7 −0.6 (4) C14—C9—C10—C11 −0.4 (5) C8—C5—C6—C7 175.8 (3) C6—C9—C10—C11 176.3 (3) C4—C5—C6—C9 −123.5 (3) C9—C10—C11—C12 1.6 (5) C8—C5—C6—C9 52.9 (3) C9—C10—C11—Cl1 −177.3 (2) O3—C3—C7—C6 2.1 (5) C10—C11—C12—C13 −1.4 (5) C2—C3—C7—C6 −177.2 (3) Cl1—C11—C12—C13 177.5 (2) O3—C3—C7—S1 −178.1 (2) C11—C12—C13—C14 0.1 (5) C2—C3—C7—S1 2.6 (4) C12—C13—C14—C9 1.1 (5) sup-5 supplementary materials C9—C6—C7—C3 119.8 (4) C10—C9—C14—C13 −0.9 (4) C5—C6—C7—C3 −1.0 (4) C6—C9—C14—C13 −177.6 (3) Hydrogen-bond geometry (Å, °) D—H···A D—H H···A D···A D—H···A N2—H2C···N1i 0.88 2.20 3.060 (4) 165. supplementary materials N2—H2D···O1ii 0.88 2.04 2.912 (4) 174. Symmetry codes: (i) −x+1, −y, −z+1; (ii) x, y, z−1. supplementary materials supplementary materials sup-6 supplementary materials Fig. 1 sup-7 supplementary materials ig. 2 2 Fig. 2 Fig. 2 sup-8
https://openalex.org/W2731340063	https://europepmc.org/articles/pmc5516050?pdf=render	English	null	Increased risk of coronary perforation during percutaneous intervention of myocardial bridge: What histopathology says	Journal of cardiovascular and thoracic research.	2,017	cc-by	3,835	Publishing Group TUOMS J Cardiovasc Thorac Res, 2017, 9(2), 108-112 doi: 10.15171/jcvtr.2017.18 http://journals.tbzmed.ac.ir/jcvtr Keywords: Keywords: Myocardial Bridge Coronary Perforation Histopathology Percutaneous Coronary Inter vention Methods: Twenty specimens of MB were obtained from dissection of 45 cadavers. Sections were stained using hematoxylin and eosin (H&E), and trichrome methods. The proximal section and the tunneled artery were compared with a normal sample in terms of the characteristics of a muscle artery. Results: The findings of this study showed an MB prevalence of 51%, as 23 out of the 45 examined cadavers were discovered to be afflicted by the MB. The intima layer in the suffering artery had gone through significant hypertrophy, while it had remained thin in the tunneled artery section. The epithelial cells under the bridge were spindle-shaped, while they were polygonal in the proximal section. In the myocardium the nuclei of the muscle fibers in the MB section were smaller than the normal section. Adventitial layer was almost normal. Conclusion: The histopathological differences between MB and proximal part of vessel combined with small vessel diameter in the tunneled segment can explain the high incidence of the LAD rupture and perforation in the section under the bridge. Please cite this article as: Pourhoseini S, Bakhtiari M, Babaee A, Ostovan MA, Eftekhar-Vaghefi SH, Ostovan N, Dehghani P. Increased risk of coronary perforation during percutaneous intervention of myocardial bridge: What histopathology says. J Cardiovasc Thorac Res 2017;9(2):108-112. doi: 10.15171/jcvtr.2017.18. Increased risk of coronary perforation during percutaneous intervention of myocardial bridge: What histopathology says Somayeh Pourhoseini1, Mohammad Bakhtiari2, Abdolreza Babaee1, Mohammad Ali Ostovan3,4, Seyed Hassa Eftekhar-Vaghefi1, Nikan Ostovan4, Pooyan Dehghani3,4* 1Department of Anatomy, School of Medicine, Kerman University of Medical Sciences, Kerman, Iran 2Department of Anatomical Science and Molecular Biology, Isfahan University of Medical Sciences, Isfahan, Iran 3Department of Cardiology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran 4Shiraz Cardiovascular Research Center, Shiraz University of Medical Sciences, Shiraz, Iran Abstract Article History: Received: 21 March 2017 Accepted: 9 June 2017 epublished: 29 June 2017 Article info Introduction: Myocardial bridge (MB) is a segment of a major epicardial coronary artery that goes intramurally under a bridge of overlying myocardium. Complications have been reported during or after stent implantation particularly coronary perforation. The aim of this study was to determine histological differences between proximal left anterior descending artery (LAD) and the tunneled segment that may have a possible role in increased risk of coronary artery perforation during percutaneous coronary intervention. © 2017 The Author (s). This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons. org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Please cite this article as: Pourhoseini S, Bakhtiari M, Babaee A, Ostovan MA, Eftekhar-Vaghefi SH, Ostovan N, Dehghani P. Increased risk of coronary perforation during percutaneous intervention of myocardial bridge: What histopathology says. J Cardiovasc Thorac Res 2017;9(2):108-112. doi: 10.15171/jcvtr.2017.18. Staining procedure I h i i The purpose of our study was to determine histological differences between proximal (LAD) and tunneled artery that may have a possible causative role in increased risk of coronary artery perforating during PCI. In the staining stage, the sections were stained using hematoxylin and eosin (H&E), and trichrome methods. For histological studies, the stained slides were studied using optic microscope equipped with camera and lens. In the investigation process, the proximal section and the tunneled artery were compared with a normal sample in terms of the characteristics of a muscle artery. Results Concerning the prevalence rate of MB in general population, the findings of this study illustrated that out of 45 cadavers examined for evidence of MB, 23 were afflicted with MB, resulting in a prevalence of 51%. For tissue processing procedure, however, only 20 MB cases were selected in line with the recommendation of the statistical adviser of the project for a sample volume of 20 MB cases. With regard to the histological findings, the histological comparisons between MB samples and normal specimens revealed that in both the proximal and the tunneled artery, the intima layer had suffered certain transformations as the intima layer in the proximal section showed remarkable hypertrophy, while in the tunneled artery section, the Fixation procedure The samples were kept in the fixative solution (10% formalin) for at least 7 days. Following this, using LECAT POLO, the samples went through tissue passaging procedure of dehydration, clearing, and impregnation for 12-16 hours. In the embedding stage, samples were vertically placed inside metal frames, and covered in melted paraffin to cool down to paraffin blocks which were kept in the refrigerator prior to sectioning. Microtome MICROM HM 235 was used to prepare 5 micro centimeter traverse sections which were fixed on slides, labeled with the samples’ information, and heated in the oven to melt the extra paraffin. The increased risk of perforation during PCI can be due to multiple factors. Thin intima and a probable smaller vessel diameter of the tunneled segment are said to be two possible causes. Over inflation of the balloon and oversizing of the stent could be another mechanism leading to coronary rupture.17 Materials and Methods Study design d h l To determine the prevalence of MB and its histological features a collection of 20 cases were needed based on Chocran’s sample size formula with 95% confidence interval and alpha level of 0.05. In other words, to come up with a reliable outcome the required sample volume was 20 cases of MB. To get access to the target sample volume of 20, 45 cadavers, from Shiraz Forensic Center were examined. The sample consisted of 24 males and 21 females in the age range between 17 and 80. Out of the 45 examined cadavers, 23 were discovered to suffer from MB. None of the 23 spotted cases had a record of heart complaints and their death had occurred due to other reasons. In line with the statistical advisory for a sample volume of 20, only 20 out of the 23 detected MB cases went through the tissue processing procedure. Tissue processing procedure Fixation procedure Percutaneous coronary intervention (PCI) with stent implantation under the MB is used mainly in patients with severe systolic and diastolic stenosis, complete occlusion at the bridged segment, resistance to drug therapies or when there is concomitant atherosclerotic lesion near the muscle bridge. Complications have been reported during or after stent implantation particularly coronary perforation during or immediately after stenting.16 In our own experience we had eight cases of PCI on muscle bridge segments that were complicated with coronary perforations. Introduction revealed an association between myocardial bridging and sudden cardiac death,8 myocardial infarction,9 arrhythmias,10 and myocardial ischemia.11 Myocardial bridge (MB), an inborn coronary abnormality,1,2 is defined as a segment of a major epicardial coronary artery, the tunneled artery, that goes intramurally under a bridge of overlying myocardium.3 MB is generally confined to the left anterior descending artery (LAD).4 Anatomically, MB is classified as superficial or deep depending on its width. The length of a typical MB is usually within 10 to 30 mm range, only rarely exceeding 40 mm.5 It is a common coronary disorder with an average prevalence of (30%) in general population,6,7 which, however varies substantially among studies, with a much higher rate at autopsy than angiography.7 In symptomatic MB cases, the occurrence of coronary artery disease is considered to be caused by the direct MB compression of the LAD.12 The segment proximal to the bridge frequently shows atherosclerotic plaque formation, although the tunneled segment is typically spared.2 Besides, the likelihood of ischemia increases with the intra-myocardial depth of the tunneled segment.12 In symptomatic MB cases, the occurrence of coronary artery disease is considered to be caused by the direct MB compression of the LAD.12 The segment proximal to the bridge frequently shows atherosclerotic plaque formation, although the tunneled segment is typically spared.2 Besides, the likelihood of ischemia increases with the intra-myocardial depth of the tunneled segment.12 With regard to the treatment, three strategies have been explored: (1) Negative inotropic and/or negative chronotropic agents i.e. beta blockers and calcium antagonists,11,13 (2) Surgical myotomy and/or coronary artery bypass graft surgery (CABG),11,14 (3) Stenting of the tunneled segment.11,15,16 With regard to the treatment, three strategies have been explored: (1) Negative inotropic and/or negative chronotropic agents i.e. beta blockers and calcium antagonists,11,13 (2) Surgical myotomy and/or coronary artery bypass graft surgery (CABG),11,14 (3) Stenting of the tunneled segment.11,15,16 Traditionally, myocardial bridging has been considered a benign condition, but symptoms such as angina-like chest pain have been reported. Also, various studies have © 2017 The Author (s). This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons. org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Histopathological aspect of myocardial bridging Tissue sampling procedures To collect the required tissue, from the supraclavicular area to the pubic symphysis, the skin was incised and the thorax was pressed aside to be able to remove the heart from the epicardium. This procedure was performed very accurately, observing all required anatomical techniques. In the next stage, the heart was closely examined and using traverse incisions, the LAD artery was followed in the ventricular groove to the apex. Samples were taken from the proximal and tunneled artery of the MB cases (Figure 1). Figure 1. (A, B) The tunneled artery, that goes intramurally through myocardium (The arrow). Figure 1. (A, B) The tunneled artery, that goes intramurally through myocardium (The arrow). Table 1. Intimal diameters of the bridged artery (µm) Mean±SD Median (min-max) P value Intimal diameter of proximal part (µm) 397.13±55.74 417.27 (300.44-459.47) 0.005* Intimal diameter of tunneled segment (µm) 62.85±2.56 62.92 (58.55-66.28) Intimal diameter difference between proximal and tunneled part (µm) 334.28±56.32 Table 1. Intimal diameters of the bridged artery (µm) J C di Th R 2017 9(2) 108 112 109 Table 1. Intimal diameters of the bridged artery (µm) Mean±SD Median (min-max) P value Intimal diameter of proximal part (µm) 397.13±55.74 417.27 (300.44-459.47) 0.005* Intimal diameter of tunneled segment (µm) 62.85±2.56 62.92 (58.55-66.28) Intimal diameter difference between proximal and tunneled part (µm) 334.28±56.32 Mean±SD Median (min-max) P value 397.13±55.74 417.27 (300.44-459.47) 0.005* 62.85±2.56 62.92 (58.55-66.28) 334.28±56.32 J Cardiovasc Thorac Res, 2017, 9(2), 108-112 109 Pourhoseini et al Figure 4. Coronary angiography shows long segment muscle bridge (A), Perforation after stent implantation (B), sealing of perforation after multiple prolonged balloon inflation (C). intima layer was thinner compared with the proximal layer, resulting in a remarkable difference between the two regions, in terms of their sizes (Table 1; Figure 2). In the proximal artery section, the majority of endothelial cells of the MB samples were spiral-shaped, while in the tunneled artery section, the endothelial cells were spindle shaped. In the majority of samples, atherosclerotic plaques were observed in the proximal artery section, while in the tunneled artery section of the samples, no plaques were observed. The Adventitial layer was almost normal (Figure 3). Figure 4. Coronary angiography shows long segment muscle bridge (A), Perforation after stent implantation (B), sealing of perforation after multiple prolonged balloon inflation (C). Figure 5. Tissue sampling procedures Coronary angiography shows significant compression of muscle bridge segment (A), Perforation and suspected proximal stent edge dissection after stenting (B), sealed perforation after Bare metal stent implantation and intermittent prolong balloon inflation (C). Concerning the myocardium, in the MB section, it was significantly different from the rest of the myocardium, as the nuclei in the muscle fibers of this section were smaller than the normal sections. Discussion h The major purpose of the histological examination of the present study was to find an explanation for the high incidence of coronary perforation during or following the stenting of the bridged segment of the coronary artery which is reported in literature.17-21 Figure 5. Coronary angiography shows significant compression of muscle bridge segment (A), Perforation and suspected proximal stent edge dissection after stenting (B), sealed perforation after Bare metal stent implantation and intermittent prolong balloon inflation (C). In our own practice we had also eight cases of coronary rupture of the bridged part during PCI. The mean age of our cases was 59.6 years including 7 females and 1 male. Five cases were complicated with cardiac tamponade needing pericardiocentesis and pigtail insertion. Three developed with intracavitary perforations with no evidence of tamponade. Figure 6. Coronary angiography reveals significant muscle bridge (A), perforation of vessel after stent implantation with pigtail insertion (B), sealing of perforation after deployment of covered stent (C). In 2 cases, the perforation was sealed with multiple prolonged balloon inflations (Figure 4). In the other, a bare metal stent was implanted because of suspicion of perforation due to edge dissection combined with repeated longstanding balloon inflations (Figure 5). We had to seal the perforation with covered stents, in the remaining five Figure 6. Coronary angiography reveals significant muscle bridge (A), perforation of vessel after stent implantation with pigtail insertion (B), sealing of perforation after deployment of covered stent (C). Figure 2. The comparisons between proximal (A) and tunneled artery in intimal layer (Figure 6). In 2008, Li et al, reported 2 cases of perforations, one was sealed with prolonged balloon inflations and the other was sent for emergency cardiac surgery due to no response to the aforementioned management.20 Shen et al presented a case of coronary perforation during PCI for muscle bridge segment, successfully managed with implanting covered stent.21 Figure 2. The comparisons between proximal (A) and tunneled artery in intimal layer In an interesting study by Haager et al,22 long term follow up of patients who underwent PCI for symptomatic myocardial bridging was investigated. They presented 11 cases out of which 4 developed with significant in stent restenosis. They concluded that high inflation pressures may be needed for optimal stent implantation and apposition. Intravascular ultrasound (IVUS) is helpful to achieve the favorable result. One of our cases with coronary perforations, developed with significant in stent restenosis after 1 year which was sent for coronary artery bypass graft surgery. Figure 3. (A) Intimal layer of a proximal part of myocardial bridge with atherosclerosis plaque (Large arrow). (B) Intimal layer of tunneled segment. Note the difference of diameters. Figure 3. (A) Intimal layer of a proximal part of myocardial bridge with atherosclerosis plaque (Large arrow). (B) Intimal layer of tunneled segment. Note the difference of diameters. This increased risk of perforation during PCI is actually a multifactorial phenomenon. Thin intima, smaller vessel J Cardiovasc Thorac Res, 2017, 9(2), 108-112 110 Histopathological aspect of myocardial bridging diameter of the tunneled segment, over inflation of the balloon and oversizing of the stent are some probable causes.17,23 To this purpose, histological differences in the proximal and tunneled artery were examined. The findings of this study showed an MB prevalence of 51%, as 23 out of the 45 examined cadavers were discovered to be afflicted by the MB. Different rates of MB prevalence has been reported in various studies, with a higher percentage reported in autopsies than conventional and even CT angiographic studies.7 This variety can be explained by the fact that CT angiography is not capable of detecting MBs thinner than 20 mm, and such MBs can be diagnosed only through autopsy.24 The prevalence of MB through autopsy is 50%-58%, indicating the highest incidence rate of MB4 compared to other techniques. (Figure 6). For instance, following autopsy examining of 90 cadavers, Ferreira et al4 reported detecting MB in 55 cases, giving a prevalence of almost 55%, in people without any previous history of heart complaints, whose death had occurred due to reasons other than heart problems. Consequently, the findings of the present study in this regard is compatible with similar studies. intima structure. These three factors can result in the over-contraction of myocardium in this section, besides inhibiting the full extension of the artery. The latter and smaller vessel diameter in the tunneled segment can explain the high incidence of the LAD rupture and perforation in the section under the bridge. References Previous studies had also reported that the endothelial cells had a helical orientation (associated with the laminar blood flow and high endothelial shear stress) under the bridge, and polymorph, flat, or polygonal shapes before the bridge (shapes associated with low endothelial shear stress).6,24 7. Harikrishnan S, Sunder KR, Tharakan J, Titus T, Bhat A, Sivasankaran S, Bimal F. Clinical and angiographic profile and follow-up of myocardial bridges: a study of 21 cases. Indian Heart J. 1999;51(5):503-7. 8. Bestetti RB, Costa RS, Kazava DK, Oliveira JS. Can isolated myocardial bridging of the left anterior descending coronary artery be associated with sudden death during exercise? Acta Cardiol. 1991;46(1):27-30. 9. Arjomand H, AlSalman J, Azain J, Amin D. Myocardial bridging of left circumflex coronary artery associated with acute myocardial infarction. J Invasive Cardiol. 2000;12(8):431-4. We also observed that in the myocardium the nuclei of the muscle fibers in the MB section were smaller than the normal section, an observation reported by other studies, too.4,6,24 There seems to be significant differences within the myocardial structure between samples taken from the bridge areas vascularized by tunneled coronaries, and the rest of the myocardium. The nuclei from the MB section fibers are always smaller than the ones from other areas.25 Furthermore, through comparing MB hearts with non-MB hearts, Brodsky found a significantly increased interstitial fibrosis in samples obtained from the anterior wall of the left ventricle as compared with equivalent samples from cases without MB.26 10. Feld H, Guadanino V, Hollander G, Greengart A, Lichstein E, Shani J. Exercise-induced ventricular tachycardia in association with a myocardial bridge. Chest. 1991;99(5):1295-6. 11. Lee MS, Chen CH. Myocardial Bridging: An Up-to-Date Review. J Invasive Cardiol. 2015;27(11):521-8. 12. Möhlenkamp S, Hort W, Ge J, Erbel R. Update on myocardial bridging. Circulation. 2002;106(20):2616-22. 13. Nair CK, Dang B, Heintz MH, Sketch MH. Myocardial bridges: effect of propranolol on systolic compression. Can J Cardiol. 1986;2(4):218-21. 14. Kracoff OH, Ovsyshcher I, Gueron M. Malignant course of a benign anomaly: myocardial bridging. Chest. 1987;92(6):1113-5. References 1. Angelini P, Velasco JA, Flamm S. Coronary anomalies: incidence,pathophysiology, and clinical relevance. Circulation. 2002;105(20):2449-54. 2. Angelini P, Trivellato M, Donis J, Leachman RD. Myocardial bridges: a review. Prog Cardiovasc Dis. 1983;26(1):75-88. 2. Angelini P, Trivellato M, Donis J, Leachman RD. Myocardial bridges: a review. Prog Cardiovasc Dis. 1983;26(1):75-88. 3. Faruqui AM, Maloy WC, Felner JM, Schlant RC, Logan WD, Symbas P. Symptomatic myocardial bridging of coronary artery. Am J Cardiol. 1978;41(7):1305-10. Our study showed that the intima layer in the suffering artery had gone through significant hypertrophy, while it had remained thin in the tunneled artery section. Previous studies confirm these findings.4,6 It has been reported that the intima layer has different characteristics before, below and after the bridge. Before the bridge, the intima layer has been reported to be about 406.6 µm wide on average, while under the bridge its width is about 66 µm.6 4. Ferreira AG Jr, Trotter SE, König B Jr, Décourt LV, Fox K, Olsen EG. Myocardial bridges: morphological and functional aspects. Br Heart J. 1991;66(5):364-7. 4. Ferreira AG Jr, Trotter SE, König B Jr, Décourt LV, Fox K, Olsen EG. Myocardial bridges: morphological and functional aspects. Br Heart J. 1991;66(5):364-7. 5. Feldman RL, Nichols WW, Pepine CJ, Conti CR. Hemodynamic significance of the length of a coronary arterial narrowing. Am J Cardiol. 1978;41(5):865-71. 5. Feldman RL, Nichols WW, Pepine CJ, Conti CR. Hemodynamic significance of the length of a coronary arterial narrowing. Am J Cardiol. 1978;41(5):865-71. 6. Dermengiu D, Vovolis I, Hostiuc S, Curca GC, Rusu MC, Luca L. Morphological features in myocardial bridging. Rom J Leg Med. 2010;18(3):163-70. 6. Dermengiu D, Vovolis I, Hostiuc S, Curca GC, Rusu MC, Luca L. Morphological features in myocardial bridging. Rom J Leg Med. 2010;18(3):163-70. Furthermore, the present study found that the epithelial cells under the bridge were spindle-shaped, while they were polygonal in the proximal section. Previous studies had also reported that the endothelial cells had a helical orientation (associated with the laminar blood flow and high endothelial shear stress) under the bridge, and polymorph, flat, or polygonal shapes before the bridge (shapes associated with low endothelial shear stress).6,24 Furthermore, the present study found that the epithelial cells under the bridge were spindle-shaped, while they were polygonal in the proximal section. Competing interests The authors declare that they have no competing interests. The authors declare that they have no competing interests. Ethical Approval This study was approved by ethics committee, Shiraz University of Medical Sciences, Shiraz, Iran. This study was approved by ethics committee, Shiraz University of Medical Sciences, Shiraz, Iran. Conclusion Based on the aforementioned discussion, it can be concluded that MB structure varies from the rest of the myocardium in that the nuclei from the bridged myocardial fibers are smaller and interstitial fibrosis is higher in this area. Besides, the bridge makes transportation in the 15. Tarantini G, Migliore F, Cademartiri F, Fraccaro C, Iliceto S. Left anterior descending artery myocardial bridging: a clinical approach. J Am Coll Cardiol. 2016;68(25):2887- 2899. doi: 10.1016/j.jacc.2016.09.973. J Cardiovasc Thorac Res, 2017, 9(2), 108-112 111 Pourhoseini et al 16. Berry JF, von Mering GO, Schmalfuss C, Hill JA, Kerensky RA. Systolic compression of the left anterior descending coronary artery: a case series, review of the literature, and therapeutic options including stenting. Catheter Cardiovasc Interv. 2002;56(1):58-63. doi:10.1002/ ccd.10151 rescue coronary rupture during percutaneous coronary intervention for myocardial bridge. Intern Med. 2009;48(12):993-6. 22. Haager PK, Schwarz ER, vom Dahl J, Klues HG, Reffelmann T, Hanrath P. Long term angiographic and clinical follow up in patients with stent implantation for symptomatic myocardial bridging. Heart. 2000;84(4):403-8. 17. Tomasevic M, Dikic M, Ostojic M. Stenting a myocardial bridge: a wrong decision in STEMI? Acta Cardiol. 2011;66(1):89-91. doi:10.2143/AC.66.1.2064974. 23. Qian J, Zhang F, Wu H, Fan B, Ge L, Lu Y, Ge J. Size of coronary artery in a myocardial bridge compared with adjacent nontunneled left anterior descending coronary artery. Am J Cardiol. 2007 Jun 15;99(12):1653-5. 18. Becher T, Baumann S, Huseynov A, Behnes M, Borggrefe M, Akin I. Coronary artery perforation in a patient with STEMI and a myocardial bridge: an increased risk for coronary artery perforation? Cardiovasc Revasc Med. 2015;16(4):246-8. doi:10.1016/j.carrev.2015.03.004. 24. Ishikawa Y, Kawawa Y, Kohda E, Shimada K, Ishii T. Significance of the anatomical properties of a myocardial bridge in coronary heart disease. Circ J. 2011;75(7):1559- 66. 19. Hering D, Horstkotte D, Schwimmbeck P, Piper C, Bilger J, Schultheiss HP. [Acute myocardial infarct caused by a muscle bridge of the anterior interventricular ramus: complicated course with vascular perforation after stent implantation]. Z Kardiol. 1997;86(8):630-8. 25. Reig J, Ruiz de Miguel C, Moragas A. Morphometric analysis of myocardial bridges in children with ventricular hypertrophy. Pediatr Cardiol. 1990;11(4):186-90. 26. Brodsky SV, Roh L, Ashar K, Braun A, Ramaswamy G. Myocardial bridging of coronary arteries: A risk factor for myocardial fibrosis? Int J Cardiol. 2008 14;124(3):391-2. 20. Li W, Li Y, Sheng L, Gong Y. Myocardial bridge: is the risk of perforation increased? Can J Cardiol. 2008;24(11):e80-1. 21. Conclusion Shen TY, Chen CC, Tseng YZ. Stent graft used to J Cardiovasc Thorac Res, 2017, 9(2), 108-112 112
https://openalex.org/W2227315955	https://europepmc.org/articles/pmc4703303?pdf=render	English	null	Spatial Biodiversity Patterns of Madagascar's Amphibians and Reptiles	PloS one	2,016	cc-by	14,471	Jason L. Brown1, Neftali Sillero2, Frank Glaw3, Parfait Bora4, David R. Vieites5, Miguel Vences6 1 Department of Zoology, Southern Illinois University, Carbondale, Illinois, United States of America, 2 Centro de Investigação em Ciências Geo-Espaciais, Alameda do Monte da Virgem, Vila Nova de Gaia, Portugal, 3 Zoologische Staatssammlung, München, Germany, 4 Département de Biologie Animale, Université d’Antananarivo, BP 906, Antananarivo, Madagascar, 5 Museo Nacional de Ciencias Naturales, MNCN–CSIC, C/José Gutierrez Abascal 2, Madrid, Spain, 6 Zoological Institute, Technische Universität Braunschweig, Braunschweig, Germany * jason.brown@siu.edu (JLB); m.vences@tu-bs.de (MV) * jason.brown@siu.edu (JLB); m.vences@tu-bs.de (MV) RESEARCH ARTICLE Abstract Madagascar has become a model region for testing hypotheses of species diversification and biogeography, and many studies have focused on its diverse and highly endemic herpeto- fauna. Here we combine species distribution models of a near-complete set of species of rep- tiles and amphibians known from the island with body size data and a tabulation of herpetofaunal communities from field surveys, compiled up to 2008. Though taxonomic revi- sions and novel distributional records arose since compilation, we are confident that the data are appropriate for inferring and comparing biogeographic patterns among these groups of organisms. We observed species richness of both amphibians and reptiles was highest in the humid rainforest biome of eastern Madagascar, but reptiles also show areas of high richness in the dry and subarid western biomes. In several amphibian subclades, especially within the Mantellidae, species richness peaks in the central eastern geographic regions while in rep- tiles different subclades differ distinctly in their richness centers. A high proportion of clades and subclades of both amphibians and reptiles have a peak of local endemism in the topo- graphically and bioclimatically diverse northern geographic regions. This northern area is roughly delimited by a diagonal spanning from 15.5°S on the east coast to ca. 15.0°S on the west coast. Amphibian diversity is highest at altitudes between 800–1200 m above sea-level whereas reptiles have their highest richness at low elevations, probably reflecting the compar- atively large number of species specialized to the extended low-elevation areas in the dry and subarid biomes. We found that the range sizes of both amphibians and reptiles strongly corre- lated with body size, and differences between the two groups are explained by the larger body sizes of reptiles. However, snakes have larger range sizes than lizards which cannot be readily explained by their larger body sizes alone. Range filling, i.e., the amount of suitable habitat occupied by a species, is less expressed in amphibians than in reptiles, possibly reflecting their lower dispersal capacity. Taxonomic composition of communities assessed by field surveys is largely explained by bioclimatic regions, with communities from the dry and especially subarid biomes distinctly differing from humid and subhumid biomes. Spatial Biodiversity Patterns of Madagascar's Amphibians and Reptiles Jason L. Brown1, Neftali Sillero2, Frank Glaw3, Parfait Bora4, David R. Vieites5, Miguel Vences6 Introduction Competing Interests: The authors have declared that no competing interests exist. Madagascar has long been renowned for its unique and diverse fauna and flora [1] and high proportion of microendemism, that is, range-restricted species characterized by exceedingly small distribution areas [2]. The island has long attracted the interest of biogeographers study- ing not only in the origins of Madagascar's biota, but also within-island distributional patterns and diversification mechanisms [2–8]. Current evidence suggests that the majority of Madagas- car's vertebrate clades, but probably also most other animals and plants, colonized Madagascar over the Cenozoic and in many cases by overseas dispersal [9–12], after a major biotic change at the K/T boundary [13, 14]. A variety of factors (i.e. river barriers and montane refugia), have subsequently influenced speciation and community assembly within Madagascar [2, 6, 15–24], and only a combination of factors can explains the complex patterns observed [25]. In addition to plants [4] and lemurs [26], the herpetofauna have historically been one of the main biogeographic model groups in Madagascar. Explicit zoogeographic regions for the island were first proposed on the basis of reptile distribution patterns [3], and further discussions and analyses of both reptile [7, 27] and amphibian patterns [28] were published later on. Various pio- neering biogeographic studies were entirely or partly based on herpetofaunal data. Some of these aimed to understand cladistic biogeographical relationships among sites in Madagascar [29, 30], defined null models of biodiversity patterns [16, 31], or modeled species’ distributions for species discovery and delimitation [32, 33]. Other herpetofauna-centered papers analyzed the impact of climate change on altitudinal distribution of montane faunas [34] and comprehensively assessed spatial and taxonomic conservation priorities in Madagascar [35–37]. Many of these works were made possible by an immense and intensified effort in inventorying these animals since the early 1990s, involving numerous survey studies across the island [38], routine application of bioacous- tic and molecular methods [39], and inclusion of undescribed candidate species in many of the assessments [40]. Hence, although it is clear that many of Madagascar's amphibian and reptile species remain scientifically undescribed, the majority of them have been genetically character- ized as candidate species [41, 42] and included in field guides [43], and thus are provisionally accessible for research and conservation. Spatial Biodiversity Patterns of Madagascar's Amphibians and Reptiles OPEN ACCESS Citation: Brown JL, Sillero N, Glaw F, Bora P, Vieites DR, Vences M (2016) Spatial Biodiversity Patterns of Madagascar's Amphibians and Reptiles. PLoS ONE 11(1): e0144076. doi:10.1371/journal.pone.0144076 Editor: Stefan Lötters, Trier University, GERMANY Editor: Stefan Lötters, Trier University, GERMANY Received: June 15, 2015 Accepted: November 12, 2015 Published: January 6, 2016 Copyright: © 2016 Brown et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Editor: Stefan Lötters, Trier University, GERMANY Received: June 15, 2015 Accepted: November 12, 2015 Published: January 6, 2016 Copyright: © 2016 Brown et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Received: June 15, 2015 Accepted: November 12, 2015 Published: January 6, 2016 Copyright: © 2016 Brown et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: Raw data are in Supporting Information files or are published in: Brown JL, Cameron A, Yoder AD, Vences M (2014). A necessarily complex model to explain the biogeography of the amphibians and reptiles of Madagascar. Nature Communications 10:5. Funding: Fieldwork of MV and PhD studies of PB were funded by the Volkswagen Foundation. Fieldwork was supported by Spanish Government grants CGL2009-10198 and CGL2013-40924-P to DRV. JLB was supported by the National Science Foundation (Grant No. 0905905). NS is supported with a research contract (IF/01526/2013) by FCT (Portugal). 1 / 26 PLOS ONE \| DOI:10.1371/journal.pone.0144076 January 6, 2016 PLOS ONE \| DOI:10.1371/journal.pone.0144076 January 6, 2016 Terminology and taxonomy The present analysis is based on distributional data of Malagasy amphibians and reptiles, from a compilation completed in 2008, and partly adjusted to account for subsequent taxonomic revisions. The compilation includes well-defined but scientifically undescribed confirmed can- didate species [40] (i.e., species characterized by a substantial genetic divergence and by addi- tional evidence for a status of independent evolutionary lineages). Given that the number of undescribed lineages keeps increasing [42] and their status is being modified in the course of taxonomic revisions, our data thus represent only a snapshot of taxonomic knowledge from 2008, with some updates; yet, our decision to include such candidate species leads to a more representative picture than the inclusion of nominal species only. Furthermore, the majority of distributional information accumulated since 2008 has not been included in our dataset. As addressed again in the Discussion, the constantly changing taxonomy in some taxa, together with incomplete distribution range information and with the exclusion of some unrevised species at the time of compilation of our distributional dataset, might have led to biased representations of species richness and endemism in some subgroups (but does not invalidate general patterns reported herein). We here use the term "reptiles" in its classical meaning, i.e., referring to all non-avian rep- tiles including squamates, chelonians and crocodylians. Given that only a limited number of turtles and one species of crocodile occur in Madagascar, our data mostly reflect the distribu- tional patterns of squamates (lizards and snakes). The single crocodile species present in Mada- gascar (Crocodylus niloticus) was not included in our analysis. Description of major biomes in Madagascar follows previous definitions of bioclimatic regions [47]. For convenience of naming particular geographic regions, we follow a previous approach [43, 48] that defined a series of regions with limits coinciding with those of major watersheds [2] (Fig 1C). Fig 1. Maps of Madagascar. (a) topography with major mountain massifs and rivers, (b) major bioclimatic zones (herein called biomes) [47], (c) geographic regions ([48], boundaries based on watersheds ([2], and (d) delimitation of northern Madagascar as used herein including the Sambirano, North, and North East regions (map also shows a few towns and nature reserves discussed in the text). Fig 1. Maps of Madagascar. Introduction The paradoxical consequence is that Madagascar hosts one of the best studied and most scientifically accessible tropical herpetofauna, despite the large amount of undescribed species that have been revealed by these studies. Notwithstanding this overall good state of knowledge, the study of classical biogeography patterns of Madagascar's herpetofauna remains patchy and elusive. Numerous studies provided information on species richness and weighted endemism, but were either based on rough dis- tribution estimates [36, 37] or targeted only particular subgroups of amphibians and reptiles [22, 31, 44, 45]. Only recently, analyses of species richness, weighted endemism and turnover based on explicit distribution models became available for all amphibians, reptiles, and selected subgroups [25]. However, these have not yet been discussed from a taxon-specific perspective. Island-wide patterns of community composition of Madagascar's amphibians and reptiles have remained largely unstudied despite the availability of numerous surveys that followed roughly similar methodological approaches [38]. The relationship of range size and body size has not been comprehensively studied in Madagascar's amphibians and reptiles, although case studies in mantellid frogs suggest that body size might be an important factor influencing gene flow and diversification [20, 46]. Here, we provide a set of analyses aimed at partly filling these gaps in knowledge and pro- viding a more complete baseline for future studies of biogeography, systematics, evolution and conservation of Madagascar's amphibians and reptiles. Our analyses include (i) calculation and comparison of species richness and endemism for various subgroups of the Malagasy herpeto- fauna, (ii) community turnover based on generalized dissimilarity modelling separately for PLOS ONE \| DOI:10.1371/journal.pone.0144076 January 6, 2016 2 / 26 Spatial Biodiversity Patterns of Madagascar's Amphibians and Reptiles amphibians and reptiles, (iii) range-body size relationships and range filling, and (iv) a com- parison of the composition of real herpetofaunal communities across Madagascar as detected by survey work. Species Distribution Models Because the distribution ranges of Madagascar's amphibians and reptiles have not been com- prehensively mapped and distribution records are scattered, we used species distribution mod- els (SDMs; also commonly called ecological niche models)[49] to obtain an estimate of the geographic ranges. The majority of our analyses are based on the SDMs compiled for a previ- ous study [25]. These were calculated from 8362 occurrence records of 745 Malagasy amphib- ian and reptile species (325 and 420 species, respectively) and were limited to species that had, at minimum, 3 unique occurrence points at the spatial resolution (0.91 km2). For original occurrence records, see supplementary materials of Brown et al. [25]. Several species (n = 19) were excluded mostly because of convoluted taxonomy or contro- versial information on their distribution ranges, leaving a total of 727 species for final analysis (yielding a more inclusive dataset compared to the 679 species included previously; Brown et al.[25]). Species distribution models were generated in MaxEnt v3.3.3e [50] using parameters as described in Brown et al. [25] that accounted for sample selection biases [25, 51, 52]. The bias file up-weighted presence-only data points with fewer neighbors in the geographic land- scape [53]. We used 19 standard variables characterizing current bioclimates for modeling (Worldclim 1.4; [54] as well as geology, aspect, elevation, solar radiation, and slope [55, 56]). All layers were projected to Africa Alber’s Equal-Area Cylindrical projection in ArcMap at a resolution of 0.91 km2. To limit over-prediction of SDMs we clipped each model as previously suggested [35]. Thus, we produced models representing suitable habitat within an area of known occurrence, based on a buffered minimum-convex-polygon of occurrence localities [25]. Continuous SDMs were converted to binary models using the ‘minimum training pres- ence’ threshold. Terminology and taxonomy (a) topography with major mountain massifs and rivers, (b) major bioclimatic zones (herein called biomes) [47], (c) geographic regions ([48], boundaries based on watersheds ([2], and (d) delimitation of northern Madagascar as used herein including the Sambirano, North, and North East regions (map also shows a few towns and nature reserves discussed in the text). doi:10.1371/journal.pone.0144076.g001 doi:10.1371/journal.pone.0144076.g001 PLOS ONE \| DOI:10.1371/journal.pone.0144076 January 6, 2016 3 / 26 Spatial Biodiversity Patterns of Madagascar's Amphibians and Reptiles Northern Madagascar has been highlighted before as a center of endemism [48] and appeared as such also in our CWE (Corrected Weighted Endemism) analyses (see below). To understand differences in community composition we separated data from communities in humid and subhumid biomes (rainforest and montane forest) in this part of Madagascar (defined as the area north of a diagonal spanning from 15.5°S on the east coast to ca. 15.0°S on the west coast, i.e., roughly from Maroantsetra to Antsohihy; Fig 1D). Then, we compared the proportion of reptiles and amphibians among these two clusters of data points. PLOS ONE \| DOI:10.1371/journal.pone.0144076 January 6, 2016 Species Richness, Corrected Weighted Endemism, and Generalized Dissimilarity Modeling Species richness (SR) and corrected weighted endemism (CWE) were calculated from different taxonomic subsets of our estimate range maps (SDMs and buffered points). We used a hexago- nal sampling grid at 5166km2, the same area used in previous studies [20, 25]. The hexagon is the most complex regular polygon and results in less orientation bias in analyses (vs. a square grid, as used in the aforementioned studies). CWE measures endemism by inversely weighting the proportion of endemics by their range size (species with smaller ranges are weighted more than those with large ranges; [57], and dividing this value by the local species richness [58]. CWE was calculated using SDMtoolbox v1 [52]. Generalized Dissimilarity Modeling (GDM; [59]) can be used to analyze and predict spatial patterns of turnover in community composition across large areas [25, 60]. To avoid computa- tional limitations associated with pairwise comparisons of large datasets, we randomly sampled 2500 points throughout Madagascar from a ca. 10 km2 grid and then measured the absence or presence of each species at each locality [25]. The 23 environmental and geography layers used for SDMs were reduced to nine vectors in a principal component analyses and these were PLOS ONE \| DOI:10.1371/journal.pone.0144076 January 6, 2016 4 / 26 Spatial Biodiversity Patterns of Madagascar's Amphibians and Reptiles sampled at the same 2500 localities. Species communities as predicted by species occurrences at each of these sites, and environmental data, were then input into a generalized dissimilarity model using the R package: GDM R distribution package v1.1 (www.biomaps.net.au/gdm/ GDM_R_Distribution_Pack_V1.1.zip). The GDM was then extrapolated based on the high resolution climate dataset [25, 59]. Classification of the GDMs was performed in SPSS v20 [61] using a two-step classification method that assesses AICc of a range of class numbers (here 2–30) to determine the optimum number of GDM classes; these were interpolated as described for the continuous model. Range Size and Body Size Relationships For descriptive range-size statistics, distribution range-sizes were sampled for all species at 0.01 degrees2 from corrected binary SDMs (or buffered point data where applicable). We sampled for each modeled species two different range size measurements: corrected range size as in the SDMs clipped by buffered minimum-convex polygons (from [25]) and uncorrected range size based on SDM prediction without such clipping. We then calculated the ratio of the corrected range size divided by the uncorrected range size as a measure of range filling, that is, the pro- portion of the suitable habitat that is occupied by the species. We furthermore extracted the maximum, minimum and mean elevations predicted for each species from the adjusted SDMs and the buffered-point maps. Subsequently, we calculated the number of species estimated by the models to occur at different altitudes, at intervals of 100 m above sea level. Body sizes of all species of Madagascar's amphibians and reptiles were compiled from the literature, mostly from a comprehensive field guide [43] and complemented with unpublished data and our own measurements where necessary (S1 Table). We used the maximum known male snout-vent length as the measurement of body size, as this variable was readily available for most species [43] and has previously been used in biogeographical and macroecological analysis [20, 62]. Although this variable ignores sexual dimorphism and different body shapes (of e.g. snakes and frogs), we are convinced it is an informative proxy in analyses over an entire and diverse herpetofauna, which in the case of Madagascar spans over three orders of magni- tude with SVL values ranging from ca. 10 mm in Stumpffia frogs to ca. 2200 mm in Acranto- phis snakes (larger crocodylians were not analyzed here). We used Statistica 7.1 (Statsoft, Tulsa, USA) to calculate and visualize correlations between range size, altitudinal range, range filling, and body size, and for additional univariate tests (t- tests) comparing species numbers between regions or between taxa. We calculated univariate linear regressions and tested for differences between reptiles and amphibians, as well as between snakes and lizards, in analyses of variance (ANCOVA) defining body size as a covari- able. Analyses of range filling were done using modeled species only; analyses of range size also included species known from only 1–2 sites. Species richness The humid rainforest biome of eastern Madagascar holds the highest concentration of species of both amphibians and reptiles, but differences are visible between the two groups (Fig 2). In reptiles, species richness (SR) is regularly distributed along the eastern rainforest band whereas the amphibian SR is concentrated in an area of the Northern Central East and Southern Cen- tral East regions. Whether the geographic gap between the two richness centers in the Northern and Southern Central East (corresponding to the two well-sampled regions around Ranoma- fana and Mantadia-Analamazaotra National Parks) reflects a real pattern or incomplete sam- pling remains uncertain with present data, although the modelling approach applied herein does account for sampling bias [25]. A closer look at independent clades of amphibians (Fig 3) suggests that the high-central SR is mainly caused by the family Mantellidae whereas the microhylid subfamily Cophylinae has a more even pattern with high SR also in rainforests of the North East. Within the Mantellidae, a central concentration of SR is found in two independent subclades (especially in Boophis and to a lesser degree in Mantidactylus) but not in a third subclade (Gephyromantis). Overall, reptile SR in Madagascar is highest in the humid biome and rather regularly distrib- uted along its entire latitudinal extension (Fig 2). Comparatively, high SR values are also found along the west coast in the dry biome, and especially in the subarid biome in the South West. Species richness is lowest on the high plateau in the Central region, and in a poorly surveyed area southwest of Mahajanga in the West. Differences among reptile clades are stronger than among amphibian clades, with some clades and subclades lacking high SR in the humid biome. Most deviant are iguanas (Fig 3), which have no rainforest representative. This is also reflected in Trachylepis skinks and Paroedura geckos, each one with only two species colonizing the humid rainforest biome. In these three groups (iguanas, Trachylepis, Paroedura), SR peaks in the dry and especially subarid biomes of the South West and West, and additionally in north- ern Madagascar for Paroedura (Fig 3). Also chameleons of the genus Furcifer have the highest richness in the dry and subarid biomes, with only few species colonizing rainforest. Herpetofaunal community analysis From the plethora of herpetofaunal surveys published for Madagascar [38], we selected 20 sur- veys that were spatially representative of the most well surveyed areas at the time these data were compiled [8, 33, 63–81]. For this dataset we tabulated the amphibian and reptile species encountered in these inventories, and separated the species records for each site in case of multi-site inventories. Given the large number of new species described from Madagascar over the last years, ascertaining the taxonomic identity of species recorded during inventories over different decades is almost impossible, and any analysis uncritically using such unpublished species lists will inevitably be flawed. However, both the overall species numbers and the assignment of species to major clades (genera, subfamilies or families) can be considered as PLOS ONE \| DOI:10.1371/journal.pone.0144076 January 6, 2016 5 / 26 Spatial Biodiversity Patterns of Madagascar's Amphibians and Reptiles rather reliable, and we therefore based our analysis on such simplified taxon lists. We assigned all species to one of four major amphibian and eight reptile categories. Amphibians were (1) hyperoliid frogs, (2) microhylid frogs of the subfamily Cophylinae, (3) microhylid frogs of the subfamilies Scaphiophryninae and Dyscophinae, (4) mantellid frogs. Reptile categories were (1) turtles and tortoises, (2) typhlopid and xenotyphlopid snakes, (3) lamprophiid snakes, (4) iguanid lizards, (5) geckos, (6) skinks, (7) gerrhosaurids, and (8) chameleons. Some other major taxa with low number of species were not included in the analysis (i.e., boid snakes, pty- chadenid and dicroglossid frogs) because they might have distorted the results due to the small sample size (1–3 species). To better understand how well these ground-truthed communities identified in survey work differ from theoretical communities as calculated by SDM overlap and used in the GDM, we compiled the latter by extracting for each survey site the theoretical communities and compar- ing its composition (in species numbers of major taxonomic categories) with the observed communities. PLOS ONE \| DOI:10.1371/journal.pone.0144076 January 6, 2016 Species richness The dwarf geckos of the genus Lygodactylus presented high SR on some central mountain massifs (Ankar- atra, Ibity, Itremo, Andringitra), which are, as well, partly important centers of SR in Trachyle- pis, Furcifer, iguanids and gerrhosaurids, but with peaks not fully coinciding among these groups. Several reptile groups have SR centers located in a small northern portion of Madagas- car (i.e., as defined here, the area north of a diagonal spanning from 15.5°S on the east coast to 6 / 26 PLOS ONE \| DOI:10.1371/journal.pone.0144076 January 6, 2016 Spatial Biodiversity Patterns of Madagascar's Amphibians and Reptiles Fig 2. Biodiversity measures for reptiles and amphibians. Species richness (SR), endemicity (corrected weighted endemism, CWE), and turnover as measured by general dissimilarity models (GDM), based on the distribution of 325 species of amphibians and 420 species of reptiles from Madagascar. Species richness scales range from low (blue) to high (red) number of species per hexagon; Local endemism values range from low (blue) to high (red). Fig 2. Biodiversity measures for reptiles and amphibians. Species richness (SR), endemicity (corrected weighted endemism, CWE), and turnover as measured by general dissimilarity models (GDM), based on the distribution of 325 species of amphibians and 420 species of reptiles from Madagascar. Species richness scales range from low (blue) to high (red) number of species per hexagon; Local endemism values range from low (blue) to high (red). Fig 2. Biodiversity measures for reptiles and amphibians. Species richness (SR), endemicity (corrected weighted endemism, CWE), and turnover as measured by general dissimilarity models (GDM), based on the distribution of 325 species of amphibians and 420 species of reptiles from Madagascar. Species richness scales range from low (blue) to high (red) number of species per hexagon; Local endemism values range from low (blue) to high (red). doi:10.1371/journal.pone.0144076.g002 ca. 15.0°S on the west coast) but the precise limits of these areas of high SR do not always coin- cide spatially. In Brookesia ground chameleons, Uroplatus geckos, and skinks, similar to some amphibians (Gephyromantis, Mantidactylus, cophylines), the peak is in the rainforests of the North East. However, in Paroedura geckos and less distinctly in gerrhosaurids, SR peaks on the western coast of northern Madagascar (i.e., the Sambirano region). PLOS ONE \| DOI:10.1371/journal.pone.0144076 January 6, 2016 Corrected weighted endemism Local endemism values, here measured and illustrated as Corrected Weighted Endemism (CWE), only partly coincide spatially with SR (Fig 2). Both in amphibians and reptiles, the PLOS ONE \| DOI:10.1371/journal.pone.0144076 January 6, 2016 PLOS ONE \| DOI:10.1371/journal.pone.0144076 January 6, 2016 7 / 26 Spatial Biodiversity Patterns of Madagascar's Amphibians and Reptiles Fig 3. Species richness. Species richness (SR) calculated separately for different clades and subclades of Malagasy amphibians a G1 includes scaphiophrynines whereas G2 includes cophylines. doi:10.1371/journal.pone.0144076.g003 Fig 3. Species richness. Species richness (SR) calculated separately for different clades and subclades of Malagasy amphibians and reptiles. Microhylidae G1 includes scaphiophrynines whereas G2 includes cophylines. doi:10.1371/journal.pone.0144076.g003 8 / 26 PLOS ONE \| DOI:10.1371/journal.pone.0144076 January 6, 2016 Spatial Biodiversity Patterns of Madagascar's Amphibians and Reptiles largest extension of high-CWE cells is in the North East Madagascar. In amphibians and to a lesser degree in reptiles, CWE peaks are also observed in the Southern and Northern Central East, and in the South East. Slight differences compared to the 679 species dataset analyzed by Brown et al. [25] are recognizable in the CWE of reptiles, especially in the South East. Amphib- ians further have a single high-CWE cell coinciding with the Isalo Massif, and reptiles have an area of high CWE in the subarid South West, with the highest values coinciding with the Oni- lahy river mouth. Comparing the major clades as well as the subclades (Fig 4) shows that high or very high CWE values in some or most grid cells in northern Madagascar are an almost general pattern, except in iguanas. Also leaf tail geckos (Uroplatus) do not show a particularly high CWE in the North (but see Discussion). The coastal areas of the South West have high CWE, especially in iguanas, but also in geckos among the major clades, and in Trachylepis skinks, Furcifer chameleons, and to a lesser degree, in Phelsuma day geckos among the subclades. An area of high CWE cells in the North West is evident in three subclades: in skinks of the subfamily Scincinae, in Furcifer chameleons, and in geckos. Similarly, a cell coinciding with the Tsingy de Bemaraha limestone massif in western Madagascar has high CWE in Boophis treefrogs, as well as in Lygodactylus and Phelsuma geckos, and Brookesia ground chameleons. Altitudinal distribution of species richness We sampled altitudinal SR of amphibians and reptiles as the number of species predicted to occur in altitudinal sections of 100 m according to their modelled distribution. As discussed below, this approach almost certainly overestimates the number of species actually occurring at a certain elevation, but represents the most objective means to assess and compare altitudinal diversity across the entire herpetofauna with current data. We therefore do not report here absolute numbers, but general trends only. According to our analyses, the SR of amphibians continuously increases with increasing ele- vation, reaching a maximum between 800‒1200 m a.s.l., with the highest value at 1000 m a.s.l. (Fig 5). From 1000 m higher, SR is negatively correlated with elevation—with distinct drops of SR values from 2000 to 2100 m a.s.l. and from 2500 to 2600 m a.s.l. The elevational SR of rep- tiles shows a different trend, with a maximum SR value in the lowlands (100 m a.s.l.) and a con- tinuous decrease of SR with increasing elevation. Again, two distinct drops of SR values are seen at high elevations, one from 2200 to 2300 m a.s.l. and one from from 2600 to 2700 m a.s.l. The differences seen between amphibians and reptiles are statistically significant, with both minimum and maximum elevation per species, and elevational range, being on average higher in amphibians (t-tests: P = 0.016, P <0.001, P < 0.001, respectively). Corrected weighted endemism This is due to the presence of species at this site which had not been recorded elsewhere at the time our dataset was compiled, although for some of them (e.g., B. tampoka) new records have in the meantime become available and there- fore, the high CWE at Bemaraha at least for Boophis will likely not be recovered in future stud- ies based on updated datasets. Geckos, in general, and especially Lygodactylus dwarf geckos, as well as skinks, also have an area of high CWE coinciding with the Central Plateau of Madagas- car around the Ankaratra-Itremo-Ibity massifs. PLOS ONE \| DOI:10.1371/journal.pone.0144076 January 6, 2016 Areas of Endemism based on Generalized Dissimilarity Modelling Generalized Dissimilarity Modelling, as applied here, reconstructs for a set of sites across the landscape the theoretical communities of species based on the overlap of their distribution ranges, and then calculates pairwise differences between these communities. On this basis, it identifies changes in the communities which reflect high species turnover, and can be PLOS ONE \| DOI:10.1371/journal.pone.0144076 January 6, 2016 9 / 26 Spatial Biodiversity Patterns of Madagascar's Amphibians and Reptiles Fig 4. Endemism. Corrected weighted endemism (CWE) calculated separately for different clades and subclades of Malagasy amp Microhylidae G1 includes scaphiophrynines whereas G2 includes cophylines. doi:10.1371/journal.pone.0144076.g004 Fig 4. Endemism. Corrected weighted endemism (CWE) calculated separately for different clades and subclades of Malagasy amphibians and reptiles. Microhylidae G1 includes scaphiophrynines whereas G2 includes cophylines. doi:10.1371/journal.pone.0144076.g004 PLOS ONE \| DOI:10.1371/journal.pone.0144076 January 6, 2016 10 / 26 Spatial Biodiversity Patterns of Madagascar's Amphibians and Reptiles Fig 5. Species richness by elevation. Number of specimens of amphibians and reptiles, predicted by the adjusted SDMs to occur at certain elevations at intervals of 100 m above sea level. Presumably due to over-prediction the inferred elevational ranges probably are larger than the realized ones, giving higher numbers of species than actually occurring in lowlands and high elevations. d i 10 1371/j l 0144076 005 Spatial Biodiversity Patterns of Madagascar's Amphibians and Reptiles Fig 5. Species richness by elevation. Number of specimens of amphibians and reptiles, predicted by the adjusted SDMs to occur at certain elevations at intervals of 100 m above sea level. Presumably due to over-prediction the inferred elevational ranges probably are larger than the realized ones, giving hi h b f i h ll i i l l d d hi h l i Fig 5. Species richness by elevation. Number of specimens of amphibians and reptiles, predicted by the adjusted SDMs to occur at certain elevations at intervals of 100 m above sea level. Presumably due to over-prediction the inferred elevational ranges probably are larger than the realized ones, giving higher numbers of species than actually occurring in lowlands and high elevations. doi:10.1371/journal.pone.0144076.g005 Fig 5. Species richness by elevation. Number of specimens of amphibians and reptiles, predicted by the adjusted SDMs to occur at certain elevations at intervals of 100 m above sea level. Presumably due to over-prediction the inferred elevational ranges probably are larger than the realized ones, giving higher numbers of species than actually occurring in lowlands and high elevations. PLOS ONE \| DOI:10.1371/journal.pone.0144076 January 6, 2016 Areas of Endemism based on Generalized Dissimilarity Modelling Fig 5. Species richness by elevation. Number of specimens of amphibians and reptiles, predicted by the adjusted SDMs to occur at certain elevations at intervals of 100 m above sea level. Presumably due to over-prediction the inferred elevational ranges probably are larger than the realized ones, giving higher numbers of species than actually occurring in lowlands and high elevations. doi:10.1371/journal.pone.0144076.g005 interpreted as boundaries of biogeographic regions. The GDMs reconstructed herein for amphibians and reptiles reflect large differences between the distributional patterns seen in the two groups. Given that amphibians are mostly distributed in the humid and subhumid biomes, with few species in dry and subarid biomes, the main GDM boundaries run in a north-south direction. In reptiles, a more complex subdivision especially of the subhumid/montane biomes is reconstructed (Fig 2). Both in amphibians and reptiles, a trend is visible of more continuous community change in low elevations along the east coast, with no latitudinal boundary in the categorical GDM of amphibians and only one for reptiles (Fig 2). On the contrary, at higher elevations a higher number of latitudinal breaks exist that mainly are located in the subhumid/montane biomes and, thus, more distinct patterns of turnover are observed. Both amphibian and reptile GDMs reconstruct a major area of turnover (corresponding to the limit between dry and subarid biomes) in the area around Morondava in the West. Also, in both amphibians and reptiles, northern Madagascar stands out separately, although its boundaries are estimated more south- wards than formally defined and as reflected by richness and endemism patterns. In amphibi- ans, a region roughly corresponding to the Tsaratanana Massif stands out as separate area of endemism. 11 / 26 PLOS ONE \| DOI:10.1371/journal.pone.0144076 January 6, 2016 Spatial Biodiversity Patterns of Madagascar's Amphibians and Reptiles Range size, range filling and body size We found a clear and highly significant correlation of range size (spatial extent of the distribu- tion area, in km2) with body size (Fig 6), measured as maximum male snout-vent length, in both amphibians (parametric correlation: r = 0.2231, P < 0.001; non parametric Spearman cor- relation: R = 0.3403, P < 0.001) and reptiles (r = 0.3538, P < 0.001; R = 0.3280, P < 0.001, respectively). Range sizes of reptiles were larger than those of amphibians (range sizes, mean ± SD: 61113 ± 83100 km2 vs. 40326 ± 53719 km2; t-test: P = 0.001) as were their body sizes (SVL 202.5 ± 280.5 mm vs. 34.7 ± 17.9 mm; t-test: P < 0.001). The large standard deviations of range size values reflect the presence of a considerable number of species with distribution areas much larger than the average, combined with a high number of microendemic species. The larger ranges of reptiles compared to amphibians are probably caused mainly by their larger body sizes. Comparing the range size values of amphibians and reptiles by ANCOVA with SVL as co-variable revealed a highly significant influence of SVL (P < 0.001), but no significant influence of the taxonomic category (P = 0.612). Within reptiles, we furthermore tested whether the larger range sizes of snakes vs. lizards (mean 98785 ± 105015 km2 vs. Range sizes of reptiles were larger than those of amphibians (range sizes, mean ± SD: 61113 ± 83100 km2 vs. 40326 ± 53719 km2; t-test: P = 0.001) as were their body sizes (SVL 202.5 ± 280.5 mm vs. 34.7 ± 17.9 mm; t-test: P < 0.001). The large standard deviations of range size values reflect the presence of a considerable number of species with distribution areas much larger than the average, combined with a high number of microendemic species. The larger ranges of reptiles compared to amphibians are probably caused mainly by their larger body sizes. Comparing the range size values of amphibians and reptiles by ANCOVA with SVL as co-variable revealed a highly significant influence of SVL (P < 0.001), but no significant influence of the taxonomic category (P = 0.612). Within reptiles, we furthermore tested whether the larger range sizes of snakes vs. lizards (mean 98785 ± 105015 km2 vs. Range size, range filling and body size 48703 ± 70307 km2; t-test: P < 0.001) were a true pattern suggestive of different barriers to dis- persal, differential dispersal capacities among the two groups, or explainable by body size influ- ences only. In this case, ANCOVA revealed a significant influence both of SVL (P < 0.001) and of taxonomic category (P < 0.001), suggesting that indeed, at similar body sizes, snakes in Madagascar appear to have larger range sizes than lizards. 48703 ± 70307 km2; t-test: P < 0.001) were a true pattern suggestive of different barriers to dis- persal, differential dispersal capacities among the two groups, or explainable by body size influ- ences only. In this case, ANCOVA revealed a significant influence both of SVL (P < 0.001) and of taxonomic category (P < 0.001), suggesting that indeed, at similar body sizes, snakes in Madagascar appear to have larger range sizes than lizards. The correlation between body size and range size is also extended to elevational ranges. In amphibians, altitudinal ranges and maximum elevation were positively correlated with SVL, while minimum elevation was negatively correlated with SVL. This suggests that larger species occur over wider elevational ranges, which probably can be explained with their larger spatial ranges (non-parametric Spearman correlations for minimum and maximum elevation, and Fig 6. Body size and range sizes correlations. Correlation of body size with range size in amphibians (black circles and solid line) and reptiles (white squares and dashed line). Both correlations are highly significant (see text). doi:10.1371/journal.pone.0144076.g006 Fig 6. Body size and range sizes correlations. Correlation of body size with range size in amphibians (black circles and solid line) and reptiles (white squares and dashed line). Both correlations are highly significant (see text). Fig 6. Body size and range sizes correlations. Correlation of body size with range size in amphibians (black circles and solid line) and reptiles (white squares and dashed line). Both correlations are highly significant (see text). PLOS ONE \| DOI:10.1371/journal.pone.0144076 January 6, 2016 12 / 26 Spatial Biodiversity Patterns of Madagascar's Amphibians and Reptiles elevational range: R = -0.215, 0.231, 0.228; P < 0.001 in all three analyses). Similar results were obtained for reptiles, albeit with slightly weaker correlation coefficients (R = -0.222, P < 0.001; R = 0.182, P < 0.001 and R = 0.142; P < 0.01). Range size, range filling and body size We inferred range filling by calculating the proportion of suitable habitat actually occupied by the species (clipped/unclipped model ratio), after excluding the non-modeled species (with 1–2 data points). Range filling differed significantly between amphibians and reptiles, with amphibians filling on average a smaller proportion of the estimated suitable range (0.472 ± 0.290 vs. 0.613 ± 0.310; t-test, P < 0.001). Both in amphibians and reptiles, range fill- ing was significantly correlated with SVL (parametric correlation; r = 0.216; P = 0.002 and r = 0.290; P < 0.001; non-parametric correlation; R = 0.255; P = 0.001 and R = 0.275; P < 0.001; Fig 7). Controlling for SVL in an ANCOVA, the difference between the taxonomic groups was maintained (i.e., both SVL and taxonomic category were significant predictors: P < 0.001 and P < 0.001). Community meta-analysis based on herpetofauna inventories For simplified comparison (all with t-tests), we summarized data for communities from the dry and subarid biomes (as arid) vs. the humid, subhumid, and montane (as moist) biomes. As expected, the number of reptile species per community is lower in the moist sites (11.7 ± 8.1 vs. 24.0 ± 11.4; P < 0.001), whereas, the number of amphibian species is higher (18.0 ± 9.8 vs. 4.9 ± 3.8; P < 0.001). Consequently, the ratio reptiles/amphibians also differs with high signifi- cance among moist and arid sites (0.39 ± 0.15 vs. 0.84 ± 0.11; P < 0.001). However, the total number of species in the herpetofaunal communities does not differ between moist and arid sites (29.8 ± 16.5 vs. 28.9 ± 13.6; P = 0.8041). Additional patterns apparent from these data are a higher proportion of reptiles in moist sites in northern Madagascar (as defined in Fig 1D). Comparing communities from moist sites in northern Madagascar vs. those from moist sites in the rest of the island, the number of amphibian species per community is on average lower in the northern regions (t-test: 15.5 ± 10.2 vs. 21.9 ± 7.9; P < 0.01), yielding also a weakly significant difference in the propor- tion among amphibians and reptiles (0.42 ± 0.16 vs. 0.35 ± 0.10; P = 0.051). For a more detailed analysis of community composition among the different biomes, we tabulated species numbers for 12 major taxonomic groups for each of the communities and performed a Principal Component Analysis (PCA) on these data (Fig 9; Table 1). Combination of the first and second principal components (PC) separates rather well the communities from moist vs. those from arid locations (i.e., humid + subhumid + montane vs. dry + subarid biomes). The first PC separates mainly communities of the humid, subhumid and montane bioclimates from those of the arid and subarid bioclimates, with a major contribution of rep- tiles (all except chameleons) and non-cophyline microhylids. The second PC separates dry from subarid climates and is mainly influenced by cophyline and mantellid frogs as well as cha- meleons. This probably reflects the almost complete absence of mantellid and cophyline frogs, and of many chameleons, from the subarid bioclimate. We used the ground-truthed encountered communities in a comparison with theoretical (model-based) communities obtained by an overlap of distribution ranges. Community meta-analysis based on herpetofauna inventories Most of the comprehensive analyses of Madagascar's biogeography in recent years have used, as original data, full distribution ranges of native species which were either derived from origi- nal records (e.g., as minimum convex polygons) or from SDMs. Such analyses estimate the number of species occurring in a certain region or site. The actual compositions of local amphibian and reptile communities are available from numerous surveys and inventories car- ried out in Madagascar over the past 25 years [38]. Such inventories yield lists of species co- occurring in one small area that can be used to calculate site similarities using parsimony analy- sis of endemism (PAE) [29, 66]. We compared community composition with data extracted from species lists of 103 sites as originally reported in the 20 selected surveys (see Materials and Methods; S2–S4 Tables). Fig 7. Range filling of reptiles and amphibians. Correlation of range filling (ratio of range sizes of clipped distribution model vs. full distribution model) with SVL, separately for amphibians (black solid circles) and reptiles (squares). Analyses carried out after removing all taxa with 1–2 data points only. doi:10.1371/journal.pone.0144076.g007 Fig 7. Range filling of reptiles and amphibians. Correlation of range filling (ratio of range sizes of clipped distribution model vs. full distribution model) with SVL, separately for amphibians (black solid circles) and reptiles (squares). Analyses carried out after removing all taxa with 1–2 data points only. Fig 7. Range filling of reptiles and amphibians. Correlation of range filling (ratio of range sizes of clipped distribution model vs. full distribution model) with SVL, separately for amphibians (black solid circles) and reptiles (squares). Analyses carried out after removing all taxa with 1–2 data points only. doi:10.1371/journal.pone.0144076.g007 Fig 7. Range filling of reptiles and amphibians. Correlation of range filling (ratio of range sizes of clipped distribution model vs. full distribution model) with SVL, separately for amphibians (black solid circles) and reptiles (squares). Analyses carried out after removing all taxa with 1–2 data points only. doi:10.1371/journal.pone.0144076.g007 doi:10.1371/journal.pone.0144076.g007 PLOS ONE \| DOI:10.1371/journal.pone.0144076 January 6, 2016 13 / 26 Spatial Biodiversity Patterns of Madagascar's Amphibians and Reptiles As expected from amphibian and reptile SR (Fig 1), geographically plotting species numbers and the proportions of amphibians vs. reptiles shows a lower proportion of amphibians in the dry, and especially in the subarid biomes, compared to the humid and subhumid biomes (Fig 8). Discussion Scope and limits of this study Community meta-analysis based on herpetofauna inventories For each of the 103 survey sites, we calculated the number of species that theoretically should occur at this site based on the clipped SDMs. Then, we calculated for various taxonomic groups a ratio of encountered species number vs. theoretical species numbers. To avoid a large number of miss- ing data and null divisions, we merged cophyline and non-cophyline microhylids and excluded hyperoliid frogs from the analysis. The obtained ratios differ among taxonomic groups (Krus- kal-Wallis-ANOVA; P = 0.018) and means range from 0.18 to 0.33. The lowest values corre- spond to lamprophiid snakes and the highest values to chelonians, iguanas, and blindsnakes. Amphibians yielded similar values as reptiles, with a lower average in microhylids than in mantellids. Spatial Biodiversity Patterns of Madagascar's Amphibians and Reptiles Fig 8. Species numbers and proportion of amphibians and reptiles recorded during herpetological inventories in Madagascar. Each pie chart represents one surveyed site (i.e., the area around one campsite at a specific elevation) and thus a community of co-occurring amphibian and reptile species. Pie area is proportional to the number of species; proportion of amphibians vs. reptiles in the community is indicated by colors. See S1 Table for a list of sites and references Fig 8. Species numbers and proportion of amphibians and reptiles recorded during herpetological inventories in Madagascar. Each pie chart represents one surveyed site (i.e., the area around one campsite at a specific elevation) and thus a community of co-occurring amphibian and reptile species. Pie area is proportional to the number of species; proportion of amphibians vs. reptiles in the community is indicated by colors. See S1 Table for a list of sites and references Fig 8. Species numbers and proportion of amphibians and reptiles recorded during herpetological inventories in Madagascar. Each pie chart represents one surveyed site (i.e., the area around one campsite at a specific elevation) and thus a community of co-occurring amphibian and reptile species. Pie area is proportional to the number of species; proportion of amphibians vs. reptiles in the community is indicated by colors. See S1 Table for a list of sites and references. doi:10.1371/journal.pone.0144076.g008 taxa [16, 22, 31, 35, 44, 45], or used the models in a hypothesis-testing framework without comparatively analyzing the patterns among groups in detail [25]. The current study is not to be seen as an exhaustive and final analysis of one or a few biogeographic questions, but rather as a baseline for future work which in part points to interesting phenomena that require in- depth study. We are aware of several restrictions in our dataset and results based on this. While our data- set was compiled and analyses being carried out, numerous novel distribution records became available which in some cases will lead to future modifications of some minor aspects of our results (e.g., Boophis CWE at Bemaraha). Most important, however, are the effects of taxo- nomic uncertainty, different evolutionary ages of species, and different species criteria applied in different groups of taxa. Therefore, the units of analysis (the species) used in this and most other biogeographical and macroecological analyses are not fully equivalent. Scope and limits of this study This study provides insights into the spatial biodiversity patterns of Malagasy amphibians and reptiles, based on explicitly modeled distributions of a near-complete set of species. We illus- trate how herpetofaunal communities are structured according to main bioclimatic regions, and demonstrate an important effect of body size on range size in these animals. Previous stud- ies have analysed spatial species richness of amphibians and reptiles in Madagascar based on unmodeled distribution areas reconstructed by expert opinion[36, 37], used only partial sets of 14 / 26 PLOS ONE \| DOI:10.1371/journal.pone.0144076 January 6, 2016 Spatial Biodiversity Patterns of Madagascar's Amphibians and Reptiles PLOS ONE \| DOI:10 1371/journal pone 0144076 January 6 2016 PLOS ONE \| DOI:10.1371/journal.pone.0144076 January 6, 2016 15 / 26 Fig 8. Species numbers and proportion of amphibians and reptiles recorded during herpetological inventories in Madagascar. Each pie chart represents one surveyed site (i.e., the area around one campsite at a specific elevation) and thus a community of co-occurring amphibian and reptile species. Pie area is proportional to the number of species; proportion of amphibians vs. reptiles in the community is indicated by colors. See S1 Table for a list of sites and references. Spatial Biodiversity Patterns of Madagascar's Amphibians and Reptiles Spatial Biodiversity Patterns of Madagascar's Amphibians and Reptiles Table 1. Results of a Principal Component Analysis of surveyed sites in Madagascar (Fig 8), based on species numbers recorded for each of 4 major amphibian and 8 major reptile groups. Principal Components with eigenvalues >1 were extracted (PC1-PC3). Component loadings with values >0.5 are in bold. PC1 PC2 PC3 Hyperoliid frogs -0.183640 0.127158 -0.957382 Microhylid frogs (non-cophylines) -0.584879 0.246071 -0.079674 Microhylid frogs (cophylines) 0.321995 -0.786310 0.045205 Mantellid frogs 0.320439 -0.748794 -0.227840 Tortoises -0.666783 0.259272 0.061954 Chameleons 0.002878 -0.794360 0.011449 Iguanas -0.738012 0.384409 0.075236 Gerrhosaurids -0.774476 -0.305005 -0.127244 Skinks -0.664216 -0.461836 0.136841 Geckos -0.761727 -0.308519 0.023604 Lamprophiid snakes -0.744714 -0.312073 -0.058414 Blindsnakes -0.822065 -0.132442 0.122052 Eigenvalue 4.423053 2.618131 1.040295 % Total variance 36.85877 21.81776 8.66912 Cumulative Eigenvalue 4.423053 7.041183 8.081478 Cumulative % 36.85877 58.67653 67.34565 doi:10.1371/journal.pone.0144076.t001 Table 1. Results of a Principal Component Analysis of surveyed sites in Madagascar (Fig 8), based on species numbers recorded for each of 4 major amphibian and 8 major reptile groups. Principal Components with eigenvalues >1 were extracted (PC1-PC3). Component loadings with values >0.5 are in bold. Table 1. Results of a Principal Component Analysis of surveyed sites in Madagascar (Fig 8), based on species numbers recorded for each of 4 major amphibian and 8 major reptile groups. Principal Components with eigenvalues >1 were extracted (PC1-PC3). Component loadings with values >0.5 are in bold. precisely dated, posing a second limitation to this study. Given continued climate change and the widespread decrease of natural vegetation over time, considering such temporal informa- tion would certainly improve the quality of the resulting models [35]. Furthermore, our method of estimating elevational species richness relies on the clipped species distribution models and therefore assesses the number of species predicted to have suit- able habitat at a certain elevation (vs. true measurements). We applied this method rather than directly extracting minimum and maximum elevations from point distributions due to issues associated with the uneven sampling of species across Madagascar that can lead to underesti- mating the altitudinal ranges of many understudied species (as the roads that provide access to habitats typically occur in regions of lower topographic complexity and lower elevation). Fur- ther, because SDMs model the species’ ecological tolerances, not geographic or altitudinal ranges, the modeled altitudinal ranges are not implicitly affected by altitudinal changes associ- ated with latitude (which would also be sensitive spatial sampling biases). Several species accepted as valid, such as the frogs Mantella viridis, M. milotympanum, M. nigricans, or the liz- ards Zonosaurus haraldmeieri and Z. trilineatus, might rather be considered as colour variants or subspecies, whereas other species contain deep mitochondrial lineages that might turn out to correspond to distinct species upon taxonomic revision. In order to include the full dataset of species and occurrence records, many of the observation records used have not been Fig 9. Results of a Principal Component Analysis of surveyed sites in Madagascar. Each dot represents the amphibian and reptile community at one site (see Fig 8 for a map of sites). Colors represent major bioclimatic subdivisions. PCA based on species numbers recorded for each of four major amphibian and eight major reptile groups (Table 1). doi:10.1371/journal.pone.0144076.g009 Fig 9. Results of a Principal Component Analysis of surveyed sites in Madagascar. Each dot represents the amphibian and reptile community at one site (see Fig 8 for a map of sites). Colors represent major bioclimatic subdivisions. PCA based on species numbers recorded for each of four major amphibian and eight major reptile groups (Table 1). Fig 9. Results of a Principal Component Analysis of surveyed sites in Madagascar. Each dot represents the amphibian and reptile community at one site (see Fig 8 for a map of sites). Colors represent major bioclimatic subdivisions. PCA based on species numbers recorded for each of four major amphibian and eight major reptile groups (Table 1). doi:10.1371/journal.pone.0144076.g009 PLOS ONE \| DOI:10.1371/journal.pone.0144076 January 6, 2016 16 / 26 PLOS ONE \| DOI:10.1371/journal.pone.0144076 January 6, 2016 Patterns of richness, endemism and turnover Amphibian SR of some clades peaks in the Central East of Madagascar (others also in the North East), while the South East in all amphibian clades is comparably species-poor. This might be due to a lower integrated survey effort in particular areas such as the Anosy Mountain chain, Kalambatritra or Befotaka-Midongy Reserve, but at least partly probably reflects a bio- logical pattern. All amphibian clades coincide in having their highest values of SR in rainforest, with differ- ences concerning the latitudinal location of the peaks. In reptiles, the pattern is more disparate among clades, as different clades have their SR peaking in the humid, subhumid, dry or subarid biomes. This reflects that some reptile clades did not or poorly adapt to rainforest [10], but diversified in other in other biomes only where they show high SR (Fig 3), together with the fact that more reptile radiations colonized Madagascar compared to amphibians. Regional endemicity in amphibians and reptiles varies among clades, but a common pattern of the majority of major clades and subclades is a high endemicity in northern Madagascar. The spatial distribution of SR in amphibians suggests a clear mid-domain effect as previ- ously postulated [16, 31]. Although this effect has been controversially discussed for the Mada- gascar example [84, 85], and a latitudinal mid-domain effect did not exert an important contribution to a multivariate model of Madagascar's amphibian richness [25], it is unequivo- cally observed that the central mid-altitudinal rainforests harbors the highest overall richness of amphibians. This is also reflected by the high numbers locally occurring in this area. Around 100 regionally sympatric amphibian species are known from comparatively small areas (less than 1000 km2, [25]) around Andasibe and Ranomafana, respectively [40]. The disparity of patterns among different amphibian clades are a strong indication for the absence of a major bias, e.g. in survey intensity, causing the overall central concentration of SR, which we therefore see as a true biological pattern characterizing some amphibian clades. In part, the high species richness in this area might be caused by local endemics as suggested by the high number of turnover boundaries inferred by GDM in the subhumid biome (Fig 2). Several areas of high species turnover identified by the GDM maps (Fig 2D and 2H) agree remarkably well with the bioclimatic zonation of Schatz [47] as represented in Fig 1B. However, it is clear that the realized elevational niches of most of Madagascar's amphibians and reptiles are nar- rower than suggested by their ecological tolerances (likely due to dispersal limitations and his- toric climate change). Due to this over-prediction, the species numbers for any elevation (Fig 5) are likely exaggerated. However, because such a bias will be equally likely for all species, we consider the general trends, and the observed differences between amphibians and reptiles, to be reliable. In some groups especially of reptiles, the effect of possibly inaccurate SDMs has been further exacerbated by taxon exclusions due to taxonomic uncertainty, leading to awkward aspects in the respective SR or CWE maps (Figs 3 and 4). For instance, the lack of CWE peaks of Uropla- tus in northern Madagascar is caused by the exclusion from our study of several of the recently identified, yet, still poorly defined candidate species of this genus [82], many of which are northern endemics. As a second example, the genus Paroedura has been subject to intense tax- onomic revisions in the past years (e.g., [83]), but much of the recent advances in knowledge on these geckos, especially in northern Madagascar, are not yet reflected in our dataset. PLOS ONE \| DOI:10.1371/journal.pone.0144076 January 6, 2016 17 / 26 Spatial Biodiversity Patterns of Madagascar's Amphibians and Reptiles One last restriction regards the comparison of different field surveys from the literature. Although most of these followed similar search methods, they differed in the number of days employed to search a particular site, and in the size of the field team. Regrettably, as a result of these factors, search effort is difficult to quantify. Variation in search effort among sites cer- tainly could have influenced the total species numbers, and to lesser degree, the proportion of different taxonomic groups that were used for our PCA. Despite these restrictions, we are convinced that the results presented here reflect true bio- logical patterns. In fact several of the major findings, such as the center of amphibian species richness in the Northern and Southern Central East, have remained stable since the pioneering study first reporting on the phenomenon [31]. This constancy in revealing the pattern is remarkable because the previous study[31] only included a fraction of the total number of amphibian species known today (97 vs. 325 amphibian species), and was not based on explicit distribution area modelling. PLOS ONE \| DOI:10.1371/journal.pone.0144076 January 6, 2016 Patterns of richness, endemism and turnover This applies in particular for the boundaries between the humid/subhumid vs. dry/subarid biomes, and even more of the dry vs. subarid biomes. This coincidence had already been remarked by Brown et al. [25] for their analysis of the full (amphibian+reptile) dataset. It should however be taken into account (see [25]) that bioclimatic data have influenced the GDM results at two ana- lytical steps: (i) in the calculation of SDMs, and (ii) in the interpolation of community PLOS ONE \| DOI:10.1371/journal.pone.0144076 January 6, 2016 18 / 26 Spatial Biodiversity Patterns of Madagascar's Amphibians and Reptiles distribution in the GDM analysis. Hence, the GDM boundaries are not fully independent from a zonation based on bioclimatic data alone. Once the distribution of Madagascar's amphibians and reptiles has been more completely mapped, it will be a fruitful perspective to analyze how closely the species turnover of these communities really matches the exact boundaries of the bioclimatic zones. The comparatively high numbers of species observed at high elevations >2000 m seems counterintuitive at first, and certainly is, in part, caused by SDM model over-prediction, con- sidering that only few montane specialists actually are found at such altitudes in the central massifs (Andringitra and Ankaratra; [66, 86]). However, at higher latitudes, and especially in the Tsaratanana Massif in northern Madagascar, rainforest extends into higher elevation and many more amphibians and reptiles can be found >2000 m a.s.l. Hence, when interpreting the graphics in Fig 5, it is important to keep in mind that these are calculated over the entire latitu- dinal and longitudinal range and considering all biomes of the island. This also provides a straightforward explanation for the higher SR of reptiles at lower elevations, and the absence of this pattern in amphibians. The arid and subarid biomes of Madagascar, with partly high rep- tile SR, but consistently low amphibian SR, are mainly made up by low elevations including almost the entire western coastline. Many reptiles occurring in these biomes contribute to the high reptile SR seen at low elevations (Fig 5). The distinct reductions of amphibian SR at the elevational steps between 2000–2100, and again from 2500–2600 m, correspond to the rainforest tree lines in the central massifs (at ca. 2000–2100 m) and in the Tsaratanana mountain (at ca. 2600 m), but also reflect simply the fact that very little surface area for occupancy is available above these altitudes. PLOS ONE \| DOI:10.1371/journal.pone.0144076 January 6, 2016 Body size influences on biogeographic pattern Low values indicate groups that are either difficult to detect during surveys, or are ecological specialists occurring patchily across their range. d i 10 1371/j l 0144076 010 Fig 10. Graph showing relative observation probability of main categories of amphibian and reptile species in survey sites across Madagascar. Bars show the percentage of species of each category found per site, relative to the respective number of species theoretically occurring at these sites based on overlap of clipped SDMs. Low values indicate groups that are either difficult to detect during surveys, or are ecological specialists occurring patchily across their range. doi:10 1371/journal pone 0144076 g010 Fig 10. Graph showing relative observation probability of main categories of amphibian and reptile species in survey sites across Madagascar. Bars show the percentage of species of each category found per site, relative to the respective number of species theoretically occurring at these sites based on overlap of clipped SDMs. Low values indicate groups that are either difficult to detect during surveys, or are ecological specialists occurring patchily across their range. Fig 10. Graph showing relative observation probability of main categories of amphibian and reptile species in survey sites across Madagascar. Bars show the percentage of species of each category found per site, relative to the respective number of species theoretically occurring at these sites based on overlap of clipped SDMs. Low values indicate groups that are either difficult to detect during surveys, or are ecological specialists occurring patchily across their range. d i 10 1371/j l 0144076 010 Fig 10. Graph showing relative observation probability of main categories of amphibian and reptile species in survey sites across Madagascar. Bars show the percentage of species of each category found per site, relative to the respective number of species theoretically occurring at these sites based on overlap of clipped SDMs. Low values indicate groups that are either difficult to detect during surveys, or are ecological specialists occurring patchily across their range. doi:10.1371/journal.pone.0144076.g010 Body size influences on biogeographic pattern The correlation between body size and range size is a well-established macroecological pattern [87, 88]. It has been previously found in Malagasy anurans [20], where small body sizes favor genetic diversification processes of anurans [20, 46]. Still, despite the availability of large num- bers of range maps, range-body size relationships remain understudied in amphibians and rep- tiles. Although intuitively obvious from the existence of many microendemic species with tiny body sizes, e.g., in Brookesia or Stumpffia [89, 90], we here provide the first comprehensive confirmation of this correlation in Malagasy amphibians and reptiles. Range sizes of snakes have previously been observed to be larger than those of lizards [91– 93] (Fig 10). We here confirm this pattern for the full assemblage of species occurring in Mada- gascar, and provide evidence that, apparently, it is not caused only by larger body sizes of snakes. Analyzing this phenomenon in more detail and testing its possible causes is a promis- ing perspective for future studies. Although our data seem to indicate that range size differ- ences between amphibians and reptiles might be caused mainly by the smaller body size of amphibians, more detailed future analysis of this pattern is warranted and should for instance seek for differences within biomes. However, the differences in range filling between the two groups are apparently not caused solely by body size differences. The lower proportion of suit- able distribution area occupied by amphibians probably reflects an overall lower vagility and dispersal capability, in turn probably caused by a higher sensitivity to microecological factors [94]. The Madagascar example, with a maximum of five clades of amphibians, but more than 15 clades of reptiles reaching the island after its geographic isolation [10], confirms that overall dispersal capacity is on average smaller in amphibians. Caution should be applied, however, when generalizing this difference because some amphibians, both in temperate regions (exam- ples in [95, 96]) and in Madagascar [97], are known to have expanded their distribution areas rapidly. 19 / 26 PLOS ONE \| DOI:10.1371/journal.pone.0144076 January 6, 2016 Spatial Biodiversity Patterns of Madagascar's Amphibians and Reptiles Fig 10. Graph showing relative observation probability of main categories of amphibian and reptile species in survey sites across Madagascar. Bars show the percentage of species of each category found per site, relative to the respective number of species theoretically occurring at these sites based on overlap of clipped SDMs. PLOS ONE \| DOI:10.1371/journal.pone.0144076 January 6, 2016 Spatial Biodiversity Patterns of Madagascar's Amphibians and Reptiles area if their required microhabitat is missing. Alternatively, species might be more difficult to observe in the wild at sites in moist vs. dry biomes. Differences between taxonomic categories in the proportion of theoretical community size vs. size of communities in the field (Fig 10) can be explained by two main factors. First, it is possible that some taxa are simply more difficult to detect than others, despite being present in similar densities at a site. Second, species belonging to some higher taxa simply might be on average rarer, occurring in lower densities or more specialized to particular microhabitats. The surveys included in our analyses were all carried out by experienced teams of researchers employing a variety of search techniques, including diurnal and nocturnal opportunistic searches and in almost all cases, pitfall trapping. Still, some taxa require a long and painstaking individualized search effort, such as small-sized leaf litter frogs, common in the Cophylinae, which often require hours searching for a single calling male specimen. The lower proportion of encountered microhylids (Fig 10) likely is the result of lower survey detectability, whereas the low number of snake observations either relates to low detectability or to a possibly lower density of these predators. Surprisingly, the detection probability of blindsnakes is compara- tively high, probably reflecting that these secretive animals are readily collected by pitfall trap- ping, or that the true distribution ranges of these animals are underestimated by our SDM approach. Conclusion and Outlook By revealing a series of biogeographic patterns in Madagascar's herpetofauna this study points to promising fields for future research. Using generalized dissimilarity modeling we found a remarkable coincidence of turnover patterns of amphibians and reptiles with bioclimatic regions. This pattern warrants further exploration using ground-truthed data of community composition across the boundary of bioclimatic zones. Northern Madagascar stands out as a center of SR and CWE for numerous amphibian and reptiles clades suggesting that surveys in many of the poorly explored northern massifs might yield novel discoveries of species unknown to science. Investigating contact and hybrid zones in northern Madagascar will yield insights into the role of in-situ speciation generating this astonishing regional diversity, possi- bly triggered by both vicariance and adaptive divergence across ecotones or elevational bands. A closer look at range size versus body size relationships will identify those taxa deviating from the general correlation, and point to intrinsic and extrinsic factors that might make these taxa particularly weak or strong in dispersal capacity. Eventually, further substantial refinement of these biogeographic studies will greatly benefit from continued survey and collection work in Madagascar, and from taxonomic revisions improving our knowledge of the baseline distribu- tional data. Supporting Information S1 Information. Figure copyright information. (DOC) S1 Information. Figure copyright information. (DOC) S1 Table. List of species, body size (maximum male snout-vent length), number of distribu- tion records, size of original and trimmed SDM, and range filling of amphibian and reptile species used for analysis. Community composition We found strong evidence that the herpetofaunal communities encountered in Madagascar on the basis of survey work have a taxonomic composition structured predominantly along a moisture gradient across the island. Communities from humid, subhumid and montane biomes differ along the main PC from those in the dry and subarid biomes. These two latter categories partly separated along the second PC axis. This finding further validates the use of bioclimatic data to interpolate theoretical community composition in the GDM analysis. Spe- cies richness of both amphibians and reptiles peaks in the humid biome, but contrary to the expectations, the actual herpetofaunal communities are not significantly more species rich in this biome or in the ecotone connecting humid, subhumid and montane biomes, when com- pared to the dry and subarid biomes. No straightforward explanation for this pattern exists. However, rainforest species might be more specialized to particular microhabitats: while occur- ring within a general rainforest area, they might not be present at particular sites within this 20 / 26 PLOS ONE \| DOI:10.1371/journal.pone.0144076 January 6, 2016 Acknowledgments A large number of students, friends and colleagues provided crucial help during fieldwork and stimulating input during various discussions, among which Franco Andreone, Alison Cam- eron, Lauren Chan, Sebastian Gehring, Steve M. Goodman, Jörn Köhler, David C. Lees, Brice P. Noonan, Maciej Pabijan, Ted Townsend, Krystal Tolley, Roger Daniel Randrianiaina, Fano- mezana Ratsoavina, Katharina C. Wollenberg, and Anne Yoder. Fieldwork of M.V. and PhD studies of P.B. were funded by the Volkswagen Foundation. Fieldwork was supported by Span- ish Government grants CGL2009-10198 and CGL2013-40924-P to DRV. J.L.B. was supported by the National Science Foundation (Grant No. 0905905). NS is supported with a research con- tract (IF/01526/2013) by FCT (Portugal). Figures are published under Creative Commons Attribution License (S1 Information). Author Contributions Conceived and designed the experiments: JLB NS FG PB DRV MV. Performed the experi- ments: JLB NS FG PB DRV MV. Analyzed the data: JLB NS PB DRV MV. Contributed reagents/materials/analysis tools: JLB NS FG DRV MV. Wrote the paper: JLB FG MV. S2 Table. Summary of survey data used in the meta-analysis and references for the original data. (DOC) 21 / 26 PLOS ONE \| DOI:10.1371/journal.pone.0144076 January 6, 2016 Spatial Biodiversity Patterns of Madagascar's Amphibians and Reptiles S3 Table. Metadata and geographical location of herpetofaunal communities used for anal- ysis. Data extracted from surveys listed in S1 Table. (DOC) S3 Table. Metadata and geographical location of herpetofaunal communities used for anal- ysis. Data extracted from surveys listed in S1 Table. (DOC) PLOS ONE \| DOI:10.1371/journal.pone.0144076 January 6, 2016 References Late Cretaceous vertebrates from Madagascar: implications for biotic change in deep time. Natural change and human impact in Madagascar. 1997:3–43. 15. Raxworthy CJ, Nussbaum RA. Systematics, speciation and biogeography of the dwarf chameleons (Brookesia; Reptilia, Squamata, Chamaeleontidae) of northern Madagascar. Journal of Zoology. 1995; 235:525–58. 16. Colwell RK, Lees DC. The mid-domain effect: geometric constraints on the geography of species rich- ness. Trends in Ecology & Evolution. 2000; 15(2):70–6. doi: 10.1016/s0169-5347(99)01767-x 17. Pastorini J, Thalmann U, Martin RD. A molecular approach to comparative phylogeography of extant Malagasy lemurs. Proceedings of the National Academy of Sciences of the United States of America. 2003; 100(10):5879–84. doi: 10.1073/pnas.1031673100 PMID: 12719521 18. Goodman SM, Ganzhorn JU. Biogeography of lemurs in the humid forests of Madagascar: the role of elevational distribution and rivers. Journal of Biogeography. 2004; 31(1):47–55. doi: 10.1111/j.1365- 2699.2004.00953.x 19. Yoder AD, Heckman KL. Mouse lemur phylogeography revises a model of ecogeographic constraint in Madagascar. Lehman SM, Fleagle JG, editors. 2006. 255–68 p. 20. Wollenberg KC, Vieites DR, Glaw F, Vences M. Speciation in little: the role of range and body size in the diversification of Malagasy mantellid frogs. BMC Evolutionary Biology. 2011; 11:217. doi: 10.1186/ 1471-2148-11-217 PMID: 21777445 21. Pearson RG, Raxworthy CJ. The evolution of local endemism in Madagascar: watershed versus cli- matic gradient hypotheses evaluated by null biogeographic models. Evolution. 2009; 63(4):959–67. doi: 10.1111/j.1558-5646.2008.00596.x PMID: 19210532 22. Townsend TM, Vieites DR, Glaw F, Vences M. Testing species-level diversification hypotheses in Mad- agascar: the case of microendemic Brookesia leaf chameleons. Systematic Biology. 2009; 58(6):641– 56. doi: 10.1093/sysbio/syp073 PMID: 20525615 23. Miraldo A, Hanski I. Competitive release leads to range expansion and rampant speciation in Malagasy dung beetles. Systematic Biology. 2014:syu011. 24. Miraldo A, Wirta H, Hanski I. Origin and diversification of dung beetles in Madagascar. Insects. 2011; 2 (2):112–27. doi: 10.3390/insects2020112 PMID: 26467617 25. Brown JL, Cameron A, Yoder AD, Vences M. A necessarily complex model to explain the biogeography of the amphibians and reptiles of Madagascar. Nature Communications. 2014; 5: e5046. 26. Martin RD. Review Lecture: Adaptive Radiation and Behaviour of the Malagasy Lemurs. 1972 1972-08- 24 00:00:00. 295–352 p. 27. Blanc CP, Paulian R. Originalité Biogeographique de la faune du sud Malgache. In: Lourenço WR, edi- tor. Biogéographie de Madagascar. Paris: ORSTOM; 1996. p. 231–44 28. Blommers-Schlösser R, Blanc CP. Amphibiens (deuxieme partie). Faune de Madagascar 1993; 75 (2):385–530. 29. Raxworthy CJ, Nussbaum RA. References 1. Goodman SM, Benstead JP. The Natural History of Madagascar: University of Chicago Press, Chi- cago; 2003. 2. Wilmé L, Goodman SM, Ganzhorn JU. Biogeographic evolution of Madagascar's microendemic biota. Science. 2006; 312(5776):1063–5. doi: 10.1126/science.1122806 PMID: 16709785 3. Angel F. Les Lézards de Madagascar: Academie Malgache; 1942. 4. Humbert H. Les territoires phytogéographiques de Madagascar. Année Biologique. 1955; 31:439–48. 5. Yoder AD, Olson LE, Hanley C, Heckman KL, Rasoloarison R, Russell AL, et al. A multidimensional approach for detecting species patterns in Malagasy vertebrates. Proceedings of the National Acad- emy of Sciences of the United States of America. 2005; 102:6587–94. doi: 10.1073/pnas.0502092102 PMID: 15851666 6. Vences M, Wollenberg KC, Vieites DR, Lees DC. Madagascar as a model region of species diversifica- tion. Trends in Ecology & Evolution. 2009; 24(8):456–65. doi: 10.1016/j.tree.2009.03.011 7. Battistini R, Richard-Vindard G. Les reptiles de Madagascar et des iles voisines. Biogeography and ecology in Madagascar: Springer; 1972. p. 501–614. 8. Paulian R. La zoogéographie de Madagascar et des îles voisines. Faune de Madagascar. 1961; 13:1– 442. 9. Yoder AD, Nowak MD. Has vicariance or dispersal been the predominant biogeographic force in Mada- gascar? Only time will tell. Annual Review of Ecology, Evolution, and Systematics. 2006; 37:405–31. 10. Crottini A, Madsen O, Poux C, Strauss A, Vieites DR, Vences M. Vertebrate time-tree elucidates the biogeographic pattern of a major biotic change around the K-T boundary in Madagascar. Proceedings of the National Academy of Sciences of the United States of America. 2012; 109(14):5358–63. doi: 10. 1073/pnas.1112487109 PMID: 22431616 11. Samonds KE, Godfrey LR, Ali JR, Goodmand SM, Vences M, Sutherland MR, et al. Spatial and tempo- ral arrival patterns of Madagascar's vertebrate fauna explained by distance, ocean currents, and ances- tor type. Proceedings of the National Academy of Sciences of the United States of America. 2012; 109 (14):5352–7. doi: 10.1073/pnas.1113993109 PMID: 22431643 22 / 26 PLOS ONE \| DOI:10.1371/journal.pone.0144076 January 6, 2016 Spatial Biodiversity Patterns of Madagascar's Amphibians and Reptiles 12. Samonds KE, Godfrey LR, Ali JR, Goodman SM, Vences M, Sutherland MR, et al. Imperfect isolation: factors and filters shaping Madagascar’s extant vertebrate fauna. PLOS ONE. 2013; 8(4):e62086. doi: 10.1371/journal.pone.0062086 PMID: 23626770 13. Krause DW, Rogers RR, Forster CA, Hartman JH, Buckley GA, Sampson SD. The Late Cretaceous vertebrate fauna of Madagascar: implications for Gondwanan paleobiogeography. GSA Today. 1999; 9 (8):1–7. 14. Krause D, Hartman J, Wells N. PLOS ONE \| DOI:10.1371/journal.pone.0144076 January 6, 2016 References Biogeographic patterns of reptiles in eastern Madagascar. In: Goodman SM, Patterson BD, editors. Natural change and human impact in Madagascar. Washington D. C.: Smithonian Institution press; 1997. p. 124–41. 30. Raxworthy CJ, Nussbaum A. Patterns of endemism for terrestrial vertebrates in eastern Madagascar. In: Lourenço W, editor. Biogéographie de Madagascar. Paris: Editions de l'ORSTOM; 1996. p. 369– 83. 31. Lees DC. The Périnet effect? Diversity gradients in an adaptive radiation of butterflies in Madagascar (Satyrinae: Mycalesina) compared with other rainforest taxa. In: Lourenço WR, editor. Biogéographie de Madagascar. Paris: Editions de l'ORSTOM; 1996. p. 479–90. 32. Raxworthy CJ, Ingram CM, Rabibisoa N, Pearson RG. Applications of ecological niche modeling for species delimitation: a review and empirical evaluation using day geckos (Phelsuma) from Madagas- car. Systematic Biology. 2007; 56(6):907–23. PMID: 18066927 33. Raxworthy CJ, Martinez-Meyer E, Horning N, Nussbaum RA, Schneider GE, Ortega-Huerta MA, et al. Predicting distributions of known and unknown reptile species in Madagascar. Nature. 2003; 426 (6968):837–41. PMID: 14685238 23 / 26 PLOS ONE \| DOI:10.1371/journal.pone.0144076 January 6, 2016 Spatial Biodiversity Patterns of Madagascar's Amphibians and Reptiles 34. Raxworthy CJ, Pearson RG, Rabibisoa N, Rakotondrazafy AM, Ramanamanjato J-B, Raselimanana AP, et al. Extinction vulnerability of tropical montane endemism from warming and upslope displace- ment: a preliminary appraisal for the highest massif in Madagascar. Global Change Biology. 2008; 14 (8):1703–20. 35. Kremen C, Cameron A, Moilanen A, Phillips SJ, Thomas CD, Beentje H, et al. Aligning conservation pri- orities across taxa in Madagascar with high-resolution planning tools. Science. 2008; 320(5873):222– 6. doi: 10.1126/science.1155193 PMID: 18403708 36. Andreone F, Cadle JE, Cox N, Glaw F, Nussbaum RA, Raxworthy CJ, et al. Species review of amphib- ian extinction risks in Madagascar: conclusions from the Global Amphibian Assessment. Conservation Biology. 2005; 19(6):1790–802. 37. Jenkins RK, Tognelli MF, Bowles P, Cox N, Brown JL, Chan L, et al. Extinction risks and the conserva- tion of Madagascar's reptiles. PLOS ONE. 2014; 9(8):e100173. doi: 10.1371/journal.pone.0100173 PMID: 25111137 38. D'Cruze N, Henson D, Olsson A, Emmett D. The importance of herpetological survey work in conserv- ing Malagasy biodiversity: are we doing enough? Herpetological Review. 2009; 40(1):19. 39. Köhler J, Vieites DR, Bonett RM, García FH, Glaw F, Steinke D, et al. New amphibians and global con- servation: a boost in species discoveries in a highly endangered vertebrate group. BioScience. 2005; 55(8):693–6. 40. Vieites DR, Wollenberg KC, Andreone F, Koehler J, Glaw F, Vences M. References Vast underestimation of Mada- gascar's biodiversity evidenced by an integrative amphibian inventory. Proceedings of the National Academy of Sciences of the United States of America. 2009; 106(20):8267–72. doi: 10.1073/pnas. 0810821106 PMID: 19416818 41. Nagy ZT, Sonet G, Glaw F, Vences M. First large-scale DNA barcoding assessment of reptiles in the biodiversity hotspot of Madagascar, based on newly designed COI primers. PLOS One. 2012; 7(3): e34506. doi: 10.1371/journal.pone.0034506 PMID: 22479636 42. Perl RB, Nagy ZT, Sonet G, Glaw F, Wollenberg KC, Vences M. DNA barcoding Madagascar’s amphib- ian fauna. Amphibia-Reptilia. 2014; 35(2):197–206. 43. Glaw F, Vences M. Field Guide to the Amphibians and Reptiles of Madagascar. Third ed. Köln: Vences and Glaw Verlag; 2007. 44. Wollenberg KC, Vieites DR, van der Meijden A, Glaw F, Cannatella DC, Vences M. Patterns of ende- mism and species richness in Malagasy cophyline frogs support a key role of mountainous areas for speciation. Evolution. 2008; 62(8):1890–907. doi: 10.1111/j.1558-5646.2008.00420.x PMID: 18485110 45. Kaffenberger N, Wollenberg KC, Köhler J, Glaw F, Vieites DR, Vences M. Molecular phylogeny and biogeography of Malagasy frogs of the genus Gephyromantis. Molecular Phylogenetics and Evolution. 2012; 62(1):555–60. doi: 10.1016/j.ympev.2011.09.023 PMID: 22019930 46. Pabijan M, Wollenberg KC, Vences M. Small body size increases the regional differentiation of popula- tions of tropical mantellid frogs (Anura: Mantellidae). Journal of Evolutionary Biology. 2012; 25 (11):2310–24. doi: 10.1111/j.1420-9101.2012.02613.x PMID: 22998688 47. Schatz GE. Endemism in the Malagasy tree flora. In: Lourenço WR, Goodman SM, editors. Diversity and Endemism in Madagascar: Société de Biogéographie, MNHN, ORSTOM; 2000. p. 1–9. 48. Boumans L, Vieites DR, Glaw F, Vences M. Geographical patterns of deep mitochondrial differentiation in widespread Malagasy reptiles. Molecular Phylogenetics and Evolution. 2007; 45(3):822–39. PMID: 17920299 49. Sillero N. What does ecological modelling model? A proposed classification of ecological niche models based on their underlying methods. Ecological Modelling. 2011; 222(8):1343–6. 50. Phillips SJ, Anderson RP, Schapire RE. Maximum entropy modeling of species geographic distribu- tions. Ecological Modelling. 2006; 190(3–4):231–59. doi: 10.1016/j.ecolmodel.2005.03.026 51. Phillips SJ, Dudik M, Elith J, Graham CH, Lehmann A, Leathwick J, et al. Sample selection bias and presence-only distribution models: implications for background and pseudo-absence data. Ecological Applications. 2009; 19(1):181–97. doi: 10.1890/07-2153.1 PMID: 19323182 52. Brown JL. SDMtoolbox: a python‐based GIS toolkit for landscape genetic, biogeographic and species distribution model analyses. Methods in Ecology and Evolution. 2014; 5:694–700. 53. Elith J, Phillips SJ, Hastie T, Dudik M, Chee YE, Yates CJ. PLOS ONE \| DOI:10.1371/journal.pone.0144076 January 6, 2016 References A statistical explanation of MaxEnt for ecolo- gists. Diversity and Distributions. 2011; 17(1):43–57. doi: 10.1111/j.1472-4642.2010.00725.x 54. Hijmans RJ, Cameron SE, Parra JL, Jones PG, Jarvis A. Very high resolution interpolated climate sur- faces for global land areas. International Journal of Climatology. 2005; 25(15):1965–78. doi: 10.1002/ joc.1276 55. Moat J, Du Puy D. Simplified Geology of Madagascar. In: Royal Botanic Gardens K, editor. 1997. PLOS ONE \| DOI:10.1371/journal.pone.0144076 January 6, 2016 24 / 26 Spatial Biodiversity Patterns of Madagascar's Amphibians and Reptiles 56. Jarvis A, Reuter HI, Nelson A, Guevara E. Hole-filled SRTM for the globe Version 4. CGIAR-CSI SRTM 90m Database2008. 57. Williams PH. Some properties of rarity scores for site-quality assessment. British Journal of Entomol- ogy and Natural History. 2000; 13:73–86. 58. Crisp MD, Laffan S, Linder HP, Monro A. Endemism in the Australian flora. Journal of Biogeography. 2001; 28(2):183–98. doi: 10.1046/j.1365-2699.2001.00524.x 59. Ferrier S, Manion G, Elith J, Richardson K. Using generalized dissimilarity modelling to analyse and predict patterns of beta diversity in regional biodiversity assessment. Diversity and Distributions. 2007; 13(3):252–64. doi: 10.1111/j.1472-4642.2007.00341.x 60. Allnutt TF, Ferrier S, Manion G, Powell GVN, Ricketts TH, Fisher BL, et al. A method for quantifying bio- diversity loss and its application to a 50-year record of deforestation across Madagascar. Conservation Letters. 2008; 1(4):173–81. doi: 10.1111/j.1755-263X.2008.00027.x 61. Corporation I. IBM SPSS Statistics for Windows. 19.0 ed. Armonk, NY: IBM Corporation; 2010. 62. Moen DS, Smith SA, Wiens JJ. Community assembly through evolutionary diversification and dispersal in Middle American treefrogs. Evolution. 2009; 63(12):3228–47. doi: 10.1111/j.1558-5646.2009.00810. x PMID: 19663988 63. Andreone F, Glaw F, Randrianirina J, Vences M. Remarkable records of amphibians and reptiles on Madagascar’s central high plateau. Tropical Zoology. 2007; 20(1):19–39. 64. Bora P, Randrianantoandro JC, Randrianavelona R, Hantalalaina EF, Andriantsimanarilafy RR, Rako- tondravony D, et al. Amphibians and reptiles of the Tsingy de Bemaraha Plateau, Western Madagas- car: Checklist, biogeography and conservation. Herpetological Conservation and Biology. 2010; 5 (1):111–25. 65. D’Cruze N, Sabel J, Green K, Dawson J, Gardner C, Robinson J, et al. The first comprehensive survey of amphibians and reptiles at Montagne des Français, Madagascar. Herpetological Conservation and Biology. 2007; 2(2):87–99. 66. Raxworthy CJ, Nussbaum RA. Montane amphibian and reptile communities in Madagascar. Conserva- tion Biology. 1996; 10(3):750–6. 67. Andreone F, Glaw F, Nussbaum R, Raxworthy C, Vences M, Randrianirina J. PLOS ONE \| DOI:10.1371/journal.pone.0144076 January 6, 2016 References Raxworthy C, Andreone F, Nussbaum R, Rabibisoa N, Randriamahazo H. Amphibians and reptiles of the Anjanaharibe-Sud Massif, Madagascar: Elevational distribution and regional endemicity. Fieldiana Zoology. 1998;(90: ):79. 79. Raxworthy C, Ramanamanjato J, Raselimanana A. Les reptiles et les amphibiens. In: Goodman S, Langrand O, editors. Inventaire Biologique Foret de Zombitse. Recherches pour le developpement Série Sciences biologiques No Spécial 1994. p. 41–57. 80. Raselimanana A. l’Herpétofaune de la forêt de Mikea. Inventaire floristique et faunistique de la forêt de Mikea: Paysage écologique et diversité biologique d’une préoccupation majeure pour la conservation. 2004:37–52. 81. Goodman SM, Ramanamanjato J-B, Raselimanana A, eds.).–Chapitre 7. Les Amphibiens et les Reptiles, pp. 110–130. In: Inventaire Biologique Foret de Vohibasia et d'Isoky-Vohimena. In: Langrand O, Goodman SM, editors. Recherches pour le developpement Série Sciences biologiques. 12; 1997. 197 p. 82. Ratsoavina FM, Raminosoa NR, Louis EE Jr, Raselimanana AP, Glaw F, Vences M. An overview of Madagascar's leaf tailed geckos (genus Uroplatus): species boundaries, candidate species and review of geographical distribution based on molecular data. Salamandra. 2013; 49(3):115–48. 83. Glaw F, Roesler H, Ineich I, Gehring P- S, Koehler J, Vences M. A new species of nocturnal gecko (Par- oedura) from karstic limestone in northern Madagascar. Zoosystematics and Evolution. 2014; 90 (2):249–59. 84. Kerr JT, Packer L. Habitat heterogeneity as a determinant of mammal species richness in high-energy regions. Nature. 1997; 385:252–4. 85. Lees DC, Colwell RK. A strong Madagascan rainforest MDE and no equatorward increase in species richness: re-analysis of 'The missing Madagascan mid-domain effect', by Kerr J.T., Perring M. & Currie D.J. (Ecology Letters 9:149–159, 2006). Ecol Lett. 2007; 10(9):E4–8; author reply E9-10. Epub 2007/ 08/01. doi: 10.1111/j.1461-0248.2007.01040.x PMID: 17663706 86. Vences M, Andreone F, Glaw F, Raminosoa N, Randrianirina JE, Vieites DR. Amphibians and reptiles of the Ankaratra Massif: reproductive diversity, biogeography and conservation of a montane fauna in Madagascar. Italian Journal of Zoology. 2002; 69(3):263–84. 87. Gaston KJ, Blackburn TM. Range size-body size relationships: evidence of scale dependence. Oikos. 1996:479–85. 88. Peters RH. The ecological implications of body size: Cambridge University Press; 1986. 89. Glaw F, Köhler J, Townsend TM, Vences M. Rivaling the world's smallest reptiles: discovery of minia- turized and microendemic new species of leaf chameleons (Brookesia) from northern Madagascar. PLOS ONE. 2012; 7(2):e31314. doi: 10.1371/journal.pone.0031314 PMID: 22348069 90. Klages J, Glaw F, Koehler J, Mueller J, Hipsley CA, Vences M. References The amphibians and rep- tiles of Nosy Be (NW Madagascar) and nearby islands: a case study of diversity and conservation of an insular fauna. Journal of Natural History. 2003; 37(17):2119–49. 68. Bora P, Otisitraka Randriambahiniarime M, Rabemananjara FC, Ravoahangimalala Ramilijaona O, Glaw F, Vences M. A rapid assessment survey of the herpetofauna at Befotaka‐Midongy National Park, south‐eastern Madagascar. Zoosystematics and Evolution. 2007; 83(2):170–8. 69. Nussbaum R, Raxworthy C, Raselimanana A, Ramanamanjato J-B. Amphibians and reptiles of the Reserve Naturelle Integrale d'Andohahela, Madagascar. Fieldiana Zoology. 1999;(94: ):155–74. 70. Rakotomalala D. Diversité des reptiles et amphibiens de la Réserve Spéciale de Manongarivo, Mada- gascar. Boissiera. 2002; 59:339–58. 71. Rakotomalala D, Raselimanana A. Les amphibiens et les reptiles des massifs de Marojejy, d’Anjana- haribe-Sud et du couloir forestier de Betaolana. In. SM Goodman et L. Wilmé (eds.) Nouveaux résultats d’inventaires biologiques faisant référence à l’altitude dans la région des massifs montagneux de Maro- jejy et d’Anjanaharibe-Sud. Recherches pour le développement, Série Sciences Biologiques. 2003;19:147–202. 72. Andreone F, Glaw F, Mattioli F, Jesu R, Schimmenti G, Randrianirina J, et al. The peculiar herpeto- fauna of some Tsaratanana rainforests and its affinities with Manongarivo and other massifs and forests of northern Madagascar. Italian Journal of Zoology. 2009; 76(1):92–110. 73. Ramanamanjato J, Rabibisoa N. Evaluation rapide de la diversité biologique des reptiles et amphibiens de la Réserve Naturelle Intégrale d’Ankarafantsika. A Biological Assessment of the Reserve Naturelle Integrale d'Ankarafantsika Alosno LE, Schulenberg T, Radilofe S and Missa O Washington DC, Con- servation International. 2002:98–104. 74. Raselimanana A. Inventaire biologique, Forêt d’Andranomay, Anjozorobe: La diversité de la faune de reptiles et d’amphibiens. Rech Dév Série Sci Biol. 1998; 13:43–59. 75. Raselimanana A. L’herpetofaune. Inventaire biologique de la réserve spéciale du Pic d’Ivohibe et du couloir forestier qui la relie au Parc National d’Andringitra (Goodman S and Rasolonandrasana BPN, eds) Recherches pour le Developpement, série Sciences Biologiques. 1999; 15:1–181. 76. Raselimanana A, Rakotomalala D, Rakotondraparany F. IX–Les reptiles et amphibiens: diversité et conservation. Inventaire biologique de la foret littorale de Tampolo (Fenoarivo Atsinanana)(Ratsirarson J & Goodman S eds) Recherches pour le developpement, série Sciences Biologiques. 1998; 14:1– 261. 25 / 26 PLOS ONE \| DOI:10.1371/journal.pone.0144076 January 6, 2016 Spatial Biodiversity Patterns of Madagascar's Amphibians and Reptiles 77. Raselimanana A, Raxworthy C, Nussbaum R. Herpetofaunal species diversity and elevational distribu- tion within the Parc National de Marojejy, Madagascar. Fieldiana Zoology. 2000:157–74. 78. PLOS ONE \| DOI:10.1371/journal.pone.0144076 January 6, 2016 References Molecular, morphological and osteologi- cal differentiation of a new species of microhylid frog of the genus Stumpffia from northwestern Mada- gascar. Zootaxa. 2013; 3717(2):280–300. 91. Anderson S, Marcus L. Aerography of Australian tetrapods. Australian Journal of Zoology. 1992; 40 (6):627–51. 92. Böhm M, Collen B, Baillie JE, Bowles P, Chanson J, Cox N, et al. The conservation status of the world’s reptiles. Biological Conservation. 2013; 157:372–85. 93. Anderson S. Aerography of North American fishes, amphibians, and reptiles. American Museum Novi- tates; no. 2802. 1984. 94. Andreone F, Randrianirina J, Lourenço W, Goodman S. Biodiversity, rainforests and herpetological communities in Madagascar: what about differences between amphibians and reptiles? Diversity and endemism in Madagascar; Mémoires de la Société de Biogéographie, Paris. 2000:217–28. 95. Zeisset I, Beebee T. Amphibian phylogeography: a model for understanding historical aspects of spe- cies distributions. Heredity. 2008; 101(2):109–19. doi: 10.1038/hdy.2008.30 PMID: 18493262 96. Vences M, Wake D. Speciation, species boundaries and phylogeography of amphibians. In: Heatwole HH, Tyler M, editors. Amphibian Biology, Systematics. 6. Chipping Norton, Australia: Surrey Beatty & Sons; 2007. p. 2613–71. 97. Rabemananjara FC, Chiari Y, Ramilijaona OR, Vences M. Evidence for recent gene flow between north-eastern and south-eastern Madagascan poison frogs from a phylogeography of the Mantella cowani group. Frontiers in Zoology. 2007; 4(1):1. 26 / 26 PLOS ONE \| DOI:10.1371/journal.pone.0144076 January 6, 2016
https://openalex.org/W2981748531	https://www.matec-conferences.org/10.1051/matecconf/201929601005/pdf	English	null	Factors Affecting Crash Frequencies: A Negative Binomial Regression Based Analysis of Indus Highway, Pakistan	MATEC web of conferences	2,019	cc-by	4,799	a Corresponding author: jingboyin@sjtu.edu.cn Factors Affecting Crash Frequencies: A Negative Binomial Regression Based Analysis of Indus Highway, Pakistan Rafi Ullah Khan1, Jingbo Yin1,a and Faluk Shair Mustafa1 1 School of Naval, Architecture, Ocean and Civil Engineering, Shanghai Jiao Tong University, Shanghai 200240, Chin Abstract. The increase in vehicular traffic have also increased the highway crash frequency with the passage of time. Improvements in highway safety is of vital importance as it could save vast life and monetary losses. The highway crash frequency analysis of major Pakistani highways is a subject less discovered and many important strategic and trade routes are not studied in this regard. This study is aimed to analyze the crash frequency and the prominent factors that cause these crashes on a 302 km section of Indus highway; one of the most important trade routes of the country. Eight years’ data from 2011 till 2018 was arranged into 19 variables where the crash frequency is set as dependent variable, while the eighteen prominent causation factors as independent variables. The tool used for analysis was negative binomial regression being run in the SPSS software. The results indicate that the driver’s behavior, understanding & risk recognition, negligence and law adherence have a significant effect on the crash frequency. Furthermore, highway crash frequency significantly increases with increase in highway segment lengths, number of lanes and lane widths. Similarly, the highway crash frequency significantly enhances when the light, pavement surface and climate condition gets deteriorated. The results of this study are of vital importance to government, transportation companies and general public in order to recognize the most important accident causing factors and devise the transport policies, rules and behaviors accordingly. © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). DP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 es/by/4 0/) , 0 0 (201 MATEC Web of Conferences 296 9) 10 ICTLE 2019 5 , 0 0 (201 MATEC Web of Conferences 296 9) 10 ICTLE 2019 5 https://doi.org/10.1051/matecconf/201929601005 1 Introduction The severity level of crashes are subject to the cumulative influences of various observed and unobserved factors [1]. These factors could either be engineering or non-engineering. The engineering factors includes geometric and structural design of the roadway, traffic operation & management and the pavement surface condition [2]. The non-engineering factors stands for the behavioral patterns observed among drivers, various other human factors, effects of environment, seasonal and diurnal variations. driver’s behavior [5]. The occurrence of a crash could be attributed to the driver being under the effect of alcohol or drugs, fatigue and carelessness. The lack of data in this regard could be attributed to the reason that it is not the responsibility of federal or state highway agencies to take into record the behavioral patterns of the drivers involved in each accident. The data on the speed, lanes, distractions, disparities, and risky behaviors of drivers could be well found in the police reports of each accident [6]. The other prominent factors that effects the intensity and frequency of highway crashes are the sudden changes in the geometries of these highways, hindrances on the road way and the prevailing climatic conditions at the site of crash [7]. The climatic or weather alterations includes the onset of rains, tempests, smog, fog, slick pavements and inadequate light situations. All these factors affect the driver’s ability to avoid any crash and collision on the road [8]. Moreover, the failures associated with brakes, axels, tires and other perfunctory culpabilities can result in crashes. An overview of the past literature shows that in analysis of the highway crashes, the most abundantly used variables are related to the engineering and design. While the effect of factors other than engineering, specifically that of driver behavior have been significantly ignored [3]. Even though the behavior oriented studies reflect that driver behavior is the most prominent contributory factor, it is still not properly incorporated into crash risk modelling [4]. The reason for this is the unavailability of a profound and reliable data collection method and source that could collect the data related to various trends in the , 0 0 (201 MATEC Web of Conferences 296 9) 10 ICTLE 2019 5 , 0 0 (201 MATEC Web of Conferences 296 9) 10 ICTLE 2019 5 https://doi.org/10.1051/matecconf/201929601005 surrounding keeps also affects the way driver perceives risk [19]. 1 Introduction The tremendous increase in the motorized vehicles, the presence of a mix of motorized and non-motorized traffic and the susceptible transport modes like motorcycles & rickshaws have increased the highway crashes manifold in Pakistan [9]. The country has to face around a hundred billion rupees of annual losses in terms of fatalities, injuries and communal defies resulting from highway crashes [10]. The highway crashes result in more than thirty thousand fatalities and around half a million injuries every year. Studies suggest among the contributory crash factors, 60% are attributed to the human factor, 30% to environmental factors, while 10% are attributed to the various mechanical faults associated with vehicles [11]. A better understanding of the contributory crash factors is pivotal in formulating better safety processes and policies. The effect of a driver’s behavior on a crash in the count related models could be replaced by substitute variables like that for over speeding [20]. Same technique could be adopted for driving under the influence of alcohol and drug [21]. Though alcohol and drugs are a type of societal crimes, however they are highly linked to the highway crashes and must be considered. In order to evaluate the highway crash frequencies and the various contributing factors, various research methods and tools have been used over the period of time. In the past these techniques were simple and based on linear regressions mostly, however with the passage of time the normality of error, residuals and the other issues associated with linear relationship between various dependent and independent variables were sorted out [22]. Therefore, to accommodate the multifaceted relationship between various variables, Poisson regression models were started using which were based on the consideration of exponential relationship between the involved variables. This study is one of the few attempts to relate the highway crash frequency to various contributory factors like roadway geometries, environmental aspects, time variation and various human behavioral patterns. This study will evaluate the connectivity between road crashes initiated by geometric and traffic factors like number, type and width of lanes, shoulders, medians, access points, U-turns, curves, speed limits and traffic percentages. While the other factors taken into account were the age, gender, behavior patterns of the drivers, specific time, weather, and season at the time of crash along the vehicle conditions. 2 Literature review Better understanding of the various contributory factors to crashes can result in better policies and measures for their reduction and management. The geometry of a road segment has a significant effect on the frequency and severity of crashes occurring in that segment [12]. The per unit length assessment of traffic crashes using Tobit regression model indicates that the pavement quality plays a key role in the crash occurrence [13]. 1 Introduction However, the dispersion of the data which required the equality of mean and variance for Poisson regression proved to be a limitation. This issue has been sorted out by the introduction of Negative binomial regression, which is considered independent of the over and under-dispersion limits. Though negative binomial regression model has some limitations associated with the multivariate variables, however it is considered very strong and efficient tool in evaluating the crash, fatality, and injuries related statistical data [2]. However, the use of this technique to evaluate the highway crashes data of Pakistan is very limited and various studies needs to be conducted evaluating various factors that affects the occurrence of crashes in land, air and maritime transportation. 3 Methodology & data collection 80 100 0.0 0.5 1.0 1.5 2.0 Crash Frequency Speed Limit (Km/h) Crash Frequency through urban areas, the number of lanes has been increased to four. The type of median varies from none to grassy, curbs and paved at places. It has a number of horizontal and vertical curves with various bridges and access points. Since the highway is passing through various rural and urban areas, the speed limits it has, are varying. Since it is a key source of goods transfer, the traffic through it has a huge portion of heavy vehicles. The data about the crashes in a specific area are collected by the regional police as per their jurisdiction, which is in the form of a properly detailed lodged FIR (First Information Report). Therefore, this study is based on the data extracted from the individual FIRs of each crash accidents in a specific segment of road from the police station of concerned jurisdiction. Figure 2. Graph between crash frequency and speed limits. These FIRs provide details on the types of vehicles involved, number plates, details on the conditions of drivers in the accident having information about the age, addiction and route or duration of driving. It also provides information on the level of experience the involved drivers had, their license details and the information about the possible causes after investigation which could have initiated these accidents. The pattern between the crash frequency and driver’s behavior, climate effect, pavement condition and vehicle issues have been depicted in the figure 3. The pattern between the crash frequency and driver’s behavior, climate effect, pavement condition and vehicle issues have been depicted in the figure 3. Figure 3. Graph between crash frequency and various factors. Climate D Behavior P Condition V Issues 0 10 20 30 40 A total of 8 years’ traffic data was taken from the year 2011 to 2018. Since crash number is a count variable, hence best analyzed through Poisson and Negative binomial regression models. Since the length of road segment with two lanes is higher than road length with four lanes, therefore the number of crashes occurring at two lanes is also higher than that occurring at four lane section as represented in figure 1. in figure 1. Figure 1. Graph between crash frequency and number of lanes. 2 4 0.0 0.5 1.0 1.5 2.0 Crash Frequency Number of Lanes Crash Frequency Figure 3. 3 Methodology & data collection The purpose of this study was to determine those geometric, human, weather and vehicle factors which played effective role in the occurrence of crashes at the selected segment of the road. The relationship between the crash occurrence frequency and the engineering and non-engineering factors have been analyzed using the negative binomial regression. For this study, a segment of the Indus highway was selected which is one of the most prominent and strategic highway of the country. This highway serves as a hinterland connectivity, as it connects the ports of the country to its major cities A study of the Florida highway states that among the human factors analysis, 94% of the fatal crashes are of the kind where drivers were under the effects of alcohol [14]. The analysis of highway accidents in Pakistan reveals that unskilled drivers, heavily overloaded vehicles, pavement surface conditions and the use of mobile phones while driving are among the significant contributory factors [15]. A study conducted through multiple linear regression indicates that among the road crashes in Jordan, pavement surface and lighting conditions have the least effect on crash occurrence [16]. However, it is a very lengthy highway with a total length of 1264 km, connecting Karachi port city to Peshawar, therefore a section of it was considered for this study which is between Peshawar and Dera Ismail Khan. The length of this section is 302 km and passes through various rural and urban areas. The selected section was divided into five segments between the prominent cities on the route. The number of lanes in this segment is mostly two, however when it passes The behavior of a driver can be elaborated by his professional & social values along proclivity towards safety and risk [17]. The way a person looks at a risk could be affected by his society and surroundings. It could be better understood by observing that some of the drivers observe and follow other drivers in the surrounding and community instead of following the highway features and rules [18]. The expectation which a community or specific group of people in the 2 , 0 0 (201 MATEC Web of Conferences 296 9) 10 ICTLE 2019 5 https://doi.org/10.1051/matecconf/201929601005 Figure 2. Graph between crash frequency and speed limits. 80 100 0.0 0.5 1.0 1.5 2.0 Crash Frequency Speed Limit (Km/h) Crash Frequency Figure 2. Graph between crash frequency and speed limits. 4 Results and discussions In order to analyze the selected variables using NBR, first the mean, standard deviation, minimum and maximum values of the dependent and independent variables have been depicted in the table 1. Table 1. Various statistical parameters of the selected Table 1. Various statistical parameters of the selected variables No Variables explanation Mean Std dev Min Max 1 Dependent Variable: Crash frequency 1.49 2.03 0.0 12 2 Proportion of Unit trucks on a segment 10.19 2.79 5 14 3 Width of lanes in meters 3.59 0.0782 3.65 3.9 4 Segment wise lanes (Numbers) 2.30 0.391 2 4 5 Length of each segment 1.11 0.789 0.15 6 6 Access points in a segment 2.09 2.87 0.0 14 7 Urban and rural specification (1 and 0 respectively) 0.44 0.55 0.0 1.0 8 Allowed speed at each segment (km/hr.) 87.89 14.87 80 100 9 U-turns provided in a segment 0.93 1.05 0.0 5.0 10 Climate Impact 0.82 0.99 0.0 1 11 Priority Negligence 2.24 1.03 3 9 12 Indecorous reversing 1.34 0.82 5 12 13 Yielding negligence 4.31 1.37 7 14 14 Vehicle control failure while driving 10.81 2.43 0.2 5 15 Inappropriate turns 8.95 1.63 0.0 6 16 Wrong way or direction 1.23 0.73 0.3 1 17 Inappropriate Maneuvering 3.78 0.058 3.0 6.0 18 Light conditions 2.96 0.89 3 4.5 19 Pavement surface 4.51 0.95 0.5 3 The results obtained after running the NBR analysis have been shown in table 2. Reference to this Table 2. 3 Methodology & data collection Graph between crash frequency and various factors. Climate D Behavior P Condition V Issues However, the utility of Poisson regression model is limited by the ratio of mean to variance of the data which has to be equal to one. While this restriction is relaxed for the negative binomial regression. The data was found to be over-dispersed, the model suitable for this study is negative binomial regression (NBR). NBR actually is an addition of the Poisson regression, which have been provided with the measure for gamma distribution error [23]. Moreover, the selection of NBR was based on the performance of both dispersion and Vuong statistic parameters. The mathematical expression to represent the negative binomial regression is given as; Figure 1. Graph between crash frequency and number of lanes. Number of Lanes g g g 𝑃𝑟𝑜𝑏[ 𝑌= 𝑦𝑖∣∣𝛆] = exp [−𝜆𝑖exp (𝛆)](𝜆𝑖)𝑦𝑖 𝑦𝑖 ! , 𝑦𝑖= 0,1,2, … (𝟏) 𝜆𝑖= exp(𝑋𝑖𝜷+ 𝛆𝒊) for the ith observation; (2) g g g 𝑃𝑟𝑜𝑏[ 𝑌= 𝑦𝑖∣∣𝛆] = exp [−𝜆𝑖exp (𝛆)](𝜆𝑖)𝑦𝑖 𝑦𝑖 ! , 𝑦𝑖= 0,1,2, … (𝟏) 𝜆𝑖= exp(𝑋𝑖𝜷+ 𝛆𝒊) for the ith observation; (2) The relationship between the crash frequency and speed limits at specific segments as per the statistical data under study shows that crashes at lower speed sections are more frequent than on high speed sections as depicted in figure 2. Where, λi is the projected mean, β is a vector of venerable strictures, xi is the factors selected as independent variables, and exp(εi) is a gamma error term with mean 1.0 and variance α2. The adding of this constraint permits the variance to vary from the mean: Var[𝑦𝑖] = E[𝑦𝑖]{1+ 𝛂E[𝑦𝑖]} = E[𝑦𝑖] + 𝛂E[𝑦𝑖]2 (3) 3 , 0 0 (201 MATEC Web of Conferences 296 9) 10 ICTLE 2019 5 https://doi.org/10.1051/matecconf/201929601005 The variable α, stands for the dispersion of the data, where if it gets equal to 0, the equation would become the Poisson regression model. table it could be seen that the number of single unit trucks are found to be significant with a t-value of 3.35 in causing highway crashes. This could be attributed to the reason that lighter trucks and vehicles have higher speeds as compared to the heavy trucks resulting in overtaking and increased crash risk. Similarly, the effect of a segment length on the crash frequency turns out to be significant with a t-stat value of 2.7. 3 Methodology & data collection The results indicate that segments with longer lengths had contributed more to the occurrence of accidents as compared to shorter segment lengths. This could be attributed to the reason that a longer length of a road segment has potentially more exposure to the different traffic types and hindrances. These sections generally have higher number of U-turns, turns & horizontal curves and entrance roads. Due to the prevalence of similar road features, drivers have the inclination to do over speeding which also increase the crash risk. The results obtained after running the NBR analysis have been shown in table 2. Reference to this 5 Conclusion The width of lanes also turns out to have significant effect on the occurrence of highway accidents with a t- value of 3.03. The results reveal that wider lanes have higher tendency of resulting in an accident as compared to the narrow or constricted lanes. Though wider lanes mean more space, decision making time and better visibility, but still the results indicate otherwise. The potential reason is that drivers on lanes wider than normal tends to increase their speed which enhances the crash risk. Moreover, in a wider lane the drivers would see space along vehicles ahead in the same lane and will tend to cross or accompany these vehicles resulting in increased endangerment. The drivers on narrow lanes remains more careful with their speed and overtaking behaviors resulting in reduced crash risk. The number of traffic has increased manifold with the passage of time and so has the highway crashes. Highway crashes result in the loss of precious lives and property, hence enhancing the traffic safety is of remarkable prominence. The statistical data of the highway crashes and the factors due to which it occurs is best analyzed using Poisson or Negative Binomial Regression for reliable results. Highway crashes have been a major concern in Pakistan for the authorities as it results in a significant annual life and monetary loss. Indus highway is one of the major trade routes of the country and its crash data have not been analyzed up till now as per the information of authors. The highway crash frequency data along its causation factors of this highway for a 302 km section from Peshawar to Dera Ismail Khan was taken and analyzed using NBR. The data was arranged as per the eighteen most important factors that affects the crash frequency and analyzed using SPSS software. The recognition, acceptance and understanding of the priorities in traffic while driving at a highway plays a very critical role in crash causation. The negligence in lane changing, passing, turning and yielding priority can result in misunderstanding and subsequently result in higher probability of crash occurrence. The t-values for priority and yield negligence were found to be 2.54 and 1.78 consequently with both of these variables being significant. Furthermore, making inappropriate turns or turns with wrong indicator also significantly increased the crash frequency with a t-value of 1.63. 4 Results and discussions Results obtained from NBR analysis Variables explanation Estimated Coefficient t-Stat p Value Constant -16.03 -3.02 0.0004 Proportion of Unit trucks on a segment 0.19 3.35 0.0007 Climate Impact 0.54 1.27 0.0013 Segment lane numbers 0.89 2.90 0.0041 Indecorous reversing 0.62 1.59 0.5803 Access points in a segment 0.08 2.270 0.0244 Vehicle control loss while driving 0.28 1.55 0.0008 Inappropriate Maneuvering 0.60 2.44 0.0035 U-turns provided in a segment 0.08 1.04 0.3016 Width of lanes in meters 3.09 3.03 0.0021 Priority Negligence 0.78 2.54 0.0020 Length of each segment 0.29 2.70 0.0059 Yielding negligence 0.82 1.78 0.0033 Urban and rural specification (1 and 0 respectively) 0.69 1.58 0.2034 Inappropriate turns 0.72 1.63 0.0062 Wrong way or opposite direction 0.40 1.25 0.0006 Allowed speed at each segment (km/hr.) 0.02 1.21 0.2309 Light conditions 0.67 1.16 0.0051 Pavement surface 0.10 1.35 0.0064 Dispersion parameter (a) 0.51 4.709 0.0012 The climate effect on the occurrence of highway crashes is found significant with a t-value of 1.27, this is because in winters there is huge fog and smog The climate effect on the occurrence of highway crashes is found significant with a t-value of 1.27, this is because in winters there is huge fog and smog The results obtained after running the NBR analysis have been shown in table 2. Reference to this 4 , 0 0 (201 MATEC Web of Conferences 296 9) 10 ICTLE 2019 5 https://doi.org/10.1051/matecconf/201929601005 conditions can result in the loss of control over a vehicle and bursting, deterioration of tires and other parts consequently resulting in high vehicular crashes. The speed limit, number of U-turns, number of access points and indecorous reversing though are very critical for traffic safety and crash frequency, but were found insignificant in the results of this study. observed on this section of road. It results in very poor visibility and consequently traffic jamming. Similarly, the loss of control on vehicle and inappropriate maneuvering with t-values of 1.55 and 2.44 respectively were also found significant. The loss of control on a vehicle yields in a very high crash risk. The careless and inappropriate maneuvering puzzles the other drivers creating unsafe crossing & braking distances and subsequently resulting in crashes. conditions can result in the loss of control over a vehicle and bursting, deterioration of tires and other parts consequently resulting in high vehicular crashes. 4 Results and discussions The speed limit, number of U-turns, number of access points and indecorous reversing though are very critical for traffic safety and crash frequency, but were found insignificant in the results of this study. 5 Conclusion Similarly, driving in the opposite direction is very dangerous and results in very high probability of head- on and sidewise collisions having a 1.25 t-value. This puts the other drivers in jeopardy of making wrong maneuvers and turns to avoid the crash and hence result in collision with other vehicles. The results indicate that the crash frequency is highly subject to the length of specific road segments, number of lanes and the width of the lanes and an increase is observed in crash frequency with increase in these parameters. Similarly, the light, pavement and climate conditions also have a significant effect on highway crashes and the number of crashes increases as the condition of above parameters gets deteriorated. Similarly, the driver behavior, attitude and law abidance also plays a key role in the occurrence of highway crashes. Negligence, lower understanding & recognition, violation of rules and carelessness significantly increases the crash frequency. Moreover, the number of lanes are also found to be significant in their effect on the crash frequency. It is attributed to the reasons that increased number of lanes would consequently result in increased traffic volumes, lane changing and higher speeds. All these reasons ultimately create circumstances in which higher crashes takes place. Similarly, urban areas are found significant in increasing crash frequencies with a t- value of 1.58. This is due to the increased volume of traffic, higher number of pedestrians, cycles, motorcycles and various carts. Another reasons which causes accidents are the encroachments in the urban areas which results in traffic jams and consequently impatience in drivers, resulting in close overtakes and higher crash frequency. References 1. M. R. R. Shaon, X. Qin, Z. Chen, and J. Zhang, “Exploration of contributing factors related to driver errors on highway segments,” Transportation Research Record, vol. 2672, no. 38, pp. 22–34, 2018. 2. M. R. R. Shaon, X. Qin, A. P. Afghari, S. Washington, and M. M. Haque, “Incorporating behavioral variables into crash count prediction by severity: A multivariate multiple risk source approach,” Accident Analysis & Prevention, vol. 129, pp. 277–288, 2019. , , pp , 3. A. P. Afghari, M. M. Haque, and S. Washington, “Applying fractional split model to examine the effects of roadway geometric and traffic characteristics on speeding behavior,” Traffic injury prevention, vol. 19, no. 8, pp. 860–866, 2018. The light conditions prevailing at the road and the surface conditions of the road are also found significant. The poor light conditions results in poor visibility and wrong judgment of the size, speed and position of the other vehicles. While the poor surface 4. S. Washington and M. Haque, “On the commonly accepted assumptions regarding observed motor vehicle crash counts at transport system locations,” 2013. 4. S. Washington and M. Haque, “On the commonly accepted assumptions regarding observed motor vehicle crash counts at transport system locations,” 2013. 5 , 0 0 (201 MATEC Web of Conferences 296 9) 10 ICTLE 2019 5 https://doi.org/10.1051/matecconf/201929601005 5. M. Shaon and R. Rahman, “Quantifying Rural Highway Safety Performance: Application of the Highway Safety Manual and Development of Mixed Distribution Statistical Models for Predicting Crash Frequency,” 2015. 15. A. Ahmed, B. A. Khan, M. B. Khurshid, M. B. Khan, and A. Waheed, “Estimating national road crash fatalities using aggregate data,” International journal of injury control and safety promotion, vol. 23, no. 3, pp. 249–254, 2016. 6. S. Box, “New Data from VTTI provides insight into cell phone use and driving distraction,” Virginia Tech Transportation Institute, vol. 27, 2009. 16. K. Jadaan, I. Al-Hyari, H. Naghawi, R. Ammourah, and Z. Al Nabulsi, “Traffic safety in Jordan: magnitude, cost and potential countermeasures,” J. of Traffic and Logistics Engineering, vol. 1, no. 1, pp. 54– 7, 2013. 7. M. R. Rahman Shaon and X. Qin, “Use of mixed distribution generalized linear models to quantify safety effects of rural roadway features,” Transportation Research Record, vol. 2583, no. 1, pp. 134–141, 2016. 17. P. M. Carter, C. R. Bingham, J. S. Zakrajsek, J. T. Shope, and T. B. References Sayer, “Social norms and risk perception: Predictors of distracted driving behavior among novice adolescent drivers,” Journal of Adolescent Health, vol. 54, no. 5, pp. S32–S41, 2014. 8. A. Montella and L. L. Imbriani, “Safety performance functions incorporating design consistency variables,” Accident Analysis & Prevention, vol. 74, pp. 133–144, 2015. 18. R. J. Schneider, A. Sanatizadeh, M. R. R. Shaon, Z. He, and X. Qin, “Exploratory analysis of driver yielding at low-speed, uncontrolled crosswalks in Milwaukee, Wisconsin,” Transportation research record, vol. 2672, no. 35, pp. 21–32, 2018. 9. I. Khan, A. Khan, F. Aziz, M. Islam, and S. Shafqat, “Factors associated with helmet use among motorcycle users in Karachi, Pakistan,” Academic emergency medicine, vol. 15, no. 4, pp. 384–387, 2008. 19. J. Moeckli and J. D. Lee, “The making of driving cultures,” Improving Traffic Safety Culture in the United States, vol. 38, no. 2, pp. 185–192, 2007. 10. I. Ahmed, T. Islam, G. Ali, and M. M. Nawaz, “Pillion riders’ cloth related injuries and helmet wearing patterns: a study of Lahore, Pakistan,” International journal of injury control and safety promotion, vol. 23, no. 4, pp. 388–394, 2016. 20. A. P. Afghari, S. Washington, M. M. Haque, and Z. Li, “A comprehensive joint econometric model of motor vehicle crashes arising from multiple sources of risk,” Analytic methods in accident research, vol. 18, pp. 1– 14, 2018. 11. D. Shinar, Traffic safety and human behavior. Emerald Publishing Limited, 2017. 12. A. Montella, L. Colantuoni, and R. Lamberti, “Crash prediction models for rural motorways,” Transportation Research Record, vol. 2083, no. 1, pp. 180–189, 2008. 21. M. Asbridge, J. A. Hayden, and J. L. Cartwright, “Acute cannabis consumption and motor vehicle collision risk: systematic review of observational studies and meta-analysis,” Bmj, vol. 344, p. e536, 2012. 13. P. C. Anastasopoulos, F. L. Mannering, V. N. Shankar, and J. E. Haddock, “A study of factors affecting highway accident rates using the random-parameters tobit model,” Accident Analysis & Prevention, vol. 45, pp. 628–633, 2012. 22. D. Lord and F. Mannering, “The statistical analysis of crash-frequency data: a review and assessment of methodological alternatives,” Transportation research part A: policy and practice, vol. 44, no. 5, pp. 291–305, 2010. 14. L. K. Spainhour, D. Brill, J. O. Sobanjo, J. Wekezer, and P. V. Mtenga, “Evaluation of traffic crash fatality causes and effects: A study of fatal traffic crashes in Florida from 1998-2000 focusing on heavy truck crashes,” 2005. 23. References W. H. Greene and C. Zhang, “Econometric analysis (Vol. 5),” Upper Saddle River, NJ: PrenticeHall, 2003. 6
https://openalex.org/W4388870326	https://www.revista.ccba.uady.mx/ojs/index.php/TSA/article/download/4749/2134	Spanish; Castilian	null	EFECTO DEL ABONADO CON BIOSÓLIDO EN EL COMPORTAMIENTO FISIOLÓGICO E INCIDENCIA DE PLAGAS EN CULTIVO DE CHILE X´CATIK (Capsicum annuum L.)	Tropical and subtropical agroecosystems	2,023	cc-by	7,091	SUMMARY Background. The use of biosolids in agriculture allows to supply essential nutrients for the plant development. Objective. To evaluate the effect of supplying swine biosolids on the physio-agronomic characteristics and incidence of pests in X'catik pepper. Methodology. The experiment was carried out under greenhouse conditions and set in a randomized block experimental design with four replicates. Three different levels of biosolids were evaluated (500, 750 and 1000 g plant-1) and the control (no supply of biosolid). Results. Plants treated with 750 g de biosólido had the highest net carbon assimilation rate (AN) and the lowest intracellular carbon (Ci), likewise, there was a trend of higher values for the yield components in this treatment. The population density of B. tabaci and the damage by Poliphagotarsonemus latus was similar among treatments. Implications. The use of swine biosolid in agriculture represent a feasible alternative to enhance the plant physiological condition and potentially the yield in horticultural crops. Conclusion. The supply of 750 g plant-1 of swine biosolid improved the physiological parameters in the X'catik pepper plants, had no effect on pest damage, but showed a strong tendency to increase yield. Keywords: Organic fertilizer; Bemisia tabaci; Polyphagotarsonemus latus; Chili production. Short Note [Nota corta] EFECTO DEL ABONADO CON BIOSÓLIDO EN EL COMPORTAMIENTO FISIOLÓGICO E INCIDENCIA DE PLAGAS EN CULTIVO DE CHILE X´CATIK (Capsicum annuum L.) † [EFFECT OF FERTILIZING WITH BIOSOLID ON THE PHYSIOLOGICAL BEHAVIOR AND INCIDENCE OF PESTS IN CROPS OF CHILE X'CATIK (Capsicum annuum L.)] Short Note [Nota corta] Fátima del R. Yam-Herrera1, Esaú Ruiz-Sánchez1, Sergio López-Vázquez1, Juan Díaz-Mayo1, J. Ismael Tucuch-Haas2, Luis Latournerie-Moreno1, and Angel M. Herrera-Gorocica1 1Tecnológico Nacional de México, Campus Conkal. Avenida Tecnológico s/n. C.P. 97345, Conkal, Yucatán, México. Email: esau.ruizi@tconkal.edu.mx 2Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias (INIFAP), Campo Experimental Mocochá. km 25 antigua carretera Mérida-Motul. CP. 97454. Mocochá, Yucatán, México. Corresponding author † Submitted January 22, 2023 – Accepted September 14, 2023. http://doi.org/10.56369/tsaes.4749 Copyright © the authors. Work licensed under a CC-BY 4.0 License. https://creativecommons.org/licenses/by/4.0/ ISSN: 1870-0462. ORCID = Esau Ruiz Sánchez: http://orcid.org/0000-0003-0245-3305 Yam-Herrera et al., 2024 Yam-Herrera et al., 2024 Tropical and Subtropical Agroecosystems 27 (2024): Art. No. 009 INTRODUCCIÓN vegetal que pudieran ser tóxicos, y/o elevar la producción de metabolitos secundarios defensivos que limitan la actividad de fitófagos (Rowen et al., 2019). Por ejemplo, varios trabajos demuestran que el estiércol ovino disminuye las poblaciones de insectos plagas en varios cultivos, como coles (Eigenbrode y Pimentel, 1988), maíz (Alle y Davis, 1996; Morales et al., 2001) y papas (Alyokhin et al., 2005); el estiércol de aves de corral también disminuye las poblaciones de insectos plaga en cultivos de brócoli (Banfield- Zanin et al., 2012), col (Stafford et al., 2012), arroz (Kajimura et al., 1995) y papa (Boiteau et al., 2008). El uso de fertilizantes químicos a gran escala ha contribuido a la perdida de la calidad del suelo, eutrofización, contaminación de agua subterránea y en general un efecto ambiental negativo (Pahalvi et al., 2021). El uso de biosólidos constituye una estrategia importante de aprovechamiento de subproductos de explotaciones ganaderas, que podría ser fuente de nutrientes a las plantas donde se usen esquemas de fertilización incompletos (Poornima et al., 2022) y de protección adicional contra insectos plaga (Alyokhin et al., 2014). Los biosólidos pecuarios contienen un valor nutrimental que potencialmente pueden mejorar la fertilidad del suelo y la producción de los cultivos (Chow y Pan, 2020). Dependiendo de su origen, los biosólidos son ricos en materia orgánica y en muchos casos de nutrientes esenciales, como de nitrógeno (N), fósforo (P) y potasio (K) (Pan et al., 2021). Lo anterior permite que estos productos sean una fuente alternativa de nutrientes para lograr disminución del uso de fertilizantes comerciales, tomando relevancia en muchas regiones agrícolas actualmente (Singh y Agrawal, 2008; Brito y de la Vega, 2015). El cultivo de chile en México tiene una gran importancia cultural, gastronómica y económica (López-Castilla et al., 2019). Existe una enorme diversidad de tipos de chile (Capsicum annuum L.) que se valoran de manera regional en zonas específicas en México (Jaiswal et al., 2021). En la península de Yucatán, C. annuum se considera una de las hortalizas de mayor importancia económica (López-Castilla et al., 2019). Uno de los tipos de chile regional, es el conocido como chile X´catik, el cual es moderadamente picante, lo que le confiere un sabor agradable, características que le confiere gran aceptación y demanda en el mercado, además también tienen el potencial para ser materia prima en la elaboración de productos industriales (Peñuela et al., 2021). INTRODUCCIÓN Este tipo de chile es cultivado en campo y en pequeñas superficies en invernaderos de mediana tecnología, donde la nutrición mineral generalmente deficiente debido al desconocimiento de los pequeños productores de los esquemas de nutrición por etapas y también al alto valor económico de los fertilizantes en el mercado (Gou et al., 2020), la formula nutrimental más empleada es de 180, 120 y 100 kg ha-1 (Gamboa- Angulo et al., 2020). El cultivo de chile X´catik también presenta otras limitantes, como son la presencia de plagas del follaje que pueden afectar la producción de frutos. En ese sentido, la mosca blanca (Bemisia tabaci Genn.) es una de las principales plagas hortícolas en invernadero y campo (Pantoja et al., 2018). Los daños directos causados por su alimentación incluyen el cierre de estomas, la formación de manchas cloróticas en las hojas (Horowitz et al., 2020). Así mismo, el daño más importante lo causa de manera indirecta, con la transmisión de begomovirus (Misal & Patil, 2022). Por su parte, también el ácaro blanco (Polyphagotarsonemus latus Banks) es una plaga fundamental en el cultivo. Se presenta en altas densidades alimentándose de las partes en crecimiento de la planta, brotes, yemas terminales y botones florales. Los daños incluyen rizado en las nervaduras de las hojas apicales, deformación de hojas desarrolladas provocando deformaciones, enanismo y una coloración verde intensa de la planta, aborto de flores (Duarte et al., 2021). En estudios previos sobre el uso de biosólidos como fertilizantes en cultivos hortícolas, se han obtenido resultados prometedores. Por ejemplo, Singh y Agrawal (2008) demostraron que el aporte de diferentes concentraciones de biosólido de origen porcino contribuye al incremento en rendimiento de varios cultivos (tomate, cebada, maíz, algodón). Además, Utria-Borges et al. (2008) señalan que la adición de biosólido de origen bovino a plántulas de tomate (S. lycopersicum) produce incremento significativo de las variables relacionadas con crecimiento y producción, como altura de la planta, diámetro de fruto, número de flores, número de frutos. Por su parte, Brito y de la Vega (2015) reportan que al aplicar los biosólidos de diversos orígenes pecuarios en plantas de S. lycopersicum se genera aumento en el diámetro de tallo, altura de la planta, número de flores, número de racimos y número de frutos; lo que provoca que el rendimiento de frutos también aumente. No obstante, Ruíz et al. (2021) no obtuvieron efectos significativos en el rendimiento del cultivo de S. RESUMEN Antecedentes. El aprovechamiento agrícola de los biosólidos permite suministrar a las plantas nutrientes para su desarrollo. Objetivo. Evaluar el efecto del abonado con diferentes niveles de biosólido porcino en el comportamiento fisiológico e incidencia de plagas en chile X´catik. Metodología. El experimento se realizó en invernadero, a través de un diseño experimental de bloques al azar con cuatro repeticiones. Se evaluaron tres diferentes niveles de biosólido (500, 750 y 1000 g planta-1) y el control (sin biosólido). Para la fertilización del cultivo se usó la fórmula 180-120- 100. Resultados. Las plantas tratadas con 750 g de biosólido tuvieron mayor tasa de asimilación neta de carbono (AN) y menor nivel de carbono intracelular (Ci), así también se observó tendencia hacia el incremento en las variables de rendimiento. La densidad poblacional de Bemisia tabaci, así como el daño por Poliphagotarsonemus latus fue similar entre los tratamientos. Implicación. El uso de biosólido porcino en la agricultura representa una opción viable para mejorar las condiciones fisiológicas y potencialmente incrementar rendimiento en hortalizas. Conclusión. La aplicación de 750 g planta-1 de biosólido porcino mejoró los parámetros fisiológicos en las plantas de chile X´catik, no tuvo efecto en el daño por plagas, pero mostró una fuerte tendencia al incremento del rendimiento. Palabras clave: Abono orgánico; Bemisia tabaco; Polyphagotarsonemus latus; Producción de chile. as clave: Abono orgánico; Bemisia tabaco; Polyphagotarsonemus latus; Producción de chile. 1 Tropical and Subtropical Agroecosystems 27 (2024): Art. No. 009 Yam-Herrera et al., 2024 Tropical and Subtropical Agroecosystems 27 (2024): Art. No. 009 INTRODUCCIÓN lycopersicum usando biosólido de caballos y carneros, Por esta razón, se requiere de evaluaciones específicas tomando en consideración el tipo de biosólido, el cultivo en cuestión y las condiciones de suelo y ambientales. Por su parte, Brito y de la Vega (2015) reportan que al aplicar los biosólidos de diversos orígenes pecuarios en plantas de S. lycopersicum se genera aumento en el diámetro de tallo, altura de la planta, número de flores, número de racimos y número de frutos; lo que provoca que el rendimiento de frutos también aumente. No obstante, Ruíz et al. (2021) no obtuvieron efectos significativos en el rendimiento del cultivo de S. lycopersicum usando biosólido de caballos y carneros, Por esta razón, se requiere de evaluaciones específicas tomando en consideración el tipo de biosólido, el cultivo en cuestión y las condiciones de suelo y ambientales. Aunque no existen reportes de los efectos de los biosólidos porcinos sobre las poblaciones de plagas, varios estudios reportan que abonar las plantas con estiércol puede limitar el crecimiento de las poblaciones de plagas, al disminuir la disponibilidad de algunos nutrientes esenciales para los fitófagos, elevar el contenido de algunos elementos en el tejido 2 Yam-Herrera et al., 2024 Tropical and Subtropical Agroecosystems 27 (2024): Art. No. 009 A la fecha no existe evidencia del uso de biosólido porcino en el cultivo de chile (Capsicum annuum L.), a pesar del potencial que pueden tener estos materiales como fuente de abono. Por los registros previos, podría especularse que el empleo de biosólidos en el cultivo de chile X´catick pudiera presentar influencia en los parámetros fisiológicos y la productividad de las plantas, así como también alterar la incidencia o severidad de las plagas y en consecuencia los daños producidos por éstas. Por lo anterior, el objetivo de este trabajo fue evaluar el efecto del abonado con diferentes niveles de biosólido porcino en el comportamiento fisio-agronómico e incidencia de plagas en chile X´catick (Capsicum annuum L.) bajo condiciones de invernadero. nuevas completamente extendidas (Garruña- Hernández et al., 2014). Las mediciones se realizaron con un medidor de gases en infrarrojo (LI6400 xt, LI- COR, Ne, E.U). Tabla 1. Análisis físico-químico del biosólido porcino. Tabla 1. Análisis físico-químico del biosólido porcino. Determinación Resultados Características físicas pH 7.20 Cond. Evaluación de densidad poblacional de Bemisia tabaci Para la densidad poblacional de Bemisia tabaci se seleccionaron ocho plantas por parcela, y la toma de datos se realizó cada 15 días por tres meses, la primera evaluación se realizó a los 103 días después del trasplante, ya que las moscas no se presentaron en las primeras etapas de crecimiento de las plantas. A cada planta se le hizo el conteo de adultos en el lado abaxial de tres hojas del estrato medio y tres del estrato superior de las plantas, contando a simple vista el número de mosquitas adultas posadas en cada hoja. Al final del muestreo se calculó la media global de todas las evaluaciones de las mosquitas adultas (Herrera- Gorocica et al., 2022) INTRODUCCIÓN Eléctrica 4.70 dSm Materia orgánica 64.6 % Macronutrientes Nitrógeno (N) 3.05 % Fósforo (P) 2.32 % Potasio (K) 0.26 % Micronutrientes Calcio (Ca) 8.87 % Magnesio (Mg) 0.96 % Sodio (Na) 0.27 % Azufre (S) 1.21 % Hierro (Fe) 6665 ppm Cobre (Cu) 524 ppm Manganeso (Mn) 511 ppm Zinc (Zn) 3606 ppm Boro (B) 26.4 ppm Metales pesados Níquel (Ni) 13.032 ppm Cobalto (Co) NA Arsénico (As) NA Bario (Ba) NA Cromo (Cr) NA Cadmio (Cd) NA Aluminio (Al) NA NA: No analizado Ubicación y preparación del Área Experimental El experimento se realizó en un invernadero rústico (temperatura 25-37°C, humedad relativa 70-90 % y fotoperiodo 14 h luz:10 h oscuridad), en el área de investigación hortícola del Instituto Tecnológico de Conkal, Yucatán, ubicado a 15 km al noreste de Mérida a 21° 4’ N y 89° 31’ O a una altitud de 10 m. Plantas de Capsicum annuum (chile X´catick criollo) de 30 días de edad se establecieron a una distancia de 0.3 m en líneas de 1.5 m de separación, que contenían camas de 50 cm de ancho, las cuales fueron abonadas 15 días antes del trasplante, con biosólido porcino proporcionado por la empresa Operadora GPM S.A. de C.V y obtenido de los lodos recuperados del tratamiento de aguas residuales con biodigestores. Se aplicaron tres niveles diferentes de biosólido por planta y se adicionó un testigo sin adición de biosólido (0 g, 500 g, 750 g, y 1000 g). Además, se proporcionó fertilización con N:P:K en proporción 180:120:100 (kg/ha-1) para el ciclo de 180 días de chile X´catick después del trasplante para todos los tratamientos. La fertilización se hizo por medio del riego, utilizando un sistema de fertirriego por goteo (cintilla) de 5/4 calibre 6000 con un gasto de 1.5 litros por hora (LPH) y con separación de 0.3 m. Las características del suelo antes de la aplicación de los fertilizantes fueron las siguientes: pH de 7.61 y 17.76% de materia orgánica; su contenido de N:P:K, fue de 68.4, 228 y 810 ppm; Na, Ca y Mg 1560, 6000 y 900 ppm. La caracterización físico-química del biosólido se realizó en el Laboratorio Fertilab (Celaya, Guanajuato, México), la cual se describe en la tabla 1. NA: No analizado Evaluación de densidad poblacional de Bemisia tabaci Variables fisiológicas Se evaluó el rendimiento total (Kg/planta), el número de frutos por planta, el peso de un fruto (g) y el diámetro polar y ecuatorial de los frutos (cm). Para ello, se seleccionaron y etiquetaron tres plantas de cada parcela, de tal manera que se pudieran evaluar las mismas plantas durante los siete meses que duró el experimento. Para las variables relacionadas con el rendimiento de fruto, se realizaron cinco cortes de frutos en función de madurez comercial. En cada corte se contabilizó el número de frutos por planta y se obtuvo el peso total de frutos por planta (rendimiento total), además se tomaron muestras de 10 frutos para obtener el promedio del peso de un fruto, el diámetro polar y el diámetro ecuatorial. Se encontró que la tasa de asimilación neta (AN) fue estadísticamente mayor en las plantas tratadas con 750 g de biosólido (23.3 mmol m-2 s-1) (gl=4, F=12.12, P=0.0001) con respecto a las plantas del resto de los tratamientos y del control (Figura 1 A). Así mismo, el tratamiento de 750 g de incorporación de biosólido presentó estadísticamente menores niveles de carbono intercelular (Ci) (gl=4, F=9.08, P=0.0001) (Figura 1B). Diseño experimental y análisis de datos incidencia se contó el número total de plantas con síntomas de toda la parcela. El porcentaje de plantas con síntomas se obtuvo con la siguiente fórmula: Incidencia = (Número de plantas con síntomas) x100/Total de plantas observadas. La severidad del daño se determinó en ocho plantas elegidas al azar en la parcela, mediante la utilización de una escala categórica de cuatro niveles, por Jiménez-Martínez et al., (2013). Nivel 0, no hay síntomas; nivel 1, débil encrespado hacia arriba en la lámina foliar de hojas nuevas y brotes nuevos; nivel 2, ondulación en hojas nuevas y viejas; nivel 3, encrespado hacia arriba y deformación en la nervadura central en forma de Zigzag; nivel 4, hojas severamente dañadas, caída de las mismas y aborto de frutos, enanismo en las plantas. Al final del muestreo se calculó la media global de todas las evaluaciones. Se utilizó un diseño experimental, bloques completos al azar, con cuatro repeticiones. Cada parcela contenía 14 plantas. Los datos de las variables fisiológicas (AN y Ci), variables de densidad poblacional de B. tabaci, variables de incidencia y severidad y rendimiento de fruto fueron analizados por medio de ANOVA y Tukey (p<0.05). La normalidad y homogeneidad de las varianzas se comprobó con las pruebas de Shapiro- Wilk y Levene respectivamente, antes del ANOVA. La variable de severidad final se analizó mediante la prueba no paramétrica de Kruskal-Wallis. Todos los análisis se realizaron con el programa estadístico InfoStat versión 2020 (Di Rienzo et al., 2020). Evaluación de las variables fisiológicas La evaluación de la tasa de asimilación neta (µmol m- 2s-1) y el carbono intercelular (µmol m-1) se realizó a los 60 días después del trasplante, entre las 8:00 h a las 10:00 h. Se eligieron tres plantas por parcela, a las cuales se les hicieron cinco mediciones en hojas Para la evaluación de daños causados por P. latus, las mediciones se hicieron en el follaje, flores y frutos a intervalos de 15 días, haciendo un total de cinco muestreos durante el ciclo del cultivo. Para la 3 Yam-Herrera et al., 2024 Tropical and Subtropical Agroecosystems 27 (2024): Art. No. 009 DISCUSIÓN Con respecto a la incidencia y severidad de P. latus, se observó alta incidencia, pero baja severidad. La incidencia final tuvo tendencia a ser menor en el tratamiento de 750 g de biosólido (93.75 ± 3.6%) comparado con el control y los otros tratamientos (100 ± 0%), aunque no se observó diferencia significativa (gl= 3,12; F=3.00, P=0.07). Sobre la severidad del daño, no se observó diferencia significativa (gl= 3,12; F=1.04, P=0.41), los grados de daño estuvieron entre 1.22 y 1.53, que se describe como débil encrespado hacia arriba en la lámina foliar de hojas nuevas y brotes nuevos. En este estudio se evaluó el efecto de la adición de tres niveles de biosólido porcino en las variables fisio- agronómicas y en el daño por plagas en chile X´catik. Se encontró que la adición de 750 g planta-1, incrementó la tasa de asimilación neta (AN) y disminuyó los valores de carbono intercelular (Ci). este estud o se eva uó e e ecto de a ad c ó de t es niveles de biosólido porcino en las variables fisio- agronómicas y en el daño por plagas en chile X´catik. Se encontró que la adición de 750 g planta-1, incrementó la tasa de asimilación neta (AN) y disminuyó los valores de carbono intercelular (Ci). Estos efectos pueden deberse a que la aplicación de los biosólidos, hasta cierto nivel, provocan aumento en la fertilidad del suelo, y en consecuencia el incremento de la absorción de nutrientes por la planta y mejora de los parámetros de intercambio de gases (Potisek- Talavera et al., 2010). El nivel más alto de biosólido evaluado en este estudio (1000 g planta-1) no produjo efecto en los parámetros fisiológicos, lo cual probablemente se debió a un incremento excesivo de elementos minerales y sales en el suelo, lo que pudo derivar en la disminución de la absorción de nutrientes del suelo, que ya no permitió el aumento en AN ni la disminución en Ci. El efecto positivo de la adición de biosólido en varios cultivos se ha documentado con anterioridad. Por ejemplo, Mohamed et al. (2018) aplicaron biosólidos (aguas residuales) en plantas de girasol (Helianthus annuus L.), donde observaron aumento en la AN. Pero también se ha reportado que aplicar abono orgánico de origen porcino en exceso, puede afectar negativamente la fisiología y crecimiento de las plantas (Chang et al., 2021; Chang et al., 2017). DISCUSIÓN Adicionar en exceso (1 kg planta-1 o más) de biosólido de origen porcino como abono vegetal puede causar efectos físico-químicos negativos al suelo, como aireación reducida, aumento en las concentraciones de sal y metales pesados (Atiyeh et al., 2000). Por ejemplo, se ha documentado que los biosólidos contienen elementos que en exceso son de alto riesgo para los cultivos, como son el Na, Fe, Cu, Evaluación de densidad poblacional de Bemisia tabaci y daño por Poliphagotarsonemus latus. Sobre la densidad poblacional de adultos de B. tabaci no se encontró diferencia significativa entre tratamientos (gl= 3,36; F=0.73, P=0.52). A B Niveles de Biosólido (g) Control 500 750 1000 AN (µmol m-2 s-1) 0 5 10 15 20 25 c c a b Niveles de Biosólido (g) Control 500 750 1000 Ci (µmol m- 1) 0 50 100 150 200 250 300 350 a a b a Figura 1. Tasa de asimilación neta (A) y carbono intercelular (B) en cultivo de Chile X´catik abonado con diferentes niveles de biosólido. Medias (± EE) con diferente letra son estadísticamente diferentes (P<0.05; Tukey). B Niveles de Biosólido (g) Control 500 750 1000 Ci (µmol m- 1) 0 50 100 150 200 250 300 350 a a b a B A A Niveles de Biosólido (g) Control 500 750 1000 AN (µmol m-2 s-1) 0 5 10 15 20 25 c c a b Figura 1. Tasa de asimilación neta (A) y carbono intercelular (B) en cultivo de Chile X´catik abonado con diferentes niveles de biosólido. Medias (± EE) con diferente letra son estadísticamente diferentes (P<0.05; Tukey). 4 Yam-Herrera et al., 2024 Tropical and Subtropical Agroecosystems 27 (2024): Art. No. 009 Parámetros de rendimiento del chile X´catik En el análisis de los parámetros del rendimiento se observó que no hubo diferencias significativas entre las plantas tratadas y las del control (Tabla 3). El rendimiento total fue similar en todos los tratamientos (gl= 3,8; F=3.08; P=0.5), con valores de 1.09 ± 0.1 a 1.43 ± 0.1 kg. El número de frutos por planta (40.3 ± 3.3 a 48.7 ± 2.6 frutos) fue similar en todos los tratamientos (gl= 3,8; F=1.21; P=0.32). El peso de un fruto (gl= 3,46; F=0.31; P=0.82) y diámetro polar del fruto (gl= 3,46; F=0.76; P=0.53) también fue similar entre los tratamientos, con valores de 27 ± 3.2 a 31.1 ± 3.1 g fruto-1 y 14.8 ± 0.2 a 15.2 ± 0.2 cm, respectivamente. Para el diámetro ecuatorial del fruto, se encontró diferencia significativa entre los tratamientos (gl= 3,46; F=3.37; P=0.02), los frutos de las plantas tratadas con 750 g de biosólido (3.9 ± 0.5 cm) tuvieron mayor diámetro que aquellos de las plantas tratadas con 500 g de biosólido planta-1. Se observó en general una tendencia que el tratamiento 750 g de biosólido tuvo las plantas con mayor peso de fruto, número de frutos, así como diámetro polar y ecuatorial de los frutos (Cuadro 2). Tabla 2. Densidad poblacional de adultos de B. tabaci por hoja e incidencia y severidad final de los síntomas asociados a la presencia de P. latus en plantas de chile X´catick abonadas con diferentes niveles de biosólido. Niveles de Biosólido Densidad de B. tabaci por hoja Incidencia final de P. latus Severidad final de P. latus Control 2.44 ± 0.25 a 100 ± 0.0 a 1.44 ± 0.16 a 500 g 2.8 ± 0.19 a 100 ± 0.0 a 1.5 ± 0.15 a 750 g 2.82 ± 0.21 a 93.8 ± 3.6 a 1.22 ± 0.16 a 1000 g 2.72 ± 0.14 a 100 ± 0.0 a 1.53 ± 0.03 a Las medias (± EE) no fueron estadísticamente diferentes (P<0.05; Tukey). Tabla 2. Densidad poblacional de adultos de B. tabaci por hoja e incidencia y severidad fina asociados a la presencia de P. latus en plantas de chile X´catick abonadas con diferentes nivele poblacional de adultos de B. tabaci por hoja e incidencia y severidad final de los síntomas ncia de P. latus en plantas de chile X´catick abonadas con diferentes niveles de biosólido. Parámetros de rendimiento del chile X´catik d D id d d B b i h j I id i fi l d P l S id d fi l d P l Tabla 3. Efecto del Biosólido en el rendimiento total, número de frutos por planta, peso de un fruto y longitud y diámetro en plantas de Chile X´catik. Niveles de Biosólido (g) Rendimiento total (kg/planta) Frutos por planta Peso de un fruto (g) Diámetro polar (cm) Diámetro ecuatorial (cm) Control 1.21 ± 0.10 a 45 ± 3.9 a 28.6 ± 31 a 14.8 ± 0.2 a 3 ± 0.3 ab 500 1.09 ± 0.10 a 40.3 ± 3.3 a 27 ± 3.2 a 14.9 ± 0.2 a 2.7 ± 0 a 750 1.43 ± 0.01 a 46.3 ± 3.4 a 31.1 ± 3.1 a 15.2 ± 0.2 a 3.9 ± 0.5 b 1000 1.42 ± 0.09 a 48.7 ± 2.6 a 29.4 ± 2.9 a 14.9 ± 0.3 a 2.9 ± 0 ab Medias (± EE) con diferente letra en una columna son estadísticamente diferentes (P<0.05; Tukey). del Biosólido en el rendimiento total, número de frutos por planta, peso de un fruto y longitud antas de Chile X´catik. Tabla 3. Efecto del Biosólido en el rendimiento total, número de frutos por planta, peso y diámetro en plantas de Chile X´catik. 5 Yam-Herrera et al., 2024 Yam-Herrera et al., 2024 Tropical and Subtropical Agroecosystems 27 (2024): Art. No. 009 absorción de elementos esenciales (Ramírez-Builes, 2007; López et al., 2008). Zn, Mn y Al, lo cual puede inducir alteración en el funcionamiento de algunas enzimas relacionadas con los procesos fisiológicos (Martínez-Martínez et al., 2018; Rusli et al., 2022). Así mismo, se ha visto que la aplicación de biosólido puede afectar negativamente la densidad aparente y la estabilidad de los agregados de suelo, lo que conduce a una alteración en la absorción de los elementos minerales por las raíces de las plantas cultivadas (Jin et al., 2015). Zn, Mn y Al, lo cual puede inducir alteración en el funcionamiento de algunas enzimas relacionadas con los procesos fisiológicos (Martínez-Martínez et al., 2018; Rusli et al., 2022). Así mismo, se ha visto que la aplicación de biosólido puede afectar negativamente la densidad aparente y la estabilidad de los agregados de suelo, lo que conduce a una alteración en la absorción de los elementos minerales por las raíces de las plantas cultivadas (Jin et al., 2015). Parámetros de rendimiento del chile X´catik Por lo tanto, la aplicación de biosólido porcino en niveles de 750 g planta-1 en chile X’catik, pudo haber favorecido la absorción de nutrientes, lo que se tradujo en aumento de la tasa de asimilación neta y disminución del carbono intercelular. Así mismo, los niveles de biosólido porcino no mostraron efectos sobre las plagas estudiadas, pero si una marcada tendencia al incremento en el rendimiento de fruto. En este estudio no se registró efecto significativo de la adición de biosólido en la densidad poblacional de B. tabaci y daño por P. latus. Existen diversos estudios con resultados contrastantes en este sentido. Por ejemplo, hay estudios que documentan el aumento en la susceptibilidad a las plagas cuando se aplican abonos orgánicos, esto debido al aumento de algunos elementos disponibles en el suelo, como nitrógeno total, que permite a las plantas mayor absorción y posterior acumulación en las hojas, mismas que se vuelven más atractivas para las plagas, al aumentar los niveles de aminoácidos libres (Balakrishnan et al., 2007; Saranraj y Stella 2012; Isman et al., 2017). Se ha documentado que B. tabaci prefiere alimentarse de las plantas de mejor calidad, es decir, con mayor contenido de nitrógeno y aminoácidos (Jiao et al., 2018). Sin embargo, existen una serie de datos que indican lo contrario, donde los abonos orgánicos reducen los daños por plagas, debido a que estos abonos favorecen la inducción de resistencia a través de mecanismos bioquímicos y morfológicos de las plantas (Chatterjee et al., 2013) o hace que las plantas sean menos atractivas para las plagas (Brown, 2010; Andrade et al., 2017). Por ejemplo, al aplicar el estiércol en plantas de calabaza aumenta el ácido fenólico en las hojas, lo que hace que se reduzca la incidencia de las plagas (Boeckler et al., 2011; Krishnaveni et al., 2019). Un trabajo similar en plantas de girasol, menciona que al aplicar estiércol se reduce la incidencia de plagas chupadoras por el aumento en el nivel de metabolitos secundarios (Ravi et al. 2006) CONCLUSIÓN La aplicación de 750 g de biosólido porcino en el cultivo de chile X´catik incrementó significativamente los valores en la tasa de asimilación neta (AN) y la disminución de los valores del carbono intercelular (Ci). La aplicación de diferentes niveles de biosólido porcino no tuvo efectos en la población de adultos de B. tabaci, ni en daño por P. latus. Sin embargo, hubo una tendencia de incremento en la producción del cultivo, con cual se evidencia el efecto benéfico que genera el uso del biosólido en la agricultura. Se sugiere realizar estudios sobre la evaluación de biosólido porcino en combinación o en aplicación alternada con algún abono orgánico o inoculante microbiano promotor de crecimiento vegetal para que la comunidad microbiana pueda aprovechar las bondades del biosólido. También es importante estudiar la concentración de elementos minerales y posibles metales pesados en el perfil de suelo durante la aplicación de biosólido a mediano y largo plazo en la producción de hortalizas. Funding. This research was funded by Tecnológico Nacional de Mexico, research project 14615.22-P. Conflict of interest. The authors declare no conflict of interest. REFERENCIAS Boeckler, G.A., Gershenzon, J., and Unsicker, S.B., 2011. Phenolic glycosides of the Salicaceae and their role as anti-herbivore defenses. Phytochemistry, 72 (13), pp. 1497-1509. https://doi.org/10.1016/j.phytochem.2011.01. 038 Ali, M., Ahmed, T., Abu-Dieyeh, M. and Al-Ghouti, M.A., 2021. Investigating the quality and efficiency of biosolid produced in qatar as a fertilizer in tomato production. Agronomy, 11 (12), pp.25-52. https://doi.org/10.3390/agronomy11122552 Boiteau, G., Lynch, D.H. and Martin, R.C., 2008. Influence of fertilization on the Colorado potato beetle, Leptinotarsa decemlineata, in organic potato production. Environmental Entomology, 37 (2), pp. 575-585. https://doi.org/10.1093/ee/37.2.575 Allee, L.L. and Davis, P.M., 1996. Effect of manure on maize tolerance to western corn rootworm (Coleoptera: Chrysomelidae). Journal of economic entomology, 89 (6), pp. 1608-1620. https://doi.org/10.1093/jee/89.6.1608 Alyokhin, A., Mota‐Sanchez, D., Baker, M., Snyder, W.E., Menasha, S., Whalon, M. and Moarsi W.F., 2014. The Red Queen in a potato field: integrated pest management versus chemical dependency in Colorado potato beetle control. Pest Management Science, 71 (3), pp. 343- 356. https://doi.org/10.1002/ps.3826 Brito, A.N., Peña, Y.J. and de la Vega Báez, D., 2015. Efecto agronómico sobre el tomate del biosólido resultante de una planta de tratamiento anaeróbico de residuales pecuario. Centro Agrícola, 42 (4), pp. 53-60. Brown, J.K., 2010. Phylogenetic biology of the Bemisia tabaci sibling species group. Bemisia: bionomics and management of a global pest, Springer, Dordrecht, pp. 31-67. https://doi.org/10.1007/978-90-481-2460- 2_2. Alyokhin, A., Porter, G., Groden, E. and Drummond, F., 2005. Colorado potato beetle response to soil amendments: a case in support of the mineral balance hypothesis? Agriculture, Ecosystems & Environment, 109 (3-4), pp. 234-244. https://doi.org/10.1016/j.agee.2005.03.005 Chang, R., Guo, Q., Pandey, P., Li, Y., Chen, Q. and Sun, Y., 2021. Pretreatment by composting increased the utilization proportion of pig manure biogas digestate and improved the seedling substrate quality. Waste Management, 129, pp. 47-53. https://doi.org/10.1016/j.wasman.2021.05.01 0 Andrade, M.C., Da, Silva, A.A., Neiva, I.P., Oliveira, I.R.C., De Castro, E.M., Francis, D.M. and Maluf, W.R., 2017. Inheritance of type IV glandular trichome density and its association with whitefly resistance from Solanum galapagense accession LA1401. Euphytica, 213 (2), pp. 1-12. https://doi.org/10.1007/s10681-016-1792-1 Chang, R.X., Michel Jr, F.C., Gan, J.J., Wang, Q., Wang, Z.Z. and Li, Y.M., 2017. Effect of single and combined herbicides in compost on growth of sensitive crops: green bean, cucumber, and tomato. Compost Science & Utilization, 25 (sup1), pp. S23-S30. https://doi.org/10.1080/1065657X.2017.1385 430 Atiyeh, R.M., Arancon, N., Edwards, C.A. and Metzger, J.D., 2000. Influence of earthworm- processed pig manure on the growth and yield of greenhouse tomatoes. Compliance with ethical standards. Not applicable. El efecto del biosólido sobre el rendimiento de fruto no fue significativo. Aunque los diferentes niveles de biosólido no mostraron efecto contundente, se vio una fuerte tendencia en el aumento del rendimiento total, peso y diámetro polar del fruto, en las plantas del tratamiento 750 g planta-1. La adición de abonos orgánicos mejora el aporte de materia orgánica y nutrientes (nitrógeno y fósforo) a las plantas, lo que se puede traducir en el incremento del rendimiento, como se ha documento en el cultivo de tomate (Stavridou et al., 2021; Tzortzakis et al., 2020; Hasnain et al., 2020). Sin embargo, cantidades altas de abonos orgánicos o biosólidos pueden tener efectos negativos en la producción de frutos (Ali et al., 2021), debido al exceso de algunos elementos que pueden llegar a niveles tóxicos para las plantas o que evitan la Data availability. The data is available upon request, with the corresponding author esau.ruiz@itconkal.edu.mx Author contribution statement (CRediT). F. del R. Yam-Herrera- Methodology, Visualization and Writing original draft., E. Ruiz-Sánchez – Conceptualization, Visualization and writing original draft., S. López-Vázquez- Methodology, and visualization., J. Díaz-Mayo – Methodology and Validation, J. I. Tucuch-Haas –Formal analysis and Writing –review and editing., L. Latournerie- Moreno - Validation, Writing –review and editing., A. M. Herrera-Gorocica - Methodology, Formal Analysis and Writing original draft. Author contribution statement (CRediT). F. del R. Yam-Herrera- Methodology, Visualization and Writing original draft., E. Ruiz-Sánchez – Conceptualization, Visualization and writing original draft., S. López-Vázquez- Methodology, and visualization., J. Díaz-Mayo – Methodology and Validation, J. I. Tucuch-Haas –Formal analysis and Writing –review and editing., L. Latournerie- Moreno - Validation, Writing –review and editing., A. M. Herrera-Gorocica - Methodology, Formal Analysis and Writing original draft. 6 Yam-Herrera et al., 2024 Tropical and Subtropical Agroecosystems 27 (2024): Art. No. 009 https://doi.org/10.1016/j.biocontrol.2012.05. 008 13. https://doi.org/10.1007/s11270-020- 04946-8 13. https://doi.org/10.1007/s11270-020- 04946-8 Herrera-Gorocica, A.M., Ruiz-Sánchez, E., Ballina- Gómez, H.S., Reyes-Solís, G. and Sánchez- Lázaro, A., 2022. Response of Bemisia tabaci Genn to the association tomato− aromatic plant. Agrociencia. 56 (4), pp. 830-853. https://doi.org/10.47163/agrociencia.v56i4.2 809 Di Rienzo, J.A., Casanoves, F., Balzarini, M.G., González, L., Tablada, M. and Robledo, C.W., 2020. InfoStat, version 2018. Centro de Transferencia InfoStat, FCA, Universidad Nacional de Córdoba, Argentina. URL http://www.infostat.com.Ar Horowitz, A.R., Ghanim, M., Roditakis, E., Nauen, R. and Ishaaya, I., 2020. Insecticide resistance and its management in Bemisia tabaci species. Journal of Pest Science, 93, pp. 893- 910. https://doi.org/10.1007/s10340-020- 01210-0 Duarte, A.D.F., Andreazza, F., Nava, D.E. and Da Cunha, U.S., 2021. Polyphagotarsonemus latus (Trombidiformes: Tarsonemidae) on Laurus nobilis (Polycarpicae: Lauraceae): Report of infestation and damage. Systematic and Applied Acarology, 26 (8), pp. 1614- 1618. https://doi.org/10.11158/saa.26.8.14 Islam, M.N., Hasanuzzaman, A.T.M., Zhang, Z.F., Zhang, Y. and Liu, T.X., 2017. High level of nitrogen makes tomato plants releasing less volatiles and attracting more Bemisia tabaci (Hemiptera: Aleyrodidae). Frontiers in plant science, 8, pp. 466. https://doi.org/10.3389/fpls.2017.00466 Eigenbrode, S.D. and Pimentel, D., 1988. Effects of manure and chemical fertilizers on insect pest populations on collards. Agriculture, Ecosystems & Environment, 20 (2), pp. 109- 125. https://doi.org/10.1016/0167- 8809(88)90151-X Jaiswal, V., Gahlaut, V., Kumar, N. and Ramchiary, N., 2021. Genetics, genomics and breeding of chili pepper Capsicum frutescens L. and other Capsicum species. Advances in Plant Breeding Strategies: Vegetable Crops. Springer, Cham, 9: Fruits and Young Shoot, pp. 59-86. https://doi.org/10.1007/978-3-030- 66961-4_2 Gamboa-Angulo, J., Ruiz-Sánchez, E., Alvarado- López, C., Gutiérrez-Miceli, F., Ruíz- Valdiviezo, V.M., Medina-Dzul, Kati., 2020. Efecto de biofertilizantes microbianos en las características agronómicas de la planta y calidad del fruto del chile xcat´ik (Capsicum annuum L.). Terra Latinoamericana, 38 (4), pp. 817-826. https://doi.org/10.28940/terra.v38i4.716 Jiao, X., Xie, W., Zeng, Y., Wang, C., Liu, B., Wang, S. and Zhang, Y., 2018. Lack of correlation between host choice and feeding efficiency for the B and Q putative species of Bemisia tabaci on four pepper genotypes. Journal of pest science, 91 (1), pp. 133-143. https://doi.org/10.1007/s10340-017-0906-4 Garruña-Hernandez, R., Orellana, R., Larque- Saavedra, A. and Canto, A., 2014. Understanding the physiological responses of a tropical crop (Capsicum chinense Jacq.) at high temperature. PLoS one, 9 (11), pp. e111402. https://doi.org/10.1371/journal.pone.011140 2 Jiménez-Martínez, E., Izaguirre, R.M. and Mario, M.C., 2013. Plaguicidas botánicos y químicos para el control del ácaro blanco (Polyphagotarsonemus latus Bank) (ACARINA: TARSONEMIDAE) en chiltoma (Capsicum annuum L.), Tisma, Masaya. La Calera, 13 (20), pp. 9-15. Kajimura, T., Widiarta, I.N., Nagai, K., Fujisaki, K. and Nakasuji, F., 1995. Effect of organic rice REFERENCIAS Bioresource Technology, 75 (3), pp. 175-180. https://doi.org/10.1016/S0960- 8524(00)00064-X Chatterjee, R., Choudhuri, P., Laskar, N. and Pundibari, C.B., 2013. Influence of nutrient management practices for minimizing whitefly (Bemisia tabaci Genn.) population in tomato (Lycopersicon esculentum Mill.). Int J Sci Environ Technol, 2 (5), pp. 956-962. Balakrishnan, N., Baskaran, R.K., and Mahadevan, N.R. 2007. Impact of manures and fertilizers on sucking pests of cotton. Annals of Plant Protection Sciences, 15 (1), pp. 235-236. Banfield-Zanin, J.A., Rossiter, J.T., Wright, D.J., Leather, S.R. and Staley, J.T., 2012. Predator mortality depends on whether its prey feeds on organic or conventionally fertilised plants. Biological control, 63 (1), pp. 56-61. Chow, H.Y. and Pan, M., 2020. Fertilization value of biosolids on nutrient accumulation and environmental risks to agricultural plants. Water, Air, & Soil Pollution, 231 (12), pp. 1- 7 Tropical and Subtropical Agroecosystems 27 (2024): Art. No. 009 Yam-Herrera et al., 2024 https://doi.org/10.1016/S0167- 8809(00)00200-0 farming on planthoppers 4. Reproduction of the white backed planthopper, Sogatella furcifera Horváth (Homoptera: Delphacidae). Population Ecology, 37, pp. 219-224. https://doi.org/10.1007/BF02515823 Pahalvi, H.N., Rafiya, L., Rashid, S., Nisar, B., and Kamili, A.N., 2021. Chemical Fertilizers and Their Impact on Soil Health. In: Dar, G.H., Bhat, R.A., Mehmood, M.A., Hakeem, K.R. (eds) Microbiota and Biofertilizers, Springer, Cham. 2, pp. 1-20. https://doi.org/10.1007/978-3-030-61010- 4_1 Krishnaveni, M., Ravi, M., Allwin, L. and Sabarinathan, K.G. 2019. Effect of organic amendments on the incidence of major pests of ash gourd, Benincasa hispida Thunb. Journal of Pharmacognosy and Phytochemistry, 8 (6), pp. 538-542. Pan, M., Yau, P.C., Lee, K.C., Zhang, H., Lee, V., Lai, C.Y. and Fan, H.J., 2021. Nutrient Accumulation and Environmental Risks of Biosolids and Different Fertilizers on Horticultural Plants. Water, Air, & Soil Pollution, 232 (12), pp. 1-16. https://doi.org/10.1007/s11270-021-05424-5 López-Castilla, L., Garruña-Hernández, R., Castillo- Aguilar, C., Martínez-Hernández, A., Ortiz- García, M. and Andueza-Noh, R.H., 2019. Structure and genetic diversity of nine important landraces of Capsicum species cultivated in the Yucatan Peninsula, Mexico. Agronomy, 9 (7), pp. 376. https://doi.org/10.3390/agronomy9070376 Pantoja, K.F., Rocha, K.C., Melo, A.M., Marubayashi, J.M., Baldin, E.L., Bentivenha, J.P. and Krause-Sakate, R., 2018. Identification of Capsicum accessions tolerant to Tomato severe rugose virus and resistant to Bemisia tabaci Middle East-Asia Minor 1 (MEAM1). Tropical Plant Pathology, 43 (2), pp. 138- 145. https://doi.org/10.1007/s40858-018- 0212-6 López, M.D., Jordán, M.J. and Pascual-Villalobos, M.J. 2008. Toxic compounds in essential oils of coriander, caraway and basil active against stored rice pests. Journal of Stored Products Research, 44 (3), pp. 273-278. https://doi.org/10.1016/j.jspr.2008.02.005 Martínez-Martínez, V., Gomez-Gil, J., Machado, M.L. and Pinto, F.A., 2018. Leaf and canopy reflectance spectrometry applied to the estimation of angular leaf spot disease severity of common bean crops. PLoS One, 13 (4), pp. 1-18. https://doi.org/10.1371/journal.pone.019607 2 Peñuela, M., Arias, L.L., Viáfara-Vega, R., Rivera Franco, N. and Cárdenas, H., 2021. Morphological and molecular description of three commercial Capsicum varieties: a look at the correlation of traits and genetic distancing. Genetic Resources and Crop Evolution, 68 (1), pp. 261-277. https://doi.org/10.1007/s10722-020-00983-8 Misal, S., Warghane, A. and Patil, G., 2022. Chilli leaf curl disease: an Indian scenario. Indian Phytopathology, 7 (5), pp. 627–637. https://doi.org/10.1007/s42360-022-00531-7 Poornima, R., Suganya, K. and Sebastian, S.P., 2022. Biosolids towards Back–To–Earth alternative concept (BEA) for environmental sustainability: a review. Environmental Science and Pollution Research, 29 (3), pp. 3246-3287. https://doi.org/10.1007/s11356- 021-16639-8 Mohamed, B., Mounia, K., Aziz, A., Ahmed, H., Rachid, B. and Lotfi, A., 2018. 13. https://doi.org/10.1007/s11270-020- 04946-8 https://doi.org/10.5377/calera.v13i20.1619 Gou, J.Y., Suo, S.Z., Shao, K.Z., Zhao, Q., Yao, D., Li, H.P. and Rensing, C., 2020. Biofertilizers with beneficial rhizobacteria improved plant growth and yield in chili Capsicum annuum L. World Journal of Microbiology and Biotechnology, 36 (6), pp. 1-12. https://doi.org/10.1007/s11274-020-02863-w Jin, V.L., Potter, K.N., Johnson, M.V.V., Harmel, D. and Arnold, J.G., 2015. Surface-applied biosolids enhance soil organic carbon and nitrogen stocks but have contrasting effects on soil physical quality. Applied and Environmental Soil Science, 2015, pp. 1-10. http://dx.doi.org/10.1155/2015/715916 Hasnain, M., Chen, J., Ahmed, N., Memon, S., Wang, L., Wang, Y. and Wang, P., 2020. The effects of fertilizer type and application time on soil properties, plant traits, yield and quality of tomato. Sustainability, 12 (21), pp. 90-65. https://doi.org/10.3390/su12219065 8 Yam-Herrera et al., 2024 Tropical and Subtropical Agroecosystems 27 (2024): Art. No. 009 https://doi.org/10.1016/S0167- 8809(00)00200-0 Sewage sludge used as organic manure in Moroccan sunflower culture: Effects on certain soil properties, growth and yield components. Science of the Total Environment, 627, pp. 681-688. https://doi.org/10.1016/j.scitotenv.2018.01.2 58 Potisek-Talavera, M.D.C., Figueroa-Viramontes, U., González-Cervantes, G., Jasso-Ibarra, R. and Orona-Castillo, I., 2010. Soil applied biosolids and its effect on soil organic matter and nutrient content. Terra Latinoamericana, 28 (4), pp. 327-333. Morales, H., Perfecto, I. and Ferguson, B., 2001. Traditional fertilization and its effect on corn insect populations in the Guatemalan highlands. Agriculture, Ecosystems & Environment, 84 (2), pp. 145-155. Ramírez-Builes, V.H., 2007. Los sistemas agroforestales en el trópico y la fertilidad del suelo. Artículo Publicado en la Revista: Investigaciones de Unisarc. 5 (2) pp. 11-21. 9 Yam-Herrera et al., 2024 Tropical and Subtropical Agroecosystems 27 (2024): Art. No. 009 Ravi, M., Dhandapani, N., Sathiah, N. and Murugan, M., 2006. Influence of organic manures and fertilizers on the incidence of sucking pests of sunflower, Helianthus annuus L. Annals of Plant Protection Sciences, 14 (1), pp. 41-44. Stafford, D.B., Tariq, M., Wright, D.J., Rossiter, J.T., Kazana, E., Leather, S.R. and Staley, J.T., 2012. Opposing effects of organic and conventional fertilizers on the performance of a generalist and a specialist aphid species. Agricultural and Forest Entomology, 14 (3), pp. 270-275. https://doi.org/10.1111/j.1461- 9563.2011.00565.x Rowen, E., Tooker, J.F. and Blubaugh, C.K., 2019. Managing fertility with animal waste to promote arthropod pest suppression. Biological Control, 134, pp. 130-140. https://doi.org/10.1016/j.biocontrol.2019.04. 012 Stavridou, E., Giannakis, I., Karamichali, I., Kamou, N.N., Lagiotis, G., Madesis, P. and Lagopodi, A.L., 2021. Biosolid-Amended Soil Enhances Defense Responses in Tomato Based on Metagenomic Profile and Expression of Pathogenesis-Related Genes. Plants, 10 (12), pp. 2789. https://doi.org/10.3390/plants10122789 Ruíz, J.L.P., Peña, Y.J., Carrera, J.S. and Santana, I.A.R., 2021. Use of biosolid as a fertilizer in the tomato culture. Universidad & Ciencia, 10 (2), pp. 1-12. Rusli, L.S., Abdullah, R., Yaacob, J.S. and Osman, N., 2022. Organic amendments effects on nutrient uptake, secondary metabolites, and antioxidant properties of Melastoma malabathricum L. Plants, 11 (2) pp. 153. https://doi.org/10.3390/plants11020153 Tzortzakis, N., Saridakis, C. and Chrysargyris, A., 2020. Treated wastewater and fertigation applied for greenhouse tomato cultivation grown in municipal solid waste compost and soil mixtures. Sustainability, 12 (10), pp. 4287. https://doi.org/10.3390/su12104287 Saranraj, P. and Stella, D., 2012. Vermicomposting and its importance in improvement of soil nutrients and agricultural crops. Novus Natural Science Research, 1 (1), pp. 14-23. Utria-Borges, E., Cabrera-Rodríguez, J.A., Reynaldo- Escobar, I.M., Morales-Guevara, D., Fernández, A.M. https://doi.org/10.1016/S0167- 8809(00)00200-0 and Toledo Toledo, E., 2008. Utilización agraria de los biosólidos y su influencia en el crecimiento de plántulas de tomate (Lycopersicon esculentum Mill). Revista Chapingo. Serie Horticultura, 14 (1), pp. 33-39. Singh, R.P. and Agrawal, M., 2008. Potential benefits and risks of land application of sewage sludge. Waste management, 28 (2), pp. 347- 358. https://doi.org/10.1016/j.wasman.2006.12.01 0 Singh, R.P. and Agrawal, M., 2008. Potential benefits and risks of land application of sewage sludge. Waste management, 28 (2), pp. 347- 358. 10
https://openalex.org/W2887938357	https://europepmc.org/articles/pmc6081435?pdf=render	English	null	ACR11 modulates levels of reactive oxygen species and salicylic acid-associated defense response in Arabidopsis	Scientific reports	2,018	cc-by	6,972	ACR11 modulates levels of reactive oxygen species and salicylic acid- associated defense response in Arabidopsis 2018 18 OPEN Received: 9 February 2018 Accepted: 27 July 2018 Published: xx xx xxxx The ACT domain (aspartate kinase, chorismate mutase and TyrA), an allosteric effector binding domain, is commonly found in amino acid metabolic enzymes. In addition to ACT domain-containing enzymes, plants have a novel family of ACT domain repeat (ACR) proteins, which do not contain any recognizable catalytic domain. Arabidopsis has 12 ACR proteins, whose functions are largely unknown. To study the functions of Arabidopsis ACR11, we have characterized two independent T-DNA insertion mutants, acr11-2 and acr11-3. RNA gel-blot analysis revealed that the expression of wild-type ACR11 transcripts was not detectable in the acr11 mutants. Interestingly, a lesion-mimic phenotype occurs in some rosette leaves of the acr11 mutants. In addition, high levels of reactive oxygen species (ROS), salicylic acid (SA), and callose accumulate in the mutant leaves when grown under normal conditions. The expression of several SA marker genes and the key SA biosynthetic gene ISOCHORISMATE SYNTHASE1 is up-regulated in the acr11 mutants. Furthermore, the acr11 mutants are more resistant to the infection of bacterial pathogen Pseudomonas syringae pathovar tomato DC3000. These results suggest that ACR11 may be directly or indirectly involved in the regulation of ROS and SA accumulation, which in turn modulates SA-associated defense responses and disease resistance in Arabidopsis. Amino acids are essential organic compounds for all life forms. The synthesis of these important molecules is tightly regulated. It is well established that many key enzymes involved in amino acid biosynthesis are subject to feedback inhibition. These feedback-regulated enzymes are usually composed of catalytic domains and allosteric domains, which are responsible for catalyzing the reaction and allosteric regulation of the enzyme activity, respec- tively1–4. Interestingly, despite being feedback-regulated by different amino acids, the allosteric domain of these enzymes shares some common features in the primary sequence and tertiary structure, which has been named the ACT domain after bacterial aspartate kinase (AK), chorismate mutase (CM) and TyrA (prephenate dehy- drogenase, PDH)5,6. AK catalyzes the first reaction of the biosynthesis of aspartate family amino acids, includ- ing lysine, methionine, and threonine. The activity of AK is feedback-regulated by lysine and threonine via the conserved ACT domain7. CM and PDH are involved in the biosynthesis of aromatic amino acids, which are feedback-regulated by phenylalanine and tyrosine through the regulatory ACT domain1,8. Feedback regulation of amino acid biosynthetic enzymes has been extensively studied in bacteria. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Institute of Plant and Microbial Biology, Academia Sinica, Taipei, 11529, Taiwan. Shashi Kant Singh, Tzu-Ying Sung and Tsui-Yun Chung contributed equally to this work. Correspondence and requests for materials should be addressed to M.-H.H. (email: ming@gate.sinica.edu.tw) Results I l i Isolation and characterization of Arabidopsis acr11 mutants. The Arabidopsis ACR11 protein is predicted to contain a transit peptide with the cleavage site located at the 52nd residue (www.cbs.dtu.dk/services/ TargetP/) followed by two ACT domains (Fig. 1a). To further characterize the functions of ACR11, we obtained two independent T-DNA insertion lines, SAIL_14H_10 and SALK_025722, from the Arabidopsis Biological Resource Center (ABRC). The acr11 homozygous mutant plants were isolated from these T-DNA insertion lines by PCR and confirmed by genomic Southern blot analysis (Supplementary Fig. S1). The T-DNA mutant of SAIL_14H_10 was previously named acr11-214. We have adopted the nomenclature and named the new allele derived from the SALK_025722 T-DNA line acr11-3. The positions of T-DNA insertion of acr11-2 and acr11-3 are shown in Fig. 1b. We used RNA gel-blot analysis to examine the expression of ACR11 in wild-type, acr11-2 and acr11-3 seedlings. Although the wild-type ACR11 transcript was not detectable in acr11-2, two faint bands, one higher and another lower than ACR11, were detected in the mutant (Fig. 1c). The identities of these two bands are unknown. By contrast, transcripts of ACR11 were not detectable in the acr11-3 mutant (Fig. 1c). The phenotypes of acr11-2 and acr11-3 are very similar. The mutant plants are smaller than wild type (Supplementary Fig. S2), and some lesions appear in the rosette leaves of acr11-2 and acr11-3 mutants when grown in soil under a 16-h light/8-h dark cycle (Fig. 1d). The lesion usually starts to develop in the rosette leaves of 3- to 4-week-old acr11 mutant plants (Fig. 1d). We used trypan blue to stain dead cells in the lesion-containing rosette leaves from acr11-2 and acr11-3. Compared with the wild type, the acr11 mutants possess more dead cells in the rosette leaves (Fig. 2). Thus, the lesions occur in some of the rosette leaves can be attributed to spontaneous cell death in the acr11 mutants. Accumulation of ROS and callose in the acr11 mutants. It is known that accumulation of ROS com- monly proceeds spontaneous cell death in plants16. We thus examined the accumulation of ROS, including H2O2, superoxide, and singlet oxygen species, in the rosette leaves of 5-week-old wild-type and acr11 mutant plants by diaminobenzidine tetrahydrochloride (DAB), nitroblue tetrazolium (NBT), and Singlet Oxygen Sensor Green (SOSG) staining, respectively. Levels of H2O2, superoxide, and singlet oxygen are significantly higher in the mutant leaves as compared with those of the wild-type (Fig. 3a–c). ACR11 modulates levels of reactive oxygen species and salicylic acid- associated defense response in Arabidopsis 2018 18 OPEN Interestingly, these enzymes are highly conserved from bacteria to plants. Most plant homologs have similar domain composition, e.g. a specific enzyme catalytic domain, followed by a general allosteric regulatory ACT domain2,3,7. It is likely that the ACT domain fused to the amino acid metabolic enzymes also serves as an allosteric ligand-binding domain in plants. Indeed, the Arabidopsis AK is feedback-inhibited by lysine and S-adenosylmethionine via the ACT domain9. The three committed enzymes in branched-chain amino acid BCAA biosynthesis, e.g. threonine deaminase, aceto- hydroxy acid synthase, and isopropylmalate synthase are feedback-regulated by branched-chain amino acids mainly via the ACT domains of these enzymes in Arabidopsis10. Furthermore, the Arabidopsis ACT domain-containing enzyme phosphoglycerate dehydrogenase is feedback regulated by its end product serine11. Thus, the ACT domain is a widespread allosteric regulatory domain that is highly conserved from bacteria to plants. Institute of Plant and Microbial Biology, Academia Sinica, Taipei, 11529, Taiwan. Shashi Kant Singh, Tzu-Ying Sung and Tsui-Yun Chung contributed equally to this work. Correspondence and requests for materials should be addressed to M.-H.H. (email: ming@gate.sinica.edu.tw) Scientific Reports \| (2018) 8:11851 \| DOI:10.1038/s41598-018-30304-0 1 www.nature.com/scientificreports/ We previously identified a novel ACT domain repeat (ACR) protein family in Arabidopsis2,12. The ACR pro- teins contain only ACT domain repeats but not any recognizable catalytic domain. The functions of these plant ACR proteins are largely unknown. Arabidopsis has 12 ACR proteins, which are further divided into 3 different groups according to their ACT domain composition and sequence homology12. Group III ACR proteins, e.g. ACR11 and ACR12, are distinct in that they contain a non-conserved N-terminal transit peptide followed by two conserved ACT domains. Indeed, the Arabidopsis ACR11 and ACR12 proteins have been demonstrated to local- ize in the chloroplast12. The ACR11 gene is specifically expressed in green tissues, and is coordinately regulated with GLN2 encoding a chloroplastic glutamine synthetase 2 (GS2) in Arabidopsis12. Recently, the Arabidopsis ACR11 protein was shown to activate the activity of GS2 and levels of glutamine were significantly reduced in the acr11 mutant13. In addition, the Arabidopsis ACR11 protein was shown to interact with ferredoxin-dependent glutamine oxoglutarate aminotransferase 1 (Fd-GOGAT1) and the activity of Fd-GOGAT was reduced in the acr11 mutants14. It has been proposed that ACR11 may stabilize Fd-GOGAT and possibly modulates its activity14. Nevertheless, the molecular mechanisms of ACR11 have yet to be elucidated. y Here, we have characterized two independent acr11 T-DNA insertion mutants in Arabidopsis. ACR11 modulates levels of reactive oxygen species and salicylic acid- associated defense response in Arabidopsis 2018 18 OPEN Interestingly, spontaneous cell death occurs in the rosette leaves of the acr11 mutants. The lesion-mimic phenotype accompa- nied by increased levels of reactive oxygen species (ROS) and salicylic acid (SA)-associated defense responses make the acr11 mutants more resistant to the bacterial pathogen Pseudomonas syringae pathvar tomato DC3000 (Pst). The homeostasis of glutamine has been proposed to modulate SA-associated redox status and defense responses in Arabidopsis15. The functions of Arabidopsis ACR11 in the interconnections of GS/Fd-GOGAT cycle, glutamine homeostasis, redox balance, ROS accumulation, and SA-associated defense responses are discussed herein. Results I l i These results suggest that the acr11 mutants have accumulated excessive amounts of ROS when grown under normal conditions. In addition, we used aniline blue to stain callose in the rosette leaves from 5-week-old wild-type and acr11 mutant plants (Fig. 4a). The mutant leaves have accumulated significant amount of callose as compared with that of the wild type (Fig. 4a,b). The expression of SA marker genes is induced in the acr11 mutants. The lesion-mimic phenotype and accumulation of ROS in the rosette leaves suggest that the SA-related signaling pathways may be activated in the acr11 mutants. To examine if the SA-associated responses are enhanced in the acr11 mutants, we used quanti- tative (q) RT-PCR analysis to measure the expression of SA marker genes in the rosette leaves of 5-week-old wild type and acr11 mutants. The selected SA maker genes include PATHOGENESIS-RELATED 1 (PR1), PR2, PR5, CALMODULIN BINDING PROTEIN 60 G (CBP60G) and two WRKY transcription factor genes, WRKY18 and WRKY53. Steady-state mRNA levels of PR1, PR2, PR5, CBP60, WRKY18, and WRKY53 were significantly higher in the acr11-2 and acr11-3 mutants as compared with those of the wild type (Fig. 5). In addition, RNA gel-blot analysis of PR1 and PR2 in Arabidopsis wild type and acr11-3 was shown in Supplementary Fig. S3. These results indicate that the SA-associated responses are constitutively activated in the acr11 mutant rosette leaves. Accumulation of SA in the acr11 mutants. Perturbation of endogenous SA levels will directly affect the SA-associated responses in plants. To test if the constitutively activated SA responses in the acr11 mutants is caused by changes of SA levels, we measured the amount of SA in the rosette leaves of 5-week-old wild-type, acr11-2 and acr11-3 mutant plants. The results indicate that the SA levels in the mutant rosette leaves are signif- icantly higher than those of the wild type (Fig. 6a). It is known that isochorismate synthase (ICS) is the major Scientific Reports \| (2018) 8:11851 \| DOI:10.1038/s41598-018-30304-0 2 www.nature.com/scientificreports/ Figure 1. Molecular and phenotypic analyses of Arabidopsis acr11 mutants. (a) Schematic diagram of the Arabidopsis ACR11 protein. TP, transit peptide. (b) Schematic diagram of the ACR11 gene and the locations of T-DNA insertion in the acr11-2 and acr11-3 mutants. (c) RNA gel-blot analysis. Total RNA extracted from 2-week-old wild type (WT), acr11-2 and acr11-3 seedlings was used to detect the expression of ACR11. The full-length blot is shown in Supplementary Fig. S5. Results I l i (d) Four-week-old Arabidopsis WT, acr11-2 and acr11-3 mutant plants grown in soil under normal conditions. Lesions appear in the rosette leaves of the acr11 mutants are indicated by white arrows. Figure 1. Molecular and phenotypic analyses of Arabidopsis acr11 mutants. (a) Schematic diagram of the Arabidopsis ACR11 protein. TP, transit peptide. (b) Schematic diagram of the ACR11 gene and the locations of T-DNA insertion in the acr11-2 and acr11-3 mutants. (c) RNA gel-blot analysis. Total RNA extracted from 2-week-old wild type (WT), acr11-2 and acr11-3 seedlings was used to detect the expression of ACR11. The full-length blot is shown in Supplementary Fig. S5. (d) Four-week-old Arabidopsis WT, acr11-2 and acr11-3 mutant plants grown in soil under normal conditions. Lesions appear in the rosette leaves of the acr11 mutants are indicated by white arrows. Figure 2. Trypan blue staining for the detection of cell death in the rosette leaves of 5-week-old Arabidopsis wild type (WT) and acr11 mutants. Scale bars are 1 mm (top) and 0.1 mm (bottom). Figure 2. Trypan blue staining for the detection of cell death in the rosette leaves of 5-week-old Arabidopsis wild type (WT) and acr11 mutants. Scale bars are 1 mm (top) and 0.1 mm (bottom). Scientific Reports \| (2018) 8:11851 \| DOI:10.1038/s41598-018-30304-0 3 www.nature.com/scientificreports/ Figure 3. Comparison of reactive oxygen species levels in the rosette leaves of 5-week-old Arabidopsis wild- type (WT) and acr11 mutant plants. (a) Staining of hydrogen peroxide by 3,3′-diaminobenzidine. (b) Staining of superoxide radical by nitroblue tetrazolium. (c) Staining of singlet oxygen species by Singlet Oxygen Sensor Green (SOSG) fluorescent dye. Scale bars are 1 mm in (a) and (b), 200 μm in (c). Figure 3. Comparison of reactive oxygen species levels in the rosette leaves of 5-week-old Arabidopsis wild- type (WT) and acr11 mutant plants. (a) Staining of hydrogen peroxide by 3,3′-diaminobenzidine. (b) Staining of superoxide radical by nitroblue tetrazolium. (c) Staining of singlet oxygen species by Singlet Oxygen Sensor Green (SOSG) fluorescent dye. Scale bars are 1 mm in (a) and (b), 200 μm in (c). Figure 4. Callose deposition in the rosette leaves of 5-week-old Arabidopsis wild type (WT) and acr11 mutants. (a) Staining of callose deposits by aniline blue. Scale bar is 100 μm. (b) Quantification of callose deposits in WT and acr11 mutant leaves. Values shown are means ± SD per 0.97 mm2 from leaves of 5 independent plants. Results I l i *P < 0.001 represents the result of Student’s t test. Figure 4. Callose deposition in the rosette leaves of 5-week-old Arabidopsis wild type (WT) and acr11 mutants. (a) Staining of callose deposits by aniline blue. Scale bar is 100 μm. (b) Quantification of callose deposits in WT and acr11 mutant leaves. Values shown are means ± SD per 0.97 mm2 from leaves of 5 independent plants. P < 0.001 represents the result of Student’s t test. enzyme involved in SA biosynthesis in Arabidopsis17. There are two ICS genes, ICS1 and ICS2, in Arabidopsis. We used qRT-PCR analysis to examine the expression of ICS1 and ICS2 in the rosette leaves of wild-type and acr11 mutant plants. The expression levels of ICS1, but not ICS2, are much higher in the acr11-2 and acr11-3 mutants as compared to the wild type (Fig. 6b). Enhanced disease resistance in the acr11 mutants. The phenotypes of lesion-mimic, enhanced SA-dependent responses, and accumulation of callose, ROS and SA suggest that the acr11 mutants may be more resistant to pathogen infection. To test this possibility, we used the virulence strain Pseudomonas syringae patho- var tomato DC3000 (Pst) to infect the rosette leaves of 5-week-old wild-type and acr11 mutant plants with syringe infiltration. The disease symptoms developed in the mutant leaves were significantly weaker than those of the wild type 3 days after inoculation (Fig. 7a). This phenotype was associated with bacterial growth in leaves infiltrated with Pst. The number of bacteria growing in the mutant leaves was significantly smaller than that of the wild type 1 to 3 days post inoculation (Fig. 7b). Scientific Reports \| (2018) 8:11851 \| DOI:10.1038/s41598-018-30304-0 4 www.nature.com/scientificreports/ Figure 5. Quantitative RT-PCR analysis of salicylic acid-responsive genes. Total RNA extracted from rosette leaves of 5-week-old Arabidopsis wild-type (WT), acr11-2, and acr11-3 mutant plants was used for qRT-PCR analysis to detect the expression of PR1, PR2, PR5, CBP60G, WRKY18 and WRKY53. Relative expression indicates the fold-change of each gene as compared to that of WT. Results shown are means ± SD from three independent experiments. P < 0.001 represents the result of Student’s t test. Figure 5. Quantitative RT-PCR analysis of salicylic acid-responsive genes. Total RNA extracted from rosette leaves of 5-week-old Arabidopsis wild-type (WT), acr11-2, and acr11-3 mutant plants was used for qRT-PCR analysis to detect the expression of PR1, PR2, PR5, CBP60G, WRKY18 and WRKY53. Results I l i Relative expression indicates the fold-change of each gene as compared to that of WT. Results shown are means ± SD from three independent experiments. P < 0.001 represents the result of Student’s t test. Figure 6. Enhanced salicylic acid (SA) accumulation is in the acr11 mutants. (a) Levels of free SA in the rosette leaves of 5-week-old Arabidopsis wild type (WT) and acr11 mutants. (b) Quantitative RT-PCR analysis of SA biosynthetic genes in the rosette leaves of 5-week-old Arabidopsis WT and acr11 mutants. The expression levels of ICS1 and ICS2 in WT were set at 1. Fold change indicates the relative expression of ICS1 and ICS2 as compared to that of WT. ICS, ISOCHORISMATE SYNTHASE. Results shown are means ± SD from three independent experiments. P < 0.001 represents the result of Student’s t test. Figure 6. Enhanced salicylic acid (SA) accumulation is in the acr11 mutants. (a) Levels of free SA in the rosette leaves of 5-week-old Arabidopsis wild type (WT) and acr11 mutants. (b) Quantitative RT-PCR analysis of SA biosynthetic genes in the rosette leaves of 5-week-old Arabidopsis WT and acr11 mutants. The expression levels of ICS1 and ICS2 in WT were set at 1. Fold change indicates the relative expression of ICS1 and ICS2 as compared to that of WT. ICS, ISOCHORISMATE SYNTHASE. Results shown are means ± SD from three independent experiments. P < 0.001 represents the result of Student’s t test. In addition to syringe infiltration on individual rosette leaves, we also inoculated whole plants with Pst by dipping. Compared with the wild type, the acr11 mutant plants showed less severe disease symptoms 3 days after dip inoculation (Supplementary Fig. S4a). The rosette leaves of the acr11 mutants had less bacterial growth as compared with that of the wild type 1 to 3 days after dip inoculation (Supplementary Fig. S4b). These results suggest that the acr11 mutants are more resistant to Pst infection. Scientific Reports \| (2018) 8:11851 \| DOI:10.1038/s41598-018-30304-0 5 www.nature.com/scientificreports/ Figure 7. Enhanced disease resistance in 5-week-old Arabidopsis acr11 mutants. (a) Symptoms of wild- type (WT), acr11-2 and acr11-3 rosette leaves 3 days after syringe infiltration with Pseudomonas syringae pv. tomato DC3000 (Pst). (b) Growth of Pst in Arabidopsis WT, acr11-2 and acr11-3 mutants. Bacterial titers were evaluated at 0 to 3 days post inoculation (dpi). Results are means ± SD from three independent experiments. Discussionh The prediction that plant ACR11 homologs contain two ACT domains is reminiscent of the recent discovery of human arginine sensor CASTOR118,19. Unlike CASTOR1, the functions of plant ACR proteins are largely unknown. Nonetheless, these proteins share a common feature that they all contain multiple ACT domains, but not any recognizable catalytic domain. The human CASTOR1 and its homolog CASTOR2 are predicted to contain 2 ACT domains18. However, crystal structure analysis of the arginine-bound CASTOR1 reveals that it is in fact composed of 4 tandem ACT domains19–21. Therefore, the real composition of ACT domains in the ACR11 protein requires further studies on the crystal structure.i p q y It is interesting that the Arabidopsis acr11 mutants have a lesion-mimic phenotype. Usually, the lesion first appears in the rosette leaves of 3- to 4-week-old acr11 mutant plants when grown in soil under a normal condi- tion. Many plant lesion-mimic mutants are associated with disease resistance22,23. The spontaneous cell death phe- notype and the induction of defense response are coordinated in the acr11 mutants. Furthermore, we have shown that the Arabidopsis acr11 mutants are more resistant to the bacterial pathogen Pst (Fig. 7 and Supplementary Fig. S4). These results suggest that the cell death pathway activated in the acr11 mutants is interconnected with the defense pathway against Pst. p y g The ACR11 protein is localized to the chloroplast12, which is also the site for SA biosynthesis and one of the major sites for ROS production inside the plant cell. The increased disease resistance of the acr11 mutants can be attributed to the accumulation of ROS and SA, which is well-documented to enhance plant immunity17,24,25. In the rosette leaves of Arabidopsis acr11 mutants, levels of ROS, SA and callose are significantly increased as com- pared to those of the wild type (Figs 3, 4 and 6). Furthermore, the expression of SA biosynthetic and responsive genes is dramatically induced in the acr11 mutant (Figs 5, 6). These results are consistent with the phenotype that the acr11 mutant is more resistant to pathogen infection. Loss-of-function in ACR11 will increase ROS levels and activate defense responses in the plant. However, if the amounts of ROS accumulated inside the cell are over the threshold, cell death will occur in the acr11 mutants. The molecular mechanisms of ACR11 in the chloroplast-triggered spontaneous cell death remain to be elucidated. Results I l i Asterisks indicate significant differences (P < 0.05; *P < 0.01; Student’s t test) compared to the WT. Figure 7. Enhanced disease resistance in 5-week-old Arabidopsis acr11 mutants. (a) Symptoms of wild- type (WT), acr11-2 and acr11-3 rosette leaves 3 days after syringe infiltration with Pseudomonas syringae pv. tomato DC3000 (Pst). (b) Growth of Pst in Arabidopsis WT, acr11-2 and acr11-3 mutants. Bacterial titers were evaluated at 0 to 3 days post inoculation (dpi). Results are means ± SD from three independent experiments. Asterisks indicate significant differences (P < 0.05; **P < 0.01; Student’s t test) compared to the WT. Discussionh This hypothesis may be partly supported by the following observations: (1) the ACR11 and GLN2 genes are coordi- nately expressed12; (2) ACR11 can activate GS2 and levels of glutamine are decreased in the acr11 mutant13; (3) ACR11 interacts with Fd-GOGAT1 and the activity of Fd-GOGAT is reduced in the acr11 mutant14. Arabidopsis Fd-GOGAT1 plays a major role in the assimilation of ammonium generated by photorespiration31. In addition to reduced levels of glutamine13, ammonium may also accumulate in the acr11 mutant. Excess amounts of ammo- nium are toxic to plants, which may activate ROS production and trigger the downstream SA-associated defense responses (Fig. 8).h GS/Fd-GOGAT cycle may affect redox balance and induce ROS production in the acr11 mutant (Fig. 8). This hypothesis may be partly supported by the following observations: (1) the ACR11 and GLN2 genes are coordi- nately expressed12; (2) ACR11 can activate GS2 and levels of glutamine are decreased in the acr11 mutant13; (3) ACR11 interacts with Fd-GOGAT1 and the activity of Fd-GOGAT is reduced in the acr11 mutant14. Arabidopsis Fd-GOGAT1 plays a major role in the assimilation of ammonium generated by photorespiration31. In addition to reduced levels of glutamine13, ammonium may also accumulate in the acr11 mutant. Excess amounts of ammo- nium are toxic to plants, which may activate ROS production and trigger the downstream SA-associated defense responses (Fig. 8).h p g The reactions catalyzed by GS/Fd-GOGAT require ATP and reducing power derived from photosynthe- sis. Thus, the GS/Fd-GOGAT cycle is a strong electron sink, which plays an important role in consuming and translocating reducing equivalents inside the plant cell. Perturbation of the GS/Fd-GOGAT cycle results in Gln-deficiency and redox imbalance, which may trigger the overproduction of ROS and enhance SA-associated defense responses in the acr11 mutant. Alternatively, ACR11 may have an effect on SA biosynthesis, and subse- quently affect the accumulation of ROS. It is known that the interplay of SA and ROS can modulate the expres- sion of defense genes32. Overproduction of SA may also result in increased levels of ROS and enhanced defense responses in the acr11 mutants. It will be interesting to investigate the role of ACR11 in the interconnection of Gln metabolism, ROS production, and SA-associated defense network in Arabidopsis. p p Fd-GOGAT1 has been shown to interact and regulate the activities of UDP-sulfoquinovose synthase and serine hydroxymethyltransferase33,34. Discussionh Interestingly, Fd-GOGAT1 plays a regulatory role when interacts with these enzymes, which is independent of its catalytic function33,34. Thus, it is possible that the ACR11/Fd-GOGAT complex may have additional functions independent of the assimilation of photorespiratory ammonium in Arabidopsis. We cannot exclude the possibility that the accumulation of ROS and SA, and the defense-related phenotypes observed in the acr11 mutants are not directly linked to the GS/Fd-GOGAT cycle. The ACR11 homologs are conserved from algae to land plants. Further studies on the functions of Arabidopsis ACR11 will provide insights into the molecular mechanism of ACR proteins in plants. Discussionh p gg p Alternatively, the increased disease resistance of the acr11 mutants can be attributed to shortage of nutrients, e.g. glutamine and its derivatives, for bacterial growth in the host plant. Pathogens have to obtain their nitrogen nutrients from the host plant. Thus, the nitrogen status of the host plant is tightly associated with pathogene- sis26,27. Interestingly, the pathogenic Pst has been shown to selectively catabolize abundant amino acids, such as glutamine, glutamate, and aspartate from the host plant28. The Arabidopsis acr11 mutants have reduced levels of glutamine13. It is possible that decreased levels of glutamine may cause shortage of nutrients for bacterial growth, and thus confers pathogen resistance in the acr11 mutants. In addition to its role in nutrition and metabolism, glutamine can also function as a signaling molecule in plants29,30. It has been shown that glutamine homeostasis can modulate SA-associated redox status and defense responses in Arabidopsis15. It is conceivable that ACR11 may be involved in the maintenance of Gln homeostasis in Arabidopsis. We propose a hypothetical working model that Gln-deficiency derived from a compromised Scientific Reports \| (2018) 8:11851 \| DOI:10.1038/s41598-018-30304-0 6 www.nature.com/scientificreports/ Figure 8. A hypothetical working model of ACR11 function in modulating reactive oxygen species (ROS) production and salicylic acid (SA)-associated defense responses. ACR11 may integrate the information of glutamine homeostasis and redox balance to modulate the activities of glutamine synthetase (GS)/ferredoxin- dependent glutamine oxoglutarate aminotransferase (Fd-GOGAT) and ROS production in the chloroplast. Overproduction of ROS activates SA biosynthesis and enhances SA-associated defense responses. In addition, ACR11 may directly affect SA biosynthesis, which in turn affects ROS production and SA-associated defense responses. Figure 8. A hypothetical working model of ACR11 function in modulating reactive oxygen species (ROS) production and salicylic acid (SA)-associated defense responses. ACR11 may integrate the information of glutamine homeostasis and redox balance to modulate the activities of glutamine synthetase (GS)/ferredoxin- dependent glutamine oxoglutarate aminotransferase (Fd-GOGAT) and ROS production in the chloroplast. Overproduction of ROS activates SA biosynthesis and enhances SA-associated defense responses. In addition, ACR11 may directly affect SA biosynthesis, which in turn affects ROS production and SA-associated defense responses. GS/Fd-GOGAT cycle may affect redox balance and induce ROS production in the acr11 mutant (Fig. 8). Methods l Plant materials and growth conditions. Arabidopsis thaliana ecotype Columbia-0 and T-DNA inser- tion mutants acr11-2 (Sail_14_H10) and acr11-3 (Salk_025722) were obtained from the Arabidopsis Biological Resource Center. Plants were grown in soil or on tissue culture plates in a controlled growth chamber on a 16-h light/8-h dark cycle at 23 °C as previously described35. Trypan blue staining and detection of ROS. Rosette leaves from 5-week-old Arabidopsis wild-type and acr11 mutant plants were used for trypan blue, DAB, NBT, and SOSG staining as previously described with minor modifications36,37. The DAB (D5637, Sigma-Aldrich) staining solution, 1.25 mg/ml, was freshly prepared in sterilized water and adjusted to pH 3.8 with KOH. Detached rosette leaves were immersed and infiltrated under vacuum with DAB staining solution and then cleared in boiling 95% (v/v) ethanol for 10 min. For NBT staining, detached rosette leaves were immersed and infiltrated under vacuum with 3.5 mg/ml NBT (N6876, Sigma-Aldrich) staining solution in 10 mM potassium phosphate buffer containing 10 mM sodium azide. After vacuum infiltration, stained leaves were bleached in boiling 95% ethanol (v/v) for 10 min. The commercially available fluorescent dye SOSG (S36002, Thermo Fisher) was used to detect singlet oxygen. Rosette leaves from Scientific Reports \| (2018) 8:11851 \| DOI:10.1038/s41598-018-30304-0 7 www.nature.com/scientificreports/ 5-week-old plants were infiltrated with a solution of 100 μM SOSG in 50 mM phosphate potassium buffer (pH 7.5). Plants were exposed to light for 30 min and infiltrated leaves were observed under a 510 META Zeiss confo- cal laser scanning microscope with excitation at 480 nm and emission at 530 nm. 5-week-old plants were infiltrated with a solution of 100 μM SOSG in 50 mM phosphate potassium buffer (pH 7.5). Plants were exposed to light for 30 min and infiltrated leaves were observed under a 510 META Zeiss confo- cal laser scanning microscope with excitation at 480 nm and emission at 530 nm. Callose staining and microscopy. Aniline blue (415049, Sigma-Aldrich) was used to stain callose deposi- tion. Rosette leaves of 5-week-old Arabidopsis were cleared overnight in 95% ethanol (v/v) at room temperature. The completely cleared leaves were rehydrated in sterilized water, and then immersed in aniline blue staining solution of 0.01% (w/v) in 0.15 M phosphate buffer, pH 9.5, for 30 min. The callose deposition was observed under a UV illumination using Zeiss Axio Scope A1 microscope. Callose deposits were quantified by the “analyze particles” function of ImageJ (http://rsb.info.nih.gov/ij/). Methods l Quantitative (q) RT-PCR and RNA gel-blot analysis. Arabidopsis total RNA was isolated using a phenol extraction protocol as previously described38. Total RNA extracted from rosette leaves of 5-week-old Arabidopsis wild-type and acr11 mutant plants was digested with DNase I and used for qRT-PCR analysis. All qRT-PCRs were performed with three biological repeats and the expression data were normalized to the nuclear gene ACTIN2 (At3g18780). The following primers were used for qRT-PCR: PR1 (At2g14610), 5′-TTCACAACCA GGCACGAGGAG-3′, 5′-GCCAGACAAGTCACCGCTACC-3′; PR2 (At3g57260), 5′-CTTGAACGTCTCGCCT CCAGTC-3′, 5′-TCCAGAAACCGCGTTCTCGATG-3′; PR5 (At1g75040), 5′-CAATTGCCCTACCACCGT CTGG-3′, 5′-CTTAGACCGCCACAGTCTCCG-3′; CBP60G (At5g26920), 5′-CGGGCGTAACACTTCTCTTC-3′, 5′-AGCTTCGGCCTTTAATTGGT-3′; WRKY18 (At4g31800), 5′-CATACGAAGGGACGCATAAC-3′, 5′-CC TTTCGTTTTTCTCCAACG-3′; WRKY53 (At4g23810), 5′-GGCAGTGTTCCAGAATCTCC-3′, 5′-GCCTCT CTCTGGGCTTATTC-3′; ACTIN2 (At3g18780), 5′-GGTAACATTGTGCTCAGTGGTGG-3′, 5′-AACGACC TTAATCTTCATGCTGC-3′; ICS1 (At1G74710), 5′-TGGCGAGGAGAGTGAATTTG-3′, 5′-TGGGTCACTT CCAGCTACTA-3′; ICS2 (At1G18870), 5′-GTTTGCGGATGTCCAGTAGAA-3′, 5′-CCACCACCAAAGAA CCCAATA-3′. For RNA gel-blot analysis, total RNA (10 μg) was separated in standard formaldehyde gel by electrophoresis and blotted onto a nylon membrane. To detect the transcripts of ACR11 and SA marker genes, digoxigenin (DIG)-labeled single-stranded DNA probes were generated by PCR using the following prim- ers: ACR11 (At1g16880), 5′-ATGGCTATGGCCT CTGCTTC-3′, 5′-GAAACTTGACTCGTCAGTTG-3′; PR1 (At2g14610), 5′- ATGAATTTTACTGGCTATTCTCG-3′, 5′-TTAGTATGGCTTCTCGTTCAC-3′; PR2 (At3g57260), 5′-ATGTCTGAATCAAGGAGCTTAGC-3′, 5′-TTAGTTGAAATTAACTTCATACTTAG-3′. DIG probe labeling, pre-hybridization, hybridization, wash conditions and detection were performed according to the Boehringer-Mannheim Genius System User’s Guide: DIG Application Manual for Filter Hybridization. Salicylic acid (SA) measurement. Rosette leaves from 5-week-old Arabidopsis wild-type and acr11 mutant plants were used for SA measurement. Sample extraction and quantitative analysis of free SA were performed as previously described39. The SA measurement was conducted by the Metabolomics Core at Academia Sinica using Ultra Performance Liquid Chromatography-High Definition Mass Spectrometry (Waters, http://www.waters.com). Pathogen infection assays. Five-week-old Arabidopsis wild-type and acr11 mutant plants were used for Pst infection assays as previously described40. Arabidopsis plants were dipped in a bacterial suspension of 107 colony-forming units (cfu)/mL in 10 mM MgCl2 containing 0.01% (v/v) Silwet L-77 for 15 min. For inoculation by syringe infiltration, 3–4 leaves per plant were infiltrated with bacterial suspension of 105 cfu/ml using a 1-ml syringe without a needle. Plants were kept at 100% relative humidity for one day after infection, and symptoms were photo- graphed 3 days post inoculation. For analysis of bacterial growth, 8 leaf discs with 0.5 cm diameter from 4 different plants sampled at 0 to 3 days after inoculation were used to measure bacterial growth as previously described40. References p , ,h y p , ( ) 2. Hsieh, M. H. & Goodman, H. M. Molecular characterization of a novel gene family encoding ACT domain repeat proteins in Arabidopsis. Plant Physiol. 130, 1797–1806 (2002). 3. Curien, G. et al. Amino acid biosynthesis: new architectures in allosteric enzymes. Plant Physiol. Biochem. 46, 325 339 (2008). 4. Lang, E. J. M., Cross, P. J., Mittelstädt, G., Jameson, G. B. & Parker, E. J. Allosteric ACTion: the varied ACT domains regulating enzymes of amino-acid metabolism. Curr. Opin. Struct. Biol. 29, 102–111 (2014). enzymes of amino-acid metabolism. Curr. Opin. Struct. Biol. 29, 102–111 (2014). 5. Aravind, L. & Koonin, E. V. Gleaning non-trivial structural, functional and evolutionary information about proteins by iter database searches. J. Mol. Biol. 287, 1023–1040 (1999).h y p 5. Aravind, L. & Koonin, E. V. Gleaning non-trivial structural, functional and evolutionary information about proteins by iterative database searches. J. Mol. Biol. 287, 1023–1040 (1999).h 6. Liberles, J. S., Thorolfsson, M. & Martinez, A. Allosteric mechanisms in ACT domain containing enzymes involved in amino acid metabolism. Amino Acids 28, 1–12 (2005).h 7. Dumas, R., Cobessi, D., Robin, A. Y., Ferrer, J. L. & Curien, G. The many faces of aspartate kinases. Arch. Biochem. Biophys. 519, 186–193 (2012). 8. Zhang, S. et al. Chorismate mutase-prephenate dehydratase from Escherichia coli. Study of catalytic and regulatory domains using genetically engineered proteins. J. Biol. Chem. 273, 6248–6253 (1998). 9. Mas-Droux, C. et al. A novel organization of ACT domains in allosteric enzymes revealed by the crystal structure of Arabidopsis aspartate kinase. Plant Cell 18, 1681–1692 (2006). 10. Xing, A. & Last, R. L. A regulatory hierarchy of the Arabidopsis branched-chain amino acid metabolic network. Plant Cell 29, 1480–1499 (2017). 1. Okamura, E. & Hirai, M. Y. Novel regulatory mechanism of serine biosynthesis associated with 3-phosphoglycerate dehydrogenase in Arabidopsis thaliana. Sci. Rep. 7, 3533 (2017).h 12. Sung, T. Y., Chung, T. Y., Hsu, C. P. & Hsieh, M. H. The ACR11 encodes a novel type of chloroplastic ACT domain repeat protein that is coordinately expressed with GLN2 in Arabidopsis. BMC Plant Biol. 11, 118 (2011). 13. Osanai, T., Kuwahara, A., Otsuki, H., Saito, K. & Hirai, M. Y. ACR11 is an activator of plastid-type glutamine synthetase GS2 in Arabidopsis thaliana. Plant and Cell Physiol. 58, 650–657 (2017). p y , ( ) 4. Takabayashi, A., Niwata, A. & Tanaka, A. h 2. Hsieh, M. H. & Goodman, H. M. Molecular characterization of a novel gene family encoding ACT domain repeat protei Arabidopsis. Plant Physiol. 130, 1797–1806 (2002). 1. Chipman, D. M. & Shaanan, B. The ACT domain family. Curr. Opin. Struct. Biol. 11, 694–700 (2001). p y ( ) 3. Curien, G. et al. Amino acid biosynthesis: new architectures in allosteric enzymes. Plant Physiol. Biochem. 46, 325–339 (2008). Scientific Reports \| (2018) 8:11851 \| DOI:10.1038/s41598-018-30304-0 Acknowledgements g We thank Ching-Chih Tseng for technical assistance, Mei-Jane Fang for confocal microscopy, Nai-Chun Lin and Erh-Min Lai for the bacterial strain. This research was supported by a grant (NSC 99-2311-B-001-007-MY3) to M.H.H. from National Science Council, Taipei, Taiwan. www.nature.com/scientificreports/ 8 Tseng C C Lee C J Chung Y T Sung T Y & Hsieh M H Differential regulation of Arabidopsis plastid gene expression and mechanisms during atrazine injury and sucrose-induced tolerance in Arabidopsis thaliana plantlets. BMC Plant Biol. 9, 28 (2009). 38. Tseng, C. C., Lee, C. J., Chung, Y. T., Sung, T. Y. & Hsieh, M. H. Differential regulation of Arabidopsis plastid gene expression and RNA editing in non-photosynthetic tissues. Plant Mol. Biol. 82, 375–392 (2013). mechanisms during atrazine injury and sucrose induced tolerance in Arabidopsis thaliana plantlets. BMC Plant Biol. 9, 28 (2009). 38. Tseng, C. C., Lee, C. J., Chung, Y. T., Sung, T. Y. & Hsieh, M. H. Differential regulation of Arabidopsis plastid gene expression and RNA editing in non-photosynthetic tissues. Plant Mol. Biol. 82, 375–392 (2013). g j y p p 38. Tseng, C. C., Lee, C. J., Chung, Y. T., Sung, T. Y. & Hsieh, M. H. Differential regulation of Arabidopsis plastid gene expression and RNA editing in non-photosynthetic tissues. Plant Mol. Biol. 82, 375–392 (2013). 38. Tseng, C. C., Lee, C. J., Chung, Y. T., Sung, T. Y. & Hsieh, M. H. Differential regulation of Arabidopsis plastid gene expression RNA editing in non-photosynthetic tissues. Plant Mol. Biol. 82, 375–392 (2013). g p y ( ) 39. Pan, X., Welti, R. & Wang, X. Quantitative analysis of major plant hormones in crude plant extracts by high-performance liquid chromatography–mass spectrometry. Nat. Protoc. 5, 986–992 (2010). Z l k b h f h fi d f h b d b g p y 9. Pan, X., Welti, R. & Wang, X. Quantitative analysis of major plant hormones in crude plant extracts by high-performance liquid chromatography–mass spectrometry. Nat. Protoc. 5, 986–992 (2010).i g p y p y 0. Zimmerli, L., Jakab, G., Metraux, J. P. & Mauch-Mani, B. Potentiation of pathogen-specific defense mechanisms in Arabidopsis by beta-aminobutyric acid. Proc. Natl. Acad. Sci. USA 97, 12920–12925 (2000). www.nature.com/scientificreports/ 5. Liu, G. et al. Amino acid homeostasis modulates salicylic acid–associated redox status and defense responses in Arabidopsis. Plan Cell 22, 3845–3863 (2010). ( ) 16. Van Breusegem, F. & Dat, J. F. Reactive oxygen species in plant cell death. Plant Physiol. 141, 384–390 (2006).h 17. Dempsey, D. A., Vlot, A. C., Wildermuth, M. C. & Klessig, D. F. Salicylic acid biosynthesis and metabolism. The Arabidopsis American Society of Plant Biologists 9, e0156 (2011).h y f g 18. Chantranupong, L. et al. The CASTOR proteins are arginine sensors for the mTORC1 pathway. Cell 165, 153–164 (2016). h 9. Saxton, R. A., Chantranupong, L., Knockenhauer, K. E., Schwartz, T. U. & Sabatini, D. M. Mechanism of arginine sensing by CASTOR1 upstream of mTORC1. Nature 536, 229–233 (2016). 0. Gai, Z. et al. Structural mechanism for the arginine sensing and regulation of CASTOR1 in the mTORC1 signaling pathway. Cel Discov. 2, 16051 (2016).i 1. Xia, J., Wang, R., Zhang, T. & Ding, J. Structural insight into the arginine-binding specificity of CASTOR1 in amino acid-dependen mTORC1 signaling. Cell Discov. 2, 16035 (2016). g g 2. Lorrain, S., Vailleau, F., Balaque, C. & Roby, D. Lesion mimic mutants: keys for deciphering cell death and defense pathways in plants? Trends Plant Sci. 8, 263–271 (2003). p 3. Bruggeman, Q., Raynaud, C., Benhamed, M. & Delarue, M. To die or not to die? Lessons from lesion mimic mutants. Front. Plan Sci. 30(6), 24 (2015). ( ) ( ) 4. Torres, M. A., Jones, J. D. G. & Dangl, J. L. Reactive oxygen species signaling in response to pathogens. Plant Physiol. 141, 373–378 (2006) 5. Dempsey, D. A. & Klessig, D. F. How does the multifaceted plant hormone salicylic acid combat disease in plants and are similar mechanisms utilized in humans? BMC Biology 15, 23 (2017).it gy 6. Seifi, H. S., Van Bockhaven, J., Angenon, G. & Hofte, M. Glutamate metabolism in plant disease and defense: friend or foe? Mol Plant Microbe Interact. 26, 475–485 (2013). ( ) 27. Fagard, M. et al. Nitrogen metabolism meets phytopathology. J. Exp. Bot. 65, 5643–5656 (2014). Preston, G. M. Pseudomonas syringae pv. tomato DC3000 uses constitutive and apoplast-induced nutrient assimilation t b li t i t th t b d t i th t t l t M l Pl t Mi b I t t 21 269 282 (2008) 28. Rico, A. & Preston, G. M. Pseudomonas syringae pv. Author Contributions M.H.H. conceived and designed the experiments. S.K.S., T.Y.S., T.Y.C., S.Y.L., S.C.L., J.C.L. and W.Y.H. conducted the experiments. S.K.S., T.Y.S., T.Y.C. and M.H.H. analyzed the data. M.H.H. wrote the manuscript. All authors read and approve the final manuscript. References Direct interaction with ACR11 is necessary for post-transcriptional control of GLU1 encoded ferredoxin-dependent glutamate synthase in leaves. Sci. Rep. 6, 29668 (2016). Scientific Reports \| (2018) 8:11851 \| DOI:10.1038/s41598-018-30304-0 Scientific Reports \| (2018) 8:11851 \| DOI:10.1038/s41598-018-30304-0 8 www.nature.com/scientificreports/ www.nature.com/scientificreports/ tomato DC3000 uses constitutive and apoplast-induced nutrient assim pathways to catabolize nutrients that are abundant in the tomato apoplast. Mol. Plant Microbe Interact. 21, 269–282 (2008). 29. Chellamuthu, V. R. et al. A widespread glutamine-sensing mechanism in the plant kingdom. Cell 159, 1188–1199 (2014). 30. Kan, C. C., Chung, T. Y., Juo, Y. A. & Hsieh, M. H. Glutamine rapidly induces the expression of key transcription facto involved in nitrogen and stress responses in rice roots. BMC Genomics 16, 731 (2015). 1. Coschigano, K. T., Melo-Oliveira, R., Lim, J. & Coruzzi, G. M. Arabidopsis gls mutants and distinct Fd-GOGAT genes: implication for photorespiration and primary nitrogen assimilation. Plant Cell 10, 741–752 (1998). ration and primary nitrogen assimilation. Plant Cell 10, 741–752 ( p p p y g 2. Herrera-Vasquez, A., Salinas, P. & Holuigue, L. Salicylic acid and reactive oxygen species interplay in the transcriptional control o defense genes expression. Front. Plant Sci. 6, 171 (2015). g 33. Jamai, A., Salomé, P. A., Schilling, S. H., Weber, A. P. & McClung, C. R. Arabidopsis photorespiratory serine hydroxymethyltransferase activity requires the mitochondrial accumulation of ferredoxin-dependent glutamate synthase. Plant Cell 21, 595–606 (2009).f y q p g y 34. Shimojima, M., Hoffmann-Benning, S., Garavito, R. M. & Benning, C. Ferredoxin-dependent glutamate synthase moonligh plant sulfolipid biosynthesis by forming a complex with SQD1. Arch. Biochem. Biophys. 436, 206–214 (2005).hi 5. Hsieh, W. Y. et al. The Arabidopsis thiamin-deficient mutant palegreen1 lacks thiamin monophosphate phosphatase of the vitamin B1 biosynthesis pathway. Plant J. 91, 145–157 (2017). 6. Pogány, M. et al. Dual roles of reactive oxygen species and NADPH oxidase RBOHD in an Arabidopsis-Alternaria pathosystem Plant Physiol. 151, 1459–1475 (2009).f y 37. Ramel, F., Sulmon, C., Bogard, M., Couee, I. & Gouesbet, G. Differential patterns of reactive oxygen species and antioxid mechanisms during atrazine injury and sucrose-induced tolerance in Arabidopsis thaliana plantlets. BMC Plant Biol. 9, 28 (20 38. Tseng, C. C., Lee, C. J., Chung, Y. T., Sung, T. Y. & Hsieh, M. H. Differential regulation of Arabidopsis plastid gene expression RNA editing in non-photosynthetic tissues. Plant Mol. Biol. 82, 375–392 (2013). y 7. Ramel, F., Sulmon, C., Bogard, M., Couee, I. & Gouesbet, G. Differential patterns of reactive oxygen species and antioxidative mechanisms during atrazine injury and sucrose-induced tolerance in Arabidopsis thaliana plantlets. BMC Plant Biol. 9, 28 (2009). Additional Information upplementary information accompanies this paper at https://doi.org/10.1038/s41598-018-30304-0.h Competing Interests: The authors declare no competing interests. Competing Interests: The authors declare no competing interests. Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Cre- ative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not per- mitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. © The Author(s) 2018 Scientific Reports \| (2018) 8:11851 \| DOI:10.1038/s41598-018-30304-0 9
https://openalex.org/W4313547822	https://www.mdpi.com/2071-1050/15/1/594/pdf?version=1672311632	English	null	Associations between the Importance of Well-Being Domains and the Subsequent Experience of Well-Being	Sustainability	2,022	cc-by	9,218	Citation: Weziak-Bialowolska, D.; Lee, M.T.; Bialowolski, P.; McNeely, E.; Chen, Y.; Cowden, R.G.; VanderWeele, T.J. Associations between the Importance of Well-Being Domains and the Subsequent Experience of Well-Being. Sustainability 2023, 15, 594. https://doi.org/10.3390/su15010594 Academic Editor: Hyo Sun Jung Received: 14 November 2022 Revised: 23 December 2022 Accepted: 26 December 2022 Published: 29 December 2022 Citation: Weziak-Bialowolska, D.; Lee, M.T.; Bialowolski, P.; McNeely, E.; Chen, Y.; Cowden, R.G.; VanderWeele, T.J. Associations between the Importance of Well-Being Domains and the Subsequent Experience of Well-Being. Sustainability 2023, 15, 594. https://doi.org/10.3390/su15010594 Keywords: well-being; character strengths; social relationships; ﬁnancial stability; health; meaning and purpose; valuing well-being Article Associations between the Importance of Well-Being Domains and the Subsequent Experience of Well-Being lowolska 1,2,* , Matthew T. Lee 1,3, Piotr Bialowolski 1,4 , Eileen McNeely 5, Ying Chen 1,6, n 1 and Tyler J. VanderWeele 1,6 Dorota Weziak-Bialowolska 1,2,* , Matthew T. Lee 1,3, Piotr Bialowolski 1,4 , Eileen McNeel Richard G. Cowden 1 and Tyler J. VanderWeele 1,6 1 Human Flourishing Program, Harvard University, Cambridge, MA 02138, USA 2 Polish Institute of Advanced Studies, Polish Academy of Sciences, 00-378 Warsaw, Poland 3 Institute for Studies of Religion, Baylor University, Waco, TX 76798, USA 4 Department of Economics, Kozminski University, 03-301 Warsaw, Poland 5 Sustainability and Health Initiative (SHINE), Department of Environmental Health, Harvard T. H. Chan School of Public Health, Boston, MA 02115, USA 6 Department of Epidemiology, Harvard T. H. Chan School of Public Health, Boston, MA 02115, USA * Correspondence: doweziak@iq.harvard.edu Abstract: Prior cross-sectional research suggests that the importance assigned to well-being domains may be associated with actual self-reported well-being in these same domains. However, cross- sectional data cannot discern directionality, leaving an open question as to whether valuing well-being leads to higher actual well-being or the other way around—higher levels of well-being lead to valuing well-being more. In the present study, we used longitudinal data from 1209 employees to examine the associations between the perceived importance of six well-being domains (emotional health, physical health, meaning and purpose, social connectedness, character strengths, and ﬁnancial stability) and subsequent well-being in these domains reported approximately 1 year later. Lagged linear regression models demonstrated that valuing character strengths and valuing social relationships were most strongly associated with subsequent well-being. None of the valuations were associated with higher subsequent emotional well-being and only one (importance of physical health) predicted physical health. We also found that higher valuations of character strengths and physical health were associated with lower ratings of subsequent ﬁnancial stability. A stronger sense of the importance of each well-being domain was predictive of subsequent character strengths. Our ﬁndings suggest that living well appears to be achieved by valuing immaterial goods, especially social connectedness and character strengths, as opposed to domains such as ﬁnancial stability or physical health. sustainability sustainability sustainability sustainability 1. Introduction Received: 14 November 2022 Revised: 23 December 2022 Accepted: 26 December 2022 Published: 29 December 2022 Research on subjective well-being is increasingly seeking to integrate multiple do- mains, reﬂecting an emerging interest in “complete well-being” [1–4]. The notion of complete well-being minimally requires “doing or being well” in all well-being domains [3]. These domains comprise emotional health, purpose in life, social connectedness, character strengths, physical health, and ﬁnancial security [3,5], and have been shown to be highly valued by people [3,6]. Most people throughout the world would also insist on including additional well-being domains, such as spirituality [7] or inner peace [8]. Consideration of community well-being, which involves more than a simple aggregation of individuals’ self-reported well-being, is also desirable [9]. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). Each individual domain of well-being has been thoroughly studied over the years, and some have been included as part of more comprehensive measures. Research on character strengths as an integral facet of well-being has emerged only recently [see for example 6]. Thus, this domain is somewhat underdeveloped relative to the others and is https://www.mdpi.com/journal/sustainability Sustainability 2023, 15, 594. https://doi.org/10.3390/su15010594 Sustainability 2023, 15, 594 2 of 13 rarely included in multidimensional measures of well-being [3,4,10]. However, it has been a perennial focus in disciplines of the humanities, such as philosophy and theology. It has also received increased attention in the social sciences in recent decades [11,12]. Although the character strengths domain has largely been avoided in some disciplines, it implicitly animates many contemporary scholarly debates [13]. The role of character strengths seems especially relevant for other domains of well-being. In particular, recent research found that it predicts future emotional health, physical health, social connectedness, and meaning and purpose [14–17]. What remains uncertain is whether valuing character strengths serves as a determinant of these other well-being domains. g Similarly, despite the fact that there is a large body of research on physical health, this domain is not usually included in measures of well-being [3]. The same is true for the ﬁnancial stability domain [3,18]. However, nearly all well-being measures include items related to emotional health. A single dimension of this domain (life satisfaction) has itself been divided into 173 distinct sub-domains [19]. Unlike most other frameworks, the framework proposed by VanderWeele [3], which informed the design of the measure that is used in this study, incorporates both the neglected and the frequently included domains of well-being. g Life circumstances may require people to make trade-offs among various well-being domains. There is evidence that some people value health over happiness and life satis- faction [20–22] or other way around—life satisfaction over health [23]; others place health above their ﬁnancial situation [24]. Nevertheless, it is generally preferable to do or be well in all well-being domains when that is possible. Prior cross-sectional research has shown that the six abovementioned domains of well- being are indeed all highly valued and that this valuation is correlated with the experience of the same set of well-being domains [6]. It remains unknown whether valuing certain well-being domains may lead to a better subsequent experience of well-being, a relationship that has not been assessed in previous research. Therefore, the current study poses two research questions: (1) How is the importance of a particular well-being domain associated with the subse- quent experience of well-being in the same domain? and (2) How is the importance of a particular well-being domain associated with the subse- quent experience of well-being in other domains? Since prior research has found that valuing domains of well-being is positively cor- related with domains of actual self-reported well-being [6], in this study we tested the following research hypotheses: H1. The importance of a particular well-being domain will be positively associated with the subsequent experience of well-being in the same domain. H2. The importance of a particular well-being domain will be positively associated with the subsequent experience of well-being in other well-being domains. H2. The importance of a particular well-being domain will be positively associated with the subsequent experience of well-being in other well-being domains. To test these hypotheses, we used longitudinal data and applied a lagged regression analytic design. Our methodological approach includes adjustment for baseline values of the outcomes, which decreases the risk of reverse causality and provides insight into time- ordered associations among variables [25]. This analytic design has the potential to offer more robust support for directionality of the associations, as it controls for the possibility that higher well-being in certain domains might also lead to valuing those domains more. 2.1. Participants Randomly sampled employees of a large, US company were invited to participate in the ﬁrst wave of the Well-Being Survey in June 2018. Out of 15,000 employees of at least 18 years of age that were invited, 2370 agreed to participate and provided responses (response rate 15.8%). The following year, participants were invited to complete the second Sustainability 2023, 15, 594 3 of 13 wave of the study (July 2019). A total of 1209 employees participated in both waves. This group constituted the analytic sample for this study. Table 1 presents the sociodemographic characteristics of the sample at baseline. Fe- males accounted for 84.5% of the sample (74.5% of employees in the organization were female). The mean age of the sample was 43.5 years (mean age of 45.6 years in the organiza- tion). Participants were mostly White and relatively well-educated ofﬁce employees, which was also consistent with the structure of employees in the target population. Approximately 72% of participants reported owning a home, 62% were married, and 48% indicated that they were caring for at least one minor child. Most participants voted in prior elections (82%). Roughly 28% of the sample reported engaging in daily spiritual practices, and about 20% attended religious services at least once a week. Nearly 10% volunteered at least once a week. The retention rate from the ﬁrst wave was 51.2%. Participants who did not complete the second wave were signiﬁcantly more likely to be young, male, non-White, not married, and not homeowners [14]. For both surveys, an email system within the organization was used to conduct a communication campaign (one week prior to survey administration), distribute letters of invitation, and send participation reminders. The surveys were also administered online, allowing participants to provide responses in a secure and anonymous space of their choice. Table 1. Participant characteristics at study baseline (N = 1209). 2.2.1. Predictor Variables We measured the self-reported importance of the following six domains of well-being with single items: emotional health (“How important is being happy and satisﬁed with life, having good mental health, and being in control and able to deal with difﬁcult emotions?”); meaning and purpose (“How important is having a sense of meaning in life, a direction to one’s activities, and pursuing what is most important?”); social connectedness (“How important is having close, meaningful, and supportive relationships and being respected by and connected to community?”); character strengths (“How important is having consistent thoughts and actions that contribute to the good of oneself and others?”); physical health (“How important is being sufﬁciently healthy to be able to carry out the important tasks in life now and into the future?”); and ﬁnancial security (“How important is having sufﬁcient ﬁnancial resources and planning so as to be able to pursue one’s life goals and not overly worry about making ends meet?”). Scores ranged from 0 “not important at all” to 10 “extremely important.” The correlation matrix for the self-reported importance variables at baseline is presented in Table S1 in the Supplementary Material. Table S2 in the Supplementary Material presents the cross-lagged correlations between the importance variables after adjusting for the control variables. Table 1. Cont. Table 1. Cont. Baseline Characteristic Statistic Spiritual practices, % Daily 28.2 Not daily but more than once a week 24.8 Once a week 8.3 1–3 times a month 12.8 Once every few months or once a year 17.8 Never 8.2 Volunteering, % More than once a week 5.03 Once a week 4.7 1–3 times a month 12.8 Once every few months or once a year 50.5 Never 26.9 Participation in community groups, % More than once a week 10.1 Once a week 8.4 1–3 times a month 15.8 Once every few months or once a year 33.9 Never 31.9 Salary (USD), mean (SD) 73,117 (34,259) Salary (USD), % <40,000 12.3 40,000–49,999 16.3 50,000–59,999 13.5 60,000–69,999 22.5 70,000–79,999 3.6 80,000–99,999 13.8 100,000–119,999 11.8 120,000–139,999 0.0 140,000+ 6.2 1 1 This table was adapted from [26]. The statistics reported in this table are based on non-imputed data. Baseline Characteristic 2.1. Participants Baseline Characteristic Statistic Gender (females), % 84.5 Age (years), mean (SD) 43.5 (10.4) Age, % ≤30 years 11.8 31–40 years 29.9 41–50 years 29.0 >50 years 29.3 Race, % White 74.3 Black or African American 12.2 Hispanic/Latino 6.7 Asian 5.1 Other 1.8 Marital status, % Single/never married 16.2 Married 62.4 Divorced 10.1 Widowed 1.3 Separated 1.3 Non-married partner 8.7 Education, % High school 7.8 Some college but no degree 22.6 Associate degree 14.0 Bachelor’s degree 35.0 Graduate school or higher 20.7 Has child dependents, % 48.1 Has older adult dependents, % 27.2 Owns a home, % 72.3 Voted in previous elections, % 82.4 Religious service attendance, % More than once a week 5.9 Once a week 14.6 1–3 times a month 11.6 Once every few months or once a year 39.8 Never 28.1 Table 1. Participant characteristics at study baseline (N = 1209). 4 of 13 Sustainability 2023, 15, 594 2.2.3. Control Variables We included a rich set of control variables that are known to affect well-being. First, we controlled for demographics including: (1) gender (male vs. female), (2) age (≤30, 31–40, 41–50, >50), (3) race (White, Black/African American, Hispanic/Latino, Asian, other), (4) educational attainment (high school, some college, associate degree, bachelor’s degree, graduate degree), (5) marital status (single/never married, married, divorced, widowed, separated, non-married partner), (6) number of child dependents under the age of 18 living in the home, (7) caring for one or more older adult dependent living in the home (yes, no), and (8) wealth [owning a house (yes, no)], and (9) income (salary based on mid-point salary bands obtained from the human resource department of the employer). A number of studies have shown that these variables are predictive of various well-being domains [27–30]. We also controlled for religious service attendance (more than once a week, once a week, 1–3 times a month, once every few months or once a year, never), spiritual practices (daily, not daily but more than once a week, once a week, 1–3 times a month, once every few months or once a year, never), volunteering (more than once a week, once a week, 1–3 times a month, once every few months or once a year, never), participation in community groups (more than once a week, once a week, 1–3 times a month, once every few months or once a year, never), and voting in the last election (yes vs. no/not sure/not registered voter). Prior research indicated that these factors can inﬂuence various domains of well-being, including both emotional and physical health [31–36]. In addition to adjusting for all of the abovementioned control variables assessed in the ﬁrst wave (the same wave as the predictor variables), we attempted to reduce the risk of reverse causality by adjusting for complete well-being assessed in the ﬁrst wave (a composite of all six well-being domains computed as an arithmetic average). 2.2.2. Outcome Variables Our analysis builds upon research that has validated a 40-item comprehensive well- being assessment [5,6], which assesses actual self-reported well-being in six domains: (1) emotional health; (2) physical health; (3) meaning and purpose; (4) character strengths; Sustainability 2023, 15, 594 5 of 13 5 of 13 (5) social connectedness; and (6) ﬁnancial security. A sample item from the emotional health domain is, “Overall, how satisﬁed are you with life as a whole these days?” Response categories ranged from 0 (not satisﬁed at all) to 10 (completely satisﬁed). A sample item from the character strengths domain is, “I get to use my strengths to help others” (0 = not true of me, 10 = completely true of me). Exact wording of all items can be found in [5]. The measure has good psychometric properties, including evidence of construct valid- ity, convergent and discriminant validity, and test–retest reliability, as well as satisfactory internal consistency for domain-speciﬁc and overall scores [5]. The utility of the overall measure and separate sub-scales has also been demonstrated in several recent studies of well-being [14,16,26]. g In this study, we calculated scores for each domain by averaging the responses across all items on a given domain. Since some items are negatively oriented, relevant items were reverse coded to ensure that a higher score implies greater well-being. 3. Results Table 2 displays the results of our primary analysis. Focusing on the outcomes by examining the table columns, we ﬁnd that none of the valuations (including importance of emotional health) predicted subsequent emotional health, all of the valuations predicted subsequent character strengths, and all but one valuation (the importance of ﬁnancial stabil- ity) predicted subsequent meaning and purpose. Additionally, valuing social connectedness and character strengths predicted the subsequent experience of social connectedness. Only the importance of physical health was associated with the subsequent experience of phys- ical health, and only two valuations were associated—both inversely—with subsequent ﬁnancial stability (the importance of character strengths and physical health). Table 2. The prospective associations between the importance of well-being domains and subsequent experience of well-being (standardized estimates/betas and 95% conﬁdence intervals, N = 1209). Table 2. The prospective associations between the importance of well-being domains and subsequent experience of well-being (standardized estimates/betas and 95% conﬁdence intervals, N = 1209). Table 2. The prospective associations between the importance of well-being domains and subsequent experience of well-being (standardized estimates/betas and 95% conﬁdence intervals, N = 1209). Emotional Health Meaning and Purpose Social Connectedness Character Strengths Financial Stability Physical Health Importance of emotional health 0.022 (−0.022; 0.067) 0.071 (0.025; 0.119) 0.041 (−0.005; 0.087) 0.079 (0.030; 0.129) −0.021 (−0.066; 0.024) 0.034 (−0.015; 0.083) Importance of meaning and purpose 0.009 (−0.038; 0.056) 0.107 * (0.059; 0.155) 0.037 (−0.011; 0.085) 0.164 * (0.113; 0.215) −0.035 (−0.081; 0.011) 0.013 (−0.039; 0.064) Importance of social connectedness 0.034 (−0.011; 0.078) 0.100 * (0.053; 0.147) 0.114 * (0.068; 0.160) 0.120 * (0.070; 0.170) −0.008 (−0.053; 0.038) 0.043 (−0.010; 0.095) Importance of character strengths 0.006 (−0.044; 0.056) 0.092 * (0.041; 0.144) 0.068 (0.020; 0.116) 0.215 * (0.164; 0.265) −0.050 * (−0.097; −0.003) −0.009 (−0.062; 0.043) Importance of ﬁnancial stability −0.011 (−0.056; 0.033) 0.016 (−0.030; 0.062) −0.011 (−0.058; 0.035) 0.147 *** (0.100; 0.195) −0.034 (−0.078; 0.011) 0.034 (−0.015; 0.084) Importance of physical health 0.035 (−0.010; 0.080) 0.049 * (0.003; 0.096) 0.023 (−0.022; 0.069) 0.135 * (0.086; 0.184) −0.059 (−0.103; −0.015) 0.060 * (0.010; 0.109) * p < 0.05, p < 0.01, * p < 0.001; The p-value cut-off for Bonferroni correction = 0.05/36 = 0.0014; Estimates signiﬁcant after correcting for multiple testing are underscored. Importance variables were measured in June 2018. Well-being outcomes were measured in July 2019. 2.3. Analytic Strategy The prospective associations were examined using a series of lagged linear regression models with control for the prior outcome and extensive set of covariates. Standardized regression estimates (betas) were presented. Consequently, a set of 36 regression models was used to regress each of the six well-being outcomes on each of the six self-reported importance measures. In particular, the association between a self-reported importance of well-being domain j and a well-being outcome k was modelled as follows: (1) WBk,j,i(T = 2) = α0,k,j + α1,k,jIWBj,i(T = 1) + α2,k,jXi(T = 1) + ηk,j,i (1) where i = 1, . . . , N; k = 1, . . . , 6; j = 1, . . . , 6. where i = 1, . . . , N; k = 1, . . . , 6; j = 1, . . . , 6. Subscript i represents an individual, the variable WB indicates one out of six (k = 1, . . . , 6) well-being outcomes, IWB is one out of six importance of well-being variables (j = 1, . . . , 6). X is a vector of control variables including the first wave (T = 1) general well-being measure. α1,k,j reflects an association between an importance of well-being predictor (j) and a subsequent Sustainability 2023, 15, 594 6 of 13 well-being outcome (k). α2,k,j is a vector that shows the association between control variables and the well-being outcome (k), and ηk,j,i is a disturbance term. well-being outcome (k). α2,k,j is a vector that shows the association between control variables and the well-being outcome (k), and ηk,j,i is a disturbance term. j All missing values on the predictor, covariate, and outcome variables were imputed using chained equations (10 datasets were generated) [37,38]. Data were arranged in a wide format as suggested by Allison [39] and all outcome, predictor, and control variables were used in the procedure. We used Rubin’s formula [40] to pool estimates derived from regression models that were performed with each of the imputed datasets. To examine the robustness of the results to missing data patterns, we reanalyzed the primary sets of models using complete cases. For both the primary and complete-case analyses, Bonferroni correction was used to correct for multiple testing. The interpretation of results focuses on unadjusted results. All statistical computations were performed using Stata 15. 3. Results We controlled for 2018 well-being levels, race, age, gender, marital status, voting, education, home ownership, salary, child dependents, older adult dependents, religious service attendance, spiritual practices, community participation, and volunteering. * p < 0.05, p < 0.01, * p < 0.001; The p-value cut-off for Bonferroni correction = 0.05/36 = 0.0014; Estimates signiﬁcant after correcting for multiple testing are underscored. Importance variables were measured in June 2018. Well-being outcomes were measured in July 2019. We controlled for 2018 well-being levels, race, age, gender, marital status, voting, education, home ownership, salary, child dependents, older adult dependents, religious service attendance, spiritual practices, community participation, and volunteering. * p < 0.05, p < 0.01, * p < 0.001; The p-value cut-off for Bonferroni correction = 0.05/36 = 0.0014; Estimates signiﬁcant after correcting for multiple testing are underscored. Importance variables were measured in June 2018. Well-being outcomes were measured in July 2019. We controlled for 2018 well-being levels, race, age, gender, marital status, voting, education, home ownership, salary, child dependents, older adult dependents, religious service attendance, spiritual practices, community participation, and volunteering. Looking at the rows, the importance of character strengths and importance of physical health were both associated with four outcomes (including a negative association with ﬁnancial stability). Valuing social connectedness was the next most consistent predictor of the outcomes, evidencing associations with subsequent meaning and purpose, social connectedness, and character strengths. The importance of ﬁnancial stability predicted only one outcome (character strengths). The examination of beta estimates measured across importance domains indicated the strongest link between importance and character strengths. The beta coefﬁcients for this outcome were the highest compared to other well-being domains. This might indicate an Sustainability 2023, 15, 594 7 of 13 especially substantial role of importance in cultivating character strengths. The weakest associations found for the set of importance indicators were with the emotional health domain, suggesting that emotional health might not be easily shaped by valuing well- being domains. The importance of speciﬁc domains can be linked to positive changes in the corre- sponding domain of well-being, but these valuations also affect other domains. In order to understand the strength of these two types of associations, we examined the summary associations between valuations and both corresponding domains and other domains. 3. Results The effects on the diagonal, which reﬂect the prospective association between a speciﬁc impor- tance domain and its corresponding subsequent well-being domain, yielded an average beta of 0.092 (absolute values were taken into account as the aim was to compare the strength of associations). The off-diagonal betas, which explain cross-relationships between importance and well-being domains, were weaker and averaged 0.052. p g g Results of the complete case analysis were very similar to those from the primary analy- sis based on multiply imputed data (see Table S3 in Supplementary Material). Directionality of all associations was preserved and standardized regression coefﬁcients were similar, with the conﬁdence intervals of comparable width. However, the association between the importance of character strengths and the subsequent experience of ﬁnancial stability no longer excluded the null in the complete-case scenario. Overall, this analysis provided evidence for the robustness of our results to missing data pattern. We also examined the correlations between the importance of domain ratings (see Table S1 in Supplementary Material). All were positive, with the ﬁnancial stability and emotional health indicators of importance evidencing the weakest correlation and character strengths importance ratings and meaning and purpose importance ratings being correlated the most. As a supple- mentary analysis, we also examined the longitudinal associations of the 2018 domain importance ratings with subsequent 2019 domain importance ratings, adjusting for control variables (see Table S2 in Supplementary Material). All associations were positive, with the weakest average association obtained for ﬁnancial stability importance. 4. Discussion There is an abundance of research on the centrality of social connections and character strengths for the ﬂourishing life [3,16,21,44], but until now the only evidence for an associa- tion between valuing these two domains and self-reported well-being in these domains was based on cross-sectional data [6]. Our longitudinal ﬁndings align with these previous results, which also found the strongest correlations between valuations and subsequent well-being for these two domains. It is not surprising that valuing social connections is associated with subsequent well- being. Although valuations were not directly tested, this is one of the primary lessons from the Harvard Grant Study, a longitudinal investigation of human development of a single cohort of men that is now in its 8th decade [45]. After spending more than $20 million to follow these men throughout the course of their lives, one of the study directors famously declared, “Happiness is love. Full stop” (Vaillant, quoted in [45]). The quality of close relationships decades earlier was the most important predictor of a range of well-being outcomes, including happiness, physical health, and longevity, leading study’s current director to declare, “Taking care of your body is important, but tending to your relationships is a form of self-care too” (Waldinger, quoted in [46]). g q Beyond tending to relationships, our ﬁndings suggest that simply valuing connect- edness is associated with an enhanced sense of purpose, the development of character strengths, and the experience of more satisfying social relationships. People who are social- ized to appreciate the importance of connectedness may be more likely, as Waldinger put it, to “tend” to their relationships in more effective ways, which could, in turn, increase the experience of well-being in a number of domains. It is interesting that the importance of connections was not associated with emotional health, but in fact none of our importance- related predictors showed evidence of association with this domain of well-being. These results add to prior evidence on the associations between mental health and temporary prior social connectedness [47], character strengths [15,16], meaning and purpose [41,48], and ﬁnancial well-being [26]. However, they also highlight a crucial difference. Although certain well-being domains may be prospectively associated with one another, the same may not be true for valuations of well-being domains. 4. Discussion Building on a foundation established by previous cross-sectional research [6], our lon- gitudinal analysis explored the prospective associations between valuing core domains of well-being and the subsequent experience of well-being in these same domains. Providing some support for our hypotheses H1 and H2, we found that most associations were positive. However, we also found that none of the valuations predicted subsequent emotional health, and two valuations were inversely associated with subsequent ﬁnancial stability (the im- portance of character strengths and physical health), which was at adds with both tested research hypotheses. Regarding our ﬁrst research question, the results indicated that the importance of a particular well-being domain is associated with subsequent experience of well-being in the same domain for all domains except emotional health and ﬁnancial stabil- ity. Regarding our second research question concerning the cross-domain associations, we found that valuing two domains—character strengths and social connectedness—yielded the strongest associations with other well-being domains, while valuing character strengths and physical health were prospectively associated with the highest number of well-being domains. Our results also showed that the strongest association was observed between the importance of character strengths and the prospective self-reports of character strengths. Additionally, self-reports of subsequent emotional health (measured in the second wave) were found to be independent of the importance attached to well-being domains in the ﬁrst wave (which provided no support for hypothesis H1), while each of the valuations pre- dicted subsequent character strengths and ﬁve out of six valuations predicted subsequent meaning and purpose (supporting research hypotheses H1 and H2). Our results indicating that valuing character strengths, emotional health, social con- nectedness, and sense of meaning and purpose predict subsequent sense of meaning and purpose in life add to evidence on the predictors of purpose and meaning in life that has Sustainability 2023, 15, 594 8 of 13 8 of 13 previously been reported, such as mental well-being, social connections, and having a sense of purpose while at work [41–43]. A particularly important contribution of the present study is the ﬁnding that importance attached to well-being domains is associated with subsequent self-reported character strengths, which has yet to receive empirical attention. 4. Discussion It may be that valuing various as- pects of well-being, while possibly leading to actions that enhance well-being, also perhaps leads to higher standards, thus making it more difﬁcult to be emotionally satisﬁed. In this vein, our results are somewhat at odds with prior research reporting that individuals are inclined to trade-off levels of happiness with levels of physical health [20], and that these trade-offs substantially depend on the individual’s own levels of happiness and health [22]. Speciﬁcally, it has been reported that unhappy people are more likely to choose unhappy lives and unhealthy people are more inclined to prefer unhealthy life [22]. Principles derived from Acceptance and Commitment Therapy [49] might partially account for the lack of associations between valuing emotional health and subsequent self-reported domains of well-being. From this perspective, feeling happy and having good mental health might result from initially prioritizing the ability to embrace negative emotions, seek deeper meaning, and especially strive to live according to deeper values. It must be noted, however, that these emotional health effects may materialize over a longer time frame than the one-year period covered by our study. These principles might, however, help to explain why valuing character strengths in our study was associated with the subsequent experience of greater meaning and purpose, enhanced social connectedness, and higher reported character strengths. g p g As we have noted, valuing emotional health was the least inﬂuential importance do- main for subsequent well-being, whereas individuals assigning value to character strengths were most likely to emerge with higher well-being. Similarly, valuing ﬁnancial stability was only associated with one subsequent well-being domain—character strengths. Long- Sustainability 2023, 15, 594 9 of 13 standing philosophical and religious traditions offer abundant reasons why placing a higher value on character development, as opposed to emotional health or ﬁnancial stability might result in beneﬁts in other domains of well-being [3,12,14]. Empirical research has been able to conﬁrm some of the pathways that we would expect in light of the wisdom of the humanities. To take just one example, it appears that it is indeed “better to give than to receive”, as a preoccupation with hedonistic enjoyment may adversely affect a sense of inner peace or personal growth, while participation in benevolent service to others is associated with a variety of positive well-being outcomes [8,35]. 4. Discussion In the same vein, we found that the increased importance of character strengths showed little evidence of association with the domains of physical health and emotional health, and was negatively associated with the ﬁnancial stability domain. We speculate that people who value “doing the right thing” might make sacriﬁces to help others. These actions, in turn, might adversely affect their well-being in one of these domains, including compromising their short-term ﬁnancial situation or sense of positive affect. Future research is needed to more fully explain these ﬁndings. In the meantime, such patterns suggest that the connection between human well- being and sustainable growth requires more direct attention, as hedonistic and material well-being might be at odds with other domains of well-being [6]. Previous research has found that a single survey item related to character strengths (“I always act to promote good in all circumstances, even in difﬁcult and challenging situations”) was associated with a variety of well-being outcomes, including mental health, physical health, social connectedness, and purpose [14,15]. This suggests that actively engaging in behaviors that reﬂect good character might be more inﬂuential on other well- being domains than simply valuing character strengths. However, we also note that our study found that valuing all six of the well-being domains predicted the subsequent experience of character strengths, which was measured by seven survey items, including an item that is similarly worded to the item used in this previous study (“I am willing to face difﬁculties in order to do what is right”). It is possible that valuing these other well-being domains may affect subsequent well-being in a variety of domains through the pathway of improved character strengths. For example, if an individual values physical health, that person might develop a disciplined commitment to exercise (e.g., running outside even when the weather is cold), which might then affect several domains of well-being (e.g., emotional or physical health) [50]. In this example, the character trait of discipline is shaped by valuing physical health. We are not able to test this hypothesis with our data, but future empirical studies could incorporate a research design that would permit the investigation of this possibility. 4.2. Limitations There are several limitations to our study. First, our self-reported data may be subject to unmeasured confounding or social desirability bias [55]. Further research might be conducted with other types of data (e.g., objective markers of health and well-being, such as health care records and clinical diagnoses). Second, we relied on single items for our measures of the importance of domains, and we hope that our ﬁndings encourage others to strengthen measurement in this area to further advance research along these lines. Third, our sample was drawn from a single workplace, whereas a randomized sample drawn from a broader community would expand the potential for generalizability. Fourth, our baseline response rate was low, and the rate of attrition at follow-up was around 50%. It is worth noting, however, that although the response rate and follow-up rate were suboptimal, participants retained in the analytic sample were similar to the employee population with respect to most major sociodemographic characteristics. Fifth, a longitudinal analysis with three waves of data would be preferable to two waves, as the prior values of outcomes could then be measured prior to the independent variables (i.e., at the pre-baseline wave rather than at the baseline wave). Adjusting for well-being levels measured simultaneously at baseline with independent variables may partially block some of the effects that the importance of the domains might have on subsequent well-being [25]. 4.1. Implications Our ﬁndings also suggest that learning to place more value on some domains of well-being, such as character strengths or social connections, might lead to the greatest increases in subsequent self-reported well-being. This provides guidance on targeted interventions. For example, if resources are limited, interventions might be designed to encourage increased valuation of one or both of these domains. Effects could then be assessed in terms of improved well-being across the full range of domains, ideally in a randomized controlled trial. Future research could also seek to disentangle the reasons why valuations are related to subsequently reported well-being. For example, people might value the domains that they are skillful at achieving or experiencing more (such as in [22] for health and happiness), leading to increases in the experience of these domains, and in turn leading to higher valuations—a virtuous cycle. The failure to achieve a high level of a speciﬁc domain might be associated with devaluing that domain, or perhaps the opposite, as an individual might more strongly desire what has been difﬁcult to obtain. Developmental stages in life might also shape these patterns. Longitudinal data with more than two waves would be required to explore how these associations might unfold across the life course. Sustainability 2023, 15, 594 10 of 13 10 of 13 Findings from this research can be also informative for social policy. High valuations of character strengths proved to be pivotal for improved meaning and purpose, and social connectedness. They were also instrumental in building character strengths, which might be indicative of a possible virtuous circle in this domain. The role of character strengths could be strengthened in the educational process with emphasis on such character strengths as moral compass, self-regulation, perseverance, and zest. The other domain with possible scope for policy actions is meaning and purpose. People striving for meaningful and purposeful life not only tend to lead such lives more often but are also able to build their character strengths. The importance of meaningful and purposeful life can be conveyed in the educational process by showing possible alleys for personal growth and present- ing their well-recognized impacts for longevity [51,52] and lower risk of cardiovascular diseases [53,54]. 5. Conclusions and E.M. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the following organizations, listed alphabetically: Aetna Inc., grant number A33796, the John Templeton Foundation, grant numbers 52125 and 61075, the Kern Family Foundation Award, grant number 2019-01467, the Levi Strauss Foundation, grant number 44057265, the Polish Institute of Advanced Studies, grant titled: “What makes us feel a sense of purpose in life?”, the Robert Wood Johnson Foundation, grant number 74275, and anonymous donors to the Sustainability and Health Initiative for NetPositive Enterprise at the Harvard T. H. Chan School of Public Health. Institutional Review Board Statement: The study was conducted in accordance with the Declaration of Helsinki and approved by the Harvard Longwood Medical Area Institutional Review Board. Informed Consent Statement: Informed consent was obtained from all subjects involved in the study Data Availability Statement: The data presented in this study are available on reasonable request from the corresponding author. The data are not publicly available due to privacy restrictions required by the organization that collected the data for this study. Conﬂicts of Interest: The author(s) disclosed receipt of the following ﬁnancial support for the research, authorship, and/or publication of this article: McNeely reports receiving grants and personal fees from Aetna Inc. and from the Levi Strauss Foundation. She also reports serving as director of SHINE at Harvard (Sustainability and Health Initiative for NetPositive Enterprise); Support is made possible through SHINE from multiple companies. VanderWeele reports receiving grants and personal fees from Aetna Inc. and from the John Templeton Foundation. Other authors have no conﬂicts of interest to declare. The research ﬁndings represent the perspective of the authors and do not reﬂect the opinions or endorsement of any organization. References 1. Keyes, C.L.M. The Mental Health Continuum: From Languishing to Flourishing in Life. J. Health Soc. Behav. 2002, 43, 207. [CrossRef] 2. Lee, M.T.; Kubzansky, L.D.; VanderWeele, T.J. Measuring Well-Being: Interdisciplinary Perspectives from the Social Sciences and the Humanities, 1st ed.; Oxford University Press: Oxford, UK, 2021. y 3. VanderWeele, T.J. On the promotion of human ﬂourishing. Proc. Natl. Acad. Sci. USA 2017, 114, 8148 3. VanderWeele, T.J. On the promotion of human ﬂourishing. Proc. Natl. Acad. Sci. USA 2017, 114, 8148–8156. [CrossRef] [PubMed] 4. Seligman, M.E.P. Flourish: A Visionary New Understanding of Happiness and Well-Being; Free Press: New York, NY, USA, 2012. 3. VanderWeele, T.J. On the promotion of human ﬂourishing. Proc. Natl. Acad. Sci. USA 2017, 114, 8148 4. Seligman, M.E.P. Flourish: A Visionary New Understanding of Happiness and Well-Being; Free Press: N 3. VanderWeele, T.J. On the promotion of human ﬂourishing. Proc. Natl. Acad. Sci. USA 2017, 114, 8148 8156. [CrossRef] [PubMed] 4. Seligman, M.E.P. Flourish: A Visionary New Understanding of Happiness and Well-Being; Free Press: New York, NY, USA, 2012. 5. Weziak-Bialowolska, D.; Bialowolski, P.; Lee, M.T.; Chen, Y.; VanderWeele, T.J.; McNeely, E. Psychometric Scales from a Comprehensive Well-Being Assessment. Front. Psychol. 2021, 12, 652209. [CrossRef] [Pub k Bialowolska, D.; Bialowolski, P.; Lee, M.T.; Chen, Y.; VanderWeele, T.J.; McNeely, E. Psychometric Properti from a Comprehensive Well-Being Assessment. Front. Psychol. 2021, 12, 652209. [CrossRef] [PubMed] 6. Lee, M.T.; Bialowolski, P.; Weziak-Bialowolska, D.; Mooney, K.; Lerner, P.E.M.; VanderWeele, T.J. Self-Assessed Importance of Domains of Flourishing: Demographics and Correlations with Well-Being. J. Posit. Psychol. 2021, 16, 137–144. [CrossRef] 7. Kee, Y. Multi-dimensional Model of Community Well-Being from a Public Service Delivery Perspective. In Handbook of Community Well-Being Research; Phillips, R., Wong, C., Eds.; International Handbooks of Quality-of-Life; Springer: Dordrecht, The Netherlands, 2017. [CrossRef] 8. Xi, J.; Lee, M.T. Inner peace as a contribution to human ﬂourishing. In Measuring Well-Being: Interdisciplinary Perspectives from the Social Sciences and the Humanities; Oxford University Press: Oxford, UK, 2021; pp. 435–481. y pp 9. Sirgy, M.J.; Widgery, R.N.; Lee, D.J.; Yu, G.B. Developing a measure of community well-being based on perceptions of impact in various life domains. Soc. Indic. Res. 2010, 96, 295–311. [CrossRef] 9. Sirgy, M.J.; Widgery, R.N.; Lee, D.J.; Yu, G.B. Developing a measure of community wel various life domains. Soc. Indic. Res. 2010, 96, 295–311. [CrossRef] 10. Hone, L.C.; Jarden, A.; Schoﬁeld, G.M.; Duncan, S. 5. Conclusions This research showed that the importance of character strengths and the importance of physical health, followed by the importance of social connectedness, were the most consistent predictors of well-being outcomes. However, we also found that none of the val- uations predicted subsequent emotional health, the importance of physical health predicted only physical health, and only two valuations were associated—both inversely—with subsequent ﬁnancial stability (the importance of character strengths and physical health). Our research provided further evidence that living well appears best achieved by valuing immaterial goods, especially social connectedness and character strengths, as opposed to domains such as ﬁnancial stability or physical health. This has important impli- cations for sustainable growth, as our main ﬁndings are consistent with a “politics of being” rather than a cultural emphasis on “having” that is associated with resource-intensive eco- nomic systems rooted in environmentally degrading forms of consumerism [56]. Character strengths emerged as the only well-being domain that was predicted by the valuation of all other well-being domains. As valuing the domains of well-being is concerned with some of the most important ends of human life, evidence of associations between these valuations and the subsequent experience of the domain of character strengths suggests that character is indeed a pathway to attaining a life of ﬂourishing. Sustainability 2023, 15, 594 11 of 13 11 of 13 Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/su15010594/s1, Table S1: Correlation matrix for the self-reported importance variables at study baseline (N = 1209); Table S2: The cross-lagged correlations adjusted for the control variables (N = 1209); Table S3: The effect of the importance of well-being domains on the subsequent experience of well-being—complete case scenario (standardized estimate and 95% conﬁdence intervals). Author Contributions: Conceptualization, M.T.L., D.W.-B., P.B., Y.C., R.G.C., E.M. and T.J.V.; method- ology, D.W.-B., M.T.L., P.B., Y.C., R.G.C., E.M. and T.J.V.; formal analysis, D.W.-B.; writing—original draft preparation, M.T.L., D.W.-B. and P.B.; writing—review and editing, D.W.-B., P.B., M.T.L., R.G.C., Y.C., E.M. and T.J.V.; project administration, E.M. and T.J.V.; funding acquisition, T.J.V. and E.M. All authors have read and agreed to the published version of the manuscript. Author Contributions: Conceptualization, M.T.L., D.W.-B., P.B., Y.C., R.G.C., E.M. and T.J.V.; method- ology, D.W.-B., M.T.L., P.B., Y.C., R.G.C., E.M. and T.J.V.; formal analysis, D.W.-B.; writing—original draft preparation, M.T.L., D.W.-B. and P.B.; writing—review and editing, D.W.-B., P.B., M.T.L., R.G.C., Y.C., E.M. and T.J.V.; project administration, E.M. and T.J.V.; funding acquisition, T.J.V. 11. Schmidt, P.F. The Character Assessment Scale: A New Tool for the Counselor. J. Pastor. Care 1980, 34, 7 10. Hone, L.C.; Jarden, A.; Schoﬁeld, G.M.; Duncan, S. Measuring ﬂourishing: The impact of operational deﬁnitions on the prevalence of high levels of wellbeing. Int. J. Wellbeing 2014, 4, 62–90. [CrossRef] References 1996 20. Adler, M.D.; Dolan, P.; Kavetsos, G. Would you choose to be happy? Tradeoffs between happiness and the other dimensions of life in a large population survey. J. Econ. Behav. Organ. 2017, 139, 60–73. [CrossRef] g p p y g 21. Benjamin, D.J.; Heffetz, O.; Kimball, M.S.; Szembrot, N. Beyond happiness and satisfaction: Toward well-being indices based on stated preference. Am. Econ. Rev. 2014, 104, 2698–2735. [CrossRef] p 22. Adler, M.D.; Dolan, P.; Henwood, A.; Kavetsos, G. “Better the devil you know”: Are stated preferences o determined by how healthy and happy people are? Soc. Sci. Med. 2022, 303, 115015. [CrossRef] 23. Balestra, C.; Boarini, R.; Tosetto, E. What Matters Most to People? Evidence from the OECD Better Life Index Users’ Responses. Soc. Indic. Res. 2018, 136, 907–930. [CrossRef] 24. Hsieh, C.-M. Counting Importance: The Case of Life Satisfaction and Relative Domain Importance. Soc. Indic. Res. 2003, 61, 227–240. [CrossRef] 25. VanderWeele, T.J.; Mathur, M.B.; Chen, Y. Outcome-wide longitudinal designs for causal inference: A new template for empirical studies. Stat. Sci. 2020, 35, 437–466. [CrossRef] 26. Bialowolski, P.; Weziak-Bialowolska, D.; Lee, M.T.; Chen, Y.; VanderWeele, T.J.; McNeely, E. The role of ﬁnancial conditions for physical and mental health. Evidence from a longitudinal survey and insurance claims data. Soc. Sci. Med. 2021, 281, 114041. [CrossRef] [PubMed] 27. Lee, M.T.; McNeely, E.; Weziak-Bialowolska, D.; Ryan, K.A.; Mooney, K.D.; Cowden, R.G.; VanderWeele, T.J. Demographic Predictors of Complete Well-Being. BMC Public Health 2022, 22, 1687. [CrossRef] [PubMed] 28. Graham, C. Does More Money Make You Happier? Why so much Debate? Appl. Res. Qual. Life 201 28. Graham, C. Does More Money Make You Happier? Why so much Debate? Appl. Res. Qual. Life 2011, 6, 219–239. [CrossRef] 29. Baranowska-Rataj, A.; Matysiak, A.; Mynarska, M. Does Lone Motherhood Decrease Women’s Happiness? Evidence from Qualitative and Quantitative Research. J. Happiness Stud. 2014, 15, 1457–1477. [CrossRef] 29. Baranowska-Rataj, A.; Matysiak, A.; Mynarska, M. Does Lone Motherhood Decrease Women’s Happiness? Evidence from Qualitative and Quantitative Research. J. Happiness Stud. 2014, 15, 1457–1477. [CrossRef] pp 30. Shaw, M. Housing and Public Health. Annu. Rev. Public Health 2004, 25, 397–418. [CrossRef] pp 30. Shaw, M. Housing and Public Health. Annu. Rev. Public Health 2004, 25, 397–418. [CrossRef] 31. Ahrenfeldt, L.J.; Möller, S.; Andersen-Ranberg, K.; Vitved, A.R.; Lindahl-Jacobsen, R.; Hvidt, N.C. Europe. Eur. J. Epidemiol. 2017, 32, 921–929. [CrossRef] 32. Daaleman, T.P.; Frey, B.B. References Measuring ﬂourishing: The impact of operational deﬁnitions on the prevalence of high levels of wellbeing. Int. J. Wellbeing 2014, 4, 62–90. [CrossRef] 12 of 13 12 of 13 Sustainability 2023, 15, 594 12. Peterson, C.; Seligman, M.E.P. Character Strengths and Virtues: A Handbook and Classiﬁcation; Oxford University Press: New York, NY, USA; American Psychological Association: Washington, DC, USA, 2004. y g g yer, A. Critiquing—And Rescuing—‘Character’. Sociology 2020, 54, 460–481. [CrossRef] y g g 13. Sayer, A. Critiquing—And Rescuing—‘Character’. Sociology 2020, 54, 460–481. [CrossRef] 14. Chen, Y.; Weziak-Bialowolska, D.; Lee, M.T.; Bialowolski, P.; McNeely, E.; VanderWeele, T.J. Longitudi domains of ﬂourishing. Sci. Rep. 2022, 12, 2740. [CrossRef] domains of ﬂourishing. Sci. Rep. 2022, 12, 2740. [CrossRef] 15. Weziak-Bialowolska, D.; Bialowolski, P.; VanderWeele, T.J.; McNeely, E. Character strengths involving an orientation to promote good can help your health and well-being. Evidence from two longitudinal studies. Am. J. Health Promot. 2021, 35, 388–398. [CrossRef] 16. Weziak-Bialowolska, D.; Lee, M.T.; Bialowolski, P.; Chen, Y.; VanderWeele, T.J.; McNeely, E. Prospective associations between strengths of moral character and health. Longitudinal evidence from survey and insurance claims data. Soc. Psychiatry Psychiatr. Epidemiol. 2022. [CrossRef] [PubMed] p 17. Weziak-Bialowolska, D.; Bialowolski, P. Can adherence to moral standards and ethical behaviors help maintain a sense of purpose in life? Evidence from a longitudinal study of middle-aged and older adults. PLoS ONE 2022, 17, e0273221. [CrossRef] [PubMed] 18 Will S S Willi A F W l h C C H M T tl W R thi ki ﬂ i hi C iti l i i ht d lit ti 17. Weziak-Bialowolska, D.; Bialowolski, P. Can adherence to moral standards and ethical behaviors help maintain a sense of purpose in life? Evidence from a longitudinal study of middle-aged and older adults. PLoS ONE 2022, 17, e0273221. [CrossRef] [PubMed] 18. Willen, S.S.; Williamson, A.F.; Walsh, C.C.; Hyman, M.; Tootle, W. Rethinking ﬂourishing: Critical insights and qualitative perspectives from the U.S. Midwest. SSM—Ment. Health 2022, 2, 100057. [CrossRef] [PubMed] g y g , , [ ] [ ] 18. Willen, S.S.; Williamson, A.F.; Walsh, C.C.; Hyman, M.; Tootle, W. Rethinking ﬂourishing: Critical insights and qualitative perspectives from the U.S. Midwest. SSM—Ment. Health 2022, 2, 100057. [CrossRef] [PubMed] The Domains of Life Satisfaction: An Attempt to Order Chaos. Soc. Indic. Res. 1996, 38, 303–328. [CrossRef] p p 19. Cummins, R.A. The Domains of Life Satisfaction: An Attempt to Order Chaos. Soc. Indic. Res. g ubin, D.B. Multiple Imputation for Non Response in Surveys; John Wiley & Sons: New York, NY, USA, 1987. References The Spirituality Index of Well-Being: A New Instrument for Health-Related. Ann. Fam. Med. 2004, 2, 499–503. [CrossRef] 33. Bradshaw, M.; Kent, B.V. Prayer, Attachment to God, and Changes in Psychological Well-Being in Later Life. J. Aging Health 2018, 30, 667–691. [CrossRef] 34. Kubzansky, L.D.; Huffman, J.C.; Boehm, J.K.; Hernandez, R.; Kim, E.S.; Koga, H.K.; Feig, E.H.; Lloyd-Jones, D.M.; Seligman, M.E.P.; Labarthe, D.R. Positive Psychological Well-Being and Cardiovascular Disease: JACC Health Promotion Series. J. Am. Coll. Cardiol. 2018, 72, 1382–1396. [CrossRef] 35. Rogers, N.T.; Demakakos, P.; Taylor, M.S.; Steptoe, A.; Hamer, M.; Shankar, A. Volunteering is associated with increased survival in able-bodied participants of the English Longitudinal Study of Ageing. J. Epidemiol. Community Health 2016, 70, 583–588. [CrossRef] 36. Santini, Z.I.; Jose, P.E.; Koyanagi, A.; Meilstrup, C.; Nielsen, L.; Madsen, K.R.; Koushede, V. Formal social participation protects physical health through enhanced mental health: A longitudinal mediation analysis using three consecutive waves of the Survey of Health, Ageing and Retirement in Europe (SHARE). Soc. Sci. Med. 2020, 251, 112906. [CrossRef] [PubMed] 37. White, I.R.; Royston, P.; Wood, A.M. Multiple imputation using chained equations: Issues and guidance for practice. Stat. Med. 2011, 30, 377–399. [CrossRef] [PubMed] 36. Santini, Z.I.; Jose, P.E.; Koyanagi, A.; Meilstrup, C.; Nielsen, L.; Madsen, K.R.; Koushede, V. Formal social participation protects physical health through enhanced mental health: A longitudinal mediation analysis using three consecutive waves of the Survey of Health, Ageing and Retirement in Europe (SHARE). Soc. Sci. Med. 2020, 251, 112906. [CrossRef] [PubMed] g g p 37. White, I.R.; Royston, P.; Wood, A.M. Multiple imputation using chained equations: Issues and guidance for practice. Stat. Med. 2011, 30, 377–399. [CrossRef] [PubMed] , , [ ] [ ] 38. Lloyd, J.E.V.; Obradovi´c, J.; Carpiano, R.M.; Motti-Stefanidi, F. Multiple imputation of missing multilevel, longitudinal data: A case when practical considerations trump best practices? J. Mod. Appl. Stat. Methods 2013, 12, 261–275. [CrossRef] 39. Allison, P. Missing Data; SAGE Publications: Thousand Oaks, CA, USA, 2001. case when practical considerations trump best practices? J. Mod. Appl. Stat. Methods 2013, 12, 261–275. [CrossRef] 39. Allison, P. Missing Data; SAGE Publications: Thousand Oaks, CA, USA, 2001. p p p J pp , , [ ] 39. Allison, P. Missing Data; SAGE Publications: Thousand Oaks, CA, USA, 2001. g 40. Rubin, D.B. References Multiple Imputation for Non Response in Surveys; John Wiley & Sons: New York, NY, USA, 1 13 of 13 13 of 13 Sustainability 2023, 15, 594 41. Steptoe, A.; Fancourt, D. An outcome-wide analysis of bidirectional associations between changes in meaningfulness of life and health, emotional, behavioural, and social factors. Sci. Rep. 2020, 10, 6463. [CrossRef] p 42. King, L.A.; Hicks, J.A.; Krull, J.L.; Del Gaiso, A.K. Positive Affect and the Experience of Meaning in Life. J. Personal. Soc. Psychol. 2006, 90, 179–196. [CrossRef] [PubMed] 43. Weziak-Bialowolska, D.; Bialowolski, P.; Sacco, P.L.; VanderWeele, T.J.; McNeely, E. Well-being in life and well-being at work: Which comes ﬁrst? Evidence from a longitudinal study. Front. Public Health 2020, 8, 103. [CrossRef] [PubMed] 44. Christakis, N.A.; Fowler, J.H. Connected: The Surprising Power of Our Social Networks and How They Shape Our Lives; Little, Brown and Co./Hachette Book Group: New York, NY, USA, 2009. p 45. Stossel, S. The Atlantic. 2012. Available online: https://www.theatlantic.com/magazine/archive/2013/ (accessed on 22 December 2022). 45. Stossel, S. The Atlantic. 2012. Available online: https://www.theatlantic.com/magazine/archive/2013/05/thanks-mom/309287/ (accessed on 22 December 2022). 46. Mineo, L. The Harvard Gazette. 2013. Available online: https://news.harvard.edu/gazette/story/ -years-harvard-study-has-been-showing-how-to-live-a-healthy-and-happy-life/ (accessed on 22 Dec 47. Trudel-Fitzgerald, C.; Zevon, E.S.; Kawachi, I.; Tucker-Seeley, R.D.; Grodstein, F.; Kubzansky, L.D. The Prospective Association of Social Integration with Life Span and Exceptional Longevity in Women. J. Gerontol. Ser. B 2020, 75, 2132–2141. [CrossRef] g p p g y 48. Weziak-Bialowolska, D.; Bialowolski, P. Bidirectional associations between meaning in life and the health, emotional ill-being, and daily life functioning outcomes among older adults. Psychol. Health 2022. [CrossRef] y g g y 49. Hayes, S.C.; Strosahl, K.D.; Bunting, K.; Twohig, M.; Wilson, K.G. What Is Acceptance and Commitment Therapy? In A Practical Guide to Acceptance and Commitment Therapy; Hayes, S.C., Strosahl, K.D., Eds.; Springer: New York, NY, USA, 2004; pp. 3–29. y g g y 49. Hayes, S.C.; Strosahl, K.D.; Bunting, K.; Twohig, M.; Wilson, K.G. What Is Acceptance and Commitment Therapy? In A Practical Guide to Acceptance and Commitment Therapy; Hayes, S.C., Strosahl, K.D., Eds.; Springer: New York, NY, USA, 2004; pp. 3–29. Ströhle, A. Physical activity, exercise, depression an 50. Ströhle, A. Physical activity, exercise, depression and anxiety disorders. J. Neural Transm. 2009, 116, 777–784. [CrossRef] 51. Boyle, P.A.; Barnes, L.L.; Buchman, A.S.; Bennett, D.A. Purpose in life is associated with mortality among community-dwelling older persons. Psychosom. Med. 2009, 71, 574–579. References [CrossRef] y y p y 51. Boyle, P.A.; Barnes, L.L.; Buchman, A.S.; Bennett, D.A. Purpose in life is associated with mortality among community-dwelling older persons. Psychosom. Med. 2009, 71, 574–579. [CrossRef] 52. Cohen, R.; Bavishi, C.; Rozanski, A. Purpose in life and its relationship to all-cause mortality and cardiovascular events: A meta-analysis. Psychosom. Med. 2016, 78, 122–133. [CrossRef] [PubMed] 52. Cohen, R.; Bavishi, C.; Rozanski, A. Purpose in life and its relationship to all-cause mortality and cardiovascular events: A meta-analysis. Psychosom. Med. 2016, 78, 122–133. [CrossRef] [PubMed] y y , , [ ] [ ] 53. Kim, E.S.; Sun, J.K.; Park, N.; Peterson, C. Purpose in life and reduced incidence of stroke in older adults: “The Health and Retirement Study”. J. Psychosom. Res. 2013, 74, 427–432. [CrossRef] [PubMed] 54. Kim, E.S.; Delaney, S.W.; Kubzansky, L.D. Sense of Purpose in Life and Cardiovascular Disease: Underlying Mechanisms and y y 53. Kim, E.S.; Sun, J.K.; Park, N.; Peterson, C. Purpose in life and reduced incidence of stroke in older adults: “The Health and Retirement Study”. J. Psychosom. Res. 2013, 74, 427–432. [CrossRef] [PubMed] 54 Ki E S D l S W K b k L D S f P i Lif d C di l Di U d l i M h i d 53. Kim, E.S.; Sun, J.K.; Park, N.; Peterson, C. Purpose in life and reduced incidence of stroke in older adults: “The Health and Retirement Study”. J. Psychosom. Res. 2013, 74, 427–432. [CrossRef] [PubMed] y J y ; Delaney, S.W.; Kubzansky, L.D. Sense of Purpose in Life and Cardiovascular Disease: Underlying Mecha rections. Curr. Cardiol. Rep. 2019, 21, 135. [CrossRef] [PubMed] Future Directions. Curr. Cardiol. Rep. 2019, 21, 135. [CrossRef] [PubMed] 55. Fisher, R.J.; Katz, J.E. Social-desirability bias and the validity of self-reported values. Psychol. Mark. 2000, 17, 105–120. [CrossRef] p 55. Fisher, R.J.; Katz, J.E. Social-desirability bias and the validity of self-reported values. Psychol. Mark. 2000, 17, 105–120. [CrossRef] p sher, R.J.; Katz, J.E. Social-desirability bias and the validity of self-reported values. Psychol. Mark. 2000, 17, 10 Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.
https://openalex.org/W4362472336	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0281856&type=printable	English	null	Targeting earlier diagnosis: What symptoms come first in Degenerative Cervical Myelopathy?	PloS one	2,023	cc-by	7,301	PLOS ONE RESEARCH ARTICLE OPEN ACCESS Citation: Munro CF, Yurac R, Moritz ZC, Fehlings MG, Rodrigues-Pinto R, Milligan J, et al. (2023) Targeting earlier diagnosis: What symptoms come first in Degenerative Cervical Myelopathy? PLoS ONE 18(3): e0281856. https://doi.org/10.1371/ journal.pone.0281856 Targeting earlier diagnosis: What symptoms come first in Degenerative Cervical Myelopathy? Colin F. MunroID1, Ratko YuracID2,3, Zipser Carl Moritz4, Michael G. Fehlings5,6,7, Ricardo Rodrigues-Pinto8,9, James Milligan10, Konstantinos Margetis11,12, Mark R. N. Kotter1,13, Benjamin M. DaviesID1,13,14* 1 Division of Neurosurgery, University of Cambridge Department of Clinical Neurosciences, Cambridge, Cambridgeshire, United Kingdom, 2 Department of Traumatology, Spine Unit, Clinica Alemana de Santiago SA, Vitacura, Santiago, Chile, 3 Department of Orthopedic and Traumatology, Desarrollo University Faculty of Medicine, Las Condes, Chile, 4 University Spine Center, Balgrist University Hospital, Zurich, Switzerland, 5 Division of Neurosurgery, Department of Surgery, University of Toronto, Toronto, Ontario, Canada, 6 Institute of Medical Science, University of Toronto, Toronto, Ontario, Canada, 7 Division of Neurosurgery, Toronto Western Hospital Krembil Neuroscience Centre, Toronto, Ontario, Canada, 8 Department of Orthopaedics, Spinal Unit (UVM), Centro Hospitalar Universita´rio do Porto EPE, Porto, Portugal, 9 Universidade do Porto Instituto de Ciencias Biomedicas Abel Salazar, Porto, Portugal, 10 McMaster University Department of Family Medicine, Hamilton, Ontario, Canada, 11 Department of Neurosurgery, Mount Sinai Hospital, New York, New York, United States of America, 12 Department of Neurosurgery, Icahn School of Medicine at Mount Sinai, New York, New York, United States of America, 13 Myelopathy.org, Charity for Degenerative Cervical Myelopathy, Cambridge, Cambridgeshire, United Kingdom, 14 AOSpine International, RECODE DCM Incubator, Diagnostic Criteria, Davos, Graubu¨nden, Switzerland a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 Abstract Editor: Andrea Martinuzzi, IRCCS Medea: Istituto di Ricovero e Cura a Carattere Scientifico Eugenio Medea, ITALY Methods An internet survey was developed, using an established list of patient-reported effects. Par- ticipants (N = 171) were recruited from an online community of people with DCM. Respon- dents selected their current symptoms and primary presenting symptom. The relationship of symptoms and their relationship to time to diagnosis were explored. This included symp- toms not commonly measured today, termed ‘non-conventional’ symptoms. Copyright: © 2023 Munro et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Background Degenerative cervical myelopathy (DCM) is a common and disabling condition. Early effec- tive treatment is limited by late diagnosis. Conventional descriptions of DCM focus on motor and sensory limb disability, however, recent work suggests the true impact is much broader. This study aimed to characterise the symptomatic presentation of DCM from the perspective of people with DCM and determine whether any of the reported symptoms, or groups of symptoms, were associated with early diagnosis. Received: April 11, 2022 Accepted: February 2, 2023 Published: March 31, 2023 Received: April 11, 2022 Accepted: February 2, 2023 Published: March 31, 2023 Peer Review History: PLOS recognizes the benefits of transparency in the peer review process; therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. The editorial history of this article is available here: https://doi.org/10.1371/journal.pone.0281856 * bd375@cam.ac.uk PLOS ONE PLOS ONE Results All listed symptoms were experienced by >10% of respondents, with poor balance being the most commonly reported (84.2%). Non-conventional symptoms accounted for 39.7% of symptomatic burden. 55.4% of the symptoms were reported as an initial symptom, with Data Availability Statement: Data cannot be shared publicly because it contains potentially identifying and sensitive patient information. Data 1 / 16 PLOS ONE \| https://doi.org/10.1371/journal.pone.0281856 March 31, 2023 PLOS ONE Targeting earlier diagnosis: What symptoms come first in Degenerative Cervical Myelopathy? neck pain the most common (13.5%). Non-conventional symptoms accounted for 11.1% of initial symptoms. 79.5% of the respondents were diagnosed late (>6 months). Heavy legs was the only initial symptom associated with early diagnosis. is available from the Myelopathy.org charity (contact via info@myelopathy.org) for researchers who meet the criteria for access to confidential data. Funding: MRNK is supported by the National Institute for Health Research (NIHR) Brain Injury MedTech Co-operative based at Cambridge University Hospitals NHS Foundation Trust and BMD via a NIHR Clinical Doctoral Research Fellowship. The views expressed in this publication are those of the authors and not necessarily those of the NHS, the National Institute for Health Research or the Department of Health and Social Care. Conclusions A comprehensive description of the self-reported effects of DCM has been established, including the prevalence of symptoms at disease presentation. The experience of DCM is broader than suggested by conventional descriptions and further exploration of non-conven- tional symptoms may support earlier diagnosis. Competing interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: CFM has declared that no competing interests exist. RY has declared that no competing interests exist. ZCM has declared that no competing interests exist. MGF currently serves as an academic editor at PLOS ONE. RRP has declared that no competing interests exist. JM has declared that no competing interests exist. KM has declared that no competing interests exist. MRNK has declared that no competing interests exist. BMD is supported by NIHR POLYFIX DCM and NIHR Clinical Doctoral Research Fellowship grants. BMD is a founder of MoveMed (a digital therapeutics platform which develops assessments and treatments using software). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. This does not alter our adherence to PLOS ONE policies on sharing data and materials. Introduction Degenerative Cervical Myelopathy (DCM) is a progressive condition that occurs when the cer- vical spinal cord is compressed by degenerative changes in surrounding structures [1]. DCM treatment is largely restricted to surgery that aims to alleviate compression of the spi- nal cord. Recent large prospective studies have demonstrated surgery is able to stop disease progression and offer a meaningful, albeit incomplete recovery [2, 3]. The amount of recovery is hypothesized to be dependent on the severity of existing damage. Consequently, time to treatment has emerged as an important predictor of treatment response [4], with the latest analysis indicating a preoperative duration of symptoms less than 4 months offers the most favourable outcome [5]. Whilst not applicable to the full spectrum of DCM, with mild forms of the disease amenable to a watch and wait approach [6], for those requiring treatment any such prompt intervention target is currently undeliverable. This is due to long delays in diagnosis, on average 2–5 years [7, 8]. This is contributing to the significant residual disability in DCM, with dependence and unemployment prevalent [9] and enabling timely treatment underpins many of the top research priorities identified by AO Spine RECODE-DCM [10]. Consequently, improving time to diagnosis is an attractive target for improving outcomes immediately in DCM. However, the factors driving missed and delayed diagnosis are poorly characterised at present and difficult to investigate. Whilst a poor awareness and understand- ing of the disease are undoubtedly factors [1, 11], the information required to support early diagnosis, including knowledge of the early symptoms, or the key differentials or at risk popu- lations, is yet to be established. Conventional descriptions of DCM have focused on motor and sensory disability to the limbs [12, 13]. This is reflected in the assessment of DCM for clinical research [14] and clinical care [15]. However, we have recently developed a long-list of patient reported symptoms in DCM [16], which broadens the potential impact of DCM. This is also a unique dataset as out- comes are based on the patient’s own wording. This study aimed to characterise the symptomatic presentation of DCM from the perspec- tive of people with cervical myelopathy [17] and determine whether any of the reported symp- toms, or groups of symptoms, were associated with early diagnosis. PLOS ONE \| https://doi.org/10.1371/journal.pone.0281856 March 31, 2023 Survey design An internet survey was developed using SurveyMonkey (California, USA), using a compre- hensive list of patient reported effects of DCM, previously established [16, 20]. In brief, this process had used semi-structured interviews with people with DCM (N = 5) and carers (N = 3) to identify effects of DCM [20]. These effects were then presented to a separate and larger cohort of sufferers via an initial internet survey (N = 224) (S1 Appendix). The survey was advertised via Myelopathy.org, an international charity for people with DCM. Respondents were asked to confirm whether they suffered from 36 shortlisted outcomes, but also given the opportunity to submit additional suggestions (S1 Table). These were then processed by investi- gators to produce a ‘longlist’ of 56 patient outcomes (S1 Table) [16]. Outcomes were generated based directly on sufferers’ wording and goes beyond the common and existing descriptions of DCM, which are the focus of current myelopathy assessment [14]. The final survey was formed of three sections. Initially participants were provided with an overview of the study and definition of DCM. By continuing into the survey, participants were confirming their diagnosis of DCM and providing informed consent to participate. Respon- dents were asked a series of sampling questions, including age, gender, length of time between symptom onset and diagnosis, length of time lived with DCM, history of surgical treatment and disease severity as measured using the p-mJOA (patient derived modified Japanese Ortho- paedic Association) score. The mJOA is the international standard for assessment of disease severity [6, 13], and the p-mJOA a validated patient reported version [21]. Finally, the list of 56 patient reported outcomes were incorporated into a matrix, with respondents asked: “What symptoms do you currently experience due to DCM?” and “Which of these was the first symp- tom you experienced as a result of DCM?”. For this latter question, only one symptom could be selected. If they experienced more than one symptom initially and could not remember which came first, they were asked to select the most significant one at the time. The survey consisted of 16 questions over 10 pages (S2 Appendix). Participation was voluntary and advertised using Myelopathy.org, an international non- profit organisation dedicated to promoting understanding and awareness of DCM, to help people with DCM, professionals and supporters. Participants from the previous (first-round) internet survey were invited by email to participate in this follow-up survey. Cambridge. It is reported in accordance with the recommendations for Conducting and REporting DElphi Studies (CREDES) [18] and Checklist for Reporting Results of Internet E-Surveys (CHERRIES) [19]. Cambridge. It is reported in accordance with the recommendations for Conducting and REporting DElphi Studies (CREDES) [18] and Checklist for Reporting Results of Internet E-Surveys (CHERRIES) [19]. Survey design Surveys could not be edited once they had been completed. No incentives were offered for completion of the surveys. IP addresses were screened to identify potential duplicate responses. If multiple entries from the same IP address were discovered, only one response was included, unless the responses gave different email addresses or had significantly different demographic data (age and sex), in which case it was assumed to be a separate individual on the same device. The more completed response of the duplicates was included, if both were equally complete, the response with the earliest end date and time was included. Methods This study aimed to determine which of the patient reported symptoms in DCM were associ- ated with earlier diagnosis. The study used a long-list of patient reported outcomes, previously established [16]. This study was conducted with ethical approval from the University of 2 / 16 PLOS ONE \| https://doi.org/10.1371/journal.pone.0281856 March 31, 2023 PLOS ONE Targeting earlier diagnosis: What symptoms come first in Degenerative Cervical Myelopathy? Analysis JASP software (Version 0.13.1) was used for statistical analysis. The Shapiro-Wilks test was used to assess for parametric distribution of numerical data sets. The Mann-Whitney U test was then used to compare the means of two non-parametric distributions whilst a Two Tailed T-test used to compare the means of two parametric distributions. Analysis of variance 3 / 16 PLOS ONE \| https://doi.org/10.1371/journal.pone.0281856 March 31, 2023 PLOS ONE Targeting earlier diagnosis: What symptoms come first in Degenerative Cervical Myelopathy? (ANOVA) was used to compare the means of 3 or more parametric distributions, whilst the Kruskal-Wallis test was used to compare the means of 3 or more non-parametric distributions. Chi-Square test for association was used to assess the relation between categorical variables. A result was taken as significant when p<0.05. Odds ratios, and their corresponding 95% confidence intervals, were used to assess the association between the presence of an initial symptom and early or late diagnosis, with a con- fidence interval not encompassing 1 taken as a significant result. Only complete surveys were used for data analysis. Surveys were categorised as incomplete (no questions answered after respondent confirmed they had DCM), partially complete (respondent only answered demographics questions) or fully complete (respondent answered all questions). The demographic characteristics of partially complete and fully complete responses were compared in order to evaluate the potential impact of missing data. Demo- graphic characteristics were also compared to the previous round. In order to assess the representation of DCM amongst the recruited cohort, current symptoms were matched to those recorded in the AO Spine prospective observational study (N = 679) of suf- ferers undergoing surgery, and their prevalence compared (S2 Table and S1 Fig). Early diagnosis was defined as <6 months from the onset of symptoms, and late diagnosis >6 months. This is based upon analysis showing treatment within 6 months is associated with improved outcomes [4, 22]. To evaluate the impact of symptoms less commonly acknowledged professionally with DCM, patient reported outcomes were divided into either ‘conventional’ or ‘non-conven- tional’ symptoms based on their acknowledgement or not within DCM review articles [12]. To evaluate the impact of key areas of effect, conventional symptoms were further subdivided into motor, sensory, pain and autonomic categories. The motor and sensory categories were then subdivided anatomically into upper and lower limb groups. Analysis This was based on the structure of the m/JOA assessment, the current gold-standard assessment for DCM [6, 13]. The non-con- ventional group was sub-divided into: sensation/pain, movement disorder, gastro-intestinal, respiratory, cranial and psychosocial. This grouping was agreed by the authors, based on com- mon systems. The final classification is split between Tables 1 and 2, showing conventional and non-conventional symptoms respectively. PLOS ONE Table 2. Survey symptom classification–non-conventional symptoms. Non-Conventional Sensation / Pain Movement Disorder Gastro-Intestinal Respiratory Cranial Psychosocial Altered temperature sensation* Leg shaking* Choking/swallowing problems* Exertional breathlessness Dizziness* Symptom variability day by day Female sexual dysfunction Muscle spasms or twitches (arms)* Constipation Difficulty breathing when lying flat Headache* Impaired cognition Hot flushes and/or sweating Hand shaking* Nausea and vomiting Tinnitus Anxiety Abdominal pain Muscle spasms or twitches (legs) Eyesight problems Fatigue Face pain Face numbness Insomnia Symptom variability hour by hour Depression/low mood Symptoms that were reported as initial symptoms. https://doi org/10 1371/journal pone 0281856 t002 Table 2. Survey symptom classification–non-conventional symptoms. Demographic information was not significantly different between complete and partially complete responses. Overall, survey respondents matched those of the previous round, with the exception of surgical history: Respondents of round 3 were more likely to have had surgery for their DCM (p = 0.04) (Table 3). Results Overall, 189 unique individuals accessed this survey. This included 78 respondents who had participated in the previous project (Fig 1). Table 1. Survey symptom classification–conventional symptoms. Conventional Motor Sensory Pain Autonomic LL Motor UL Motor UL Sensory LL Sensory Poor balance Clumsiness* Pins and needles in your hand* Leg numbness* Neck pain* Difficulty emptying bladder* Lack of control of legs* Reduced dexterity* Lhermitte’s phenomena* Pins and needles in your leg* Shoulder pain* Faecal incontinence Leg stiffness* Reduced grip strength* Hand numbness* Neck stiffness* Urinary incontinence Heavy legs* Arm stiffness* Arm numbness* Neck clicking* Waking to go to the toilet Dragging legs* Pins and needles in your arm* Back pain* Erectile Dysfunction Falls* Arm pain* Allodynia Leg pain Symptoms that were reported as initial symptoms. https://doi.org/10.1371/journal.pone.0281856.t001 PLOS ONE \| https://doi.org/10.1371/journal.pone.0281856 March 31, 2023 4 / 16 e 1. Survey symptom classification–conventional symptoms. Symptoms that were reported as initial symptoms. 4 / 16 Targeting earlier diagnosis: What symptoms come first in Degenerative Cervical Myelopathy? PLOS ONE \| https://doi.org/10.1371/journal.pone.0281856 March 31, 2023 Current symptoms All symptoms were currently experienced by at least 10% of respondents, with each respon- dent on average experiencing 27 different symptoms. The most commonly reported individual symptoms were poor balance (84.2%), clumsiness (80.7%), neck stiffness (78.4%), reduced grip strength (76.6%), and hand numbness (75.4%) (Fig 2). Current symptom burden closely matched that experienced by sufferers participating in the AO Spine prospective observational study (S1 Fig). The proportion of all reported symptoms attributable to each symptom group is shown in Fig 3. Conventional symptoms account for 60.3% of symptomatic burden and non-conven- tional symptoms account for 39.7%. Fig 1. Flowchart of the study design. The findings from previous semi-structured (Round 1) interviews [20] and paired internet survey (Round 2) [16], were used to produce this survey (Round 3). https://doi.org/10.1371/journal.pone.0281856.g001 Fig 1. Flowchart of the study design. The findings from previous semi-structured (Round 1) interviews [20] and paired internet survey (Round 2) [16], were used to produce this survey (Round 3). https://doi.org/10.1371/journal.pone.0281856.g001 Fig 1. Flowchart of the study design. The findings from previous semi-structured (Round 1) interviews [20] and paired internet survey (Round 2) [16], were used to produce this survey (Round 3). https://doi.org/10.1371/journal.pone.0281856.g001 5 / 16 PLOS ONE \| https://doi.org/10.1371/journal.pone.0281856 March 31, 2023 Targeting earlier diagnosis: What symptoms come first in Degenerative Cervical Myelopathy? PLOS ONE \| https://doi.org/10.1371/journal.pone.0281856 March 31, 2023 https://doi.org/10.1371/journal.pone.0281856.t003 Initial symptoms Of the 56 listed symptoms, 31 (55.4%) were reported as an initial symptom. The most com- monly reported individual initial symptoms were: neck pain (13.5%), shoulder pain (8.8%), pins and needles in the hand (7.0%), arm pain (5.3%) and Lhermitte’s phenomena (5.3%) (Fig 4). The percentage of respondents with an initial symptom from each symptom category is shown in Fig 5. Conventional symptoms account for 88.9% of initial symptoms and non-con- ventional symptoms account for 11.1%. PLOS ONE PLOS ONE Table 3. Comparison of demographics of survey respondents in round 2 and round 3. Respondent Demographics: Round 2 (N = 224): Round 3 (N = 171): P value Mean age (years) 56.6 53.9 0.63 Female/Male (%) 75.9/24.1 73.9/26.3 0.62 Surgery/No Surgery (%) 62.1/38.0 71.9/28.1 0.04* Mean time to diagnosis (years) 4.9 3.9 0.23 Early/Late Diagnosis (%) 21.0/79.0 20.5/79.5 0.90 Mean length of time with DCM (years) 8.2 6.8 0.43 Mean total mJOA 11.6 11.5 0.93 Mean upper limb motor mJOA 3.6 3.6 0.99 Mean lower limb motor mJOA 4.3 4.2 0.67 Mean upper limb sensory mJOA 1.7 1.7 0.50 Mean sphincter dysfunction mJOA 2.1 2.1 0.93 Table 3. Comparison of demographics of survey respondents in round 2 and round 3. Fig 2. Bar chart of the prevalence of symptoms. The number of respondents reporting each symptom are displayed at the end of the bar. https://doi.org/10.1371/journal.pone.0281856.g002 Fig 2. Bar chart of the prevalence of symptoms. The number of respondents reporting each symptom are displayed at the end of the bar. Fig 2. Bar chart of the prevalence of symptoms. The number of respondents reporting each symptom are displayed at the end of the bar. https://doi org/10 1371/journal pone 0281856 g002 https://doi.org/10.1371/journal.pone.0281856.g002 6 / 16 PLOS ONE \| https://doi.org/10.1371/journal.pone.0281856 March 31, 2023 PLOS ONE Targeting earlier diagnosis: What symptoms come first in Degenerative Cervical Myelopathy? Fig 3. Pie chart illustrating the proportion of overall symptoms reported that were attributable to each symptom category. Conventional symptom segments are illustrated with lines and non-conventional symptom segments with dots. https://doi org/10 1371/journal pone 0281856 g003 Fig 3. Pie chart illustrating the proportion of overall symptoms reported that were attributable to each symptom category. Conventional symptom segments are illustrated with lines and non-conventional symptom segments with dots. Fig 3. Pie chart illustrating the proportion of overall symptoms reported that were attributable to each symptom category. Conventional symptom segments are illustrated with lines and non-conventional symptom segments with dots. https://doi.org/10.1371/journal.pone.0281856.g003 PLOS ONE \| https://doi.org/10.1371/journal.pone.0281856 March 31, 2023 Association between initial symptoms and time to diagnosis The mean time to diagnosis was 46.4 months, with 20.5% (35/171) diagnosed early (within 6 months) and 79.5% (136/171) diagnosed late (after 6 months). Fig 6 shows the association between individual, initial symptoms and the likelihood of an early or late diagnosis. Heavy legs was the only symptom significantly associated with early diagnosis (95% Confidence Interval <1). No initial symptoms were significantly associated with late diagnosis. Initial symptoms were categorised into the predefined symptom domains. Fig 7 shows their association with early or late diagnosis. No initial symptom group significantly favoured an 7 / 16 PLOS ONE \| https://doi.org/10.1371/journal.pone.0281856 March 31, 2023 PLOS ONE Targeting earlier diagnosis: What symptoms come first in Degenerative Cervical Myelopathy? Fig 4. Bar chart of the prevalence of initial symptoms. The number of respondents reporting each initial symptom are displayed at the end of the bar. https://doi.org/10.1371/journal.pone.0281856.g004 nitial symptoms. The number of respondents reporting each initial symptom are displayed at the end of the bar. Fig 4. Bar chart of the prevalence of initial symptoms. The number of respondents reporting each initial symptom are displayed at the end of the bar. https://doi.org/10.1371/journal.pone.0281856.g004 https://doi.org/10.1371/journal.pone.0281856.g004 early or late diagnosis, although there was a trend for non-conventional cranial symptoms to be associated with a late diagnosis. Discussion This is the first study to explore the patient reported experience of DCM at presentation. The findings of this survey indicate that patients experience a far greater breadth of symptoms than are commonly considered in textbooks [12], or evaluated in clinical research [14, 22] or clini- cal care [15]. Whilst this study indicates that a sub-selection may be particularly relevant for detection, they remain diverse and non-specific. Only ‘heavy legs’ appeared specific for early diagnosis. PLOS ONE \| https://doi.org/10.1371/journal.pone.0281856 March 31, 2023 Limitations The findings of this study must be considered in the context of its methodology. As an open, internet recruited survey of self-selected people with DCM, reporting their retrospective 8 / 16 PLOS ONE \| https://doi.org/10.1371/journal.pone.0281856 March 31, 2023 PLOS ONE Targeting earlier diagnosis: What symptoms come first in Degenerative Cervical Myelopathy? Fig 5. Pie chart illustrating the proportion of initial symptoms that were attributable to each symptom category. Conventional symptom segments are illustrated with lines and non-conventional symptom segments with dots. Categories with N 1 have been left out for the purposes of clear illustration. Fig 5. Pie chart illustrating the proportion of initial symptoms that were attributable to each symptom category. Conventional symptom segments are illustrated with lines and non-conventional symptom segments with dots. Categories with N 1 have been left out for the purposes of clear illustration. https://doi.org/10.1371/journal.pone.0281856.g005 experience, the results are at risk of sampling and recall bias. That said, a number of design fac- tors and findings are reassuring that this is unlikely to be significant. Firstly, internet surveys are increasingly recognised as an effective means of reaching repre- sentative samples of a disease [23–26]. For example, they are the mainstay of core-outcome set initiatives to define symptom burden and a recent exercise in inflammatory bowel disease, using cross-validation with an individual’s health records, found self-reporting is accurate [27]. Further, in our survey participants were presented with a description of DCM, and asked to confirm their diagnosis, making it unlikely people without a diagnosis of DCM participated. Secondly, the disease characteristics of participants, including symptom profiles, matched those identified within the high-quality AO Spine observational studies [5] (S1 Fig). Further, whilst respondents were more likely to be female, as has been the case with previous surveys using Myelopathy.org, a DCM charity [9, 16], the cohort demographics also matched the clini- cal series [5] (S2 Table). This inconsistency is thought to reflect the increased participation of women in online health-communities [28]. Traditionally a DELPHI process will recruit respondents only once, inviting and measuring dropout for each consecutive round. As an additional adjunct, the third-round survey was an PLOS ONE \| https://doi.org/10.1371/journal.pone.0281856 March 31, 2023 9 / 16 PLOS ONE Targeting earlier diagnosis: What symptoms come first in Degenerative Cervical Myelopathy? Fig 6. Odds of early diagnosis using individual symptoms. A forest plot of individual odds ratios for each initial symptom. Limitations Error bars represent 95% confidence intervals of the odds ratio. Fig 6. Odds of early diagnosis using individual symptoms. A forest plot of individual odds ratios for each initial symptom. Error bars represent 95% confidence intervals of the odds ratio. https://doi.org/10.1371/journal.pone.0281856.g006 https://doi.org/10.1371/journal.pone.0281856.g006 open survey to improve the response rate. However, the consistent sampling demographics provide some reassurance that this is unlikely to have influenced the findings. Due to the very varied nature of DCM presentation, the absolute numbers of respondents presenting with any one symptom at presentation was often small. This likely resulted in the study being underpowered to find significant associations between initial symptoms and early or late diagnosis. It was therefore also felt unsuitable to explore with modelling. Thus, one needs to be careful to avoid a Type II error in concluding that, with the exception of heavy legs, no initial symptoms are associated with either early or late diagnosis. Interpretation Pain and upper limb sensory symptoms predominate initially. Whilst there was sub- stantial variability in the individual presenting symptoms, the majority (59.1%) of respondents first experienced a symptom which could be classified as a conventional pain or upper limb sensory symptom, indicating a potential focus point. Behrbalk et al (2013) in their description PLOS ONE \| https://doi.org/10.1371/journal.pone.0281856 March 31, 2023 10 / 16 PLOS ONE Targeting earlier diagnosis: What symptoms come first in Degenerative Cervical Myelopathy? Fig 7. Odds of early diagnosis using symptom categories. A forest plot of individual odds ratios for each symptom category. Error bars represent 95% confidence intervals of the odds ratio. Categories with N 1 have been left out for the purposes of clear illustration. Fig 7. Odds of early diagnosis using symptom categories. A forest plot of individual odds ratios for each symptom category. Error bars represent 95% confidence intervals of the odds ratio. Categories with N 1 have been left out for the purposes of clear illustration. https://doi.org/10.1371/journal.pone.0281856.g007 https://doi.org/10.1371/journal.pone.0281856.g007 of diagnostic delay, found 43% of patients were initially diagnosed and sometimes treated for carpal tunnel syndrome [7]. Nevertheless, the diagnostic utility of these symptoms is unclear, given the one year incidence for neck pain, in the general population, ranges from 10 to 21% [29] and that general practitioners are typically consulted 7 times a week for neck or upper extremity complaints of various causes [30]. Whilst pain is a common prompt to seek health- care assessment in general [31, 32], its experience here was not associated with earlier diagno- sis in this series. Consequently, the absence of these symptoms may instead be useful for ruling out DCM, given their high sensitivity, but low specificity, for the condition [33, 34]. Are lower limb symptoms helpful for diagnosis? Gait dysfunction is considered one of the earliest clinical manifestations of DCM [35]. It was the most common (60%) first symptom of myelopathy in a prospective observational study of asymptomatic spinal cord compression [36], and in their review of the diagnostic accuracy of DCM symptoms, Mizer et al [34] found that difficulty in walking for 15 minutes was one of 4 symptoms with a positive likelihood ratio for DCM greater than 5. Gait dysfunction was not individually matched in this survey. The ‘conventional lower limb motor symptoms’ group would contribute to gait dysfunction and made up 18.7% of initial symptoms. PLOS ONE \| https://doi.org/10.1371/journal.pone.0281856 March 31, 2023 Conclusions This study has re-confirmed that patients describe a varied experience of DCM, much broader than conventional descriptions, which is also the case from the outset; Non-conventional symptoms comprised 40% of a patient’s symptom burden and were experienced by all individ- uals. Early symptoms most commonly relate to pain or upper limb sensation, although indi- vidually heavy legs were the only single symptom associated with early diagnosis. Understanding how these symptoms can be used to distinguish and diagnose DCM early will require further research, including into their sensitivity and specificity individually but also in combination. This is an active goal of the RECODE-DCM Diagnostic Criteria Incuba- tor, an international working group hosted by Myelopathy.org. Parties interested in support- ing this consortia are welcomed. Non-conventional symptoms are common and overlooked. Could they have a role in Non-conventional symptoms are common and overlooked. Could they have a role in early detection? In this study, symptoms which were not cited in narrative review articles on DCM, were categorised as “non-conventional”. This included a number of controversial symptoms, including headache, vision and hearing impairments and dizziness. A number of hypotheses are proposed, including altered signalling via the sympathetic chain (the so called Barre´-Lie´ou "syndrome") or facial symptoms via involvement of the spinal nucleus of the tri- geminal nerve [38, 39]. However, these remain hypotheses, as whilst there are numerous descriptions of their association, particularly in the context of cervical spondylosis, the evi- dence base linking the two remains of low quality [40–43] and as standard DCM assessments do not capture these symptoms, high quality series cannot comment. However, they do dem- onstrate the high prevalence of co-morbidities in these patient groups, and it is possible these experiences are secondary to different disease processes [5]. Nevertheless, whilst using similar sampling techniques, this is the second cohort in which we have described prevalent non-conventional symptoms [16]. Specifically, in this study 100% of respondents reported at least one of the 26 listed “non-conventional” symptoms and 39.7% of overall symptomatic burden was attributable to non-conventional symptoms. 11.1% of patients reported non-conventional symptoms as the first manifestation of their disease, with the vast majority (89.5%) of these being cranial or movement disorder symptoms, and whilst not shown to have statistical significance in this study, presenting with cranial symptoms was associated with a higher probability of late diagnosis. Although controversial, this prevalence could have significant value for the detection, and subsequent earlier diagnosis, of DCM, as these symptoms will not occur with many differen- tials, for example carpal tunnel syndrome. We note, whilst not as comprehensive in their development of associated symptoms, a screening questionnaire based solely on symptoms for the detection of DCM (sensitivity 93.5%; specificity 67.3%) found that the odds ratio of chest tightness, a non-conventional symptom not identified by this study, in myelopathy patients compared with controls was 22.9 [44]. Supporting the proposition that non-conventional symptoms may have a role to play in the earlier diagnosis of DCM. S2 Appendix. Copy of round 3 internet survey. (DOCX) Interpretation Of the individual symptoms, only ‘heavy legs’ was associated with early diagnosis. This is a similar finding to that of Hilton et al. who found that subjective imbalance was the only symptom associated with a shorter referral time between primary and secondary assess- ment [15]. Whilst this could be confirmation of its importance to detection, it could also repre- sent a selection bias by professionals: driven by the socio-economic consequence of falls [37], their investigation has well-defined pathways from primary to secondary care [15]. This con- trasts with the lack of a clear or unified referral pathway for most DCM symptoms, with patients being seen by neurologists, orthopaedics, pain specialists, rheumatologists and geria- tricians, contributing to delays in assessment and treatment [8]. 11 / 16 PLOS ONE \| https://doi.org/10.1371/journal.pone.0281856 March 31, 2023 PLOS ONE Targeting earlier diagnosis: What symptoms come first in Degenerative Cervical Myelopathy? Author Contributions Conceptualization: Mark R. N. Kotter, Benjamin M. Davies. Data curation: Colin F. Munro. Formal analysis: Colin F. Munro. Funding acquisition: Mark R. N. Kotter, Benjamin M. Davies. Methodology: Benjamin M. Davies. Conceptualization: Mark R. N. Kotter, Benjamin M. Davies. Data curation: Colin F. Munro. Project administration: Benjamin M. Davies. Resources: Mark R. N. Kotter, Benjamin M. Davies. Resources: Mark R. N. Kotter, Benjamin M. Davies. Supervision: Mark R. N. Kotter, Benjamin M. Davies. Writing – original draft: Colin F. Munro, Benjamin M. Davies. Writing – review & editing: Colin F. Munro, Ratko Yurac, Zipser Carl Moritz, Michael G. Fehlings, Ricardo Rodrigues-Pinto, James Milligan, Konstantinos Margetis, Mark R. N. Kotter, Benjamin M. Davies. S2 Table. Comparison of demographics between survey cohort and AO Spine observa- tional study. Aside from gender and time to diagnosis, the cohorts are closely matched. (DOCX) S1 Fig. Symptom frequency comparison with Tetreault et al. 2018. Comparison of matched symptoms between this study (white) and the AO Spine prospective observational study of suf- ferers undergoing surgical treatment for DCM (grey). Error bars represent 95% confidence intervals. (DOCX) Supporting information S1 Appendix. Copy of round 2 internet survey. (DOCX) S1 Appendix. Copy of round 2 internet survey. (DOCX) S2 Appendix. Copy of round 3 internet survey. (DOCX) 12 / 16 PLOS ONE \| https://doi.org/10.1371/journal.pone.0281856 March 31, 2023 PLOS ONE Targeting earlier diagnosis: What symptoms come first in Degenerative Cervical Myelopathy? S1 Table. Short and longlisted survey outcomes. *Outcomes added after respondent sugges- tion in round 2. 2 shortlisted symptoms (italicised) were expanded “anatomically” for the long- list. (DOCX) PLOS ONE \| https://doi.org/10.1371/journal.pone.0281856 March 31, 2023 References 1. Davies BM, Mowforth OD, Smith EK, Kotter MR. Degenerative cervical myelopathy. BMJ. 2018 Feb 22; 360:k186. https://doi.org/10.1136/bmj.k186 PMID: 29472200 2. Fehlings MG, Wilson JR, Kopjar B, Yoon ST, Arnold PM, Massicotte EM, et al. Efficacy and safety of surgical decompression in patients with cervical spondylotic myelopathy: results of the AOSpine North America prospective multi-center study. J Bone Joint Surg Am. 2013 Sep 18; 95(18):1651–8. https:// doi.org/10.2106/JBJS.L.00589 PMID: 24048552 3. Fehlings MG, Ibrahim A, Tetreault L, Albanese V, Alvarado M, Arnold P, et al. A global perspective on the outcomes of surgical decompression in patients with cervical spondylotic myelopathy: results from the prospective multicenter AOSpine international study on 479 patients. Spine. 2015 Sep 1; 40 (17):1322–8. https://doi.org/10.1097/BRS.0000000000000988 PMID: 26020847 4. Tetreault LA, Karpova A, Fehlings MG. Predictors of outcome in patients with degenerative cervical spondylotic myelopathy undergoing surgical treatment: results of a systematic review. Eur Spine J Off Publ Eur Spine Soc Eur Spinal Deform Soc Eur Sect Cerv Spine Res Soc. 2015 Apr; 24 Suppl 2:236– 51. https://doi.org/10.1007/s00586-013-2658-z PMID: 23386279 5. Tetreault L, Wilson JR, Kotter MRN, Coˆte´ P, Nouri A, Kopjar B, et al. Is Preoperative Duration of Symp- toms a Significant Predictor of Functional Outcomes in Patients Undergoing Surgery for the Treatment of Degenerative Cervical Myelopathy? Neurosurgery. 2018 Nov 16; PLOS ONE \| https://doi.org/10.1371/journal.pone.0281856 March 31, 2023 13 / 16 PLOS ONE Targeting earlier diagnosis: What symptoms come first in Degenerative Cervical Myelopathy? 6. Fehlings MG, Tetreault LA, Riew KD, Middleton JW, Aarabi B, Arnold PM, et al. A Clinical Practice Guideline for the Management of Patients With Degenerative Cervical Myelopathy: Recommendations for Patients With Mild, Moderate, and Severe Disease and Nonmyelopathic Patients With Evidence of Cord Compression. Glob Spine J [Internet]. 2017 Sep 5 [cited 2017 Oct 5]; Available from: http:// journals.sagepub.com/doi/metrics/10.1177/2192568217701914 PMID: 29164035 7. Behrbalk E, Salame K, Regev GJ, Keynan O, Boszczyk B, Lidar Z. Delayed diagnosis of cervical spon- dylotic myelopathy by primary care physicians. Neurosurg Focus. 2013 Jul; 35(1):E1. https://doi.org/10. 3171/2013.3.FOCUS1374 PMID: 23815245 8. Hilton B, Tempest-Mitchell J, Davies B, Kotter M. Route to diagnosis of degenerative cervical myelopa- thy in a UK healthcare system: a retrospective cohort study. BMJ Open. 2019 May 5; 9(5):e027000. https://doi.org/10.1136/bmjopen-2018-027000 PMID: 31061045 9. Pope DH, Mowforth OD, Davies BM, Kotter MRN. Diagnostic Delays Lead to Greater Disability in Degenerative Cervical Myelopathy and Represent a Health Inequality. Spine. 2020 Mar 15; 45(6):368– 77. https://doi.org/10.1097/BRS.0000000000003305 PMID: 31658234 10. References Davies BM, Khan DZ, Mowforth OD, McNair AGK, Gronlund T, Kolias AG, et al. RE-CODE DCM (REsearch Objectives and Common Data Elements for Degenerative Cervical Myelopathy): A Consen- sus Process to Improve Research Efficiency in DCM, Through Establishment of a Standardized Dataset for Clinical Research and the Definition of the Research Priorities. Glob Spine J. 2019 May; 9(1 Suppl):65S–76S. https://doi.org/10.1177/2192568219832855 PMID: 31157148 11. Waqar M, Wilcock J, Garner J, Davies B, Kotter M. Quantitative analysis of medical students’ and physi- cians’ knowledge of degenerative cervical myelopathy. BMJ Open. 2020 Jan 12; 10(1):e028455. https://doi.org/10.1136/bmjopen-2018-028455 PMID: 31932384 12. Davies BM, Munro CF, Kotter MR. A Novel Insight Into the Challenges of Diagnosing Degenerative Cer- vical Myelopathy Using Web-Based Symptom Checkers. J Med Internet Res. 2019 Jan 11; 21(1): e10868. https://doi.org/10.2196/10868 PMID: 30300137 13. Tetreault L, Kopjar B, Nouri A, Arnold P, Barbagallo G, Bartels R, et al. The modified Japanese Ortho- paedic Association scale: establishing criteria for mild, moderate and severe impairment in patients with degenerative cervical myelopathy. Eur Spine J Off Publ Eur Spine Soc Eur Spinal Deform Soc Eur Sect Cerv Spine Res Soc. 2017 Jan; 26(1):78–84. https://doi.org/10.1007/s00586-016-4660-8 PMID: 27342612 14. Davies BM, McHugh M, Elgheriani A, Kolias AG, Tetreault LA, Hutchinson PJA, et al. Reported Out- come Measures in Degenerative Cervical Myelopathy: A Systematic Review. PloS One. 2016; 11(8): e0157263. https://doi.org/10.1371/journal.pone.0157263 PMID: 27482710 15. Hilton B, Tempest-Mitchell J, Davies B, Kotter M. Assessment of degenerative cervical myelopathy dif- fers between specialists and may influence time to diagnosis and clinical outcomes. PloS One. 2018; 13 (12):e0207709. https://doi.org/10.1371/journal.pone.0207709 PMID: 30557368 16. Davies BM, Munro C, Khan DZ, Fitzpatrick SM, Hilton B, Mowforth OD, et al. Outcomes of Degenerative Cervical Myelopathy From The Perspective of Persons Living With the Condition: Findings of a Semi- structured Interview Process With Partnered Internet Survey. Glob Spine J. 2020 Nov 18;2192568220953811. https://doi.org/10.1177/2192568220953811 PMID: 33203262 17. Boerger TF, Davies BM, Sadler I, Sarewitz E, Kotter MRN. Patient, sufferer, victim, casualty or person with cervical myelopathy: let us decide our identifier. Integr Healthc J. 2020 Jun 1; 2(1):e000023. 18. Ju¨nger S, Payne SA, Brine J, Radbruch L, Brearley SG. Guidance on Conducting and REporting DElphi Studies (CREDES) in palliative care: Recommendations based on a methodological systematic review. Palliat Med. 2017 Sep; 31(8):684–706. https://doi.org/10.1177/0269216317690685 PMID: 28190381 19. Eysenbach G. Improving the quality of Web surveys: the Checklist for Reporting Results of Internet E- Surveys (CHERRIES). J Med Internet Res. References Bot SDM, van der Waal JM, Terwee CB, van der Windt D a. WM, Schellevis FG, Bouter LM, et al. Inci- dence and prevalence of complaints of the neck and upper extremity in general practice. Ann Rheum Dis. 2005 Jan; 64(1):118–23. https://doi.org/10.1136/ard.2003.019349 PMID: 15608309 31. Finley CR, Chan DS, Garrison S, Korownyk C, Kolber MR, Campbell S, et al. What are the most com- mon conditions in primary care? Systematic review. Can Fam Physician Med Fam Can. 2018 Nov; 64 (11):832–40. PMID: 30429181 32. Ma¨ntyselka¨ P, Kumpusalo E, Ahonen R, Kumpusalo A, Kauhanen J, Viinama¨ki H, et al. Pain as a rea- son to visit the doctor: a study in Finnish primary health care. Pain. 2001 Jan; 89(2–3):175–80. https:// doi.org/10.1016/s0304-3959(00)00361-4 PMID: 11166473 33. Cook C, Roman M, Stewart KM, Leithe LG, Isaacs R. Reliability and diagnostic accuracy of clinical spe- cial tests for myelopathy in patients seen for cervical dysfunction. J Orthop Sports Phys Ther. 2009 Mar; 39(3):172–8. https://doi.org/10.2519/jospt.2009.2938 PMID: 19252263 34. Mizer A, Bachmann A, Gibson J, Donaldson MB. Self-report and subjective history in the diagnosis of painful neck conditions: A systematic review of diagnostic accuracy studies. Musculoskelet Sci Pract. 2017 Oct; 31:30–44. https://doi.org/10.1016/j.msksp.2017.06.002 PMID: 28644963 35. Kalsi-Ryan S, Karadimas SK, Fehlings MG. Cervical spondylotic myelopathy: the clinical phenomenon and the current pathobiology of an increasingly prevalent and devastating disorder. Neurosci Rev J Bringing Neurobiol Neurol Psychiatry. 2013 Aug; 19(4):409–21. https://doi.org/10.1177/ 1073858412467377 PMID: 23204243 36. Kadanka Z, Adamova B, Kerkovsky M, Kadanka Z, Dusek L, Jurova B, et al. Predictors of symptomatic myelopathy in degenerative cervical spinal cord compression. Brain Behav. 2017 Sep; 7(9):e00797. https://doi.org/10.1002/brb3.797 PMID: 28948090 37. Radcliff KE, Curry EP, Trimba R, Walker JB, Purtill JJ, Austin MS, et al. High Incidence of Undiagnosed Cervical Myelopathy in Patients With Hip Fracture Compared With Controls. J Orthop Trauma. 2016 Apr; 30(4):189–93. https://doi.org/10.1097/BOT.0000000000000485 PMID: 26562581 38. Mowforth OD, Davies BM, Kotter MR. ‘I am not delusional!’ Sensory dysaesthesia secondary to degen- erative cervical myelopathy. BMJ Case Rep. 2019 Apr 11; 12(4). 39. Rao R. Neck pain, cervical radiculopathy, and cervical myelopathy: pathophysiology, natural history, and clinical evaluation. J Bone Joint Surg Am. 2002 Oct; 84-A(10):1872–81. https://doi.org/10.2106/ 00004623-200210000-00021 PMID: 12377921 40. Muheremu A, Sun Y, Yan K, Yu J, Zheng S, Tian W. Effect of Anterior Cervical Discectomy and Fusion on Patients with Atypical Symptoms Related to Cervical Spondylosis. J Neurol Surg Part Cent Eur Neu- rosurg. 2016 Sep; 77(5):395–9. References 2004 29; 6(3):e34. https://doi.org/10.2196/jmir.6.3.e34 PMID: 15471760 20. Khan DZ, Fitzpatrick SM, Hilton B, McNair AG, Sarewitz E, Davies BM, et al. Prevailing Outcome Themes Reported by People With Degenerative Cervical Myelopathy: Focus Group Study. JMIR Form Res. 2021 Feb 3; 5(2):e18732. https://doi.org/10.2196/18732 PMID: 33533719 21. Rhee JM, Shi WJ, Cyriac M, Kim JY, Zhou F, Easley KA, et al. The P-mJOA: A Patient-derived, Self- reported Outcome Instrument for Evaluating Cervical Myelopathy: Comparison with the mJOA. Clin Spine Surg. 2018; 31(2):E115–20. https://doi.org/10.1097/BSD.0000000000000591 PMID: 29088009 22. Davies BM, McHugh M, Elgheriani A, Kolias AG, Tetreault L, Hutchinson PJA, et al. The reporting of study and population characteristics in degenerative cervical myelopathy: A systematic review. PloS One. 2017; 12(3):e0172564. https://doi.org/10.1371/journal.pone.0172564 PMID: 28249017 23. Ekman A, Dickman PW, Klint A, Weiderpass E, Litton J-E. Feasibility of using web-based question- naires in large population-based epidemiological studies. Eur J Epidemiol. 2006; 21(2):103–11. https:// doi.org/10.1007/s10654-005-6030-4 PMID: 16518678 PLOS ONE \| https://doi.org/10.1371/journal.pone.0281856 March 31, 2023 14 / 16 PLOS ONE Targeting earlier diagnosis: What symptoms come first in Degenerative Cervical Myelopathy? 24. van Gelder MMHJ Bretveld RW, Roeleveld N. Web-based questionnaires: the future in epidemiology? Am J Epidemiol. 2010 Dec 1; 172(11):1292–8. https://doi.org/10.1093/aje/kwq291 PMID: 20880962 24. van Gelder MMHJ Bretveld RW, Roeleveld N. Web-based questionnaires: the future in epidemiology? Am J Epidemiol. 2010 Dec 1; 172(11):1292–8. https://doi.org/10.1093/aje/kwq291 PMID: 20880962 25. Smith B, Smith TC, Gray GC, Ryan MAK, Millennium Cohort Study Team. When epidemiology meets the Internet: Web-based surveys in the Millennium Cohort Study. Am J Epidemiol. 2007 Dec 1; 166 (11):1345–54. https://doi.org/10.1093/aje/kwm212 PMID: 17728269 26. Touvier M, Me´jean C, Kesse-Guyot E, Pollet C, Malon A, Castetbon K, et al. Comparison between web- based and paper versions of a self-administered anthropometric questionnaire. Eur J Epidemiol. 2010 May; 25(5):287–96. https://doi.org/10.1007/s10654-010-9433-9 PMID: 20191377 27. Kelstrup AM, Juillerat P, Korzenik J. The accuracy of self-reported medical history: a preliminary analy- sis of the promise of internet-based research in Inflammatory Bowel Diseases. J Crohns Colitis. 2014 May; 8(5):349–56. https://doi.org/10.1016/j.crohns.2013.09.012 PMID: 24183653 28. Bidmon S, Terlutter R. Gender Differences in Searching for Health Information on the Internet and the Virtual Patient-Physician Relationship in Germany: Exploratory Results on How Men and Women Differ and Why. J Med Internet Res. 2015 Jun 22; 17(6):e156. https://doi.org/10.2196/jmir.4127 PMID: 26099325 29. Hoy DG, Protani M, De R, Buchbinder R. The epidemiology of neck pain. Best Pract Res Clin Rheuma- tol. 2010 Dec; 24(6):783–92. https://doi.org/10.1016/j.berh.2011.01.019 PMID: 21665126 30. 44. Kobayashi H, Kikuchi S, Otani K, Sekiguchi M, Sekiguchi Y, Konno S. Development of a self-adminis- tered questionnaire to screen patients for cervical myelopathy. BMC Musculoskelet Disord. 2010 Nov 22; 11:268. https://doi.org/10.1186/1471-2474-11-268 PMID: 21092213 Targeting earlier diagnosis: What symptoms come first in Degenerative Cervical Myelopathy? PLOS ONE \| https://doi.org/10.1371/journal.pone.0281856 March 31, 2023 S ONE Targeting earlier diagnosis: What symptoms come first in Degenerative Cervical Myelopathy? PLOS ONE Targeting earlier diagnosis: What symptoms come first in Degenerative Cervical Myelopathy? References https://doi.org/10.1055/s-0036-1582015 PMID: 27168319 41. Pearce JMS. Barre´-Lie´ou ‘syndrome’. J Neurol Neurosurg Psychiatry. 2004 Feb; 75(2):319. 42. Sun Y, Muheremu A, Yan K, Yu J, Zheng S, Tian W. Effect of double-door laminoplasty on atypical symptoms associated with cervical spondylotic myelopathy/radiculopathy. BMC Surg. 2016 May 10; 16 (1):31. https://doi.org/10.1186/s12893-016-0146-1 PMID: 27160834 43. Sun Y-Q, Zheng S, Yu J, Yan K, Tian W. Effect of total disc replacement on atypical symptoms associ- ated with cervical spondylosis. Eur Spine J Off Publ Eur Spine Soc Eur Spinal Deform Soc Eur Sect Cerv Spine Res Soc. 2013 Jul; 22(7):1553–7. https://doi.org/10.1007/s00586-013-2785-6 PMID: 23653130 15 / 16 PLOS ONE \| https://doi.org/10.1371/journal.pone.0281856 March 31, 2023 PLOS ONE Targeting earlier diagnosis: What symptoms come first in Degenerative Cervical Myelopathy? 16 / 16
https://openalex.org/W4288704555	https://amt.copernicus.org/articles/15/4751/2022/amt-15-4751-2022.pdf	English	null	Reply on RC2	null	2,022	cc-by	13,051	Correspondence: Sara Martínez-Alonso (sma@ucar.edu) We use AirCore measurements as the reference to evaluate the error introduced by this approach in cloudy TROPOMI retrievals over land after accounting for TROPOMI’s verti- cal sensitivity to CO (relative bias and its standard devia- tion = 2.02 % ± 11.13 %). We also quantify the null-space error, which accounts for differences between the shape of TROPOMI reference proﬁles and that of AirCore measured proﬁles (for TROPOMI cloudy enull = 0.98 % ± 2.32 %). MOPITT retrieval biases in that region. TROPOMI can re- trieve CO under both clear and cloudy conditions. The lat- ter is achieved by quantifying interfering trace gases and parameters describing the cloud contamination of the mea- surements together with the CO column; then, the reference CO proﬁles used in the retrieval are scaled based on esti- mated above-cloud CO rather than on estimated total CO. We use AirCore measurements as the reference to evaluate the error introduced by this approach in cloudy TROPOMI retrievals over land after accounting for TROPOMI’s verti- cal sensitivity to CO (relative bias and its standard devia- tion = 2.02 % ± 11.13 %). We also quantify the null-space error, which accounts for differences between the shape of TROPOMI reference proﬁles and that of AirCore measured proﬁles (for TROPOMI cloudy enull = 0.98 % ± 2.32 %). Atmos. Meas. Tech., 15, 4751–4765, 2022 https://doi.org/10.5194/amt-15-4751-2022 © Author(s) 2022. This work is distributed under the Creative Commons Attribution 4.0 License. Atmos. Meas. Tech., 15, 4751–4765, 2022 https://doi.org/10.5194/amt-15-4751-2022 © Author(s) 2022. This work is distributed under the Creative Commons Attribution 4.0 License. Correspondence: Sara Martínez-Alonso (sma@ucar.edu) Correspondence: Sara Martínez-Alonso (sma@ucar.edu) Received: 16 February 2022 – Discussion started: 25 February 2022 Revised: 29 July 2022 – Accepted: 2 August 2022 – Published: 22 August 2022 Received: 16 February 2022 – Discussion started: 25 February 2022 Revised: 29 July 2022 – Accepted: 2 August 2022 – Published: 22 August 2022 Abstract. AirCore in situ vertical proﬁles sample the at- mosphere from near the surface to the lower stratosphere, making them ideal for the validation of satellite tropospheric trace gas data. Here we present intercomparison results of AirCore carbon monoxide (CO) measurements with re- spect to retrievals from MOPITT (Measurements of Pollution In The Troposphere; version 8) and TROPOMI (TROPO- spheric Monitoring Instrument), on board the NASA Terra and ESA Sentinel 5-Precursor satellites, respectively. Mean MOPITT/AirCore total column bias values and their stan- dard deviation (0.4 ± 5.5, 1.7 ± 5.6, and 0.7 ± 6.0 for MO- PITT thermal-infrared, near-infrared, and multispectral re- trievals, respectively; all in %) are similar to results obtained in MOPITT/NOAA aircraft ﬂask data comparisons from this study and from previous validation efforts. MOPITT CO re- trievals are systematically validated using in situ vertical pro- ﬁles from a variety of aircraft campaigns. Because most air- craft vertical proﬁles do not sample the troposphere’s entire vertical extent, they must be extended upwards in order to be usable in validation. Here we quantify, for the ﬁrst time, the error introduced in MOPITT CO validation by the use of shorter aircraft vertical proﬁles extended upwards by ana- lyzing validation results of MOPITT with respect to full and truncated AirCore CO vertical proﬁles. Our results indicate that the error is small, affects mostly upper tropospheric re- trievals (at 300 hPa: ∼2.6, 0.8, and 3.2 percent points for MOPITT thermal-infrared, near-infrared, and multispectral, respectively), and may have resulted in the overestimation of MOPITT retrieval biases in that region. TROPOMI can re- trieve CO under both clear and cloudy conditions. The lat- ter is achieved by quantifying interfering trace gases and parameters describing the cloud contamination of the mea- surements together with the CO column; then, the reference CO proﬁles used in the retrieval are scaled based on esti- mated above-cloud CO rather than on estimated total CO. 1 Introduction Tropospheric CO (carbon monoxide) is mostly produced by incomplete combustion of fuels, biomass burning, and atmo- spheric oxidation of CH4 (methane) and other hydrocarbons. Its main sink is oxidation by OH (the hydroxyl radical) (Spi- vakovsky et al., 2000; Lelieveld et al., 2016). CO is of great importance in understanding climate and for monitoring and predicting air quality because it has an indirect positive radia- tive forcing (Szopa et al., 2021) and is an excellent tracer for identifying pollution sources, transport, and sinks. A long, 1Atmospheric Chemistry Observations and Modeling (ACOM), National Center for Atmospheric Research (NCAR), 1Atmospheric Chemistry Observations and Modeling (ACOM), National Center for Atmospheric Research (NCAR), B ld CO USA 1Atmospheric Chemistry Observations and Modeling (ACOM), National Center for Atmospheric Research (NCAR), Boulder, CO, USA 2Global Monitoring Laboratory (GML), National Oceanic and Atmospheric Administration, Boulder, CO, USA 3Cooperative Institute for Research in Environmental Sciences (CIRES), University of Colorado, Boulder, CO, USA 4SRON Netherlands Institute for Space Research, Leiden, the Netherlands Evaluation of MOPITT and TROPOMI carbon monoxide retrievals using AirCore in situ vertical proﬁles Sara Martínez-Alonso1, Merritt N. Deeter1, Bianca C. Baier2,3, Kathryn McKain2,3, Helen Worden1, Tobias Borsdorff4, Colm Sweeney3, and Ilse Aben4 2.1 AirCore The AirCore (Tans, 2009; Karion et al., 2010; Tans, 2022) is an innovative atmospheric sampling system comprised of a long tubing coil that is used to passively sample the at- mosphere on high-altitude balloons. Before launch the Air- Core is ﬁlled with a gas mixture of known composition: the “ﬁll gas”, which is comprised of ambient levels of CO2 (car- bon dioxide) and CH4 but is spiked with high CO mole frac- tions. With one end closed and the other open to the out- side air, the AirCore evacuates the ﬁll gas as the balloon as- cends to ∼30 km above mean sea level. Once the AirCore is released from the balloon, it collects a continuous sam- ple of ambient air as it descends from the altitude ceiling to the ground. Upon landing, the open end of the coil is auto- matically closed, thus preserving the sample air inside. Mix- ing (which is only a result of molecular diffusion and Taylor dispersion) is relatively insigniﬁcant: ∼0.5 m in both direc- tions over the 4 h typically needed to retrieve and analyze the air sample; thus, in the case of the NOAA AirCore de- sign used for this analysis, approximately 100 discrete sam- ples can be measured in these 100 m long, uniform-diameter tubing coils (Tans, 2022). The quantiﬁed altitude uncertainty of trace gas proﬁles retrieved using this technique, provided in the data ﬁles, is dependent upon the bi-directional diffu- sion of molecules of a gas of interest in the AirCore sample and is larger at higher altitudes because the air sampled ﬁrst (i.e., that in the stratosphere) has a longer diffusion time in the tubing coil. While not empirically quantiﬁed in NOAA AirCore CO proﬁles presented here, others have quantiﬁed the uncertainty in AirCore altitude registration due to incor- rect assumptions in the AirCore tubing pressure equilibrium during balloon descent (Wagenhaeuser et al., 2021). NOAA proﬁles attempt to correct for pressure disequilibrium in the AirCore tubing and for its effect on the total mass of air en- TROPOMI (the TROPOspheric Monitoring Instrument) (Veefkind et al., 2012), onboard the ESA Sentinel-5 Precur- sor platform, measures CO, among other species, at high spa- tial resolutions. Unlike MOPITT, TROPOMI can retrieve CO under both clear and cloudy conditions. S. Martínez-Alonso et al.: AirCore CO for satellite validation S. Martínez-Alonso et al.: AirCore CO for satellite validation S. Martínez-Alonso et al.: AirCore CO for satellite validation consistent global tropospheric CO record allows for the de- tection of spatial, seasonal, and long-term trends as well as for the placement of individual CO-emitting events into con- text, which is key to a better understanding of their signiﬁ- cance. face to the lower stratosphere. Because of its ability to sam- ple such a large vertical range, AirCore is a great candidate for validating tropospheric satellite instruments. The AirCore atmospheric sampling system consists of an airborne coiled tube, typically ﬂown on a balloon and ﬁlled with a gas of known composition, which is evacuated during ascent; once the balloon’s altitude ceiling is reached, the now-empty tube starts a parachute-based descent, during which it ﬁlls with the air it encounters. After recovery, the whole-air sample collected is analyzed in the laboratory for various long-lived atmospheric trace gases. The MOPITT (Measurements of Pollution In The Tro- posphere) instrument (Drummond and Mand, 1996; Drum- mond et al., 2010), onboard NASA’s Terra satellite, pro- vides the longest global record of tropospheric CO avail- able to date (2000–present). The MOPITT dataset is con- sistent and, thus, useful in climate and air quality analy- ses because it is systematically validated with respect to both aircraft data (Emmons et al., 2004; Deeter et al., 2010, 2012, 2013, 2014, 2017, 2018, 2019) and ground- based measurements (Buchholz et al., 2017; Hedelius et al., 2019). The vertical extent of most aircraft in situ proﬁles that are used to validate satellite retrievals of tropospheric trace gases is largely determined by the range of the aircraft used to collect them and is often not sufﬁcient to sample in its entirety the vertical column sensed by the satellite in- struments. In those cases, the aircraft measurements closest to the tropopause and modeled a priori vertical proﬁles of the species of interest are used to extend upwards the mea- sured aircraft proﬁles to allow for comparison to satellite- retrieved values (see, for example, Martínez-Alonso et al., 2014, 2020); the error associated with this approach is un- known. In the following sections we describe the datasets used in this study (Sect. 2), detail the methodology used in the analy- ses outlined above (Sect. 3), present our results (Sect. 4), and discuss their relevance (Sect. 5). In Sect. 6 we offer conclu- sions. Published by Copernicus Publications on behalf of the European Geosciences Union. Published by Copernicus Publications on behalf of the European Geosciences Union. 4752 2.1 AirCore It is also possi- ble that chemical interactions or measurement interferences from other trace gas species or incorrect AirCore sample end- member assumptions have been made. Given these uncer- tainties and the number of independent CO measurements in each AirCore sample, we derive an estimated AirCore XCO uncertainty of ∼1.3 ppb (2 sigma), equivalent to ∼1.8 %. Figure 1. AirCore vertical proﬁle locations listed from west to east. USA: Edwards Air Force Base (California), Boulder (Colorado), Lamont (Oklahoma), and Park Falls (Wisconsin). France: Traînou. Finland: Sodankylä. New Zealand: Lauder. tering the AirCore at each altitude through comparisons of modeled pressure equilibrium and that measured in situ be- tween ambient air and the closed end of the AirCore. This uncertainty is largest at lower atmospheric pressures (i.e., at more than ∼20 km above mean sea level) than at higher ones. We hypothesize that this potential uncertainty compo- nent is likely to be of smaller magnitude than that calculated for CO diffusion at altitudes up to 15–20 km (above which a higher uncertainty is likely, but these portions of the CO pro- ﬁles are discarded, as described below). Therefore, we be- lieve this potential uncertainty component would have a neg- ligible inﬂuence on the results presented here. AirCore sam- ple trace gas proﬁles retrieved are calibrated and traceable to World Meteorological Organization standard scales. The AirCore trace gas measurements have been rigorously evalu- ated and have shown comparable repeatability (precision) to those from aircraft data collected from continuous analyzers and sampled in silicate glass ﬂasks (Karion et al., 2010). The most recent AirCore dataset (13 August 2021 version; Baier et al., 2021) contains over 130 vertical proﬁles of CO, CO2, CH4, temperature, and relative humidity acquired at several locations worldwide (Fig. 1) between January 2012 and July 2021. Unlike most aircraft vertical proﬁles, AirCore proﬁles sample from near the surface to the lower stratosphere and, therefore, do not need to be extended upwards with their clos- est measurement to the tropopause and a priori values in or- der to represent the full tropospheric column as measured by satellite instruments. The NOAA AirCore systems are typically deployed in pairs on the same balloon ﬂight string. We have quantiﬁed the repeatability of retrieved CO proﬁles by comparing the 41 pairs of AirCore proﬁles launched simultaneously (i.e., with zero minutes lag time) and from the same site. 2.1 AirCore TROPOMI retrieves parameters describing the cloud contamination of the mea- surement (cloud height, cloud optical thickness) and inter- fering trace gases together with the CO column. TROPOMI retrievals are based on the proﬁle-scaling method (Borsdorff et al., 2014; Landgraf et al., 2016). Under cloudy conditions, the scaling of the proﬁle is estimated by the CO concentra- tion in higher altitudes in the atmosphere instead of by the real total CO column; this is fully described by the total col- umn AK (averaging kernel) supplied with the data product. The error introduced by this approach on cloudy TROPOMI retrievals over bodies of water has been previously quantiﬁed (on the order of a few percent; Martínez-Alonso et al., 2020). Errors over land could in theory be larger, since most pollu- tion sources are on land and close to the surface (i.e., below cloud top). Here we use AirCore data (Tans, 2009; Karion et al., 2010) to estimate for the ﬁrst time the error introduced in MOPITT validation results by the use of shorter aircraft vertical CO proﬁles extended upwards. We also investigate the error in- troduced by clouds on TROPOMI land CO retrievals by com- paring them to AirCore vertical proﬁles. AirCore provides calibrated, high-precision measurements of CO and other long-lived species along vertical proﬁles from near the sur- Atmos. Meas. Tech., 15, 4751–4765, 2022 https://doi.org/10.5194/amt-15-4751-2022 4753 S. Martínez-Alonso et al.: AirCore CO for satellite validation Figure 1. AirCore vertical proﬁle locations listed from west to east. USA: Edwards Air Force Base (California), Boulder (Colorado), Lamont (Oklahoma), and Park Falls (Wisconsin). France: Traînou. Finland: Sodankylä. New Zealand: Lauder. other trace gas proﬁles (Karion et al., 2010). The top- and bottommost portions of AirCore CO proﬁles used for this correction are thus discarded, resulting in CO proﬁles that extend from the near-surface to between 15 and 20 km above mean sea level. CO in AirCore samples is measured by cav- ity ringdown-spectroscopy (CRDS) at a precision typically less than 5 ppb (Karion et al., 2013) for ∼0.5 Hz measure- ments. AirCore CO is, however, still considered a develop- mental product due to its use for correcting end-member mix- ing in other trace gas proﬁles. Comparisons of stratospheric AirCore CO proﬁles have sometimes shown differences up to ∼15 ppb, which could be a result of AirCore tubing sur- face interactions, uncertainties in the altitude registration of the AirCore CO proﬁle, or diffusion effects. S. Martínez-Alonso et al.: AirCore CO for satellite validation S. Martínez-Alonso et al.: AirCore CO for satellite validation S. Martínez-Alonso et al.: AirCore CO for satellite validation Figure 2. Differences between CO vertical proﬁles acquired by pairs of AirCore systems deployed simultaneously and from the same location. Pink: biases for each AirCore pair. Black: mean of all biases. Gray: mean ±1 standard deviation (SD). ity to CO near the surface in some land observations (Wor- den et al., 2010). MOPITT CO proﬁles are provided for 10 levels (surface, 900 hPa, ..., 100 hPa), where each retrieval level corresponds to a uniformly weighted layer immediately above that level (Deeter et al., 2013). MOPITT retrievals are performed under clear conditions only, allowing ≤5 % cloud areal coverage inside the ﬁeld of view. Here we use level 2 TIR, NIR, and multispectral standard archival ﬁles (Deeter et al., 2017) from MOPITT version 8 (Deeter et al., 2019). ity to CO near the surface in some land observations (Wor- den et al., 2010). MOPITT CO proﬁles are provided for 10 levels (surface, 900 hPa, ..., 100 hPa), where each retrieval level corresponds to a uniformly weighted layer immediately above that level (Deeter et al., 2013). MOPITT retrievals are performed under clear conditions only, allowing ≤5 % cloud areal coverage inside the ﬁeld of view. Here we use level 2 TIR, NIR, and multispectral standard archival ﬁles (Deeter et al., 2017) from MOPITT version 8 (Deeter et al., 2019). 2.3 TROPOMI TROPOMI is a push-broom imaging spectrometer in a sun- synchronous orbit at 824 km of altitude and with a 13:30 LST Equator-crossing time (Veefkind et al., 2012). Because of its wide (2600 km) swath width, it provides quasi-global daily coverage. Its spatial resolution at nadir has been near 7 × 5.5 km2 (across × along track) since 6 August 2019, down from around 7×7 km2 before that date. A change in the Copernicus Sentinel-5P operations scenario resulted in this resolution improvement (Landgraf et al., 2021). TROPOMI measures radiance in the ultraviolet, visible, and reﬂected- infrared; total CO column values are retrieved from the latter (from a ∼2.3 µm band, like MOPITT). CO retrievals over land are obtained in both clear and cloudy conditions; the latter is possible by retrieving effective parameters (cloud height and optical thickness) that describe the cloud contam- ination of the measurements simultaneously with the trace gas columns (Landgraf et al., 2016) and then approximat- ing partial CO columns under cloud tops with scaled refer- ence proﬁles from the global chemical transport model TM5 (Krol et al., 2005). Even though reﬂected-infrared radiances are used, this approach allows for the retrieval of CO over bodies of water if clouds are present; in their absence, most of the incoming radiation is absorbed by the water. We have used, for any given day, TROPOMI data ﬁles from the most recent processor version available (1 January–1 April 2000), either ofﬂine or reprocessed. Figure 2. Differences between CO vertical proﬁles acquired by pairs of AirCore systems deployed simultaneously and from the same location. Pink: biases for each AirCore pair. Black: mean of all biases. Gray: mean ±1 standard deviation (SD). main similarly low to those obtained with the most restricted colocation thresholds. Increasing biases near the surface with larger colocation distances is consistent with CO values be- ing more variable near the surface, where emissions take place. Horizontal displacements between the start and end of AirCore proﬁles are on average 26 km ± 10 km, similar to those between the start and end of NOAA aircraft ﬂask proﬁles (14 km ± 18 km at the Park Falls, Wisconsin site; 53 km ± 28 km at the East Trout Lake, Saskatchewan site). 2.1 AirCore Each proﬁle was resampled to a common 20 000-level vertical grid, and intra-pair differences were calculated. Figure 2 shows that, at most altitudes, mean differences are well below ±2 ppb (av- erage 0.03 ppb). Mean differences at the top of the proﬁles (between 50 and 70 hPa, approximately) are slightly larger (5–15 ppb); this is consistent with the higher uncertainty in AirCore stratospheric CO retrievals described by Chen et al. (2022). We have compared the AirCore-retrieved CO proﬁles to colocated CO vertical proﬁles from the NOAA aircraft ﬂask dataset (GLOBALVIEWplus v2.0 ObsPack; Sweeney et al., 2021) in order to quantify biases of the former with respect to the latter (∼accuracy). The NOAA aircraft ﬂask dataset (or “aircraft dataset” for simplicity) has been described in detail by Sweeney et al. (2015) and used extensively in MO- PITT validation (Deeter et al., 2019, and references therein). We analyzed the two datasets in their entirety (i.e., all sites and dates). Only proﬁles from the Southern Great Plains site (in Oklahoma, USA; 36.607◦N, −97.489◦E) acquired be- tween January 2012 and July 2018 satisﬁed the different colocation criteria imposed. The averaged biases (AirCore minus aircraft data) for the ﬁve available colocated pairs ac- quired less than 2 h and 15 km apart range between approxi- mately −6 and +6 ppb (near 750 and 920 hPa, respectively), with a 0.6 ppb overall average bias (Fig. 3a). Allowing larger distances between colocated pairs results in more colocated pairs and a slight increase in biases closer to the surface: up to 13 ppb for colocation distance < 25 km (Fig. 3b) and up to 20 ppb for colocation distance < 50 km (Fig. 3c) at ∼875 hPa in both cases. Biases at lower pressure levels re- Current techniques for retrieving trace gas proﬁles rely on the use of a CO-spiked ﬁll gas – all of which except ∼1 % evacuates during balloon ascent – and a high CO mole fraction “push gas” that follows the AirCore sample during analysis. Both mixtures are used to identify the be- ginning and end of the air sample collected but affect the topmost (stratospheric) and bottommost (near-surface) por- tions of the proﬁle through end-member mixing. With this method, CO is used to correct for “end-member” mixing in https://doi.org/10.5194/amt-15-4751-2022 Atmos. Meas. Tech., 15, 4751–4765, 2022 4754 S. Martínez-Alonso et al.: AirCore CO for satellite validation S. Martínez-Alonso et al.: AirCore CO for satellite validation 4755 Figure 3. Differences between colocated CO vertical proﬁles from AirCore and NOAA aircraft ﬂask campaign (AirCore minus aircraft data). Black lines show the bias for each colocated pair, while averaged biases from all pairs are shown in red. Colocation criteria and number of colocated pairs are indicated in each panel. Figure 3. Differences between colocated CO vertical proﬁles from AirCore and NOAA aircraft ﬂask campaign (AirCore minus aircraft data). Black lines show the bias for each colocated pair, while averaged biases from all pairs are shown in red. Colocation criteria and number of colocated pairs are indicated in each panel. ori (Rodgers and Connor, 2003). The MOPITT algorithm is based on optimal estimation, as developed by Rodgers (2000); thus, for MOPITT, (Martínez-Alonso et al., 2020). AirCore proﬁles were inter- polated to match the MOPITT a priori 35-level vertical grid, which preserves high vertical resolution in the troposphere. Empty levels at the bottom of each interpolated proﬁle (lev- els with no CO value) were ﬁlled with the interpolated mea- surement closest to the surface. Empty levels between the top of the interpolated proﬁle and the tropopause would usu- ally be ﬁlled with the interpolated measurement closest to the tropopause; however, because all AirCore proﬁles reached the tropopause, this step was not necessary. Finally, empty levels above the tropopause were ﬁlled with colocated MO- PITT a priori CO values. The now complete AirCore proﬁles were interpolated to match the 10-level vertical grid of the MOPITT retrievals. Total CO column values were derived from the vertical proﬁles as follows: (1) Cret = Ca + Ac(Xtrue −Xa), (1) where Cret is the retrieved total column value, Ca is the a priori total column value, Ac is the total column averaging kernel, Xtrue is the true proﬁle value (i.e., the actual atmo- spheric composition at the time and location of the remote observation, approximated in practice with in situ measure- ments), and Xa is the a priori proﬁle value. Ac is unitless, and all other variables are expressed in column density, i.e., molecules per unit area. By applying Eq. (1) to the in situ proﬁle, we can simulate the effects of the remote sounder retrieval and produce a “smoothed” version of the in situ measurement, which can then be directly compared to the sounder retrieval. 3 Methodology MOPITT, onboard NASA’s Terra satellite, is a cross-track scanning gas correlation radiometer (Drummond and Mand, 1996; Drummond et al., 2010; Worden et al., 2013). From its sun-synchronous orbit at 705 km of altitude and 10:30 LST (local standard time) Equator-crossing time, it provides global coverage approximately every 3 d with a 22 × 22 km2 footprint at nadir. It measures radiances in two spectral bands: one in the near-infrared (NIR, at ∼2.3 µm), the other in the thermal-infrared (TIR, at ∼4.7 µm). Tropospheric CO proﬁles and total CO column values are derived separately from measurements in each of these two bands as well as from their combined multispectral radiances (TIR+NIR). MOPITT is currently the only satellite instrument capable of multispectral CO retrievals, which have enhanced sensitiv- We compare tropospheric total CO column retrievals from MOPITT and TROPOMI with respect to their colocated AirCore counterparts. Additional comparisons of colocated MOPITT and truncated AirCore vertical proﬁles were also performed. Colocation criteria required that observations from the two instruments involved were acquired within ≤12 h from each other and that their horizontal distance was ≤50 km, which are the same thresholds routinely used in MOPITT validations over land (e.g., Deeter et al., 2019). Comparisons of remote sounder retrievals obtained with optimal estimation-based methods and in situ measure- ments must take into account the characteristics of the retrieval, e.g., its averaging kernels, or AK, and a pri- https://doi.org/10.5194/amt-15-4751-2022 Atmos. Meas. Tech., 15, 4751–4765, 2022 S. Martínez-Alonso et al.: AirCore CO for satellite validation 4755 4.1.1 Effect of extending shorter aircraft proﬁles upwards As stated earlier, aircraft proﬁles used in MOPITT valida- tion do not, in most cases, sample the entire troposphere due to limitations in the maximum altitude reachable by the sam- pling aircraft. The aircraft proﬁles in these cases are extended to the tropopause using the interpolated aircraft measurement closest to the tropopause and above the tropopause using a priori data from the CAM-chem model (Community At- mosphere Model with chemistry, Lamarque et al., 2012) for the same location and month of the aircraft proﬁle to be ex- tended. The error introduced in validation by extending air- craft proﬁles upwards was expected to be small, but quanti- fying it had not been possible in the past due to the lack of suitable in situ measurements intrinsic to this problem. The AirCore dataset brings, for the ﬁrst time, the opportunity to quantify this error. To this effect, we simulated a shorter Air- Core dataset by truncating all AirCore proﬁles at 7000 m, i.e., slightly above the 400 hPa pressure threshold, which must be reached by aircraft proﬁles to be usable in MOPITT valida- tion. The truncated AirCore proﬁles were then extended up- wards using the closest measurement to the tropopause and a priori CO data and compared to the MOPITT dataset, as described earlier. For consistency, we constrained this analy- sis to the period between January 2012 and December 2019. Results are summarized in Table 2 and Fig. 4. Biases between the three MOPITT variants (TIR, NIR, and multispectral) relative to AirCore are well below the MOPITT 10 % target accuracy (Francis et al., 2017) in all cases. MOPITT TIR partial column biases range from −2.4 % at 600 hPa and 0.9 % at 300 hPa; their mean is −0.85 %. MOPITT TIR total column bias is below 0.1 × 1017 molec. cm−2. MOPITT NIR partial column biases are even smaller, ranging between 0.2 % at 100 hPa and 1.3 % at 900, 800, 500, and 400 hPa, with a 1.1 % mean. The NIR total column bias is 0.3×1017 molec. cm−2. Partial col- umn biases for the MOPITT multispectral variant are be- tween −5.8 % and 2.4 % at 500 and 300 hPa, respectively; the mean is −1.64 %. MOPITT multispectral total column bias is 0.1 × 1017 molec. cm−2. 4.1 MOPITT vs. AirCore CO values from the MOPITT version 8 and AirCore datasets were compared following the standard validation procedure, as described above. Additionally, for reference, we per- formed an analogous comparison of MOPITT data with re- spect to the NOAA aircraft ﬂask dataset traditionally used in MOPITT validation. To avoid ambiguities, we constrained both sets of comparisons to the period between January 2012 (the start of the AirCore dataset) and December 2019 (the most recent aircraft data ofﬁcially available at the time of writing). Figure 4 summarizes CO bias values obtained in these comparisons; those values are also shown in Table 1, where full MOPITT validation results for the 2000–2018 pe- riod with respect to the aircraft dataset (from Deeter et al., 2019) are included for reference. (4) (4) enull = (I −Ac)Xtrue, (4) enull = (I −Ac)Xtrue, where I (a vector of ones) is the total column operator. The TROPOMI null-space error is indicative of differences be- tween the shape of TROPOMI CO reference proﬁles and that of true CO proﬁles, which may result in differences between the true and TROPOMI-retrieved total CO column values. This error is only important when total column TROPOMI AKs are not used in retrieval comparisons or validations; oth- erwise, enull is, by deﬁnition, zero. where I (a vector of ones) is the total column operator. The TROPOMI null-space error is indicative of differences be- tween the shape of TROPOMI CO reference proﬁles and that of true CO proﬁles, which may result in differences between the true and TROPOMI-retrieved total CO column values. This error is only important when total column TROPOMI AKs are not used in retrieval comparisons or validations; oth- erwise, enull is, by deﬁnition, zero. MOPITT partial column biases with respect to both the AirCore and the aircraft datasets follow very similar vertical patterns (Fig. 4). The MOPITT multispectral variant displays the most extreme bias values. TIR and multispectral biases are close to zero near the surface, become negative between the surface and 500 hPa, and then become positive between 300 and 100 hPa. In general, MOPITT biases with respect to AirCore and aircraft data are similar in the low-mid tro- posphere (i.e., between the surface and 500 hPa) for the TIR and multispectral variants. In contrast, biases for these two variants are closer to zero in the upper troposphere (300– 100 hPa) when AirCore is involved. The NIR variant shows very small positive biases at all pressure levels for both Air- Core and aircraft data. S. Martínez-Alonso et al.: AirCore CO for satellite validation C = 2.12 × 1013 n X i=1 ∆pixi, (3) (3) (3) The Rodgers and Connor (2003) methodology is not ap- plicable to TROPOMI retrievals because the TROPOMI al- gorithm is not based on the optimal-estimation method but rather on Tikhonov regularization (Vidot et al., 2012; Bors- dorff et al., 2014; Landgraf et al., 2016, and references therein). For TROPOMI, where C is the total column value in molec. cm−2, the con- stant 2.12 × 1013 is in molec. cm−2 hPa−1 ppb−1, n is the number of partial columns in the proﬁle, 1pi is the thick- ness of partial column i in hPa, and xi is the mean volume mixing ratio (VMR) for the layer above level i reported in ppb units. The derivation of Eq. (3) can be found in Deeter (2009). (2) Cret = AcXtrue. (2) Cret = AcXtrue. Statistical values from the comparison of the satellite datasets (MOPITT, TROPOMI) with respect to AirCore in situ measurements were then calculated (satellite minus Air- Core). Applying Eq. (2) to the in situ proﬁle results in a retrieval- simulated (smoothed) version of the in situ measurement, which can be directly compared to the TROPOMI retrieval. Prior to obtaining smoothed AirCore total CO columns, complete (e.g., from the surface to the top of the atmo- sphere) AirCore CO proﬁles were generated following the standard method for MOPITT validation with aircraft data Additionally, we calculated the error introduced by ap- proximating TROPOMI’s partial columns below cloud-top with the TROPOMI reference proﬁles by calculating the null-space error (enull) of the TROPOMI retrieval process https://doi.org/10.5194/amt-15-4751-2022 Atmos. Meas. Tech., 15, 4751–4765, 2022 4756 S. Martínez-Alonso et al.: AirCore CO for satellite validation S. Martínez-Alonso et al.: AirCore CO for satellite validation (Borsdorff et al., 2014; Landgraf et al., 2016): (Borsdorff et al., 2014; Landgraf et al., 2016): MOPITT NIR partial column biases range between 0.1 % at 100 hPa and 1.8 % at 900 hPa; the mean is 0.84 %. MO- PITT NIR total column bias is 0.3×1017 molec. cm−2. MO- PITT multispectral total column biases range from −5.6 % at 600 hPa to 7.8 % at 200 hPa, with a −0.23 % mean. MOPITT multispectral total column bias is 0.2 × 1017 molec. cm−2. Partial column SD values range from 1.6 % (NIR, 100 hPa) to 16.5 % (TIR+NIR, 300 hPa), with a mean of 7.5 %. For total column, the SD values are between 1.1 and 1.4 × 1017 molec. cm−2 (mean = 1.23×1017 molec. cm−2). These statistical results are in good agreement with the values re- ported by Deeter et al. (2019) for the 2000–2018 period (Ta- ble 1). S. Martínez-Alonso et al.: AirCore CO for satellite validation Table 1. Statistics from the comparison of MOPITT CO with respect to AirCore and to NOAA aircraft ﬂask data for the 2012–2019 period. Statistics from the validation of MOPITT CO with respect to aircraft data for 2000–2018 (Deeter et al., 2019) are also included for reference. Column bias and SD are provided in percent (%) and in units of 1017 molec. cm−2 (in parentheses). Column bias r from VMR values (in parentheses) are lower than r from percent values because VMR results are presented in terms of log(VMR) after subtracting the a priori values (see Deeter et al., 2017); thus, the a priori variability does not contribute to the correlation. Partial column relative bias and SD in %. Partial column results only shown for even pressure levels, for simplicity. S. Martínez-Alonso et al.: AirCore CO for satellite validation Total column Surface 800 hPa 600 hPa 400 hPa 200 hPa MOPITT TIR vs AirCore Bias 0.4 (<0.1) −0.6 −1.4 −2.4 −1.1 0.6 (2012–2019) SD 5.5 (0.9) 4.3 5.7 6.7 10.0 6.9 r 0.84 (0.71) 0.82 0.74 0.63 0.52 0.49 MOPITT TIR vs aircraft Bias 0.7 (0.1) 0.0 −1.3 −1.7 1.5 3.0 (2012–2019) SD 6.3 (1.2) 5.1 5.8 6.8 9.8 7.5 r 0.95 (0.84) 0.80 0.83 0.83 0.74 0.56 MOPITT TIR vs aircraft Bias (0.2) 0.5 −0.7 −1.3 1.6 3.0 (2000–2018) SD (1.4) 5.7 7.2 8.3 11.2 8.3 r (0.82) 0.74 0.77 0.80 0.72 0.54 MOPITT NIR vs AirCore Bias 1.7 (0.3) 1.1 1.3 1.2 1.3 0.8 (2012–2019) SD 5.6 (1.0) 4.6 5.4 5.4 5.8 4.0 r 0.84 (0.30) 0.42 0.37 0.32 0.32 0.46 MOPITT NIR vs aircraft Bias 1.3 (0.3) 0.9 0.9 0.8 0.9 0.5 (2012–2019) SD 6.0 (1.1) 5.5 5.8 5.7 6.0 4.5 r 0.93 (0.57) 0.57 0.61 0.62 0.62 0.60 MOPITT NIR vs aircraft Bias (0.1) 0.1 −0.1 −0.2 −0.1 −0.4 (2000–2018) SD (1.3) 6.3 6.5 6.2 6.6 4.8 r (0.60) 0.60 0.62 0.64 0.64 0.61 MOPITT TIR+NIR vs AirCore Bias 0.7 (0.1) −0.5 −2.5 −5.7 −3.0 2.3 (2012-2019) SD 6.0 (1.0) 8.1 8.6 8.8 10.3 11.5 r 0.86 (0.73) 0.67 0.63 0.46 0.43 0.16 MOPITT TIR+NIR vs aircraft Bias 0.9 (0.2) −0.1 −3.4 −5.6 0.2 7.8 (2012-2019) SD 7.2 (1.4) 9.9 9.6 8.6 12.4 13.4 r 0.94 (0.83) 0.66 0.73 0.80 0.66 0.30 MOPITT TIR+NIR vs aircraft Bias (0.2) −0.1 −2.7 −5.1 0.2 6.7 (2000-2018) SD (1.6) 9.8 11.7 10.6 14.1 14.7 r (0.81) 0.62 0.68 0.76 0.64 0.30 Biases between the three MOPITT variants and truncated AirCore are well below the MOPITT 10 % target accuracy. MOPITT TIR partial column biases range from −1.4 % at 600–700 hPa and 3.5 % at 300 hPa; their mean is 0.25 %. MOPITT TIR total column bias is 0.2 × 1017 molec. cm−2. MOPITT NIR partial column biases range between 0.4 % at 100 hPa and 2.1 % at 900 hPa, with a 1.73 % mean. The NIR total column bias is 0.4 × 1017 molec. cm−2. Par- tial column biases for MOPITT multispectral are between −4.9 % and 5.6 % at 600 and 300 hPa, respectively; the mean is −0.55 %. MOPITT multispectral total column bias is 0.2 × 1017 molec. cm−2. 4.1.1 Effect of extending shorter aircraft proﬁles upwards Partial column standard de- viation (SD) values range from 1.4 (for MOPITT TIR at 100 hPa) to 13.5 % (TIR+NIR, 300 hPa), with a mean of 6.9 %. For total column, the SD values are between 0.9 and 1.0 × 1017 molec. cm−2 (mean = 0.97 × 1017 molec. cm−2). Biases between the three MOPITT variants and aircraft proﬁles are also analyzed here for reference; next we de- scribe results for the same time period covered by AirCore. MOPITT TIR partial column biases range from −1.7 % at 700 and 600 hPa and 3.9 % at 300 hPa, with a 0.3 % mean. MOPITT TIR total column bias is 0.1 × 1017 molec. cm−2. https://doi.org/10.5194/amt-15-4751-2022 Atmos. Meas. Tech., 15, 4751–4765, 2022 4757 S. Martínez-Alonso et al.: AirCore CO for satellite validation Partial column SD values range from 1.4 % (NIR, 100 hPa) to 11.6 % (TIR+NIR, 300 hPa), with a mean of 6.2 %. For total column, the SD values are between 0.8 and 0.9 × 1017 molec. cm−2 (mean = 0.84 × 1017 molec. cm−2). In general, MOPITT multispectral prod- ucts exhibit more extreme retrieval errors compared to TIR and NIR retrievals because the effects of potential biases be- tween measured and calculated radiances are ampliﬁed in the multispectral version of the retrieval algorithm. This ampliﬁ- cation is done intentionally to boost the inﬂuence of the NIR radiances on the retrieval. In addition, multispectral retrievals are generally less stable than TIR and NIR retrievals because there is a greater chance that the radiances used in the re- trieval will not be internally consistent (Deeter et al., 2012). Figure 4 shows that biases between MOPITT and trun- cated AirCore partial columns differ from the MOPITT/Air- Core biases described in Sect. 4.1. In general, biases between all three MOPITT variants and truncated AirCore proﬁles ap- pear to shift to the right (i.e., increase slightly) with respect S. Martínez-Alonso et al.: AirCore CO for satellite validation S. Martínez-Alonso et al.: AirCore CO for satellite validation Figure 4. CO biases for the 2012–2019 period from the comparison of MOPITT with respect to NOAA aircraft ﬂask data (blue), AirCore proﬁles (pink), and truncated AirCore proﬁles extended upwards (purple). (a) For MOPITT TIR. (b) For MOPITT NIR. (c) For MOPITT multispectral. Column bias values are provided in percent (%) and in units of 1017 molec. cm−2 (in parentheses). The ±10 % CO bias range is equal to the MOPITT target accuracy (Francis et al., 2017). Figure 4. CO biases for the 2012–2019 period from the comparison of MOPITT with respect to NOAA aircraft ﬂask data (blue), AirCore proﬁles (pink), and truncated AirCore proﬁles extended upwards (purple). (a) For MOPITT TIR. (b) For MOPITT NIR. (c) For MOPITT multispectral. Column bias values are provided in percent (%) and in units of 1017 molec. cm−2 (in parentheses). The ±10 % CO bias range is equal to the MOPITT target accuracy (Francis et al., 2017). Table 2. Statistics from the comparison of MOPITT CO with respect to truncated AirCore proﬁles extended upwards. Column bias and SD are provided in percent (%) and in units of 1017 molec. cm−2 (in parentheses). Column bias r from VMR values (in parentheses) are lower than r from percent values because VMR results are presented in terms of log(VMR), after subtracting the a priori values (see Deeter et al., 2017); thus, the a priori variability does not contribute to the correlation. Partial column relative bias and SD in %. Partial column results only shown for even pressure levels, for simplicity. Table 2. Statistics from the comparison of MOPITT CO with respect to truncated AirCore proﬁles extended upwards. Column bias and SD are provided in percent (%) and in units of 1017 molec. cm−2 (in parentheses). Column bias r from VMR values (in parentheses) are lower than r from percent values because VMR results are presented in terms of log(VMR), after subtracting the a priori values (see Deeter et al., 2017); thus, the a priori variability does not contribute to the correlation. Partial column relative bias and SD in %. Partial column results only shown for even pressure levels, for simplicity. S. Martínez-Alonso et al.: AirCore CO for satellite validation Total column Surface 800 hPa 600 hPa 400 hPa 200 hPa MOPITT TIR vs truncated AirCore Bias 1.5 (0.2) −0.4 −1.0 −1.4 1.1 2.3 (2012–2019) SD 4.8 (0.8) 4.0 5.0 5.5 8.5 6.1 r 0.87 (0.78) 0.84 0.78 0.73 0.67 0.63 MOPITT NIR vs truncated AirCore Bias 2.4 (0.4) 1.8 2.0 1.8 2.0 1.4 (2012–2019) SD 5.1 (0.9) 4.4 5.1 5.0 5.3 3.8 r 0.86 (0.38) 0.46 0.42 0.39 0.39 0.49 MOPITT TIR+NIR vs truncated AirCore Bias 1.5 (0.2) −0.8 −2.5 −4.9 −0.5 4.8 (2012–2019) SD 4.8 (0.8) 8.0 8.5 7.7 6.9 11.0 r 0.89 (0.82) 0.68 0.64 0.56 0.77 0.34 Total column Surface 800 hPa 600 hPa 400 hPa 200 hPa https://doi.org/10.5194/amt-15-4751-2022 https://doi.org/10.5194/amt-15-4751-2022 Atmos. Meas. Tech., 15, 4751–4765, 2022 4758 5 Discussion Here we have evaluated the repeatability and biases of in situ AirCore CO vertical proﬁles. We have compared the AirCore proﬁles to the MOPITT version 8 CO dataset to assess their performance in validation efforts and to quantify errors intro- duced in validation by the use of CO vertical proﬁles lacking upper tropospheric in situ measurements, a common issue in aircraft datasets. Finally, we have used AirCore data to esti- mate the error introduced by clouds in TROPOMI land CO retrievals. g p We analyzed separately TROPOMI clear and cloudy data. TROPOMI clear-sky and clear-sky-like observations are de- ﬁned by aerosol optical thickness < 0.5 and cloud altitude values < 500 m; they correspond to TROPOMI quality assur- ance (QA) value = 1.0. TROPOMI observations with mid- level clouds are those with aerosol optical thickness ≥0.5 and cloud altitude values < 5000 m; QA = 0.7 (Landgraf et al., 2021). Comparisons of clear/cloudy TROPOMI total CO column values with respect to their colocated AirCore counterparts are summarized in Fig. 5 and Table 3. Under clear conditions, TROPOMI has similarly low bias values (1.27 % and 1.61 %) with respect to both unsmoothed and smoothed AirCore total CO column values; the latter ac- count for TROPOMI vertical sensitivity to CO, as shown in Eq. (2). The Pearson correlation coefﬁcient (R) values (0.81 and 0.82) indicate a slight improvement in the ﬁt when the AK are applied. The slope of the ﬁtted line remains un- changed (0.96). Under cloudy conditions, the change in bi- ases is also small (1.03 % and 2.02 % for unsmoothed and smoothed AirCore values, respectively); the R values (0.74 and 0.76) and slope of the linearly ﬁtted line (0.80 and 0.83) show larger improvement of the ﬁt when the TROPOMI AK are accounted for. Figure 6 shows that, overall, the distribu- tion of bias values is mostly symmetrical with respect to the zero % bias value; i.e., relative biases show no obvious latitu- dinal dependence, although the latitudinal coverage of avail- able AirCore data is limited. From CO proﬁles acquired by pairs of AirCore systems deployed simultaneously and from the same site, we have es- timated that the average repeatability at most altitudes is well below ± 2 ppb (Fig. 2). S. Martínez-Alonso et al.: AirCore CO for satellite validation S. Martínez-Alonso et al.: AirCore CO for satellite validation mean and SD values of enull in this case are 0.36 % ± 0.66 %, or 0.61 ± 1.14 × 1016 molec. cm−2 (Fig. 7a). In cloudy con- ditions, TROPOMI is more sensitive to CO above the clouds than to CO below them; in these cases, if the shape of the TROPOMI reference proﬁles does not properly represent that of the actual CO proﬁles, a null-space error is introduced. Our results indicate that the relative mean and SD values of enull are in this case slightly larger: 0.98 % ± 2.32 %, or 1.65 ± 4.15 × 1016 molec. cm−2 (Fig. 7b). mean and SD values of enull in this case are 0.36 % ± 0.66 %, or 0.61 ± 1.14 × 1016 molec. cm−2 (Fig. 7a). In cloudy con- ditions, TROPOMI is more sensitive to CO above the clouds than to CO below them; in these cases, if the shape of the TROPOMI reference proﬁles does not properly represent that of the actual CO proﬁles, a null-space error is introduced. Our results indicate that the relative mean and SD values of enull are in this case slightly larger: 0.98 % ± 2.32 %, or 1.65 ± 4.15 × 1016 molec. cm−2 (Fig. 7b). (Martínez-Alonso et al., 2020). In that same study, cloudy TROPOMI CO retrievals over bodies of water were also val- idated with respect to ATom (Atmospheric Tomography mis- sion; Wofsy et al., 2018) aircraft proﬁles. The results showed that the enull (null-space error) of the proﬁle scaling retrieval over water is very small (2.16 % with respect to the in situ measurements). The authors concluded that, since there are no major emission sources over water, CO values closer to the surface (most likely to be below cloud-top) are well char- acterized by the scaled reference proﬁles. Larger errors could occur, however, in cloudy TROPOMI land retrievals, par- ticularly near CO emission sources, if not accounting for the TROPOMI AK. Their analysis could not be extended over land because the ATom campaign was designed to sam- ple the troposphere mostly over oceans. Here we extend the Martínez-Alonso et al. (2020) analysis by characterizing the error introduced by clouds in land TROPOMI CO retrievals using CO proﬁles from the AirCore dataset. 4.2 TROPOMI vs AirCore to the MOPITT/AirCore biases. For MOPITT TIR, biases in- crease mostly in the upper troposphere, by up to 2.6 p.p. (per- cent points) at 300 hPa, and mimic very closely in sign and magnitude those between MOPITT and the aircraft data. For MOPITT NIR, biases increase almost uniformly at all pres- sure levels, by 0.2–0.8 p.p. For MOPITT multispectral, the change in bias is larger (up to 3.2 p.p. at 300 hPa), mimick- ing once more the MOPITT/aircraft biases. For the total CO column, the biases between MOPITT and truncated AirCore increase by 0.20, 0.10, and 0.10 × 1017 molec. cm−2 (for the TIR, NIR, and multispectral variants, respectively). TROPOMI retrieves total CO column values from solar re- ﬂected radiances over land (under clear and cloudy con- ditions) and water (cloudy only). During TROPOMI, re- trieval parameters describing the cloud contamination of the measurement and interfering trace gases are quan- tiﬁed together with the CO column. The reference CO proﬁles used in the retrieval are scaled based on esti- mated above-cloud CO rather than based on estimated to- tal CO (Landgraf et al., 2016). A previous comparison of clear MOPITT and TROPOMI total CO column re- trievals showed good agreement between the two datasets Atmos. Meas. Tech., 15, 4751–4765, 2022 https://doi.org/10.5194/amt-15-4751-2022 4759 S. Martínez-Alonso et al.: AirCore CO for satellite validation S. Martínez-Alonso et al.: AirCore CO for satellite validation Table 3. Summary of statistics from the comparison of total CO column values from TROPOMI (under either clear or cloudy conditions) and AirCore. Bias and SD values are provided in percent (%) and in units of 1017 molec. cm−2 (in parentheses). Table 3. Summary of statistics from the comparison of total CO column values from TROPOMI (under either clear or cloudy conditions) and AirCore. Bias and SD values are provided in percent (%) and in units of 1017 molec. cm−2 (in parentheses). Total column Total column “smoothed” TROPOMI vs AirCore Bias 1.27 (0.08) 1.61 (0.14) clear SD 9.32 (1.43) 9.04 (1.38) r 0.81 0.82 TROPOMI vs AirCore Bias 1.03 (0.02) 2.02 (0.19) cloudy SD 11.37 (1.83) 11.13 (1.77) r 0.74 0.76 ure 5. Comparison of total CO column values from TROPOMI and AirCore for the November 2017–July 2021 period. Top row panels (a) (b) are both for TROPOMI clear-sky and clear-sky-like observations (i.e., QA = 1.0). Bottom row panels (c) and (d) are for TROPOMI rvations with mid-level clouds (i.e., QA = 0.7). Left column panels (a) and (c) show unsmoothed AirCore data. Right column panels nd (d) show smoothed AirCore data to account for TROPOMI vertical sensitivity to CO. Bias values are provided in percent (%) and in of molec. cm−2. Total column Total column “smoothed” Figure 5. Comparison of total CO column values from TROPOMI and AirCore for the November 2017–July 2021 period. Top row panels (a) and (b) are both for TROPOMI clear-sky and clear-sky-like observations (i.e., QA = 1.0). Bottom row panels (c) and (d) are for TROPOMI observations with mid-level clouds (i.e., QA = 0.7). Left column panels (a) and (c) show unsmoothed AirCore data. Right column panels (b) and (d) show smoothed AirCore data to account for TROPOMI vertical sensitivity to CO. Bias values are provided in percent (%) and in units of molec. cm−2. In order to investigate the effects of extending up- wards shorter tropospheric aircraft CO proﬁles used in validation, we have simulated a truncated version of the AirCore CO dataset, which we have compared to MO- PITT retrievals. Differences between MOPITT/AirCore and MOPITT/truncated-AirCore biases (Fig. 4) are small: < 1 p.p. on average. We observe that, for the TIR and multi- for all MOPITT variants. 5 Discussion Our analysis shows lower repeatabil- ity values (5–15 ppb) between 50 and 70 hPa, consistent with higher uncertainty in AirCore stratospheric CO retrievals at- tributable to AirCore surface effects, chemical interactions or measurement interferences from other trace gas species, or incorrect AirCore sample end-member assumptions (Chen et al., 2022). Colocated (<2 h and <15 km apart) CO proﬁles from AirCore and NOAA aircraft ﬂask proﬁles indicate that AirCore biases are between −6 and +6 ppb, with a 0.6 ppb overall average bias (Fig. 3a). g g Our MOPITT comparisons show that AirCore provides validation results that are analogous in magnitude and sign to those from the NOAA aircraft ﬂask dataset (Fig. 4); in all cases, biases are well below the MOPITT 10 % target accu- racy (Francis et al., 2017). MOPITT/AirCore and MOPIT- T/aircraft biases between the surface and 500 hPa differ very slightly (by < 0.5 p.p. on average for all MOPITT variants). The same is true for MOPITT NIR/AirCore biases at all pres- sure levels. Between 400 and 200 hPa, though, AirCore is closer to MOPITT than the aircraft dataset is by (on average) 2.7 p.p. (TIR) and 4.7 p.p. (multispectral). Larger biases be- tween MOPITT and the aircraft dataset at that pressure range are consistent with the fact that proﬁles from the latter do not, in most cases, reach above 400 hPa and have to be extended upwards. In contrast, the low MOPITT/AirCore biases indi- cate good agreement between the two datasets and imply that previous validation results may have overestimated the mag- nitude of MOPITT retrieval biases in that upper tropospheric region. Further up in the troposphere, at 100 hPa, both MO- PITT/AirCore and MOPITT/aircraft biases approximate zero The TROPOMI null-space error (enull) quantiﬁes the dif- ference between the shapes of TROPOMI CO reference pro- ﬁles and true CO proﬁles, which may result in differences between true and retrieved total CO column values. We have calculated enull values between TROPOMI and Air- Core proﬁles over land using Eq. (4); results as a func- tion of latitude are shown in Fig. 7. Under clear conditions, the TROPOMI total column averaging kernel Ac closely matches the total column operator I such that, according to Eq. (4), the null-space error e is close to zero. The relative https://doi.org/10.5194/amt-15-4751-2022 Atmos. Meas. Tech., 15, 4751–4765, 2022 4760 S. Martínez-Alonso et al.: AirCore CO for satellite validation S. Martínez-Alonso et al.: AirCore CO for satellite validation spectral variants, MOPITT/truncated-AirCore biases depart from MOPITT/AirCore biases to mimic the MOPITT/air- craft biases. These results reinforce our interpretation regard- ing previous validation efforts having slightly overestimated the magnitude of MOPITT retrieval biases in the upper tropo- sphere due to the use of shorter tropospheric aircraft CO pro- ﬁles. While always small, the effects are relatively stronger (2 to 3 p.p.) at 400–200 hPa; more modest effects can also be seen at other pressure levels. This is because, at any given pressure level P , the MOPITT CO retrievals are sensitive not only to CO at that level but also to CO at other levels. That is, the MOPITT AK (with which the AirCore proﬁles are convolved prior to bias calculations) are not delta func- tions peaking at level P but rather are curves of increasing amplitude towards level P . We also observe that, for the NIR variant, the effects are similar at all pressure levels. This is consistent with the MOPITT NIR retrievals being sensitive to total CO column only, i.e., the MOPITT NIR AK are not curve-like but rather are ﬂat. Our ﬁndings support the results of Tang et al. (2020), where in situ aircraft proﬁles extended with reanalysis data were compared to MOPITT multispec- tral retrievals. The authors found good agreement between MOPITT and the extended aircraft proﬁles at the surface layer; at upper levels (400 and 200 hPa), biases increased due to limited aircraft observations. S. Martínez-Alonso et al.: AirCore CO for satellite validation S. Martínez-Alonso et al.: AirCore CO for satellite validation Figure 6. Latitudinal distribution of bias values between TROPOMI and AirCore cloudy observations over land. the other quality-of-ﬁt indicators (R and linear ﬁt slope) show that TROPOMI CO retrievals are closer in value to unsmoothed AirCore CO under clear conditions. Borsdorff et al. (2018) reported a similarly small difference in bias (0.2 ppb, equivalent to ∼0.25 p.p.) between clear and cloudy TROPOMI CO observations with respect to in situ ground measurements over nine remote sites. Our results indicate that TROPOMI/AirCore biases for cloudy observations over land do not show obvious latitudinal effects (Fig. 6). The spread in biases shown in this ﬁgure may reﬂect differences in the actual CO concentrations observed by each of the two instruments, which may be up to 12 h and 50 km apart. y p p The null-space error (enull) quantiﬁes the expected dif- ference between the true CO column and the retrieved TROPOMI CO column due to differences between the shape of the true proﬁle and that of the TROPOMI reference pro- ﬁle. It is only relevant when the TROPOMI CO retrievals are compared with respect to other reference measurements without accounting for the sensitivity loss caused by clouds; enull can be completely avoided by using the TROPOMI to- tal column AK provided in the data product. Our null-space error calculations using AirCore CO data show that the mag- nitude of the error introduced in cloudy TROPOMI CO re- trievals over land by using scaled reference proﬁles is very small (0.98 % ± 2.32 %, or 1.65 ± 4.15 × 1016 molec. cm−2) and slightly skewed towards positive values (Fig. 7b). These observations are in agreement with results reported by Martínez-Alonso et al. (2020) in their analysis of cloudy CO observations from TROPOMI and ATom-4 over bod- ies of water (relative mean and SD values 2.16 % ± 2.23 %, or 3.70 ± 3.75 × 1016 molec. cm−2). The prevalence of pos- itive null-space error values suggests that, on average, the TROPOMI reference proﬁles analyzed may have too much CO near the surface, thus resulting in TROPOMI retrievals that may overestimate the below-cloud partial column. No latitudinal dependence was observed in the null-space er- ror values in this analysis nor in the Martínez-Alonso et al. (2020) study. Figure 6. Latitudinal distribution of bias values between TROPOMI and AirCore cloudy observations over land. S. Martínez-Alonso et al.: AirCore CO for satellite validation Because MOPITT proﬁles are less sensitive to CO at/above 100 hPa, the a priori dominates re- trievals at that pressure level, leading to low biases (both MOPITT/AirCore and MOPITT/aircraft biases) due to can- cellation of the dominant a priori term in the difference of retrieved and smoothed in situ proﬁles. for all MOPITT variants. Because MOPITT proﬁles are less sensitive to CO at/above 100 hPa, the a priori dominates re- trievals at that pressure level, leading to low biases (both MOPITT/AirCore and MOPITT/aircraft biases) due to can- cellation of the dominant a priori term in the difference of retrieved and smoothed in situ proﬁles. Atmos. Meas. Tech., 15, 4751–4765, 2022 https://doi.org/10.5194/amt-15-4751-2022 4761 S. Martínez-Alonso et al.: AirCore CO for satellite validation S. Martínez-Alonso et al.: AirCore CO for satellite validation S. Martínez-Alonso et al.: AirCore CO for satellite validation Figure 7. Latitudinal distribution of null-space error between TROPOMI and AirCore observations over land. (a) For clear observations. (b) For cloudy observations. Figure 7. Latitudinal distribution of null-space error between TROPOMI and AirCore observations over land. (a) For clear observations. (b) For cloudy observations. forts are being made towards solving these problems in the near future. performance of its reference proﬁles. Our null-space error calculations show that the magnitude of the error introduced in cloudy TROPOMI retrievals over land by using scaled reference proﬁles is very small (∼0.98 %), does not show latitudinal dependencies, and is slightly skewed towards positive values. While the AirCore dataset spans a ∼10-year time frame, it is still rather limited geographically; more latitudinally widespread measurements are needed to study whether there are substantial latitudinal dependencies in the TROPOMI retrievals. We have validated a temporal subset (2012–2019) of the MOPITT version 8 data with respect to AirCore proﬁles by applying the procedure used in previous MOPITT validation efforts with respect to aircraft in situ measurements (Deeter et al., 2019, and references therein). As a reference, we have also validated the same MOPITT temporal subset with re- spect to NOAA aircraft ﬂask proﬁles. The resulting MOPIT- T/AirCore and MOPITT/aircraft biases are very similar and align well with the full MOPITT validation results reported by Deeter et al. (2019). Data availability. AirCore data from the 13 August 2021 version are publicly available from the NOAA Global Moni- toring Laboratory upon request: https://doi.org/10.15138/6AV0- MY81 (Baier et al., 2021). NOAA aircraft ﬂask data version 2.0 from the 9 February 2021 version were obtained from https://doi.org/10.7289/V5N58JMF (Sweeney et al., 2021). MOPITT data from version 8 can be downloaded from https://doi.org/10.5067/TERRA/MOPITT/MOP02T_L2.008 (Ziskin, 2019c) (TIR), https://doi.org/10.5067/TERRA/ MOPITT/MOP02N_L2.008 (Ziskin, 2019b) (NIR), and https://doi.org/10.5067/TERRA/MOPITT/MOP02J_L2.008 (Ziskin, 2019a) (TIR+NIR). TROPOMI level 2 CO retrievals for 7 November 2017 to 27 June 2018 were downloaded from https://s5pexp.copernicus.eu/ (last access: 27 November 2019) (ESA, 2018a); retrievals for dates after 28 June 2018 were down- loaded from https://s5phub.copernicus.eu/ (last access: 9 February 2021) (ESA, 2018b). We ﬁnd MOPITT/AirCore biases at 400–200 hPa to be smaller than their MOPITT/aircraft counterparts; it is also at that pressure range that MOPITT/AirCore and MOPITT/truncated-AirCore biases differ the most. 6 Conclusions AirCore is a novel airborne sampler suited for the valida- tion of satellite retrievals of tropospheric CO and, potentially, other relevant tropospheric gases and parameters such as CO2, CH4, temperature, and relative humidity because, un- like most aircraft platforms, it samples continuously from the lower stratosphere to near the surface. According to our anal- ysis, the mean bias (with respect to the NOAA aircraft ﬂask dataset) and repeatability of CO AirCore measurements are near 0.6 and 0.03 ppb, respectively. AirCore measurements from near the surface are currently being discarded because they are affected by end-member mixing with spiked CO push gas; higher stratospheric uncertainties of up to ∼15 ppb (this study; Chen et al., 2022) have also been identiﬁed. Ef- Finally, we have used the AirCore dataset to investigate cloud effects in TROPOMI total CO column retrievals over land. The mean relative bias between clear TROPOMI and smoothed AirCore (1.61 %) is only slightly smaller than that between cloudy TROPOMI and smoothed AirCore (2.02 %) (Fig. 5b and d). Mean relative biases between TROPOMI and unsmoothed AirCore are 1.27 % and 1.03 % (for clear and cloudy TROPOMI retrievals, respectively); we note that, although both are very small and differ by only 0.24 p.p., the mean bias is higher for clear observations. However, https://doi.org/10.5194/amt-15-4751-2022 Atmos. Meas. Tech., 15, 4751–4765, 2022 4762 S. Martínez-Alonso et al.: AirCore CO for satellite validation mann, R., Hase, F., Blumenstock, T., Mahieu, E., and Langerock, B.: Validation of MOPITT carbon monoxide using ground-based Fourier transform infrared spectrometer data from NDACC, At- mos. Meas. Tech., 10, 1927–1956, https://doi.org/10.5194/amt- 10-1927-2017, 2017. mann, R., Hase, F., Blumenstock, T., Mahieu, E., and Langerock, B.: Validation of MOPITT carbon monoxide using ground-based Fourier transform infrared spectrometer data from NDACC, At- mos. Meas. Tech., 10, 1927–1956, https://doi.org/10.5194/amt- 10-1927-2017, 2017. ysis. SMA wrote the original draft. All authors contributed to the review and editing of this paper. Competing interests. At least one of the (co-)authors is a member of the editorial board of Atmospheric Measurement Techniques. The peer-review process was guided by an independent editor, and the authors also have no other competing interests to declare. Chen, H., Hooghiem, J., Brownlow, R., Kivi, R., Heikkinen, P., Leuenberger, M., Nyfeler, P., Ramonet, M., Lopez, M., Engel, A., Wagenhaeuser, T., Laube, J., Baier, B., Sweeney, C., Danis, F., and Crevoisier, C.: Towards accurate vertical proﬁle measure- ments of greenhouse gases using AirCore, in preparation, 2022. Deeter, M. N.: MOPITT Measurements Of Pollution In The Tropo- sphere validation version 4 product users guide, Tech. rep., At- mospheric Chemistry Division, National Center for Atmospheric Research, 2009. Disclaimer. Publisher’s note: Copernicus Publications remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. Deeter, M. N., Edwards, D. P., Gille, J. C., Emmons, L. K., Francis, G., Ho, S. P., Mao, D., Masters, D., Worden, H., Drummond, J. R., and Novelli, P. C.: The MOPITT ver- sion 4 CO product: Algorithm enhancements, validation, and long-term stability, J. Geophys. Res.-Atmos., 115, D07306, https://doi.org/10.1029/2009JD013005, 2010. Acknowledgements. NCAR internal reviews provided by Gene Francis and Wenfu Tang are greatly appreciated. This paper ben- eﬁted from helpful comments from two anonymous reviewers. Sentinel-5 Precursor is part of the EU Copernicus program, and Copernicus (modiﬁed) Sentinel data for 2017–2021 have been used. Deeter, M. N., Worden, H. M., Edwards, D. P., Gille, J. C., and Andrews, A. E.: Evaluation of MOPITT re- trievals of lower-tropospheric carbon monoxide over the United States, J. Geophys. Res.-Atmos., 117, D13306, https://doi.org/10.1029/2012JD017553, 2012. Financial support. This material is based upon work supported by the National Center for Atmospheric Research (NCAR), which is a major facility sponsored by the National Science Foundation (grant no. 1852977). The NCAR MOPITT project is supported by the Na- tional Aeronautics and Space Administration (NASA) Earth Ob- serving System (EOS) Program. S. Martínez-Alonso et al.: AirCore CO for satellite validation AirCore work was funded in part by NASA ROSES award 80NSSC18K0898. The NOAA Coopera- tive Agreement with CIRES is NA17OAR4320101. Deeter, M. N., Martínez-Alonso, S., Edwards, D. P., Emmons, L. K., Gille, J. C., Worden, H. M., Pittman, J. V., Daube, B. C., and Wofsy, S. C.: Validation of MOPITT Version 5 thermal- infrared, near-infrared, and multispectral carbon monoxide pro- ﬁle retrievals for 2000–2011, J. Geophys. Res.-Atmos., 118, 6710–6725, https://doi.org/10.1002/jgrd.50272, 2013. Deeter, M. N., Martínez-Alonso, S., Edwards, D. P., Emmons, L. K., Gille, J. C., Worden, H. M., Sweeney, C., Pittman, J. V., Daube, B. C., and Wofsy, S. C.: The MOPITT Version 6 product: al- gorithm enhancements and validation, Atmos. Meas. Tech., 7, 3623–3632, https://doi.org/10.5194/amt-7-3623-2014, 2014. Review statement. This paper was edited by Frank Hase and re- viewed by two anonymous referees. Deeter, M. N., Edwards, D. P., Francis, G. L., Gille, J. C., Martínez-Alonso, S., Worden, H. M., and Sweeney, C.: A climate-scale satellite record for carbon monoxide: the MO- PITT Version 7 product, Atmos. Meas. Tech., 10, 2533–2555, https://doi.org/10.5194/amt-10-2533-2017, 2017. S. Martínez-Alonso et al.: AirCore CO for satellite validation Both pieces of evidence indicate that extending upwards shorter aircraft proﬁles (i.e., aircraft proﬁles that sample up to the re- quired 400 hPa MOPITT validation threshold but not above it) results in small validation errors in the upper troposphere (up to 2–3 p.p. in the 400–200 hPa range) and, thus, in a slight overestimation of MOPITT retrieval biases in that region. Our TROPOMI/AirCore analysis shows that the TROPOMI approach to retrieve total CO column val- ues under cloudy conditions results in small biases over land (1 %–2 %); similarly, small biases over bodies of water had been previously reported by Martínez-Alonso et al. (2020). We must keep in mind, however, that this study’s results may be representative of unpolluted areas only. AirCore in situ measurements are commonly per- formed away from CO emission sources such as heavily populated areas, industrial regions, or active ﬁres, where CO concentrations at the boundary layer (and, thus, most likely below cloud-top) would be more variable and, thus, could depart from the TROPOMI reference proﬁle values. AirCore measurements near CO emission sources would be needed to fully evaluate the TROPOMI approach and the Author contributions. SMA deﬁned the concept and methodology of the paper, performed the formal analysis of data sets, conducted software development, and oversaw data presentation. MND pro- vided additional software for MOPITT validation. SMA, MND, and HW oversaw the MOPITT data analysis. TB and IA oversaw the TROPOMI data analysis. BCB and CS oversaw the AirCore data analysis. KM and CS oversaw the NOAA aircraft ﬂask data anal- Atmos. Meas. Tech., 15, 4751–4765, 2022 https://doi.org/10.5194/amt-15-4751-2022 4763 S. Martínez-Alonso et al.: AirCore CO for satellite validation S. Martínez-Alonso et al.: AirCore CO for satellite validation 4764 Lelieveld, J., Gromov, S., Pozzer, A., and Taraborrelli, D.: Global tropospheric hydroxyl distribution, budget and reactivity, Atmos. Chem. Phys., 16, 12477–12493, https://doi.org/10.5194/acp-16- 12477-2016, 2016. Emmons, L., Deeter, M., Gille, J., Edwards, D., Attie, J., Warner, J., Ziskin, D., Francis, G., Khattatov, B., Yudin, V., Lamarque, J., Ho, S., Mao, D., Chen, J., Drummond, J., Novelli, P., Sachse, G., Coffey, M., Hannigan, J., Gerbig, C., Kawakami, S., Kondo, Y., Takegawa, N., Schlager, H., Baehr, J., and Ziereis, H.: Validation of Measurements of Pollution in the Troposphere (MOPITT) CO retrievals with aircraft in situ proﬁles, J. Geophys. Res.-Atmos., 109, D03309, https://doi.org/10.1029/2003JD004101, 2004. Martínez-Alonso, S., Deeter, M., Worden, H., Borsdorff, T., Aben, I., Commane, R., Daube, B., Francis, G., George, M., Landgraf, J., Mao, D., McKain, K., and Wofsy, S.: 1.5 years of TROPOMI CO measurements: comparisons to MOPITT and ATom, At- mos. Meas. Tech., 13, 4841–4864, https://doi.org/10.5194/amt- 13-4841-2020, 2020. ESA: Sentinel-5P Expert Users Data Hub, ESA [data set] https:// s5pexp.copernicus.eu/ (last access: 27 November 2019), 2018a. ESA: Sentinel-5P Pre-Operations Data Hub, ESA [data set], https: //s5phub.copernicus.eu/ (last access: 9 February 2021), 2018b. Martínez-Alonso, S., Deeter, M. N., Worden, H. M., Gille, J. C., Emmons, L. K., Pan, L. L., Park, M., Manney, G. L., Bernath, P. F., Boone, C. D., Walker, K. A., Kolonjari, F., Wofsy, S. C., Pittman, J., and Daube, B. C.: Comparison of upper tropospheric carbon monoxide from MOPITT, ACE-FTS, and HIPPO-QCLS, J. Geophys. Res.-Atmos., 119, 14144–14164, https://doi.org/10.1002/2014JD022397, 2014. Francis, G. L., Deeter, M. N., Martínez-Alonso, S., Gille, J. C., Ed- wards, D. P., Mao, D., Worden, H. M., and Ziskin, D.: Measure- ment Of Pollution In The Troposphere algorithm theoretical ba- sis document. Retrieval of carbon monoxide proﬁles and column amounts from MOPITT observed radiances (Level 1 to Level 2), Tech. rep., Atmospheric Chemistry Observations and Modeling Laboratory, National Center for Atmospheric Research, 2017. Rodgers, C. and Connor, B.: Intercomparison of remote sounding instruments, J. Geophys. Res.-Atmos., 108, 4116, https://doi.org/10.1029/2002JD002299, 2003. Hedelius, J. K., He, T.-L., Jones, D. B. A., Baier, B. C., Buchholz, R. R., De Mazière, M., Deutscher, N. M., Dubey, M. K., Feist, D. G., Grifﬁth, D. W. T., Hase, F., Iraci, L. T., Jeseck, P., Kiel, M., Kivi, R., Liu, C., Morino, I., Notholt, J., Oh, Y.-S., Ohyama, H., Pollard, D. F., Rettinger, M., Roche, S., Roehl, C. S. Martínez-Alonso et al.: AirCore CO for satellite validation M., Schneider, M., Shiomi, K., Strong, K., Sussmann, R., Sweeney, C., Té, Y., Uchino, O., Velazco, V. A., Wang, W., Warneke, T., Wennberg, P. O., Worden, H. M., and Wunch, D.: Evaluation of MOPITT Version 7 joint TIR–NIR XCO retrievals with TCCON, At- mos. Meas. Tech., 12, 5547–5572, https://doi.org/10.5194/amt- 12-5547-2019, 2019. Rodgers, C. D.: Inverse Methods for Atmospheric Sounding, World Scientiﬁc, 1st edition, https://doi.org/10.1142/3171, 2000. Spivakovsky, C., Logan, J., Montzka, S., Balkanski, Y., Foreman- Fowler, M., Jones, D., Horowitz, L., Fusco, A., Brenninkmei- jer, C., Prather, M., Wofsy, S., and McElroy, M.: Three- dimensional climatological distribution of tropospheric OH: Up- date and evaluation, J. Geophys. Res.-Atmos., 105, 8931–8980, https://doi.org/10.1029/1999JD901006, 2000. Sweeney, C., McKain, K., Higgs, J., Wolter, S., Crotwell, A., Neff, D., Dlugokencky, E., Petron, G., Madronich, M., Moglia, E., Crotwell, M., and Mund, J.: NOAA Carbon Cy- cle and Greenhouse Gases Group aircraft-based measurements of CO2, CH4, CO, N2O, H2, and SF6 in ﬂask-air sam- ples taken since 1992, Tech. rep., NOAA Earth System Re- search Laboratories, Global Monitoring Laboratory [data set], https://doi.org/10.7289/V5N58JMF, 2021. Karion, A., Sweeney, C., Tans, P., and Newberger, T.: AirCore: An Innovative Atmospheric Sampling System, J. Atmos. Ocean. Tech., 27, 1839–1853, https://doi.org/10.1175/2010JTECHA1448.1, 2010. Karion, A., Sweeney, C., Wolter, S., Newberger, T., Chen, H., An- drews, A., Koﬂer, J., Neff, D., and Tans, P.: Long-term green- house gas measurements from aircraft, Atmos. Meas. Tech., 6, 511–526, https://doi.org/10.5194/amt-6-511-2013, 2013. Sweeney, C., Karion, A., Wolter, S., Newberger, T., Guenther, D., Higgs, J. A., Andrews, A. E., Lang, P. M., Neff, D., Dlu- gokencky, E., Miller, J. B., Montzka, S. A., Miller, B. R., Masarie, K. A., Biraud, S. C., Novelli, P. C., Crotwell, M., Crotwell, A. M., Thoning, K., and Tans, P. P.: Seasonal cli- matology of CO2 across North America from aircraft mea- surements in the NOAA/ESRL Global Greenhouse Gas Ref- erence Network, J. Geophys. Res.-Atmos., 120, 5155–5190, https://doi.org/10.1002/2014JD022591, 2015. Krol, M., Houweling, S., Bregman, B., van den Broek, M., Segers, A., van Velthoven, P., Peters, W., Dentener, F., and Bergamaschi, P.: The two-way nested global chemistry-transport zoom model TM5: algorithm and applications, Atmos. Chem. Phys., 5, 417– 432, https://doi.org/10.5194/acp-5-417-2005, 2005. Crotwell, A. M., Thoning, K., and Tans, P. P.: Seasonal cli- matology of CO2 across North America from aircraft mea- surements in the NOAA/ESRL Global Greenhouse Gas Ref- erence Network, J. Geophys. Res.-Atmos., 120, 5155–5190, https://doi.org/10.1002/2014JD022591, 2015. Lamarque, J.-F., Emmons, L. K., Hess, P. References Baier, B., Sweeney, C., Tans, P., Newberger, T., Higgs, J., Wolter, S., and NOAA Global Monitoring Laboratory: NOAA AirCore at- mospheric sampling system proﬁles (Version 20210813), NOAA GML [data set], https://doi.org/10.15138/6AV0-MY81, 2021. Deeter, M. N., Martínez-Alonso, S., Andreae, M. O., and Schlager, H.: Satellite-Based Analysis of CO Seasonal and Interannual Variability Over the Amazon Basin, J. Geophys. Res.-Atmos., 123, 5641–5656, https://doi.org/10.1029/2018JD028425, 2018. Borsdorff, T., Hasekamp, O. P., Wassmann, A., and Landgraf, J.: Insights into Tikhonov regularization: application to trace gas column retrieval and the efﬁcient calculation of total column averaging kernels, Atmos. Meas. Tech., 7, 523–535, https://doi.org/10.5194/amt-7-523-2014, 2014. Deeter, M. N., Edwards, D. P., Francis, G. L., Gille, J. C., Mao, D., Martínez-Alonso, S., Worden, H. M., Ziskin, D., and Andreae, M. O.: Radiance-based retrieval bias mitigation for the MOPITT instrument: the version 8 product, Atmos. Meas. Tech., 12, 4561– 4580, https://doi.org/10.5194/amt-12-4561-2019, 2019. Borsdorff, T., aan de Brugh, J., Hu, H., Hasekamp, O., Sussmann, R., Rettinger, M., Hase, F., Gross, J., Schneider, M., Garcia, O., Stremme, W., Grutter, M., Feist, D. G., Arnold, S. G., De Maz- ière, M., Kumar Sha, M., Pollard, D. F., Kiel, M., Roehl, C., Wennberg, P. O., Toon, G. C., and Landgraf, J.: Mapping car- bon monoxide pollution from space down to city scales with daily global coverage, Atmos. Meas. Tech., 11, 5507–5518, https://doi.org/10.5194/amt-11-5507-2018, 2018. Drummond, J. and Mand, G.: The measurements of pol- lution in the troposphere (MOPITT) instrument: Over- all performance and calibration requirements, J. Atmos. Ocean. Tech., 13, 314–320, https://doi.org/10.1175/1520- 0426(1996)013<0314:TMOPIT>2.0.CO;2, 1996. Drummond, J. R., Zou, J., Nichitiu, F., Kar, J., Deschambaut, R., and Hackett, J.: A review of 9-year performance and opera- tion of the MOPITT instrument, Adv. Space Res., 45, 760–774, https://doi.org/10.1016/j.asr.2009.11.019, 2010. p g Buchholz, R. R., Deeter, M. N., Worden, H. M., Gille, J., Edwards, D. P., Hannigan, J. W., Jones, N. B., Paton-Walsh, C., Grifﬁth, D. W. T., Smale, D., Robinson, J., Strong, K., Conway, S., Suss- Buchholz, R. R., Deeter, M. N., Worden, H. M., Gille, J., Edwards, D. P., Hannigan, J. W., Jones, N. B., Paton-Walsh, C., Grifﬁth, D. W. T., Smale, D., Robinson, J., Strong, K., Conway, S., Suss- Atmos. Meas. Tech., 15, 4751–4765, 2022 https://doi.org/10.5194/amt-15-4751-2022 S. Martínez-Alonso et al.: AirCore CO for satellite validation sphere (MOPITT) carbon monoxide retrievals over urban ver- sus non-urban regions, Atmos. Meas. Tech., 13, 1337–1356, https://doi.org/10.5194/amt-13-1337-2020, 2020. Worden, H. M., Deeter, M. N., Edwards, D. P., Gille, J. C., Drummond, J. R., and Nedelec, P.: Observations of near- surface carbon monoxide from space using MOPITT mul- tispectral retrievals, J. Geophys. Res.-Atmos., 115, D18314, https://doi.org/10.1029/2010JD014242, 2010. sphere (MOPITT) carbon monoxide retrievals over urban ver- sus non-urban regions, Atmos. Meas. Tech., 13, 1337–1356, https://doi.org/10.5194/amt-13-1337-2020, 2020. Tans, P. P. : System and method for providing vertical proﬁle measurements of atmospheric gases, United States Patent US 7597014 B2, 2009. Worden, H. M., Deeter, M. N., Frankenberg, C., George, M., Nichi- tiu, F., Worden, J., Aben, I., Bowman, K. W., Clerbaux, C., Coheur, P. F., de Laat, A. T. J., Detweiler, R., Drummond, J. R., Edwards, D. P., Gille, J. C., Hurtmans, D., Luo, M., Martínez-Alonso, S., Massie, S., Pﬁster, G., and Warner, J. X.: Decadal record of satellite carbon monoxide observations, At- mos. Chem. Phys., 13, 837–850, https://doi.org/10.5194/acp-13- 837-2013, 2013. Tans, P.: Fill dynamics and sample mixing in the AirCore, At- mos. Meas. Tech., 15, 1903–1916, https://doi.org/10.5194/amt- 15-1903-2022, 2022. Veefkind, J. P., Aben, I., McMullan, K., Forster, H., de Vries, J., Otter, G., Claas, J., Eskes, H. J., de Haan, J. F., Kleipool, Q., van Weele, M., Hasekamp, O., Hoogeveen, R., Landgraf, J., Snel, R., Tol, P., Ingmann, P., Voors, R., Kruizinga, B., Vink, R., Visser, H., and Levelt, P. F.: TROPOMI on the ESA Sentinel-5 Precursor: A GMES mission for global observations of the atmospheric composition for climate, air quality and ozone layer applications, Remote Sens. Environ., 120, 70–83, https://doi.org/10.1016/j.rse.2011.09.027, 2012. Ziskin, D.: Measurements Of Pollution In The Troposphere (MOPITT) Level 2 Derived CO (Near and Thermal Infrared Radiances) (MOP02J) V008, NASA Atmospheric Sciences Data Center [data set] https://doi.org/10.5067/TERRA/MOPITT/ MOP02J_L2.008, 2019a. Ziskin, D.: Measurements Of Pollution In The Troposphere (MOPITT) Level 2 Derived CO (Near Infrared Radiances) (MOP02N) V008, NASA Atmospheric Sciences Data Center [data set] https://doi.org/10.5067/TERRA/MOPITT/MOP02N_ L2.008, 2019b. Vidot, J., Landgraf, J., Hasekamp, O. P., Butz, A., Galli, A., Tol, P., and Aben, I.: Carbon monoxide from shortwave infrared re- ﬂectance measurements: A new retrieval approach for clear sky and partially cloudy atmospheres, Remote Sens. Environ., 120, 255–266, https://doi.org/10.1016/j.rse.2011.09.032, 2012. Wagenhäuser, T., Engel, A., and Sitals, R.: Testing the altitude at- tribution and vertical resolution of AirCore measurements with a new spiking method, Atmos. Meas. Tech., 14, 3923–3934, https://doi.org/10.5194/amt-14-3923-2021, 2021. S. Martínez-Alonso et al.: AirCore CO for satellite validation G., Kinnison, D. E., Tilmes, S., Vitt, F., Heald, C. L., Holland, E. A., Lauritzen, P. H., Neu, J., Orlando, J. J., Rasch, P. J., and Tyndall, G. K.: CAM-chem: description and evaluation of interactive at- mospheric chemistry in the Community Earth System Model, Geosci. Model Dev., 5, 369–411, https://doi.org/10.5194/gmd-5- 369-2012, 2012. Szopa, S., Naik, V., Adhikary, B., Artaxo, P., Berntsen, T., Collins, W., Fuzzi, S., Gallardo, L., Kiendler-Scharr, A., Klimont, Z., Liao, H., Unger, N., and Zanis, P.: Short-Lived Climate Forcers, in Climate Change: The Physical Science Basis. Con- tribution of Working Group I to the Sixth Assessment Re- port of the Intergovernmental Panel on Climate Change, Cam- bridge University Press, Cambridge, United Kingdom and New York, NY, USA, https://www.ipcc.ch/report/ar6/wg1/downloads/ report/IPCC_AR6_WGI_Chapter06.pdf (last access: 17 August 2022), 2021. Landgraf, J., aan de Brugh, J., Scheepmaker, R., Borsdorff, T., Hu, H., Houweling, S., Butz, A., Aben, I., and Hasekamp, O.: Car- bon monoxide total column retrievals from TROPOMI short- wave infrared measurements, Atmos. Meas. Tech., 9, 4955– 4975, https://doi.org/10.5194/amt-9-4955-2016, 2016. p g Landgraf, J., Borsdorff, T., Langerock, B., and Keppens, A.: S5P mission performance centre carbon monoxide (L2 CO) readme, Tech. Rep. 02.02.00, 2021-07-05, Netherlands Institute for Space Research (SRON), 2021. Tang, W., Worden, H. M., Deeter, M. N., Edwards, D. P., Em- mons, L. K., Martínez-Alonso, S., Gaubert, B., Buchholz, R. R., Diskin, G. S., Dickerson, R. R., Ren, X., He, H., and Kondo, Y.: Assessing Measurements of Pollution in the Tropo- https://doi.org/10.5194/amt-15-4751-2022 Atmos. Meas. Tech., 15, 4751–4765, 2022 4765 S. Martínez-Alonso et al.: AirCore CO for satellite validation Ziskin, D.: Measurements Of Pollution In The Troposphere (MO- PITT) Level 2 Derived CO (Thermal Infrared Radiances) (MOP02T) V008, NASA Atmospheric Sciences Data Center [data set] https://doi.org/10.5067/TERRA/MOPITT/MOP02T_ L2.008, 2019c. Wofsy, S., Afshar, S., Allen, H., Apel, E., Asher, E., Barletta, B., Bent, J., Bian, H., Biggs, B., Blake, D., Blake, N., Bourgeois, I., Brock, C., Brune, W., Budney, J., Bui, T., Butler, A., Campuzano- Jost, P., Chang, C., Chin, M., Commane, R., Correa, G., Crounse, J., Cullis, P., Daube, B., Day, D., Dean-Day, J., Dibb, J., Digangi, J., Diskin, G., Dollner, M., Elkins, J., Erdesz, F., Fiore, A., Flynn, C., Froyd, K., Gesler, D., Hall, S., Hanisco, T., Hannun, R., Hills, A., Hintsa, E., Hoffman, A., Hornbrook, R., Huey, L., Hughes, S., Jimenez, J., Johnson, B., Katich, J., Keeling, R., Kim, M., Kupc, A., Lait, L., Lamarque, J.-F., Liu, J., Mckain, K., Mclaugh- lin, R., Meinardi, S., Miller, D., Montzka, S., Moore, F., Morgan, E., Murphy, D., Murray, L., Nault, B., Neuman, J., Newman, P., Nicely, J., Pan, X., Paplawsky, W., Peischl, J., Prather, M., Price, D., Ray, E., Reeves, J., Richardson, M., Rollins, A., Rosenlof, K., Ryerson, T., Scheuer, E., Schill, G., Schroder, J., Schwarz, J., St.Clair, J., Steenrod, S., Stephens, B., Strode, S., Sweeney, C., Tanner, D., Teng, A., Thames, A., Thompson, C., Ullmann, K., Veres, P., Vizenor, N., Wagner, N., Watt, A., Weber, R., Weinzierl, B., Wennberg, P., Williamson, C., Wilson, J., Wolfe, G., Woods, C., and Zeng, L.: ATom: Merged Atmospheric Chem- istry, Trace Gases, and Aerosols (1.5), ORNL Distributed Ac- tive Archive Center, https://doi.org/10.3334/ORNLDAAC/1581, 2018. Atmos. Meas. Tech., 15, 4751–4765, 2022 S. Martínez-Alonso et al.: AirCore CO for satellite validation Wofsy, S., Afshar, S., Allen, H., Apel, E., Asher, E., Barletta, B., Bent, J., Bian, H., Biggs, B., Blake, D., Blake, N., Bourgeois, I., Brock, C., Brune, W., Budney, J., Bui, T., Butler, A., Campuzano- Jost, P., Chang, C., Chin, M., Commane, R., Correa, G., Crounse, J., Cullis, P., Daube, B., Day, D., Dean-Day, J., Dibb, J., Digangi, Diskin, G., Dollner, M., Elkins, J., Erdesz, F., Fiore, A., F C., Froyd, K., Gesler, D., Hall, S., Hanisco, T., Hannun, R., Hills, A., Hintsa, E., Hoffman, A., Hornbrook, R., Huey, L., Hughes, S., Jimenez, J., Johnson, B., Katich, J., Keeling, R., Kim, M., Kupc, A., Lait, L., Lamarque, J.-F., Liu, J., Mckain, K., Mclaugh- lin, R., Meinardi, S., Miller, D., Montzka, S., Moore, F., Morgan, E., Murphy, D., Murray, L., Nault, B., Neuman, J., Newman, P., Ni l J P X P l k W P i hl J P h M P i y, J., Pan, X., Paplawsky, W., Peischl, J., Prather, M., Pri D., Ray, E., Reeves, J., Richardson, M., Rollins, A., Rosenlof, K., Ryerson, T., Scheuer, E., Schill, G., Schroder, J., Schwarz, J., St.Clair, J., Steenrod, S., Stephens, B., Strode, S., Sweeney, C., Tanner, D., Teng, A., Thames, A., Thompson, C., Ullmann, Atmos. Meas. Tech., 15, 4751–4765, 2022 https://doi.org/10.5194/amt-15-4751-2022
https://openalex.org/W4313261734	https://raep.emnuvens.com.br/raep/article/download/2239/530	Portuguese	null	Financial Education and Sustainability: A Conceptual Framework	Administração	2,022	cc-by	13,002	Administração: Ensino e Pesquisa Administração: Ensino e Pesquisa artigos articles 510 Financial Education and Sustainability: a conceptual framework 510–534 Set-Dez 2022 DOI 10.13058/raep.2022.v23n3.2239 ISSN 2358-0917 DOI 10.13058/raep.2022.v23n3.2239 ISSN 2358-0917 DOI 10.13058/raep.2022.v23n3.2239 ISSN 2358-0917 Educação financeira e sustentabilidade: um framework conceitual Performance Evaluation in the University Context: an investigation of literature from the Constructivist perspective Renally Fernandes Couto \| Kettrin Farias Bem Maracajá \| Petruska de Araújo Machado 511 511 511 ABSTRACT ABSTRACT Financial education and sustainability are highly relevant as- pects of the contemporary context and for the direction of so- ciety. This research stems from the theoretical gap identified between these fields and the research problem sought to un- derstand which dimensions they involved. The study assumes an exploratory nature and a qualitative-descriptive approach since the intention is to deepen the discussions of a little-ex- plored problem and not to carry out objective observations of reality. A conceptual framework was proposed with the dimen- sions and their relationships, based on Skinner’s concept of triple contingency (1969), containing seven dimensions. For the antecedent factor, financial education and sustainability dimen- sions were assumed and considered attributes of knowledge, understanding, and awareness. As for the behavior factor, the dimensions of personal and domestic budget, credit and indeb- tedness, savings and investment, and consumption and waste were attributed, considering attributes of practices and beha- viors. Finally, the consequent factor included the dimension of future and legacy and considered the attributes of expectations and assessment of consequences. The framework does not in- tend to predict behaviors but to present dimensions in which they can be structured. This work intends to encourage future research involving financial education and sustainability, simul- taneously; preparing a fertile environment for the emergence of better approaches. The main limitation of this study is its strictly theoretical character; therefore, it is suggested that in subsequent research, measurable attributes be listed for the dimensions, making it possible to test the model in different types of samples and contexts. Keywords: Financial education; Sustainability; Conceptual fra- mework Keywords: Financial education; Sustainability; Conceptual fra- mework Financial Education and Sustainability: a conceptual framework Renally Fernandes Couto Kettrin Farias Bem Maracajá Petruska de Araújo Machado RESUMO A educação financeira e a sustentabilidade são aspectos de extrema relevância para o contexto contemporâneo e para os rumos da sociedade. Esta pesquisa decorre da lacuna teórica identificada entre esses campos e o problema da pesquisa buscou compreender quais as dimensões por eles envolvi- das. O estudo assume natureza exploratória e abordagem qualitativa-descritiva, já que o intuito é aprofundar as discus- sões de um problema pouco explorado, e não realizar cons- tatações objetivas da realidade. Foi proposto um framework conceitual com as dimensões e suas relações, baseado no conceito de tríplice contingência de Skinner (1969), contendo sete dimensões. Para o fator antecedente foram assumidas as dimensões de educação financeira e sustentabilidade e considera atributos de conhecimentos, compreensão e cons- ciência. Já para o fator de comportamento foram atribuídas as dimensões de orçamento pessoal e doméstico, crédito e endividamento, poupança e investimento e consumo e resí- duos, considerando atributos de práticas e comportamentos. Por fim, o fator consequente contou com a dimensão de futu- ro e legado e considerou os atributos de expectativas e ava- liação de consequências. O framework não pretende prever comportamentos, mas apresentar dimensões nos quais eles podem se estruturar. A pretensão deste trabalho é encora- jar pesquisas futuras envolvendo a educação financeira e a sustentabilidade, simultaneamente; preparando um ambiente fértil para o surgimento de melhores abordagens. A principal limitação deste estudo está no seu caráter estritamente teó- rico; por isso, é sugerido que nas pesquisas subsequentes sejam elencados atributos mensuráveis para as dimensões, tornando possível o teste do modelo em diferentes tipos de amostras e contextos.i RESUMO Recebido em: 11/07/2022 Aprovado em: 20/12/2022 Renally Fernandes Couto renally456@gmail.com Mestrado Universidade Federal de Campina Grande Campina Grande / PA – Brasil Kettrin Farias Bem Maracajá kettrin.farias@uaac.ufcg.edu.br Doutorado Universidade Federal de Campina Grande Campina Grande / PA – Brasil Petruska de Araújo Machado petruskamachado@gmail.com Doutorado Universidade Federal do Rio Grande do Norte João Pessoa / PA – Brasil Palavras-chave: Educação financeira; sustentabilidade; fra- mework conceitual. Administração: Ensino e Pesquisa Rio de Janeiro v. 23 nº 3 p. DOI 10.13058/raep.2022.v23n3.2239 ISSN 2358-0917 Introdução Desde a eclosão do termo sustentabilidade (World Commission on Environment and Development [WCED], 1987), ciência e sociedade têm buscado formas de conciliar o crescimento econômico com o desenvolvimento sustentável (Sharachchandra, 1991; Bettencourt et al., 2007; Murray, Skene & Haynes, 2017). O termo sustentabilidade tem a sua origem na raiz “sustain” que significa suportar, assegurar, equilibrar. Esta mesma ideia se firma na comunidade cientí- fica, uma vez que sustentabilidade representa o equilíbrio mútuo entre economia, sociedade e meio ambiente, formando os três pilares do modelo Triple Bottom Line (TBL) (WCED, 1987; Elkington, 1994). Nesse sentido, para que haja sustentabilida- Administração: Ensino e Pesquisa Rio de Janeiro v. 23 nº 3 p. 510–534 Set-Dez 2022 DOI 10.13058/raep.2022.v23n3.2239 ISSN 2358-0917 Administração: Ensino e Pesquisa Rio de Janeiro v. 23 nº 3 p. 510–534 Set-Dez 2022 DOI 10.13058/raep.2022.v23n3.2239 ISSN 2358-0917 Administração: Ensino e Pesquisa Rio de Janeiro v. 23 nº 3 p. 510–534 Set-Dez 2022 DOI 10.13058/raep.2022.v23n3.2239 ISSN 2358-0917 Educação financeira e sustentabilidade: um framework conceitual Performance Evaluation in the University Context: an investigation of literature from the Constructivist perspective Renally Fernandes Couto \| Kettrin Farias Bem Maracajá \| Petruska de Araújo Machado Educação financeira e sustentabilidade: um framework conceitual Performance Evaluation in the University Context: an investigation of literature from the Constructivist perspective Renally Fernandes Couto \| Kettrin Farias Bem Maracajá \| Petruska de Araújo Machado 512 de, o tripé deve estar equilibrado; se o nível de atenção e relevância atribuídos a cada pilar for diferente, o resultado se traduzirá em problemas sociais, disfunções econômicas, degradações e catástrofes ambientais, entre outros (Khandelwal & Darbha, 2021). É impossível refutar a centralidade assumida pelo pilar econômico na contemporaneidade. A maior evidência disso é que nações e indivíduos precisam se adequar às ferramentas monetárias e mercadológicas de intercâmbio pré- estabelecidas; e de modo contrário, serão excluídos ou marginalizados do sistema (Moro & Hofmann, 2012). A insuficiência de compreensão e criticidade acerca de tais mecanismos e seus impactos, contribuem para que os indivíduos internalizem, deficiente e equivocadamente, conceitos como desenvolvimento econômico, segurança e independência financeira, qualidade de vida, sustentabilidade, e muitos outros, resultando em práticas econômicas, sociais e ambientais que cada vez mais desestabilizam a equação do TBL. As camadas menos favorecidas da sociedade são as que mais se aproxi- mam das vulnerabilidades consequentes da atividade econômica. Introdução Somando-se o ambiente de insegurança financeira, social e ambiental à carência de competências financeiras, decorrentes da inexistência ou insuficiência de canais universais e de- mocráticos de ensino-aprendizagem acerca do principal recurso desse sistema - o dinheiro - podem ser considerados causas e consequências dessa espiral negativa (West & Friedline, 2016). A difusão da educação financeira associada à educação ambiental e à sustentabilidade pode servir como dispositivo de enfrentamento para que o indivíduo vulnerável assuma um papel de protagonismo. Assim, a gestão das finanças pessoais torna-se uma ferramenta de segurança e longevidade para o alcance do bem-estar e da qualidade de vida individual e, consequentemente, coletiva (Anderson et al., 2013). O crescimento exponencial das discussões sobre educação financeira e sus- tentabilidade, verificado tanto no contexto científico quanto no social, incita muitos questionamentos pertinentes. Esta pesquisa concentra-se em investigar “Quais as dimensões envolvidas na relação entre educação financeira e sustentabilida- de?”; sendo assim, seu objetivo é propor um framework que possibilite ampliar as discussões, bem como, compreender melhor a relação entre educação financeira e sustentabilidade na perspectiva do comportamento. Administração: Ensino e Pesquisa Rio de Janeiro v. 23 nº 3 p. 510–534 Set-Dez 2022 DOI 10.13058/raep.2022.v23n3.2239 ISSN 2358-0917 Administração: Ensino e Pesquisa Rio de Janeiro v. 23 nº 3 p. 510–534 Set-Dez 2022 DOI 10.13058/raep.2022.v23n3.2239 ISSN 2358-0917 Administração: Ensino e Pesquisa Rio de Janeiro v. 23 nº 3 p. 510–534 Set-Dez 2022 DOI 10.13058/raep.2022.v23n3.2239 ISSN 2358-0917 DOI 10.13058/raep.2022.v23n3.2239 ISSN 2358-0917 Educação financeira e sustentabilidade: um framework conceitual Performance Evaluation in the University Context: an investigation of literature from the Constructivist perspective Renally Fernandes Couto \| Kettrin Farias Bem Maracajá \| Petruska de Araújo Machado Educação financeira e sustentabilidade: um framework conceitual Performance Evaluation in the University Context: an investigation of literature from the Constructivist perspective Renally Fernandes Couto \| Kettrin Farias Bem Maracajá \| Petruska de Araújo Machado 513 513 Este estudo é também um convite à reflexão acerca das carências e deficiên- cias existentes na educação básica e nos demais canais de difusão da educação financeira e da sustentabilidade, bem como suas práticas articuladas, seja no am- biente escolar, familiar e na própria sociedade. Considerando que uma das respon- sabilidades das ciências sociais é contribuir para a melhoria contínua da vida em sociedade, os achados deste trabalho poderão contribuir para o desenvolvimento de práticas pedagógicas, políticas públicas, ações da iniciativa privada e sensibili- zar demais públicos interessados. Revisão da Literatura A educação, de maneira geral, possui um papel extremamente importante para o desenvolvimento da sociedade, uma vez que objetiva preparar o indivíduo para a vida. Nesse sentido, emerge a importância da formação do cidadão global (Davies, Evans & Reid, 2005), um indivíduo capaz de perceber, refletir, compreender, criticar e agir, de acordo com o mundo plural à sua volta; ciente dos múltiplos contex- tos que o envolvem, sejam sociais, políticos, econômicos, ambientais ou culturais (Campos, Teixeira & Coutinho, 2005; Rosseto et al., 2020). A alfabetização financeira, decorrente da internalização de elementos da edu- cação financeira, é considerada uma habilidade essencial para a vida adulta, dada a conjuntura social, política e econômica contemporânea (Huston, 2010; Potrich, Vieira, & Kirch, 2015). O tópico vem ganhando relevância na comunidade científica, evidenciado através do crescimento exponencial das produções científicas nas últi- mas décadas (López-Medina et al., 2021; Couto, Maracajá & Machado, 2022). Tal interesse ainda não se traduz em solidez conceitual, pois os pesquisado- res ainda divergem ou aplicam ambiguidades na abordagem de termos e na apli- cação de nomenclaturas (Hung, Parker & Yoong, 2009). No entanto, massivas pes- quisas apontam para a importância da alfabetização financeira para a capacidade de tomar melhores decisões de consumo, de gerenciar as finanças pessoais e de compreender os produtos financeiros, seus riscos e oportunidades, objetivando a melhoria do bem-estar financeiro (Organization for Economic Co-Operation and De- velopment [OECD], 2013). Administração: Ensino e Pesquisa Rio de Janeiro v. 23 nº 3 p. 510–534 Set-Dez 2022 DOI 10.13058/raep.2022.v23n3.2239 ISSN 2358-0917 Administração: Ensino e Pesquisa Rio de Janeiro v. 23 nº 3 p. 510–534 Set-Dez 2022 DOI 10.13058/raep.2022.v23n3.2239 ISSN 2358-0917 Administração: Ensino e Pesquisa Rio de Janeiro v. 23 nº 3 p. Revisão da Literatura 510–534 Set-Dez 2022 DOI 10.13058/raep.2022.v23n3.2239 ISSN 2358-0917 DOI 10.13058/raep.2022.v23n3.2239 ISSN 2358-0917 Educação financeira e sustentabilidade: um framework conceitual Performance Evaluation in the University Context: an investigation of literature from the Constructivist perspective Renally Fernandes Couto \| Kettrin Farias Bem Maracajá \| Petruska de Araújo Machado Educação financeira e sustentabilidade: um framework conceitual Performance Evaluation in the University Context: an investigation of literature from the Constructivist perspective Renally Fernandes Couto \| Kettrin Farias Bem Maracajá \| Petruska de Araújo Machado 514 A socialização da educação financeira alinhada à sustentabilidade tem o potencial de imbuir no indivíduo conhecimentos essenciais para coexistir no con- temporaneidade, sem perder a capacidade crítica de questionar e transformar o ambiente à sua volta; para Christensen et al. (1996) isso compõe o desenvolvimento sustentável, na ausência de consenso. Nesse sentido, seria possível conciliar os objetivos da alfabetização financeira com os da sustentabilidade: pensar e explorar o mundo, suprir as necessidades e buscar o bem-estar nas gerações do presente, sem afetar a capacidade das gerações futuras de fazerem o mesmo por si (Elking- ton, 1994; Becker, 1995; Oliveira Filho, 2004; Zozzoli, 2008). Ainda são incipientes os trabalhos que abordam educação financeira e susten- tabilidade, simultaneamente. No entanto, é possível visualizar a existência de quatro blocos de perspectivas predominantes de pesquisas, identificados através da co- -ocorrência de palavras-chave mais utilizadas nos trabalhos que envolvem os dois campos entre 1995 e 2021, presentes na Web of Science, apresentados na Figura 1. Figura 1 Posição atual da co-ocorrência de palavras-chave em artigos sobre Figura 1 Posição atual da co-ocorrência de palavras-chave em artigos sobre DOI 10.13058/raep.2022.v23n3.2239 ISSN 2358-0917 Figura 1 Posição atual da co-ocorrência de palavras-chave em artigos sobre Figura 1 Posição atual da co-ocorrência de palavras-chave em artigos sobre d ã fi i t t bilid d W b f S i (1995 2021) Figura 1 Posição atual da co-ocorrência de palavras-chave em artigos sobre educação financeira e sustentabilidade na Web of Science (1995-2021). Fonte: Elaborado pelas autoras. educação financeira e sustentabilidade na Web of Science (1995-2021). Fonte: Elaborado pelas autoras. Fonte: Elaborado pelas autoras. O primeiro bloco aborda os elementos culturais e comportamentais que in- fluenciam na prática da gestão das finanças pessoais; grande parte da pesquisa investiga e compara como determinados grupos gerenciam suas finanças e se pre- param para o futuro e a aposentadoria, dadas as influências à sua volta (Bucher-Ko- enen & Lusardi, 2011a; Atkinson & Messy, 2011; Morgan & Trinh, 2020; Herrador-Al- caide, Montserrat & Topa, 2021). Já o segundo bloco abrange as questões relacionadas ao corpo de conheci- mentos, à composição dos conteúdos, o papel e as ferramentas didáticas da edu- Administração: Ensino e Pesquisa Rio de Janeiro v. 23 nº 3 p. 510–534 Set-Dez 2022 DOI 10.13058/raep.2022.v23n3.2239 ISSN 2358-0917 Administração: Ensino e Pesquisa Rio de Janeiro v. 23 nº 3 p. 510–534 Set-Dez 2022 Administração: Ensino e Pesquisa Rio de Janeiro v. 23 nº 3 p. 510–534 Set-Dez 2022 DOI 10.13058/raep.2022.v23n3.2239 ISSN 2358-0917 Educação financeira e sustentabilidade: um framework conceitual Performance Evaluation in the University Context: an investigation of literature from the Constructivist perspective Renally Fernandes Couto \| Kettrin Farias Bem Maracajá \| Petruska de Araújo Machado Educação financeira e sustentabilidade: um framework conceitual Performance Evaluation in the University Context: an investigation of literature from the Constructivist perspective Renally Fernandes Couto \| Kettrin Farias Bem Maracajá \| Petruska de Araújo Machado 515 cação financeira. É, portanto, o bloco de investigação mais antigo e com o maior quantitativo de publicações do campo, visando responder questões em aberto e preencher lacunas conceituais (Tennyson, Sharon & Nguyen, 2005; Huston, 2010; Fernandes, Lynch & Netemeyer, 2014; Rebello, Harres & Rocha Filho, 2015; Lucey, White & André; 2020). O terceiro bloco é o segundo mais tradicional do campo e trata das ferramen- tas de transição entre teoria e prática da educação financeira, envolvendo temas como planejamento financeiro, poupança, investimento, crédito e o impacto dos aspectos econômicos na vida social (Fox, Bartholomae & Lee, 2005; Gristein-Weiss, Charles & Curley, 2007; Birkenmaier, Curley & Kelly, 2012. Gristein-Weiss, et. al., 2015; Rosales-Pérez, et. al., 2021). Figura 1 Posição atual da co-ocorrência de palavras-chave em artigos sobre Por fim, o quarto bloco é o que possui o menor volume de pesquisas e suas investigações discutem a educação financeira de uma forma mais interdisciplinar; é justamente neste bloco onde as discussões envolvendo a sustentabilidade se concentram (Lyons, Rachlis & Scherpf, 2007; Brennan & Coppack, 2008; Hira, 2012; Kindle, 2010; 2013; Helm et al., 2019; Anderson et al., 2013; Ianole et al., 2020; Ló- pez-Medina, 2021). Mesmo com a amplificação das discussões e investigações acerca da edu- cação financeira, são escassos os registros que envolvem este campo com a sus- tentabilidade. São ainda mais raros os trabalhos que fazem uma ponte equilibrada com os pilares do TBL, principalmente o ambiental. Portanto, faz-se necessário esquadrinhar em quais aspectos estes campos se permutam no comportamento humano; é, justamente, o esforço empreendido neste trabalho. Métodos A motivação desta pesquisa decorre justamente da escassez de investigação cien- tífica envolvendo campos tão necessários como os de educação financeira e sus- tentabilidade na vida das pessoas na atualidade. O questionamento orientador (Gil, 2007) deste trabalho é: “Quais as dimensões envolvidas na relação entre edu- cação financeira e sustentabilidade?”. A proposta de um framework conceitual (Quivy & Campenhoudt, 1995) responde a esta questão, uma vez que facilita a com- Administração: Ensino e Pesquisa Rio de Janeiro v. 23 nº 3 p. 510–534 Set-Dez 2022 DOI 10.13058/raep.2022.v23n3.2239 ISSN 2358-0917 Administração: Ensino e Pesquisa Rio de Janeiro v. 23 nº 3 p. 510–534 Set-Dez 2022 DOI 10.13058/raep.2022.v23n3.2239 ISSN 2358-0917 Administração: Ensino e Pesquisa Rio de Janeiro v. 23 nº 3 p. 510–534 Set-Dez 2022 DOI 10.13058/raep.2022.v23n3.2239 ISSN 2358-0917 Educação financeira e sustentabilidade: um framework conceitual Performance Evaluation in the University Context: an investigation of literature from the Constructivist perspective Renally Fernandes Couto \| Kettrin Farias Bem Maracajá \| Petruska de Araújo Machado Educação financeira e sustentabilidade: um framework conceitual Performance Evaluation in the University Context: an investigation of literature from the Constructivist perspective Renally Fernandes Couto \| Kettrin Farias Bem Maracajá \| Petruska de Araújo Machado 516 preensão do problema investigado e amplia discussões para trabalhos futuros e, portanto, é o objetivo desta pesquisa. Logo, esta pesquisa assume natureza exploratória, dado o objetivo de compre- ender um problema pouco investigado, do qual se tem muitas dúvidas, ou que não foi abordado anteriormente (Sampieri, Collado & Lucio, 2006). A abordagem é qualitati- va-descritiva, uma vez que o seu foco está no aprofundamento das discussões e não em constatações objetivas da realidade (Gil, 2007; Silveira & Córdova, 2009). Os frameworks conceituais podem ser narrativas ou estruturas gráfico-visuais que representam fatores-chave, construtos ou variáveis e suas supostas relações, em um fragmento da realidade, fato ou fenômeno (Le Moigne, 1977; Miles & Hu- berman, 1994; Shehabuddeen et al., 1999). Esta representação é frequentemente utilizada na pesquisa científica quando há complexidade conceitual, inconsistência teórica ou falta de consenso da comunidade; portanto, se adequa ao objetivo desta pesquisa (Crossan, Lane & White, 1999; Morgan & Morrison, 1999). Figura 2 Processo de desenvolvimento do framework. Figura 2 Processo de desenvolvimento do framework. Fonte: Elaborado pelas autoras. Fonte: Elaborado pelas autoras. Logo, têm-se: a) a identificação do fenômeno de interesse - a relação entre educação financeira e sustentabilidade, e b) a identificação do objetivo da pes- quisa - propor de um framework conceitual envolvendo as dimensões de relação entre educação financeira e sustentabilidade. Portanto, na seção subsequente os elementos que compõem o modelo serão indicados e descritos; posteriormente, serão apresentadas as suas relações e, finalmente, assumidas as premissas-chaves e subjacentes, que poderão ser utilizadas em estudos futuros. Métodos O modelo proposto se baseia no conceito de tríplice contingência, onde um comportamento, ou a falta dele, depende de um contexto antecedente, formado por fatores ambientais, e de um contexto consequente, formado pelas expectativas e consequências capazes (Skinner, 1969); logo: Sa + R = Sc Onde: Sa = Estímulo antecedente, R = Resposta, comportamento, Sc = Estímulo consequente. É importante salientar que o comportamento de um indivíduo é fruto de um processo interativo multifacetado, através do qual o indivíduo monta seu repertório comportamental (Gomes et al., 2019); logo, o mesmo não deve ser avaliado atra- vés de uma lente mecânico-determinista (Skinner, 1935; 1938; Todorov & Morei- ra, 2009). Nesse sentido, o desenvolvimento do modelo ilustrado na Figura 2 não pretende expôr determinantes do comportamento de um indivíduo, mas apontar dimensões nas quais existem muitos fatores capazes de influenciar o seu repertório comportamental. Administração: Ensino e Pesquisa Rio de Janeiro v. 23 nº 3 p. 510–534 Set-Dez 2022 DOI 10.13058/raep.2022.v23n3.2239 ISSN 2358-0917 Administração: Ensino e Pesquisa Rio de Janeiro v. 23 nº 3 p. 510–534 Set-Dez 2022 DOI 10.13058/raep.2022.v23n3.2239 ISSN 2358-0917 Administração: Ensino e Pesquisa Rio de Janeiro v. 23 nº 3 p. 510–534 Set-Dez 2022 DOI 10.13058/raep.2022.v23n3.2239 ISSN 2358-0917 DOI 10.13058/raep.2022.v23n3.2239 ISSN 2358-0917 Educação financeira e sustentabilidade: um framework conceitual Performance Evaluation in the University Context: an investigation of literature from the Constructivist perspective Renally Fernandes Couto \| Kettrin Farias Bem Maracajá \| Petruska de Araújo Machado Figura 2 Processo de desenvolvimento do framework. Fonte: Elaborado pelas autoras. Educação financeira e sustentabilidade: um framework conceitual Performance Evaluation in the University Context: an investigation of literature from the Constructivist perspective Renally Fernandes Couto \| Kettrin Farias Bem Maracajá \| Petruska de Araújo Machado 517 Apresentação e interpretação dos resultados Propõe-se sete dimensões para os três fatores do framework, distribuídas confor- me Tabela 1; considerando conhecimentos, compreensão e consciência acerca da educação financeira e da sustentabilidade para determinar o contexto ante- cedente. Este, por sua vez, condiciona os níveis, volumes e perfis de orçamento pessoal e doméstico, de tomada de crédito, de endividamento, de poupança, de investimento, de consumo e da produção de resíduos. Enquanto o contexto consequente é formado pelas expectativas em relação ao futuro, às incertezas e ao legado. Administração: Ensino e Pesquisa Rio de Janeiro v. 23 nº 3 p. 510–534 Set-Dez 2022 DOI 10.13058/raep.2022.v23n3.2239 ISSN 2358-0917 Tabela 1 Descrição dos elementos dimensionados do framework Fator Dimensão Atributos Antecedente Educação financeira Conhecimentos, compreensão e consciência Sustentabilidade Conhecimentos, compreensão e consciência bela 1 Descrição dos elementos dimensionados do framework Tabela 1 Descrição dos elementos dimensionados do framework Administração: Ensino e Pesquisa Rio de Janeiro v. 23 nº 3 p. 510–534 Set-Dez 2022 ISSN 2358-0917 Educação financeira e sustentabilidade: um framework conceitual Performance Evaluation in the University Context: an investigation of literature from the Constructivist perspective Renally Fernandes Couto \| Kettrin Farias Bem Maracajá \| Petruska de Araújo Machado Educação financeira e sustentabilidade: um framework conceitual Performance Evaluation in the University Context: an investigation of literature from the Constructivist perspective Renally Fernandes Couto \| Kettrin Farias Bem Maracajá \| Petruska de Araújo Machado 518 Comportamento Orçamento pessoal e doméstico Comportamentos e práticas Crédito e endividamento Comportamentos e práticas Poupança e investimento Comportamentos e práticas Consumo e resíduos Comportamentos e práticas Consequente Futuro e legado Expectativas e avaliação de consequências Comportamento Orçamento pessoal e doméstico Comportamentos e práticas Crédito e endividamento Comportamentos e práticas Poupança e investimento Comportamentos e práticas Consumo e resíduos Comportamentos e práticas Consequente Futuro e legado Expectativas e avaliação de consequências Comportamento É válido salientar que as relações investigadas ocorrem em um macroambiente multifacetado e subjetivo; à vista disso, serão melhor compreendidas se avaliadas em conjunto com os contextos socioeconômicos e culturais. Cada dimensão será apresentada individualmente, para que seja possível justificar sua alocação no modelo. DOI 10.13058/raep.2022.v23n3.2239 ISSN 2358-0917 EDUCAÇÃO FINANCEIRA A educação financeira é imprescindível para a vida de um indivíduo, pois ao longo de sua vida este será responsável por tomar diversas decisões financei- ras e realizar inúmeros intercâmbios que envolvem recursos financeiros (Bernheim, Garrett & Maki, 2001; Lusardi & Mitchell, 2007b; Remund, 2010; Hastings, Madrian & Skimmyhorn, 2013). Nesse sentido, obter orientação, principalmente nas bases familiares e escolares, é imprescindível para a minimização de vulnerabilidades cau- sadas pelo analfabetismo financeiro na vida adulta, não excluindo os demais canais de ensino-aprendizagem (Lusardi, Mitchell & Curto, 2010; Huston, 2010; Flores, Vieira & Coronel, 2013). A socialização do conhecimento financeiro, que culmina no comportamento financeiro (Zhu & Chou, 2018) deve levar em consideração múltiplos fatores, es- pecialmente, socioeconômicos e demográficos (Lusardi & Mitchell, 2011a; 2014; Atkinson & Messy, 2012; Brown & Graf, 2013). Também é importante que a prática da gestão, bem como a busca de conhecimentos que reforcem o entendimento da gestão financeira, seja perene na vida do indivíduo, com o intuito de não defasar a curva de conhecimento ao longo do tempo e manter o domínio sobre o assunto (Fernandes, Lynch & Netemeyer, 2014). De acordo com o framework, faz-se necessário a existência de um currículo de educação financeira que envolva: noções de economia - a estrutura dos pilares Administração: Ensino e Pesquisa Rio de Janeiro v. 23 nº 3 p. 510–534 Set-Dez 2022 DOI 10.13058/raep.2022.v23n3.2239 ISSN 2358-0917 Administração: Ensino e Pesquisa Rio de Janeiro v. 23 nº 3 p. 510–534 Set-Dez 2022 DOI 10.13058/raep.2022.v23n3.2239 ISSN 2358-0917 Administração: Ensino e Pesquisa Rio de Janeiro v. 23 nº 3 p. 510–534 Set-Dez 2022 DOI 10.13058/raep.2022.v23n3.2239 ISSN 2358-0917 Educação financeira e sustentabilidade: um framework conceitual Performance Evaluation in the University Context: an investigation of literature from the Constructivist perspective Renally Fernandes Couto \| Kettrin Farias Bem Maracajá \| Petruska de Araújo Machado Educação financeira e sustentabilidade: um framework conceitual Performance Evaluation in the University Context: an investigation of literature from the Constructivist perspective Renally Fernandes Couto \| Kettrin Farias Bem Maracajá \| Petruska de Araújo Machado 519 macroeconômicos: monetário, fiscal e cambial, suas composições e principais fun- ções, matemática financeira, orçamento pessoal/doméstico, tipos de empréstimos, tipos financiamentos (de bens móveis, imóveis e de serviços - em especial, os tipos mais comuns como: financiamentos de carros, casas e estudantil), cartão de cré- dito, empréstimos poupança, investimentos, seguros, declaração de imposto de renda e aposentadoria. EDUCAÇÃO FINANCEIRA Dado que estes são elementos com os quais o indivíduo interage quando obtém autonomia sobre suas finanças. SUSTENTABILIDADE A economia e a sociedade vêm se desenvolvendo a partir da exploração dos recursos finitos do nosso planeta; no entanto, as alterações ecossistêmicas causa- das por essa exploração alertam para a necessidade de alcançar formas mais sus- tentáveis de viver (Dunlap, et al., 2000). Muitas escalas de sustentabilidade apontam que, de maneira geral, a idade e o nível de exposição às questões ambientais são fatores de interferência no desenvolvimento da consciência ambiental; sendo a pri- meira, inversamente e a segunda, diretamente proporcional (Dunlap & Van Liere, 2008; Jones & Dunlap, 2010). Qualquer intervenção humana provoca impactos que podem ameaçar a qualidade da vida, individual e coletiva, e a própria existência humana (Hines, Hungerford & Tomera, 1987; Kollmuss & Agyeman, 2002). Para boa parte dos pes- quisadores, desenvolver indivíduos e, consequentemente, empresas ambiental- mente responsáveis é um dos principais objetivos da educação ambiental (Gough & Gough 2010). Deste modo, os conceitos de sustentabilidade devem ser traba- lhados para que o indivíduo se sinta pertencente à uma biosfera na qual coexistem diversos tipos de vida, e Economia e contemporaneidade são facetas às quais a educação ambiental está, irremediavelmente, vinculada (Jickling & Wals, 2008; Hursh, Henderson & Greenwood, 2015). Sendo assim, de acordo com o modelo proposto, a educação ambiental e a sustentabilidade são, além de pertinentes, essenciais e estão em consonância com a educação financeira, com o intuito de influenciar positivamente o comportamento e as escolhas dos indivíduos, sejam os hábitos de consumo, o modo de produzir e lidar com os resíduos e a percep- ção do mundo à sua volta. Administração: Ensino e Pesquisa Rio de Janeiro v. 23 nº 3 p. 510–534 Set-Dez 2022 DOI 10.13058/raep.2022.v23n3.2239 ISSN 2358-0917 Administração: Ensino e Pesquisa Rio de Janeiro v. 23 nº 3 p. 510–534 Set-Dez 2022 DOI 10.13058/raep.2022.v23n3.2239 ISSN 2358-0917 DOI 10.13058/raep.2022.v23n3.2239 ISSN 2358-0917 Administração: Ensino e Pesquisa Rio de Janeiro v. 23 nº 3 p. 510–534 Set-Dez 2022 Educação financeira e sustentabilidade: um framework conceitual Performance Evaluation in the University Context: an investigation of literature from the Constructivist perspective Renally Fernandes Couto \| Kettrin Farias Bem Maracajá \| Petruska de Araújo Machado Educação financeira e sustentabilidade: um framework conceitual Performance Evaluation in the University Context: an investigation of literature from the Constructivist perspective Renally Fernandes Couto \| Kettrin Farias Bem Maracajá \| Petruska de Araújo Machado 520 ORÇAMENTO PESSOAL E DOMÉSTICO O orçamento doméstico refere-se ao cômputo e acompanhamento das ren- das e despesas pessoais ou da família, envolvendo salários e ordenados, gastos com alimentação, saúde, educação, vestuário, lazer e demais dispêndios com a aquisição de produtos e serviços; tudo isso com o objetivo de buscar um equilíbrio saudável para a vida financeira (Ferreira, 2008; Pereira, 2011). O orçamento compõe o tripé da educação financeira (Tomášková, Mohelská & Němcová, 2011), e, por ser uma das principais ferramentas para a alfabetização financeira e para a prática da gestão das finanças, é proposto em uma dimensão à parte. Utilizar o orçamento para a gestão das finanças pessoais é a melhor forma de identificar momentos de escassez ou abundância de recursos com antecedência, e assim, fazer uso dos instrumentos financeiros disponíveis com maior efetividade (Lusardi & Mitchell, 2007a; Deaton, 2019; Kurowski, 2021). O ambiente doméstico é terreno fértil para o desenvolvimento da maioria dos hábitos de um indivíduo. Logo, tomar esse ambiente como ponto de partida para o gerenciamento das finanças pode ser um fator multiplicador de conhecimentos e hábitos, uma vez que consi- dera e envolve todas as pessoas do domicílio, independente da idade e do nível de geração de renda e despesas. A construção e o acompanhamento de um orçamento, leva em consideração as noções de planejamento, raciocínio lógico, regularidade, do todo e das partes, de fontes e de aplicações de recursos, de desperdício, de trabalho em equipe, de incerteza, e muitos outros aspectos que contribuem para a perspectiva das finan- ças sustentáveis. Condutas financeiramente saudáveis são capazes de propiciar segurança e bem-estar para o indivíduo e sua família. Além disso, esse ganho de eficiência financeira também é capaz de produzir benefícios ambientais, quando gera economia de recursos naturais decorrentes da diminuição de desperdícios, dos níveis de consumo por impulso, da produção de resíduos, da exposição à vul- nerabilidades causadas pelo endividamento, do aumento do consumo consciente e do reuso, por exemplo. CRÉDITO E ENDIVIDAMENTO Tomar crédito é dispor imediatamente de recursos, o que permite antecipar a fruição de bens e serviços, o que, automaticamente, gera uma obrigação futura do Administração: Ensino e Pesquisa Rio de Janeiro v. 23 nº 3 p. 510–534 Set-Dez 2022 DOI 10.13058/raep.2022.v23n3.2239 ISSN 2358-0917 Administração: Ensino e Pesquisa Rio de Janeiro v. 23 nº 3 p. 510–534 Set-Dez 2022 Administração: Ensino e Pesquisa Rio de Janeiro v. 23 nº 3 p. 510–534 Set-Dez 2022 DOI 10.13058/raep.2022.v23n3.2239 ISSN 2358-0917 DOI 10.13058/raep.2022.v23n3.2239 ISSN 2358-0917 Educação financeira e sustentabilidade: um framework conceitual Performance Evaluation in the University Context: an investigation of literature from the Constructivist perspective Renally Fernandes Couto \| Kettrin Farias Bem Maracajá \| Petruska de Araújo Machado Educação financeira e sustentabilidade: um framework conceitual Performance Evaluation in the University Context: an investigation of literature from the Constructivist perspective Renally Fernandes Couto \| Kettrin Farias Bem Maracajá \| Petruska de Araújo Machado 521 521 devedor para com o credor (Marques & Frade, 2003). Existem diversas categorias e tipos de crédito ofertados aos consumidores; nesse sentido, a tomada de crédito é positiva tanto para o desenvolvimento da economia, quanto para a qualidade de vida das pessoas (Silva, Silva Neto & Araújo, 2017). Os antecedentes decisórios da tomada de crédito por parte do consumidor devem considerar a finalidade e o valor do crédito, as garantias associadas, o prazo, as taxas praticadas, as formas e condições de pagamento, bem como, a capaci- dade de arcar com o que foi negociado; tudo isso exige uma pesquisa extensiva sobre as condições disponíveis no mercado (Levine, 2005; Lusardi & Tufano, 2009; Gathergood, 2012). Frequentemente, condições predatórias são intencionalmente ofertadas a pú- blicos que, em sua grande maioria, não foram educados financeiramente ou estão em posição de vulnerabilidade, como: jovens, idosos e indivíduos de baixa renda (Hill & Kozup, 2007). Ao tomar crédito sem análise prévia dos múltiplos fatores que o envolvem, os indivíduos estarão muito mais propensas a caírem no superendivi- damento e na inadimplência, o que inviabiliza a transformação de suas realidades individuais e coletivas (Stango & Zinman, 2009; Agarwal & Mazumder, 2013; Gerardi et al., 2013; Zinman, 2015). Os danos do endividamento e da inadimplência, além do déficit de renda destinado ao suprimento das necessidades básicas do indivíduo e seus dependen- tes financeiros, ainda geram encarecimento do crédito para o público correlato. CRÉDITO E ENDIVIDAMENTO A impossibilidade ou dificuldade de acessar bens e serviços que dependam direta ou indiretamente do sistema financeiro, como: empreender, alugar e financiar imóveis, fome, doenças nutricionais e psicossomáticas são exemplos da espiral negativa altamente complexa gerada por esse panorama financeiro. POUPANÇA E INVESTIMENTO Na perspectiva inversa, uma gestão superavitária das finanças gera o que conhecemos como poupança; ou seja, quando um indivíduo acumula patrimônio a partir das diferenças positivas entre suas rendas e despesas. O acúmulo de pou- pança pode ter diversas finalidades, que podem ir desde a mitigação de insegu- ranças, aposentadoria e planejamento futuro (Cagetti, 2003) até à remuneração de capital ocioso e a conquista de um objetivo específico. Administração: Ensino e Pesquisa Rio de Janeiro v. 23 nº 3 p. 510–534 Set-Dez 2022 DOI 10.13058/raep.2022.v23n3.2239 ISSN 2358-0917 Administração: Ensino e Pesquisa Rio de Janeiro v. 23 nº 3 p. 510–534 Set-Dez 2022 DOI 10.13058/raep.2022.v23n3.2239 ISSN 2358-0917 Administração: Ensino e Pesquisa Rio de Janeiro v. 23 nº 3 p. 510–534 Set-Dez 2022 DOI 10.13058/raep.2022.v23n3.2239 ISSN 2358-0917 Administração: Ensino e Pesquisa Rio de Janeiro v. 23 nº 3 p. 510–534 Set-Dez 2022 DOI 10.13058/raep.2022.v23n3.2239 ISSN 2358-0917 DOI 10.13058/raep.2022.v23n3.2239 ISSN 2358-0917 Educação financeira e sustentabilidade: um framework conceitual Performance Evaluation in the University Context: an investigation of literature from the Constructivist perspective Renally Fernandes Couto \| Kettrin Farias Bem Maracajá \| Petruska de Araújo Machado Educação financeira e sustentabilidade: um framework conceitual Performance Evaluation in the University Context: an investigation of literature from the Constructivist perspective Renally Fernandes Couto \| Kettrin Farias Bem Maracajá \| Petruska de Araújo Machado 522 Níveis mais altos de alfabetização financeira levam a tendências mais altas de poupar (Cole, Sampson & Zia, 2011; Fun & Zhang, 2021). E, do ponto de vista eco- nômico, a poupança é um fator estabilizante (Lusardi & Mitchell, 2011b). No entanto, dados os seus níveis de literacia financeira, os indivíduos ainda poupam de maneira ineficiente (Lusardi, 2000; Kaiser & Menkhoff, 2017), o que torna complexa a missão de potencializar a poupança através do ato de investir. Investimento significa a opção ou oportunidade de projetar e realizar uma quantia de recursos financeiros a partir de uma grandeza de recursos menor (Torres & Barros, 2014). Tanto os investimentos produtivos, quanto os financeiros, compar- tilham da promessa ou expectativa de remuneração em troca da tomada de risco (Trinh, Morgan & Sonobe, 2020). Resumidamente, os investimentos produtivos referem-se à decisão de abrir ou ampliar um negócio e consistem na oferta de produtos e serviços a um público consumidor em troca de uma remuneração (Nugent & Yhee, 2002). POUPANÇA E INVESTIMENTO Já os investi- mentos financeiros consistem na aplicação de recursos superavitários por parte de um investidor em ativos ou derivativos ofertados por um tomador deficitário, em tro- ca ou na expectativa de remuneração; tudo através de um ambiente intermediado e fiscalizado (Santos & Santos, 2005). A proficiência e a prática da gestão dos recursos financeiros influenciam na quantidade e na qualidade da poupança das pessoas, que, por sua vez, é terreno fértil para o investimento. Em consonância, investidores com níveis mais altos de educação financeira são menos propensos a utilizarem estratégias ingênuas em seus investimentos (Hibbert, Lawrence & Prakash, 2012). Portanto, em escala in- dividual, o ato de investir pode contribuir para superar vulnerabilidades (Lyons & Kass-Hanna, 2019; López-Rodríguez & López-Ordoñez, 2022) e em escala alargada tem potencial para ajudar a quebrar o ciclo geracional de pobreza (Moreno-García, Santillán & Gutiérrez-Delgado, 2017). Ampliar as discussões entre educação financeira e sustentabilidade na pers- pectiva dos investimentos tem grande valor, principalmente para as populações financeiramente vulneráveis, pois refletirá diretamente nas suas decisões. Na pers- pectiva dos investimentos produtivos, os impactos vão desde à expectativa de melhor gerenciamento dos negócios, muitas vezes iniciados na informalidade, na Administração: Ensino e Pesquisa Rio de Janeiro v. 23 nº 3 p. 510–534 Set-Dez 2022 DOI 10.13058/raep.2022.v23n3.2239 ISSN 2358-0917 Administração: Ensino e Pesquisa Rio de Janeiro v. 23 nº 3 p. 510–534 Set-Dez 2022 DOI 10.13058/raep.2022.v23n3.2239 ISSN 2358-0917 Administração: Ensino e Pesquisa Rio de Janeiro v. 23 nº 3 p. 510–534 Set-Dez 2022 Educação financeira e sustentabilidade: um framework conceitual Performance Evaluation in the University Context: an investigation of literature from the Constructivist perspective Renally Fernandes Couto \| Kettrin Farias Bem Maracajá \| Petruska de Araújo Machado Educação financeira e sustentabilidade: um framework conceitual Performance Evaluation in the University Context: an investigation of literature from the Constructivist perspective Renally Fernandes Couto \| Kettrin Farias Bem Maracajá \| Petruska de Araújo Machado 523 redução da mortalidade de empresas, na busca por soluções internas, produtos e serviços sustentáveis que contribuam com o desempenho financeiro, entre outras possibilidades. No que diz respeito às contribuições dos dois campos para os investimentos financeiros, os impactos podem influenciar a dispersão de renda, a ampliação do mercado de valores mobiliários, o aumento da oferta de produtos financeiros ver- des, a captação de recursos para empresas/iniciativas sustentáveis, bem como, o surgimento de novos produtos e serviços financeiros sustentáveis. POUPANÇA E INVESTIMENTO A movimentação dos players no mercado financeiro está fortemente relacionada aos objetivos dos seus investidores; logo, a declaração de intenções de retornos consistentes atrela- dos a benefícios ambientais e sociais pode revolucionar o entendimento acerca dos investimentos nesta geração. CONSUMO E RESÍDUOS O ato de consumir é extremamente complexo e envolve fatores de cunho econômico, psicológico, emocional e sócio-cultural, entre outros (Mancebo, et al., 2002; Aquino & Tomassini, 2009). Atualmente, as pesquisas sobre o comportamen- to do consumidor investigam as nuances do consumo, muito mais envoltos por de- sejos do que por necessidades (Belk, Ger & Askegaard, 2003; Charles-Leija, Aboites & Llamas, 2018). No contexto contemporâneo, além dos fatores econômicos e sociais, é im- possível dissociar o consumo dos fatores ambientais, principalmente quando se trata dos recursos naturais empreendidos na produção dos bens e serviços e dos resíduos deles decorrentes (Grimm, et al., 2008; He, et al., 2020). Um dos grandes desafios da atualidade é, justamente, encontrar um ponto de interseção entre os interesses de lucratividade dos produtores, da satisfação dos consumidores e do equilíbrio ambiental (Hira, 2012). Logo, trabalhar a educação financeira na perspectiva da alavancagem do processo de consumo não só é ineficiente, mas também nocivo para a qualidade de vida individual e coletiva (Halilovic, et al., 2019). A gestão dos recursos financei- ros e o ato de consumir estão fortemente relacionados ao TBL, por isso é impres- cindível fomentar uma educação financeira sustentável para direcionar o consumo Administração: Ensino e Pesquisa Rio de Janeiro v. 23 nº 3 p. 510–534 Set-Dez 2022 DOI 10.13058/raep.2022.v23n3.2239 ISSN 2358-0917 Administração: Ensino e Pesquisa Rio de Janeiro v. 23 nº 3 p. 510–534 Set-Dez 2022 DOI 10.13058/raep.2022.v23n3.2239 ISSN 2358-0917 Administração: Ensino e Pesquisa Rio de Janeiro v. 23 nº 3 p. 510–534 Set-Dez 2022 Educação financeira e sustentabilidade: um framework conceitual Performance Evaluation in the University Context: an investigation of literature from the Constructivist perspective Renally Fernandes Couto \| Kettrin Farias Bem Maracajá \| Petruska de Araújo Machado Educação financeira e sustentabilidade: um framework conceitual Performance Evaluation in the University Context: an investigation of literature from the Constructivist perspective Renally Fernandes Couto \| Kettrin Farias Bem Maracajá \| Petruska de Araújo Machado Educação financeira e sustentabilidade: um framework conceitual Performance Evaluation in the University Context: an investigation of literature from the Constructivist perspective Renally Fernandes Couto \| Kettrin Farias Bem Maracajá \| Petruska de Araújo Machado 524 consciente. CONSUMO E RESÍDUOS Essa forma cidadã de consumir é uma expressão de valores, pois a opção de consumir, reduzir ou renunciar determinados produtos e serviços leva em consideração se a produção, a distribuição e os impactos dos mesmos estão em consonância com a sustentabilidade ou a justiça social, por exemplo (Willis & Schor, 2012; Anderson et al., 2013). Os problemas envolvendo a tríplice sustentável no contexto atual necessitam de mudanças de postura de todos os players envolvidos - consumidores, produ- tores, governos e demais instituições. A educação financeira sustentável tem po- tencial para aumentar os níveis do consumo consciente através do orçamento e do planejamento das compras (preços, prazos, vida-útil, formas e condições de paga- mento), mitigar os riscos do consumo por impulso, dos desperdícios e até modificar a relação dos indivíduos com seus resíduos. Os resíduos sólidos refletem as preferências e a forma como a sociedade pensa e age em relação ao consumo e ao meio-ambiente (Silva, Barbieri & Monte- -Mór, 2012). Isso pode ser interpretado como uma resposta para o comportamento produtivo das empresas, seja ele positivo ou negativo (Stock & Mulki, 2009). Pro- blemas de saúde pública, distúrbios sociais e desequilíbrios ambientais decorrentes dos problemas gerados pelos resíduos sólidos interferem diretamente na qualida- de de vida. Então, a existência de planos e ações que integrem as empresas, os governos e os consumidores/cidadãos é crucial, uma vez que todos são parte do problema e, portanto, da solução. Mudanças relacionadas aos hábitos de consumo e ao volume e descarte de resíduos sólidos podem pressionar o mercado a desenvolver conexões baseadas em valores mais alinhados entre empresa-cliente. A expressão dessa mudança de pos- tura pode acontecer, por exemplo, através de um P&D e uma engenharia cada vez mais sustentável aplicada aos produtos e serviços, de um aumento do envolvimento do consumidor nas ações de sustentabilidade e na estruturação de sua cadeia pro- dutiva envolvendo logística reversa. Já os governos e as instituições precisam se adaptar às novas condições sociais, econômicas e ambientais, através da legislação e dos mecanismos de fiscalização e controle, contribuindo para a harmonia do TBL, de modo que o empreendedorismo, o consumo e a gestão das finanças possam ser praticados e potencializados numa perspectiva cada vez mais sustentável. Administração: Ensino e Pesquisa Rio de Janeiro v. 23 nº 3 p. 510–534 Set-Dez 2022 DOI 10.13058/raep.2022.v23n3.2239 ISSN 2358-0917 Administração: Ensino e Pesquisa Rio de Janeiro v. 23 nº 3 p. CONSUMO E RESÍDUOS 510–534 Set-Dez 2022 DOI 10.13058/raep.2022.v23n3.2239 ISSN 2358-0917 Administração: Ensino e Pesquisa Rio de Janeiro v. 23 nº 3 p. 510–534 Set-Dez 2022 Administração: Ensino e Pesquisa Rio de Janeiro v. 23 nº 3 p. 510–534 Set-Dez 2022 Educação financeira e sustentabilidade: um framework conceitual Performance Evaluation in the University Context: an investigation of literature from the Constructivist perspective Renally Fernandes Couto \| Kettrin Farias Bem Maracajá \| Petruska de Araújo Machado Educação financeira e sustentabilidade: um framework conceitual Performance Evaluation in the University Context: an investigation of literature from the Constructivist perspective Renally Fernandes Couto \| Kettrin Farias Bem Maracajá \| Petruska de Araújo Machado 525 FUTURO E LEGADO As ações que o indivíduo pratica no presente dependem tanto do seu con- texto de influência, quanto do que ele anseia e espera para o seu futuro; esta ideia é válida para a gestão das finanças pessoais (Hastings, Madrian & Skimmyhorn, 2013) e para qualidade de vida no envelhecimento (Pillemer et al., 2010). A forma como as pessoas gerenciam suas finanças, honram seus compromissos e se preparam para enfrentar as incertezas do futuro é defendida, inclusive, como um atributo de caráter do ator financeiro (Maman & Rosenhek, 2019). Em todo o mundo, o debate envolvendo os sistemas previdenciários e o futu- ro das finanças dos indivíduos vem se amplificando (Hibbert, Lawrence & Prakash, 2012). As famílias com as menores rendas nos sistemas previdenciários são as que possuem o menor nível de letramento financeiro, de forma que estas são as que mais carecem de educação financeira para enfrentar contextos econômicos cada vez mais complexos, conforme o avanço da tecnologia e o amadurecimento etário (Berry, Karlan & Pradhan, 2018). E, piorando o cenário, o endurecimento de regras de aposentadoria estão, progressivamente, transferindo para os próprios indivíduos as responsabilidades sobre as provisões para a velhice e retirando do Estado e dos empregadores (Bucher-Koenen & Lusardi, 2011; Sekita, 2011). Por sua vez, o meio ambiente também é um elemento indispensável para a qualidade de vida, especialmente na perspectiva do envelhecimento, quando se torna essencial gozar de um ambiente agradável e saudável (Pillemer et al., 2010). As mudanças no contexto demográfico, que englobam o aumento da expectativa de vida, precisam ser consideradas para o delineamento de políticas públicas, de iniciativas privadas e do terceiro setor. O envelhecimento engloba muitos fatores, sejam eles sociais, culturais, biológicos e psicológicos, que são afetados direta- mente pelo meio-ambiente (Filiberto et al., 2010). Outro fator relevante é o desenvolvimento de novos hábitos e de estilos de vida que uma população assume à medida que envelhece, contribuindo para os problemas ambientais (Rosenbloom, 2001; Wright, Caserta & Lund, 2003). Logo, a forma como os indivíduos interagem com suas finanças e com o meio ambiente corrobora para a formação do seu contexto do presente e do futuro. Portanto, ter propósitos financeiros e ambientais alinhados propiciarão ao indivíduo um ambiente saudável para envelhecer gozando de tranquilidade e qualidade de vida, perpetu- Administração: Ensino e Pesquisa Rio de Janeiro v. 23 nº 3 p. FUTURO E LEGADO 510–534 Set-Dez 2022 DOI 10.13058/raep.2022.v23n3.2239 ISSN 2358-0917 Administração: Ensino e Pesquisa Rio de Janeiro v. 23 nº 3 p. 510–534 Set-Dez 2022 DOI 10.13058/raep.2022.v23n3.2239 ISSN 2358-0917 DOI 10.13058/raep.2022.v23n3.2239 ISSN 2358-0917 Administração: Ensino e Pesquisa Rio de Janeiro v. 23 nº 3 p. 510–534 Set-Dez 2022 Educação financeira e sustentabilidade: um framework conceitual Performance Evaluation in the University Context: an investigation of literature from the Constructivist perspective Renally Fernandes Couto \| Kettrin Farias Bem Maracajá \| Petruska de Araújo Machado 526 ando um círculo virtuoso por meio do exemplo para as gerações futuras, e assim, deixando sua contribuição para a posteridade. ando um círculo virtuoso por meio do exemplo para as gerações futuras, e assim, deixando sua contribuição para a posteridade. O FRAMEWORK Diante do exposto, é possível concluir que as sete dimensões do modelo podem desenvolver uma relação de mutualidade, e os processos de reforço, mo- dificação, diminuição ou extinção dos comportamentos são livres para ocorrer em qualquer dos três fatores de contingência, conforme Figura 3. Figura 3 Framework das dimensões entre educação financeira e sustentabi- Figura 3 Framework das dimensões entre educação financeira e sustentabi- Figura 3 Framework das dimensões entre educação financeira e sustentabi- lidade. Fonte: Elaboração própria, baseado em Skinner (1969). Fonte: Elaboração própria, baseado em Skinner (1969). Nesse sentido, as estruturas do comportamento dependem da percepção do agente em relação aos benefícios, prejuízos e encaixe de contextos que cada bloco dimensional tem para a sua vida e seus objetivos. Quando um indivíduo vul- nerável obtêm perícia acerca das contingências que cercam a sua vida e seus com- portamentos financeiros, sociais, econômicos e ambientais, será muito mais capaz de desenvolver autoconhecimento, autocontrole e autonomia. Em uma conjuntura onde o poderio econômico, concentrado nas mãos de poucos, dita as regras e os rumos da vida de muitos, obter mais controle sobre as rédeas da vida pode ser sinônimo de revolução. Administração: Ensino e Pesquisa Rio de Janeiro v. 23 nº 3 p. 510–534 Set-Dez 2022 DOI 10.13058/raep.2022.v23n3.2239 ISSN 2358-0917 Administração: Ensino e Pesquisa Rio de Janeiro v. 23 nº 3 p. 510–534 Set-Dez 2022 DOI 10.13058/raep.2022.v23n3.2239 ISSN 2358-0917 DOI 10.13058/raep.2022.v23n3.2239 ISSN 2358-0917 Administração: Ensino e Pesquisa Rio de Janeiro v. 23 nº 3 p. 510–534 Set-Dez 2022 Educação financeira e sustentabilidade: um framework conceitual Performance Evaluation in the University Context: an investigation of literature from the Constructivist perspective Renally Fernandes Couto \| Kettrin Farias Bem Maracajá \| Petruska de Araújo Machado Educação financeira e sustentabilidade: um framework conceitual Performance Evaluation in the University Context: an investigation of literature from the Constructivist perspective Renally Fernandes Couto \| Kettrin Farias Bem Maracajá \| Petruska de Araújo Machado Educação financeira e sustentabilidade: um framework conceitual Performance Evaluation in the University Context: an investigation of literature from the Constructivist perspective Renally Fernandes Couto \| Kettrin Farias Bem Maracajá \| Petruska de Araújo Machado Educação financeira e sustentabilidade: um framework conceitual Performance Evaluation in the University Context: an investigation of literature from the Constructivist perspective Renally Fernandes Couto \| Kettrin Farias Bem Maracajá \| Petruska de Araújo Machado 527 Considerações finais 510–534 Set-Dez 2022 Educação financeira e sustentabilidade: um framework conceitual Performance Evaluation in the University Context: an investigation of literature from the Constructivist perspective Renally Fernandes Couto \| Kettrin Farias Bem Maracajá \| Petruska de Araújo Machado Educação financeira e sustentabilidade: um framework conceitual Performance Evaluation in the University Context: an investigation of literature from the Constructivist perspective Renally Fernandes Couto \| Kettrin Farias Bem Maracajá \| Petruska de Araújo Machado 528 sustentável, além do ganho de eficiência, será cada vez mais capaz de perceber, criticar e se tornar um contribuinte ativo para a melhoria contínua da socieidade e do planeta de recursos finitos que o envolve. Todo esforço aqui empreendido avança a discussão da gestão dos recursos financeiros com a sustentabilidade, dois campos tratados de forma muito incipiente pela comunidade científica. A maior pretensão assumida nesta pesquisa, portanto, é a de encorajar pesquisas futuras envolvendo a educação financeira e a susten- tabilidade, simultaneamente; preparando um ambiente fértil para o surgimento de melhores abordagens sobre os problemas por elas envolvidos. A limitação deste trabalho está no seu caráter estritamente teórico e, por isso, incapaz de proporcio- nar recortes e inferências precisos. Portanto, é sugerido que nas pesquisas subse- quentes sejam elencados atributos mensuráveis para as dimensões do framework, tornando possível o teste do modelo em diferentes tipos de amostras e contextos. Considerações finais Esta pesquisa decorre da lacuna teórica identificada entre os campos de educa- ção financeira e sustentabilidade, ambos de extrema relevância, tanto no ambiente científico, quanto social. O problema da pesquisa buscou compreender quais as di- mensões envolvidas na relação entre educação financeira e sustentabilidade; deste modo, foi proposto um framework conceitual, propondo tais dimensões e suas rela- ções através do conceito de tríplice contingência de Skinner (1969). O modelo compreende que a afinidade que um indivíduo possui com a edu- cação financeira e a sustentabilidade influenciam no contexto que antecede o comportamento, através dos conhecimentos, da internalização de conceitos e na formação da sua consciência. Este panorama reforça, ou não, comportamentos e práticas de orçamento, de tomada de crédito e endividamento, de poupança e in- vestimento, de consumo e de produção e descarte de resíduos. Todo esse processo culmina nas perspectivas de qualidade de vida que o indivíduo espera para o seu futuro, bem como, na construção do seu legado. E, uma vez que o indivíduo assume consciência de seus anseios e expectativas, a per- cepção de consequências sobre os seus atos terá potencial para reforçar, diminuir, modificar ou extinguir determinados comportamentos, que beneficiem ou prejudi- quem o seu presente, seu futuro e o seu espólio, sejam eles financeiros, sociais ou ambientais. Diante disso, o framework, não pretende ser uma previsão comportamen- tal, onde indivíduos sob idênticos fatores antecedentes reproduzem os mesmos comportamentos e obtêem as mesmas consequências e percepções; ou, inver- samente, comportamentos e consequências semelhantes derivam das mesmas condições sine qua non. É sim, um esforço inicial para a compreensão da interdis- ciplinaridade dos dois campos e um estímulo para a reflexão e o desenvolvimento de estudos futuros. Dado que a economia é uma construção social que impacta o meio ambien- te, é pertinente trabalhar a educação financeira e a sustentabilidade com o TBL, pois eles coexistem. A literacia financeira, por si só, tem o papel de fazer com que o indivíduo desempenhe melhor a gestão dos seus recursos financeiros, obtendo protagonismo econômico. Já, quando instruído a partir de uma educação financeira Administração: Ensino e Pesquisa Rio de Janeiro v. 23 nº 3 p. 510–534 Set-Dez 2022 DOI 10.13058/raep.2022.v23n3.2239 ISSN 2358-0917 Administração: Ensino e Pesquisa Rio de Janeiro v. 23 nº 3 p. 510–534 Set-Dez 2022 DOI 10.13058/raep.2022.v23n3.2239 ISSN 2358-0917 Administração: Ensino e Pesquisa Rio de Janeiro v. 23 nº 3 p. Referências Journal of Busi- ness & Economic Statistics, 21(3), 339–353. doi:10.1198/073500103288619007 Campos, C. R., Teixeira, James. & Coutinho, C. Q. S. (2015). Reflexões sobre a educação financeira e suas interfaces com a educação matemática e a educação crítica. III Fórum de Discussão: Parâmetros Balizadores da Pesquisa em Educação Matemática no Brasil. Educ. Matem. Pesq., São Paulo, v.17, n.3, pp.556-577. Charles-Leija, H., Aboites, G. & Llamas, I. (2018). Una revisión de aportaciones que contribuyeron al estu- dio de la utilidad y la felicidad en la economía. Revista de Analisis Economico. XXXIII. 57-76. doi:0.24275/ uam/azc/dcsh/ae/2018v33n84/Charles. stensen, N. L., Bartuska, A. M., Brown, J. H., Carpenter, S., D’Antonio, C., Francis, R., Franklin, J Christensen, N. L., Bartuska, A. M., Brown, J. H., Carpenter, S., D’Antonio, C., Francis, R., Franklin, J. F MacMahon J A Noss R F Parsons D J Peterson C H Turner M G & Woodmansee R G MacMahon, J. A., Noss, R. F., Parsons, D. J., Peterson, C. H., Turner, M. G., & Woodmansee, R. G F., MacMahon, J. A., Noss, R. F., Parsons, D. J., Peterson, C. H., Turner, M. G., & Woodmansee, R. G. (1996). The Report of the Ecological Society of America Committee on the Scientific Basis for Ecosystem Management Ecological Applications 6(3) 665–691 doi:10 2307/2269460 (1996). The Report of the Ecological Society of America Committee on the Scientific Basis for Ecosystem Management. Ecological Applications, 6(3), 665–691. doi:10.2307/2269460 Management. Ecological Applications, 6(3), 665–691. doi:10.2307/2269460 Cole,, S., Sampson, T., & Zia, B. (2011). Prices or Knowledge? What Drives Demand for Financial Services in Emerging Markets? The Journal of Finance, 66(6), 1933–1967. doi:10.1111/j.1540-6261.2011.01696.x Couto, R. F., Maracajá, K. F. B. & Machado, P. A. (2022) Bibliometric analysis of studies in financial ed- ucation and sustainability. Research, Society and Development, [S. l.], v. 11, n. 10, p. e395111033014, 2022. doi:10.33448/rsd-v11i10.33014. Crossan, M., Lane, H. & White, R. (1999). An organizational learning framework: from intuition to institu- tion. Academy of Management Review, v. 24, n. 3, p. 522-537. Davies, I., Evans, M., & Reid, A. (2005). Globalizing citizenship education? A critique of “global education” and “citizenship education.” British Journal of Educational Studies, 53(1), 66–89. doi:10.1111/j.1467- 8527.2005.00284.x Deaton, A. (2019). The Analysis of Household Surveys : A Microeconometric Approach to Development Policy. Washington, DC: World Bank. ISBN (paper): 978-1-4648-1331-3 ISBN (electronic): 978-1-4648- 1352-8 doi:10.1596/ 978-1-4648-1331-3 1352-8 doi:10.1596/ 978-1-4648-1331-3 Dunlap, R. E., Van Liere, K. D., Mertig, A. G., & Jones, R. E. (2000). Referências Anderson, L., Ostrom, A. L., Corus, C., Fisk, R. P., Gallan, A. S., Giraldo, M., … Williams, J. D. (2013). Transformative service research: An agenda for the future. Journal of Business Research, 66(8), 1203– 1210. doi:10.1016/j.jbusres.2012.08. Anderson, L., Ostrom, A. L., Corus, C., Fisk, R. P., Gallan, A. S., Giraldo, M., … Williams, J. D. (2013). Transformative service research: An agenda for the future. Journal of Business Research, 66(8), 1203– 1210. doi:10.1016/j.jbusres.2012.08. Aquino, R. D. & Tomassini, R. (2009). O Consumo para os Estudantes de Administração: Uma Aplicação da Teoria do Núcleo Central de Representações Sociais. XXXIII EnANPAD Atkinson, A. & Messy, F. (2011). Assessing financial literacy in 12 countries: An OECD/INFE inter- national pilot exercise. Journal of Pension Economics and Finance, 10(4), 657-665. doi:10.1017/ S1474747211000539 Atkinson, A., & Messy, F. (2012). Measuring Financial Literacy: Results of the OECD / International Net- work on Financial Education (INFE) Pilot Study. doi:10.1787/20797117. Becker, C. D. (1995). Human Ecology and Resource Sustainability: The Importance of Institutional Diversity. Annual Review of Ecology and Systematics. 26. 113-133. doi:10.1146/annurev.ecolsys.26. 1.113. Belk, R. W., Ger, G., & Askegaard, S. (2003). The Fire of Desire: A Multisited Inquiry into Consumer Pas- sion. Journal of Consumer Research, 30(3), 326–351. doi:10.1086/378613 Bettencourt, L., Lobo, J., Helbing, D., Kühnert, C. & West, G. (2007). Growth, Innovation, Scaling, and the Pace of Life in Cities. Proceedings of the National Academy of Sciences of the United States of America. doi:104. 7301-6. 10.1073/pnas.0610172104. Birkenmaier, J., Curley, J. & Kelly, P. (2012). Credit Building in IDA Programs Early Findings of a Longitu- dinal Study. Research on Social Work Practice. 22. 605-614. doi:10.1177/1049731512453208. Administração: Ensino e Pesquisa Rio de Janeiro v. 23 nº 3 p. 510–534 Set-Dez 2022 DOI 10.13058/raep.2022.v23n3.2239 ISSN 2358-0917 Educação financeira e sustentabilidade: um framework conceitual Performance Evaluation in the University Context: an investigation of literature from the Constructivist perspective Renally Fernandes Couto \| Kettrin Farias Bem Maracajá \| Petruska de Araújo Machado 529 Brennan, C. & Coppack, M. (2008). Consumer Empowerment: Global Context, UK Strategies and Vul- nerable Consumers. International Journal of Consumer Studies. 32. 306 - 313. doi:10.1111/j.1470- 6431.2007.00640.x. Brown, M., & Graf, R. (2013). Financial Literacy and Retirement Planning in Switzerland. Numeracy. 6. doi:10.5038/1936-4660.6.2.6. Bucher-Koenen, T. & Lusardi, A. (2011). Financial literacy and retirement planning in Germany. Journal of Pension Economics and Finance, 10(4), 565-584. doi:10.1017/S1474747211000485 Cagetti, M. (2003). Wealth Accumulation Over the Life Cycle and Precautionary Savings. Educação financeira e sustentabilidade: um framework conceitual Performance Evaluation in the University Context: an investigation of literature from the Constructivist perspective Renally Fernandes Couto \| Kettrin Farias Bem Maracajá \| Petruska de Araújo Machado Referências New Trends in Measuring Environmen- tal Attitudes: Measuring Endorsement of the New Ecological Paradigm: A Revised NEP Scale. Journal of Social Issues, 56(3), 425–442. doi:10.1111/0022-4537.00176 Dunlap, R & Van Liere, K. (2008). The “New Environmental Paradigm”. The Journal of Environmental Ed- ucation. 40. 19-28. doi:10.3200/JOEE.40.1.19-28. Jones, R. & Dunlap, R. (2010). The Social Bases of Environmental Concern: Have They Changed Over Time?. Rural Sociology. 57. 28 - 47. doi:10.1111/j.1549-0831.1992.tb00455.x. Administração: Ensino e Pesquisa Rio de Janeiro v. 23 nº 3 p. 510–534 Set-Dez 2022 DOI 10.13058/raep.2022.v23n3.2239 ISSN 2358-0917 Administração: Ensino e Pesquisa Rio de Janeiro v. 23 nº 3 p. 510–534 Set-Dez 2022 DOI 10.13058/raep.2022.v23n3.2239 ISSN 2358-0917 Educação financeira e sustentabilidade: um framework conceitual Performance Evaluation in the University Context: an investigation of literature from the Constructivist perspective Renally Fernandes Couto \| Kettrin Farias Bem Maracajá \| Petruska de Araújo Machado 530 Elkington, J. (1998). Partnerships from cannibals with forks: The triple bottom line of 21st-century busi- ness. Environmental Quality Management, 8(1), 37–51. doi:10.1002/tqem.3310080106 Fernandes, D., Lynch, J. G. Jr. & Netemeyer, R. G. (2014). Financial Literacy, Financial Education and Downstream Financial Behaviors. Forthcoming in Management Science. Available at SSRN: https://ssrn. com/abstract=2333898 Filiberto, D., Wethington, E., Pillemer, K., Wells, N., Wysocki, M., & Parise, J. (2008). Older People and Climate Change: Vulnerability and Health Effects. Generations. 33. 19-25. Flores, S. A. M., Vieira, K. M., & Coronel, D. A. (2013). Influência de fatores comportamentais na propen- são ao endividamento. Faces: Revista de Administração, 12(1), 13-35. Fox, J., Bartholomae, S. & Lee, J. (2005). Building the Case for Financial Education. Journal of Consumer Affairs. 39. 195 - 214. doi:10.1111/j.1745-6606.2005.00009.x. Fan, L. and Zhang, L. (2021), The Influence of Financial Education Sources on Emergency Savings: The Role of Financial Literacy. Fam Consum Sci Res J, 49: 344-361. https://doi.org/10.1111/fcsr.12400 Gathergood, J. (2012). Self-control, financial literacy and consumer over-indebtedness. Journal of Eco- nomic Psychology, 33(3), 590–602. doi:10.1016/j.joep.2011.11.006 Gil, A. C. (2007). Métodos e técnicas de pesquisa social. 5. ed. São Paulo: Atlas, 1999. Como elaborar projetos de pesquisa. 4. ed. São Paulo: Atlas. Gomes, F., Brito, P., Tives, H., Fagundes, F. & Canedo, E.D. (2019). Aplicação da Tríplice Contingência da Análise Comportamental na Gamificação do Módulo de Ensino e Aprendizagem da Lógica Propo- sicional. Anais do XXX Simpósio Brasileiro de Informática na Educação (SBIE 2019), doi:10.5753/cbie. sbie.2019.942. Grimm, N. B., Faeth, S. H., Golubiewski, N. E., Redman, C. Educação financeira e sustentabilidade: um framework conceitual Performance Evaluation in the University Context: an investigation of literature from the Constructivist perspective Renally Fernandes Couto \| Kettrin Farias Bem Maracajá \| Petruska de Araújo Machado Referências Journal of Consumer Affairs, 44: 296-316. doi:10.1111/ j.1745-6606.2010.01170.x Ianole, R.; Hubona, G.; Druica, E. & Basu, C. (2020). Understanding sources of financial well-being in Ro- mania: A prerequisite for transformative financial services. Journal of Services Marketing. ahead-of-print. doi:10.1108/JSM-02-2019-0100. Jickling, B., & Wals, A. E. J. (2008). Globalization and environmental education: looking beyond sustain- able development. Journal of Curriculum Studies, 40(1), 1–21. doi:10.1080/00220270701684667 Kaiser, T., & Menkhoff, L. (2017). Does Financial Education Impact Financial Literacy and Financial Be- havior, and If So, When?. The World Bank Economic Review, 31(3), 611–630. doi:10.1093/wber/lhx018 Khandelwal, N. & Darbha, G. K. (2021). A decade of exploring MXenes as aquatic cleaners: Covering a broad range of contaminants, current challenges, and future trends, Chemosphere, Volume 279, ISSN 0045-6535, doi:10.1016/j.chemosphere.2021.130587. Kindle, P. (2010). Student Perceptions of Financial Literacy: Relevance to Practice. Journal of Social Service Research. 36. 470-481. doi:10.1080/01488376.2010.510951. Kindle, P. (2010). Student Perceptions of Financial Literacy: Relevance to Practice. Journal of Social Service Research. 36. 470-481. doi:10.1080/01488376.2010.510951. Kindle, P. (2013). The Financial Literacy of Social Work Students. Journal of social work education. 49. 397-407. doi:10.1080/10437797.2013.796853. Kindle, P. (2013). The Financial Literacy of Social Work Students. Journal of social work education. 49. 397-407. doi:10.1080/10437797.2013.796853. Kollmuss, A., & Agyeman, J. (2002). Mind the Gap: Why do people act environmentally and what are the barriers to pro-environmental behavior? Environmental Education Research, 8(3), 239–260. doi:10.1080/13504620220145401 Kollmuss, A., & Agyeman, J. (2002). Mind the Gap: Why do people act environmentally and what are the barriers to pro-environmental behavior? Environmental Education Research, 8(3), 239–260. doi:10.1080/13504620220145401 owski, Ł. (2021). Household’s Overindebtedness during the COVID-19 Crisis: The Role of Debt and Kurowski, Ł. (2021). Household’s Overindebtedness during the COVID-19 Crisis: The Role of Debt and Financial Literacy. Risks, 9(4), 62. doi:10.3390/risks9040062 Kurowski, Ł. (2021). Household’s Overindebtedness during the COVID-19 Crisis: The Role of Debt and Financial Literacy. Risks, 9(4), 62. doi:10.3390/risks9040062 Financial Literacy. Risks, 9(4), 62. doi:10.3390/risks9040062 Levine, R. (2005). Finance and Growth: Theory and Evidence. Handbook of Economic Growth, in: Philippe Aghion & Steven Durlauf (ed.), Handbook of Economic Growth, edition 1, volume 1, chapter 12, pages 865-934, Elsevier. doi:10.1016/S1574-0684(05)01012-9 Levine, R. (2005). Finance and Growth: Theory and Evidence. Handbook of Economic Growth, in: Philippe Aghion & Steven Durlauf (ed.), Handbook of Economic Growth, edition 1, volume 1, chapter 12, pages 865-934, Elsevier. doi:10.1016/S1574-0684(05)01012-9 López-Medina, T., Mendoza-Ávila, I., Contreras-Barraza, N., Salazar, G. & Vega, A. (2021). Referências L., Wu, J., Bai, X., & Briggs, J. M. (2008). Global Change and the Ecology of Cities. Science, 319(5864), 756–760. doi:10.1126/science.1150195 Grinstein-Weiss, M., Charles, P. & Curley, J. (2007). Asset Building in Rural Communities: The Expe- rience of Individual Development Accounts*. Rural Sociology. 72. 25 - 46. doi:10.1526/00360110778 1147383. Grinstein-Weiss, M.; Guo, S.; Reinertson, V. & Russell, B. (2015). Financial Education and Savings Out- comes for Low-Income IDA Participants: Does Age Make a Difference?. Journal of Consumer Affairs. 49. doi:10.1111/joca.12061. comes for Low-Income IDA Participants: Does Age Make a Difference?. Journal of Consumer Affairs. 49. doi:10.1111/joca.12061. Halilovic, S., Zaimovic, A., Berilo, A. A., & Zaimovic, T. (2019). Financial Literacy Assessment in Bosnia and Herzegovina. Procedia Computer Science, 158, 836-843. doi:10.1016/j.procs.2019.09.121 Hastings, J. S., Madrian, B. C., & Skimmyhorn, W. L. (2013). Financial Literacy, Financial Education, and Economic Outcomes. Annual Review of Economics, 5(1), 347–373. doi:10.1146/annurev-econom- ics-082312-125807 He, H., Reynolds, C. J., Hadjikakou, M., Holyoak, N., & Boland, J. (2020). Quantification of indirect waste generation and treatment arising from Australian household consumption: A waste input-output analysis. Journal of Cleaner Production, 258, 120935. doi:10.1016/j.jclepro.2020.120935 Herrador-Alcaide, T.; Montserrat, H. & Topa, G. (2021). A model for personal financial planning to- wards retirement. Journal of Business Economics and Management. 22. 482-502. doi:10.3846/ jbem.2020.13978. Administração: Ensino e Pesquisa Rio de Janeiro v. 23 nº 3 p. 510–534 Set-Dez 2022 DOI 10.13058/raep.2022.v23n3.2239 ISSN 2358-0917 Administração: Ensino e Pesquisa Rio de Janeiro v. 23 nº 3 p. 510–534 Set-Dez 2022 DOI 10.13058/raep.2022.v23n3.2239 ISSN 2358-0917 Administração: Ensino e Pesquisa Rio de Janeiro v. 23 nº 3 p. 510–534 Set-Dez 2022 DOI 10.13058/raep.2022.v23n3.2239 ISSN 2358-0917 531 Hibbert, A. M., Lawrence, E. R., & Prakash, A. J. (2012). The Role of Financial Education in the Management of Retirement Savings. Journal of Behavioral Finance, 13(4), 299–307. doi:10.1080/15427560.2012.7357 Hines, J. M., Hungerford, H. R., & Tomera, A. N. (1987). Analysis and Synthesis of Research on Re- sponsible Environmental Behavior: A Meta-Analysis. The Journal of Environmental Education, 18(2), 1–8. doi:10.1080/00958964.1987.9943 Hira, T. K. (2012). Promoting sustainable financial behavior: implications for education and research. In- ternational Journal of Consumer Studies. ISSN 1470-6423 doi:10.1111/j.1470-6431.2012.01115.x Hofmann, R. M. & Moro, M. L. F. (2012). Educação matemática e educação financeira: perspectivas para a ENEF. Zetetiké. 20. 37-54. doi:10.20396/zet.v20i38.8646609. Hung, A., Parker, A. & Yoong, J. (2009). Defining and Measuring Financial Literacy. RAND Corporation Publications Department, Working Papers. 708. doi:10.2139/ssrn.1498674. Huston, S. J. (2010), Measuring Financial Literacy. Educação financeira e sustentabilidade: um framework conceitual Performance Evaluation in the University Context: an investigation of literature from the Constructivist perspective Renally Fernandes Couto \| Kettrin Farias Bem Maracajá \| Petruska de Araújo Machado Referências Bibliometric Mapping of Research Trends on Financial Behavior for Sustainability. Sustainability. 14. 117. doi:10.3390/ su14010117. López-Medina, T., Mendoza-Ávila, I., Contreras-Barraza, N., Salazar, G. & Vega, A. (2021). Bibliometric Mapping of Research Trends on Financial Behavior for Sustainability. Sustainability. 14. 117. doi:10.3390/ su14010117. López-Rodríguez, C. E., & López-Ordoñez, D. A. (2022). Financial education in Colombia: Challenges from the perception of its population with socioeconomic vulnerability. Economics and Sociology, 15(1), 193-204. doi:10.14254/2071-789X.2022/15-1/12 López-Rodríguez, C. E., & López-Ordoñez, D. A. (2022). Financial education in Colombia: Challenges from the perception of its population with socioeconomic vulnerability. Economics and Sociology, 15(1), 193-204. doi:10.14254/2071-789X.2022/15-1/12 Administração: Ensino e Pesquisa Rio de Janeiro v. 23 nº 3 p. 510–534 Set-Dez 2022 DOI 10.13058/raep.2022.v23n3.2239 ISSN 2358-0917 Administração: Ensino e Pesquisa Rio de Janeiro v. 23 nº 3 p. 510–534 Set-Dez 2022 DOI 10.13058/raep.2022.v23n3.2239 ISSN 2358-0917 Educação financeira e sustentabilidade: um framework conceitual Performance Evaluation in the University Context: an investigation of literature from the Constructivist perspective Renally Fernandes Couto \| Kettrin Farias Bem Maracajá \| Petruska de Araújo Machado 532 Lucey, T. A., White, E. S., & André, A. (2020). Teacher´s Interpretation of the Cultural Relevancy of Mon- eySKILLS. Education and Urban Society, 53(2), 185â€“205. doi:10.1177/0013124520920586 Lusardi, A. (2000). Saving for Retirement: The Importance of Planning. Journal TIAA-CREF. Institute Re- search Dialogue Lusardi, A. & Mitchell, O. S. (2007a). Baby Boomer retirement security: The roles of planning, finan- cial literacy, and housing wealth. Journal of Monetary Economics, 54(1), 205–224. doi:10.1016/j.jmone- co.2006.12. Lusardi, A. & Mitchelli, O. S. (2007b). Financial Literacy and Retirement Preparedness: Evidence and Im- plications for Financial Education. Business Economics, 42(1), 35–44. doi:10.2145/20070104 Lusardi, A. & Mitchell, O. S. (2011a). Financial Literacy and Retirement Planning in the United States. J l f P i E i d Fi 10 509 525 d i 10 2139/ 1810550 Lusardi, A. & Mitchell, O. S. (2011a). Financial Literacy and Retirement Planning in the United States. Journal of Pension Economics and Finance. 10. 509-525. doi:10.2139/ssrn.1810550. Lusardi, A. & Mitchell, O. S. (2011a). Financial Literacy and Retirement Planning in the United States. Lusardi, A. & Mithcell, O. (2011b). Financial literacy around the world: An overview. Journal of Pension Economics and Finance, 10(4), 497-508. doi:10.1017/S1474747211000448 Lusardi, A. & Mithcell, O. (2011b). Financial literacy around the world: An overview. Journal of Pension Economics and Finance, 10(4), 497-508. doi:10.1017/S14747472110004 Lusardi, A. & Mitchell, O. S. (2014). The Economic Importance of Financial Literacy: Theory and Evidence. Educação financeira e sustentabilidade: um framework conceitual Performance Evaluation in the University Context: an investigation of literature from the Constructivist perspective Renally Fernandes Couto \| Kettrin Farias Bem Maracajá \| Petruska de Araújo Machado Referências Journal of Economic Literature, 52 (1): 5-44. doi:10.1257/jel.52.1.5 Lusardi, A. & Mitchell, O. S. (2014). The Economic Importance of Financial Literacy: Theory and Evidence. Journal of Economic Literature, 52 (1): 5-44. doi:10.1257/jel.52.1.5 Lusardi, A., Mitchell, O. S., & Curto, V. (2010). Financial Literacy among the Young. Journal of Consumer Affairs, 44(2), 358–380. doi:10.1111/j.1745-6606.2010.01173.x Lusardi, A. & Tufano, P. (2009). Debt Literacy, Financial Experiences, and Overindebtedness. National Bureau of Economic Research. doi:10.3386/w14808. Lyons, A. C.; Rachlis, M. & Scherpf, E. (2007). What’s in a score? Differences in consumers’ credit knowl- edge using OLS and quantile regressions. Journal of Consumer Affairs, vol. 41, no. 2, ISSN 0022-0078 L A & K H J (2019) Fi i l I l i Fi i l Lit d E i ll V l bl Lyons, A. C.; Rachlis, M. & Scherpf, E. (2007). What’s in a score? Differences in consumers’ credit knowl- edge using OLS and quantile regressions. Journal of Consumer Affairs, vol. 41, no. 2, ISSN 0022-0078 Lyons, A & Kass-Hanna, J. (2019). Financial Inclusion, Financial Literacy and Economically Vulnerable Lyons, A. C.; Rachlis, M. & Scherpf, E. (2007). What’s in a score? Differences in consumers’ credit knowl- edge using OLS and quantile regressions. Journal of Consumer Affairs, vol. 41, no. 2, ISSN 0022-0078 Lyons, A & Kass-Hanna, J. (2019). Financial Inclusion, Financial Literacy and Economically Vulnerable Populations in the Middle East and North Africa. Emerging Markets Finance and Trade. 57. 2699-2738. g g q g , , , Lyons, A & Kass-Hanna, J. (2019). Financial Inclusion, Financial Literacy and Economically Vulnerable Populations in the Middle East and North Africa. Emerging Markets Finance and Trade. 57. 2699-2738. 10.1080/1540496X.2019.1598370. Lyons, A & Kass-Hanna, J. (2019). Financial Inclusion, Financial Literacy and Economically Vulnerable Populations in the Middle East and North Africa. Emerging Markets Finance and Trade. 57. 2699-2738. 10.1080/1540496X.2019.1598370. y ( ) y y Populations in the Middle East and North Africa. Emerging Markets Finance and Trade. 57. 2699-2738. 10.1080/1540496X.2019.1598370. Mancebo, D., Oliveira, D. M., Teixeira, J. G. & Silva, L. V. (2002). Consumo e subjetividade: trajetórias teóricas. Estud. psicol. (Natal). Vol. 7(2):325-332. doi:10.1590/S1413-294X2002000200013 Mancebo, D., Oliveira, D. M., Teixeira, J. G. & Silva, L. V. (2002). Consumo e subjetividade: trajetórias teóricas. Estud. psicol. (Natal). Vol. 7(2):325-332. doi:10.1590/S1413-294X2002000200013 Marques, M. L. M. & Frade, C. (2003). Regular o sobreendividamento (Relatório de Pesquisa). Coimbr Portugal, Centro de Estudos Sociais, Faculdade de Economia, Universidade de Coimbra. Referências 510–534 Set-Dez 2022 DOI 10.13058/raep.2022.v23n3.2239 ISSN 2358-0917 DOI 10.13058/raep.2022.v23n3.2239 ISSN 2358-0917 DOI 10.13058/raep.2022.v23n3.2239 ISSN 2358-0917 Educação financeira e sustentabilidade: um framework conceitual Performance Evaluation in the University Context: an investigation of literature from the Constructivist perspective Renally Fernandes Couto \| Kettrin Farias Bem Maracajá \| Petruska de Araújo Machado 533 O.; Gelcich, S.; Jouffray, J., Leach, M., Österblom, H. (2020). Principles for knowledge co-production in sustainability research. Nature Sustainability. 3. doi:10.1038/s41893-019-0448-2. Nugent, J. B. & Yhee, S. (2002). Small and Medium Enterprises in Korea: Achievements, Constraints and Policy Issues. Small Business Economics. 18. 85-119. doi:10.1023/A:1015181911497. Oliveira Filho, J. E. (2004). Gestão ambiental e sustentabilidade: um novo paradigma eco-econômico para as organizações modernas, Domus: Pol., soc., Cidade. Salvador, v. 1, n. 1, p. 92-113. jan.,/jun. Pillemer, K., Wells, N. M., Wagenet, L. P., Meador, R. H., & Parise, J. T. (2010). Environmental Sus- tainability in an Aging Society: A Research Agenda. Journal of Aging and Health, 23(3), 433–453. doi:10.1177/0898264310381278 Potrich, A. C. G., Vieira, K. M., & Kirch, G. (2015). Determinantes da Alfabetização Financeira: Análise da Influência de Variáveis Socioeconômicas e Demográficas. Revista Contabilidade & Finanças, 26(69), 362–377. doi:10.1590/1808-057x201501040 Quivy, R. & Campenhoudt, L. V. (1995). Manuel de recherche en sciences sociales. Paris: Dunod. Rebello, A., Harres, S. & Rocha Filho, J. (2015). Educação financeira: uma proposta pedagógica para alunos do ensino médio politécnico. HOLOS. 6. 308. doi:10.15628/holos.2015.3645. Rebello, A., Harres, S. & Rocha Filho, J. (2015). Educação financeira: uma proposta pedagógica p alunos do ensino médio politécnico. HOLOS. 6. 308. doi:10.15628/holos.2015.3645. Rosales-Pérez, A. M., Fernández-Gámez, M. A., Torroba-Díaz, M. & Molina-Gómez, J. (2021). A Study of the Emotional Intelligence and Personality Traits of University Finance Students. Education Sciences, 11(1), 25. doi:10.3390/educsci11010025 Rosenbloom, S. (2001). Sustainability and automobility among the elderly: An international assessment. Transportation. 28. 375-408. doi:10.1023/A:1011802707259. Rosseto, J. C., Schneider, T., Quartieri, M. T., Oliveira, E. C. (2020). Educação financeira crítica: uma prática pedagógica para a educação de jovens e adultos. Revista Eletrônica de Educação Matemática - REVEMAT, Florianópolis, v. 15, p. 01-24, jan./dez. Universidade Federal de Santa Catarina. ISSN 1981- 1322. doi:10.5007/1981-1322.2020.e74215 Sampieri, R. H., Collado, C. F. & Lucio, P. B. (2006). Metodologia de pesquisa. 3ª ed. São Paulo: Mc- Graw-Hill. Santos, J. O. & Santos, J. A. R. (2005). Mercado de capitais: racionalidade versus emoção. Revis- ta Contabilidade & Finanças., v. 16, n. 37. Epub 09 Jun 2011. ISSN 1808-057X. doi:10.1590/S1519- 70772005000100008. Savoia, J. R. Educação financeira e sustentabilidade: um framework conceitual Performance Evaluation in the University Context: an investigation of literature from the Constructivist perspective Renally Fernandes Couto \| Kettrin Farias Bem Maracajá \| Petruska de Araújo Machado Referências g Portugal, Centro de Estudos Sociais, Faculdade de Economia, Universidade de Coimbra. Moreno-García, E., Santillán, A. G. & Gutiérrez-Delgado, L. (2017). Nivel de educación financiera en es- cenarios de educación superior. Un estudio empírico con estudiantes del área económico-administrativa. Moreno-García, E., Santillán, A. G. & Gutiérrez-Delgado, L. (2017). Nivel de educación financiera en es- cenarios de educación superior. Un estudio empírico con estudiantes del área económico-administrativa. ista Iberoamericana de Educación Superior. 8. doi:10.22201/iisue.20072872e.2017.22.234. Revista Iberoamericana de Educación Superior. 8. doi:10.22201/iisue.20072872e.2017.22.234. Morgan, M. S. & Morrison, M. (1999). Model as Mediators: perspectives on natural and social science. Cambridge University Press, New York. p Morgan, M. S. & Morrison, M. (1999). Model as Mediators: perspectives on natural and social science. Cambridge University Press, New York. Morgan, P & Trinh, L. (2020). Financial Literacy, Financial Inclusion, and Savings Behavior in Laos. Journal of Asian Economics. 68. doi:101197. 10.1016/j.asieco.2020.101197 Morgan, P & Trinh, L. (2020). Financial Literacy, Financial Inclusion, and Savings Behavior in Laos. Journal of Asian Economics. 68. doi:101197. 10.1016/j.asieco.2020.101197 Murray, A., Skene, K. & Haynes, K. (2017). The Circular Economy: An Interdisciplinary Exploration of the Concept and Application in a Global Context. Journal of Business Ethics. doi:10.1007/s10551-015- 2693-2. Murray, A., Skene, K. & Haynes, K. (2017). The Circular Economy: An Interdisciplinary Exploration of the Concept and Application in a Global Context. Journal of Business Ethics. doi:10.1007/s10551-015- 2693-2. Norström, A., Cvitanovic, C., Löf, M., West, S., Wyborn, C., Balvanera, P., Bednarek, A., Bennett, E., Biggs, R., De Bremond, A., Campbell, B. M.; Canadell, J.; Carpenter, S.; Folke, C.; Fulton, E.; Gaffney, Norström, A., Cvitanovic, C., Löf, M., West, S., Wyborn, C., Balvanera, P., Bednarek, A., Bennett, E., Biggs, R., De Bremond, A., Campbell, B. M.; Canadell, J.; Carpenter, S.; Folke, C.; Fulton, E.; Gaffney, Norström, A., Cvitanovic, C., Löf, M., West, S., Wyborn, C., Balvanera, P., Bednarek, A., Bennett, E., Biggs, R., De Bremond, A., Campbell, B. M.; Canadell, J.; Carpenter, S.; Folke, C.; Fulton, E.; Gaffney, Administração: Ensino e Pesquisa Rio de Janeiro v. 23 nº 3 p. 510–534 Set-Dez 2022 DOI 10.13058/raep.2022.v23n3.2239 ISSN 2358-0917 Administração: Ensino e Pesquisa Rio de Janeiro v. 23 nº 3 p. Referências F.; Saito, A. T.; Santana, F. A. (2007). Paradigmas da educação financeira no Brasil. RAP Rio de Janeiro 41(6):1121-41, Nov./Dez, doi: 10.1590/S0034-76122007000600006 Sharachchandra, M., L. (1991). Sustainable development: A critical review. World Development, Volume 19, Issue 6, Pages 607-621, ISSN 0305-750X. doi:10.1016/0305-750X(91)90197-P. Shehabuddeen, N., Probert, D., Phaal, R., & Platts, K.W. (1999). Representing and Approaching Complex Management Issues: Part 1 - Role and Definition. Silva, H., Barbieri, A. F. & Monte-Mór, R. L. (2012). Demografia do consumo urbano: um estudo sobre a geração de resíduos sólidos domiciliares no município de Belo Horizonte. Revista Brasileira de Estudos de População, [online], v. 29, n. 2 pp. 421-449. Epub 15 Jan 2013. ISSN 1980-5519. doi:10.1590/S0102- 30982012000200012. Silva, H., Barbieri, A. F. & Monte-Mór, R. L. (2012). Demografia do consumo urbano: um estudo sobre a geração de resíduos sólidos domiciliares no município de Belo Horizonte. Revista Brasileira de Estudos de População, [online], v. 29, n. 2 pp. 421-449. Epub 15 Jan 2013. ISSN 1980-5519. doi:10.1590/S0102- 30982012000200012. Silva, J. G., Silva Neto, O. S. & Araújo, R. C. C. (2017). Educação Financeira de Servidores Públicos: Há- bitos de Consumo, Investimento e Percepção de Risco. Revista Evidenciação Contábil &Amp; Finanças, 5(2), 104–120. Administração: Ensino e Pesquisa Rio de Janeiro v. 23 nº 3 p. 510–534 Set-Dez 2022 DOI 10.13058/raep.2022.v23n3.2239 ISSN 2358-0917 Administração: Ensino e Pesquisa Rio de Janeiro v. 23 nº 3 p. 510–534 Set-Dez 2022 Educação financeira e sustentabilidade: um framework conceitual Performance Evaluation in the University Context: an investigation of literature from the Constructivist perspective Renally Fernandes Couto \| Kettrin Farias Bem Maracajá \| Petruska de Araújo Machado 534 Silveira, D. T. & Córdova, F. P. (2009). A pesquisa científica. In T. E. Gerhardt, & D. T. Silveira (Orgs.), Métodos de pesquisa. (Coord. Universidade Aberta do Brasil – UAB/UFRGS e Curso de Graduação Tec- nológica – Planejamento e Gestão para o Desenvolvimento Rural - SEAD/UFRGS). Porto Alegre: Editora da UFRGS. Skinner, B. F. (1935). The Generic Nature of the Concepts of Stimulus and Response. The Journal of General Psychology, 12(1), 40–65. doi:10.1080/00221309.1935.9920087 Skinner, B. F. (1938). The behavior of organisms. New York: Appleton-Century. ISBN 978-0-9964539-0-5 Skinner, B. F. (1969). Contingencies of reinforcement: A theoretical analysis. New York: Appleton-Centu- Skinner, B. F. (1938). The behavior of organisms. New York: Appleton-Century. ISBN 978-0-9964539-0-5 Skinner, B. F. (1938). The behavior of organisms. New York: Appleton-Century. ISBN 978-0-9964539-0-5 Skinner, B. F. (1969). Contingencies of reinforcement: A theoretical analysis. Educação financeira e sustentabilidade: um framework conceitual Performance Evaluation in the University Context: an investigation of literature from the Constructivist perspective Renally Fernandes Couto \| Kettrin Farias Bem Maracajá \| Petruska de Araújo Machado Administração: Ensino e Pesquisa Rio de Janeiro v. 23 nº 3 p. 510–534 Set-Dez 2022 Referências New York: Appleton-Centu- ry-Crofts. ISBN: 978-0-9899839-3-8 Skinner, B. F. (1969). Contingencies of reinforcement: A theoretical analysis. New York: Appleton-Centu- ry-Crofts. ISBN: 978-0-9899839-3-8 Stock, J. R. & Mulki, J. (2009). Product Returns Processing: An Examination of Practices of Man- ufacturers, Wholesalers/Distributors, and Retailers. Journal of Business Logistics. 30. 33 - 62. doi:10.1002/j.2158-1592.2009.tb00098.x. Tennyson, Sharon & Nguyen, C. (2005). State Curriculum Mandates and Student Knowledge of Personal Finance. Journal of Consumer Affairs - J CONSUM AFF. 35. 241-262. doi:10.1111/j.1745-6606.2001. tb00112.x. Todorov, J. C. & Moreira, M. B. (2009). Psicologia, comportamento, processos e interações. Psicologia: Reflexão e Crítica, 22(3), 404–412. doi:10.1590/s0102-79722009000300011 Torres, I. A & Barros, F. S. (2014). Investimentos financeiros: uma análise dos alunos investidores de uma Instituição de ensino superior de Brasília – DF, Universitas Gestão e TI, Brasília, v. 4, n. 1, p. 39-53, jan./ jun. 2014 doi: 10.5102/un.gti.v4i1.2804 Trinh, L. Q., Morgan, P. J., & Sonobe, T. (2020). Investment behavior of MSMEs during the downturn periods: Empirical evidence from Vietnam. Emerging Markets Review, 100739. doi:10.1016/j.eme- mar.2020.100739 West, S., & Friedline, T. (2016). Coming of Age on a Shoestring Budget: Financial Capability and Financial Behaviors of Lower-Income Millennials. Social Work, 61(4), 305–312. doi:10.1093/sw/sww057 Behaviors of Lower-Income Millennials. Social Work, 61(4), 305–312. doi:10.1093/sw/sww057 Willis, M. M., & Schor, J. B. (2012). Does Changing a Light Bulb Lead to Changing the World? Political Action and the Conscious Consumer. The ANNALS of the American Academy of Political and Social Sci- ence, 644(1), 160–190. doi:10.1177/0002716212454831 Willis, M. M., & Schor, J. B. (2012). Does Changing a Light Bulb Lead to Changing the World? Polit Wright, S., Caserta, M. & Lund, D. (2003). Older Adults’ Attitudes, Concerns, and Support for Envi- ronmental Issues in the “New West”. International journal of aging & human development. 57. 151-79. doi:10.2190/Y73Y-0RK9-RP0J-E7HH. Zozzoli, J. C. J. (2009). Marca e comunicação ambiental. In: Anais XXXI Congresso Brasileiro de Ciências da Comunicação - Intercom: Natal-RN. Zozzoli, J. C. J. (2009). Marca e comunicação ambiental. In: Anais XXXI Congresso Brasileiro de Ciências da Comunicação - Intercom: Natal-RN. Zhu, A. Y. F. & Chou, K.. (2018). Financial literacy of Hong Kong adolescents: Testing the validity of a scale and evaluating two conceptual models. Youth & Society. doi:10.1177/0044118X17753813 Zhu, A. Y. F. & Chou, K.. (2018). Financial literacy of Hong Kong adolescents: Testing the validity of a scale and evaluating two conceptual models. Youth & Society. doi:10.1177/0044118X17753813 Administração: Ensino e Pesquisa Rio de Janeiro v. Referências 23 nº 3 p. 510–534 Set-Dez 2022
https://openalex.org/W3199390165	https://link.springer.com/content/pdf/10.1007/s10539-021-09815-0.pdf	English	null	Unknotting reciprocal causation between organism and environment	Biology & philosophy	2,021	cc-by	15,543	* Jan Baedke jan.baedke@rub.de Biology & Philosophy (2021) 36:48 https://doi.org/10.1007/s10539-021-09815-0 Biology & Philosophy (2021) 36:48 https://doi.org/10.1007/s10539-021-09815-0 1 Department of Philosophy I, Ruhr University Bochum, Universitätsstrasse 150, 44801 Bochum, Germany Jan Baedke, Alejandro Fábregas-Tejeda and Guido I. Prieto have contributed equally to this work. Jan Baedke1 · Alejandro Fábregas‑Tejeda1 · Guido I. Prieto1 Received: 20 January 2021 / Accepted: 9 August 2021 / Published online: 20 September 2021 © The Author(s) 2021 Keywords Reciprocal causation · Niche construction · Organism · Environment · Experiential niche construction Introduction ‘Reciprocity’ has become a buzzword in current biological parlance, especially in debates about the purported reciprocal nature of many developmental and evo- lutionary processes (e.g., Laland et al. 2011, 2013; Moczek 2015; Schwab et al. 2019; Uller and Laland 2019). Some biologists contend that the study of complex feedbacks between developing organisms and their environments, which have protracted evolutionary consequences, brings about important reconfigurations to standard evolutionary theory (see Laland et al. 2011, 2013, 2015). At the same time, a philosophical debate surrounding the novelty, limits and scope of the theoretical tenet of ‘reciprocal causation’ has emerged (e.g., Dickins and Barton 2013; Scholl and Pigliucci 2015; Svensson 2018; Baedke 2019; Fábregas-Tejeda and Vergara-Silva 2018a), primarily evaluating the concept’s epistemological, explanatory, and heuristic roles in biological practice (Buskell 2019). From the scientific side, niche construction theory (NCT; sensu Odling-Smee et al. 2003) has stood at the forefront of this debate (Uller and Helanterä 2019), but allied fields and research areas such as ecological evolutionary developmental biol- ogy (eco-evo-devo) and developmental systems theory have also contributed (see Schwab et al. 2019; Baedke and Gilbert 2020). A common misassumption in this debate is that the reasoning about recipro- cal interactions in evolution is a fairly recent development in the biosciences. However, reciprocity has been theorized in many ways throughout the history of biology. Firstly, a diversity of relata have been conceptualized as engaged in reciprocal relationships: for instance, organism-environment, gene-environment, gene-population, or population-population. Secondly, different kinds of reciprocal relationships have been posited for these relata. For example, for the organism- environment relationship, biologists have postulated ontological co-constitution, mutual structural fitting, concomitant reaction, and reciprocal causation, among others, as different hallmarks of reciprocity (for a different historical taxonomy, see Di Paolo 2020). Ontological co-constitution holds that organisms and their environments are commingled and form a single interacting system that cannot be meaningfully disentangled (e.g., Haldane 1884, 1935; Levins and Lewontin 1985; Griffiths and Gray 1994; Oyama 2000; Walsh 2015:181, 2021; for an overview, see Baedke 2019; Pearce 2020). The view of mutual structural fitting between organism and environment can be found in Lawrence J. Abstract In recent years, biologists and philosophers of science have argued that evolution- ary theory should incorporate more seriously the idea of ‘reciprocal causation.’ This notion refers to feedback loops whereby organisms change their experiences of the environment or alter the physical properties of their surroundings. In these loops, in particular niche constructing activities are central, since they may alter selection pressures acting on organisms, and thus affect their evolutionary trajectories. This paper discusses long-standing problems that emerge when studying such recipro- cal causal processes between organisms and environments. By comparing past approaches to reciprocal causation from the early twentieth century with contempo- rary ones in niche construction theory, we identify two central reoccurring problems: All of these approaches have not been able to provide a conceptual framework that allows (i) maintaining meaningful boundaries between organisms and environments, instead of merging the two, and (ii) integrating experiential and physical kinds of reciprocal causation. By building on case studies of niche construction research, we provide a model that is able to solve these two problems. It allows distinguishing between mutually interacting organisms and environments in complex scenarios, as well as integrating various forms of experiential and physical niche construction. Keywords Reciprocal causation · Niche construction · Organism · Environment · Experiential niche construction 1 Department of Philosophy I, Ruhr University Bochum, Universitätsstrasse 150, 44801 Bochum, Germany 456789) 3 48 Page 2 of 29 J. Baedke et al. Introduction Henderson (1913), who argued that, besides considering organisms as structurally adapted to their surroundings, the specific physico-chemical properties of the environment should also be regarded as adapted, for they support the development of life: “The fitness of the environ- ment is one part of a reciprocal relationship of which the fitness of the organ- ism is the other” (Henderson 1913:271). Concomitant reaction refers to an event that concurrently alters an organism and its environment. It resembles John Stuart Mill’s (1843) law of coexistence. An example is the competitive exclusion prin- ciple in ecology, which states that species that have similar niches cannot stably coexist in the same place when their common resources are limiting (see Raerinne 1 3 Unknotting reciprocal causation between organism and… Page 3 of 29 48 and Baedke 2015). Finally, reciprocal causation is usually defined as a feedback loop between two interacting, yet separate entities or processes: “According to the causal construal, organisms cause changes to the features of the environment external to them. […] These modified physical conditions, in turn, redound upon the organisms” (Walsh 2015:180). Here, from this vast array of views on reciprocity in ecology and evolution, we will focus on organism-environment reciprocal causation, given that this has been the main target of recent heated discussions about NCT and the so-called ‘Extended Evolutionary Synthesis’ (Laland et al. 2015, 2017; see also Fábregas-Tejeda and Vergara-Silva 2018b). In recent years, some evolutionary biologists have high- lighted reciprocal causation as a theoretical and modeling principle due to its alleged empirical aptness, claiming that reciprocal interactions are ubiquitous in evolution- ary processes. Moreover, it could be used to explain stable, biased selection pres- sures acting on organisms, and provide a conceptual framework that could poten- tially extend the explanatory power and limited practices of standard evolutionary theory (Buskell 2019; see also Baedke et al. 2020a). Nevertheless, as Buskell (2019) appositely points out, this concept has remained ambiguous and it is still in need of further philosophical clarification. i In this paper, we provide guidance to unknot organism-environment reciprocal causation in current evolutionary and philosophical debates. We identify reoccur- ring problems in these views and introduce a model for solving them. We begin by providing a brief overview of some theorizations of organism-environment reciproc- ity in early twentieth-century biology. Introduction Our historical narrative contextualizes the study of reciprocity in novel ways by drawing on sources hitherto under-explored by scholars and by explaining the decline of studies on organism-environment reciproc- ity in evolutionary biology in the second half of the twentieth-century. We identify two main theoretical problems in these early conceptual frameworks that contributed to this development: They could not (i) maintain (meaningful) epistemic boundaries between organisms and environments, but instead merged the two, and (ii) failed to integrate physical kinds of reciprocal causation with those in which organisms’ experience of the environment plays a key role. These shortcomings led to methodo- logical intractability and untranslatability to experimental interventions. Next, we show how these problems are reoccurring in present-day debates on reciprocal cau- sation in evolutionary biology, especially in NCT. In this sense, we demonstrate that a better historical understanding of former debates on reciprocal causation can be a guide for exposing unsolved challenges in contemporary scientific practice. Finally, we propose a general conceptual and visual model of reciprocal causation that tack- les these long-standing problems. By applying it to a number of niche construc- tion (NC) cases, we illustrate how this model allows us to (i) distinguish between mutually interacting organisms and environments, and (ii) integrate various forms of experiential and physical NC. By doing so, we try to bridge the gap between con- ceptual frameworks of NC and scientific practice. In particular, we show that the organism-environment boundary is an epistemic necessity to understand complex causality in NC processes, including the causal roles each component plays in them. O d l di t l i l i t i bl i l l ti b Our model disentangles seemingly inextricable reciprocal relations by identifying characteristic causal patterns of different evolutionary relevant 1 1 3 3 Page 4 of 29 J. Baedke et al. 48 organism-environment interactions. This allows correlating different types of NC within one common causal framework. In this context, it is important to stress that we do not propose a new taxonomy of types of NC here. Rather, we move in a dif- ferent direction: towards integrating and making sense of different processes that involve reciprocal causation between organism and environment. Introduction Our model is able to identify and distinguish between different kinds of reciprocal causal interactions, clarify their characteristic causal patterns and integrate them even in complex evo- lutionary scenarios that have been difficult to conceptualize thus far (e.g., those that involve interactions of two or more species, or those in which physical and experi- ential views of the environment must be integrated). This approach secures the com- parability of NC methodologies and opens avenues for formalization to advance the debates in the field. 1 3 Organism‑environment reciprocity in historical context A simplified schema of the ‘function-circle.’ The organism (subject) perceives a ‘carrier of a feature’ of the environment (object) in its ‘per- ception world’ and reciprocally acts on and shapes the same environment (as a carrier of this effect) in its ‘effect world.’ (After Uexküll 1928:158) Fig. 1 Uexküll’s model of recip- rocal organism-environment interaction. A simplified schema of the ‘function-circle.’ The organism (subject) perceives a ‘carrier of a feature’ of the environment (object) in its ‘per- ception world’ and reciprocally acts on and shapes the same environment (as a carrier of this effect) in its ‘effect world.’ (After Uexküll 1928:158) connected with its own environment, which it subjectively perceives and acts upon. Thus, the environment contains a ‘perception world,’ which is experienced through sense receptors and processed neurally, and an ‘effect world,’ in which the organism causally acts on the environment through various traits and behaviors. The organ- ism’s perceptions and actions together create a causal feedback loop or ‘function- circle’ (Funktionskreis) between the perception world and the effect world (see Fig. 1). For example, when organisms explore new environments, they attempt to establish new functional circles (e.g., by changing nutritional habits). If this action is not rewarded (i.e., a functional circle cannot be closed, e.g., when the new diet is not nutritious), the organism gives up this environment (switching back to old nutritional habits or exploring new ones). During this process, the environment causes the organism to rewire neural networks and physiological receptor-reaction connections. Uexküll’s view of reciprocity conceptualizes the organism-environment relation- ship as highly individualized and mediated through experience (see Brentari 2015). It is likely because of this that it had only limited influence on evolutionary biology. One of the few scholars that drew extensively on Uexküll was Adolf Meyer-Abich (1943, 1964). His evolutionary reciprocity theory of holobiosis anticipated central elements of Lynn Margulis’ theory of endosymbiosis and introduced the holobiont concept around 50 years before her (Baedke et al. 2020b). Meyer-Abich argued that macroevolutionary change is driven by reciprocal processes between increasingly integrated organisms, which first constitute symbiotic partners, then holobionts, and finally systems of organs in a larger whole. At the crucial evolutionary stage of holo- biosis, the formerly independent units can no longer develop on their own. Holo- bionts are able to realize novel traits and explore new environments. Organism‑environment reciprocity in historical context In order to clarify the concept of reciprocal causation, especially in NCT, we first need to understand how it was introduced and applied in the history of evolutionary thought. In fact, clarifying this poorly understood conceptual history sheds light on the theoretical challenges that reciprocal causation approaches to evolution still face today. Svensson (2018) argues that the ideas of causal reciprocity, and of organisms as active modifiers of their selection pressures, mainly stem from the dialectical biol- ogy of Levins and Lewontin (1985; see also Lewontin 1983). However, these ideas are considerably older and theoretically richer. Already Haeckel (1866:I, 154; Ger- man original) claimed that “reciprocity of every single individual with its entire sur- rounding leads to the adaptation of its individual characters.” While he did not spell out this notion in detail, other scholars in the late nineteenth century and particularly the early twentieth century did. Especially organism-centered approaches to evolu- tion, such as organicism, (German) holistic biology, and dialectical materialism (see Nicholson and Gawne 2015; Baedke 2019), developed sophisticated views of organ- ism-environment reciprocity. For example, J.S. Haldane (1884:32–33) highlighted: “The organism is thus no more determined by the surroundings than it at the same time determines them. The two stand to one another, not in the relation of cause and effect, but in that of reciprocity.” Holistic thinkers like Haldane expanded Immanuel Kant’s view of reciprocity on the organization of organisms to the relation between organism and environment. As Whitehead (1929: 185) puts it: “The relation of part to whole has the special reciprocity associated with the notion of organism […]; but this relation reigns throughout nature.” He contended that evolutionary research should acknowl- edge that “organisms can create their own environment” (Whitehead 1929: 140). Let us take a brief look at three early advocates of causal reciprocity–Jakob von Uexküll, Adolf Meyer-Abich, and Conrad Hal Waddington–to see how the above general ideas were developed into theoretical frameworks for biology. Uexküll (1928) conceptualized the environment (Umwelt) as constructed through the sen- sorial exploration of each organism. He argued that each organism is reciprocally 1 3 Unknotting reciprocal causation between organism and… Page 5 of 29 48 Page 5 of 29 48 48 Fig. 1 Uexküll’s model of recip- rocal organism-environment interaction. 1 In a similar manner, American pragmatists like John Dewey defended a view of organism-environment inseparability (Pearce 2014, 2020). They often adopted an ontological reading where organism and envi- ronment could not be disentangled, unless abstracted. For example, Dewey claimed in his ‘The Reflex Arc Concept in Psychology’ that stimulus and response are not separate entities (see Pearce 2020:180). In this paper we do not discuss this kind of ontological co-constitution between organism and environ- ment (what Pearce labels the ‘dual aspects view’), but reciprocal causation. Organism‑environment reciprocity in historical context For example, in ‘lichen holobiosis,’ through the increasing interaction between green algae and/ or blue-green algae and fungi, a new metabolic unit with new traits (novel metabo- lites and reproductive bodies) emerges over evolutionary time. This unit has a new energy-status, which allows accessing an unexplored ecological niche. In short, during holobiosis symbiotic organisms create new environments, which they both act on and experience in a novel manner. This view integrates previously distinct 1 3 3 3 48 Page 6 of 29 48 Page 6 of 29 J. Baedke et al. 48 ‘function circles’ through organism-organism reciprocal causation (Meyer-Abich 1942). Organisms do not only construct their own individual environment (sensu Uexküll), but also construct integrated and shared environments for one another. Another evolutionary take on reciprocal causation was developed by Conrad Hal Waddington. He argued that evolution involves changes in explorative behaviors (what he called ‘exploitive systems;’ see Waddington 1957:104–108, Waddington 1959a). These include the influence exerted by the organism on its environment, leading to feedback loops with selection pressures: “Animals […] themselves select the particular habitat in which their life will be passed. Thus the animal by its behav- iour contributes in a most important way to determining the nature and intensity of the selective pressures which will be exerted on it” (Waddington 1959a:1635–1636). Waddington urged biologists to abandon two common views on the organism-envi- ronment relationship: First, “[n]atural selection is far from being as external a force as the conventional picture might lead one at first sight to believe” (1959a:1636). Second, “we have to think in terms of circular and not merely unidirectional causal sequences” (Waddington 1959b:400). As we will see below, it was these very two views, namely constructivism instead of externalism and causal reciprocity instead of unidirectional causality, that came under attack in evolutionary biology. Theoretical problems and decline of organism‑environment reciprocity Uexküll’s ‘sense physi- ological’ characterization of environment was deemed too narrow. Authors argued that the environment experienced by any organism (e.g., through its sensory recep- tors) is not the whole (relevant) environment for it (e.g., Hartmann 1950). Although not experienced, physical environmental factors can have causal consequences over organisms and their evolutionary trajectories. Moreover, the notion of experience was a heavily debated topic (e.g., Bierens de Haan 1947) and most researchers ada- mantly opposed to studying anything reminiscent of subjectivity (e.g., Tinbergen 1963). In sum, theories of organisms-environment reciprocity failed in providing a clear understanding of how experienced and physical environments are related, and how they can be integrated under one conceptual framework. f (ii) Another main problem was that, for many biologists, the concept of ‘environ- ment’ defended by reciprocity theories had shortcomings. Uexküll’s ‘sense physi- ological’ characterization of environment was deemed too narrow. Authors argued that the environment experienced by any organism (e.g., through its sensory recep- tors) is not the whole (relevant) environment for it (e.g., Hartmann 1950). Although not experienced, physical environmental factors can have causal consequences over organisms and their evolutionary trajectories. Moreover, the notion of experience was a heavily debated topic (e.g., Bierens de Haan 1947) and most researchers ada- mantly opposed to studying anything reminiscent of subjectivity (e.g., Tinbergen 1963). In sum, theories of organisms-environment reciprocity failed in providing a clear understanding of how experienced and physical environments are related, and how they can be integrated under one conceptual framework. The decline of organism-environment reciprocity views in the second half of the twentieth century was also part of a more general trend that displaced the organism as the central, causally efficacious unit of biology, and supplanted it with the gene (Nicholson 2014; Baedke 2019). At least two stands can be identified in this trend: The marginalization of views of organism-environment reciprocity and a shift to study reciprocity in other relata; and the (re-)establishment of externalism as one of the main explanatory principles in evolutionary biology, thereby excluding notions of ‘individual experienced environments.’ First, a move away from studying organism-environment reciprocity was wel- comed by classical and population geneticists. Clear boundaries between organisms and environments are a stipulation for fruitful research and “a practically and theo- retically valuable abstraction” in those fields (Haldane 1936:349). Moreover, main- stream evolutionary biology was dominated by an asymmetric, unidirectional view of the organism-environment relationship (e.g., Williams 1992:484). Theoretical problems and decline of organism‑environment reciprocity Traditional approaches of organism-environment reciprocal causation suffered from two main problems, already hinted by some scientists at the time, which contributed to their demise in the second half of the twentieth century: (i) Scholars were unsuc- cessful in retaining meaningful boundaries between organism and environment, and thus frequently proposed views of reciprocity in which organism and environment are merged, making them intractable for empirical studies; and (ii) there was lit- tle integration of experiential and physical views of the environment impinging on organisms. As we will see below, these problems are re-emerging in today’s debate regarding reciprocal causation and NCT. (i) Many early twentieth-century authors held holistic viewpoints apropos the organism-environment relationship in which it is impossible “to distinguish sepa- rately the factors concerned,” and life is regarded as an “integrated unity cov- ering both organism and its environment” (Haldane 1935:12; see also Uexküll 1909:196).1 In that same line, Waddington (1957:189) claimed that “organism and environment are not two separable things.” But this stance of organism-environment inseparability, in which reciprocal interactions constitute each other into a single 1 1 3 Unknotting reciprocal causation between organism and… Page 7 of 29 48 system, was also heavily criticized. Against Haldane, for instance, Joseph Needham (1936:10–11) highlighted: “we easily see that his demand for the unification of the organism and its surroundings is a methodologically impossible aim, for if no line can be drawn between organism and immediate surroundings, no better line can be drawn between immediate surroundings and far-off surroundings.” According to Needham, biologists need individuation criteria in order to carry on their research, otherwise they are unable to tell apart any organic system from another or envi- ronmental features relevant to the organism from those irrelevant to it. This was a common problem of strong views of reciprocity. For example, dialectical biologist Marcel Prenant (1943:61) spoke of an “organism-environment complex” in which causes and effects become indistinguishable. In general, this shared position led sci- entists to blur the boundary between organism and environment, resulting in meth- odological problems for biological practice. In that vein, many biologists saw organ- ism-environment reciprocity as intractable (or harder to assay) in empirical studies, or maintained that explanations appealing to this principle have particular epistemic demands that call for different research strategies (e.g., Labouvie 1974). f (ii) Another main problem was that, for many biologists, the concept of ‘environ- ment’ defended by reciprocity theories had shortcomings. Theoretical problems and decline of organism‑environment reciprocity The organism was downplayed as a causal agent that constructs its environment, and thus its own development and evolution. Around the same time, gene-environment reciprocity was increasingly considered in population genetics (e.g., Haldane 1946; Lerner 1950; Falconer 1952), and studied 1 3 48 Page 8 of 29 J. Baedke et al. 48 Page 8 of 29 48 Page 48 through statistical tools like path analysis (Wright 1960). New models of reciprocal interactions of genes-populations and genes-environments (e.g., Fisher 1930; Kirk- patrick 1982; for overviews, see Reznick 2013 and Svensson 2018) focused, among other things, on positive and negative frequency-dependent selection (e.g., Fisher 1930; Wright 1969; Charlesworth 1971), population regulation by genetic feedbacks (e.g., Pimentel 1968), and eco-evolutionary dynamics (e.g., Thompson 1998). Their advantages notwithstanding, the vast majority of these evolutionary models did not encompass organism-environment reciprocal causation, despite what some scientists claim (Brodie III 2005; Svensson 2018). Reciprocity was usually studied for other relata, but not for whole organisms and their environments. Second, an overt ‘externalist logic’ in evolutionary explanations reigned supreme (see Godfrey-Smith 1996; Walsh 2015), and the main concepts of environment employed in the field attest to that shift (see Brandon 1990; see also Antonovics et al. 1988): Views of ‘individual environments,’ like that of Uexküll, were replaced by populational vantage points. Brandon (2012) contends that, although no two organisms will ever interact with the same environment, they need to be seen as members of a homogeneous, shared selective environment (i.e. factors external to a population that affect its members’ relative reproductive success). Explaining evolu- tionary changes in genotype frequencies requires that two different genotypes share an external selective environment to which one is better adapted than the other (see also Walsh 2021). This externalist logic impoverished the diverse roles previously granted to the environment in development and evolution. Evolutionists commonly overestimated the causal influence of the selective environment on organisms, and increasingly downgraded the influence of the non-selective environment: “Environment vari- ance is a source of error that reduces precision in genetical studies, and the aim […] is therefore to reduce it as much as possible” (Falconer 1960:140). Theoretical problems and decline of organism‑environment reciprocity Waddington (1957:189) bitterly complained about this drawback: “Any further influence which the environment might have was degraded to the status of mere ‘noise’ in the system of genetic determination.” In the next section, we will turn to new views defending organism-environment reciprocal causation that are currently emerging in evolutionary biology. With a focus on NCT and the novel taxonomies of types of NC that have been recently pro- posed, we will show that the same problems (i) and (ii) that plagued early organism- environment reciprocity perspectives are still lingering in the field. 1 3 Niche construction theory and reoccurring problems The idea of organism-environment reciprocity has recently made a comeback in evolutionary biology (Laland et al. 2011, 2013, 2015; Mesoudi et al. 2013; for dis- cussion, see Fábregas-Tejeda and Vergara-Silva 2018a, b; Svensson 2018; Buskell 2019; Baedke 2019). This development has been stirred by a growing interest to again center evolutionary reasoning on the organism (Nicholson 2014; Laland et al. 2013, 2015, 2017; Moczek 2015; Sultan 2015; Walsh 2015), including organisms’ phenotypic plasticity and NC behaviors, which, as feedback circles, modify the 1 3 Unknotting reciprocal causation between organism and… Page 9 of 29 48 natural selection pressures impinging upon them (Lewontin 1983; Sterelny 2001; Odling-Smee et al. 2003; 2011; Chiu and Gilbert 2015, 2020). NCT argues that organisms are not only effects of adaptation, but also causal starting points of evolu- tionary trajectories. This view of reciprocal causation has recently led to a number of different taxonomies distinguishing diverse kinds of feedback processes in NC.2 f According to Aaby and Ramsey (2019), during NC, organisms alter the feature- factor relationship between themselves and their environments in three different ways: by affecting environmental factors, by changing their features (traits), or by modifying the relation between organismic features and environmental factors. They call these external, constitutive and relational NC, respectively (for definitions, see Table 1, 1–3). In contrast, Chiu (2019) distinguishes physical NC from experiential NC (see Table 1:4–5), depending on whether organisms change the intrinsic physical properties of the environment or the way the environment is experienced, without, however, physically changing it. In this taxonomy, relocational NC and mediational NC are subtypes of experiential NC. These taxonomies of types of NC–distinguishing physical/external, constitu- tive, relational, and experiential (including relocational and mediational) NC–have enriched our understanding of reciprocal organism-environment interactions in development and evolution. At the same time, however, despite these conceptual improvements the current debate about different types of NC still faces the same problems that theories of reciprocity could not solve in the early twentieth century: participants in this debate usually (i) do not spell out on what grounds meaningful boundaries between organisms and environments can be maintained and exploited for research purposes, and/or (ii) provide no guidance for how to integrate experien- tial and physical forms of reciprocal causation. (i) By building on a rather externalist view of the niche (Brandon 1990), early NCT constructed organisms and environment as separate entities (Odling-Smee et al. 1996, 2003). 2 We focus on the works of Aaby and Ramsey (2019) and Chiu (2019). Both can be seen as conceptual updates to the original taxonomy of NC types by Odling-Smee et al. (2003:44–45). We should stress, however, that outside of NCT and its taxonomies, there exists a broader trend to study organism-environ- ment reciprocity in evolutionary biology and ecology (from ecosystem engineering and habitat choice, to developmental plasticity and active sensory mediation of environmental cues). Niche construction theory and reoccurring problems Discounting relocation, every instance of NC was construed as an alteration of the physical properties of the environment. The role of organis- mal experience as an active causal factor in reciprocal interactions was not a cen- tral element of early NCT. Afterwards, influenced by developmental systems the- ory (Oyama 2000; Griffiths and Gray 2001) and its prominent assumption that “[t] here is no distinction between organism and environment” (Griffiths and Gray 2001: 207), more constructivist views of the environment emerged in NCT. Similar to J.S. Haldane’s or Uexküll’s earlier ideas, present-day strands of NCT often highlight the centrality of the organism in evolution, but, at the same time, merge organisms with environments and argue, more generally, that they are “engaged in reciprocally caused relationships […], entwined in, to coin a very apt phrase from developmental 1 3 48 Page 10 of 29 48 Page 10 of 29 J. Baedke et al. 48 3 Table 1 Taxonomies of niche construction Types of niche construction Characterization Examples Additional references Aaby and Ramsey (2019) 1. External niche construction Organisms modify factors in their external environments Dam-building in beavers or soil-process- ing in earthworms External NC is equivalent to ‘perturbational NC’ (sensu Odling-Smee et al. 2003) 2. Constitutive niche construction A change in an organism’s trait/capacity affects how the organism reacts to its environment or acts upon it, without modifications in the external environ- ment A holometabolous insect develops, and through successive stages in its life cycle, opens different environment experiences Constitutive NC is also related to phenotypic plasticity and ‘constructive development’ (Laland et al. 2015; see also Walsh 2015) 3. Relational niche construction A change in the relations between organ- isms and environments, without trans- forming organisms’ constitution nor the physical conditions of their environments Mice pile up to keep warm by affecting the rates of heat loss due to physical proximity, but not by changing the actual physical temperature of the nest Relational NC also includes cases of reloca- tional NC (sensu Odling-Smee et al. 2003) Chiu (2019) 4. Physical niche construction Organisms modify the physical factors in their environments Building of nests, burrows, and mounds by animals; the alteration of nutrient cycling by plants Physical NC is equivalent to ‘external/per- turbational NC’ 5. Experiential niche construction a. Relocational b. Mediational Changes in the experienced environment without modifications to intrinsic factors of the physical environment. Niche construction theory and reoccurring problems This can happen either by relocation (organisms actively changing place) or by determin- ing how the intrinsic properties of the environment are experienced (through mediational mechanisms) Fluctuating temperatures in an environ- ment can be experienced as constant if the organism has compensating mecha- nisms of temperature regulation. A specific case of this is the phenomenon of leaf ‘wilting’ in plants Experiential NC has been discussed, e.g., by Levins and Lewontin (1985:98–106); Lewontin (2000:55–68); Sultan (2015); Walsh (2015) 1 Unknotting reciprocal causation between organism and… 48 Page 11 of 29 systems theory, ‘cycles of contingency’” (Laland and O’Brien 2011:193; see also Laland and Brown 2018:127).fi Sonia Sultan holds that we have to accept the “difficulty of identifying a mean- ingful boundary between the organism and the environment [because] individual phenotypes inevitably affect both the external environment and the organism’s expe- rience of that environment” (Sultan 2015:44–45). For example, she highlights that, when day length changes, pregnant meadow voles (Microtus pennsylvanicus) alter a key hormonal signal in their offspring. Due to a change in parental experience autumn-born pups develop thicker coats compared to summer-born pups, and this affects how they experience and interact with their environment. By acknowledging such reciprocal connections between the organism’s experience and external envi- ronment, Sultan expresses discomfort with the organism-environment distinction. Interestingly, she develops her critique along the same arguments about the intercon- nectedness of organisms’ ‘perception world’ and ‘effect world’ that already made Uexküll (1909:196) contend that the environment forms an “inextricable whole” with the organism. Walsh (2021) links another inseparability view to reciprocal causation. He argues that “[t]wo causes, x and y, are reciprocal just if the effect that x has on y at time t is dependent upon the effect that y has on x at time t” (original emphasis). He asserts that when an environment’s effect on an organism is a concomitant consequence of the organism’s effect on that environment, one cannot explain the dynamics of the overall system by apportioning it to the effects of each component, thereby making the system inseparable. This view is, strictly speaking, not one of reciprocal causa- tion, but one of synchronic ontological co-constitution, similar to that of Haldane (1884, 1935). However, Walsh understands his inseparability thesis to also be of relevance for temporally expanded processes of reciprocal NC (e.g., Laland et al. 3 Below we do not aim at solving cases of synchronic inseparability, i.e. ontological co-constitution. Rather, within a Humean view of causation (in which effects follow causes over time), we address cases of NC in which diachronic organism-environment relations are considered to be inextricable. We take these problems to be the cornerstone of current explanatory efforts in evolutionary biology that link con- structive behaviors and environmental properties, rather than the metaphysical thesis of co-constitution. Niche construction theory and reoccurring problems 2016).3 These recent arguments for organism-environment inseparability open the way for anti-individualistic positions in NCT (see Baedke 2019). These views are meth- odologically problematic given that NCT needs to be able to clearly individuate the organism and its causal roles. This is because this approach tries to conceptualize and study the organism as a causal agent, different from its environment, which actively molds its own niche and evolutionary trajectory. Moreover, as noted in the previous section (see Needham 1936:10–11), the holistic stance of organism- environment inseparability is troublesome because it does not solve, ipso facto, the problem of ‘individuation’ of biological units. Claiming that organism and environ- ment form an inextricable unit still carries the burden of proof of how to individuate such a system (i.e., how to separate it from other systems). Individuation is thus una- voidable for distinguishing between proximal and distal environments of a particular 1 3 3 Page 12 of 29 48 J. Baedke et al. 48 system (e.g., what elements of the physical world are included in this system and in what spatio-temporal scales) as well as for clarifying the units of physiological and ecological interaction, for recognizing which biological systems can legitimately claimed to be ‘causal agents,’ for distinguishing between one or more conspecifics in a community or population, and for individuating the components in multi-species collectives such as holobionts. In that sense, the holistic view of organism-envi- ronment inseparability does not address the problem of individuation of biological units, but simply pushes it aside. (ii) Besides the old inseparability problem, a second challenge reoccurs in today’s reciprocity views on NC. It concerns the lack of integration between approaches of physical and experiential NC. While already Lewontin (Levins and Lewontin 1985:98–106; Lewontin 2000:55–68) (re)introduced the idea of experiential NC, this concept has not had a major impact on NCT to this day (see Chiu 2019). For example, critics have contended that ‘mediational NC’ is nothing but a phenotypic response to selective environmental pressures and that changes in environmen- tal experiences should not be mixed up with changes in the environment (Bran- don 1990; Godfrey-Smith 1996, 2001). Doing so might result in defending anti- realistic and insoluble holistic positions in which organisms subjectively construct environments. 4 We understand integration of experiential and physical NC to depend, first, on identifying and distin- guishing the two (rather than substituting one by the other), and, second, on providing clear guidance and criteria on how both types of NC work together and are linked in complex evolutionary scenarios. 5 Although we are not trying to define a particular boundary between organisms and environments, our model offers a way of demarcating biological individuals from their surroundings by clarifying the causal structure of each interacting component. For a similar strategy, see Sterner (2017). Niche construction theory and reoccurring problems While, in recent years, we have seen a number of approaches that try to strengthen the idea of experiential mediation of the environment in evolution (Walsh 2015, 2021; Sultan 2015; Chiu 2019; Chiu and Gilbert 2020), at least some of them suf- fer from the older Uexküllian stance of reinterpreting the whole environment of the organism as nothing but the experienced environment. For those that want to avoid this step, arguments have to be provided for how exactly the evolutionary relevant physical environment of organisms differs from their experienced one, why certain downstream physical effects on the environment can be distinguished and excluded from the processes of experiential NC, and how exactly experience affects evolution (e.g., population dynamics) differently from physical effects on selection pressures. These challenges, together with older anti-holistic objections, still lead to a lack of integration between approaches of physical/external and experiential NC, and of the application of experiential NC in evolutionary theory more generally.4 Here, we do not argue for a conceptualization of the environment as “a wholly external, autonomous, causally unified entity” (Walsh 2021:3). In fact, we side with Walsh in pointing out that this construal of the environment is an abstraction that, according to the historical narrative of the previous section, solidified in evolution- ary biology after the decline of organism-environment reciprocity theories in the second half of the twentieth century. Nevertheless, contra Walsh (2021), we con- tend that the proper apportioning and disentanglement of causal contributions in protracted interactions between organisms and environments is something important and feasible in the study of complex evolutionary scenarios. 1 3 Unknotting reciprocal causation between organism and… Page 13 of 29 48 We will now introduce a model that aims at solving these two long-standing problems of organism-environment reciprocal causation. It allows distinguishing between organisms and environments in all of the types of NC described in current taxonomies. To do so, this model does not develop a particular theory of biological boundaries.5 However, it implies rejecting the general idea that boundaries between organisms and environments collapse due to reciprocal interactions over develop- mental and evolutionary time. In fact, it makes possible maintaining epistemically set boundaries between the two even in complex reciprocal evolutionary dynamics. In addition, it not only countenances distinguishing different types of experiential and physical NC, but provides a shared conceptual and visual framework that makes it possible to relate and integrate the two. Unknotting reciprocal causation in physical niche construction Previous conceptualizations of the relationship between organisms and environ- ments have been strongly driven by visual approaches, especially by diagrams rep- resenting their cyclical connection (see, e.g., Fig. 1; see also Di Paolo 2020). We tie in with this tradition, as visual representations can indeed play powerful epistemic and heuristic roles. They allow organizing and guiding scientific and philosophical reasoning by supporting the articulation of novel concepts, pointing to relations and anomalies not expressed verbally in theories, and encouraging the clarification of implicit assumptions. In addition, they can guide scientific practice by coordinating methodological strategies and facilitating formalization (Waddington 1977; Griese- mer 1991; Baedke and Schöttler 2017). Despite these general advantages, past and present visual models of the relation- ship between organisms and their environments typically show some limitations. For example, in line with the long-standing problems discussed above, they usually depict organism and environment as partaking in a seemingly inextricable recipro- cal loop (see Fig. 2a; see also Levis and Pfennig 2017; Laland et al. 2017; Di Paolo 2020). Therefore, a first step towards unknotting organism-environment reciprocal interactions would require distinguishing between the two components. This means that some causal processes occurring in the organism are relatively autonomous from the environment, and vice versa (see Toepfer 2012). Thus, in addition to causal pathways connecting organism and environment, we have to incorporate others that start and end within the limits of the organism and within the limits of the environ- ment (see Fig. 2b). The resulting model of reciprocal causation, nevertheless, is still conceptually intractable as such depictions of loops cannot convey an understanding of how causal processes in and between organism and environment occur over time. A second unknotting step, then, consists of unrolling the organism-environment cycle to make explicit the sequential character in which reciprocal causation occurs 1 3 3 48 Page 14 of 29 48 Page 14 of 29 J. Baedke et al. 48 (see Fig. 2c). In that sense, if we epistemically grant that organism and environment are distinct, we can articulate the complex causal relations between them as well as how they are modified through each component and unfold over life histories.i (see Fig. 2c). Unknotting reciprocal causation in physical niche construction In that sense, if we epistemically grant that organism and environment are distinct, we can articulate the complex causal relations between them as well as how they are modified through each component and unfold over life histories.i i We propose a refined version of this ‘open-loop’ model to represent organism- environment reciprocity (see Fig. 2d). Its components are two ordered series of states of the organism and the environment (O and E, respectively), and arrows rep- resenting causal processes between their states, as well as between successive states of the organism and successive states of the environment.6 For instance, the interac- tion between organism and environment at state n (symbolized as On and En) caus- ally contributes to the next state of the organism, the environment, or both (On+1 and En+1). Accordingly, the diagram includes an arrow from En to On+1, one from On to En+1, or both. If we represent all the possible causal relations, we obtain a basic unknotted causal model of organism-environment reciprocity (see Fig. 2d).7 The causal relations between, say, an organism state and a successive environment state can be conceptualized as being invariant under a range of counterfactual inter- ventions on the organism state. In short, an arrow connecting two states of organism and environment indicates that these states are causally related in an interventionist sense (Woodward 2003). This kind of diagram is rather unilluminating unless we weigh the arrows accord- ing to their relevance for a particular causal explanation. This process leads to the identification of certain invariant causal paths that connect particular changes in states of the organism and the environment. In this view, some arrows (but not oth- ers) represent meaningful explanantia addressing regularly occurring causal cou- plings of organisms and environments. The precise interpretation of the states and relations depends on the causal narrative a scientist is putting forward, given a par- ticular research case. It should be kept in mind that highlighting certain pathways (i.e. including them in an explanans) does not imply that non-highlighted causal arrows do not exist, but instead that we can (to some degree) abstract from their causal effects. Thus, the totality of causal relations is always at play. f Let us now illustrate this visual and conceptual model of reciprocal causation by applying it to a well-known case of external/physical NC (sensu Chiu 2019; Aaby and Ramsey 2019). 6 Please note that although time is implicit in these causal arrows, a series of states should not be inter- preted as a series of regular time intervals, since the time scale at which organismic and environmental processes occur might vary within one series. 7 Krakauer et al. (2020) recently proposed a similar diagram, but with the different aim of identifying biological individuals as quantitative patterns of information flow. For a different diagram of organism- environment relationships, see Ay and Löhr (2015). In addition, our causal diagrams should not be understood as representing inter-level relations as those traced in neo-mechanistic approaches of explana- tion. In more complex scenarios of NC (see Figs. 3–5 below) it is neither clear what is a higher or lower level nor whether experience qualifies as a level of organization at all. Unknotting reciprocal causation in physical niche construction Reef-building corals dramatically alter the physical and chemi- cal conditions of their environments mainly by secreting calcium carbonate skel- etons. These accumulate and form complex structures that constitute habitats for hundreds of other species (Jones et al. 1994). These environmental changes have reciprocal effects on corals. For instance, some macroalgae and sponges that settle 1 1 3 Unknotting reciprocal causation between organism and… Page 15 of 29 48 48 Fig. 2 Unknotting organism-environment reciprocal causation. a Organism (O) and environment (E) par- take in a loop of reciprocal interaction. b Additional loops represent intrinsic causal processes in the organism and environment. c Organism-environment interactions as in (b) but depicted in a sequential manner. d Model of organism-environment reciprocal causation with a succession of states. Subscripts indicate different states of the organism and the environment, and arrows represent causal contribu- tions. e Application of the model (d) to the case of physical niche construction in reef-building corals. The highlighted arrows and states of corals (O) and their environment (E) constitute a causal path that matches a sequence of steps (1–3) in a causal narrative. For details, see text Fig. 2 Unknotting organism-environment reciprocal causation. a Organism (O) and environment (E) par- take in a loop of reciprocal interaction. b Additional loops represent intrinsic causal processes in the organism and environment. c Organism-environment interactions as in (b) but depicted in a sequential manner. d Model of organism-environment reciprocal causation with a succession of states. Subscripts indicate different states of the organism and the environment, and arrows represent causal contribu- tions. e Application of the model (d) to the case of physical niche construction in reef-building corals. The highlighted arrows and states of corals (O) and their environment (E) constitute a causal path that matches a sequence of steps (1–3) in a causal narrative. For details, see text in coral reefs compete with corals for space, light, or food resources (see Sultan 2015 and references therein). This NC case is represented in Fig. 2e. The causal nar- rative underlying the explanation of physical NC in these corals is highlighted in the figure (sequence 1–3). Corals (On−1) in a given environment (En−1) secrete calcium carbonate. Experienced environments and niche construction In recent years, there have been increasing efforts to show that experienced envi- ronments and experiential NC do not, in fact, necessarily open the door to spuri- ous, subjectivist, anti-realist and insoluble holistic views on the organism-environ- ment relation (Walsh 2015, 2021; Sultan 2015; Chiu 2019). In addition, outside the NCT literature there exists a large body of fruitful ecological projects that study, for example, how landscape features drive avoidance behaviors through experiences of fear and disgust (Sheriff and Thaler 2014; Gaynor et al. 2019). While these devel- opments suggest that problems of integrating physical and experiential reciprocal processes between organism and environment might not be as widespread in ecol- ogy as they are in evolutionary biology, unfortunately, these new conceptual frame- works have not yet exerted influence on evolutionary methodologies. For instance, it remains largely unclear how shifts in organisms’ experiences can change selection pressures acting on them or establish individualized niches (but see Sultan 2015; Chiu and Gilbert 2020; Müller et al. 2020). We believe this is not least due to the fact that advocates of experiential NC often endorse a view of organism-environ- ment inseparability (Levins and Lewontin 1985; Sultan 2015; Walsh 2015, 2021), which can make methodological implementation difficult. fi We try to bridge this gap between conceptual frameworks of experiential NC, on the one hand, and scientific practice, on the other, by integrating experienced envi- ronments into the above model–however, without merging the whole environment with the organism. In our model, the ‘experienced environment,’ variable Ex (see Fig. 3a), represents a mediating interface between organism and physical environ- ment. It constitutes the sum of environmental cues (temperature, pressure, location, etc.) that can causally affect this interface and thus the organism.8 Ex is meant to convey four basic ideas. First, what is a cue depends on the organism’s sensory sys- tem and the way the organism modulates its behavior to choose certain environmen- tal factors (see Sultan 2015). Second, experienced cues are transduced into chemical and cellular processes (which regulate, e.g., gene expression patterns or microbiome composition), and finally lead to metabolic, morphological or behavioral changes. Third, a difference in Ex between two organisms living in the same environment E means that E is experienced differently by each organism (e.g., as favorable or unfavorable, as stressful or non-stressful). Unknotting reciprocal causation in physical niche construction This (1) causes changes in the species composition of the environment, which in the next state (En) includes some species that newly compete with corals (On), thus having (2) causal downstream effects on corals’ survival, development, or reproduction, i.e., the next state of the corals (On+1). Corals, in turn, may respond to these environmental changes by (3) modifying the environment in a new round of NC. This simplified narrative could be enriched by adding intermediate steps within the causal path. Depending on the case under study, the sequence could also start with an environmental change that alters the organism, with that organism affecting again the environment at a later time point. in coral reefs compete with corals for space, light, or food resources (see Sultan 2015 and references therein). This NC case is represented in Fig. 2e. The causal nar- rative underlying the explanation of physical NC in these corals is highlighted in the figure (sequence 1–3). Corals (On−1) in a given environment (En−1) secrete calcium carbonate. This (1) causes changes in the species composition of the environment, which in the next state (En) includes some species that newly compete with corals (On), thus having (2) causal downstream effects on corals’ survival, development, or reproduction, i.e., the next state of the corals (On+1). Corals, in turn, may respond to these environmental changes by (3) modifying the environment in a new round of NC. This simplified narrative could be enriched by adding intermediate steps within the causal path. Depending on the case under study, the sequence could also start with an environmental change that alters the organism, with that organism affecting again the environment at a later time point. In the next section, we will explore how our model can be expanded to incorpo- rate the causal processes of experiential NC. This extension will be crucial to tackle the second problem of reciprocity theories, namely how to integrate physical and experiential NC. 1 3 48 Page 16 of 29 J. Baedke et al. 8 In some organisms, environmental cues are compared with internal organismic cues (e.g., body growth, change in hormone levels, hunger, etc.) to adjust physiological or behavioral responses. Experienced environments and niche construction Individual experiences are then directly linked to the ecological performance of these organisms in E, and hence affect their distribution and potentially their evolutionary trajectories. Finally, and most impor- tantly, a change in Ex means a change in the relation of the organism to its physical 1 3 Unknotting reciprocal causation between organism and… 48 Page 17 of 29 environment, without alterations of the intrinsic properties of the external environ- ment (Chiu 2019).9f Let us now discuss how this expanded model represents different types of expe- riential NC, i.e. constitutive, mediational, relational and relocational NC.10 Organ- isms that conduct constitutive NC actively construct the experiences of their envi- ronments through their plastic behavior and development. Aaby and Ramsey (2019) exemplify constitutive NC with a lion (On−1) that changes its size, strength, and coordination as it develops (see Fig. 3b, sequence 1–3). Through this maturation and changes in bodily constitution (1), the experienced space of possible prey (Exn) is transformed (2). The ingestion of larger prey then affects the constitution of the lion (3), which has ecological downstream effects. Aaby and Ramsey (2019:11) assert that “the development of the lion is thus partly responsible for the construction of its niche.” In short, experienced environments are in part created by intrinsic develop- mental processes.f Different from that, mediational NC describes changes in the impact and sig- nificance of the environment for the organism that are due to novel ways of how the environment is experienced, but without physical modifications of it (see Chiu 2019). Mediational NC can happen within or across generations, but all instances show the same causal pattern (see Fig. 3c, sequence 1–3).11 An example of media- tional NC is nutrient foraging in Arabidopsis thaliana (see Giehl and von Wirén 2014; Sultan 2015). Plants are able to respond to nutrient shortage or localized nutri- ent availability by altering their root system architecture to efficiently explore certain zones of the soil that contain limited resources. As Sultan (2015:80) notes: “plants are able to experience an environment that is consistently high in nutrients despite this resource patchiness because of the developmental and physiological plasticity of root systems.” Low levels of nutrients in the soil (En−1) produce a shift in the way the plant (On−1) experiences the environment (Exn−1), i.e. as unfavorable (1). This causes developmental and physiological changes in the plant, i.e. 9 In many cases of experiential NC, however, there are also causal downstream effects on the physical environment. 10 For reasons of simplicity, in the examples discussed in this section we do not consider (and abstract from) direct physical effects on the organism that are not mediated through organisms’ experience (but see Fig. 5b below). 11 An example of transgenerational mediational NC is the case of pregnant meadow voles discussed above. Here a change in day length during pregnancy leads to pups with thicker coats that are born in autumn, which again affects how they experience their environment. Experienced environments and niche construction primary or lateral root elongation (2), which then affect how the plant experiences its environment, i.e. as nutrient-rich (3). This experience can then stop further physiological changes in the plant (4). 11 An example of transgenerational mediational NC is the case of pregnant meadow voles discussed above. Here a change in day length during pregnancy leads to pups with thicker coats that are born in autumn, which again affects how they experience their environment. 1 3 J. Baedke et al. 48 Page 18 of 29 48 Page 18 of 29 Fig. 3 Experienced environments and niche construction (I). a Basic model of reciprocal causation between organism (O) and environment (E) including experienced environment (Ex). b Application of the model to an example of constitutive NC, in which the development of a lion (O) changes its experi- ence (Ex) of potential prey in its environment (E). c Example of mediational NC, where the experience (Ex) of low levels of nutrients in the soil (E) causes physiological and developmental changes in a plant (O), which subsequently change the plant’s experience. For the numerated sequence of steps along these paths, see text Fig. 3 Experienced environments and niche construction (I). a Basic model of reciprocal causation between organism (O) and environment (E) including experienced environment (Ex). b Application of the model to an example of constitutive NC, in which the development of a lion (O) changes its experi- ence (Ex) of potential prey in its environment (E). c Example of mediational NC, where the experience (Ex) of low levels of nutrients in the soil (E) causes physiological and developmental changes in a plant (O), which subsequently change the plant’s experience. For the numerated sequence of steps along these paths, see text Yet another causal pattern comes to light in the case of relational NC (Aaby and Ramsey 2019), where organism-organism interaction mediates changes in the expe- rienced environment, without organisms modifying their constitution or their envi- ronment. These biotic interactions can be represented as two interlinked sequences of reciprocal organism-environment interactions (see Fig. 4a, sequence 1–3). One example is mice that pile up to keep warm (Aaby and Ramsey 2019). If their nest gets cold, mice (On−1 and O’n−1) physically interact with one another, which affects the rates of heat loss just by existing in physical proximity (1). 12 Note that in this example the environments Ex and Ex’ are the same. 13 Walsh (2015, 2021) has defended the view that every instance of physical NC is one of experiential NC (but not vice versa). For his theory that relies on the notion of affordances, all changes in the physical environment are experienced in some way or another by organisms. Affordances emerge from organism- environment systems as a whole, providing organisms different ‘graspable tools’ to react to (for discus- sions on the evolutionary roles of affordances in NCT, see Heras-Escribano 2020). 14 Additionally, in the history of life some physical properties of the environment emerged before becoming experienced cues of organisms. For example, in the global NC process in which cyanobacteria made oxygen accumulate in the ocean/atmosphere (as a byproduct of photosynthesis), oxygen was not immediately an experienced cue for all life forms present at that time. Experienced environments and niche construction Baedke et al. This model has several advantages. First, as the above examples show, it allows representing highly different forms of experiential NC in a single conceptual and visual framework. It makes it possible to identify these forms through their charac- teristic causal patterns, which emerge from particular reciprocal organism-environ- ment interactions. Second, this model does not need to merge organism’s physical and experienced environments, but can keep them conceptually distinct. It rec- ognizes that there are cases of physical NC that are not experiential NC, and vice versa.13 In other words, it avoids the persistent stance that reduces all ecologically and evolutionary relevant physical factors to individual experienced environments. As an alternative, we hold that there are instances of relevant physical effects on the organism that are not directly experienced by it. For example, some wavelengths of radiation cannot be experienced by most organisms but can cause evolutionary- relevant genetic mutations in them. In the case of many mammals, ionizing radiation affects DNA directly, without receptor or sensory mediation, which can have con- sequences in their evolutionary trajectories (e.g., Adewoye et al. 2015; Kesäniemi et al. 2020).14l Third, and related to the previous point, this model avoids an inflationary con- strual of organismic experience, in which every single influence on the organism causing some internal reaction is mediated by inbuilt sensory filters. Instead, it pre- supposes that Ex shows a sufficient degree of sensory specificity (which evolved biologically or was acquired through biological or cultural transmission) for detect- ing particular changes in the environment in such a way that the organism can react functionally to them in a (minimally) directed manner. Such reactions range from purposeful behavioral responses to environmentally-induced developmental biases. Finally, this model allows clearly distinguishing not only physical and experienced environments, but the causal roles different types of experiential and physical NC play as well. Even more importantly, as we show below, it can integrate experi- ential and physical NC in a common framework applicable to complex empirical scenarios. Experienced environments and niche construction This causes mice to experience their environment (Exn and Ex’n) as warmer than it actually is (2), as this behavior does not actually change the physical temperature of the nest.12 The 1 3 Unknotting reciprocal causation between organism and… 48 Page 19 of 29 individual experience of mice then again modulates their behavior to continue or stop piling up (3). Finally, our model can represent relocational NC as a break in the continuity of the sequence of environmental states. This occurs during an experience-mediated transition of the organism from one physical environment to a new one. One exam- ple is the migration of anadromous fish from the ocean to rivers to spawn (Fig. 4b, sequence 1–5). Migrating behavior (On) can be triggered by fish’s experience (Exn−1) of certain environmental conditions in the ocean (En−1), like population density, food availability or climatic variability (1). This exposes the fish to a series of transitional environments (Eτ) during migration (2), the experience of which (Exτ) continuously modulates its behavior (3). Finally, after continuous migration (4), the experience (Exn+1) of a favorable environment, a certain river (E’n+1), triggers a change in fish’s behavior, which stops migrating and chooses that environment to spawn (5). Fig. 4 Experienced environments and niche construction (II). a Example of relational NC. In a cold nest, mice (O and O’) pile up to change the experience of the temperature of their environment (Ex and Ex’), but not the actual temperature in the nest (E and E’). b Example of relocational NC. Environmental cues (E) cause migration of anadromous fish from the ocean to rivers, during which fish experience (Exτ) dif- ferent transitional environments (Eτ), out of which the organism chooses one (E’) in which it spawns. See details in the text Fig. 4 Experienced environments and niche construction (II). a Example of relational NC. In a cold nest, mice (O and O’) pile up to change the experience of the temperature of their environment (Ex and Ex’), but not the actual temperature in the nest (E and E’). b Example of relocational NC. Environmental cues (E) cause migration of anadromous fish from the ocean to rivers, during which fish experience (Exτ) dif- ferent transitional environments (Eτ), out of which the organism chooses one (E’) in which it spawns. See details in the text 1 3 1 3 48 Page 20 of 29 48 Page 20 of 29 J. Integrating different types of niche construction Our visual and conceptual model can identify the precise causal patterns which are characteristic of each type of NC identified by current taxonomies. This allows drawing clear-cut distinctions between different kinds of causal narratives about NC 1 1 3 Unknotting reciprocal causation between organism and… Page 21 of 29 48 processes. However, each of these narratives on their own have limitations when applied on more complex phenomena, in which two or more organisms (from dif- ferent species) interact in reciprocal ways that include more than one type of NC. In such cases, the present taxonomies do not provide clear guidelines on how to inte- grate different NC processes, especially if they include both physical and experien- tial forms of NC. We hold that our model serves as a tool for relating and integrating different kinds of NC. This enables addressing more comprehensive causal explana- tions of complex reciprocal organism-environment interactions, which are the norm rather than the exception in ecological and evolutionary scenarios. We illustrate this with two examples.il Our first example is the acceleration of flower production in Solanum melongena (eggplant) as a consequence of active leaf damage by Bombus terrestris bumblebees (Pashalidou et al. 2020). When faced with a shortage of pollen, bumblebee workers actively damage the leaves of flowerless plants, which accelerates flower production. In this way, bumblebees increase the local availability of their nutritional resources (see Fig. 5a, sequence 1–7). A shortage of pollen in the environment of the bumble- bees (En−2) is experienced (Exn−2) by them (On−2) as nutrient scarcity (1). This mod- ifies the behavior of bumblebees (2), which start damaging the leaves of eggplants (3). The damaged plants (O’n) experience their environment as threatening (4) and thus alter their constitution by allocating resources to the production of flowers (5). Here, we see two different physiological processes at play, with distinct locations in the plant and different functions: plant experience refers to a particular sensory- cue interface which detects with sufficient specificity damage to leaves, and plant constitution refers to the morphogenetic processes that lead to flower production in meristems. The availability of flowers, in turn, alters the behavior of bumblebees, which cease damaging the plants and start collecting pollen (6). Further reciprocal interactions between bumblebees and plants could then continue (7). Integrating different types of niche construction This case can be framed as physical NC: bumblebees actively modify their envi- ronment (which includes plants) and the consequences of their activity reciprocally impact them. But it is important to note that also experience is included here as a key component, contrary to the way physical/external NC has traditionally been con- ceptualized. In addition, and more importantly, this case of reciprocal interaction is only possible by virtue of the NC capacities of plants. Indeed, from the perspective of the eggplant, this case is an instance of mediational NC, whereby plants accel- erate flower production due to a change in their experienced environment, without altering intrinsic features of the environment. In short, a proper understanding of this case requires the integration of two kinds of NC and of experience into physical NC within one causal explanation. Our model allows achieving such integrations. We now move to a more challenging example: the transition to herbivory in ruminants and the role symbiotic microbes play in this evolutionary shift (Chiu and Gilbert 2020). This example involves two processes. On the one hand, microbes colonize the digestive system of the animal and help develop the rumen, which is the organ that houses them and allows them to function. On the other hand, these developmental changes cause a dietary shift to herbivory in the animal, which now perceives plants as edible. According to Chiu and Gilbert (2020), the former is an instance of physical NC (though they label it as ‘perturbational,’ following 1 3 3 8 Page 22 of 29 48 Page 22 of 2 J. Baedke et al. 48 Odling-Smee et al. 2003), whereby microbes help construct their own environ- ment–the rumen. The latter is an instance of mediational NC, i.e. a change in the animal’s experience of its environment without physically changing it. Further, Chiu and Gilbert state that (a) ruminants also engage in physical NC by contributing to the development of their microbes’ niche, and that (b) the holobiont as a whole (i.e. the ruminant host plus its microbiome), and not just the animal, is the unit that engages in mediational NC. Taken together, these statements are problematic, since (a) implies that microbes and host are separate units, each partaking in the environ- ment of the other, whereas (b) assumes that both the host and its microbiome behave as one unit that shares an experience of the environment. Integrating different types of niche construction Let us represent the case by means of our model (Fig. 5b, sequence 1–6). We consider the rumen microbes, taken as a collective, and the animal host as distinct organisms (O’ and O, respectively). The rumen is the microbiome’s environment (E’) and the host’s environment is its external environment (E). After colonizing the rumen during birth, the microbes (O’n−2) proliferate and release compounds (particularly butyrate) that cause the growth and differentiation of this organ (1). The altered rumen (E’n−1) provides a suitable environment for further microbial proliferation and diversification of the microbial community (2). The development of the microbial community modifies the constitution of the animal (3) and this, in turn, leads the animal to start perceiving the plants in its environment as edible and digestible (4). The dietary shift of the animal alters its development (5) and, consequently, reacts upon the rumen microbiome’s composition and diversity (6). Further downstream reciprocal processes would, for instance, include microbes con- tinuing to mold the rumen and causally contribute to the animal’s development by producing cellulose-digesting enzymes that allow the host to digest plant material, or by neutralizing plant defense chemicals that would otherwise be toxic for the host (Chiu and Gilbert 2020). What we can learn from the application of our model to this complex case is that we can trace these different but entangled processes of NC only by considering the microbes as organisms distinct from their host and by studying the reciprocal interactions of these components with their unique environments individually. If, on the contrary, we consider the holobiont as an indivisible whole, we would not be allowed to identify the NC activities of the microbes, simply because NC is a dialec- tical relation between organisms and environments, and we would only be left with the environment of the holobiont (not of microbes). Consequently, embracing the ‘holobiont-as-individual’ view would imply collapsing O’ into O, thus reducing our diagram to steps 3–6. The result of this operation would be indistinguishable from a case of constitutive NC (see, e.g., the causal pattern in the development of lions; Fig. 3b). To sum up, these examples show that our model allows for the conceptual and visual integration of different kinds of physical and experiential NC. 1 3 Integrating different types of niche construction The model provides a consistent framework that can relate and compare causal explanations of organism-environment reciprocity in multi-species interaction networks, which are more complex and heterogeneous than those described by current NC taxonomies. More generally, it calls attention to implicit assumptions and overlooked questions in NCT. For instance, regarding the second example, we may ask what exactly an 1 3 Unknotting reciprocal causation between organism and… Page 23 of 29 48 Page 23 of 29 48 48 Fig. 5 Integrating different kinds of niche construction. a Causal diagram of the acceleration of flower production in Solanum melongena (O’) as a consequence of active leaf damage by Bombus terrestris bumblebees (O). Bumblebees’ behavior is triggered by the perceived (Ex) shortage of pollen in the envi- ronment (E). Flower production in plants is mediated by their perception (Ex’) of their environment (E’) as threatening. In this case, both organisms are part of each other’s (partly overlapping) environments that causally affect them. b Causal diagram of the transition to herbivory in ruminants (O) in a given environment (E). This transition is partly explained by changes in the rumen (E’) driven by microbes (O’), which cause the animal to experience plants as edible (Ex). For details, see text Fig. 5 Integrating different kinds of niche construction. a Causal diagram of the acceleration of flower production in Solanum melongena (O’) as a consequence of active leaf damage by Bombus terrestris bumblebees (O). Bumblebees’ behavior is triggered by the perceived (Ex) shortage of pollen in the envi- ronment (E). Flower production in plants is mediated by their perception (Ex’) of their environment (E’) as threatening. In this case, both organisms are part of each other’s (partly overlapping) environments that causally affect them b Causal diagram of the transition to herbivory in ruminants (O) in a given Fig. 5 Integrating different kinds of niche construction. a Causal diagram of the acceleration of flower production in Solanum melongena (O’) as a consequence of active leaf damage by Bombus terrestris bumblebees (O). Bumblebees’ behavior is triggered by the perceived (Ex) shortage of pollen in the envi- ronment (E). Flower production in plants is mediated by their perception (Ex’) of their environment (E’) as threatening. In this case, both organisms are part of each other’s (partly overlapping) environments that causally affect them. b Causal diagram of the transition to herbivory in ruminants (O) in a given environment (E). Integrating different types of niche construction This transition is partly explained by changes in the rumen (E’) driven by microbes (O’), which cause the animal to experience plants as edible (Ex). For details, see text environment is for a holobiont, and what causal role the microbial environment plays in the host’s environment. Also, what does it tell us to see similar causal pat- terns arising on the level of individual organisms (e.g., constitutive NC) compared to those in integrated collectives, where causal roles and phases of the whole causal path are distributed across different organisms? In the final section, we discuss the heuristic roles of our model for scientific research. Conclusions and outlook In this paper we identified long-standing problems about reciprocal causation in evolutionary theory, and offered solutions to them. We first showed that, contrary to what is assumed in current evolutionary debates, theoretical viewpoints that argue 3 3 48 Page 24 of 29 J. Baedke et al. 48 Page 24 of 29 48 for organism-environment reciprocal causation have a long pedigree in the history of evolutionary thought, especially in early twentieth-century. We then recounted how accounts of organism-centered reciprocity were marginalized in the second half of the twentieth century, and how the study of reciprocity between other causal relata (e.g., gene-environment, gene-population) gained the upper hand. This shift was accompanied by a consistent avoidance of the concept of ‘individual environments’ and the establishment of externalism as an important explanatory principle in evolu- tionary biology. The decline of traditional approaches of organism-environment reciprocity was also related to two, still today unsolved theoretical problems in these views: (i) Meaningful boundaries between organism and environment were often blurred, which leads to each component’s intractability for empirical studies, and (ii) there was little theoretical integration of experiential and physical views of the environ- ment. The latter prevented the clarification of the manifold causal consequences that take place through reciprocal organism-environment interactions, as it cannot spell out the exact causal links between organism-driven changes in experienced and physical environments. We showed that these problems are re-emerging in today’s debate on reciprocal causation in ecological and evolutionary processes. It is impor- tant to mention that their reappearance is not restricted to new approaches that combine ideas from developmental systems theory and NCT. Instead, more gener- ally, these problems result from certain views of causal reciprocity (wherein organ- isms are merged with their environments and all physical interactions are under- stood as mediated through experience) that are present in a number of evolutionary frameworks. In addition, we presented a conceptual and visual model that is able to tackle the aforementioned problems. Instead of scattering causes and effects across inex- tricable organism-environment systems, we showed that the organism-environment boundary is an epistemic necessity to understand the complex causality and causal contributions of each component in reciprocal NC processes. Our model unknots seemingly inextricable reciprocal causation by underscoring the sequential character of these interactions. Then, it clarifies the kind of interactions by identifying charac- teristic causal patterns in sequences of organism-environment relations. 1 3 Conclusions and outlook This allows mapping all types of reciprocal causation currently identified in different NC tax- onomies (external or physical, constitutive, mediational, relational and relocational NC) onto one common framework. In turn, this makes possible to identify and dis- tinguish the causal patterns different kinds of reciprocal processes play in ecology and evolution. Most importantly, with respect to the above two problems, (i) our model does not require the assumption that reciprocally interacting organisms and environments are inseparable. It allows distinguishing between organisms and envi- ronments by clarifying each component’s specific causal role in their mutual interac- tions. In addition, (ii) it can integrate cases of organisms’ (experience and) experien- tial NC with those of physical NC. This allows applying the present framework even to highly complex empirical scenarios, in which both processes work together. It also demystifies the role of organismic experience in ecology and evolution. i We have not attempted to provide a metaphysical account of reciprocal causa- tion, but an epistemic tool that can partition causal contributions of organisms and 1 3 Unknotting reciprocal causation between organism and… Page 25 of 29 48 environments. This should allow biologists to develop conceptually well-grounded and feasible methodologies for probing into complex evolutionary settings. We contend that no matter what one’s ontological position regarding the nature of the organism-environment relationship is, one needs to provide an answer on how to translate this position into scientific practice. We addressed this challenge not by drawing organism-environment boundaries per se, but by tracing and distinguish- ing different kinds of relations between organism and environment and to apportion their causal contributions. This makes it possible to theoretically sustain epistemi- cally set boundaries even in complex reciprocal evolutionary dynamics, in which other approaches would rather drop the idea of boundaries altogether (i.e., cases in which, e.g., many organisms from different species and their different experiences and environments are involved). Besides these conceptual and theoretical advantages, our model may also play important heuristic roles in experimental research on NC. By building on (and being compatible with) interventionist views of causation (Woodward 2003), it allows the clear identification of causal paths and relevant counterfactual dependencies between organisms and environments, which could help designing experimental set- ups and selecting suitable variables to intervene on. Of course, more fine-grained manipulations or simulations would require formalization and mathematical mod- eling. Acknowledgements We thank Grant Ramsey, Bendik Aaby, Rose Trappes, Azita Chellappoo, and Daniel Brooks for constructive comments on earlier versions of this paper. We express gratitude to two anonymous reviewers of this journal for the comments and feedback provided. Moreover, we thank the attendees of the online workshop ‘Niche Construction and Other Mechanisms in Ecology and Evolution’ (Bielefeld University, 2020) and of a session at the Oslo Meeting of the International Society for the His- tory, Philosophy, and Social Studies of Biology (ISHPSSB, 2019) for their useful feedback on presenta- tions on this topic. We gratefully acknowledge the financial support from the German Research Founda- tion (DFG; project no. BA 5808/2-1). Declarations Conflict of interest The authors declare no conflict of interest. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Com- mons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licen ses/by/4.0/. Conclusions and outlook Recently, several mathematical models have been proposed to address recipro- cal interactions between organisms and environments, and some of them explicitly deal with NC (e.g., Torres et al. 2009). Following Lewontin’s (1983) early proposal, some formalizations represent organism-environment interaction by means of sys- tems of coupled differential equations (e.g Gurney and Lawton 1996; Krakauer et al. 2009; Tanaka et al. 2020), while others incorporate elements of game theory (Fort 2020) or make use of causal graph theory (e.g., Ay and Löhr 2015; Otsuka 2015). By drawing on the latter set of approaches as well as interventionist interpretations of causality in path analysis (Pearl 2009), we will explore the formalization of our conceptual model elsewhere. In fact, the diagrams presented in this paper can be seen as directed acyclic graphs, whereby the states of the organism and the environ- ment are vertices and the causal relations are edges. For now, we ask philosophers of biology and evolutionary biologists to become aware of (and finally overcome) long-standing problematic assumptions about recip- rocal causation between organisms and environments. These relations can, in fact, be conceptually clarified and investigated. Therefore, we need to unknot the seem- ingly inextricable bundle between organisms and environments, and integrate organ- ismic experience more seriously to understand how organisms shape their ecological contexts as well as their evolutionary trajectories. Acknowledgements We thank Grant Ramsey, Bendik Aaby, Rose Trappes, Azita Chellappoo, and Daniel Brooks for constructive comments on earlier versions of this paper. We express gratitude to two anonymous reviewers of this journal for the comments and feedback provided. Moreover, we thank the attendees of the online workshop ‘Niche Construction and Other Mechanisms in Ecology and Evolution’ (Bielefeld University, 2020) and of a session at the Oslo Meeting of the International Society for the His- tory, Philosophy, and Social Studies of Biology (ISHPSSB, 2019) for their useful feedback on presenta- tions on this topic. We gratefully acknowledge the financial support from the German Research Founda- tion (DFG; project no. BA 5808/2-1). 1 3 J. Baedke et al. 48 Page 26 of 29 48 Funding Open Access funding enabled and organized by Projekt DEAL. References Aaby BH, Ramsey G (2019) Three kinds of niche construction. Br J Philos. https://doi.org/10.1093/bjps/ axz054f Adewoye AB, Lindsay SJ, Dubrova YE et al (2015) The genome-wide effects of ionizing radiation on mutation induction in the mammalian germline. Nat Commun 6:6684. https://doi.org/10.1038/ ncomms7684 Antonovics J, Ellstrand NC, Brandon RN (1988) Genetic variation and environmental variation: expecta- tions and experiments. In: Gottlieb LD, Jain SK (eds) Plant evolutionary biology. Springer, Nether- lands, Dordrecht, pp 275–303 pp Ay N, Löhr W (2015) The Umwelt of an embodied agent—a measure-theoretic definition. Theory Biosci 134(3):105–116 Baedke J (2019) O organism, where art thou? Old and new challenges for organism-centered biology. J Hist Biol 52(2):293–324 Baedke J, Schöttler T (2017) Visual metaphors in the sciences: the case of epigenetic landscape images Gen Philos Sci 48(2):173–194 Baedke J, Fábregas-Tejeda A, Vergara-Silva F (2020a) Does the extended evolutionary synthesis enta extended explanatory power? Biol Philos 35(1):20. https://doi.org/10.1007/s10539-020-9736-5 Baedke J, Fábregas-Tejeda A, Nieves Delgado A (2020b) The holobiont concept before Margulis. J Exp Zool B Mol Dev Evol 334(3):149–155 Baedke J, Gilbert SF (2020) Evolution and development. In: Zalta EN (ed) The Stanford encyclopedia of philosophy, Fall 2020. Metaphysics Research Lab, Stanford University. https://stanford.library. sydney.edu.au/archives/fall2020/entries/evolution-development/ Bierens de Haan JA (1947) Animal psychology and the science of animal behaviour. Behavio 1(1):71–80 Brandon RN (1990) Adaptation and environment. Princeton University Press, Princeton Brandon RN (2012) The concept of the environment in evolutionary theory. In: Kabasenche WP, O’Rourke M, Slater MH (eds) The environment: philosophy, science, and ethics. MIT Press, Cam- bridge, MA, pp 19–35 g pp Brentari C (2015) Jakob von Uexküll: the discovery of the Umwelt between biosemiotics and theoretical biology. Springer, Dordrecht Brodie ED III (2005) Caution: niche construction ahead. Evolution 59(1):249–251 Buskell A (2019) Reciprocal causation and the extended evolutionary synthesis. Biol Theory 14(4):267–279 Charlesworth B (1971) Selection in density-regulated populations. Ecology 52(3):469–474i Chiu L (2019) Decoupling, commingling, and the evolutionary significance of experiential niche con- struction. In: Uller T, Laland KN (eds) Evolutionary causation: biological and philosophical reflec- tions. MIT Press, Cambridge, MA, pp 299–322 Chiu L (2019) Decoupling, commingling, and the evolutionary significance of experiential niche con- struction. In: Uller T, Laland KN (eds) Evolutionary causation: biological and philosophical reflec- tions. References MIT Press, Cambridge, MA, pp 299–322 1 3 Unknotting reciprocal causation between organism and… Page 27 of 29 48 Chiu L, Gilbert SF (2015) The birth of the holobiont: multi-species birthing through mutual scaffolding and niche construction. Biosemiotics 8(2):191–210 Chiu L, Gilbert SF (2020) Niche construction and the transition to herbivory: phenotype switching and the organization of new nutritional modes. In: Levine H, Jolly MK, Kulkarni P, Nanjundiah V (eds) Phenotypic switching. Academic Press, Cambridge, MA, pp 459–482 Di Paolo EA (2020) Picturing organisms and their environments: interaction, transaction, and constit tion loops. Front Psychol 11:1912. https://doi.org/10.3389/fpsyg.2020.01912 Dickins TE, Barton RA (2013) Reciprocal causation and the proximate–ultimate distinction. Biol Phil 28(5):747–756 Fábregas-Tejeda A, Vergara-Silva F (2018a) Hierarchy theory of evolution and the extended evolutionary synthesis: some epistemic bridges, some conceptual rifts. Evol Biol 45(2):127–139 y p g p ( ) Fábregas-Tejeda A, Vergara-Silva F (2018b) The emerging structure of the extended evolutionary synthe- sis: where does Evo-Devo fit in? Theory Biosci 137(2):169–184 Fábregas-Tejeda A, Vergara-Silva F (2018b) The emerging structure of t sis: where does Evo-Devo fit in? Theory Biosci 137(2):169–184 i Falconer DS (1952) The problem of environment and selection. Am Nat 86(830):293–298 Falconer DS (1960) Introduction to quantitative genetics. Ronald Press Company, New York Fisher RA (1930) The genetical theory of natural selection. Clarendon Press, Oxfordi Fort H (2020) Combining niche and game theories to address interspecific cooperation in ecologic communities. Community Ecol 21(1):13–24 y Gaynor KM, Brown JS, Middleton AD, Power ME, Brashares JS (2019) Landscapes of fear: spatial pat- terns of risk perception and response. Trends Ecol Evol 34:355–368 Giehl RFH, von Wirén N (2014) Root nutrient foraging. Plant Physiol 166(2):509–517 Godfrey-Smith P (1996) Complexity and the function of mind in nature. Cambridge University Pres Cambridge Godfrey-Smith P (2001) On the status and explanatory structure of developmental systems theory. In: Oyama S, Griffiths P, Gray RD (eds) Cycles of contingency. MIT Press, Cambridge, MA, pp 283–297 Griesemer JR (1991) Must scientific diagrams be eliminable? The case of path analysis. Biol Phil 6(2):155–180fi Griffiths PE, Gray RD (1994) Developmental systems and evolutionary explanation. J Phil 91(6):277–304 Griffiths P, Gray R (2001) Darwinism and developmental systems. In: Oyama S, Griffiths P, Gray R (eds) Cycles of contingency: developmental systems and evolution. References 48 Laland KN, Sterelny K, Odling-Smee J, Hoppitt W, Uller T (2011) Cause and effect in biology revisited: is mayr’s proximate-ultimate dichotomy still useful? Science 334(6062):1512–1516f Laland KN, Odling-Smee J, Hoppitt W, Uller T (2013) More on how and why: cause and effect in biology revisited. Biol Philos 28(5):719–745 Laland KN, Uller T, Feldman MW, Sterelny K, Müller GB, Moczek A, Jablonka E, Odling-Smee J (2015) Th d d l i h i i i d di i P R l S B Laland KN, Uller T, Feldman MW, Sterelny K, Müller GB, Moczek A, Jablonka E, Odling-Smee J (2015) The extended evolutionary synthesis: its structure, assumptions and predictions. Proc Royal Soc B 282(1813):20151019. https://doi.org/10.1098/rspb.2015.1019 Laland K, Matthews B, Feldman MW (2016) An introduction to niche construction theory. Evol Ec 30(2):191–202 Laland K, Odling-Smee J, Endler J (2017) Niche construction, sources of selection and trait coevolution. Interf Focus 7(5):20160147. https://doi.org/10.1098/rsfs.2016.0147 p g Lerner IM (1950) Population genetics and animal improvement. Cambridge University Press, Cambridge Levins R, Lewontin R (1985) The dialectical biologist. Harvard University Press, Cambridge, MA Levis NA, Pfennig DW (2017) Organisms and their environment: an evolving relationship. Evolutio 71(2):503–504 Lewontin RC (1983) Gene, organism, and environment. In: Bendall D (ed) Evolution: from molecules to men. Cambridge University Press, Cambridge, pp 273–285 Lewontin RC (2000) The triple helix: gene, organism, and environment. Harvard University Press, Cam- bridge, MA É Mesoudi A, Blanchet S, Charmantier A, Danchin É, Fogarty L, Jablonka E, Laland KN, Morgan TJH, Müller GB, Odling-Smee FJ, Pujol B (2013) Is non-genetic inheritance just a proximate mechanism? A cor- roboration of the extended evolutionary synthesis. Biol Theory 7(3):189–195 Meyer-Abich A (1942) Kant und das biologische Denken. Acta Biotheor 6(3):185–211 Meyer-Abich A (1943) Beiträge zur Theorie der Evolution der Organismen. I. Das typologische Grundgesetz und seine Folgerungen für Phylogenie und Entwicklungsphysiologie. Acta Biotheor 7(1):1–80 Meyer-Abich A (1964) The historico-philosophical background of the modern evolution-biology. E. J. Brill, Leiden Mill JS (1843) A system of logic, ratiocinative and inductive, being a connected view of the principles of evidence, and the methods of scientific investigation, vol 2. John W Parker, West Strand i Moczek AP (2015) Re-evaluating the environment in developmental evolution. Front Ecol Evol 3:7. https:// doi.org/10.3389/fevo.2015.00007 Müller C, Caspers BA, Gadau J, Kaiser S (2020) The power of infochemicals in mediating individualized niches. Trends Ecol Evol 35(11):981–989 Needham J (1936) Order and life. References MIT Press, Cambridge, MA, pp 195–218 Gurney WSC, Lawton JH (1996) The population dynamics of ecosystem engineers Gurney WSC, Lawton JH (1996) The population dynamics of ecosystem engineers. Oikos 76(2):273–28 ey WSC, Lawton JH (1996) The population dynamics of ecosystem engineers. Oikos 76(2):273–283 kel E (1866) Generelle Morphologie der Organismen, 2 vols. Reimer, Berlin urney WSC, Lawton JH (1996) The population dynamics of ecosystem engineers. Oikos 76(2):273 283 aeckel E (1866) Generelle Morphologie der Organismen, 2 vols. Reimer, Berlin Haeckel E (1866) Generelle Morphologie der Organismen, 2 vols. Reimer, Berlin Haldane JS (1884) Life and mechanism. Mind 9(33):27–47 Haldane JS (1935) The physiology of Descartes and its modern developments. Acta Biotheor 1(1):5–16 ( ) p y gy p ( ) Haldane JBS (1936) Some principles of causal analysis in genetics. Erkenntnis 6:346–357 Haldane JBS (1936) Some principles of causal analysis in genetics. Erkenntnis 6:346–357 The interaction of nature and nurture. Ann Eugen 13 Hartmann N (1950) Philosophie der Natur: abriss der speziellen Kategorienlehre. De Gruyter, Berlinii Henderson LJ (1913) The fitness of the environment: an inquiry into the biological significance of th properties of matter. Macmillan, New Yorkf Heras-Escribano M (2020) The evolutionary role of affordances: ecological psychology, niche constru tion, and natural selection. Biol Philos 35(2):30. https://doi.org/10.1007/s10539-020-09747-1 Jones CG, Lawton JH, Shachak M (1994) Organisms as ecosystem engineers. Oikos 69(3):373–386 Kesäniemi J, Lavrinienko A, Tukalenko E et al (2020) Exposure to environmental radionuclides alters mito chondrial DNA maintenance in a wild rodent. Evol Ecol 34:163–174 Kesäniemi J, Lavrinienko A, Tukalenko E et al (2020) Exposure to environmen chondrial DNA maintenance in a wild rodent. Evol Ecol 34:163–174 chondrial DNA maintenance in a wild rodent. Evol Ecol 34:163–174 Kirkpatrick M (1982) Sexual selection and the evolution of female choice. Evolution 36(1):1–12 Krakauer DC, Page KM, Erwin DH (2009) Diversity, dilemmas, and monopolies of niche construction. A Nat 173(1):26–40 Krakauer D, Bertschinger N, Olbrich E, Flack JC, Ay N (2020) The information theory of individuality. The- ory Biosci 139(2):209–223 Labouvie EW (1974) Developmental causal structures of organism-environment interactions. Hum Dev 17(6):444–452 Laland K, Brown G (2018) The social construction of human nature. In: Lewens T, Hannon E (eds) Why w disagree about human nature. Oxford University Press, Oxford, pp 127–144 Laland KN, O’Brien MJ (2011) Cultural niche construction: an introduction. Biol Theory 6(3):191–202 1 3 48 Page 28 of 29 48 Page 28 of 29 J. Baedke et al. References Front Genet 9:735 https://doi.org/10.3389/fgene.2018.00735 Schwab DB, Casasa S, Moczek AP (2019) On the reciprocally causal and constructive nature of develop- l l i i d b F G 9 735 h //d i /10 3389/f 2018 00735 Sheriff MJ, Thaler JS (2014) Ecophysiological effects of predation risk; an integration across disciplines. Oecologia 176:607–611 erelny K (2001) Niche construction, developmental systems and the extended replicator. In: Oyama S, fi Sterelny K (2001) Niche construction, developmental systems and the extended replicator. In: Oyama S Griffiths P, Gray RD (eds) Cycles of contingency. MIT Press, Cambridge, MA, pp 333–350 Sterelny K (2001) Niche construction, developmental systems and the extended replicator. In: Oyama Griffiths P, Gray RD (eds) Cycles of contingency. MIT Press, Cambridge, MA, pp 333–350 Griffiths P, Gray RD (eds) Cycles of contingency. MIT Press, Cambridge, MA, pp 333–350 fi Sterner B (2017) Individuality and the control of life cycles. In: Lidgard S, Nyhart LK (eds) Biological individuality: integrating scientific, philosophical, and historical perspectives. University of Chicago Press, Chicago, pp 84–108 Sultan SE (2015) Organism and environment: ecological development, niche construction, and adaptation. Oxford University Press, Oxford Svensson EI (2018) On reciprocal causation in the evolutionary process. Evol Biol 45(1):1–14 Tanaka MM, Godfrey-Smith P, Kerr B (2020) The dual landscape model of adaptation and niche constru tion. Philos Sci 87(3):478–498 ( ) Thompson JN (1998) Rapid evolution as an ecological process. Trends Ecol Evol 13(8):329 Tinbergen N (1963) On aims and methods of ethology. Z Tierpsychol 20(4):410–433 Toepfer G (2012) Teleology and its constitutive role for biology as the science of organized systems in nature. Stud Hist Philos Biol Biomed Sci 43(1):113–119 Toepfer G (2012) Teleology and its constitutive role for biology as the science of organized systems in natur Toepfer G (2012) Teleology and its constitutive role for biology as the science of organiz St d Hi t Phil Bi l Bi d S i 43(1) 113 119 Toepfer G (2012) Teleology and its constitutive role for b Stud Hist Philos Biol Biomed Sci 43(1):113–119 Stud Hist Philos Biol Biomed Sci 43(1):113–119 Stud Hist Philos Biol Biomed Sci 43(1):113–119 Torres J-L, Pérez-Maqueo O, Equihua M, Torres L (2009) Quantitative assessment of organism–environment couplings. Biol Philos 24(1):107–117 Uller T, Helanterä H (2019) Niche construction and conceptual change in evolutionary biology. Br J Philos Sci 70(2):351–375l Uller T, Laland KN (2019) Evolutionary causation: biological and philosophical reflections. References Yale University Press, Yale Nicholson DJ (2014) The return of the organism as a fundamental explanatory concept in biology. Philos Compass 9(5):347–359 Nicholson DJ, Gawne R (2015) Neither logical empiricism nor vitalism, but organicism: what the philosophy of biology was. Hist Philos Life Sci 37(4):345–381 gy ( ) Odling-Smee FJ, Laland KN, Feldman MW (1996) Niche construction. Am Nat 147:641–64 Odling-Smee FJ, Laland KN, Feldman MW, Feldman MW (2003) Niche construction: the neglected process in evolution Princeton University Press Princeton Odling-Smee FJ, Laland KN, Feldman MW, Feldman MW (2003) Niche construction: the neglected pro Odling-Smee FJ, Laland KN, Feldman MW, Feldman MW ( in evolution. Princeton University Press, Princeton dling-Smee FJ, Laland KN, Feldman MW, Feldman MW (2003) Niche construction: the neglected process in evolution. Princeton University Press, Princeton Otsuka J (2015) Using causal models to integrate proximate and ultimate causation. Biol Philos 30(1):19–3 Oyama S (2000) The ontogeny of information: developmental systems and evolution. Duke University Pres Durham Pashalidou FG, Lambert H, Peybernes T, Mescher MC, Moraes CMD (2020) Bumble bees damage plant leaves and accelerate flower production when pollen is scarce. Science 368(6493):881–884 l Pearce T (2014) The dialectical biologist, circa 1890: John Dewey and the Oxford Hegelians. J Hist Philos 52:747–778 Pearce T (2020) Pragmatism’s evolution: organism and environment in american philosophy. University Chicago Press, Chicago Pearl J (2009) Causality. Cambridge University Press, Cambridge Pimentel D (1968) Population regulation and genetic feedback: evolution provides foundation for control of herbivore, parasite, and predator numbers in nature. Science 159(3822):1432–1437 ite, and predator numbers in nature. Science 159(3822) Prenant M (1943) Biology and marxism. International Publishers, New York Raerinne J, Baedke J (2015) Exclusions, explanations, and exceptions: on the causal and lawlike status of the competitive exclusion principle. Philos Theory Biol 7:e602 https://doi.org/10.3998/ptb.6959004.0007. 002 3 Unknotting reciprocal causation between organism and… 48 Page 29 of 29 Reznick DN (2013) A critical look at reciprocity in ecology and evolution: introduction to the symposium. Am Nat 181(S1):S1–S8 Scholl R, Pigliucci M (2015) The proximate–ultimate distinction and evolutionary developmental biology: causal irrelevance versus explanatory abstraction. Biol Philos 30(5):653–670 Schwab DB, Casasa S, Moczek AP (2019) On the reciprocally causal and constructive nature of develop- mental plasticity and robustness. References MIT Pres Cambridge, MA von Uexküll J (1909) Umwelt und Innenwelt der Tiere. Springer, Berlin von Uexküll J (1928) Theoretische Biologie. Springer, Berlin Heidelberg, Berlin Waddington CH (1957) The strategy of the genes: a discussion of some aspects of theoretical biology. Allen and Unwin, London Waddington CH (1959a) Evolutionary systems–animal and human. Nature 183(4676):1634–1638 Waddington CH (1959b) Evolutionary adaptation. In: Tax S (ed) Evolution after Darwin, the Univ Waddington CH (1959b) Evolutionary adaptation. In: Tax S (ed) Evolution af Chicago centennial. University of Chicago Press, Chicago, pp 381–402 Chicago centennial. University of Chicago Press, Chicago, pp 381–402 Waddington CH (1977) Tools for thought. Jonathan Cape, Frogmore Walsh DM (2015) Organisms, agency, and evolution. Cambridge University Press, Cambridge Walsh DM (2021) Environment as abstraction. Biol Theory. https://doi.org/10.1007/s13752-020-003 Williams GC (1992) Gaia, nature worship and biocentric fallacies. Q Rev Biol 67(4):479–486 Woodward J (2003) Making things happen. Oxford University Press, Oxford Wright S (1960) The treatment of reciprocal interaction, with or without lag, in path analysis. Biometrics 16(3):423–445 Wright S (1960) The treatment of reciprocal interaction, with or without lag, in path analysis. Biometri 16(3):423–445 Wright S (1969) Evolution and the genetics of populations, vol 2: theory of gene frequencies. University of Chicago Press, Chicago Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 1 3
https://openalex.org/W4383912870	https://www.researchsquare.com/article/rs-3131002/latest.pdf	English	null	Kardar--Parisi--Zhang Roughening Associated with Nucleation-Limited Steady Crystal Growth	Research Square (Research Square)	2,023	cc-by	10,631	Kardar--Parisi--Zhang Roughening Associated with Nucleation-Limited Steady Crystal Growth Noriko Akutsu (  nori3@phys.osakac.ac.jp ) Osaka Electro-Communication University ABSTRACT The roughness of crystal surfaces and the shape of crystals play an important role in multiscale phenomena. For example, the roughness of a crystal surface affects the frictional and optical properties of the material such as ice or silica. For the roughness of a crystal surface, theoretical studies based on the symmetry principle predicted that the growing surfaces of crystal growth would be classiﬁed in the universal class of Kardar-Parisi-Zhang (KPZ) but experiments only rarely observe KPZ properties. To ﬁll this the gap, extensive numerical calculations of the crystal growth rates and the surface roughness (surface width) are performed for a lattice model with a size in the nanoscale range using the Monte Carlo method. The results indicate that a (001) surface is smooth within the single nucleation growth region. In contrast, the same surface is atomically smooth but thermodynamically rough in the poly-nucleation growth region in conjunction with a KPZ roughness exponent. Inclined surfaces are known to become Berezinskii–Kosterlitz–Thouless (BKT) rough surfaces both at and near equilibrium. The two types of steps associated with the (001) and (111) terraces were found to induce KPZ surface roughness, while the interplay between steps and multilayered islands promoted BKT roughness. Kardar–Parisi–Zhang Roughening Associated with Nucleation-Limited Steady Crystal Growth Noriko Akutsu1,* 1Faculty of Engineering, Osaka Electro-Communication University, Hatsu-cho, Neyagawa, Osaka 572-8530, Japan *nori3@phys.osakac.ac.jp g, Osaka Electro-Communication University, Hatsu-cho, Neyagawa, Osaka 572-8530, Japa ac jp DOI: https://doi.org/10.21203/rs.3.rs-3131002/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. R d F ll Li License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Additional Declarations: No competing interests reported. Version of Record: A version of this preprint was published at Scienti¦c Reports on September 26th, 2023. See the published version at https://doi.org/10.1038/s41598-023-43002-3. Introduction Surface roughness is a complex phenomenon to analyze even in the case that the length scale is limited to less than 2 µm1. This topic is also complicated by the possibility of two types of roughness: atomic2,3 and thermodynamic4,5. At equilibrium, there will be a thermal roughening transition temperature, TR, for a two-dimensional (2D) surface in a 3D system. For a given temperature, T < TR, the surface will be smooth while, at TR < T, the surface will be rough with W 2 diverging logarithmically from L. The latter is characteristic behavior linked to the Berezinskii–Kosterlitz–Thouless (BKT) universality class6,7. This thermodynamic roughening transition is directly connected to a shape transition occurring at equilibrium and referred to as the faceting transition8–12. In the case of kinetic roughening based on studies of crystal growth, kinetically rough surfaces are thought to grow via an adhesive process13–18. While, in the ﬁeld of statistical mechanics, the term kinetic roughening is used to refer to Kardar–Parisi–Zhang (KPZ) roughening19. The Family–Vicsek scaling relationship20–26 has been widely used to describe the surface width, W(L,t), of ﬂuctuating surfaces and the concept of a self-afﬁne surface (or interface) has been successfully developed. The Family–Vicsek scaling relationship for a surface can be expressed as W(L,t) ∼Lα f(L−zt), z = α/β, W(L,t) ∼Lα as t →∞, (1) W(L,t) ∼Lα f(L−zt), z = α/β, W(L,t) ∼Lα as t →∞, (1) where α, β and z are the roughness, growth and dynamic exponents, respectively. In the non-equilibrium steady state, the surface width is characterized by the roughness exponent α. Based on the symmetry principle, a surface growth equation including a nonlinear term obtained from the KPZ model19 can be derived as where α, β and z are the roughness, growth and dynamic exponents, respectively. In the non-equilibrium steady state, the surface width is characterized by the roughness exponent α. Based on the symmetry principle, a surface growth equation including a nonlinear term obtained from the KPZ model19 can be derived as ∂ht ∂t = v0 +ν∇2ht + λ 2 (∇ht)2 +ηt (2) where ht is the surface height at time t, v0 is the constant surface velocity, ν > 0 is a coefﬁcient related to surface tension, λ is a coefﬁcient for the nonlinear term and ηt is white noise in space and time. Introduction In the case of a 2D surface in a 3D system, the values of these exponents are predicted to be α = 0.3869, β = 0.2398 and z = 1.613125,26 (KPZ-rough surface). The experimentally determined values of various systems such as directed polymers are known to agree with these exponents, indicating that these systems belong to the KPZ universality class. However, in the case of crystal growth, the observed roughness exponents tend to differ from those predicted by the KPZ model21,26,27, with the exception of several special surface systems28,29. where ht is the surface height at time t, v0 is the constant surface velocity, ν > 0 is a coefﬁcient related to surface tension, λ is a coefﬁcient for the nonlinear term and ηt is white noise in space and time. In the case of a 2D surface in a 3D system, the values of these exponents are predicted to be α = 0.3869, β = 0.2398 and z = 1.613125,26 (KPZ-rough surface). The experimentally determined values of various systems such as directed polymers are known to agree with these exponents, indicating that these systems belong to the KPZ universality class. However, in the case of crystal growth, the observed roughness exponents tend to differ from those predicted by the KPZ model21,26,27, with the exception of several special surface systems28,29. 1 0 1 2 0 0.05 0.1 0 1 2 0 0.05 0.1 0 1 2 0 0.05 0.1 0.15 L 80√2 160√2 240√2 320√2 L 80√2 160√2 240√2 320√2 L 80√2 160√2 240√2 320√2 (a) (b) (c) Δμ/ε Δμ/ε Δμ/ε V W²/ "nL W / L0.385 p = 0 p = 0 p = 0 Figure 1. Surface growth velocity and scaled surface widths at the (001) surface (p = 0) as functions of ∆µ. (a) Surface growth velocity with unit of a/τ, where a (= 1) is the unit height and τ is the time interval for 1 MCS/site. Line: V = 0.0643∆µ/ε −0.0412. (b) The squared surface width, W 2 = ⟨[h(⃗x)−⟨h⟩]2⟩, scaled by the logarithm of the system size, L. (c) The surface width scaled by Lα with the roughness exponent α ≈0.385 determined from the KPZ model. kBT/ε = 0.4. Introduction For this purpose, extensive numerical data on growth rate and surface roughness of planar or inclined surfaces were collected using the Monte Carlo method for a non-equilibrium steady state. Introduction 0 1 2 0 0.05 0.1 L 80√2 160√2 240√2 320√2 (a) Δμ/ε V p = 0 0 1 2 0 0.05 0.1 0.15 L 80√2 160√2 240√2 320√2 (b) Δμ/ε W²/ "nL W / L0 385 p = 0 0 1 2 0 0.05 0.1 L 80√2 160√2 240√2 320√2 (c) Δμ/ε W / L p = 0 (b) (c) (a) (b) Figure 1. Surface growth velocity and scaled surface widths at the (001) surface (p = 0) as functions of ∆µ. (a) Surface growth velocity with unit of a/τ, where a (= 1) is the unit height and τ is the time interval for 1 MCS/site. Line: V = 0.0643∆µ/ε −0.0412. (b) The squared surface width, W 2 = ⟨[h(⃗x)−⟨h⟩]2⟩, scaled by the logarithm of the system size, L. (c) The surface width scaled by Lα with the roughness exponent α ≈0.385 determined from the KPZ model. kBT/ε = 0.4. (a) (b) (c) Overhead view Side view [100] [010] Figure 2. Images of simulated p = 0 surfaces. Upper ﬁgures: overhead views. Lower ﬁgures: side views showing the height along the lower perimeters of the upper ﬁgures for∆µ/ε = (a)0.8,(b)1.4and(c)2.6. kBT/ε = 0.4. L = 320× √ 2. ∆µcr/ε = 0.336. To better indicate the shapes of the steps on the crystal surfaces, the surface height is represented by 10 degrees of brightness, with a brighter color corresponding to a greater height. (b) (c) Figure 2. Images of simulated p = 0 surfaces. Upper ﬁgures: overhead views. Lower ﬁgures: side views showing the height along the lower perimeters of the upper ﬁgures for∆µ/ε = (a)0.8,(b)1.4and(c)2.6. kBT/ε = 0.4. L = 320× √ 2. ∆µcr/ε = 0.336. To better indicate the shapes of the steps on the crystal surfaces, the surface height is represented by 10 degrees of brightness, with a brighter color corresponding to a greater height. The aim of the present work is to clarify what makes the growing crystal surface KPZ rough using the RSOS model with and without surface steps. For this purpose, extensive numerical data on growth rate and surface roughness of planar or inclined surfaces were collected using the Monte Carlo method for a non-equilibrium steady state. The aim of the present work is to clarify what makes the growing crystal surface KPZ rough using the RSOS model with and without surface steps. KPZ Roughening on a (001) Surface Monte Carlo results the 2D critical nucleus sizes at the edges of the straight (01) steps were less than a for 1 < ∆µ/ε. In these processes, an atom (that is, the growth unit) attached at the edges of the steps associated with an island will increase the island’s size on average in the case of 1 < ∆µ/ε. The attachment of an adatom to the (001) surface, which is also regarded as an island, will increase its size on average for 2 < ∆µ/ε. the 2D critical nucleus sizes at the edges of the straight (01) steps were less than a for 1 < ∆µ/ε. In these processes, an atom (that is, the growth unit) attached at the edges of the steps associated with an island will increase the island’s size on average in the case of 1 < ∆µ/ε. The attachment of an adatom to the (001) surface, which is also regarded as an island, will increase its size on average for 2 < ∆µ/ε. Figures 1 (b) and (c) provide the scaled surface width data. Near equilibrium and for ∆µ/ε < 0.55, W = 0 and the surface is atomically and thermodynamically smooth. However, in the region deﬁned by 0.55 ≤∆µ/ε < 2.0, the surface width, W, increases as the system size, L, increases, meaning that the surface is thermodynamically rough. To our surprise, in the present work the roughened surface was found to have a KPZ roughness exponent. In the non- equilibrium steady state, the roughness exponent α determines the universality class for a 2D growing surface (Eq. (1)). In Fig. 1 (c) the surface widths are scaled by Lα and exhibit good agreement with the KPZ roughness exponent (α ≈0.385). Herein, the KPZ roughening point is designated as ∆µ(001) KPZ = 0.55ε while the end of the KPZ rough surface is designated as ∆µ(001) KtoB = 2.0ε. In Figs. 2 (a), (b) and (c), images acquired at 4 × 108 MCS/site are shown for ∆µ/ε = 0.8, 1.4, and 2.6, respectively. From Figs. 2 (a) and (b) it is evident that poly-nucleated multilayer islands appeared on the (001) surface and the perimeter of each island is also apparent. At ∆µ/ε = 0.8 (Fig. 2 (a)), island-on-island structures can be seen, otherwise referred to as multilayer islands32, having distorted square morphologies. The side view presented in Fig. KPZ Roughening on a (001) Surface Monte Carlo results The model we simulated is the restricted solid-on-solid (RSOS) model (Eq. (7)) with the driving force for the crystal growth ∆µ. The RSOS model was simulated using the Monte Carlo method with the Metropolis algorithm. Details of the model and the Monte Carlo method are given in the “Methods” section. Figure 1 presents the Monte Carlo results for the surface growth velocity, V, and the scaled surface width, W, with regard to the (001) surface. Figure 1 (a) indicates that the surface grows exponentially with respect to ∆µ during the 2D nucleation process for ∆µ/ε < 2.0. In contrast, for 2.0 ≤∆µ, the surface grows linearly via an adhesive growth process. Because the temperature value of kBT/ε = 0.4 is far less than the thermal roughening temperature of kBTR/ε = 1.57830,31, the (001) surface is atomically and thermodynamically smooth at equilibrium. It should be noted that the 2D critical nucleus sizes on the (001) surface were determined to be 2a and a for ∆µ/ε = 1 and 2, respectively, assuming that each nucleus was a square. In addition, 2/13 -5 -10 -15 1 2 -5 -10 -15 1 2 0 L 80√2 160√2 240√2 320√2 L 80√2 160√2 240√2 320√2 ε/Δμ ln (V ) ε/Δμ ln (V/v ) s (2/3) (a) (b) A B C A B C Figure 3. Surface growth velocity data for a 2D nucleation process. The points A, B and C indicate ∆µ(001) KtoB , ∆µ(001) kr and ∆µ(001) KPZ , respectively. (a) Dotted line: y = −8.45x+5.72 where y = ln(V) and x = 1/∆µ. Dashed line: y = −8.62x+4.76. (b) Dashed line y = −3.67x−0.373 where y = ln(V/v2/3 s ). p = 0. kBT/ε = 0.4. -5 -10 -15 1 2 0 L 80√2 160√2 240√2 320√2 ε/Δμ ln (V/v ) s (2/3) (b) A B C -5 -10 -15 1 2 L 80√2 160√2 240√2 320√2 ε/Δμ ln (V ) (2/3) (a) A B C 0 (b) Figure 3. Surface growth velocity data for a 2D nucleation process. The points A, B and C indicate ∆µ(001) KtoB , ∆µ(001) kr and ∆µ(001) KPZ , respectively. (a) Dotted line: y = −8.45x+5.72 where y = ln(V) and x = 1/∆µ. Dashed line: y = −8.62x+4.76. (b) Dashed line y = −3.67x−0.373 where y = ln(V/v2/3 s ). p = 0. kBT/ε = 0.4. KPZ Roughening on a (001) Surface Monte Carlo results 2 (b) also clearly indicates an island-on-island structure associated with ∆µ/ε = 1.4. These islands are known to coalesce to complete the growth layer15–17, and so a self-afﬁne surface exhibiting KPZ roughness was formed based on the island-on-island structure. It should be noted that this KPZ rough surface represents a type of faceted rough surface that is atomically smooth but thermodynamically rough. The authors previously proposed the new concept of a so-called faceted rough surface and provided numerical evidence related to kinetic roughening1 based on evaluating the surface roughness of surface systems between the atomic and mesoscopic length scales. A faceted rough surface is deﬁned as being atomically smooth but thermodynamically rough, even though such surfaces grow via a 2D poly-nucleation process. Our prior work1 using the Monte Carlo method demonstrated that an inclined surface meeting the criteria will be thermodynamically rough with a roughness exponent of α = 0.60 in the non-equilibrium steady state. This result provided evidence for the possibility of a large roughness exponent. ∆µ(001) KPZ is also a crossover point between the single and poly-nucleation growth processes. For small ∆µ values, V will be proportional to the single nucleation rate per area, In ∝exp(−G∗/kBT), according to the relationship V = L2In. Here, G∗ is the free energy change for the formation of a critical nucleus. Based on the thermodynamics of a 2D island, G∗/kBT can be expressed as G∗/kBT = γ2 s,total/(4SkBT∆µ) ≡g∗/∆µ, where γs,total is the total step free energy at the perimeter of the 2D equilibrium crystal shape with the Lagrange multiplier being 1 and S is the corresponding area33 (please see “Analysis of 2D single nucleation” subsection in the “Methods” section). In this work, γs,total and S were calculated based on the 2D Ising model using the imaginary path-weight random walk method34,35. Then, γs,totala/ε = 6.441 and S/a2 = 3.001 were determined at kBT/ε = 0.4. On this basis, a value of g∗/ε = 8.640 was obtained. Figure 3 plots ln(V) as a function of ε/∆µ and ln(V/v2/3 s ) as a function of ε/∆µ based on the Monte Carlo results in Fig. 1 (a), where vs is the step velocity (Eq. (12) in the “Methods” section). For ∆µ < ∆µ(001) KPZ , the slopes of the lines obtained using different system sizes are in good agreement and are close to the value of 8.640 calculated using the Ising model. KPZ Roughening on a (001) Surface Monte Carlo results For larger ∆µ, 3/13 0.05 -0.05 0.1 0.05 0 0 0 0.5 1 0 1 2 0.15 0.1 0.05 0 0 1 2 p 0 0.247 0.530 1.061 Δμ/ε 0.08 0.2 0.8 1.4 2.2 Δμ/ε 0.2 0.8 1.4 2.2 <110> <001> (a) (b) p Δμ/ε V V/V1.061 (c) Figure 4. The effects of slope, p and ∆µ, on surface growth velocity. (a) The surface growth velocity scaled by V1.061 (the surface growth velocity at p = 1.061) as a function of the slope value. Line: Eq. (6). Symbols: L = 240 √ 2a, 160 √ 2a and 80 √ 2a with a = 1. Note that V is independent of system size. (b) A polar graph of surface velocity normal to the inclined surface, Vn = V/√g, where g = 1+ p2 x + p2 y. Taking angle θ = 0 as the ⟨001⟩direction, Vn are plotted from the origin to the normal direction of the surface between the ⟨¯1¯11⟩( -54.74◦) and ⟨111⟩(54.74◦) directions. Dark shaded area: surface orientations less than -54.74◦and larger than 54.74◦. Light shaded area: terrace-step-kink (TSK) regions with 0.9 < \|p\|. L = 240 √ 2. (c) Surface growth velocity as a function of ∆µ for several slopes. L = 160 √ 2a with a = 1. kBT/ε = 0.4. ∆µcr/ε = 0.3. 0.05 -0.05 0.1 0.05 0 0 Δμ/ε 0.2 0.8 1.4 2.2 <110> <001> (b) 0 0.5 1 0 1 2 Δμ/ε 0.08 0.2 0.8 1.4 2.2 (a) p V/V1.061 0.15 0.1 0.05 0 0 1 2 p 0 0.247 0.530 1.061 Δμ/ε V (c) (c) (b) Figure 4. The effects of slope, p and ∆µ, on surface growth velocity. (a) The surface growth velocity scaled by V1.061 (the surface growth velocity at p = 1.061) as a function of the slope value. Line: Eq. (6). Symbols: L = 240 √ 2a, 160 √ 2a and 80 √ 2a with a = 1. Note that V is independent of system size. (b) A polar graph of surface velocity normal to the inclined surface, Vn = V/√g, where g = 1+ p2 x + p2 y. Taking angle θ = 0 as the ⟨001⟩direction, Vn are plotted from the origin to the normal direction of the surface between the ⟨¯1¯11⟩( -54.74◦) and ⟨111⟩(54.74◦) directions. Dark shaded area: surface orientations less than -54.74◦and larger than 54.74◦. Light shaded area: terrace-step-kink (TSK) regions with 0.9 < \|p\|. KPZ Roughening on a (001) Surface Monte Carlo results L = 240 √ 2. (c) Surface growth velocity as a function of ∆µ for several slopes. L = 160 √ 2a with a = 1. kBT/ε = 0.4. ∆µcr/ε = 0.3. the logarithm of V/v2/3 s was plotted against 1/∆µ (Fig. 3 (b)) because V ∝(vs)2/3I1/3 n (3) for 2D poly-nucleation16,17. The Monte Carlo results obtained for 0.55 < ∆µ/ε < 1.2 form a suitably straight line for L = 320 √ 2, 240 √ 2 and 160 √ 2. The least squares ﬁt to these values gave a slope of −3.67, the absolute value of which is larger than the expected value of g∗/3 = 2.88 based on 2D poly-nucleation theory16,17 Eq. (3). In contrast, in the case of 1.2 < ∆µ/ε, the Monte Carlo data deviate from a straight line. Here it is important to recall that ∆µ(001) kr = 1.15ε36 for a relatively high growth velocity, V (Fig. 1 (a)). Since the critical size of a square on a (001) terrace is less than 2 but larger than 1 for 1.0 < ∆µ/ε < 2.0, a single atom or a dimer on the (001) terrace detaches on average. However, the critical size of a square at a step edge is less than 1 for 1.0 < ∆µ/ε. Consequently, an adatom attached to an island edge does not detach on average. As is evident from Fig. 1 (b), for 2.0 ≤∆µ, the surface approaches BKT roughness as ∆µ is increased. In addition, at ∆µ/ε = 2.6 (Fig. 2 (c)), island-on-island structures are still seen but the size of the islands decreases and the side view of the surface indicates a greater number of ﬁne irregularities. Furthermore, the surface velocity in this region increases linearly as ∆µ increases (Fig. 1 (a)). Based on the results concerning this structure and the surface growth velocity data, the surface can be said to be kinetically, atomically and thermodynamically rough. For large ∆µ, various types of kinetic roughening are known to occur in the crystal growth ﬁeld15–17,32. Herein, the so called kinetic roughening point, ∆µc, is deﬁned as πγ2 s /(3kBT∆µc) ∼g∗/(3∆µc) ∼1. Consequently, we have ∆µc/ε ∼2.88, which is a large value compared with the Monte Carlo result of ∆µ(001) KtoB = 2.0 at which adhesive growth starts for ∆µ(001) KtoB < ∆µ. It should also be noted that the size of the critical nucleus is 1 at ∆µ/ε = 2.0 if we assume that each island is square. Therefore, ∆µc/ε = ∆µ(001) KtoB = 2.0 is adopted as the deﬁnition of ∆µc. AKPZ criteria Wolf38 reported the criteria for the classiﬁcation of surface width as follows: λ˜xλ˜y > 0 W ∝Lα (algebraic rough) λ˜xλ˜y ≤0 α = 0, W 2 ∝lnL. (5) For the limit p →0, the surface growth velocity on an inclined surface can be expressed as V ≈vsp for ∆µ < ∆µ(001) KPZ (Figs. 4 (a) and (b)). In the case of this ∆µ range, the surface grows based on a TSK process. In contrast, for ∆µ(001) KPZ < ∆µ < ∆µ(001) KtoB , islands are frequently formed on the (001) terraces. Using a magniﬁed version of Fig. 4 (a), we conﬁrmed that V = V0 +cpp2 +O(p3) for p →0, where cp is a positive coefﬁcient at ∆µ/ε = 0.8 with L = 320 √ 2. Note that this effect of the slope on V in the vicinity of p = 0 is revisited in the subsection titled ”Kinetic shape changes of a crystallite.” ( ) ( ) For ∆µ(001) KPZ < ∆µ < ∆µ(001) KtoB , ∂2V/∂p2 > 0 and (∂V/∂p)/p > 0 near p = 0, giving a coefﬁcient value of λxλy > 0. Hence, the surface should be algebraically rough and the Monte Carlo data also show KPZ roughening on the surface. (001) (001) (001) In the case of ∆µ(001) KtoB < ∆µ, we have λxλy > 0 for the same reason as in the case of ∆µ(001) KPZ < ∆µ < ∆µ(001) KtoB . Consequently, the surface should be algebraically rough but the Monte Carlo results suggest BKT roughening. Therefore, we conclude that the AKPZ criteria (Eq. (5)) are partly consistent with the Monte Carlo results for a (001) surface. In the case of ∆µ(001) KtoB < ∆µ, we have λxλy > 0 for the same reason as in the case of ∆µ(001) KPZ < ∆µ < ∆µ(001) KtoB . Consequently, the surface should be algebraically rough but the Monte Carlo results suggest BKT roughening. Therefore, we conclude that the AKPZ criteria (Eq. (5)) are partly consistent with the Monte Carlo results for a (001) surface. AKPZ criteria Here it is helpful to examine the extent of agreement between the Monte Carlo results and the KPZ equation. The crossover from BKT roughness to KPZ roughness on a surface can be discussed using the arguments proposed in Refs. [37] and [38]. The relationship between the surface velocity and the ﬂuctuation width was discussed by Wolf38 using the renormalization group method. The values of λ˜x and λ˜y are given by λ˜x = ∂2V/∂p2, λ˜y = (∂V/∂p)/p, (4) 4/13 0 1 2 0.1 0.2 0.3 0 1 2 0.1 0.2 0.3 0 1 2 0.6 0.2 0.8 0.4 0 0 1 2 0 1 2 0 1 2 0.1 0.12 0.14 0.16 0.18 0.05 0.1 0.15 0.2 0.1 0.15 L 80√2 160√2 240√2 320√2 L 80√2 160√2 240√2 320√2 L 80√2 160√2 240√2 320√2 L 80√2 160√2 240√2 320√2 L 80√2 160√2 240√2 320√2 L 80√2 160√2 240√2 320√2 (c) (b) (a) gW ²/ "n（L） gW ²/ "n（L） gW ²/ "n（L） √g W/L 0.33 √g W/L 0.385 √g W/L 0.33 Δμ/ε Δμ/ε Δμ/ε Figure 5. Scaled surface widths as functions of the driving force with (a) p = 3 √ 2/4 ≈1.061, (b) p = 3 √ 2/8 ≈0.530 and (c) p = 7 √ 2/40 ≈0.247. The upper subﬁgures show gW 2 scaled by lnL. The lower subﬁgures show √gW scaled by Lα. kBT/ε = 0.4. ∆µcr/ε = 0.3. 0 1 2 0.6 0.2 0.8 0.4 0 0 1 2 0.05 0.1 0.15 0.2 L 80√2 160√2 240√2 320√2 L 80√2 160√2 240√2 320√2 (b) ² （） gW ²/ "n（L） √g W/L 0.33 √g W/L 0.385 Δμ/ε 0 1 0.1 0.2 0.3 0 1 2 0.6 0.2 0.8 0.4 0 0 1 0 1 2 0.05 0.1 0.15 0.2 0.1 0.15 L 80√2 160√2 240√2 320√2 L 80√2 160√2 240√2 320√2 L 80√2 160√2 240√2 320√2 L 80√2 160√2 240√2 320√2 (c) (b) gW ²/ "n（L） gW ²/ "n（L） √g W/L 0.33 √g W/L 0.385 Δ Δμ/ε (b) (a) (c) Figure 5. Scaled surface widths as functions of the driving force with (a) p = 3 √ 2/4 ≈1.061, (b) p = 3 √ 2/8 ≈0.530 and (c) p = 7 √ 2/40 ≈0.247. The upper subﬁgures show gW 2 scaled by lnL. The lower subﬁgures show √gW scaled by Lα. kBT/ε = 0.4. ∆µcr/ε = 0.3. where p is the slope in the ˜x direction. Inclined surfaces This section clariﬁes the difference in surface roughness between a terrace-step-kink (TSK)39,40 model surface and a surface with terrace roughness for inclined surfaces. In the ideal TSK model, an inclined surface consists of a train of elementary steps having unit heights with no islands or negative islands (that is, clusters of adholes) on the terraces. Figure 5 summarizes the effect of ∆µ on the scaled surface width for several surface slopes and system sizes at a temperature of kBT/ε = 0.4. Here, the upper subﬁgures show the squared surface widths scaled by lnL while the lower subﬁgures present the surface widths scaled by Lα. In Fig. 6, the calculated scaled surface widths for several ∆µ and system sizes at a temperature kBT/ε = 0.4 are plotted as functions of slope. The upper subﬁgures show the squared surface widths scaled by lnL while the lower subﬁgures show the surface width scaled by Lα with α = 0.385 (that is, is the value of the KPZ roughness exponent). 5/13 0 0.5 1 0 0.5 1 0 0.5 1 0 0.5 1 0 0.5 1 0 0.5 1 0 0.5 1 0 0.5 1 0 0.1 0.05 0 0 0.1 0.2 0.3 0.1 0.2 0.3 0.3 0.4 0.2 0.6 0.1 0.2 0.1 0.15 0.15 0.05 0.1 0.15 0.05 0.1 0.05 1.5 1.5 1.5 0.05 1.5 0.8 0 0.4 1.5 1.5 L 80√2 160√2 240√2 L 80√2 160√2 240√2 L 80√2 160√2 240√2 320√2 L 80√2 160√2 240√2 320√2 L 80√2 160√2 240√2 320√2 L 80√2 160√2 240√2 320√2 L 80√2 160√2 240√2 320√2 L 80√2 160√2 240√2 320√2 (a) (b) (c) gW ²/ "n（L） p p p √g W/L 0.385 (d) p Figure 6. Scaled surface widths as functions of the slope. (a) ∆µ/ε = 0.2. (b) ∆µ/ε = 0.8. (c) ∆µ/ε = 1.4. (d) ∆µ/ε = 2.2. The upper subﬁgures show gW 2 scaled by lnL. The lower subﬁgures show √gW scaled by Lα with a KPZ roughness exponent of α ≈0.385. kBT/ε = 0.4. ∆µcr/ε = 0.3. Surfaces with almost ideal TSK structures Hereafter, we consider an inclined surface for which ∆µcr < ∆µ. As is evident from Fig. 4 (a), in the case of large surface slopes (0.9 < p) near the (111) surface, the surface growth velocities are in good agreement with one another because the surfaces have almost the ideal TSK structure due to the RSOS restriction36,44,45. Figure 4 (c) indicates the effect of ∆µ on V and demonstrates that V for p = 1.061 plateaus at 0.8 ≤∆µ/ε. Physically, surface growth occurs via the attachment and detachment of the atoms (here represented as cubes or growth units) at the step edges. The lower subﬁgure in Fig. 5 (a) demonstrates that, at p = 1.061, a stepped surface without terrace islands becomes algebraically rough at ∆µcr < ∆µ. Here, the roughness exponent is α = 0.33 and so is slightly smaller than the expected value for a KPZ roughened surface. From Figs. 6 (b), (c) and (d), it is apparent from the surface widths for surfaces with 0.9 < p < 1.25 that the surfaces are algebraically rough. In addition, the roughness exponent appears to gradually decrease as p increases. At this point, it is helpful to ascertain agreement with the AKPZ criteria in Eq. (5). Since ∂(V/V1.601)/∂p < 0 and ∂2V/V1.601/∂p2 < 0, λ˜xλ˜y > 0 based on Eq. (5). Hence, the surfaces should be algebraically rough, which is consistent with the Monte Carlo results for 0.9 < p. For the limit p → √ 2, the present numerical results conﬁrm that ∂2(V/V1.601)/∂p2 →0 and that V = vneg s ( √ 2−p), where vneg s is the step velocity for a negative step, meaning a step associated with a (111) terrace. These results demonstrate that the contribution of the nonlinear terms in the KPZ or AKPZ equation are reduced as p approaches √ 2. On this basis, we conclude that the effects of the slope and ∆µ on the surface width for 0.9 < p as obtained using the Monte Carlo method are consistent with the KPZ or AKPZ criteria. Kinetic shape changes of a crystallite Figure 4 (c) presents results for surfaces with p = 0.247 and 0.530 and shows that V increases steeply for ∆µ(001) kr < ∆µ as ∆µ increases, except for the surface for which p = 1.061. This steep increase in V (other than the above exception) provides evidence that island formation on (001) terraces resulting from the 2D nucleation process causes a steep increase in V as well as is also the case for p = 0. ( ) When assessing crystal growth, it is interesting that the anisotropy with respect to V for ∆µ < ∆µ(001) KPZ is large compared with that in the corresponding Wulff ﬁgure showing the polar graph of the surface tension46. This can be seen from Fig. 4 (b). The signiﬁcant anisotropy of V indicates that the crystallite grows such that it has a wider (001) facet compared with the equilibrium shape for ∆µ < ∆µ(001) KPZ . This effect occurs because there are two kinds of steps around p ∼0.7 associated with two kinds of terraces: the (001) and (111) terraces. As noted in the previous section, steps with (111) terraces and (001) side surfaces can be considered as negative steps. In this scenario, steps with small p values will grow to the right (e.g. see Fig. 7) whereas negative steps with large p values will grow to the left. For p values of 0.5 ∼0.8, a surface having a mixture of steps including negative steps will grow in both directions (see the Supplementary Movie 147 ). In the case of ∆µ < ∆µ(001) KPZ , the surface velocity, V, can be written as V = vsp w(001) +vneg s ( √ 2−p)w(111), w(001) = ( √ 2−p)/ √ 2, w(111) = p/ √ 2, 2−p)/ √ 2, w(111) = p/ √ 2, (6) (6) where w(001) and w(111) are the statistical weights for the number of steps determined by the surface slope, p. Because vs is approximately equivalent to vneg s (see Eq. (12)) for ∆µ < ∆µ(001) KPZ , the slope dependence of V is almost symmetrical along with p = 1/ √ 2 (Fig. 4 (a)). Figure 4 (a) plots the line obtained from Eq. (6) with V1.061 = (vs +vneg s )3/(8 √ 2) and this line is seen to be in good agreement around both limits p →0 and p → √ 2. Inclined surfaces 0 0.5 1 0 0.5 1 0 0.1 0.05 0 0.1 0.05 L 80√2 160√2 240√2 L 80√2 160√2 240√2 (a) gW ²/ "n（L） p √g W/L 0.385 0 0.5 1 0 0.5 1 0 0.5 1 0 0.5 1 0.1 0.2 0.3 0.3 0.4 0.2 0.6 0.1 0.2 0.1 0.15 0.15 0.05 1.5 1.5 1.5 0.05 1.5 0.8 0 0.4 L 80√2 160√2 240√2 320√2 L 80√2 160√2 240√2 320√2 L 80√2 160√2 240√2 320√2 L 80√2 160√2 240√2 320√2 (c) p (d) p (b) (c) (d) (a) p p Figure 6. Scaled surface widths as functions of the slope. (a) ∆µ/ε = 0.2. (b) ∆µ/ε = 0.8. (c) ∆µ/ε = 1.4. (d) ∆µ/ε = 2.2. The upper subﬁgures show gW 2 scaled by lnL. The lower subﬁgures show √gW scaled by Lα with a KPZ roughness exponent of α ≈0.385. kBT/ε = 0.4. ∆µcr/ε = 0.3. (a) (b) (c) Overhead view Side view [100] [010] (a’) (b’) (c’) Overhead view Side view [100] [010] Figure 7. Images of simulated inclined surfaces. kBT/ε = 0.4. ∆µcr/ε = 0.3. (a) and (a’) ∆µ/ε = 0.8. (b) and (b’) ∆µ/ε = 1.4. (c) and (c’) ∆µ/ε = 2.2. (a), (b) and (c) Nstep = 8. (a) L = 320 √ 2. (b) and (c) L = 240 √ 2. (a’), (b’) and (c’) p = 7 √ 2/40 ≈0.247 surface. Nstep = 28. L = 80× √ 2. (c) (b’) (c’) Figure 7. Images of simulated inclined surfaces. kBT/ε = 0.4. ∆µcr/ε = 0.3. (a) and (a’) ∆µ/ε = 0.8. (b) and (b’) ∆µ/ε = 1.4. (c) and (c’) ∆µ/ε = 2.2. (a), (b) and (c) Nstep = 8. (a) L = 320 √ 2. (b) and (c) L = 240 √ 2. (a’), (b’) and (c’) p = 7 √ 2/40 ≈0.247 surface. Nstep = 28. L = 80× √ 2. 6/13 The logarithmic divergence of gW 2 for an inclined surface at equilibrium is well established4,5,36,41,42. Here, g = 1+ p2 x + p2 y is the ﬁrst fundamental quantity in the differential geometry10,43. For all slopes near equilibrium, the surface with ∆µ/ε ≤0.3 exhibits BKT roughening (see Fig. 5 and Fig. 6 (a)). Hence, the present work conﬁrms that the crossover point between BKT and KPZ roughening of an inclined surfaces is ∆µcr/ε = 0.3. Kinetic shape changes of a crystallite It should also be noted that the values obtained using the Monte Carlo method for p ∼0.7 are higher than those produced by Eq. (6). ( ) For ∆µ(001) KPZ < ∆µ, because ∂V/∂p = 0 at p = 0, the planar shape on the (001) surface becomes unstable in conjunction with inﬁnitesimally small ﬂuctuations in the slope. Hence, a kinetic version of the faceting transition is expected to occur at ∆µ(001) KPZ . For ∆µ(001) KPZ < ∆µ < ∆µ(001) kr , V continues to exhibit a high degree of anisotropy. Hence, the (001) surface should not be planar but rather should exhibit some small degree of curvature. For ∆µ(001) kr < ∆µ, the anisotropy in V is drastically reduced in the vicinity of the (001) surface. In particular, in the region ∆µ(001) KtoB < ∆µ, adatoms on the (001) terraces are so large that the elementary step as the edge of the (001) terrace cannot be well deﬁned (see Figs. 7 (c) and (c’)), similar to the behavior of a surface with TR < T. 7/13 able 1. Characteristic driving forces and slopes (L/( √ 2a) ≥240, a = 1) for kBT/ε = 0.4. Table 1. Characteristic driving forces and slopes (L/( √ 2a) ≥240, a = 1) for kBT/ε = 0.4. Symbol value/ε p description (001) surface ∆µ(001) KPZ 0.55 0 Smooth to KPZ rough surface transition point. For ∆µ < ∆µ(001) KPZ , V = vsp (p →0); whereas for ∆µ(001) KPZ < ∆µ, V = V0 +cpp2 (0 < cp, p →0). This change is explained by the AKPZ criteria. ∆µ(001) kr 36 1.15 0 For ∆µ(001) kr < ∆µ, V0 becomes relatively large. ∆µ(001) KtoB 2.0 0 Crossover point between a 2D poly-nucleation process (a KPZ roughened surface) and an adhesive growth process with kinetic and atomic roughening (a BKT roughened surface). Inclined surface ∆µcr 0.3 0 < p < √ 2 Crossover point from a BKT roughened to algebraic or KPZ roughened surface36. ∆µ(p<0.4) KtoB 1.15 0.247 Crossover point from a KPZ roughened to BKT roughened surface. This crossover is explained by the competition between the step growth velocity and 2D nucleation rate on terraces. p(p<0.4) KtoB dependent 0 ∼0.17 Crossover point from KPZ or algebraic roughened to BKT roughened surface. on ∆µ This crossover is explained by the AKPZ criteria. Kinetic shape changes of a crystallite p(p<0.4) BtoK dependent 0.1 ∼0.4 Crossover point from BKT to algebraic or KPZ roughened surface. on ∆µ This crossover is explained by the competition between the step growth velocity and 2D nucleation rate on terraces. Table 1. Characteristic driving forces and slopes (L/( √ 2a) ≥240, a = 1) for kBT/ε = 0.4. Nonlinear effect For p < 0.4, the roughness change is complex due to the interplay between multilayered islands and steps. Near p = 0, the surfaces are algebraically rough and √gW decreases as p increases, as shown in Figs. 6 (b)–(d). With increases in p, a transition to a BKT roughened surfaces occurs near p ∼0.05 that is dependent on ∆µ. Here, the crossover point for p is denoted as p(p<0.4) KtoB . For ∆µ/ε = 0.8, 1.4 and 2.2, p(p<0.4) KtoB ≈0.017, 0.051 and 0.073, respectively. In the vicinity of p = 0.3, there is another transition to an algebraically roughened surface and this crossover point is denoted as p(p<0.4) BtoK . For ∆µ/ε = 0.8, 1.4 and 2.2, p(p<0.4) BtoK = 0.11, 0.28 and 0.30, respectively. Both p(p<0.4) KtoB and p(p<0.4) BtoK increase as ∆µ increases. to to to Here, it is helpful to assess the level of consistency between Monte Carlo results and the AKPZ criteria. Using the data in Fig. 4 (a), the inﬂection point with respect to p was calculated based on the Monte Carlo data. This process gave 0.056, 0.17 and 0.14 for ∆µ/ε = 0.8, 1.4 and 2.2, respectively and these inﬂection points are close to those for p(p<0.4) KtoB . That is, when p < p(p<0.4) KtoB , ∂2V/∂p2 > 0. On the basis of the criteria given by Eq. (5), the surface should therefore be algebraically rough. Whereas, if p(p<0.4) KtoB < p < 0.4, ∂2V/∂p2 < 0 and Eq. (5) suggests that the surface should exhibit BKT roughening. Figures 7 (a), (b) and (c) demonstrate the coalescence of terrace islands to steps and this process enhances the step ﬂuctuations for small values of p (Nstep = 8). Therefore, we conclude that the nonlinear effect associated with surface growth makes the inclined surface near p = 0 algebraically rough. Here, it is helpful to assess the level of consistency between Monte Carlo results and the AKPZ criteria. Using the data in Fig. 4 (a), the inﬂection point with respect to p was calculated based on the Monte Carlo data. This process gave 0.056, 0.17 and 0.14 for ∆µ/ε = 0.8, 1.4 and 2.2, respectively and these inﬂection points are close to those for p(p<0.4) KtoB . That is, when p < p(p<0.4) KtoB , ∂2V/∂p2 > 0. On the basis of the criteria given by Eq. Nonlinear effect (5), the surface should therefore be algebraically rough. Whereas, if p(p<0.4) KtoB < p < 0.4, ∂2V/∂p2 < 0 and Eq. (5) suggests that the surface should exhibit BKT roughening. Figures 7 (a), (b) and (c) demonstrate the coalescence of terrace islands to steps and this process enhances the step ﬂuctuations for small values of p (Nstep = 8). Therefore, we conclude that the nonlinear effect associated with surface growth makes the inclined surface near p = 0 algebraically rough. Physically, the inﬂection point can be explained by an effect in which the inclined steps hinder the formation and free growth of multilayered islands. Figures 7 (a’), (b’) and (c’) present images of the surfaces for p = 0.247 (Nstep = 28) and it is apparent that these surfaces appear different from those in (a), (b) and (c). In Figs. 7 (a’), (b’) and (c’), fewer multilayered islands appear than in Fig. 2 and so it is evident that the KPZ structure was changed to a BKT structure. In the case of p(p<0.4) BtoK < p, the transition from BKT roughened to algebraically roughened cannot be explained by the KPZ criteria. The following subsection discusses the origin of this crossover point. For p < 0.4, the roughness change is complex due to the interplay between multilayered islands and steps. Near p = 0, the surfaces are algebraically rough and √gW decreases as p increases, as shown in Figs. 6 (b)–(d). With increases in p, a transition to a BKT roughened surfaces occurs near p ∼0.05 that is dependent on ∆µ. Here, the crossover point for p is denoted as p(p<0.4) KtoB . For ∆µ/ε = 0.8, 1.4 and 2.2, p(p<0.4) KtoB ≈0.017, 0.051 and 0.073, respectively. In the vicinity of p = 0.3, there is another transition to an algebraically roughened surface and this crossover point is denoted as p(p<0.4) BtoK . For ∆µ/ε = 0.8, 1.4 and 2.2, p(p<0.4) BtoK = 0.11, 0.28 and 0.30, respectively. Both p(p<0.4) KtoB and p(p<0.4) BtoK increase as ∆µ increases. Conclusions • The effects of the slope value on V and √gW are not equivalent but are approximately similar. Competition between step growth velocity and nucleation rate on the terrace p p g y From Fig. 5 (c) it is apparent that, for ∆µ/ε ∼1.5 at p = 0.247, the surface width becomes BKT rough. A peak around ∆µ/ε ∼0.8 is also apparent and was originally attributed to a kinetic roughening transition, although this conclusion was later found to be incorrect. In Fig. 7 (a’), the surface structure at ∆µ/ε = 0.8 is shown and this surface appears to be a TSK-type 8/13 stepped surface. Few adatoms or adholes are seen on terraces in this area and so the peak in Fig. 5 (c) cannot be the result of kinetic roughening. stepped surface. Few adatoms or adholes are seen on terraces in this area and so the peak in Fig. 5 (c) cannot be the result of kinetic roughening. The surface structure at ∆µ/ε = 1.4 is presented in Fig. 7 (b’). Here, the elementary steps show numerous overhang structures and there are small numbers of islands or negative islands on the terraces. Where poly-nucleated clusters on the terraces merge with growing steps, the step edges have generated overhang structures. Because islands having different heights or negative islands cannot merge completely with steps, the higher islands or lower negative islands act as obstacles to the growing steps. In this manner, ﬂuctuations of the steps are reduced by the multi-height islands or negative islands. For ∆µ(001) kr < ∆µ, there is a non-negligible reduction in step ﬂuctuations that produces a BKT roughened surface. As a result, the surface width decreases to form a peak as seen in Fig. 5 (c). We denote this crossover point for ∆µ as ∆µ(p<0.4) BtoK and note that ∆µ(p<0.4) BtoK is close to ∆µ(001) kr when p = 0.247. Conclusions The following are the conclusions obtained from the present study of (001) surfaces. The following are the conclusions obtained from the present study of (001) surfaces. • Monte Carlo results for 0 ≤∆µ < ∆µ(001) KPZ show that the surface remains smooth during a single nucleation process. In contrast, in the case of ∆µ(001) KPZ ≤∆µ < ∆µ(001) KtoB , the surface undergoes KPZ roughening and grows via a 2D poly- nucleation process. The multilayer islands were found to be essential for the formation of the self-afﬁne surface structure. • For ∆µ(001) KtoB ≤∆µ, the surface undergoes BKT roughening and is also kinetically and atomically rough with adhesive growth. The steps on the surface are difﬁcult to deﬁne, similar to the case of a rough surface at temperatures deﬁned by TR < T. • For ∆µ(001) KtoB ≤∆µ, the surface undergoes BKT roughening and is also kinetically and atomically rough with adhesive growth. The steps on the surface are difﬁcult to deﬁne, similar to the case of a rough surface at temperatures deﬁned by TR < T. The conclusions for inclined surfaces are as follows. • In the case of 0 < p < √ 2, an inclined surface will exhibit BKT roughening for ∆µ < ∆µcr, where ∆µcr36 is a crossover point between BKT and algebraically rough surfaces. • The roughness of inclined surfaces varies in a complex manner depending on the values of ∆µ and slope, p, due to the interplay between step growth and the formation of multilayered islands on (001) terraces. The crossover points between BKT and algebraically rough surfaces are summarized in Table 1. • The surface growth velocity, V, exhibits greater anisotropy than that associated with surface tension for ∆µ < ∆µ(001) KPZ due to the possibility of two kinds of steps: those with (001) terraces and those with (111) terraces. The growth shape of a crystallite involves a wider facet area than that at equilibrium. For ∆µ(001) KPZ < ∆µ, the non-equilibrium KPZ roughening transition induces a kinetic change in the crystallite shape on the nanoscale such that the (001) facets have very slightly curved surfaces. For ∆µ(001) KtoB < ∆µ, the anisotropy of V is drastically reduced such that the growth shape is expected to be nearly spherical. • The effects of the slope value on V and √gW are not equivalent but are approximately similar. RSOS model RSOS model The surface energy of a surface with an orientation close to (001) exhibiting (001) terrace roughness can be expressed by the discrete Hamiltonian36 RSOS model The surface energy of a surface with an orientation close to (001) exhibiting (001) terrace roughness can be expressed by the discrete Hamiltonian36 HRSOS = N εsurf +∑ n,m ε[\|h(n+1,m)−h(n,m)\|+\|h(n,m+1)−h(n,m)\|]−∑ n,m ∆µ h(n,m), (7) (7) where h(n,m) is the surface height at site (n,m), N is the total number of lattice points, εsurf is the surface energy per unit cell on the planar (001) surface and ε is the microscopic ledge energy associated with nearest neighbor (nn) interactions. The summation with respect to (n,m) is over all sites on the square lattice. The RSOS condition, meaning that the height difference between nearest neighbor sites is restricted to {0,±1}, is required implicitly. In this equation, ∆µ is the driving force for crystal growth, deﬁned as µambient −µcrystal, where µambient and µcrystal are the chemical potentials of the ambient phase and the crystal, respectively. In the case that the ambient phase is an ideal solution, ∆µ = kBT lnC/Ceq48, where kB is the Boltzmann constant, 9/13 T is temperature, C is the concentration of the solute and Ceq is the concentration of the solute at saturation. If the ambient phase is an ideal gas, ∆µ = kBT lnP/Peq49, where P is the gas pressure and Peq is the gas pressure at equilibrium. T is temperature, C is the concentration of the solute and Ceq is the concentration of the solute at saturation. If the ambient phase is an ideal gas, ∆µ = kBT lnP/Peq49, where P is the gas pressure and Peq is the gas pressure at equilibrium. Since the RSOS model is a coarse grained model used for the purpose of ﬁrst principles quantum mechanical calculations, εsurf and ε relate to the surface free energy in the atomic model including the entropy for lattice vibrations and distortions50. Hence, these variables are affected by temperature. However, the present work assumes constant values for εsurf and ε in all calculations. It should be noted that the RSOS model employed in the present work30,51 is equivalent to a 19-vertex model and, because the latter represents a non-integrable system, the RSOS model cannot be solved exactly using the Bethe Ansatz approach52. This is one of the reasons why the present work chose to study the RSOS model numerically. Monte Carlo calculations In this work, the surface conﬁguration was updated using the Metropolis algorithm and the energy difference, ∆E, was calculated based on Eq. (7). The ﬁrst 2×108 Monte Carlo steps per site (MCS/site) were ignored and each quantity was averaged over the subsequent 2×108 MCS/site. The number of steps, Nstep, was ﬁxed to give a surface slope p = Nstepa/L, where a = 1 is a lattice constant. The surface growth velocity, V, was calculated as V = (⟨h(t + ˜τ)⟩−⟨h(t)⟩)/˜τ, where ˜τ is set to 2 × 108 MCS/site. en considering an inclined surface, the squared surface width was calculated as When considering an inclined surface, the squared surface width was calculated as gW 2 = ⟨⟨[h(˜x, ˜y,t)−⟨h(˜x,t)⟩˜y]2⟩˜y⟩˜x, (8) where W is a surface width normal to the inclined surface, g is a geometrical factor deﬁned as 1+ p2 x + p2 y with px = ∂⟨h⟩/∂x and py = ∂⟨h⟩/∂y43, ˜x and ˜y are the [110] and [¯110] directions, respectively, and ⟨·⟩˜y or ⟨·⟩˜x are the averages over the ˜y or ˜x directions. where W is a surface width normal to the inclined surface, g is a geometrical factor deﬁned as 1+ p2 x + p2 y with px = ∂⟨h⟩/∂x and py = ∂⟨h⟩/∂y43, ˜x and ˜y are the [110] and [¯110] directions, respectively, and ⟨·⟩˜y or ⟨·⟩˜x are the averages over the ˜y or ˜x directions. Periodic boundary conditions were adopted in the vertical ([¯110]) direction. In the horizontal ([110]) direction, periodic boundary conditions were adopted while also adding the number of steps, Nstep. Crystal growth proceeds by the attachment/detachment of speciﬁc units. As such, the number of units in the crystal does not have to be conserved during the process, making this a non-conserved system. The present work also did not include unit exchange on the surface, meaning that surface diffusion was neglected. At equilibrium, the unit attachment rate will equal the detachment rate. The attachment rate automatically increases whereas the detachment rate decreases as ∆µ increases. RSOS model y p y y At equilibrium, the RSOS model is equivalent to that previously used to determine roughness exponents by Amar and Family37,53. It is also important to note that a model used in the ﬁeld of nonlinear dynamics with the restriction of the height difference being an integer is also sometimes referred to as the RSOS model but is known as the absolute SOS (ASOS) model in the ﬁeld of roughening transition studies4,54. During the present work, surface diffusion, volume diffusion and elastic effects were not taken into consideration. Analysis of 2D single nucleation To obtain the nucleation barrier for a non-spherical shape, we introduce the scaling parameter λ. Assuming the shape of the critical nucleus is similar to the 2D equilibrium crystal shape (ECS), γs,total is deﬁned as the total step free energy of the ECS with the Lagrange multiplier being 1. If S is the area corresponding to the ECS, the island formation free energy, G, is given by (9) G = −λ 2∆µS+λγs,total, ˆS = λ 2S, ˆγs,total = λγs,total, G = −λ 2∆µS+λγs,total, ˆS = λ 2S, ˆγs,total = λγs,total, G = −λ 2∆µS+λγs,total, ˆS = λ 2S, ˆγs,total = λγs,total, (9) γs,total, ˆS = λ 2S, ˆγs,total = λγs,total, (9 where ˆS is the area of the island and ˆγs,total is the total step free energy at the perimeter of the island. In the case that the island is the critical nucleus, ∂G/∂λ = 0 and we have where ˆS is the area of the island and ˆγs,total is the total step free energy at the perimeter of the island. In the case that the island is the critical nucleus, ∂G/∂λ = 0 and we have λc = γs,total/(2S∆µ), G∗= λ 2 c S∆µ. λc = γs,total/(2S∆µ), G∗= λ 2 c S∆µ. Since G∗/kBT ≡g∗/∆µ, we can write λc = γs,total/(2S∆µ), G∗= λ 2 c S∆µ. Since G∗/kBT ≡g∗/∆µ, we can write g∗= γ2 s,total/(4SkBT). (10) Since G∗/kBT ≡g∗/∆µ, we can write Since G∗/kBT ≡g∗/∆µ, we can write (11) g∗= γ2 s,total/(4SkBT). g∗= γ2 s,total/(4SkBT). Data availability The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. R f The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Estimation of vs Estimation of vs To obtain the explicit form for vs, this work used parameters that provided the best least squares ﬁt to the Monte Carlo results in Fig. 4 (c) for a negative step velocity vneg s at p = 1.061. Here, a negative step is deﬁned as a step with a (111) terrace and (001) side surface, such that vneg s = (a1x+a2x2 +a3x3 +a4x4 +a5x5 +a6x6)/( √ 2−p), x = ∆µ/ε, p = 3/(2 √ 2) ≈1.061, a1 = 0.15676, a2 = −0.19464, a3 = 0.13698, a4 = −0.055575, a5 = 0.012133, a6 = −0.0011059. (12) (a1x+a2x2 +a3x3 +a4x4 +a5x5 +a6x6)/( √ 2−p), x = ∆µ/ε, p = 3/(2 √ 2) ≈1.061, (12) 15676, a2 = −0.19464, a3 = 0.13698, a4 = −0.055575, a5 = 0.012133, a6 = −0.0011059. (12 10/13 The vs value at p = 0.09 was conﬁrmed to equal vneg s at p = 1.061 within a difference of 5%. The results obtained with p = 1.061 were employed to determine vs because there was a lack of nucleation on the (111) terraces for negative steps due to the RSOS restriction. References 1. Akutsu, N. Sci. Rep. 2021, 11, 3711, 1-11. 1. Akutsu, N. Sci. Rep. 2021, 11, 3711, 1-11. 2. Nishinaga, T.; Sasaoka, C.; Chernov, A.A. A numerical analysis for the supersaturation distribution around LPE macrostep. Morphology and Growth Unit of Crystals; Sunagawa, I., Ed.; Terra Scientiﬁc Publishing Company: Tokyo, Japan, 1989. 3. Abraham, F.F.; Broughton, J.Q. Pulsed Melting of Silicon (111) and (100) Surfaces Simulated by Molecular Dynamics. Phys. Rev. Lett. 1986 56, 734–737. 4. Weeks, J.D. The roughening transition. Ordering in Strongly Fluctuation Condensed Matter Systems; Riste, T., Ed. Plenum, New York, NY, USA; London, UK, 1980; p. 293. 5. van Beijeren, H. Exactly Solvable Model for the Roughening Transition of a Crystal Surface. Phys. Rev. Lett. 1977, 38, 993–996. 6. Berezinskii, V. L., Destruction of Long-range Order in One-dimensional and Two-dimensional Systems having a Continuous Symmetry Group I. Classical Systems. Sov. Phys. JETP 1971, 32, 493- 500. 7. Kosterlitz, J.M.; Thouless, D.J. Ordering, metastability and phase transitions in two-dimensional systems. J. Phys. C 1973, 6, 1181–1203. 8. Jayaprakash, C.; Saam, W.F.; Teitel, S. Roughening and facet formation in crystals. Phys. Rev. Le 9. Rottman, C.; Wortis, M. Statistical mechanics of equilibrium crystal shapes: Interfacial phase diagrams and phase transitions. Phys. Rep. 1984, 103, 59–79. 10. Akutsu, N.; Akutsu, Y. Roughening, faceting and equilibrium shape of two-dimensional anisotropic interface. I. Thermo- dynamics of interface ﬂuctuations and geometry of equilibrium crystal shape. J. Phys. Soc. Jpn. 1987, 56, 1443–1453. 11. Akutsu, N.; Akutsu, Y. Equilibrium Crystal Shape: Two Dimensions and Three Dimensions. J. Phys. Soc. Jpn. 1987, 56, 2248–2251. 12. Akutsu, Y.; Akutsu, N.; Yamamoto, T. Universal jump of Gaussian curvature at the facet edge of a crystal. Phys. Rev. Lett. 1988, 61, 424–427. 13. Burton, W.K.; Cabrera, N.; Frank, F.C. The growth of crystals and the equilibrium structure of their surfaces. Philos. Trans. Roy. Soc. Lond. A, 19514, 243, 299–358. 14. Cahn, J. W. Theory of crystal growth and interface motion in crystalline materials. Acta Metallurgica 1960, 8, 554–562. 15. A. Ookawa, Crystal Growth. Sy¯okab¯o, Tokyo, 1977, in Japanese. 15. A. Ookawa, Crystal Growth. Sy¯okab¯o, Tokyo, 1977, in Japanese. 15. A. Ookawa, Crystal Growth. Sy¯okab¯o, Tokyo, 1977, in Japanese. 16. Saito, Y. Statistical Physics of Crystal Growth. World Scientiﬁc, New Jersey, London, Singapore, Hong Kong, 1996. 16. Saito, Y. Statistical Physics of Crystal Growth. World Scientiﬁc, New Jersey, London, Singa 17. Pimpinelli, A.; Villain, J. Physics of Crystal Growth. References Cambridge University Press, Cambridge, New York, Port Chester, Mellbourne, Sydney,, 1998. 18. Uwaha, M. Crystal Growth Mechanisms. Kyoritsu Publishing, Tokyo, 2002, in Japanese. Crystal Growth Mechanisms. Kyoritsu Publishing, Tokyo, 2002, in Japanese. 19. Kardar, M.; Parisi,G.; Zhang, Y.-C., Dynamic Scaling of Growing Interfaces. Phys. Rev. Lett., 1986, 56, 889–892. 20. Vicsek, T. Surface Disordering: Growth, Roughening, and Phase Transitions, p. 155, eds. Jullien, R.; Kertesz, J. Meakin, P.; Wolf, D. E. Nova Science, New York, 1992. 21. Barabasi, A.L.; Stanley, H.E. Fractal Concepts in Surface Growth. Cambridge University Press, Cambridge, New York, Port Chester, Mellbourne, Sydney, 1995. 22. Krug, J.; Spohn, H. Solids Far From Equilibrium, p.479, ed. Godr`eche, C. Cambridge University Press, Cambridge, New York, Port Chester, Mellbourne, Sydney, 1991. 23. Krug, J. Origins of scale invariance in growth processes. Adv. Phys., 1977, 46, 139–282. 23. Krug, J. Origins of scale invariance in growth processes. Adv. Phys., 1977, 46, 139–282. 11/13 24. Takeuchi, K. A., Crossover from Growing to Stationary Interfaces in the Kardar–Parisi–Zhang Class. Phys. Rev. Lett., 2013, 110, 210604. 25. Pagnani, A.; Parisi, G., Numerical estimate of the Karder–Parisi–Zhang universality class in (2+1) dimensions. Phys. Rev. Lett., 2015, 92, 010101. 26. Takeuchi, K. A., An appetizer to modern developments on the Kardar–Parisi–Zhang universality class. Physica A, 2018, 504, 77–105. 27. Krim, J.; Palasantzas, G., Experimental observations of self-afﬁne scaling and kinetic roughening at sub-micron length scales. Int. J. Mod. Phys. B 1995, 9, 599–632. 28. Gupta, I.; Mohanty, B. C., Dynamics of surface evolusion in semiconductor thin ﬁlms grown from a chemical bath. Sci. Rep. 2016, 6, 33136. 29. Almeid, R.A.L.; Ferreira, S.O.; Ferraz, I.; Oliveira,T.J. Initial pseudo-steady state & asymptotic KPZ universality in semiconductor on poymer deposition. Sci. Rep., 2017, 7 3773. 30. den Nijs, M., Corrections to scaling and self-duality in the restricted solid-on-solid model. J. Phys. A, Math. Gen. 1985, 18, L549–L556. 31. Akutsu, N. Faceting diagram for sticky steps. AIP Adv. 2016, 6, 035301. Faceting diagram for sticky steps. AIP Adv. 2016, 6, 035301. 32. Markov, I.V. Crystal Growth for Beginners: Fundamentals of Nucleation, Crystal Growth and Epitaxy, 2nd ed.. World Scientiﬁc, Singapore, 2003. 33. Akutsu, N. Height of a faceted macrostep for sticky steps in a step-faceting zone. Phys. Rev. Mater. 2018, 2, 023603. 34. Akutsu, Y.; Akutsu, N. Interface tension, equilibrium crystal shape, and imaginary zeros of partition function: Planar Ising systems. Phys. Rev. Lett. 1990, 64, 1189–1192. 35. 48. Widom, B. Statistical Mechanics: A Concise Introduction for Chemists; Cambridge University Press, Cambridge, New York, Port Chester, Mellbourne, Sydney, 2002. References Akutsu, N.; Akutsu, Y. Statistical mechanical calculation of anisotropic step stiffness of a two-dimensional hexagonal lattice-gas model with next-nearest-neighbor interactkions: application to Si(111) surface. J. Phys.: Condens. Matter 1999, 11, 6635–6652. 36. N. Akutsu, ”Crossover from BKT-Rough to KPZ-Rough Surfaces for Interface-Limited Crystal Growth/Recession”, Sci. Rep., 2020, 10, 13057, 1–11. 37. Krug, J.; Spohn, H. Mechanism for Rough-to Rough transitions in Surface Growth. Phys. Rev. Le 37. Krug, J.; Spohn, H. Mechanism for Rough-to Rough transitions in Surface Growth. Phys. Rev. Lett., 1990, 64, 2332–2332. 38. Wolf, D.E., Kinetic Roughening of Vicinal Surface. Phys. Rev. Lett., 1991, 67, 1783–1786. 38. Wolf, D.E., Kinetic Roughening of Vicinal Surface. Phys. Rev. Lett., 1991, 67, 1783–1786. 38. Wolf, D.E., Kinetic Roughening of Vicinal Surface. Phys. Rev. Lett., 1991, 67, 1783–1786. 39. Gruber, E.E.; Mullins, W.W. On the theory of anisotropy of crystalline surface tension. J. Phys. Chem. Solids 1967, 28, 875-887. 40. Pokrovsky, V.L.; Talapov, A.L. Ground state, spectrum, and phase diagram of two-dimensional incommensurate crystals. Phys. Rev. Lett. 1979, 42, 65–67. 41. Yamamoto, T.; Akutsu, Y.; Akutsu, N. Fluctuation of a Single Step on the Vicinal Surface –Universal and Non-Universal Behaviors. J. Phys. Soc. Jpn., 1994, 63, 915–925. 42. Akutsu, Y.; Akutsu, N.; Yamamoto, T. Logarithmic step ﬂuctuations in vicinal surface: a Monte Carlo study. J. Phys. Soc. Jpn. 1994, 63, 2032–2036. 43. Kreyszig, E. Introduction to Differential Geometry and Riemannian Geometry (University of Tronto Press, 1968). 44. Akutsu, N.; Akutsu, Y. Slope–Temperature Faceting Diagram for Macrosteps at Equilibrium. Sci. Rep., 2022, 12, 17037, 1–11. 45. Akutsu, N. Relationship Between Macrostep Height and Surface Velocity for a Reaction-Limited Crystal Growth Process. Cryst. Growth Des. 2019, 19, 2970–2978. 46. Akutsu, N. Proﬁle of a Faceted Macrostep Caused by Anomalous Surface Tension. Adv. Condens. Matter Phys. 2017, 2017, 2021510. 47. Supplementaly movie 1. See Supplemental Material at [URL will be inserted by publisher] for data representing the growth of inclined surfaces having steps with (001) or (111) terraces (that is, negative steps), with p = 5 √ 2/4, L = 40 √ 2, kBT/ε = 0.4 and ∆µ/ε = 1.0. Here, the height of the (001) surface is expressed in the same manner as in the images shown as Fig. 7. The (111) layers are indicated by applying alternating blue and pink coloration. 48. Widom, B. Statistical Mechanics: A Concise Introduction for Chemists; Cambridge University Press, Cambridge, New York, Port Chester, Mellbourne, Sydney, 2002. 47. Supplementaly movie 1. See Supplemental Material at [URL will be inserted by publisher] for data representing the growth of inclined surfaces having steps with (001) or (111) terraces (that is, negative steps), with p = 5 √ 2/4, L = 40 √ 2, kBT/ε = 0.4 and ∆µ/ε = 1.0. Here, the height of the (001) surface is expressed in the same manner as in the images shown as Fig. 7. The (111) layers are indicated by applying alternating blue and pink coloration. Acknowledgements The author wishes to acknowledge the encouragement provided by Prof. T. Sasada, Prof. T. Koshikawa, Prof. Y. Kangawa and Prof. T. Ohachi. This work was supported by a KAKENHI Grant-in-Aid (no. JP22K03487) from the Japan Society for the Promotion of Science (JSPS). This work was also supported in part by a Collaborative Research Program (2022S3–CD–1, 2023S3–CD–1) of the Research Institute for Applied Mechanics, Kyushu University. The author wishes to acknowledge the encouragement provided by Prof. T. Sasada, Prof. T. Koshikawa, Prof. Y. Kangawa and Prof. T. Ohachi. This work was supported by a KAKENHI Grant-in-Aid (no. JP22K03487) from the Japan Society for the Promotion of Science (JSPS). This work was also supported in part by a Collaborative Research Program (2022S3–CD–1, 2023S3–CD–1) of the Research Institute for Applied Mechanics, Kyushu University. References 12/13 49. Akutsu, N. Equilibrium Crystal Shape of Planar Ising Antiferromagnets in External Fields. J. Phys. Soc. Jpn. 1992, 61, 477–498. 50. Kempisty, P.; Kangawa, Y. Evolution of the free energy of the GaN(0001) surface based on ﬁrst-principles phonon calculations. Phys. Rev. B 2019, 100, 085304. 51. Akutsu, Y.,Exact Landau Free-Energy of Solvable N-State Vertex Model. J. Phys. Soc. Jpn., 19 51. Akutsu, Y.,Exact Landau Free-Energy of Solvable N-State Vertex Model. J. Phys. Soc. Jpn., 1989 58, 2219–2222. 52. Bethe, H. A. Zur theorie der metalle. Zeit. f¨ur Physik 1931, 71 205–226. gy y p 52. Bethe, H. A. Zur theorie der metalle. Zeit. f¨ur Physik 1931, 71 205–226. 52. Bethe, H. A. Zur theorie der metalle. Zeit. f¨ur Physik 1931, 71 205–226. 53. Amar, J. G.; Family, F. Phase Transition in a Restricted Solid-onSolid Surface-Growth Model in 2+1 Dimensions. Phys. Rev. Lett., 1990, 64, 543–546. 54. M¨uller-Krumbhaar, H. Monte Carlo Simulation of Crystal Growth. Monte Carlo Mehtods in Statistical Mechanics; Binder, K. Ed. Springer-Verlag; Berlin, Heidelberg, Germany; New York, USA, 1979; p.261–299. Author contributions statement N.A. conceived and conducted the Monte Carlo calculations and analyzed the results. Additional information Supplementary information is available for this paper at a URL. Supplementary information is available for this paper at a URL. Competing ﬁnancial interests: The authors declare no competing interests. Competing ﬁnancial interests: The authors declare no competing interests. 13/13 Supplementary Files This is a list of supplementary ¦les associated with this preprint. 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https://openalex.org/W3031476593	https://zenodo.org/record/3733004/files/11_IJRG20_B03_3144.pdf	English	null	WAGE LEGAL SYSTEM BASED ON THE CONCEPT OF THE PANCASILA WELFARE STATE	International journal of research - granthaalayah	2,020	cc-by	7,546	Abstract Indonesia as a Welfare State based on Pancasila is tasked with carrying out people's prosperity, the state is allowed to interfere in the private lives of its citizens, one of which interferes in the Employment field including in the wage field, which has a variety of main problems including: Low wages for lower workers, lowest wage gap and highest, Variation in the wage component, Unclear relationship between wages and productivity, Workers' living needs, Social inequality, Work performance, and Human values and self-esteem. So, the purpose of this article is to analyze the legal system of remuneration based on theories and viewpoints of legal goals. The conclusion of this article is that the legal system of remuneration needs to be assessed based on theories of legal objectives such as ethical theory, utility theory and theorizing theory and based on legal objectives from the point of view, namely the point of view of positive-normative or dogmatic juridical law, where the objective of wage law is at emphasize the legal certainty, the point of view of legal philosophy, where the goal of wage law is emphasized in terms of justice, and the viewpoint of the sociology of law, where the purpose of wage law is emphasized on the benefit aspect. The results of the study are based on the theories and the objectives of the legal field, it is expected to be able to describe the wage legal system that can be in line with the ultimate goal of industrial relations, namely “the welfare of all parties (employers and workers)” or the wage legal system that fulfills a decent living for humanity that correlates with company productivity or profit. Keywords: Country; Pancasila; Welfare; Wages Law. Cite This Article: Ahmad Hunaeni Zulkarnaen. (2020). “WAGE LEGAL SYSTEM BASED ON THE CONCEPT OF THE PANCASILA WELFARE STATE.” International Journal of Research - Granthaalayah, 8(3), 86-97. https://doi.org/10.5281/zenodo.3733004. Cite This Article: Ahmad Hunaeni Zulkarnaen. (2020). “WAGE LEGAL SYSTEM BASED ON THE CONCEPT OF THE PANCASILA WELFARE STATE.” International Journal of Research - Granthaalayah, 8(3), 86-97. https://doi.org/10.5281/zenodo.3733004. Social WAGE LEGAL SYSTEM BASED ON THE CONCEPT OF THE PANCASILA WELFARE STATE Ahmad Hunaeni Zulkarnaen 1 1 Postgraduate Law, Universitas Suryakancana, Indonesia ISSN- 2350-0530(O), ISSN- 2394-3629(P) Index Copernicus Value (ICV 2018): 86.20 DOI: 10.5281/zenodo.3733004 ISSN- 2350-0530(O), ISSN- 2394-3629(P) Index Copernicus Value (ICV 2018): 86.20 DOI: 10.5281/zenodo.3733004 [Zulkarnaen , Vol.8 (Iss.3): March 2020] Social 1. Introduction The concept of the state of Indonesia, is a democratic rule of law based on Pancasila, this can be seen in the formulation of paragraph 4 (four) of the Preamble of the 1945 Constitution which states that than that ... to the Almighty God, just and civilized humanity, the Indonesian Unity and Society which is led by wisdom in Consultation or Representation, and by realizing a social justice for all Indonesian people. Http://www.granthaalayah.com ©International Journal of Research - GRANTHAALAYAH [86] ISSN- 2350-0530(O), ISSN- 2394-3629(P) Index Copernicus Value (ICV 2018): 86.20 DOI: 10.5281/zenodo.3733004 [Zulkarnaen , Vol.8 (Iss.3): March 2020] In addition to adhering to the concept of a democratic rule of law based on Pancasila, Indonesia also adheres to the concept of a Prosperity State (Wohlfaart Staats). The state of Indonesia which protects all of the Indonesian people and all of Indonesia's blood and to promote public welfare …… .. The type of Indonesian Prosperity State is "organizing people's prosperity based on Pancasila and the 1945 Constitution". In the concept of the state of Indonesia as a Prosperity State, the state is the only tool for carrying out people's prosperity. Here the state is active in organizing the prosperity of its citizens for the benefit of all the people and the state, in the type of prosperity the duty of the state (Indonesia) is merely to carry out the people's prosperity as much as possible [1]. Because the state is active in organizing the prosperity of its citizens for the benefit of all the people and the state, for that the Indonesian state is allowed to interfere in the private lives of its citizens, one of which, is that the Indonesian state interferes in the field of Manpower including in the area of wages. The existence of interference by the Indonesian state in the area of wages is motivated by wage problems that always arise which are triggered by conflicts of interest between employers and workers. The main problems of remuneration include a) Low wages for lower workers; b) The lowest and highest wage gap; c) Variation in the wage component; d) Unclear relationship between wages and productivity [2]. 1. Introduction For this reason, in this scientific paper, the author tries to explain the correlation of the purpose of wage law which refers to several theories of the purpose of law, so that the outcomes of this scientific paper are the wage problems as described above, so that the wage legal system can be used as an instrument to realize the goal of Indonesia as a Prosperity State in an effort to realize prosperity or welfare for all Indonesian people, namely a wage legal system that meets a decent living for humanity which correlates with productivity or corporate profits. 2.1. Indonesia Is A Welfare State Based on Pancasila If we trace the discussion in the sessions of the Indonesian Independence Preparatory Agency for Investigation (BPUPKI), we will find an opinion that wishes that an independent Indonesian state to be formed is a welfare state, a state that is sovereign of the people, a country that wants to realize justice, a state that guarantees health the people, a country that guarantees the freedom of the people to associate, gather and express opinions. This was explained by M. Yamin, Soekarno, Hatta and others [3]. M. Yamin, among others, said: “........ that the state to be formed is only for the entire people, for the benefit of the entire nation which will stand strong within its own state [4]. He further added: The people's welfare which is the basis and purpose of an independent Indonesian state is to summarize community justice or social justice [5]. 2.1. Indonesia Is A Welfare State Based on Pancasila On the same occasion Sukarno said: "People who had felt themselves lacking in food, lacked clothes, created a new world in which there was justice, under the leadership of Ratu Adil, therefore if we really truly understood, remembered, loved the people: Indonesia, let us accept the Http://www.granthaalayah.com ©International Journal of Research - GRANTHAALAYAH [87] [Zulkarnaen , Vol.8 (Iss.3): March 2020] ISSN- 2350-0530(O), ISSN- 2394-3629(P) Index Copernicus Value (ICV 2018): 86.20 DOI: 10.5281/zenodo.3733004 principle of the rechtsvaardigheid sociale, that is not only political equality, even though on the economic field we must hold equality, meaning the best possible joint welfare [6] " ISSN- 2350-0530(O), ISSN- 2394-3629(P) Index Copernicus Value (ICV 2018): 86.20 [Zulkarnaen , Vol.8 (Iss.3): March 2020] DOI: 10.5281/zenodo.3733004 principle of the rechtsvaardigheid sociale, that is not only political equality, even though on the economic field we must hold equality, meaning the best possible joint welfare [6] " principle of the rechtsvaardigheid sociale, that is not only political equality, even though on the economic field we must hold equality, meaning the best possible joint welfare [6] " Furthermore, it is good to see that Sukiman's opinion which is more concerned with power is in the people rather than the formal form of the state, whether a republic or kingdom; he said: For me about the republic or kingdom, in this day and age, it is a matter of "etiquette" only, because in it the guaranteed or recognized power that is in the people is the sovereignty of the people, even in a state in the form of a kingdom; in modern times there is no longer a form of kingdom "despotisch" or "feudalisch" as before especially for our nation that already has a level of civilization that is not inferior to some other countries on earth [7]. If we pay attention to this, it can be concluded that the state desired by the Indonesian people is a country that guarantees the welfare of the people, which guarantees justice and human rights. Such a state cannot be otherwise a rule of law [8]. If the concept of continental European rule of law and the concept of the Anglo-Saxon rule of law are based on individualistic liberalism, then the concept of the rule of law of Indonesia is based on the Indonesian way of life, Pancasila [9]. 2.1. Indonesia Is A Welfare State Based on Pancasila Political organizations are always closely Http://www.granthaalayah.com ©International Journal of Research - GRANTHAALAYAH [88] [Zulkarnaen , Vol.8 (Iss.3): March 2020] ISSN- 2350-0530(O), ISSN- 2394-3629(P) Index Copernicus Value (ICV 2018): 86.20 DOI: 10.5281/zenodo.3733004 monitored, even broadcasting religion (Islam) continues to be overshadowed by the Dutch secret service (PID) [14] [Zulkarnaen , Vol.8 (Iss.3): March 2020] DOI: 10.5281/zenodo.3733004 monitored, even broadcasting religion (Islam) continues to be overshadowed by the Dutch secret service (PID) [14] What is the nature of the state in the view of the Indonesian people? According to Padmo Wahjono, the state is "the life of a group of Indonesian people who, thanks to the grace of God Almighty, are driven by a noble desire for a free national life" this is the nature of the state according to the ideals of the Pancasila state [15]. Based on the explanation above, Indonesia adheres to the concept of a welfare state, which means that people's welfare is the basis and objective of an independent Indonesian state. In short, community justice or social justice, or Indonesia adheres to the concept of the State of Material Law (based on Pancasila), a material state law is a development of the state formal law, in the formal state of law the actions of the authorities must be based on the law or the principle of legality must apply, in the material state law in matters of urgency and in the interests of citizens, the authorities are justified to act in violation of the law (the principle of opportunity) [16]. The development of society and the needs of the community (Indonesia) are not enough if only formally regulated by the principle of legality, as a result the formal legal state has been criticized quite sharply in the Netherlands, so Scheltema considers that there are many policy actions from the government in various provisions. This is made possible by the delegation of legislative power to the government in making its implementing regulations, and the existence of freisermessen allows the government to guarantee fairer order in an effort to meet the needs of the community [17]. The purpose of delegation by forming this law, is because the task of state administrators is no longer just to maintain the existing order, but also to issue a fair order. 2.1. Indonesia Is A Welfare State Based on Pancasila The difference in this case mainly lies in the problem of the position of the individual against society and the rights and obligations of individuals towards the community, this difference is due to the influence of worldview and historical background of the Indonesian nation. Therefore, the concept of the rule of law in Indonesia is also different from the concept of a liberal rule of law. The concept of the Indonesian rule of law is a democratic rule of law based on Pancasila [10]. Indonesia formed its country by proclamation which is an embodiment of a one-goal agreement, the country desired by the Indonesian people, the answer was found in the Preamble to the 1945 Constitution paragraph 2 (two), namely: “............. the Indonesian state, which is independent, united, sovereign, just and prosperous [11]”. And if the sentence is connected to the state's purpose contained in paragraph 4 which reads: “............ protect all the people of Indonesia and all of Indonesia's blood and to promote public welfare, educate the nation's life, and participate in carrying out world order, based on independence, eternal peace and social justice [12]”. Then it can be concluded that the state that was intended to be formed (at that time) by the Indonesian nation was a “welfare state” This was revealed by Sukarno in the BPUPKI (Indonesian Investigation Preparatory Agency Preparatory Agency) session on June 1, 1945, as follows: " The people want prosperity, people who were previously lacking in food, clothing, creating a new world in which there is justice, under the leadership of Ratu Adil, therefore, if we really truly understand, remember, love the people of Indonesia, let us accept the principle of things rechtvaardigheid sociale (social justice), that is not only political equality, but also on the economic field, we must hold equality, meaning the best common welfare [13]. In Western literature the "welfare state" is called verzorggingsstaat or sociale rechtsstaat. Prosperity means material and spiritual prosperity, the choice of the Indonesian people directly to the welfare state, because of the bitter experience of the Indonesian people under the Dutch colonialism which not only lacked food and clothing. But it also lacks spiritual well-being, because the Indonesian people have almost no freedom. 2.2. Government Interventions in The Wage Sector Wages are one of the most sensitive aspects of work relations and industrial relations. Between 70% -80% of cases that occur in employment relations and industrial relations contain wage problems and various related aspects, such as benefits, wage increases, wage structures, wage scales and so on [23]. For this reason, government intervention is needed, because the government is very interested in harmonizing wages that fulfill a decent life for humanity and achieving work productivity, by taking into account: a) The living needs of workers; b) Social inequality; c) Job performance; and d) Human values and dignity [24]. The government adopted a Minimum Wage Determination policy that was based on the Minimum Physical Needs (KFM) developing into the Minimum Living Needs (KHM), applied on a micro- regional basis with the intention of: a) as a safety net; b) As a means to improve the lives of the lowest groups; c) as a tool for income distribution; d) Wages above the minimum wage are regulated internally in the company [25]. As explained above, government policies in the area of wages are motivated by wage problems that always arise which are triggered by conflicts of interest between employers and workers. The main problems of remuneration include a) Low wages for lower workers; b) The lowest and highest wage gap; c) Variation in the wage component; d) Unclear relationship between wages and productivity [26]. 2.1. Indonesia Is A Welfare State Based on Pancasila For example: 1) Article 42 paragraph (1) of Law Number 13 of 2003 concerning Manpower (UUK), concerning the need for permits to use foreign workers; 2) Article [89] Http://www.granthaalayah.com ©International Journal of Research - GRANTHAALAYAH [Zulkarnaen , Vol.8 (Iss.3): March 2020] ISSN- 2350-0530(O), ISSN- 2394-3629(P) Index Copernicus Value (ICV 2018): 86.20 DOI: 10.5281/zenodo.3733004 59 paragraph (1) of the Law on Manpower, regarding the provisions on making certain time work agreements (PKWT); 3) Article 153 paragraph (1) UUK, prohibition on terminating work relations (PHK) on certain cases; 4) Article 13 Regulation of the Minister of Manpower Number PER-01 / MEN / 1999, concerning prohibitions for employers who pay wages lower than the Minimum Wage [21]. ISSN- 2350-0530(O), ISSN- 2394-3629(P) Index Copernicus Value (ICV 2018): 86.20 DOI: 10.5281/zenodo.3733004 [Zulkarnaen , Vol.8 (Iss.3): March 2020] 59 paragraph (1) of the Law on Manpower, regarding the provisions on making certain time work agreements (PKWT); 3) Article 153 paragraph (1) UUK, prohibition on terminating work relations (PHK) on certain cases; 4) Article 13 Regulation of the Minister of Manpower Number PER-01 / MEN / 1999, concerning prohibitions for employers who pay wages lower than the Minimum Wage [21]. 59 paragraph (1) of the Law on Manpower, regarding the provisions on making certain time work agreements (PKWT); 3) Article 153 paragraph (1) UUK, prohibition on terminating work relations (PHK) on certain cases; 4) Article 13 Regulation of the Minister of Manpower Number PER-01 / MEN / 1999, concerning prohibitions for employers who pay wages lower than the Minimum Wage [21]. While the law which is facultative or regelendrecht or aanvul-lenrecht (the law that regulates / complements), the law can be ruled out in its implementation. For example: 1) Article 51 paragraph (1) of the Manpower Act, regarding employment agreements can be written or unwritten; 2) Article 60 paragraph (1) of the Manpower Act, regarding work agreements for an indefinite period of time may require a trial period of 3 (three) months; 3) Article 16 Government Regulation No. 8 of 1981 concerning employers' freedom to pay salaries in the usual place [22]. 2.1. Indonesia Is A Welfare State Based on Pancasila For this reason, a wider scope of freedom of action by the government is needed, namely through increasing the provision of freisermessen to the government to organize a welfare state based on Pancasila [18]. In the concept of a prosperous state or wohlfaartstaats the state fully serves the community, the state is the only means for carrying out people's prosperity. Here the state is active in organizing the prosperity of its citizens for the benefit of all the people and the country. So in this type of prosperity the task of the state is merely to carry out the people's prosperity as much as possible [19]. Because the state is active in organizing the prosperity of its citizens for the benefit of all the people and the country, the Indonesian state is allowed to interfere in the private lives of its citizens, one of which, is that the Indonesian state interferes in the field of Manpower including in the wage field for example with the Government Regulation No. 08 of 1981 regarding Wage Protection, so specifically labor law or wage law originally based on the contents of the rule of law included in the category of Private (civil) Law Rule entered into the Public Law Rule, because in order to tackle certain employment problems government (state) intervention requires, among others : 1) In the form of: a) Licenses involving the field of employment; b) Determination of minimum wages (wage protection); c) Problems in resolving industrial relations disputes or termination of employment, and so on. 2) The existence of the application of sanctions against labor violations or criminal acts [20]. Budiono divides the nature of Labor Law into 2 (two), which are imperative and facultative. Law which is imperative or dwingwnrecht (law forcing), meaning that law must be obeyed absolutely, must not be violated. 2.3. The Purpose of Legal Wages and Legal Theories The definition of wages according to Government Regulation No. 08 of 1981 concerning Wage Protection, is: “An acceptance in return from employers to workers for a job or service that has been or will be done, expressed or valued in the form of money that has been determined according to an agreement or legislation, regulations, and are paid on the basis of a work agreement between the employer and the worker, including benefits for both the laborers themselves and their families”. According to Article 1 point 30 of Law Number 13 Year 2003 concerning Manpower (UUK), wages are: “Workers' rights are received and expressed in money as compensation from employers [90] Http://www.granthaalayah.com ©International Journal of Research - GRANTHAALAYAH [Zulkarnaen , Vol.8 (Iss.3): March 2020] ISSN- 2350-0530(O), ISSN- 2394-3629(P) Index Copernicus Value (ICV 2018): 86.20 DOI: 10.5281/zenodo.3733004 or employers to workers who are determined and paid according to an agreements, agreements or laws and regulations, including benefits for workers and their families for work or services that have been or will be performed”. [Zulkarnaen , Vol.8 (Iss.3): March 2020] or employers to workers who are determined and paid according to an agreements, agreements or laws and regulations, including benefits for workers and their families for work or services that have been or will be performed”. or employers to workers who are determined and paid according to an agreements, agreements or laws and regulations, including benefits for workers and their families for work or services that have been or will be performed”. Based on Article of the 1945 Constitution it is stated that wages must fulfill a decent living for humanity. Thus, the fulfillment of a decent wage for livelihoods and humanity is a concept of remuneration that is constitutionally applicable in Indonesia. Therefore, manpower law defines wages as basic rights of workers that must be fulfilled by employers. If employers do not pay workers' wages, this is a violation of human rights, and is a crime that can be convicted. The concept of wages in Indonesia today is shifting from civil rights to violations of human rights that are criminal in nature [27]. According to Suwarto [28], with the above definition of wages, on one hand wages are workers 'rights and employers' obligations, on the other hand workers are obliged to give time, energy and thoughts to work or provide services. 1) Ethical Theory ) y Ethical theory holds that the purpose of law (wage law: the author) is to bring about justice, according to Aristotle, justice means giving everyone what is part of or their rights (ius suum cuique tribuere). Part of the rights of each person is not the same, Aristotle distinguishes the existence of 2 (two) types, namely: a) Distributive justice, is justice that gives each person (worker) a part or ration (his salary) in accordance with his services, which is the principle of distributive justice rather than equality of parts, but comparability, meaning that by not ignoring the principle of remuneration must fulfill a decent living for humanity, workers with higher work productivity must be given higher wages than workers with lower productivity. Workers who have longer service periods, naturally receive higher wages than workers whose tenure has not been long, workers whose positions are higher or have greater responsibilities, receive higher wages than workers with lower positions and responsibilities smaller answer; b) Commutative justice, justice that gives to every person (worker) the same amount without regard to his services, which becomes the principle of commutative justice is the principle of equality [29]. For example, every worker has the right to get a minimum wage of a City or Regency Minimum Wage (UMK). 2.3. The Purpose of Legal Wages and Legal Theories In addition, we also hold that wages also have a social nature, where the amount of wages and benefits must be able to meet the needs of families or wages must meet a decent living for humanity, namely meeting the needs of home, board, food, education, health, recreation and others -other. So that the wage legal system can overcome wage problems that always arise that are triggered by conflicts of interest between employers and workers as described above. The purpose of wage law can refer to the purpose of law, many theories about the purpose of law, including the purpose of wage law. 3) Theory of Protection y Law (wage law) aims to provide protection or protect people (employers and workers), means to protect people (employers and workers) in the passive and active sense [32]: y Law (wage law) aims to provide protection or protect people (employers and workers), means to protect people (employers and workers) in the passive and active sense [32]: • Protecting humans (employers and workers) in the passive sense, ie preventing arbitrary acts and violations of rights. Wage law to protect workers from arbitrary acts and violations of rights by employers, for example the amount of wages given by employers does not violate Government Regulation No. 08 of 1981 concerning Wage Protection. For workers, wages are a source of income that can be used to make ends meet. Therefore, in accordance with the objectives of someone working, then through increasing one's welfare wages can be increased. Because if wages get bigger, the greater the chance for someone to be able to meet and improve their standard of living, such as meeting the needs for clothing, food, shelter, health, recreation and others. For employers, the salary is not only as a production cost but also as an instrument to increase productivity, work ethic, work discipline of workers in an effort to increase productivity and company profits from time to time. There are several factors that affect the provision of wages at the company level, namely: a) Education and training; b) Labor market conditions; c) Proportion of wage costs with other costs; d) The use of technology; e) Company capability; f) The ability of workers' organizations; g) Government policies and interventions [33]. • Protect humans in the active sense, that is: • Protect humans in the active sense, that is: 1) Includes all efforts to create social conditions that open the widest possible way and encourage humans to continuously humanize themselves, the purpose of the law is to create humane social conditions that enable social processes to take place naturally, so that every human being has a fair opportunity broad to develop the potential (talents and abilities) of humanity as a whole [34]. 2) Utility Theory ) Ut ty eo y Law (wage law) aims to realize what is useful or useful (doelmatig) for people (employers and workers), which is to realize happiness as much as possible for as many people (entrepreneurs and workers). Only in order does everyone (employers and workers) get the chance to realize happiness as much as possible [30]. Based on the theory of utility, the objective of wage law must be to make as much useful or useful happiness as possible for all parties involved in industrial relations [91] Http://www.granthaalayah.com ©International Journal of Research - GRANTHAALAYAH [Zulkarnaen , Vol.8 (Iss.3): March 2020] ISSN- 2350-0530(O), ISSN- 2394-3629(P) Index Copernicus Value (ICV 2018): 86.20 DOI: 10.5281/zenodo.3733004 (employers and workers), for wage law employers who are useful and useful, which can increase worker productivity in efforts to increase productivity or profit of the company, for wage law workers who are useful and useful is a wage law that can improve the welfare of workers and their families. This is in accordance with the ultimate goal of industrial relations arrangements (wage arrangements) conveyed by Suwarto, is to improve the welfare of all parties (employers and workers). Achieving this requires an increase in productivity (company profits) from time to time that correlates with the welfare of workers and their families [31]. 3) Theory of Protection Based on the explanation above, the objective of wage law is to cover all efforts to create conditions of industrial relations that open the widest possible way and encourage all parties (employers and workers) to continuously humanize themselves, the purpose of wage law is to create conditions of industrial relations that humane that allows the processes of industrial relations to take place naturally, so that every party in industrial relations (employers and workers) fairly has the broad opportunity to develop their full potential (talents and abilities) for humanity, that is, a continual increase in productivity or profits companies that correlate with the welfare of workers or laborers and their families. 2) Maintaining and developing humane morality and the noble moral ideals of the people based on God (see explanation of the 1945 Constitution), the wage legal system can increase worker productivity which correlates with company productivity or profit, because the wage legal system can maintain and develop mind humanitarian character and 2) Maintaining and developing humane morality and the noble moral ideals of the people based on God (see explanation of the 1945 Constitution), the wage legal system can increase worker productivity which correlates with company productivity or profit, because the wage legal system can maintain and develop mind humanitarian character and Http://www.granthaalayah.com ©International Journal of Research - GRANTHAALAYAH [92] [Zulkarnaen , Vol.8 (Iss.3): March 2020] ISSN- 2350-0530(O), ISSN- 2394-3629(P) Index Copernicus Value (ICV 2018): 86.20 DOI: 10.5281/zenodo.3733004 ISSN- 2350-0530(O), ISSN- 2394-3629(P) Index Copernicus Value (ICV 2018): 86.20 DOI: 10.5281/zenodo.3733004 noble moral ideals of the people based on God, namely the wage legal system that can meet the needs of workers and their families, including meeting the needs of clothing, shelter, food, health, education, saving, recreation and others, while still can increase company productivity or profit from time to time [35]. noble moral ideals of the people based on God, namely the wage legal system that can meet the needs of workers and their families, including meeting the needs of clothing, shelter, food, health, education, saving, recreation and others, while still can increase company productivity or profit from time to time [35]. 3) Theory of Protection True peace (in industrial relations) will be realized if the members of the community (all parties involved in an industrial relationship) can feel inner peace, peace there will be if the community members (all parties involved in an industrial relationship) feel confident: 1) The survival of the exercise of rights does not depend on mere physical or non-physical strength (but depends on the productivity or profit of the company which correlates with the productivity or welfare of workers; 2) As long as they do not feel violating the rights or harming others without worrying the True peace (in industrial relations) will be realized if the members of the community (all parties involved in an industrial relationship) can feel inner peace, peace there will be if the community members (all parties involved in an industrial relationship) feel confident: 1) The survival of the exercise of rights does not depend on mere physical or non-physical strength (but depends on the productivity or profit of the company which correlates with the productivity or welfare of workers; 2) As long as they do not feel violating the rights or harming others, without worrying the community members (all parties involved in an industrial relationship): a) can freely carry out what he believes to be true (for example, workers carry out work in accordance with competencies and job descriptions or procedures set by the company); b) can freely develop their talents and pleasures (for example workers can increase their competence and the company can increase profits); c) feels that he will always receive fair, humane, fair and civilized treatment, even when he has made a mistake, that is, the worker or laborer is considered a subject of production or an employer partner, a partner in the production process, a partner in the company's profits, even a partner in the case of the company loss. Partners in profits, namely workers get a humane wage, wages that can meet the needs of workers and families, namely the needs of home, board, food, education, health, recreation and others [37]. 3) Theory of Protection p y p 3) Efforts to realize shelter, are businesses: a) Order and order (in an industrial relationship); b) True peace, peaceful peace (industrial peace); c) Justice (wage justice) includes, distributive justice, commutative, protective justice (social justice theory); d) Welfare and social justice (for all parties involved in industrial relations, for wage workers to improve the welfare of workers or laborers and their families, wage employers can motivate workers or laborers to improve work productivity, work ethics and work discipline) ; e) Maintenance and development of morals (noble character and ideals) based on the Almighty God for all parties involved in an industrial relationship [36]. 2.4. The Purpose of Legal Wages from Various Perspectives From the perspective of positive-normative or dogmatic juridical jurisprudence, where the purpose of law is emphasized on legal certainty. From the perspective of legal philosophy, where the purpose of law is emphasized in terms of justice. Enforce the same that should be treated the same (MSE). Not applying the same for something that should not be the same (old employees should have received a higher wage than new employees or fair contribution) [38]. The purpose of wage law according to various points of view, namely certainty and fairness is in line with the ultimate goal of industrial relations arrangements delivered by Suwarto, namely improving welfare for all parties (employers or workers), to achieve this it is necessary to increase productivity from time-to-time. Productivity can be achieved if there is work and business peace (Industrial peace) within the company, understanding of work and business peace (Industrial peace), including “rights and obligations (employers, workers) guaranteed and implemented [39]” which is a condition of working conditions (especially in the area of wages), which are divided Http://www.granthaalayah.com ©International Journal of Research - GRANTHAALAYAH [93] [Zulkarnaen , Vol.8 (Iss.3): March 2020] ISSN- 2350-0530(O), ISSN- 2394-3629(P) Index Copernicus Value (ICV 2018): 86.20 DOI: 10.5281/zenodo.3733004 into 2 (two) outlines, namely wage arrangements called labor legislation and terms of employment [40]. [Zulkarnaen , Vol.8 (Iss.3): March 2020] into 2 (two) outlines, namely wage arrangements called labor legislation and terms of employment [40]. Wages are work norms (labor legislation), are workers' wage arrangements contained in legislation. According to Suwarto [41] this work norm, is imperative which must be implemented because it is mandatory, so it is binding on all companies, so that it is minimal macro. Macro in the sense of binding all companies without exception both the place, size, type of business, the nature of the legal entity, etc., and minimal in the sense that in practice the matters regulated can be carried out better or larger depending on the ability and will of the company as a individual. this is in accordance with the purpose of wage law based on the perspective of positive-normative or juridical dogmatic jurisprudence, where wage legal objectives are emphasized on legal certainty, for example employers are prohibited from paying wages to their workers or laborers lower than the minimum city or regency minimum wage (Vide Article 90 paragraph (1) Manpower Law). 2.4. The Purpose of Legal Wages from Various Perspectives Wages that are in terms of employment (terms of employment), are wage arrangements that have not been regulated or not regulated by statutory regulations. According to Suwarto [42] the arrangement (wage) is conditional micro. Micro in the sense is regulated only for certain companies individually. Conditional in the sense of arrangement (wage: author) adjusted to the condition or ability of the company concerned, the form of remuneration is a work requirement, can be set forth in a Work Agreement (PK), Company Regulations (PP) and Collective Labor Agreement (PKB). Examples of wages that are micro conditional, namely each company has its own wage system, both in determining the size of the basic wage, the distance between the highest wage with the lowest wage and the level of wages according to skills or achievements and years of service. In addition to wages, companies usually provide benefits. Allowances are provided for various purposes and purposes, both as an appreciation for the responsibilities incurred (position allowance) as an incentive to increase discipline (attendance benefits), in addition to adjusting price differences for those who work in certain places (expensiveness benefits) and others [43]. In determining the size of the basic wage, the distance between the highest wage with the lowest wage and the level of wages according to skills or achievements and years of service or in providing benefits according to the conditions or abilities of each company concerned, meaning that each company will be different or it is not the same in determining the size of the basic wage, the distance between the highest wage and the lowest wage and in determining wage levels according to skills or achievements and years of service or in providing benefits to the workers or laborers. From the standpoint of legal sociology, where the purpose of law (wages) is emphasized in terms of benefits [44], namely the benefits of wages for workers or laborers, employers and the government. Wage benefits for workers or laborers are a source of income that can be used to meet their daily needs. Therefore, in accordance with one's goals of work, then through increasing one's welfare wages can be increased, the greater the wages, the greater the chance for someone to be able to meet and improve their standard of living, such as meeting the needs for clothing, food, shelter, health, recreation etc. [ 45]. 2.4. The Purpose of Legal Wages from Various Perspectives For workers or laborers is a source of income to meet the needs of themselves and their families. Therefore, the level of wages must be able to meet their minimum needs. Psychologically wages can also create satisfaction for workers or laborers. Institutional Http://www.granthaalayah.com ©International Journal of Research - GRANTHAALAYAH [94] [Zulkarnaen , Vol.8 (Iss.3): March 2020] ISSN- 2350-0530(O), ISSN- 2394-3629(P) Index Copernicus Value (ICV 2018): 86.20 DOI: 10.5281/zenodo.3733004 work performance for workers or laborers is also reflected among other things in the wage level. In addition, the macro level of wages also affects the overall purchasing power of the community [46]. ISSN- 2350-0530(O), ISSN- 2394-3629(P) Index Copernicus Value (ICV 2018): 86.20 DOI 10 5281/ d 3733004 [Zulkarnaen , Vol.8 (Iss.3): March 2020] work performance for workers or laborers is also reflected among other things in the wage level. In addition, the macro level of wages also affects the overall purchasing power of the community [46]. work performance for workers or laborers is also reflected among other things in the wage level. In addition, the macro level of wages also affects the overall purchasing power of the community [46]. For employers, wages are the cost of production. Therefore, every time an increase in wages means an increase in costs. But in human resource management wages must be seen as investment or human investment, i.e. increases in wages or labor welfare can be seen as improvements or improvements in the quality of human resources or workers, the results of which will be obtained later. If wages and welfare are better, it is possible to improve health and nutrition, improve skills through additional education, training, reading, discipline improvement, improvement of work conditions, increased morale, work calm and others. These factors will encourage an increase in work productivity [47], which leads to an increase in productivity or profits of the company. Therefore, wages should be associated with work productivity, which basically the level of productivity must be higher than the level of wages. As such, wages are one way to motivate workers to increase productivity and work ethic [48]. 3. Conclusions Indonesia adheres to the concept of a welfare state, meaning that people's welfare is the basis and goal of an independent Indonesian state in an effort to realize community justice or social justice, the state as the only tool to realize active state social justice in carrying out social justice for all Indonesian people, therefore the state allowed to interfere in the private lives of its citizens including in the field of employment, one of which is in the wage field, considering that wages also have a social nature, where the amount of wages and benefits must be able to meet the needs of workers and families, wages must meet a decent living for humanity, that is, meeting needs for clothing, housing, food, education, health, recreation and others. With the social legal system of remuneration, the legal system of remuneration is expected to be able to overcome the problem of remuneration that always arises which is triggered by a conflict of interest between employers and workers, the cause of a conflict of interest due to: a) Low wages for lower workers; b) The lowest and highest wage gap; c) Variation in the wage component; d) Unclear relationship between wages and productivity. Efforts to overcome conflicts of interest as described above, the formation of a wage legal system needs to be assessed based on theories and points of view of legal objectives. From theories of legal purposes such as: ethical theory, utility theory and guardian theory. Establishment of remuneration system based on legal objectives from the point of view, such as: From the perspective of positive-normative or dogmatic juridical law, where the goal of remuneration is emphasized on legal certainty, the perspective of legal philosophy, where the objective of remuneration law is emphasized in terms of justice, and the viewpoint of legal sociology, where the purpose of wage law is emphasized on the benefit side. [1] Ni’matul Huda, Negara Hukum, Demokrasi Judicial Review, UII Press Yogyakarta, Yogyakarta, 2005. [2] Aloysius Uwiyono,. Dkk, Asas-Asas Hukum Perburuhan, PT RajaGrafindo Persada, Cetakan ke 3 (tiga), Depok, 2018. [3] Azary, Negara Hukum Indonesia, Analisis Yuridis Normatif Tentang Unsur-Unsurnya, UI Press, Jakarta, 1995. References [3] Azary, Negara Hukum Indonesia, Analisis Yuridis Normatif Tentang Unsur-Unsurnya, UI Press, Jakarta, 1995. Http://www.granthaalayah.com ©International Journal of Research - GRANTHAALAYAH [95] [Zulkarnaen , Vol.8 (Iss.3): March 2020] ISSN- 2350-0530(O), ISSN- 2394-3629(P) Index Copernicus Value (ICV 2018): 86.20 DOI: 10.5281/zenodo.3733004 [4] M. Yamin dalam Azary, Negara Hukum Indonesia, Analisis Yuridis Normatif Tentang Unsur Unsurnya, UI Press, Jakarta, 1995. [5] M. Yamin dalam Azary, Negara Hukum Indonesia, Analisis Yuridis Normatif Tentang Unsur Unsurnya, UI Press, Jakarta, 1995. [6] M. Yamin dalam Azary, Negara Hukum Indonesia, Analisis Yuridis Normatif Tentang Unsur Unsurnya, UI Press, Jakarta, 1995. [7] Azary, Negara Hukum Indonesia, Analisis Yuridis Normatif Tentang Unsur-Unsurnya, UI Press, Jakarta, 1995. [8] Azary, Negara Hukum Indonesia, Analisis Yuridis Normatif Tentang Unsur-Unsurnya, UI Press, Jakarta, 1995. [9] Azary, Negara Hukum Indonesia, Analisis Yuridis Normatif Tentang Unsur-Unsurnya, UI Press, Jakarta, 1995. [10] Azary, Negara Hukum Indonesia, Analisis Yuridis Normatif Tentang Unsur-Unsurnya, UI Press, Jakarta, 1995. [11] Azary, Negara Hukum Indonesia, Analisis Yuridis Normatif Tentang Unsur-Unsurnya, UI Press, Jakarta, 1995. [12] Azary, Negara Hukum Indonesia, Analisis Yuridis Normatif Tentang Unsur-Unsurnya, UI Press, Jakarta, 1995. [13] Azary, Negara Hukum Indonesia, Analisis Yuridis Normatif Tentang Unsur-Unsurnya, UI Press, Jakarta, 1995. [13] Azary, Negara Hukum Indonesia, Analisis Yuridis Normatif Tentang Unsur Unsurnya, UI Press, Jakarta, 1995. [14] Azary, Negara Hukum Indonesia, Analisis Yuridis Normatif Tentang Unsur-Unsurnya, UI Press, J k 1995 [14] Azary, Negara Hukum Indonesia, Analisis Yuridis Normatif Tentang Unsur-Unsu Jakarta, 1995. [14] Azary, Negara Hukum Indonesia, Analisis Yuridis Normatif Tentang Unsur-Unsurnya, UI Press, Jakarta, 1995. [15] Azary, Negara Hukum Indonesia, Analisis Yuridis Normatif Tentang Unsur-Unsurnya, UI Press, Jakarta 1995 , [15] Azary, Negara Hukum Indonesia, Analisis Yuridis Normatif Tentang Unsur-Unsurnya, UI Press, Jakarta, 1995. , [16] Abu Daud Busroh, Ilmu Negara, Cetakan Pertama, Bumi Aksara, Jakarta, 1990. [17] Azary, Negara Hukum Indonesia, Analisis Yuridis Normatif Tentang Unsur-Unsurnya, UI Press, Jakarta, 1995. [18] Ni’matul Huda, Negara Hukum, Demokrasi Judicial Review, UII Press Yogyakarta, Yogyakarta, 2005 [19] Padmo Wahjono, Ilmu Negara Suatu Sistematik dan Penjelasan 14 Teori Ilmu Negara dari Jellinek, Melati Study Group, Jakarta, 1977. [20] Abdul Khakim, Pengantar Hukum Ketenagakerjaan, Berdasarkan Undang-Undang Nomor 13 Tahun 2003, PT Citra Aditya Bakti, Bandung, 2003. [21] Abdul Rachmad Budiono, Hukum Perburuhan di Indonesia, Cet I, PT Raja Grafindo Persada, Jakarta, 1995. [22] Abdul Rachmad Budiono, Hukum Perburuhan di Indonesia, Cet I, PT Raja Grafindo Persada, Jakarta, 1995. [23] Suwarto, Hubungan Industrial Dalam Praktek, Asosiasi Hubungan Industrial Indonesia, Jakarta, 2003. References [24] Aloysius Uwiyono,. Dkk, Asas-Asas Hukum Perburuhan, PT RajaGrafindo Persada, Cetakan ke 3 (tiga), Depok, 2018. [25] Aloysius Uwiyono,. Dkk, Asas-Asas Hukum Perburuhan, PT RajaGrafindo Persada, Cetakan ke 3 (tiga), Depok, 2018. g p [26] Aloysius Uwiyono,. Dkk, Asas-Asas Hukum Perburuhan, PT RajaGrafindo Persada, Cetakan ke 3 (tiga), Depok, 2018. [27] Al i U i Dkk A A H k P b h PT R j G fi d P d C k k 3 [26] Aloysius Uwiyono,. Dkk, Asas-Asas Hukum Perburuhan, PT RajaGrafindo Persada, Cetakan ke 3 (tiga), Depok, 2018. [27] Aloysius Uwiyono,. Dkk, Asas-Asas Hukum Perburuhan, PT RajaGrafindo Persada, Cetakan ke 3 (tiga), Depok, 2018. [27] Aloysius Uwiyono,. Dkk, Asas-Asas Hukum Perburuhan, PT RajaGrafindo Persada, Cetakan ke 3 (tiga) Depok 2018 g p [27] Aloysius Uwiyono,. Dkk, Asas-Asas Hukum Perburuhan, PT RajaGrafindo Persad (tiga), Depok, 2018. [28] Suwarto, Hubungan Industrial Dalam Praktek, Asosiasi Hubungan Industrial Indonesia, Jakarta, 2003. Http://www.granthaalayah.com ©International Journal of Research - GRANTHAALAYAH [96] [Zulkarnaen , Vol.8 (Iss.3): March 2020] ISSN- 2350-0530(O), ISSN- 2394-3629(P) Index Copernicus Value (ICV 2018): 86.20 DOI: 10.5281/zenodo.3733004 [29] Tim Pengajar FH Unpar, Pengantar Ilmu Hukum, Universitas Katolik Parahyangan, Bandung, 1995. [30] Tim Pengajar FH Unpar, Pengantar Ilmu Hukum, Universitas Katolik Parahyangan, Bandung, 1995. o, Hubungan Industrial Dalam Praktek, Asosiasi Hubungan Industrial Indonesia, 2003. [31] Suwarto, Hubungan Industrial Dalam Praktek, Asosiasi Hubungan Industrial Indon [32] Tim Pengajar FH Unpar, Pengantar Ilmu Hukum, Universitas Katolik Parahyangan, Bandung, 1995. [33] Widodo Suryandono dalam Aloysius Uwiyono., Dkk, Asas-Asas Hukum Perburuhan, Edisi Kedua, PT RajaGrafindo Persada, Depok, 2018. [34] Tim Pengajar FH Unpar, Pengantar Ilmu Hukum, Universitas Katolik Parahyangan, Bandung, 1995. [35] Tim Pengajar FH Unpar, Pengantar Ilmu Hukum, Universitas Katolik Parahyangan, Bandung, 1995. [36] Tim Pengajar FH Unpar, Pengantar Ilmu Hukum, Universitas Katolik Parahyangan, Bandung, 1995. [37] Tim Pengajar FH Unpar, Pengantar Ilmu Hukum, Universitas Katolik Parahyangan, Bandung, 1995. [38] Hyronimus Rhiti, Filsafat Hukum, edisi lengkap, (dari klasik sampai postmodernisme), Universitas Atma Jaya, Yogyakarta, Yogyakarta, 2011. [39] Suwarto, Hubungan Industrial Dalam Praktek, Asosiasi Hubungan Industrial Indonesia, Jakarta, 2003. [40] Suwarto, Hubungan Industrial Dalam Praktek, Asosiasi Hubungan Industrial Indonesia, Jakarta, 2003. [41] Suwarto, Hubungan Industrial Dalam Praktek, Asosiasi Hubungan Industrial Indonesia, Jakarta, 2003. [42] Suwarto, Hubungan Industrial Dalam Praktek, Asosiasi Hubungan Industrial Indonesia, Jakarta, 2003. [43] Suwarto, Hubungan Industrial Dalam Praktek, Asosiasi Hubungan Industrial Indonesia, Jakarta, 2003. [44] Hyronimus Rhiti, Filsafat Hukum, edisi lengkap, (dari klasik sampai postmodernisme), Universitas Atma Jaya, Yogyakarta, Yogyakarta, 2011. References [45] Suwarto, Hubungan Industrial Dalam Praktek, Asosiasi Hubungan Industrial Indonesia, Jakarta, 2003. [46] Suwarto, Hubungan Industrial Dalam Praktek, Asosiasi Hubungan Industrial Indonesia, Jakarta, 2003. [47] Suwarto, Hubungan Industrial Dalam Praktek, Asosiasi Hubungan Industrial Indonesia, Jakarta, 2003. [48] Widodo Suryandono dalam Aloysius Uwiyono., Dkk, Asas-Asas Hukum Perburuhan, Edisi Kedua, PT RajaGrafindo Persada, Depok, 2018. [97] Http://www.granthaalayah.com ©International Journal of Research - GRANTHAALAYAH
https://openalex.org/W2906554332	https://www.scielo.br/j/sdeb/a/9rS4Mf7fFSFsYvxWjwZFd9h/?lang=pt&format=pdf	Portuguese	null	A crise mundial de 2008 e o golpe do capital na política de saúde no Brasil	Saúde em Debate	2,018	cc-by	6,522	1 Universidade Federal da Bahia (UFBA), Instituto de Humanidades, Artes e Ciências Professor Milton Santos (IHAC) – Salvador (BA), Brasil. Orcid: https://orcid. org/0000-0002-8080- 9146 carment@ufba.br 2 Universidade Federal da Bahia (UFBA), Instituto de Saúde Coletiva (ISC) – Salvador (BA), Brasil. Orcid: https://orcid. org/0000-0003-0783- 262X jairnil@ufba.br 11 11 ARTIGO DE OPINIÃO \| OPINION ARTICLE 1 Universidade Federal da Bahia (UFBA), Instituto de Humanidades, Artes e Ciências Professor Milton Santos (IHAC) – Salvador (BA), Brasil. Orcid: https://orcid. org/0000-0002-8080- 9146 carment@ufba.br The global crisis of 2008 and the coup of capital in Brazilian health policy Carmen Fontes de Souza Teixeira1, Jairnilson Silva Paim2 Carmen Fontes de Souza Teixeira1, Jairnilson Silva Paim2 DOI: 10.1590/0103-11042018S201 DOI: 10.1590/0103-11042018S201 RESUMO O objetivo do artigo foi analisar a conjuntura posterior às eleições presidenciais de 2014, discutindo possíveis relações com a crise econômica mundial e com os desdobramentos do golpe de 2016 na saúde. Trata-se de um artigo de opinião que contemplou a análise dos principais fatos políticos do período. Os resultados ressaltam que a financeirização da saúde, o ajuste fiscal, a restauração do neoliberalismo e o clientelismo político da direita têm gerado o desmonte do Sistema Único de Saúde (SUS), vis-à-vis alguma resistência de frentes e mo vimentos sociais progressistas. Conclui-se pela necessidade de acúmulo de energias políticas para alterar a correlação de forças na atual conjuntura. KEYWORDS Health policy. Unified Health System. Health care reform. PALAVRAS-CHAVE Política de saúde. Sistema Único de Saúde. Reforma dos serviços de saúde. ABSTRACT The objective of the article is to analyze the conjuncture after the 2014 presidential elections, by discussing possible connections with the global economic crisis and the unfolding of the 2016’s coup d’etat in the health context. It is an opinion article that contemplated the analysis of the main political facts of the period above mentioned. The results highlight that the financialization of health, the fiscal adjustment, the restoration of neoliberalism, and the politi cal clientelism of the right-wing political party have caused the breakdown of the SUS (Unified Health System), facing some resistance of fronts and progressive social movements. It is con cluded the need for accumulation of political energies to change the correlation of forces in the current conjuncture. KEYWORDS Health policy. Unified Health System. Health care reform. KEYWORDS Health policy. Unified Health System. Health care reform. Este é um artigo publicado em acesso aberto (Open Access) sob a licença Creative Commons Attribution, que permite uso, distribuição e reprodução em qualquer meio, sem restrições, desde que o trabalho original seja corretamente citado. SAÚDE DEBATE \| RIO DE JANEIRO, V. 42, NÚMERO ESPECIAL 2, P. 11-21, OUTUBRO 2018 Teixeira CFS, Paim JS 12 Introdução desde a aprovação da Constituição Federal de 1988, e expressa no subfinanciamento, na articulação público-privada, na gestão e na desvalorização dos trabalhadores de saúde. Desse modo, várias perguntas emergem na conjuntura recente: quais os desdobramen tos da crise de 2008 no Brasil? Até que ponto essa crise pôde ser adiada mediante políticas anticíclicas dos governos Lula e Dilma? Será que se expressou em crises econômica, ética e política desde 2014? Quais as repercussões nas políticas sociais e no SUS, especialmente depois do golpe de 2016? A crise mundial de 2008 tem sido reconhe cida como manifestação de contradições do capitalismo na qual se destacam a queda tendencial da taxa de lucro, os problemas gerados pelo subprime (modalidade de em préstimos como crédito de risco) e a espe culação imobiliária nos Estados Unidos e em países europeus. A resposta dos países domi nantes a essa crise foi a estatização das dívidas para salvar os bancos e seus executivos, assim como a ênfase em políticas neoliberais e o reforço na financeirização da economia, am pliando as críticas e os combates contra as políticas universais e o Welfare State. Nessa perspectiva, o objetivo do presente estudo é analisar a conjuntura posterior às eleições presidenciais de 2014, discutindo possíveis relações com a crise econômica mundial e com certos desdobramentos do golpe de 2016 na política de saúde. Aproveitando essa oportunidade, o capital realizou uma ofensiva contra a saúde1. Sistemas nacionais de saúde sofreram com prometimento da universalidade, aprofun dando políticas de competição regulada e comercialização2, bem como fortalecendo as tendências de segmentação e de mercantili zação na saúde. Assim, interesses de mercado impuseram políticas de austeridade fiscal neoliberais na Europa pela troika (Fundo Monetário Internacional – FMI, Banco Central Europeu e Comissão Europeia), com impacto negativo nas instituições de saúde estruturadas no século XX3. Houve cortes com restrições de serviços, ampliação de copagamentos, transferência de custos para os usuários, diminuição de responsabi lidades por parte do Estado e aumento nas formas de privatização. Na América Latina, a privatização observada nos sistemas de saúde do Chile, da Colômbia, do México e da Argentina não se mostrou efetiva, aumentan do a barreira de acesso e custos administrati vos, enquanto no caso colombiano apontava para a insolvência1. Alguns antecedentes De Sarney a Dilma, o orçamento da segurida de social não foi adotado, e suas fontes de re cursos foram segmentadas e desviadas para financiar a economia. Parte do financiamen to da seguridade social foi capturada pela área econômica dos diferentes governos, e a questão social passou a ser conduzida por meio de programas emergenciais e políticas focalizadas, defendidas por especialistas e preconizadas por instituições internacionais como o Banco Mundial (BM). Cabe reconhe cer, entretanto, que os programas de transfe rência condicionada de renda4, a exemplo do ‘Bolsa Família’, apresentaram efeitos positi vos na redução da pobreza e da desigualdade social, com consequente geração de dividen dos políticos-ideológicos. Nem a sociedade nem o Estado brasileiro têm apostado no projeto da Reforma Sanitária Brasileira (RSB) e optado pelo SUS como políti ca prioritária, sendo boicotado sucessivamente por vários governos desde a Constituição de 19884. Grandes desafios continuavam postos para a RSB e para a consolidação do SUS, Tal ofensiva em outros países não deve escamotear determinantes histórico-es truturais internos que conformaram uma sociedade extremamente desigual como a brasileira, nem obscurecer a crise do Sistema Único de Saúde (SUS), observada SAÚDE DEBATE \| RIO DE JANEIRO, V. 42, NÚMERO ESPECIAL 2, P. 11-21, OUTUBRO 2018 13 A crise mundial de 2008 e o golpe do capital na política de saúde no Brasil enquanto sistema de saúde público, universal, igualitário, integral e de qualidade. configurando uma porta aberta para as parcerias público-privadas (PPP), tercei rizações, OS, Organização da Sociedade Civil de Interesse Público (Oscip)6 etc. Costa, Bahia e Scheffer5, ao analisarem posições ambíguas do governo Dilma em relação à articulação público-privada, de nunciaram desde então ameaças contra o SUS decorrentes de pressões dos que apos tavam na privatização, tanto nos setores à direita quanto à esquerda do espectro político. Alertaram que mais subsídios e desonerações fiscais para a expansão do mercado de assistência médica suplementar deparavam-se com um acúmulo de experi ências negativas de consumidores iludidos de que o mercado seria capaz de atender às suas necessidades. Desde aquela época, não se vislumbrava um cenário otimista para a sustentabilidade do SUS. Mesmo se conse guindo mais recursos, outras lutas seriam necessárias para evitar a adoção do modelo americano e para não permanecer refém da indústria de equipamentos e de medicamen tos, dos hospitais privados e do corporativis mo de profissionais. O ‘ensaio desenvolvimentista’, a polarização das eleições e o golpe do capital 11-21, OUTUBRO 2018 Teixeira CFS, Paim JS 14 Paradoxalmente, após a vitória aperta da do segundo turno, a presidente adotou a política econômica defendida pelo seu oponente durante a campanha. Essa opção, obviamente, ia no sentido contrário das ex pectativas dos seus eleitores, fragilizando a sua base social e a sua sustentação política. O capital financeiro inicialmente parecia preferir Dilma à instabilidade, mas não foi acompanhado por empresários industriais. Essas frações se articularam depois e deram o golpe de 2016, liderado pelo vice-presi dente e por parte dos seus ministros, com a “ajuda do Congresso [...] e de uma mídia e uma Justiça partidarizada”9(49). de Dilma/Guido Mantega e as manifestações de junho de 2013, incluindo posteriormente o golpe do capital. Os limites do neodesenvolvimentismo geraram espaço para a política, aproveitado pelos conservadores e pela direita desde as Jornadas de Junho de 2013. O capital in dustrial afastou-se de Dilma, alinhando-se ao bloco rentista de oposição, devido aos seguintes motivos: 1) Mistura do capital da indústria e das finanças; 2) Pleno emprego, força dos sindicatos e elevação dos salários reais; 3) Ideologia anti-intervencionista; 4) Correlação de forças internacional; 5) Abertura de excessivas frentes de luta7. As políticas de austeridade implanta das pelos ministros Joaquim Levi e Nelson Barbosa, em 2015, foram radicalizadas pelo governo interino, a partir de 12 de maio de 2016, e expandidas com a consumação final do golpe de 31 de agosto. Com efeito, uma soma fabulosa do orçamento tem sido apro priada pelo capital financeiro. Em 2014, quase 1 trilhão de reais (R$ 978 bilhões) do orça mento da União executado foi destinado ao pagamento da dívida pública (45,11%). Para a saúde, coube apenas 3,98%. Correspondiam às parcelas informadas pelo governo a título de ‘juros’ (R$ 170 bi) e ‘amortizações’ (R$ 808 bi), ou seja, 12 vezes do que foi destina do à educação, 11 vezes à saúde, mais que o dobro dos gastos com a Previdência Social. Entre 2010 e 2014, o governo gastou só em juros R$ 700 bilhões; e com o Bolsa Família, R$ 103 bilhões10. O ‘ensaio desenvolvimentista’, a polarização das eleições e o golpe do capital Do lado do governo, foi elaborada a chamada Nova Matriz econômica (NME), integrando as seguintes medidas: redução dos juros; uso in tensivo do Banco Nacional de Desenvolvimento Econômico e Social (BNDES); aposta na rein dustrizalização; desonerações; plano para infraestrutura; reforma do setor elétrico; desva lorização do real para facilitar as exportações; controle de capitais; e proteção ao produto na cional. Contudo o agravamento da crise inter nacional, desde 2011, ao lado de uma possível redução das taxas de lucro, levou os principais grupos empresariais brasileiros, liderados pelos bancos privados, a demandar do governo federal políticas de austeridade no sentido con trário ao ensaio desenvolvimentista, visando aprofundar o ajuste recessivo, aumentar o de semprego e conter o ciclo grevista, a fim de impor uma série de reformas antipopulares, como a da previdência e a trabalhista8(59-60). O ‘ensaio desenvolvimentista’, a polarização das eleições e o golpe do capital Nos anos de 2011 e 2012, os desenvolvimen tistas do governo Dilma atuaram sobre o mercado financeiro, ampliando espaços para bancos públicos e reduzindo os juros, mas o Banco Central voltou a elevar a taxa Selic em abril de 2013. Assim, o governo devolveu ao mercado o controle sobre a política econô mica, comprometendo a retomada do desen volvimento e o avanço progressista. Nessa conjuntura, o capital financeiro, aparente mente diferenciado do capital industrial e da fração organizada dos trabalhadores que formavam a coalizão produtivista, aliou-se à classe média tradicional, reforçando a co alizão rentista. Enquanto isso, o subproleta riado permanecia sob as asas do lulismo, que arbitrava as forças sociopolíticas7. Assim, a crise do SUS antecedeu a crise mundial e as crises econômica, políti ca e ética constatadas no Brasil a partir de 2014. Aliás, as forças sociais e polí ticas que formularam a RSB e o SUS já acumulavam derrotas desde o período pós-constituinte4. Entretanto, nas pri meiras duas décadas desse período, as medidas contra o SUS limitavam-se ao Executivo; até quando o Congresso ex tinguiu a Contribuição Provisória sobre Movimentação Financeira (CPMF) em 2007. Já na década seguinte, o Executivo e o Legislativo rejeitaram a proposta de 10% das receitas brutas da União para a saúde, ignorando o movimento Saúde+10. Juntos comprometeram a sustentabilidade eco nômica do SUS e permitiram a abertura da saúde ao capital estrangeiro. Em 2014, chegou a vez do Judiciário, reconhecendo a constitucionalidade das Organizações Sociais (OS) no Supremo Tribunal Federal (STF) depois de 17 anos de protelação e Entre o maio do ensaio desenvolvimentis ta – quando a presidente Dilma fez uma forte crítica aos juros praticados pelos bancos no País e anunciou, no Dia dos Trabalhadores de 2012, a redução nas taxas de juros – e as Jornadas de Junho de 2013, algo precisa ser entendido e explicado. Nesse interregno, os comentaristas econômicos, economistas, representantes do mercado financeiro e a grande mídia anunciavam a possível volta da inflação. Ademais, o governo federal combinou com os estados e municípios para adiarem o reajuste das tarifas de transportes coletivos até junho de 2013 para que os au mentos não incidissem sobre as taxas de in flação. Esse fato pode ser um dos elementos que faltavam para entender o nexo entre a crise mundial de 2008, a política econômica SAÚDE DEBATE \| RIO DE JANEIRO, V. 42, NÚMERO ESPECIAL 2, P. Repercussões das crises e do golpe na saúde O cerco a favor da reversão neoliberal contou com apoio da burguesia, classe média, setores da classe trabalhadora, além de personalida des influentes sobre Dilma. Com as eleições de 2014, o choque recessivo e a opção de acelerar o lulismo terminaram por produzir a pior reces são desde 19927. A partir de então, aumentou a oposição do capital e dos seus representantes ao governo Dilma, aprofundando a polarização nas eleições de 2014. Vale recordar os vários golpes desferidos contra o SUS desde 2014: 1) Abertura da saúde ao capital estrangeiro; 2) Projeto de Lei (PL) para obrigatoriedade de planos privados de saúde para empregados, exceto os domésticos; 3) PL das Terceirizações; 4) Prorrogação da Desvinculação das SAÚDE DEBATE \| RIO DE JANEIRO, V. 42, NÚMERO ESPECIAL 2, P. 11-21, OUTUBRO 2018 A crise mundial de 2008 e o golpe do capital na política de saúde no Brasil 15 Receitas da União (DRU), acrescida da Desvinculação das Receitas dos Estados (DRE) e da Desvinculação das Receitas dos Municípios (DRM); 5) Proposta de Emenda Constitucional (PEC 241) da Câmara dos Deputados e PEC 55 do Senado (Novo Regime Fiscal); 6) Planos populares; 7) Rejeição da Emenda Popular Saúde + 10; 8) Orçamento impositivo; 9) Reconhecimento da constitucionalidade das OS; 10) Proposta de Cobertura Universal em Saúde; 11) Agenda Brasil com cobrança de serviços no SUS; 12) Novos pacotes de ajuste; 13) Saúde, educação e ciência e tecnologia como moeda de troca político-partidária. Cumpre registrar que parte dessas iniciativas já foi efetivada (1,3,4,5,7,8,9,13), enquanto outras ainda pairam como ameaças concretas (2,6,10,11,12). Receitas da União (DRU), acrescida da Desvinculação das Receitas dos Estados (DRE) e da Desvinculação das Receitas dos Municípios (DRM); 5) Proposta de Emenda Constitucional (PEC 241) da Câmara dos Deputados e PEC 55 do Senado (Novo Regime Fiscal); 6) Planos populares; 7) Rejeição da Emenda Popular Saúde + 10; 8) Orçamento impositivo; 9) Reconhecimento da constitucionalidade das OS; 10) Proposta de Cobertura Universal em Saúde; 11) Agenda Brasil com cobrança de serviços no SUS; 12) Novos pacotes de ajuste; 13) Saúde, educação e ciência e tecnologia como moeda de troca político-partidária. Cumpre registrar que parte dessas iniciativas já foi efetivada (1,3,4,5,7,8,9,13), enquanto outras ainda pairam como ameaças concretas (2,6,10,11,12). estudadas e denunciadas pelo movimento sanitário. Repercussões das crises e do golpe na saúde Trata-se, agora, de uma articulação público-privada específica, via empresas, que realizam a intermediação da assistência, ou seja, instituições financeiras que adminis tram planos de saúde e se inserem no jogo especulativo das bolsas de valores. Esse pro cesso está acompanhado do financiamento de campanhas de parlamentares e dirigentes do executivo, da cooptação de ministros da Fazenda, do Planejamento, da Casa Civil e da Saúde, assim como da captura da Agência Nacional de Saúde Suplementar (ANS), isto é, da ocupação de cargos de direção da ANS por empresários de planos de saúde, expres são do circuito entre gestores e técnicos da burocracia estatal e empresas do setor saúde (‘porta giratória’) e da produção e reprodu ção da corrupção11,12. Para além do plano fenomênico, houve modificações nas relações entre o Estado e o capital que vão além do financiamento das políticas sociais e do SUS. Uma dessas regras foi a mencionada abertura da saúde ao capital estrangeiro, aprovada no Congresso nacional e não vetada pela Presidência da República, o que gerou questionamentos de entidades do movimento sanitário. O ‘austericídio’ e a saúde O ‘austericídio’ produzido pela implemen tação das medidas preconizadas na Emenda Constitucional 95 (EC-95), que congela os gastos públicos na área social para os pró ximos 20 anos, deve produzir resultados nefastos na saúde. Com o aumento da popu lação e duplicação do percentual de idosos, ao tempo em que os recursos para a saúde serão pré-fixados pela inflação, certamen te será drasticamente reduzido o gasto per capita com saúde. Isto, em um cenário epi demiológico que demandará exatamente o contrário, ou seja, o aumento do valor per capita investido em saúde ante os problemas e necessidades da população, e diante da ten dência ao aumento dos custos da assistência, por conta da incorporação de tecnologias de alta densidade de capital e de insumos ad quiridos, em grande parte, em dólar. Não foi apenas o então presidente da Câmara de Deputados nem mesmo o Congresso Nacional que impuseram essas derrotas, mas, especialmente, as operadoras de planos de saúde que financiaram as cam panhas das eleições de 2010 e 2014. Portanto, o determinante fundamental desses retro cessos está representado pelo capital finan ceiro que define, direta e indiretamente, as políticas públicas no Brasil e no mundo. Essa financeirização não é só uma hiper trofia do capital financeiro, nem uma pato logia do sistema capitalista, mas um novo padrão sistêmico de acumulação de riqueza do capitalismo contemporâneo11. Esta vai mais além que a privatização da saúde na infraestrutura, produção e consumo de ser viços e gestão do sistema. Essas modalidades de privatização já eram bem conhecidas, Além disso, formou-se uma ‘bomba-reló gio’ com a epidemia anunciada de diabetes e hipertensão de crianças e adultos jovens de hoje com obesidade e sobrepeso; com a epidemia atual de acidentes de motocicletas; com uma geração de crianças com Síndrome SAÚDE DEBATE \| RIO DE JANEIRO, V. 42, NÚMERO ESPECIAL 2, P. 11-21, OUTUBRO 2018 Teixeira CFS, Paim JS 16 Neurológica da Zika; com pacientes sofren do problemas articulares decorrentes da Chikungunya; com taxas elevadas de mor bimortalidade por câncer; com prevalências elevadas de transtornos mentais, especial mente depressão; com o crescimento de vio lências e acidentes; com o envelhecimento da população; e com a tripla carga de doenças. qual o gasto social não deve pressionar o orçamento fiscal14. O ‘austericídio’ e a saúde Entretanto, quando res surge a crise, esses recursos têm sido usados para socorrer a economia, seja comprando dólar nas crises cambiais, seja desonerando empresas e empresários, seja repassando bilhões do Tesouro Nacional para o BNDES viabilizar oferta de juros subsidiados. Diante dessa conjuntura, as perspectivas para as políticas sociais se apresentavam bastante sombrias. No caso da saúde, está em curso um processo de americanização do sistema, tendo como cenário a expansão do mercado de serviços de saúde e subsídios, in clusive assimilando a proposta de ‘cobertura universal em saúde’15. A proposta de planos de saúde acessíveis, por sua vez, expressa um movimento do capital na saúde que já vinha se conformando no País, corroendo a possibilidade de manu tenção do SUS, enquanto um sistema público e universal de saúde. Essa proposta, mesmo que não entregue o que promete e iluda seus possíveis consumidores, é consistente com as políticas delineadas pelo governo e com os documentos divulgados pelos empresários da saúde desde as eleições de 2014. Novas bases sociais, forças políticas e estratégias? Se os gastos federais cresceram mais que as receitas nos últimos anos, não foi por causa da saúde. Os recursos aplicáveis a ela estiveram estáveis em relação ao Produto Interno Bruto (PIB), aproximadamente 1,7% deste. Representavam gastos que beneficia riam milhões de brasileiros, ao contrário de subsídios e desonerações. A guinada adotada pelo Partido dos Trabalhadores (PT) desde o final da década de 198016 e, especialmente, a partir de 2003 o deslocou para o liberalismo social, vinculado ao pragmatismo político. Se este liberalismo social, sob o pretexto do realismo político, foi capaz de legitimar parcialmente o lulismo, possibilitando a vitória do PT nas eleições presidenciais de 2006, 2010 e 2014, de outro modo comprometeu as políticas universais, a exemplo do SUS, desconstruiu a seguridade social concebida pela Constituição Cidadã e deu prosseguimento ao seu desmonte inicia do pelos governos Collor e FHC. O curso e a agenda do retrocesso A mobilidade na base da estrutura social verifi cada até 2014, com reforço do contingente de trabalhadores e um realinhamento político -eleitoral traduzido pelo lulismo, quando bur gueses e proletários são substituídos por ricos e pobres no discurso político, não parece ter resultado em um avanço progressista. A conjuntura delineada depois do golpe de 2016 possibilitou a implantação de parte do primeiro projeto e expressa um cenário ainda mais regressivo, extremamente grave e preocupante diante das medidas contrárias aos direitos sociais anteriormente assegura dos pela Constituição de 1988. Vão contra os interesses nacionais e contra a democracia, além de comprometerem a vida e a saúde da classe trabalhadora e da população mais pobre deste país. Em 2015, dois grandes projetos disputa ram ideologicamente a cena brasileira: a) Uma Ponte para o Futuro; b) O Brasil que que remos. O primeiro, formulado pela Fundação Ulisses Guimarães do então Partido do Movimento Democrático Brasileiro (PMDB), também conhecido como ‘Plano Temer’, contém uma agenda ultraliberal na perspectiva econômica e regressiva no que diz respeito aos direitos sociais. O segundo, articulado por pesquisadores, intelectuais e lideranças sindicais de São Paulo vinculados à Plataforma Social, foi debatido por diversas entidades do movimento da RSB, a exemplo do Centro Brasileiro de Estudos de Saúde (Cebes) e da Associação Brasileira de Saúde Coletiva (Abrasco). Este projeto, apoiado pelas forças progressistas, representava uma proposta alternativa ao projeto conservador, esboçando uma agenda de curto prazo cen trada nas seguintes medidas: 1) Preservar o emprego e a renda; 2) Desarmar a armadilha recessiva; 3) Aprender com as lições da ex periência internacional; 4) Baixar os juros; 5) Recompor a capacidade de financiamen to do Estado; 6) Destravar os investimentos públicos e privados; 7) Fortalecer o mercado interno; 8) Preservar os gastos sociais. A proposta explicitava, ainda, as seguintes A financeirização e a hegemonia do capital financeiro, o ajuste fiscal violento (EC-95), a restauração radical do neoliberalismo e o clien telismo político da direita conservadora estão possibilitando o desmonte do SUS, ainda que gerando algum grau de resistência mediante frentes de movimentos sociais progressistas, conquistas de aparelhos da sociedade civil e algumas incursões no parlamento. Esse ajuste significa mais que uma ameaça ao SUS universal ou à garantia de recursos do Estado para saúde e educação. Trata-se de um atentado do Executivo e do Legislativo à democracia e contra gerações de brasileiros nos próximos 20 anos. O curso e a agenda do retrocesso O governo Temer implementou, o mais rápido possível, a sua agenda de retrocesso por meio do corte das políticas sociais, recuo da legislação ambiental, retirada dos direitos trabalhistas e previdenciários, entrega do patrimônio público, tributação regressiva, restrição ao pensamento crítico e “na arbitra riedade escancarada da força policial”13(36). Os projetos de reforma progressista, na cional, desenvolvimentista, democrático e redistributivo vinculados à Constituição Cidadã, como o ‘Esperança e Mudança’ e o ‘Democrático-popular’, foram derrotados por diferentes modos4. Até mesmo o neode senvolvimentismo foi substituído por refor mas conservadoras. Novas ‘crises’ da previdência têm sido fabricadas para justificar as reformas nos governos de Fernando Henrique Cardoso (FHC), Lula, Dilma e Temer, acionando o suposto deficit como pretexto. De acordo com os tecnoburocratas, os recursos da União de veriam reforçar o capitalismo, desonerando o capital e prevalecendo uma regra pétrea entre os economistas do poder segundo a Além disso, os três poderes da República têm atuado na contramão da RSB e dos prin cípios e diretrizes do SUS. Constata-se uma ação do Estado contra o SUS, negligenciando SAÚDE DEBATE \| RIO DE JANEIRO, V. 42, NÚMERO ESPECIAL 2, P. 11-21, OUTUBRO 2018 A crise mundial de 2008 e o golpe do capital na política de saúde no Brasil 17 diretrizes; a) Defesa da Constituição de 1988; b) Preservar a inclusão social; c) Enfrentar as desigualdades e discriminações históricas; d) Enfrentar as desigualdades da renda e do patrimônio; e) Enfrentar as desigualdades na oferta de serviços e universalizar a ci dadania. Além disso, propunha ‘uma ampla mobilização social com o objetivo claro de lutar contra o atual modelo econômico’, tra duzido na política de ajuste fiscal na qual os povos pagam a conta e as classes dominan tes se acertam em torno de uma agenda de desconstrução dos direitos sociais, do meio ambiente e de culturas17(49). o princípio constitucional segundo o qual a ‘saúde é direito de todos e dever do Estado’. O movimento sanitário, mesmo mobilizando su jeitos sociais em defesa de um sistema de saúde universal, dispõe de bases de apoio insuficien tes, pois não conquistou, efetivamente, traba lhadores, classe média e setores populares. Diferentemente do que muitos imagi navam, a conjuntura que se foi desenhando desde as Jornadas de Junho de 2013 tem imposto um conjunto de derrotas ao SUS. O curso e a agenda do retrocesso Do cadinho desses economistas, surgiu essa monstruosida de na área econômica do governo que não tem paralelo em nenhum país do mundo. O Parlamento abdicou de suas responsabilida des e prerrogativas no que tange à situação fiscal do País em duas décadas. O Presidente da República, mesmo o SAÚDE DEBATE \| RIO DE JANEIRO, V. 42, NÚMERO ESPECIAL 2, P. 11-21, OUTUBRO 2018 Teixeira CFS, Paim JS 18 eleito em 2018, fica impedido de estabele cer uma política econômica por dez anos no mínimo, podendo ser o prazo estendido por mais uma década. Esvazia-se a democracia, pois não adianta o eleitor fazer a escolha de um dado programa de candidato. A dita dura de Pinochet, que foi o laboratório das políticas neoliberais desde os anos 1970 e 1980, e a intervenção recente da troika sobre a Grécia não ousaram tanto. Assim, não adianta o povo votar no presidente, nos de putados e nos senadores da sua preferência, pois, a não ser que ocorra uma mudança na correlação de forças, possibilitando os votos parlamentares necessários para a aprovação de uma emenda constitucional substitutiva à EC-95 ou um questionamento admitido pelo Supremo Tribunal Federal, dificilmente poderá ocorrer uma alteração dessa políti ca definida por um governo sem votos que assumiu o poder em 31 de agosto de 2016. A ‘Agenda Estratégica para a Saúde no Brasil’18, composta por 5 diretrizes voltadas para uma política de saúde ‘5 estrelas’ e subs crita por 9 importantes entidades vinculadas ao movimento sanitário, tem sido propos ta para os debates nas eleições de 2018. Do mesmo modo, a ‘Tese do Cebes 2018-2019’19 apresenta uma análise da conjuntura inter nacional, nacional e setorial, destacando a tensão entre o capitalismo e a democracia, o golpe de 2016 e destruição da cidadania, do direito à saúde, do universalismo e da inte gralidade, com o fortalecimento da ideia de saúde como mercadoria. Propõe um con junto de medidas em defesa da democracia e contra o capitalismo, explicitando as bases e princípios da entidade por democracia e saúde, assim como os caminhos para a luta. Não cabe, portanto, pensar estratégias se toriais diante da gravidade da situação atual. Não há viabilidade na reversão desse quadro, com garantia do financiamento público ao SUS, sem uma grande mobilização demo crática e popular com atores sociais capazes de incluir temas na agenda do Estado. O curso e a agenda do retrocesso Tanto o direito à saúde quanto o SUS, para serem preservados, supõem lutas conjuntas em defesa da seguridade social e dos direitos civis, políticos, sociais e ambientais, com outras frentes de forças que defendam a democracia, os direitos humanos e as con quistas sociais e se oponham ao ‘novo regime fiscal’, à Reforma da Previdência e à Reforma Trabalhista. Apesar de várias palavras de ordem e propostas que surgem em busca de saídas imediatas, ainda não se vislumbra um cenário que não seja o da resistência e do acúmulo de energias políticas para alterar a correlação de forças na conjuntura. De positivo, nota-se uma crescente indig nação das pessoas e da sociedade civil organi zada quando tomam conhecimento do que as forças políticas e econômicas que assaltaram o poder estão fazendo contra o País e contra o seu povo. Desse modo, setores democráticos e populares cada vez mais discutem a situação, mobilizam-se, articulam-se e organizam-se para resistir ao golpe, a exemplo do Fórum da RSB, que agrega entidades e instituições comprometidas com a democracia, com a RSB e com o SUS e participa da Frente Brasil Popular e da Frente Povo sem Medo, A difusão de denúncias e críticas desde o início do governo Temer indica a potenciali dade de mobilizar consciências críticas acerca de uma concepção ampla de saúde e de forta lecer um convite para a ação, resistindo contra o golpe continuado. Estimula a construção de uma pauta democrática para a articulação política, partindo da bandeira de ‘NENHUM DIREITO A MENOS’ com um alargamento de demandas e lutas por outras gerações de direi tos: direito a um ambiente saudável, direito à cidade, direito ao lazer, direito ao gozo estético, direito à felicidade etc. Comentários finais É possível sistematizar na análise dessa con juntura pelo menos três vias de ataque ao SUS. A primeira, a via ideológica, quando a mídia, políticos, gestores, economistas, SAÚDE DEBATE \| RIO DE JANEIRO, V. 42, NÚMERO ESPECIAL 2, P. 11-21, OUTUBRO 2018 19 A crise mundial de 2008 e o golpe do capital na política de saúde no Brasil profissionais de saúde e segmentos da classe média defendiam um SUS pobre para pobres. A segunda, a via política, quando o Legislativo e o Executivo aprovaram a parti cipação do capital estrangeiro na saúde, ter ceirizações e planos privados de saúde, além das propostas de obrigatoriedade para todos os trabalhadores e dos ‘planos populares’. A terceira, a via econômica, mediante o subfi nanciamento crônico, DRU, DRE, DRM, sub sídios ao setor privado e a EC-95. A materialidade e a institucionalida de construídas nas últimas três décadas sugerem que não é qualquer golpe que pode acabar com o SUS. Algum SUS pode perma necer, mesmo que seja um arremedo, um simulacro: um SUS que se confunde com ‘saúde pública’ destinado exclusivamente aos pobres, ao controle de epidemias e ao atendimento do que não interessa à iniciati va privada; um SUS de uma ‘saúde pública’ confinada às ações preventivas e assisten ciais para os que não têm acesso ao mercado e apoio ao setor privado nos procedimentos de alto custo; um SUS distante das diretri zes da igualdade, da universalidade e da integralidade propostas pela RSB e conce bidas pela saúde coletiva. Essas vias de reprodução do golpe do capital na saúde representaram um desas tre para os direitos civis, políticos e sociais e um ataque à democracia, à Constituição de 1988 e às conquistas sociais e ambien tais. Esse golpe de empresários, urdido pela Federação das Indústrias do Estado de São Paulo (Fiesp), Confederação Nacional da Indústria (CNI), Confederação Nacional da Agricultura (CNA), Federação Brasileira de Bancos (Febraban) etc., usou a mídia e parte da classe média para realizar uma intervenção parlamentar com a chancela do Judiciário. Na saúde, o subfinanciamen to constitucionalizado por intermédio da EC-95 (teto dos gastos) se apresenta como um dos mecanismos mais drásticos para o desmonte do SUS. Resignar-se a essa tendência significa minar o caráter solidário de um sistema de saúde universal para o País. Comentários finais A repug nância diante dos ataques do governo sem votos das cidadãs e cidadãos, ilegítimo, e da direção impressa à política setorial pelo Ministério da Saúde exige a revitalização das instâncias de controle social, os diálo gos do movimento da RSB com distintos parceiros, tais como o Conselho Nacional de Saúde (CNS), Conselho Nacional de Secretários de Saúde (Conass), Conselho Nacional de Secretarias Municipais de Saúde (Conasems), Associação Nacional do Ministério Público de Defesa da Saúde (Ampasa), Confederação Nacional dos Bispos do Brasil (CNBB), Ordem dos Advogados do Brasil (OAB), entidades re presentativas de profissionais de saúde e movimentos sociais comprometidos com a democracia e com a luta contra o fascismo, pela garantia dos direitos sociais, contra o racismo e contra todas as formas de discri minação. Nessa perspectiva, apresenta-se a continuidade e a intensificação da luta em diversos espaços políticos, enfren tando os desafios da radicalização da de mocracia para a construção de um novo Estado de Bem-Estar Social no Brasil. A tensão entre conservação e mudança, entretanto, permite considerar a possibi lidade de surgimento de sujeitos da antí tese4 capazes de criar fatos políticos e de estimular a tomada de consciência crítica, contribuindo para o fortalecimento de mo vimentos sociais comprometidos com um projeto contra-hegemônico que contemple, além dos interesses específicos dos traba lhadores, as lutas contra o sexismo, racismo, discriminação sexual e defesa do ambiente, entre outras. Esta pluralidade de vozes na democracia pode reforçar a cadeia de equi valências entre as lutas contra as distintas formas de opressão e subordinação nas re lações sociais, aproveitando-se dos distin tos antagonismos presentes na sociedade contemporânea20. SAÚDE DEBATE \| RIO DE JANEIRO, V. 42, NÚMERO ESPECIAL 2, P. 11-21, OUTUBRO 2018 20 Teixeira CFS, Paim JS Colaboradores Paim JS contribuiu substancialmente para a concepção, planejamento, análise e interpre tação dos dados, elaboração e revisão crítica da versão preliminar e participou da aprova ção da versão final do manuscrito. s Teixeira CF contribuiu significativamente na revisão crítica do conteúdo e participou da aprovação da versão final do manuscrito. Referências 1. Conill EM. A importância da continuidade dos sis temas nacionais europeus para as políticas de saú de na América Latina. Cad Saúde Pública. 2014; 30(11):2253-2255. 6. Marques T, Mendes A. Uma decisão favorá vel às OSS: impasses à construção do SUS [inter net]. Domingueira da Saúde 012/2015. 2015 jun 28 [acesso em 2017 ago 18]. Disponível em: http:// idisa.org.br/img/File/Domingueira%20da%20 Sa%C3%BAde%20-%20012%202015%20-%20 28%2006%202015.pdf. 6. Marques T, Mendes A. Uma decisão favorá vel às OSS: impasses à construção do SUS [inter net]. Domingueira da Saúde 012/2015. 2015 jun 28 [acesso em 2017 ago 18]. Disponível em: http:// idisa.org.br/img/File/Domingueira%20da%20 Sa%C3%BAde%20-%20012%202015%20-%20 28%2006%202015.pdf. 2. Giovanella L, Stegmüller K. The financial crisis and health care systems in Europe: universal care under threat? Trends in health sector reforms in Germany, the United Kingdom, and Spain. Cad Saúde Pública. 2014; 30(11):2263-2281. 2. Giovanella L, Stegmüller K. The financial crisis and health care systems in Europe: universal care under threat? Trends in health sector reforms in Germany, the United Kingdom, and Spain. Cad Saúde Pública. 2014; 30(11):2263-2281. 7. Singer AE. A (falta de) base política para o ensaio desenvolvimentista. In: Singer A, Loureiro I, orga nizadores. As contradições do lulismo: a que ponto chegamos? São Paulo: Boitempo; 2016. p. 21-54. 3. Ottersen OP, Dasgupta J, Blouin C, et al. The politi cal origins of health inequity: prospects for change. Lancet Commissions [internet]. 2014 fev [acesso em 2018 jul 5]; 383(9917):630-667. Disponível em: ht tps://www.thelancet.com/journals/lancet/article/ PIIS0140-6736(13)62407-1/abstract. 8. Braga R. O fim do lulismo. In: Jinkings I, Doria K, Cleto M. Por que gritamos golpe?: para entender o impeachment e a crise política no Brasil. São Paulo: Boitempo; 2016. p. 55-60. 8. Braga R. O fim do lulismo. In: Jinkings I, Doria K, Cleto M. Por que gritamos golpe?: para entender o impeachment e a crise política no Brasil. São Paulo: Boitempo; 2016. p. 55-60. 4. Paim JS. A Constituição Cidadã e os 25 anos do Sis tema Único de Saúde. Cad Saúde Pública. 2013; 29(10):1927-1953. 9. Oliveira C. O ódio como discurso político propaga do nas redes e nas ruas a serviço do golpe. In: Rovai R, organizador. Golpe 16. São Paulo: Publisher Bra sil; 2016. p. 41-54. 5. Costa AM, Bahia L, Scheffer M. Onde foi parar o so nho do SUS? Le Monde Diplomatique Bras. 2013; 69:30-31. 10. Scaff FF. A DRU, os direitos sociais e o pagamento dos juros da dívida [internet]. Consultor Jurídico. Referências SAÚDE DEBATE \| RIO DE JANEIRO, V. 42, NÚMERO ESPECIAL 2, P. 11-21, OUTUBRO 2018 21 A crise mundial de 2008 e o golpe do capital na política de saúde no Brasil Grupos Dirigentes do PT (1979-1998) [tese]. Nite rói: Universidade Federal Fluminense; Programa de Pós-Graduação em História; 2005. Grupos Dirigentes do PT (1979-1998) [tese]. Nite rói: Universidade Federal Fluminense; Programa de Pós-Graduação em História; 2005. Grupos Dirigentes do PT (1979-1998) [tese]. Nite rói: Universidade Federal Fluminense; Programa de Pós-Graduação em História; 2005. Grupos Dirigentes do PT (1979-1998) [tese]. Nite rói: Universidade Federal Fluminense; Programa de Pós-Graduação em História; 2005. 2015 jul 14 [acesso em 2015 jul 22]. Disponível em: https://www.conjur.com.br/2015-jul-14/contas-vis ta-dru-direitos-sociais-pagamento-juros-divida. 11. Sestelo JAF. Planos e seguros de saúde do Brasil de 2000 a 2015 e a dominância financeira [tese]. Rio de Janeiro: Universidade Federal do Rio de Janeiro; Centro de Ciências da Saúde; Instituto de Estudos de Saúde Coletiva; 2017. 648 p. 17. Brasil Debate, Fórum 21, Fundação Perseu Abramo, et al. Por um Brasil justo e democrático: o Brasil que queremos: subsídios para um projeto de desenvol vimento nacional. [sem local]: Brasil Debate; 2015 [acesso em 2017 jul 22]. v. 2. Disponível em: http:// plataformapoliticasocial.com.br/por-um-brasil -justo-e-democratico-2. 12. Monteiro MG. Trayectoria y cambios de dirección en las políticas públicas: análisis de la reforma del sistema sanitario brasileño (1975-2015) [tese]. Bar celona: Universitad Autónoma de Barcelona; De partamento de Ciencia Política y Derecho Público; 2016. 327 p. 18. Associação Brasileira de Saúde Mental, Associa ção Brasileira de Saúde Coletiva, Centro Brasileiro de Estudos em Saúde, et al. SUS igual para todos: Agenda Estratégica para a Saúde no Brasil: 5 diretri zes de uma política de saúde 5 estrelas para pobres e ricos. Rio de Janeiro: Abrasco; Cebes; 2011. 18. Associação Brasileira de Saúde Mental, Associa ção Brasileira de Saúde Coletiva, Centro Brasileiro de Estudos em Saúde, et al. SUS igual para todos: Agenda Estratégica para a Saúde no Brasil: 5 diretri zes de uma política de saúde 5 estrelas para pobres e ricos. Rio de Janeiro: Abrasco; Cebes; 2011. 13. Miguel LF. A democracia na encruzilhada. In: Jinkings I, Doria K, Cleto M. Por que gritamos gol pe?: para entender o impeachment e a crise política no Brasil. São Paulo: Boitempo; 2016. p. 31-37. 19. Centro Brasileiro de Estudos de Saúde. Cebes na luta: transformar e radicalizar a Democracia para assegurar Direitos Sociais e Saúde: Teses do CEBES 2018-2019. Referências Rio de Janeiro: Cebes; 2018. 19. Centro Brasileiro de Estudos de Saúde. Cebes na luta: transformar e radicalizar a Democracia para assegurar Direitos Sociais e Saúde: Teses do CEBES 2018-2019. Rio de Janeiro: Cebes; 2018. 14. Fagnani E. Política Social no Brasil (1964-2002): Entre a Cidadania e a Caridade [tese]. Campinas: Universidade Estadual de Campinas; Instituto de Economia; 2005. 587 p. 20. Laclau EE, Mouffe C. Hegemonia y estrategia so cialista: hacia una radicalización de la democracia. 3. ed. Buenos Aires: Fondo de Cultura Económica; 2010. 15. Titelman D, Centrángolo O, Acosta OL. Universal Health Coverage in Latin American Countries: how to improve solidarity-based schemes. Lancet. 2015 abr; 385(9975):1359-1363. 16. Coelho E. Uma esquerda para o capital. Crise do Marxismo e Mudanças nos Projetos Políticos dos 16. Coelho E. Uma esquerda para o capital. Crise do Marxismo e Mudanças nos Projetos Políticos dos SAÚDE DEBATE \| RIO DE JANEIRO, V. 42, NÚMERO ESPECIAL 2, P. 11-21, OUTUBRO 2018
https://openalex.org/W2954133775	https://www.frontiersin.org/articles/10.3389/fnut.2019.00109/pdf	English	null	Cohort Description of the Madagascar Health and Environmental Research–Antongil (MAHERY–Antongil) Study in Madagascar	Frontiers in nutrition	2,019	cc-by	6,670	CLINICAL STUDY PROTOCOL published: 19 July 2019 doi: 10.3389/fnut.2019.00109 Edited by: Kathleen L. Hefferon, Cornell University, United States Edited by: Kathleen L. Hefferon, Cornell University, United States 1 Department of Nutrition, Harvard T.H. Chan School of Public Health, Boston, MA, United States, 2 Department of Environmental Health, Harvard T.H. Chan School of Public Health, Boston, MA, United States, 3 Madagascar Health and Environmental Research, Maroantsetra, Madagascar, 4 Department of Anthropology, Montclair State University, Montclair, NJ, United States, 5 Department of Organismic and Evolutionary Biology, Harvard University, Cambridge, MA, United States, 6 ARS Western Human Nutrition Research Center, United States Department of Agriculture, Davis, CA, United States, 7 Department of Nutrition, University of California, Davis, Davis, CA, United States, 8 CH Dyson School of Applied Economics & Management, Cornell University, Cornell, NY, United States, 9 National Center for Socio-Environmental Synthesis (SESYNC), Annapolis, MD, United States, 10 Cooper/Smith, Washington, DC, United States, 11 Service de District de la Santé Publique de Maroantsetra, Ministère de la Santé Publique d’Analanjirofo, Maroantsetra, Madagascar, 12 Direction Generale d’Antananarivo, Ministère de la Santé Publique, Antananarivo, Madagascar, 13 Department of Immunology and Infectious Diseases, Harvard T.H. Chan School of Public Health, Boston, MA, United States, 14 Infectious Disease Initiative, Broad Institute of MIT and Harvard, Cambridge, MA, United States, 15 College of Natural, Behavioral, and Health Sciences, Simmons University, Boston, MA, United States Reviewed by: Ranjay K. Singh, Central Soil Salinity Research Institute (ICAR), India Samuel Sellers, University of Washington, United States Vik Mohan, Blue Ventures, United Kingdom Correspondence: Christopher D. Golden golden@hsph.harvard.edu The Madagascar Health and Environmental Research-Antongil (MAHERY-Antongil) study cohort was set up in September 2015 to assess the nutritional value of seafood for the coastal Malagasy population living along Antongil Bay in northeastern Madagascar. Over 28 months of surveillance, we aimed to understand the relationships among different marine resource governance models, local people’s ﬁsh catch, the consumption of seafood, and nutritional status. In the Antongil Bay, ﬁsheries governance takes three general forms: traditional management, marine national parks, and co-management. Traditional management involves little to no involvement by the national government or non-governmental organizations, and focuses on culturally accepted Malagasy community practices. Co-management and marine national parks involve management support from either an non-govermental organization (NGO) or the national government. Received: 02 February 2019 Accepted: 01 July 2019 Published: 19 July 2019 Received: 02 February 2019 Accepted: 01 July 2019 Published: 19 July 2019 Cohort Description of the Madagascar Health and Environmental Research–Antongil (MAHERY–Antongil) Study in Madagascar Christopher D. Golden 1,2,3, Cortni Borgerson 3,4, Benjamin L. Rice 3,5, Lindsay H. Allen 6,7, Evelin Jean Gasta Anjaranirina 3, Christopher B. Barrett 8, Godfred Boateng 1, Jessica A. Gephart 9, Daniela Hampel 6,7, Daniel L. Hartl 5, Erwin Knippenberg 10, Samuel S. Myers 2, Dera H. Ralalason 11, Herlyne Ramihantaniarivo 12, Hervet Randriamady 3, Setareh Shahab-Ferdows 6, Bapu Vaitla 1, Sarah K. Volkman 13,14,15 and Miadana Arisoa Vonona 3 Five communities of varying governance strategies were enrolled into the study including 225 households and 1031 individuals whose diets, resource acquisition strategies, ﬁsheries and agricultural practices, and other social, demographic and economic indicators were measured over the span of 3 years. Clinical visits with each individual were conducted at two points during the study to measure disease and nutritional status. By analyzing differences in ﬁsh catch arising from variation in governance (in addition to Specialty section: This article was submitted to Nutrition and Sustainable Diets, a section of the journal Frontiers in Nutrition Citation: Golden CD, Borgerson C, Rice BL, Allen LH, Anjaranirina EJG, Barrett CB, Boateng G, Gephart JA, Hampel D, Hartl DL, Knippenberg E, Myers SS, Ralalason DH, Ramihantaniarivo H, Randriamady H, Shahab-Ferdows S, Vaitla B, Volkman SK and Vonona MA (2019) Cohort Description of the Madagascar Health and Environmental Research–Antongil (MAHERY–Antongil) Study in Madagascar. Front. Nutr. 6:109. doi: 10.3389/fnut.2019.00109 July 2019 \| Volume 6 \| Article 109 Frontiers in Nutrition \| www.frontiersin.org 1 Golden et al. MAHERY-Antongil Cohort Study intra-annual seasonal changes and minor inter-annual changes), the project will allow us to calculate the public health value of sustainable ﬁsheries management approaches for local populations. There is hope that coastal zones that are managed sustainably can increase the productivity of ﬁsheries, increasing the catch of seafood products for poor, undernourished populations. intra-annual seasonal changes and minor inter-annual changes), the project will allow us to calculate the public health value of sustainable ﬁsheries management approaches for local populations. There is hope that coastal zones that are managed sustainably can increase the productivity of ﬁsheries, increasing the catch of seafood products for poor, undernourished populations. Keywords: food security, micronutrient deﬁciencies, ﬁsheries, malnutrition, health impact assessment, seafood INTRODUCTION Using this variation in ﬁsheries management, we formed the cohort to understand the contribution of wild captured seafood in supporting the nutritional well-being of local people. The cohort also provided an opportunity to understand the interactive dynamics among subsistence ﬁshing, dietary intake, nutritional status, and the incidence of intestinal parasites and malaria. To account for seasonal changes in dietary patterns and disease status, our team characterized the diets of individuals over long time scales, similar to past MAHERY cohort studies (18). In the context of the very high prevalence of stunting (linear growth retardation) in Madagascar—over 50% of children under 5 years of age are stunted (19)—understanding the relationship of diets to physical, nutritional and developmental outcomes across the lifecourse is critical. This is particularly the case in low-income countries, where deﬁciencies of iron, zinc, and vitamins A, D, and B vitamins (especially B12) cause a range of poor health outcomes, including increased morbidity and mortality, poor child development and pregnancy outcomes, and low micronutrients in maternal milk (20, 21). The Madagascar Health and Environmental Research-Antongil (MAHERY-Antongil) study cohort was set up in September 2015 to assess the nutritional value of seafood for the coastal Malagasy population living along Antongil Bay in northeastern Madagascar. We aimed to understand the relationships among diﬀerent marine resource governance models, local people’s ﬁsh catch, the consumption of seafood, and nutritional status (1–3). Marine conservation has been hypothesized to increase ﬁsheries productivity and to increase the catch of seafood for local consumption (4–6). The direct impacts of ﬁsheries management on human nutrition have been relatively understudied, however, with many long-lasting policy narratives lacking a rigorous evidence base (1, 7). Several studies have created a foundation to assess this relationship [e.g., (3, 8)]. Certain studies have examined the contribution of ﬁsh to overall dietary patterns [e.g., (9, 10)], and in some cases to nutrient intake, including micronutrients, vitamins, and protein [e.g., (11)] or linking to nutritional status as proxied by anthropometry [e.g., (12, 13)]. Our study diﬀers in that it connects ﬁsheries management practices to dietary patterns, nutrient intakes, anthropometric measures, and clinical biomarkers of micronutrient, vitamin, and fatty acid status. Socio-Economic Status In 2015, we began a prospective cohort study in ﬁve remote rural communities (hereafter referred to as Communities 1–5 or C1– 5) along the coast of Antongil Bay in northeastern Madagascar. The ﬁsheries of Communities 1 and 2 are traditionally managed without governmental or NGO oversight, co-management, or control. The ﬁsheries of Communities 3 and 4 are LMMAs co- managed by WCS since 2007. Community 5 is located near a marine national park (oﬃcially designated in 1997), where the national government installed a protected area strictly limiting access to the local ﬁshery. In terms of exclusive access to ﬁshing rights, Community 5 had exclusive “sea tenure”— no other communities can ﬁsh inside the marine national park—whereas Communities 1–4 did not have sea tenure, and ﬁshing from adjacent villages was common. We assessed socio-economic status through: (1) weekly recalls of each household’s food-related expenditures (this did not include the consumption of home-grown foods); and (2) monthly recalls of (a) the amount (and source) of all cash income earned by members of each household, (b) the number of select commercial assets owned by members of each household (e.g., motorcycles, bicycles, radios, laptops/tablets, ﬂashlights, ﬁshing nets, boats, livestock), and (c) the amount of “luxury” goods consumed or used by each household during the prior day, week, and/or month (e.g., sugar, coﬀee, oil, salt, petrol). At the start of the study, we also recorded the primary economic activities of each household, their access to primary and secondary education, medical care, potable water, public safety services (e.g., police oﬃcers, court), and a list of market goods available from local stores. On a monthly basis, we conducted a morbidity recall to estimate the incidence of certain types of illnesses. The cohort in Communities 1–5 was maintained from September 2015 until October 2017. Using a community-wide comprehensive census that we created between May to June 2015, we assigned each household a number and randomly selected households to be included in the research. The C1 sample contained 25 households (out of a total of 360 households), and C2–5 contained 50 households each (out of a total of 260, 634, 180, and 98 households, respectively). The C1 household sample was less because C1 only had a half-time local assistant to monitor the population, whereas C2-5 had full time assistants present to assist with the research. At several points, households in the initial enrollment were lost. Study Site The Antongil Bay of northeastern Madagascar is characterized by lowland and litoral rainforest, a high prevalence of malnutrition, and a human population of rural agriculturalists and ﬁsherfolk predominantly of Betsimisaraka ethnicity. We selected this study site for two primary reasons: (1) it is a coastal population with heavy subsistence reliance on local seafood; and (2) it oﬀers the opportunity to study varying marine resource management systems. Market access is limited and domesticated meats and aquaculture products are an infrequent luxury in this region (22, 23). Thus, the consumption of seafood from wild capture ﬁsheries using nets, lines, and shore gleaning, can provide crucial micronutrients that are otherwise unavailable in the diet (2). Globally, beginning in the 1980s, decentralized management of marine systems and coastal zones became a preferred way to co-manage environments joining local communities with engagement and support from an non-govermental organization (NGO) (14, 15). This structure of governance is often called a locally managed marine area (LMMA). LMMAs devolve centralized government control over coastal zones to local authorities. In theory, they allow local communities to prevent overexploitation and reinforce sustainable management by tapping their unsurpassed knowledge of the local resource and ability to monitor human activity to assert control over their own resources. The goal of LMMAs is to simultaneously protect biodiversity and increase ﬁsheries productivity in support of local human health and livelihoods. Communities perceive that LMMAs confer beneﬁts (16), and we sought to assess whether these governance systems provide beneﬁts to human nutrition and health. Marine conservation and ﬁsheries management improvements could be viewed as a public health nutrition intervention; targeted conservation eﬀorts can rehabiliate ﬁsh stocks and increase ﬁsheries productivity, including reef systems in Madagascar (17), potentially creating synergies between marine conservation, and human well-being. In the Antongil Bay, ﬁsheries governance takes three general forms: traditional management, marine national parks, and co-management. Traditional management involves little to no involvement by the national government or non-governmental organizations, and focuses on culturally accepted Malagasy community practices that tend not to restrict ﬁshing gear or protect certain locations from harvest. In terms of formal restrictions on capture, traditional management tends to be the least strict of the three management forms. Marine national parks are managed by the national government and often July 2019 \| Volume 6 \| Article 109 Frontiers in Nutrition \| www.frontiersin.org 2 MAHERY-Antongil Cohort Study Golden et al. Data Collection Throughout the 28 month study, our team used a mixed-methods approach to collect a variety of information spanning social, environmental, and clinical health data (Figure 2). We surveyed all individuals in the study once per month over the duration of the study. For children who were too young to answer their own questions (typically under 5 years of age), we accepted proxy responses from primary caregivers. Study Site to moving villages or absence due to attending school in another area. Our ﬁnal sample population included 779 individuals of both sexes from birth to 91 years of age that enrolled and completed the clinical aspects of the study. involve “no-take zones” enforced by local government agents; they are the strictest form of ﬁsheries management. Co- management is often enabled through ﬁnancing and training by a NGO partner. The Wildlife Conservation Society (WCS) has been implementing co-management eﬀorts through a series of LMMAs. We hypothesized that co-management would lead to greater subsistence catch and more consumption of seafood in comparison to traditionally managed communities and communities adjacent to a marine national park. This hypothesis is rooted in evidence from the Paciﬁc that found that periodically harvested closures can maximize harvest eﬃciency and ﬁsheries yield beyond that achievable by no-take permanent closures or no management (24). Socio-Economic Status First, we lost several households in the period between initial enrollment and the start of our pilot period, largely due to some households moving outside the study area. These households were replaced through randomization prior to beginning the pilot period. The initial pilot period (from September 2015 until April 2016) began with 225 households and a total of 1,031 individuals, during which we ﬁnalized enrollment and began collecting preliminary baseline survey information. During this pilot period, 35 of the initial 225 households withdrew and we went from 1,031 to 878 individuals (Figure 1). Repeated socio-economic and dietary surveys of the cohort of 878 individuals were conducted prior to beginning the survey portion of the clinical study in May 2016 and well before our ﬁrst collection of clinical samples between July–August 2017. Following ﬁnal enrollment, we had ∼6.4% of the overall clinical enrollment subjects withdraw due to survey fatigue and/or fear of needles and 4.9% of the enrollment was lost to follow-up due Frontiers in Nutrition \| www.frontiersin.org Dietary Intake d di We measured dietary intake through monthly 24-h and prior- week recall assessments and food frequency surveys. These permit the calculation of the frequency, seasonality, and diversity of diets, though not the volume of foods or nutrients consumed. To estimate weights, we visited the same six households in each community, without prior notice, once per week and observed them during breakfast, lunch, and dinner to determine approximate weights of foods that were recorded in the dietary intake qualitative assessments. Prior to cooking, a research assistant from the community measured the quantity (to the nearest gram) of all foods (grains, vegetables, meats, etc.) consumed during mealtime. All meat weights were dressed weights after skinning, feathering, and cleaning had taken place. All weigt measurements were carried out using an EatSmart Precision Pro Digital Kitchen Scale, calibrated with a Chrome 100g calibration weight to assure proper scale functioning during the visits. The assistant recorded the individual IDs of all household members, as well as the number, age, and sex of all household guests present during each meal to account for variation in household attendance at meal times. Allocating the weight of household meals to individuals will follow procedures created in past work from this region (22). For food consumption outside of household meals, we collected an individual 24-h recall survey of foods eaten by each household member. For July 2019 \| Volume 6 \| Article 109 3 MAHERY-Antongil Cohort Study Golden et al. FIGURE 1 \| Consort ﬁgure of the MAHERY-Antongil study population. FIGURE 1 \| Consort ﬁgure of the MAHERY-Antongil study population. instance, cookies, coﬀee, honey, alcohol, insects, fruits, etc. that tend to be eaten outside of scheduled meals are accounted for in these six households by surveying each individual once per week throughout the duration of the study. specimens of each species caught by that ﬁsherman on that day, we also measured individual ﬁsh length (recorded in centimeters) and weight (grams). We completed monthly recall surveys of the number of over 75 species of seafood that household members ate during the prior week. We recorded whether each species eaten by that household was caught by a household member, received as a gift, and/or purchased. If caught, households speciﬁed both the method used to capture that ﬁsh and the distance of capture from the household (in minutes). If purchased, households speciﬁed the cost per unit. Dietary Intake d di During these interviews, we recorded the total amount of money earned from the sale of seafood, the percentage of seafood that was sold locally or exported, their total time invested in ﬁshing and indirect ﬁshing-related activities (e.g., net repair), and the total amount spent on all ﬁshing related activities (e.g., materials and/or services) during the prior month. Fisheries Activities We assessed intra-annual seasonal diﬀerences in ﬁsheries catch through weekly shore-side catch surveys, monthly household recall surveys, and the seasonal mapping of ﬁshing ranges. At each of the ﬁve sites, we conducted the catch surveys of all ﬁsherman, once per week, following established methodological procedures [see (25)]. During these surveys we recorded, for each ﬁsherman/team of ﬁshermen sighted, the time they spent ﬁshing (time left shore, time returned to shore, and tidal stage), the number of boats in each group, the number of ﬁsherman in each boat, the name of the location where they ﬁshed and distance from shore (in minutes), and the method they used for catching seafood (e.g., net, line, spear, trap, hand, or other). Fishermen using nets reported the number and size (mesh size, net length, and net height) of all nets used during that outing. Those using lines, spears, and/or aquatic traps also reported line length, weight, hook size and style, spear style, and/or the number and style of traps used, respectively. A research assistant from the community measured the number of all seafood species (including species of marine invertebrates, ﬁsh, reptiles, and mammals; hereafter referred to as “ﬁsh”) caught by each ﬁsherman or team of ﬁshermen, the total weight sold, and the total weight intended to be eaten by that ﬁsherman’s family. For each individual species of ﬁsh caught, we also recorded the total number of that species, their total weight, grams sold, grams eaten by ﬁsherman’s family/household, and the cost of one individual of that species of ﬁsh on that given day. For ﬁve Clinical Visits and Biological Samples Following procedures from past MAHERY cohorts (18), we conducted the same protocols for two clinical visits (August/September 2016 and November/December 2016), each separated by 2–3 months to account for two distinct ﬁshing seasons (cold/wet and hot/dry, respectively). The cold/wet season corresponds to rough seas and less ﬁsh catch and consumption. The hot/dry season corresponds to smoother seas and high ﬁsh catch and consumption, often driven by the seasonal ﬂow of pelagic ﬁsh into the area. Clinical visits were deﬁned as a consultation with a doctor and the collection of blood and/or other biological samples. The household was notiﬁed ∼1 week in advance of their scheduled date. Every household visited our health center in their respective community. Frontiers in Nutrition \| www.frontiersin.org Fisheries Activities On the evening prior to the subject’s blood draw, each individual scheduled for July 2019 \| Volume 6 \| Article 109 4 Golden et al. MAHERY-Antongil Cohort Study FIGURE 2 \| Data and samples collected during the MAHERY-Antongil cohort study. subjects arrived, we applied a 5% lidocaine topical anesthetic cream to the area where the needle would be injected to minimize the amount of discomfort from blood collection. We drew blood via venipuncture using 21Gx1.5′′ safety needles that was collected into S-Monovette R⃝(Sarstedt, North Carolina) venous blood collection tubes (7.5 ml 15 × 92 mm, Lithium Heparin). Smaller children and infants would have their blood drawn using 23Gx1.5′′ safety needles. All blood collection materials were designed for trace metal analysis. Once blood was collected, we inverted tubes three times to properly activate the lithium heparin and attached a Haemo-DiﬀR⃝with a smear edge so that we could apply a drop of blood onto a slide to create a thin blood smear for microscopy. Another blood drop was applied to a rapid diagnostic test (several brands all provided freely by the Malagasy Ministry of Health) via a capillary tube for immediate malaria diagnosis. A blood drop was then inserted into a HemoCue Hb 201 microcuvette via a capillary tube to test for levels of hemoglobin. Finally, several blood drops were applied on Whatman ﬁlter paper FTA cards (two spots per individual) and one drop on OmegaQuant ﬁlter paper treated with HUFASaveTM. Dried blood from the Whatman FTA was used for DNA preservation/extraction and genotyping the following morning came to the health centers etablished for this study to have their height and weight measured and to answer all of the questions in our health survey. We did this to streamline activities the following morning and to remind all individuals that they needed to fast prior to their blood draw. The health survey comprised several questions concerning morbidity recalls, bednet usage, vitamin intake, and medication usage (including deworming medicine). Women of reproductive age were asked about pregnancy and breastfeeding. For anthropometry, we used an ETEKCITY electronic personal digital scale to measure body weight. We measured small children in their mother’s arms and then subtracted the mother’s independent weight. Frontiers in Nutrition \| www.frontiersin.org DISCUSSION Variable Outcome Sex (% female) 49.4 Age (median years; min-max) 16.0; (0.1–91.0) HOUSEHOLD MEDIAN ANNUAL INCOME (CURRENT INTERNATIONAL DOLLARS)* All communities $6,840 Community 1 $4,940 Community 2 $6,580 Community 3 $6,590 Community 4 $7,910 Community 5 $4,430 STUNTING AMONG CHILDREN ≤AGE 5 (% SEVERE; TOTAL) Both sexes (n = 184) 23.9; 44.2 Females (n = 99) 18.2; 37.4 Males (n = 85) 28.2; 51.9 UNDERWEIGHT AMONG CHILDREN ≤AGE 5 (% SEVERE; TOTAL) Both sexes (n = 184) 2.7; 19.6 Females (n = 99) 2.0; 14.1 Males (n = 85) 3.7; 25.9 WASTING AMONG CHILDREN ≤AGE 5 (% SEVERE; TOTAL)** Both sexes (n = 184) 2.2; 3.6 Females (n = 99) 1.0; 2.0 Males (n = 85) 3.7; 6.2 Reproductive aged women (women ages 15–49 as % of all women, n = 209) 46.7 Pregnant women (% of women of ages 15–49, n = 9) 4.7 Lactating women (% of women of ages 15–49, n = 27) 13.4 Current international dollars adjusted for purchasing power parity. Stunting, underweight, and wasting all based on WHO Multicentre Growth Reference Study Group (27). TABLE 1 \| Subject population description of key variables and outcomes. y g Fecal samples were provided by each individual enrolled in the study on roughly the same day as clinical visits so that our team could assess the presence and levels of intestinal parasites. Each subject was given a sterile polypropylene screw cap feces collection tube (Sarstedt, Sparks, NV; ref. 80.623). The subject was instructed to defecate on top of the waxy side of a banana leaf and collected three small spoonfuls of feces, which were transferred into the collection tube. Once the samples were returned to our local research team (typically 10 min to 10 h after collection), we added 3 mL of 97% ethanol. All samples were kept at ambient temperature prior to being stored in a −23◦C freezer within 14 days of collection, and then shipped on dry ice before being stored at −80◦C after shipment to the Harvard T.H. Chan School of Public Health. At one time point in March 2017, we administered a ﬁnger prick to evaluate the prevalence of malaria using a rapid diagnostic test. This permitted analysis of malarial infection across all three seasons in northeastern Madagascar. We also collected ﬁngernail samples at this time. Fisheries Activities We measured height using a Seca Road Rod and infant length using a Quick Medical Starters Measure Mat, mid-upper arm circumference (MUAC) of all children 5 years of age and under and the cranial circumferences of all children 2 years of age and under. These anthropometric measurements were collected every 3 months outside of the clinical visits as well. The following morning, ∼24 individuals (typically belonging to 2–6 households) arrived at ∼4:15–5:00 a.m., a time chosen to ensure subjects were in a fasting state, as well as to not interfere with the busy daily schedules of the subjects. When July 2019 \| Volume 6 \| Article 109 5 Golden et al. MAHERY-Antongil Cohort Study avoid contamination. These ﬁngernails will be tested for their carbon and nitrogen isotopic signature as well as for mercury and arsenic content. of Plasmodium infections, among other disease analyses. Dried blood from OmegaQuant ﬁlter paper was used to characterize fatty acid proﬁles for each individual following established protocols (26). Two health care professionals from the local Maroantsetra hospital worked simultaneously on all blood draws. All blood tubes were stored in the dark inside portable refrigerators and kept at 5◦C prior to centrifugation. Within 25 min of drawing, all tubes were spun in the centrifuge. INITIAL RESULTS We found a high prevalence of stunting, wasting, and underweight throughout the study population (Table 1). The population is heavily left-skewed with more than 50% of the population being ≤16 years of age, indicating rapid population growth in this region. Households are generally very poor, though far wealthier than non-coastal geographically adjacent populations in the Makira Natural Park (18). Laboratory analyses are still in progress; baseline point-of-care results are shown in Table 1. Centrifugation of the lithium heparin tubes permitted the separation of plasma from pelleted red blood cells following centrifugation. We centrifuged tubes at 3,300 RPM for 10 min using the Block Scientiﬁc Octafuge Plus Centrifuge (Block Scientiﬁc, Inc., Bellport, NY). Following centrifugation, all plasma was pipetted into 1.8 mL cryo-tubes (also trace metal free). These cryo-tubes were then placed in groups of 2–4 inside a section of nylon pantyhose (which is resistant to degradation in liquid nitrogen) and dropped into a liquid nitrogen tank for ﬂash freezing. We obtained plasma samples from 745 individuals over the course of the study. We were unable to obtain venous blood samples from some of the individuals due to unwillingness (n = 29) and technical diﬃculties (n = 5), the latter primarily from unsusccessful attempts to ﬁnd a vein (Figure 1). Frontiers in Nutrition \| www.frontiersin.org DISCUSSION The main strength of this cohort is its detailed longitudinal dietary data that correspond to a suite of nutritional biomarkers and disease targets. The study was purposefully designed to understand the nutritional contribution of seafood to people living in isolated, seafood-dependent regions of Madagascar—a context comparable to many other areas of sub-Saharan Africa All frozen plasma was shipped on dry ice from Madagascar to the Western Human Nutrition Research Center (WHNRC/USDA) via World Courier. At the WHNRC, aliquots of plasma were analyzed for zinc, copper, and iron by inductively coupled plasma-atomic emission spectroscopy (ICP-AES); retinol, β-carotene, α-tocopherol using high performance liquid chromatography with diaode array detection (HPLC-DAD); and vitamin B12, ferritin, transferrin receptors, and inﬂammation markers (C-reactive protein and α-1-acid glycoprotein) by automated bioanalyzers (Roche e411 and Integra 400). TABLE 1 \| Subject population description of key variables and outcomes. Variable Outcome Sex (% female) 49.4 Age (median years; min-max) 16.0; (0.1–91.0) HOUSEHOLD MEDIAN ANNUAL INCOME (CURRENT INTERNATIONAL DOLLARS) All communities $6,840 Community 1 $4,940 Community 2 $6,580 Community 3 $6,590 Community 4 $7,910 Community 5 $4,430 STUNTING AMONG CHILDREN ≤AGE 5 (% SEVERE; TOTAL) Both sexes (n = 184) 23.9; 44.2 Females (n = 99) 18.2; 37.4 Males (n = 85) 28.2; 51.9 UNDERWEIGHT AMONG CHILDREN ≤AGE 5 (% SEVERE; TOTAL) Both sexes (n = 184) 2.7; 19.6 Females (n = 99) 2.0; 14.1 Males (n = 85) 3.7; 25.9 WASTING AMONG CHILDREN ≤AGE 5 (% SEVERE; TOTAL)** Both sexes (n = 184) 2.2; 3.6 Females (n = 99) 1.0; 2.0 Males (n = 85) 3.7; 6.2 Reproductive aged women (women ages 15–49 as % of all women, n = 209) 46.7 Pregnant women (% of women of ages 15–49, n = 9) 4.7 Lactating women (% of women of ages 15–49, n = 27) 13.4 Current international dollars adjusted for purchasing power parity. *Stunting, underweight, and wasting all based on WHO Multicentre Growth Reference Study Group (27). TABLE 1 \| Subject population description of key variables and outcomes. 2. Golden CD, Allison EH, Cheung WW, Dey MM, Halpern BS, McCauley DJ, et al. Fall in ﬁsh catch threatens human nutrition. Nature. (2016) 534:317–20. doi: 10.1038/534317a 3. Kawarazuka N. The Contribution of Fish Intake, Aquaculture, and Small- Scale Fisheries to Improving Food and Nutrition Security: A Literature Review. Penang: WorldFish Center Working Paper (2010). DISCUSSION The index ﬁnger of the left hand was preferentially used, though sometimes multiple ﬁngers were used to collect an adequate sample. Fingernails of each individual in the study were clipped and weighed on American Weigh Signature Series Digital Pocket Scales (American Weigh Scales, Atlanta, GA) to obtain ∼30 mg of ﬁngernails. These ﬁngernails were then placed into a Staples #1 Coin Envelope and folded closed without using the glue seal to July 2019 \| Volume 6 \| Article 109 6 MAHERY-Antongil Cohort Study Golden et al. ETHICS STATEMENT All households were recruited and enrolled, and each individual consented or assented, following our IRB approved study (Protocol #15-2230, Committee on the Use of Human Subjects, Oﬃce of Human Research Administration at the Harvard T.H. Chan School of Public Health). Consent forms were read by literate study members and our team read the form contents to illiterate studymembers. After a discussion of the study materials, consent and permission were attained for all participants. The study was also reviewed and approved by the Malagasy Ministry of Health, and conducted in concert with the regional medical inspector of the Maroantsetra region of Madagascar. Study subjects were not compensated for their participation. However, the population did receive beneﬁts for participating by having access to healthcare professionals working in their community. FUNDING The work was supported by the UK Government Department for Environment, Food & Rural Aﬀairs’ Darwin Initiative [22- 016] (co-I CG), the Wellcome Trust Our Planet, Our Health program [Grant number: 106864MA] (PI: CG), the Rockefeller Foundation through support to the Planetary Health Alliance, and Cornell University. Analytical and conceptual support was provided by the National Socio-Environmental Synthesis Center (SESYNC) under funding received from the National Science Foundation DBI-1052875 (PI: CG). The aforementioned funding included ﬁnancial support for nutritional analyses conducted by the USDA/ARS Western Human Nutrition Research Center, and additional laboratory analyses of malaria genetics and genomics was supported by the Harvard Malaria Initiative and funded by the Bill and Melinda Gates Foundation and the National Institutes of Health (PI, DLH: NIH grant AI106734 and PIs, DLH, and Dyann L. Wirth: NIH grant AI099105). ACKNOWLEDGMENTS We extend our deepest gratitude to many of the Malagasy research assistants from the MAHERY team. Without their assistance, and the tremendous support we received from local communities, this work would never have been possible. We would also like to thank Madagascar’s Ministry of Public Health and the Maroantsetra District Public Health Service for their logistical and intellectual support. We also extend our thanks to Stephanie D’Agata and Tolojanahary Rakotonirina (unfortunately this manuscript will be published post-humously for Tolotra). DATA AVAILABILITY All point-of-care health results (anemia and malaria) were provided by our team’s health professionals to the study subjects including access to free treatment and referrals to local clinics. All health results from laboratory analyses that were conducted outside of the study area were returned to participants. The health data collected through this study will not be made open access for public use but collaboration is welcomed. Please contact Dr. Christopher Golden (golden@hsph.harvard.edu) for more information. AUTHOR CONTRIBUTIONS and small island developing states of the Paciﬁc (2), particularly in the context of rising metabolic disease and hypertension indicated in Madagascar (28). Another strength of the study is that we sampled a population of both sexes and all ages that was randomized within communities to determine whether impacts were consistent across demographic characteristics, socio-economic status, and ﬁsheries governance regimes (2). CG designed the study, led the ﬁeld research and data analysis, and drafted the manuscript. CB, BR, EA, GB, HRan, and MV co-led the ﬁeld research, supporting data collection, database synthesis, and analytical support. GB and BV led the database synthesis, and analytical support. BR, DLH, and SV designed and led the malaria research embedded within the cohort. CB, EK, and JG designed a module of the research on environmental shocks and agricultural and health outcomes. LA, DH, and SS-F analyzed blood samples and supported the study design for nutritional biomarkers. SM supported the study design of the cohort and the analysis. DR and HRam supported the study design, ﬁeld logistics, health system referrals, permitting, and translation of research into policy. All authors contributed to revising and approving the manuscript. A primary weakness of the study is that the duration (28 months) and observational nature is insuﬃcient to understand the causal relationship between ﬁsheries governance strategies, ﬁsh catch, and subjects’ nutritional or health status. However, we can create scenarios of the impacts of ﬁsheries governance on ﬁsh catch by assuming that the scientiﬁc basis for marine conservation is valid. For instance, a global meta-analysis of hundreds of marine protected areas around the world found roughly 1.6 times greater ﬁsh biomass in marine protected areas than in unprotected areas (29). We can also use seasonal variation in ﬁsh catch to infer the eﬀects of reduced productivity of ﬁsheries in the future. REFERENCES 1. Béné C, Arthur R, Norbury H, Allison EH, Beveridge M, Bush S, et al. Contribution of ﬁsheries and aquaculture to food security and poverty reduction: assessing the current evidence. World Dev. (2016) 79:177–96. doi: 10.1016/j.worlddev.2015.11.007 July 2019 \| Volume 6 \| Article 109 Frontiers in Nutrition \| www.frontiersin.org 7 MAHERY-Antongil Cohort Study Golden et al. 4. Anderson JL, Anderson CM, Chu J, Meredith J, Asche F, Sylvia G, et al. The ﬁshery performance indicators: a management tool for triple bottom line outcomes. PLoS ONE. (2015) 10:e0122809. doi: 10.1371/journal.pone.0122809 20. Black R. Micronutrient deﬁciency – an underlying cause of morbidity and mortality. Bull World Health Organ. (2003) 8:79. 21. Tulchinsky TH. Micronutrient deﬁciency conditions: global health issues. Publ Health Rev. (2010) 32:243–55. doi: 10.1007/BF033 91600 5. Costello C, Ovando D, Clavelle T, Strauss CK, Hilborn R, Melnychuk MC, et al. Global ﬁshery prospects under contrasting management regimes. Proc Natl Acad Sci USA. 113:5125–9. doi: 10.1073/pnas.1520420113 22. Golden CD, Gupta AC, Vaitla B, Myers SS. Ecosystem services and food security: assessing inequality at community, household and individual scales. Environ Conserv. (2016) 43:381–8. doi: 10.1017/S0376892916000163 6. Ojeda-Martinez C, Bayle-Sempere JT, Sanchez-Jerez P, Salas F, Stobart B, Goni R, et al. Review of the eﬀects of protection in marine protected areas: current knowledge and gaps. Anim Biodiv Conserv. (2011) 34:191–203. 23. Golden CD, Seto KL, Dey MM, Dey MM, Chen OL, Gephart JA, et al. Does aquaculture support the needs of nutritionally vulnerable nations? Front Mar Sci. (2017) 4:159. doi: 10.3389/fmars.2017.00159 7. Mascia MB, Claus CA, Naidoo R. Impacts of marine protected areas on ﬁshing communities. Conserv Biol. (2010) 24:1424–9. doi: 10.1111/j.1523-1739.2010.01523.x 24. Carvalho PG, Jupiter SD, Januchowski-Hartley FA, Goetze J, Claudet J, Weeks R, et al. Optimized ﬁshing through periodically harvested closures. J Appl Ecol. (2019). doi: 10.1111/1365-2664.13417 8. Thilsted SH, Thorne-Lyman A, Webb P, Bogard JR, Subasinghe R, Phillips MJ, et al. Sustaining healthy diets: The role of capture ﬁsheries and aquaculture for improving nutrition in the post-2015 era. Food Policy. (2016) 61:126–31. doi: 10.1016/j.foodpol.2016.02.005 25. McClanahan TR, Hicks CC, Darling ES. Malthusian overﬁshing and eﬀorts to overcome it on Kenyan coral reefs. Ecol Appl. (2008) 18:1516–29. doi: 10.1890/07-0876.1 9. Alva S, Johnson K, Jacob A, D’Agnes H, Mantovani R, Evans T. Marine protected areas and children’s dietary diversity in the Philippines. Pop Environ. (2016) 37:341–61. doi: 10.1007/s11111-015-0240-9 26. Harris WS, Del Gobbo L, Tintle NL. REFERENCES The Omega-3 Index and relative risk for coronary heart disease mortality: estimation from 10 cohort studies. Atherosclerosis. (2017) 262:51–4. doi: 10.1016/j.atherosclerosis.2017.05.007 10. Cabral RB, Geronimo RC. How important are coral reefs to food security in the Philippines? Diving deeper than national aggregates and averages. Mar Policy. (2018) 91:136–41. doi: 10.1016/j.marpol.2018.02.007 27. WHO Multicentre Growth Reference Study Group. WHO Child Growth Standards: Length/Height-for-Age, Weight-for-Age, Weight-for-Length, Weight-for-Height and Body Mass Index-for-Age: Methods and Development. Geneva: World Health Organization (2006). Available online: http://www. who.int/childgrowth/standards/Technical_report.pdf (accessed November 27, 2017). 11. Roos N, Mazharul Islam M, Thilsted SH. Small ﬁsh is an important dietary source of vitamin A and calcium in rural Bangladesh. Int J Food Sci Nutr. (2003) 54:329–39. doi: 10.1080/09637480120092125 12. Fisher B, Ellis AM, Adams DK, Fox HE, Selig ER. Health, wealth, and education: the socioeconomic backdrop for marine conservation in the developing world. Mar Ecol Prog Ser. (2015) 530:233–42. doi: 10.3354/meps11232 28. Manus MB, Bloomﬁeld GS, Leonard AS, Guidera LN, Samson DR, Nunn CL. High prevalence of hypertension in an agricultural village in Madagascar. PLoS ONE. 2018; 13:e0201616. doi: 10.1371/journal.pone.0201616 p 13. Merten S, Haller T. Property rights, food security and child growth: dynamics of insecurity in the Kafue Flats of Zambia. Food Policy. (2008) 33:434–43. doi: 10.1016/j.foodpol.2008.01.004 29. Gill DA, Mascia MB, Ahmadia GN, Glew L, Lester SE, Barnes M, et al. Capacity shortfalls hinder the performance of marine protected areas globally. Nature. (2017) 543:665–9. doi: 10.1038/nature21708 14. Cinner JE, Daw TM, McClanahan TR, Muthiga N, Abunge C, Hamed S, et al. Transitions toward co-management: the process of marine resource management devolution in three east African countries. Glob Environ Change (2012) 22:651–8. doi: 10.1016/j.gloenvcha.2012.03.002 Conﬂict of Interest Statement: EK was employed as a graduate student at Cornell University at time of study and writing, and has since moved to Cooper/Smith. Conﬂict of Interest Statement: EK was employed as a graduate student at Cornell Conﬂict of Interest Statement: EK was employed as a graduate student at Cornell University at time of study and writing, and has since moved to Cooper/Smith. This has no impact on the research conducted. 15. Jupiter SD, Cohen PJ, Weeks R, Tawake A, Govan H. Locally-managed marine areas: multiple objectives and diverse strategies. Paciﬁc Conserv Biol. (2014) 20:165–79. Frontiers in Nutrition \| www.frontiersin.org July 2019 \| Volume 6 \| Article 109 REFERENCES doi: 10.1071/PC140165 The remaining authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. 16. McClanahan TR, Cinner JE, Abunge C, Rabearisoa A, Mahatante P, Ramahatratra F, et al. Perceived beneﬁts of ﬁsheries management restrictions in Madagascar. Ecol Soc. (2014) 19:5. doi: 10.5751/ES-06080-190105 The handling editor declared a shared aﬃliation, though no other collaboration, with one of the authors CB. 17. McClanahan TR, Maina JM, Graham NAJ, Jones KR. Modeling reef ﬁsh biomass recovery potential, and management priorities in the Western Indian Ocean. PLoS ONE. (2016) 11:e0154585. doi: 10.1371/journal.pone.0154585 Copyright © 2019 Golden, Borgerson, Rice, Allen, Anjaranirina, Barrett, Boateng, Gephart, Hampel, Hartl, Knippenberg, Myers, Ralalason, Ramihantaniarivo, Randriamady, Shahab-Ferdows, Vaitla, Volkman and Vonona. This is an open- access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Copyright © 2019 Golden, Borgerson, Rice, Allen, Anjaranirina, Barrett, Boateng, Gephart, Hampel, Hartl, Knippenberg, Myers, Ralalason, Ramihantaniarivo, Randriamady, Shahab-Ferdows, Vaitla, Volkman and Vonona. This is an open- access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. 18. Golden CD, Anjaranirina EJG, Fernald LC, Hartl DL, Kremen C, Milner DA Jr, et al. Cohort proﬁle: the Madagascar Health and Environmental Research (MAHERY) study in north-eastern Madagascar. Int J Epidemiol. (2017) 46:1747–8d. doi: 10.1093/ije/dyx071 19. United Nations Development Programme. Human Development Report 2016: Human Development for Everyone. New York, NY: UNDP (2016). Available online at: http://hdr.undp.org/sites/default/ﬁles/2016_human_ development_report.pdf (accessed November 27, 2017). July 2019 \| Volume 6 \| Article 109 Frontiers in Nutrition \| www.frontiersin.org 8
https://openalex.org/W4286829479	https://www.scielo.br/j/aabc/a/9K6VPdtdPP5n4kbgRBbqCDP/?lang=en&format=pdf	English	null	Arbuscular mycorrhizal fungi community in coffee agroforestry, consortium and monoculture systems	Anais da Academia Brasileira de Ciências	2,022	cc-by	8,962	WELLUMA T. BARROS, PATRÍCIA A.B. BARRETO-GARCIA, ORIVALDO JOSÉ SAGGIN JÚNIOR, RAFAEL N. SCORIZA & MAICON S. DA SILVA Abstract: Understanding the effects of different production systems on arbuscular mycorrhizal fungi (AMF) can help to interpret interactions between their components and to defi ne management strategies. As a result, our study was conducted on soils under three coffee production systems (one homogeneous and two heterogeneous) and in a native forest located in the Bahia state, Brazil. This study aimed to answer the following questions: 1) Does the organization and management of the coffee production system affect the occurrence and diversity of AMF?; and 2) Is the seasonality effect similar between systems? To do so, soil samples (0-10 cm depth) were collected at two times of the year (rainy and dry). Number of spores (NS) and average richness did not show differences between the systems, only between seasons. There was a reduction in NS in the dry season (1.4 and 2.7 spores g-1 soil) in relation to the rainy season (3.8 to 12.5 spores g-1 soil). The infl uence of coffee production systems was observed in the presence and absence of some AMF species. The AMF community was shown to be related to the plant species composition of the system, which was refl ected in the dissimilarity of heterogeneous systems in relation to the coffee monoculture system. Key words: Grevillea robusta, Coffea arabica, Musa spp., Mycorrhizae, seasonality. An Acad Bras Cienc (2022) 94(3): e20201228 DOI 10.1590/0001-3765202220201228 Anais da Academia Brasileira de Ciências \| Annals of the Brazilian Academy of Sciences Printed ISSN 0001-3765 I Online ISSN 1678-2690 www.scielo.br/aabc \| www.fb.com/aabcjournal INTRODUCTION gaining importance nationally and worldwide. Agroforestry systems (AFS) are considered the land use models which most ecologically resemble native forests (Nair 1993, Gama- Rodrigues 2004, Miccolis et al. 2016). In these systems there is the association of agricultural crops with tree components which enables an increase in the entry of organic matter into the soil, and as a consequence favors improving its chemical, physical and biological characteristics. In addition, AFS can contribute to greater diversity in the microbial community and soil fauna, which act as biological control agents and soil conditioners (Young 1994). Coffee (Coffea arabica L. and C. canephora Pierre) is widely cultivated in Brazil and constitutes one of the most important products for the national and world economy, giving the country the title of largest producer and exporter of coffee in the world. The state of Bahia is the fourth largest coffee producer among the Brazilian states, with relevant participation in regional development with an annual production of about 3.8 million 60-kilo sacks of coffee (CONAB 2020). The coffee production system most adopted is monoculture in full sun. However, adopting systems which optimize land use and enable biological and socioeconomic benefi ts has been An Acad Bras Cienc (2022) 94(3) An Acad Bras Cienc (2022) 94(3) MYCORRHIZAL FUNGI IN COFFEE PRODUCTION SYSTEMS WELLUMA T. BARROS et al. Understanding the effects of different production systems on soil quality can assist in interpreting interactions between its components and in defining management strategies (Marshall 2000). Arbuscular mycorrhizal fungi (AMF) are among the biological attributes of the soil which are considered sensitive to changes in the environment. These organisms form symbiotic associations in the roots of host plants (Pereira et al. 2018). Thus, plants are able to meet the demands of the AMF for carbon compounds through this relationship (Moreira & Siqueira 2006, Ghazanfar et al. 2016), while fungi favor absorption of nutrients from the soil (Mergulhão et al. 2014). In addition, AMF provide several other benefits such as favoring moisture retention, aggregate formation, soil stability (Nobre et al. 2015) and stimulating the primary defense system of plants to attack pathogens (Mechri et al. 2014), which increases their tolerance to biotic stress caused by diseases (Calvo-Polanco et al. 2016, Meddad- Hamza et al. 2017). influences the AMF community according to its characteristics (Posada et al. 2016). INTRODUCTION For example, a homogeneous (monoculture) system tends to provide a less favorable environment to root colonization and diversity of AMF species when compared to heterogeneous systems such as AFS or a native forest (Siqueira et al. 2010, Prates Júnior et al. 2019). This is because the species composition of the system interferes with plant- fungus interactions because AMF occurrence and distribution are conditioned by the existence of suitable hosts (Verbruggen et al. 2012) and by the release of root exudates (Ajeesh et al. 2015). In addition, implementing management techniques such as soil movement also affects the AMF as it causes hyphae disruption, and as a consequence propagule and spore exposure, thus decreasing their infectious capacity (Jasper et al. 1991, Kabir et al. 1997, Caproni et al. 2003, Hu et al. 2015). Several studies on the AMF community have been carried out in Brazil on monoculture crop systems, agroforestry systems and native forests (Loss et al. 2009, Ferreira et al. 2012, Costa et al. 2013, Santos et al. 2014, Lima et al. 2015, Souza et al. 2016, Durazzini et al. 2016, Pereira et al. 2018, Martins et al. 2019). However, studies comparing different coffee production systems are still scarce (Bonfim et al. 2010, Durazzini et al. 2016), especially those which evaluate native forest as a reference system. The occurrence of AMF is regulated by several biotic and abiotic factors which influence the abundance and survival of infectious propagules (Mello et al. 2012) and the richness of communities (Sousa et al. 2014, Ferreira et al. 2018), altering the root colonization process in plants (Rocha et al. 2020, Moreira et al. 2019). Among these factors there are climatic conditions, the cultivation system organization (homogeneous or heterogeneous) and the adopted management (Martínez-García et al. 2012, Carrenho et al. 2010). Given the above, our study aimed to answer the following questions: 1) Does the organization and management of the coffee production system affect the occurrence and diversity of AMF community?; and 2) Is the seasonality effect similar between systems? To do so, the AMF community in three coffee production systems (one homogeneous and two heterogeneous) and in a native forest (which was used as a reference) were evaluated. An Acad Bras Cienc (2022) 94(3) e20201228 2 \| 15 Area descriptions The study was conducted in the district of Lucaia, municipality of Planalto, Southwest region of the state of Bahia, Brazil. Three coffee production systems and a natural vegetation area were evaluated: (1) AFS - Coffea arabica L. with Grevillea robusta agroforestry system, 17 years old and spacing 3.5 x 15.0 m (between trees) and 1.5 x 2.5 m (among coffee trees) (14° 44’ 58” S and 40° 32’ 21” W); (2) BC - Coffea arabica L. with banana (Musa spp.) consortium, aged 17 years old, including drastic coffee pruning (Stumping) at the age of eight, and established in 1.5 x 4 spacing, 0 m (among coffee trees) and 1.0 x 16.0 m (among banana trees) (14° 45’ 01” S and 40° 31’ 24” W); and (3) MC - Coffea arabica L. monoculture, 15 years old, with two stumpings and 1.5 x 2.5 m spacing (14° 45’ 08” S and 40° 32’ 27” W); and (4) NF - native forest, which was used as a reference system and is located in an area adjacent to the coffee systems (14° 44’ 52” S and 40° 31’ 21” W). The region has a tropical altitude climate (Cwb) according to the Köppen classification, with an average altitude of 923 meters above sea level (SEI 2013), average annual temperature of 19.2°C and an average annual rainfall of 750 mm. The monthly rainfall data from September 2017 to July 2018 are shown in Figure 1. The soil in the studied areas is classified as Oxisol according to the USDA-Natural Resources Conservation Service classification (Soil Survey Staff, 2014), and dystrophic Yellow Latossol according to the Brazilian Classification System (Santos et al. 2018a). INTRODUCTION It was assumed that the production system causes different The climate directly controls forming an association and establishing AMF communities due to temperature variations and water availability, and indirectly according to the plants’ demand for water and nutrients which is higher at certain times of the year (Santos et al. 2014). Similarly, the cultivation system also MYCORRHIZAL FUNGI IN COFFEE PRODUCTION SYSTEMS WELLUMA T. BARROS et al. magnitudes of change in the structure and composition of the AMF community according to its organization and management. cover-maintenance fertilization (32 Mg ha-1 of bovine manure). Soil tillage with plowing, harrowing and furrowing, planting fertilization (20 Mg ha-1 of simple superphosphate) and annual maintenance (17 Mg ha-1 of urea and 33 Mg ha-1 of NPK 20-00-20) were adopted in the BC and MC systems. Maintenance was performed twice a year in all systems with clearing to control spontaneous herbs. An Acad Bras Cienc (2022) 94(3) e20201228 3 \| 15 Soil and litter sampling First, four plots of 20 m x 20 m (400 m2) were demarcated randomly in each system, ensuring a minimum distance of 10 m between plots. The soil and litter collections were carried out in the months of December 2017 (beginning of the rainy season) and April 2018 (beginning of the dry season). The native forest fragment has vegetation classified as Semi-deciduous Seasonal Forest and a total area of about 30 hectares. It is a forest with relatively low arboreal stratum (between 10 and 15 m high), with a predominance of the Parapiptadenia and Anadenanthera genera (IBGE 2012) and intermediate regeneration stage according to criteria described in CONAMA Resolution #01/1994 (Brasil 1994), since it has not been submitted to any intervention for over 20 years. Random soil sampling was performed after removing (cleaning) the litter, collecting 10 individual samples (depth 0-10 cm) which were gathered to form a composite sample from each plot. The accumulated surface litter was collected with a square wooden template of 0.25 m2 (0.5 m x 0.5 m) which was randomly thrown over the area of each plot. The litter samples were dried in an oven at 65ºC, then weighed on a precision scale (0.01g) and the dry mass results were converted to Mg ha-1. The AFS was established from opening furrows with planting fertilization (20 Mg ha-1 of simple superphosphate) and annual organic MYCORRHIZAL FUNGI IN COFFEE PRODUCTION SYSTEMS WELLUMA T. BARROS et al. Figure 1. Monthly rainfall recorded at the station closest to the study site (municipality of Vitória da Conquista, Bahia, Brazil), from September 2017 to July 2018 (Source: INMET 2020). Figure 1. Monthly rainfall recorded at the station closest to the study site (municipality of Vitória da Conquista, Bahia, Brazil), from September 2017 to July 2018 (Source: INMET 2020). The soils were chemically characterized according to Table I following the procedures described by EMBRAPA (2017): pH in water; extractable P and K by Mehlich-1; Ca2+, Mg2+ and Al3+ exchangeable with 1 mol L-1 of KCl; and organic matter by oxidation with 0.4 mol L-1 of K2Cr2O7. Data analysis The number of AMF spores counted in 50 cm3 of soil was transformed into the abundance of spores g-1 of soil. Furthermore, total spore richness, mean richness and occurrence frequency have been calculated in each of the four plots (repetitions) per site. The obtained data were analyzed for normality (Shapiro-Wilk) and homogeneity (Cochran and Bartlett test) of the error variances, and converted when necessary. Parametric data were subjected to analysis of variance (ANOVA) when found, according to a completely randomized design (CRD). Multiple comparisons of the means were performed between times and between treatments by the Tukey test at 5% significance when ANOVA showed a significant result in the F-test (p < 5%). The analyzes were performed using the Assistat® v.7.7 statistical software program. Spore extraction, counting and identification In which: AFS = agroforestry system coffee with Grevillea robusta, BC = banana coffee, MC = monoculture coffee, NF = native forest, SOM = soil organic matter, H+Al = potential acidity, SB = sum of soil bases, E = effective soil CEC, soil moisture = moisture at the time of collection. III). Of this total, 15 species occurred in the rainy season and ten species in the dry season. This was reflected in greater total species richness in the rainy season for all studied systems (Table II), although the average richness only showed differences between seasons in the NF. Following this same pattern, a reduction in the number of spores (NS) was observed in the dry season for most systems. The total density in the rainy season varied from 3.8 to 12.5 spores g-1 soil, while the density in the dry season was between 1.4 and 2.7 spores g-1 soil (Table II). The presence-absence of the AMF species (occurrence or non-occurrence of species, respectively), accumulated litter and soil moisture data were complementarily submitted to a principal component analysis (PCA) using the Addinsoft XLSTAT® Version 2020.1.3 (1995- 2020) program. This analysis was performed to synthesize the multidimensional variation of the treatments in a diagram and order them into the components according to their similarities around the measured soil variables. The interrelationships between attributes of soil, litter, spore numbers and AMF richness were analyzed using Pearson’s 5% correlation using the SAEG® v.9.1 program. Although no significant variations were observed between the systems regarding the number of spores and AMF richness (Table II), significant correlations were observed between mean species richness and soil pH (r = -0.66; p < 0.05), litter (r = 0.67; p<0.05), soil moisture (r = 0.87; p<0.05) and SOM (r = 0.70; p<0.05). In addition, differences were observed regarding the presence and absence of AMF species (Table III). The Acaulospora denticulata, Acaulospora mellea, Acaulospora scrobiculata and Claroideoglomus etunicatum species only occurred in coffee production systems. Glomus macrocarpum and Sclerocystis clavispora occurred in all systems studied at both times of the year. Acaulospora tuberculata, Gigaspora sp. and Racocetra persica exclusively occurred in Spore extraction, counting and identification First, 50 g of each soil sample were used to extract the arbuscular mycorrhizal fungi (AMF) spores, adopting an adapted procedure described for nematodes according to the wet- sieving methodology (Gerdemann & Nicolson 1963) and centrifugation in density gradient with water and 45% sucrose (Jenkins 1964). Next, spore counting and species identification were performed using a stereoscopic microscope, referring to the Schenck & Pérez manual (1988) and the international collection website of AMF - INVAM (2020, https://invam.wvu.edu/). An Acad Bras Cienc (2022) 94(3) e20201228 4 \| 15 MYCORRHIZAL FUNGI IN COFFEE PRODUCTION SYSTEMS WELLUMA T. BARROS et al. Table I. Chemical attributes and humidity of Dystrophic Yellow Latossol (depth 0-10 cm) under three coffee production systems and in native forest. Table I. Chemical attributes and humidity of Dystrophic Yellow Latossol (depth 0-10 cm) under three coffee production systems and in native forest. System pH P SOM K Ca Mg H+Al SB E Soil moisture Moist Dry H2O mg dm-3 g dm-3 ------ cmolc dm-3 ------ AFS 6.2 41.5 21.0 0.5 3.8 2.6 2.7 6.9 7.0 12.9 13.0 BC 6.2 26.5 17.0 0.6 5.0 2.2 3.3 7.7 7.8 12.6 14.2 MC 7.2 27.0 15.5 0.7 4.0 2.8 1.4 7.4 7.4 12.7 13.0 NF 5.5 3.5 30.0 0.2 4.0 3.0 7.1 7.1 7.3 18.1 19.7 In which: AFS = agroforestry system coffee with Grevillea robusta, BC = banana coffee, MC = monoculture coffee, NF = native forest, SOM = soil organic matter, H+Al = potential acidity, SB = sum of soil bases, E = effective soil CEC, soil moisture = moisture at the time of collection. oforestry system coffee with Grevillea robusta, BC = banana coffee, MC = monoculture coffee, NF = native organic matter, H+Al = potential acidity, SB = sum of soil bases, E = effective soil CEC, soil moisture = moisture ction In which: AFS = agroforestry system coffee with Grevillea robusta, BC = banana coffee, MC = monoculture coffee, NF = native forest, SOM = soil organic matter, H+Al = potential acidity, SB = sum of soil bases, E = effective soil CEC, soil moisture = moisture at the time of collection. RESULTS The variation pattern in litter accumulation between the systems was the same at both times of the year (Table II). The highest value was observed in the native forest (12.26 Mg ha- 1), followed by AFS (6.07 Mg ha-1), which was not distinguished from the consortium (3.66 Mg ha- 1), which in turn was similar to monoculture (0.78 Mg ha-1). A total of 16 AMF species were identified and presented different occurrence frequencies according to the systems and time of year (Table An Acad Bras Cienc (2022) 94(3) e20201228 5 \| 15 MYCORRHIZAL FUNGI IN COFFEE PRODUCTION SYSTEMS WELLUMA T. BARROS et al. Table II. Accumulated litter (Mg ha-1), average number of spores (in 50 g of soil) and richness of arbuscular mycorrhizal fungi species in three coffee production systems and in native forest at two times of the year. Systems Litter NS TR AR Rainy Dry Rainy Dry Rainy Dry Rainy Dry AFS 6.16 Ab 5.98 Ab 202.5 Aa 70.25 Ba 9 5 4.5 Aa 2.75 Aa BC 3.93 Abc 3.38 Abc 626.75 Aa 126.75 Ba 10 6 5.25 Aa 3.25 Aa MC 0.84 Ac 0.72 Ac 193.00 Aa 136.75 Aa 10 10 5.50 Aa 4.25 Aa NF 14.17 Aa 10.34 Ba 474.25 Aa 81.75 Ba 11 6 5.50 Aa 3.25 Ba In which: Litter = accumulated dry phytomass on the soil, NS = number of spores, TR = total richness, AR = average richness. Different capital letters within rows compare season for each measurement, and different lower case letters in columns indicate differences using the Tukey test at 5% significance. Table II. Accumulated litter (Mg ha-1), average number of spores (in 50 g of soil) and richness of arbuscular mycorrhizal fungi species in three coffee production systems and in native forest at two times of the year. In which: Litter = accumulated dry phytomass on the soil, NS = number of spores, TR = total richness, AR = average richness. Different capital letters within rows compare season for each measurement, and different lower case letters in columns indicate differences using the Tukey test at 5% significance. In which: Litter = accumulated dry phytomass on the soil, NS = number of spores, TR = total richness, AR = average richness. Different capital letters within rows compare season for each measurement, and different lower case letters in columns indicate differences using the Tukey test at 5% significance. RESULTS the native forest during the rainy season. On the other hand, Claroideoglomus etunicatum only occurred in monoculture in the dry season, and Sieverdingia tortuosa occurred in all systems in the rainy season and in almost all of them in the dry season. therefore the most prevalent for differentiating the native forest, AFS and BC) were: litter, moisture and A. scrobiculata, A. tuberculata, Am. Leptoticha, Gigaspora sp., R. persica (Figure 2a, Table IV). In turn, the variables most strongly associated with PC2 and consequently with MC were A. denticulata, A. foveata, A. mellea, Glomus sp.1 and Glomus sp. (Figure 2a, Table IV). The most abundant genera in the two seasons considering all the studied systems were Acaulospora and Glomus (Table III), representing approximately 56% of the total number of AMF identified. The PCA for the dry season presented eigenvalues of 52.3% (PC1) and 40.4% (PC2). In addition to the A. mellea and A. scrobiculata species, litter and moisture were among the variables most associated with PC1 in the dry season following a similar pattern to the rainy season. The most important variables for PC2 were the Am. Leptoticha, C. pellucida, Cl. etunicatum and G. glomerulatum species (Figure 2c, Table IV). When analyzed together using PCA, the accumulated litter, soil moisture and presence and absence of AMF species explained more than 86% of the variation between treatments using the first two principal components in the two studied seasons (87.0% in the rainy season) and 92.6% in the dry season) (Figure 2). The graphic dispersion of the treatments in relation to the axes showed a similar pattern between the two periods (Figure 2b and 2d), with isolation of the NF (next to the principal component 1, PC1) and the MC (next to the principal component 2, PC2), which were in different quadrants. It also showed clustering of AFS and BC, which were located in the same quadrant between PC1 and PC2. An Acad Bras Cienc (2022) 94(3) e20201228 6 \| 15 DISCUSSION The greater litter accumulation in the NF can be attributed to the species composition and diversity in the native ecosystem which enables greater plant residue additions. This highlights the significant contribution of the tree component to the litter supply and also explains the fact that the AFS has the second most significant accumulation, although without distinction from the BC. Likewise, the smaller Eigenvalues of 63.5% for PC1 and 23.5% for PC2 were verified in the rainy season. The variables most associated with PC1 (and An Acad Bras Cienc (2022) 94(3) e20201228 6 \| 15 MYCORRHIZAL FUNGI IN COFFEE PRODUCTION SYSTEMS WELLUMA T. BARROS et al. Table III. Frequency of occurrence (%) of arbuscular mycorrhizal fungi species at two times of the year in three coffee production systems and in native forest. Species AFS BC MC NF Rainy season Acaulospora denticulata Sieverding & Toro. 0 25 25 0 Acaulospora foveata Trappe & Janos. 0 25 25 25 Acaulospora mellea Spain & Schenck. 25 75 0 0 Acaulospora scrobiculata Trappe. 25 25 50 0 Acaulospora tuberculata Janos & Trappe. 0 0 0 25 Ambispora leptoticha (Schenck & Smith) Morton & Redecker. 50 100 75 0 Cetraspora pellucida (T.H. Nicolson & N.C. Schenck) Oehl, F.A. Souza & Sieverd. 25 25 50 25 Gigaspora sp. 0 0 0 50 Glomus glomerulatum Sieverding. 50 0 75 50 Glomus macrocarpum Tulasne & Tulasne. 100 100 100 100 Glomus sp.1 0 0 50 25 Glomus sp. 25 25 0 100 Racocetra persica Oehl, Souza & Sieverd. 0 0 0 25 Sclerocystis clavispora (Trappe) Almeida & Schenck. 100 100 75 75 Sieverdingia tortuosa Schenck & Smith. 50 25 25 50 Dry season Acaulospora mellea Spain & Schenck 50 75 25 0 Acaulospora scrobiculata Trappe 25 25 25 0 Ambispora leptoticha (Schenck & Smith) Morton & Redecker 0 0 50 25 Cetraspora pellucida (T.H. Nicolson & N.C. Schenck) Oehl, F.A. Souza & Sieverd 0 0 25 25 Claroideoglomus etunicatum (W.N. Becker & Gerdemann) C. Walker & A. Schüßler 0 0 25 0 Glomus glomerulatum Sieverding 0 0 50 0 Glomus macrocarpum Tulasne & Tulasne 100 100 100 100 Glomus sp.1 25 25 25 50 Sclerocystis clavispora (Trappe) Almeida & Schenck 50 100 50 75 Sieverdingia tortuosa Schenck & Smith 25 0 50 50 and or with banana in relation to the system in full sun. An Acad Bras Cienc (2022) 94(3) e20201228 8 \| 15 DISCUSSION Table IV. Factor loadings and variability explained by the axes in the principal component analysis (PCA) of the presence-absence of arbuscular mycorrhizal fungi, litter and soil moisture in three coffee production systems and in native forest in the rainy and dry seasons in Planalto, Bahia, Brazil. Variables/ Treatments Rainy season Variables/ Treatments Dry season PC1 PC2 PC3 PC1 PC2 PC3 Factor loadings Factor loadings Litter 0.912 0.409 -0.042 Litter -0.828 -0.516 -0.221 Moisture 0.994 0.074 -0.083 Moisture -0.989 -0.090 0.117 A. denticulata -0.612 -0.616 -0.497 A. mellea 0.998 0.028 0.050 A. foveata 0.288 -0.679 -0.675 A. scrobiculata 0.998 0.028 0.050 A. mellea -0.623 0.762 -0.177 Am. leptoticha -0.592 0.789 -0.165 A. scrobiculata -0.994 -0.032 0.102 C. pellucida -0.592 0.789 -0.165 A. tuberculata 0.994 0.032 -0.102 Cl. etunicatum 0.314 0.939 -0.141 Am. leptoticha -0.994 -0.032 0.102 G. glomerulatum 0.314 0.939 -0.141 G. glomerulatum 0.431 -0.201 0.880 S. tortuosa 0.386 0.591 0.709 Gigaspora sp 0.994 0.032 -0.102 Glomus sp1 0.623 -0.762 0.177 Glomus sp. 0.275 0.912 -0.306 R. persica 0.994 0.032 -0.102 Variability % 63.519 23.461 13.020 Variability % 52.278 40.345 7.377 Cumulative % 63.519 86.980 100.000 Cumulative % 52.278 92.623 100.000 the studied conditions. However, variations in NS were observed by Bonfim et al. (2010) and Durazzini et al. (2016) when comparing agroforestry coffee systems with monoculture coffee systems. no significant differences in soil moisture were observed between the seasons of the year in all studied sites (Table I), which must be related to the fact that the soil was sampled on only one date, and therefore did not reflect average humidity conditions. According to Mangan et al. (2004), seasonality affects the occurrence of AMF as the species produce their spores at different times of the year, and these become physiologically active in seasons which are more conducive to their development. Reductions in the number of spores in the dry season were also observed by Khaekhum et al. (2017) and Ramos-Zapata et al. (2011) in eucalyptus stands and coastal dunes, respectively. The occurrence of the Acaulospora denticulata, Acaulospora mellea, Acaulospora scrobiculata and Claroideoglomus etunicatum species only in the coffee production systems is in line with the results found by Fernandes & Siqueira (1989), who observed the occurrence of these same species (except Acaulospora denticulate) in coffee plantations in the south of Minas Gerais. DISCUSSION amount of litter stocked in the monoculture compared to NF and AFS is explained by the system’s homogeneous characteristic which provides less diversity and less litter. In evaluating different coffee production systems, Meylan et al. (2017) observed a greater amount of litter in the shaded systems with Erythrina Litter accumulation was maintained in most of the studied systems when comparing the seasons, with the exception of the native forest which showed a significant increase in the rainy season (Table II). The fact that it only varied in the NF is related to the typical seasonal pattern An Acad Bras Cienc (2022) 94(3) e20201228 7 \| 15 MYCORRHIZAL FUNGI IN COFFEE PRODUCTION SYSTEMS WELLUMA T. BARROS et al. Figure 2. Diagram of the ordering of variables and treatments in the rainy season (a and b) and in the dry season (c and d) produced by the principal components analysis of the presence-absence of arbuscular mycorrhizal fungi, litter and soil moisture in three coffee production systems and in native forest in Planalto, Bahia, Brazil. Figure 2. Diagram of the ordering of variables and treatments in the rainy season (a and b) and in the dry season (c and d) produced by the principal components analysis of the presence-absence of arbuscular mycorrhizal fungi, litter and soil moisture in three coffee production systems and in native forest in Planalto, Bahia, Brazil. and branches falling by mechanical action (Dias & Oliveira-Filho 1997, Vendrami et al. 2012). of semi-deciduous seasonal forests, with litter deposition peaks coinciding with the end of the dry season as a vegetation response to climatic variation (Dias & Oliveira-Filho 1997, Santos Neto et al. 2015, Barreto-Garcia et al. 2019), which in turn is reflected in greater litter accumulations at the beginning of the rainy season. These larger accumulations are usually associated with the influence of rain which creates more favorable conditions for leaf renewal and due to leaves The reduction in the number of species, NS and richness in the dry season indicates that only the species which are more resistant to water deficit conditions would present reproduction and dispersion structures in the dry season. This denotes that the AMF community becomes less complex in low water availability conditions, thus preserving the most tolerant species (Santos et al. 2014). Despite this, An Acad Bras Cienc (2022) 94(3) e20201228 8 \| 15 MYCORRHIZAL FUNGI IN COFFEE PRODUCTION SYSTEMS WELLUMA T. BARROS et al. DISCUSSION This reveals a high adaptation of these species to the edaphoclimatic conditions prevalent in coffee ecosystems (Theodoro et al. 2003). For example, Acaulospora mellea was one of the species most commonly found in coffee The absence of variation in NS and average richness between systems suggests that the coffee production systems did not cause changes in these attributes for the AMF community under An Acad Bras Cienc (2022) 94(3) e20201228 9 \| 15 MYCORRHIZAL FUNGI IN COFFEE PRODUCTION SYSTEMS WELLUMA T. BARROS et al. plantations in Colombia and Mexico (Posada et al. 2016). negative correlation was observed between mean species richness and soil pH. According to Zhu et al. (2007), soil pH is a factor which directly or indirectly influences AMF diversity since it can compromise the nutrient availability for the fungus or for the plant. The presence of Glomus macrocarpum and Sclerocystis clavispora in all systems studied and at both times of the year suggests that these fungi have adapted well to the conditions of all studied systems. Glomus macrocarpum is usually reported as a species with a high capacity to adapt to stress and climatic variations, and therefore it is commonly found in different environmental conditions (Carvalho et al. 2012, Ferreira et al. 2012, Carneiro et al. 2015, Silva et al. 2016). On the other hand, the occurrence of Sclerocystis clavispora is more common in the dry season (Al-Yahya’Ei et al. 2011, Silva et al. 2016, 2019). The occurrence of Acaulospora denticulata, Acaulospora foveata, Acaulospora mellea, Acaulospora tuberculata, Gigaspora sp., Glomus sp. and Racocetra persica species only in the rainy season (Table III) shows that water availability was a limiting factor to sporulation. In turn, the occurrence of Sieverdingia tortuosa in all systems in the rainy season and in almost all the systems in the dry season is explained by the fact that this species is considered generalist, and can therefore occur in preserved or disturbed natural environments and in times with high or low water availability (Santos et al. 2014, Silva et al. 2016). The exclusivity of Acaulospora tuberculata, Gigaspora sp. and Racocetra persica in the native forest is possibly related to the characteristics of this environment which is more biologically complex than coffee systems, has higher levels of organic matter in the soil and is less subject to temperature and moisture variations. This would favor the survival of more demanding species in climate and soil conditions. An Acad Bras Cienc (2022) 94(3) e20201228 10 \| 15 MYCORRHIZAL FUNGI IN COFFEE PRODUCTION SYSTEMS MYCORRHIZAL FUNGI IN COFFEE PRODUCTION SYSTEMS WELLUMA T. BARROS et al. CONCLUSIONS (Tables I and II). The native forest provides greater litter (Table II) and organic matter accumulation in the soil (Table I) due to not suffering anthropic influence and presenting a great diversity of plant species, whereas monoculture causes smaller organic residue entry and a less diverse litter due to its homogeneity characteristic, in addition to presenting only one host species. This would be influencing the occurrence of some AMF species. An example of these are the Gigaspora sp., A. tuberculata, and Racocetra Perssica species which were found exclusively in the native forest, the Glomus sp. species which was only absent in the MC, and Am. Leptoticha and A. scrobiculata which did not occur in the NF. In line with this explanation, significant positive correlations were found between mean species richness and litter, soil moisture and SOM. According to Verbruggen et al. (2012), the occurrence and distribution of AMF species are related to contemporary ecological processes such as the existence of one or more hosts, and environmental factors such as organic matter content, soil temperature and moisture which act on the fungal community, conditioning its abundance and diversity. Although not presenting an effect on spore density and average species richness, coffee production systems cause changes in the presence or absence of arbuscular mycorrhizal fungi (AMF) species. The AMF community was shown to be related to the species composition of the productive system, which was reflected in a similar influence by the heterogeneous systems (agroforestry coffee-grevillea system and banana-coffee consortium), and distinct from the coffee monoculture and native forest in terms of effect on the fungal community. The species distribution and number of spores was shown to be influenced by climatic conditions with a reduction in the dry season, but without differentiation between the studied systems. DISCUSSION Several records of the occurrence of these species are found in the literature (Santos et al. 2014, 2018b, Pereira et al. 2018, Silva et al. 2019). The greater abundance of the Acaulospora and Glomus genera can be attributed to the fact that they produce smaller spores and in greater quantity, being less influenced by seasonal changes when compared to other genera such as Gigaspora, which have larger spores (Sousa et al. 2014). These genera are generally found with great frequency in a wide range of forest ecosystems (Davison et al. 2015, Soteras et al. 2015, Silva et al. 2016, Bonfim et al. 2016, Araújo et al. 2019, Pagano et al. 2019, Becerra et al. 2019) and also in agricultural ecosystems (Oehl et al. 2017, Cristo et al. 2018, Vieira et al. 2020). The presence of Claroideoglomus etunicatum only in the dry season is also in agreement with several studies which found the occurrence of this species being associated with water restriction periods, including in studies by Pedone-Bonfim et al. (2018) and Teixeira-Rios et al. (2013) in dry tropical forests, and Sousa et al. (2013) in cultivated areas in the semi-arid region of Brazil. On the other hand, the fact that this species only occurred in the MC suggests that the system provided some factor favorable to its occurrence or sporulation, such as the pH which was relatively higher in this system (Table I). Corroborating this hypothesis, a significant A similar dispersion pattern of treatments between the rainy season (Figure 2a and 2b) and dry season (Figure 2c and 2d) in the PCA with AFS and BC clustering and MC and NF isolation demonstrates that AMF dynamics in the studied systems remain between the seasons. The dissimilarity of NF and MC (Figures 2b and 2d) can be attributed to differences in the litter and soil moisture accumulation in these treatments An Acad Bras Cienc (2022) 94(3) e20201228 10 \| 15 REFERENCES Brasília: Diário Oficial da União, 1684-1685 p. EMBRAPA. 2017. Serviço Nacional de Pesquisa do Solo. Manual de métodos de análises de solo, 3ª ed., Brasília: Embrapa, 575 p. CALVO-POLANCO M, SÁNCHEZ-CASTRO I, CANTOS M, GARCÍA JL, AZCÓN R, RUIZ-LOZANO JM, BEUZÓN CR & AROCA R. 2016. Effects of different arbuscular mycorrhizal fungal backgrounds and soils on olive plants growth and water relation properties under well-watered and drought conditions. Plant Cell & Environ 39: 2498-2514. FERNANDES AB & SIQUEIRA JO. 1989. Micorrizas vesicular- arbusculares em cafeeiros da região sul do Estado de Minas Gerais. Pesqui Agropecu Bras 24: 1489-1498. FERREIRA DA, CARNEIRO MAC & SAGGIN JUNIOR OJ. 2012. Fungos micorrízicos arbusculares em um Latossolo Vermelho sob manejos e usos no Cerrado. Rev Bras Cienc Solo 36: 51-61. FERREIRA DA, CARNEIRO MAC & SAGGIN JUNIOR OJ. 2012. Fungos micorrízicos arbusculares em um Latossolo Vermelho sob manejos e usos no Cerrado. Rev Bras Cienc Solo 36: 51-61. CAPRONI AL, FRANCO AA, BERBARA RLL, TRUFEM SB, GRANHAI JRDO & MONTEIRO AB. 2003. Ocorrência de fungos micorrízicos arbusculares em áreas revegetadas após mineração de bauxita em Porto Trombetas, Pará. Pesq Agropec Bras 38: 1409-1418. FERREIRA PFA, SILVA LC, MARTINEZ HAR, FERREIRA KAL & NOBRE CP. 2018. Efeito da sazonalidade na comunidade de Fungos Micorrízicos Arbusculares em áreas com Mimosa caesalpiniifolia. R Tróp Ci Agr Biol 10: 105-113. CARNEIRO M AC, FERREIRA DA, SOUZA EDD, PAULINO HB, JUNIOR OJS & SIQUEIRA JO. 2015. Arbuscular mycorrhizal fungi in soil aggregates from fields of “murundus” converted to agriculture. Pesq Agropec Bras 50: 313-321. GAMA-RODRIGUES AC. 2004. Ciclagem de nutrientes em sistemas agroflorestais na região tropical: funcionalidade e sustentabilidade. In: Sistemas agroflorestais, tendência da agricultura ecológica nos trópicos: sustento da vida e sustento de vida. Ilhéus: Sociedade Brasileira de Sistemas Agroflorestais, p. 67-87. CARRENHO R, GOMES-DA-COSTA SM, BALOTA EL & COLOZZI- FILHO A. 2010. Fungos micorrízicos arbusculares em agroecossistemas Brasileiros. In: Micorrizas: 30 anos de pesquisa no Brasil. 7th ed., Lavras: Editora UFLA, p. 215-278. GERDEMANN JW & NICOLSON TH. 1963. Spores of mycorrhizal endogone species extracted from soil by wet-sieving and decanting. Trans Br Mycol Soc 46: 235-244. CARVALHO F, SOUZA FA, CARRENHO R, MOREIRA FMS, JESUS EC & FERNANDES GW. 2012. The mosaic of habitats in the high-altitude Brazilian rupestrian fields is a hotspot for arbuscular mycorrhizal fungi. Agric Ecosyst Environ Appl Soil Ecol 52: 9-19. REFERENCES AJEESH R, VIKAS K, SANTOSHKUMAR AV & SURENDRA GKH. 2015. Arbuscular Mycorrhizal Fungi (AMF) for Quality Seedling Production. Res J Agriculture Forestry Sci 3: 22-40. AL-YAHYA’EI MN, OEHL F, VALLINO M, LUMINI E, REDECKER D, WIEMKEN A & BONFANTE P. 2011. Unique arbuscular mycorrhizal fungal communities uncovered in date palm plantations and surrounding desert habitats of Southern Arabia. Mycorrhiza 21: 195-209. In turn, the ASF and BC grouping (Figures 2b and 2d) can be explained by the fact that these systems are made up of more than one plant species. Thus, the vegetation structure and composition (with the presence of the arboreal component in the AFS and the banana tree in the BC) would provide a specific environment for the AMF, with more diversified litter and a more balanced microclimate when compared to the MC. In other words, the heterogeneous systems would be exercising a similar influence in the AMF community, while the MC (as previously discussed) is distinguished by being composed of a single plant species presenting restriction in the entrance and diversity of organic residues and being more prone to disturbance. ARAÚJO TM, DA SILVA K, PEREIRA GMD, CURCINO A, STÜRMER SL & GOMIDE PHO. 2019. Diversity of Arbuscular Mycorrhizal Fungi in Agroforestry, Conventional Plantations and Native Forests in Roraima State, Northern Brazil. J Agric Sci 11: 282-290. BARRETO-GARCIA PAB, OLIVEIRA MF, OLIVEIRA FGR & LACERDA LRL. 2019. Edge Effect on the Litter Production of a Semi- Deciduous Seasonal Forest Fragment. Floresta Ambient 26: 1- 9. BECERRA AG, DIVÁN A & RENISON D. 2019. Bare soil cover and arbuscular mycorrhizal community in the first montane forest restoration in Central Argentina. Restor Ecol 27: 804-812. BONFIM JA, MATSUMOTO SN, LIMA JM, CÉSAR FRCF & SANTOS MAF. 2010. Fungos micorrízicos arbusculares (FMA) e aspectos Fisiológicos em cafeeiros cultivados em sistema agroflorestal e a pleno sol. Bragantia 69: 201-206. An Acad Bras Cienc (2022) 94(3) e20201228 11 \| 15 MYCORRHIZAL FUNGI IN COFFEE PRODUCTION SYSTEMS WELLUMA T. BARROS et al. DURAZZINI AM, TEIXEIRA MA & ADAMI AA. 2016. Quantificação de esporos de fungos micorrízicos arbusculares (FMAs) em solo sob diferentes cultivos de cafeeiros. Rev Agrogeoambiental 8: 83-91. BONFIM JA, VASCONCELLOS RLF, STÜRMER SL & CARDOSO EJBN. 2016. Arbuscular mycorrhizal fungi in the Brazilian Atlantic forest: A gradient of environmental restoration. Appl Soil Ecol 71: 7-14. BRASIL. 1994. Resolução CONAMA nº. 1, de 31 de Janeiro de 1994. REFERENCES in microbial communities and carbohydrate profiles induced by the mycorrhizal fungus (Glomus intraradices) in rhizosphere of olive trees (Olea europaea L.). Appl Soil Ecol 75: 124-133. in microbial communities and carbohydrate profiles induced by the mycorrhizal fungus (Glomus intraradices) in rhizosphere of olive trees (Olea europaea L.). Appl Soil Ecol 75: 124-133. INVAM. 2020. International culture collection of (vesicular) arbuscular mycorrhizal fungi. Disponivel em: http:// www.invam.caf.wvu.edu/ Acesso em: maio. 2020. MEDDAD-HAMZA A, HAMZA N, NEFFAR S, BEDDIAR A, GIANINAZZI S & CHENCHOUNI H. 2017. Spatiotemporal variation of arbuscular mycorrhizal fungal colonization in olive (Olea europaea L.) roots across a broad mesic-xeric climatic gradient in North Africa. Sci Total Environ 583: 176-189. JASPER DA, ABBOTT LK & ROBSON AD. 1991. The effect of soil disturbance on vesicular-arbuscular mycorrhizal fungi in soils from different vegetation types. New Phytol 118: 471-476. MELLO CMA SILVA IR, PONTES JS, GOTO BT, SILVA GA & MAIA LC. 2012. Diversidade de fungos micorrízicos arbusculares em área de Caatinga, PE, Brasil. Acta Bot Bras 26: 938-943. JENKINS WR. 1964. A rapid centrifugal-flotation technique for separating nematodes from soil. Plant Dis 28: 692. KABIR Z, O’HALLORAN IP, FYLES JW & HAMEL C. 1997. Seasonal changes of arbuscular mycorrhizal fungi asaffected by tillage pratices and fertilization: hyphaldensity and mycorrhizal root colonization. Plant Soil 192: 285-293. MERGULHÃO ACES, SILVA MV, LYRA MCCP, FIGUEIREDO MVB, SILVA MLRB & MAIA LC 2014. Caracterização morfológica e molecular de fungos micorrízicos arbusculares isolados de áreas de mineração de gesso, Araripina, PE, Brasil. Hoehnea 41: 393-400. KHAEKHUM S, LUMYONG S, KUYPER TW & BOONLUE S. 2017. Species richness and composition of arbuscular mycorrhizal fungi occurring on eucalypt trees (Eucalyptus camaldulensis Dehnh.) in rainy and dry season. Curr Res Environ Appl Mycol 7: 282-292. MEYLAN L, GARY C, ALLINNE C, ORTIZ J, JACKSON L & RAPIDEL B. 2017. Evaluating the effect of shade trees on provision of ecosystem services in intensively managed coffee plantations. Agric Ecosyst Environ 245: 32-42. LIMA KB, NETTO AFR, MARTINS MA & FREITAS MSM. 2015. Crescimento, acúmulo de nutrientes e fenóis totais de mudas de cedro-australiano (Toona ciliata) inoculadas com fungos micorrízicos. Ciência Florestal 25: 853-862. LIMA KB, NETTO AFR, MARTINS MA & FREITAS MSM. 2015. Crescimento, acúmulo de nutrientes e fenóis totais de mudas de cedro-australiano (Toona ciliata) inoculadas com fungos micorrízicos. Ciência Florestal 25: 853-862. MICCOLIS A, PENEIREIRO FM & MARQUES HR. 2016. REFERENCES Restauração ecológica com Sistemas Agroflorestais: Como conciliar conservação com produção: Opções para Cerrado e Caatinga. Brasília: Instituto Sociedade, População e Natureza – ISPN/Centro Internacional de Pesquisa Agorflorestal – ICRAF, 266 p. LOSS A, ANGELINI GAR, PEREIRA ACC, LÃ RR, MAGALHÃES MOL, SILVA EMR & SAGGIN JUNIOR OJ. 2009. Atributos químicos do solo e ocorrência de fungos micorrízicos sob áreas de pastagem e sistema agroflorestal, Brasil. Acta Agron. 58: 91-95 LOSS A, ANGELINI GAR, PEREIRA ACC, LÃ RR, MAGALHÃES MOL, SILVA EMR & SAGGIN JUNIOR OJ. 2009. Atributos químicos do solo e ocorrência de fungos micorrízicos sob áreas de pastagem e sistema agroflorestal, Brasil. Acta Agron. 58: 91-95 MOREIRA FMS & SIQUEIRA JO. 2006. Microbiologia e bioquímica do solo, 2ª ed., Lavras: Editora UFLA, 744 p. MOREIRA FW, DE OLIVEIRA CM, MAIA JLZ & OLIVEIRA LA. 2019. Fungos micorrízicos arbusculares nas plantas e características químicas dos solos de clareiras da Província Petrolífera de Urucu, Am. Rev Ibero-Am Ciênc Ambient 10: 56-68. MANGAN SA, EOM AH, ADLER GH, YAVITT JB & HERRE EA. 2004. Diversity of arbuscular mycorrhizal fungi across a fragmented forest in Panama: insular spore communities differ from mainland communities. Oecologia 141: 687-700. NAIR PKR. 1993. An Introduction do Agroforestry, 1st ed., Dordrecht: Springer Science & Business Media, 499 p. MARSHALL VG. 2000. Impacts of forest harvesting on biological processes in northern forest soils. For Ecol Manag 1331: 43-60. MARSHALL VG. 2000. Impacts of forest harvesting on biological processes in northern forest soils. For Ecol Manag 1331: 43-60. NOBRE CP, LÁZARO, ML, SANTO MME, PEREIRA MG & BERBARA RLL. 2015. Agregação, glomalina e carbono orgânico na chapada do Araripe, Ceará, Brasil. Rev Caatinga 28: 138-147. MARTÍNEZ-GARCÍA LB, MIRANDA JD & PUGNAIRE FI. 2012. Impacts of changing rainfall patterns on mycorrhizal status of a shrub from arid environments. Eur J Soil Biol 50: 64-67. OEHL F, LACZKO E, OBERHOLZER HR, JANSA J & EGLI S. 2017. Diversity and biogeography of arbuscular mycorrhizal fungi in agricultural soils. Biol Fertil Soils 53:777-797. MARTINS EM, SILVA ERD, CAMPELLO EFC, LIMA SSD, NOBRE CP, CORREIA MEF & RESENDE ASD. 2019. O uso de sistemas agroflorestais diversificados na restauração florestal na Mata Atlântica. Ciência Florestal 29: 632-648. PAGANO MC, SILVA DK, SILVA GA & MAIA LC. 2019. Tropical Dry Forest Compared to Rainforest and Associated Ecosystems in Brazil. In: Mycorrhizal Fungi in South America. Cham: Springer, Cham, Suíça, p. 177-192. REFERENCES GHAZANFAR B, ZHIHUI C, WU C, LIU H, LI H, REHMAN RNU, AHMAD I & KHAN AR. 2016. Glomus etunicatum root inoculation and foliar application of acetyl salicylic acid induced nacl tolerance by regulation of Nac1 & Lenhx1 gene expression and improved photosynthetic performance in tomato seedlings. Pak J Bot 48: 1209-1217. CONAB – COMPANHIA NACIONAL DE ABASTECIMENTO. 2020. Acompanhamento da safra brasileira: café – Safra 2020, nº 1, Brasília: Companhia Nacional de Abastecimento (Conab), 62 p. HU J, YANG A, WANG J, ZHU A, DAI J, WONG MH & LIN X. 2015. Arbuscular mycorrhizal fungal species composition, propagule density, and soil alkaline phosphatase activity in response to continuous and alternate no-tillage in Northern China. Catena 133: 215-220. COSTA RSC, MENDES AM, RODRIGUES VGS & LEÔNIDAS FC. 2013. Micorrizas arbusculares em sistemas agroflorestais. Porto Velho: Embrapa, 18 p. CRISTO SC, FORS RO & CARVALHO AG. 2018. Diversity of arbuscular mycorrhizal fungi in pasture areas in the Serra do Itajaí National Park. Rev Bras Cienc Agrar 13: 1-7. IBGE - INSTITUTO BRASILEIRO DE GEOGRAFIA E ESTATÍSTICA. 2012. Manual técnico da vegetação brasileira: sistema fitogeográfico, inventário das formações florestais e campestres, técnicas e manejo de coleções botânicas, procedimentos para mapeamentos. Rio de Janeiro-RJ: IBGE - Diretoria de Geociências, (Manuais Técnicos de Geociências, 1), 271 p. DAVISON J, MOORA M, OPIK M, ADHOLEYA A, AINSAAR L, BÂ A, BURLA S, DIEDHIOU AG, HIIESALU I & JAIRUS T. 2015. Global assessment of arbuscular mycorrhizal fungus diversity reveals very low endemism. Science 349: 970-973. DAVISON J, MOORA M, OPIK M, ADHOLEYA A, AINSAAR L, BÂ A, BURLA S, DIEDHIOU AG, HIIESALU I & JAIRUS T. 2015. Global assessment of arbuscular mycorrhizal fungus diversity reveals very low endemism. Science 349: 970-973. DIAS HCT & OLIVEIRA-FILHO AT. 1997. Variação temporal e espacial da produção de serapilheira em uma área de floresta estacional semidecídua Montana em Lavras-MG. Rev Arvore 21: 11-26. DIAS HCT & OLIVEIRA-FILHO AT. 1997. Variação temporal e espacial da produção de serapilheira em uma área de floresta estacional semidecídua Montana em Lavras-MG. Rev Arvore 21: 11-26. INMET - INSTITUTO NACIONAL DE METEOROLOGIA. 2020. Banco de Dados Meteorológicos para Ensino e Pesquisa — BDMEP. Disponível em: http://www.inmet.gov.br/portal/ Rev Arvore 21: 11-26. An Acad Bras Cienc (2022) 94(3) e20201228 12 \| 15 MYCORRHIZAL FUNGI IN COFFEE PRODUCTION SYSTEMS WELLUMA T. BARROS et al. index.php?r=bdmep/bdmep. Acesso em: 02 de junho de 2020. REFERENCES MECHRI B, MANGA AG, TEKAYA M, ATTIA F, CHEHEB H, MERIEM FB, BRAHAMD M, BOUJNAHD D & HAMMAMI M. 2014. Changes An Acad Bras Cienc (2022) 94(3) e20201228 13 \| 15 MYCORRHIZAL FUNGI IN COFFEE PRODUCTION SYSTEMS WELLUMA T. BARROS et al. PEDONE-BONFIM MVL, DA SILVA DKA, MAIA LC & YANO-MELO AM. 2018. Mycorrhizal benefits on native plants of the Caatinga, a Brazilian dry tropical forest. Symbiosis 74: 79-88. PEDONE-BONFIM MVL, DA SILVA DKA, MAIA LC & YANO-MELO AM. 2018. Mycorrhizal benefits on native plants of the Caatinga, a Brazilian dry tropical forest. Symbiosis 74: 79-88. aplicados à ciclagem de nutrientes. Floresta e Ambient 2: 1-18. SEI. 2013. Estatística dos municípios baianos: Território de Identidade - Vitória da Conquista. Publicações SEI. Salvador 4: 433-452. SEI. 2013. Estatística dos municípios baianos: Território de Identidade - Vitória da Conquista. Publicações SEI. Salvador 4: 433-452. PEREIRA JES, GARCIA P, SCORIZA RN, SAGGIN JUNIOR OJ & GOMES VS. 2018. Arbuscular mycorrhizal fungi in soils of arboreal Caatinga submitted to forest management. Rev. Bras. Cienc. Agrar 13: 1-6. PEREIRA JES, GARCIA P, SCORIZA RN, SAGGIN JUNIOR OJ & GOMES VS. 2018. Arbuscular mycorrhizal fungi in soils of arboreal Caatinga submitted to forest management. Rev. Bras. Cienc. Agrar 13: 1-6. SILVA CFD, PEREIRA MG, SANTOS VLD, MIGUEL DL & SILVA EMRD. 2016. Fungos micorrízicos arbusculares: composição, comprimento de micélio extrarradicular e glomalina em áreas de Mata Atlântica, Rio de Janeiro. Ciência Florestal 26: 419-430. POSADA RH, DE PRAGER MS, HEREDIA-ABARCA G & SIEVERDING E. 2016. Effects of soil physical and chemical parameters, and farm management practices on arbuscular mycorrhizal fungi communities and diversities in coffee plantations in Colombia and Mexico. Agrofor Syst 92: 555-574. POSADA RH, DE PRAGER MS, HEREDIA-ABARCA G & SIEVERDING E. 2016. Effects of soil physical and chemical parameters, and farm management practices on arbuscular mycorrhizal fungi communities and diversities in coffee plantations in Colombia and Mexico. Agrofor Syst 92: 555-574. SILVA FF, SANTOS TA, JESUS EC & CHAER GM. 2019. Caracterização de rizóbios e fungos micorrízicos arbusculares em áreas impactadas pela exploração de piçarra na Caatinga. Rev Caatinga 32: 995-1004. PRATES JÚNIOR P, MOREIRA BC, SILVA MDCS, VELOSO TGR, STÜRMER SL, FERNANDES RBA & KASUYA MCM. 2019. Agroecological coffee management increases arbuscular mycorrhizal fungi diversity. Plos One 14: 1-9. SIQUEIRA JO, SOUZA FA, CARDOSO EJBN & TSAI SM. 2010. Micorrizas: 30 anos de pesquisas no Brasil. Lavras: Editora UFLA, 716 p. REFERENCES SOTERAS F, GRILLI G, COFRÉ MN, MARRO N & BECERRA A. 2015. Arbuscular mycorrhizal fungal composition in high montane forests with different disturbance histories in central Argentina. Appl Soil Ecol 85: 30-37. RAMOS-ZAPATA JA, ZAPATA-TRUJILLO R, ORTÍZ-DÍAZ JJ & GUADARRAMA P. 2011. Arbuscular mycorrhizas in a tropical coastal dune system in Yucatan, Mexico. Fungal Ecol 4: 256-261. SOUSA CS, MENEZES RSC, SAMPAIO EVSB, LIMA FS, OEHL F & MAIA LC. 2013 Arbuscular mycorrhizal fungi within agroforestry and traditional land use systems in semi-arid Northeast Brazil. Acta Sci Agron 35: 307-314. SOUSA CS, MENEZES RSC, SAMPAIO EVSB, LIMA FS, OEHL F & MAIA LC. 2013 Arbuscular mycorrhizal fungi within agroforestry and traditional land use systems in semi-arid Northeast Brazil. Acta Sci Agron 35: 307-314. ROCHA LPM, MOREIRA FW, OLIVEIRA CM & OLIVEIRA LA. 2020. Ocorrência de fungos micorrízicos arbusculares em um plantio de cupuaçu na estrada de Balbina, Amazonas. Rev Ibero-Am Ciênc Ambient 11: 78-84. SOUSA CS, MENEZES RSC, SAMPAIO EVSB, LIMA FS, MAIA LC & OEHL F. 2014. Arbuscular mycorrhizal fungi in successional stages of Caatinga in the semi-arid region of Brazil. Ciência Florestal 24: 137-148. SOUSA CS, MENEZES RSC, SAMPAIO EVSB, LIMA FS, MAIA LC & OEHL F. 2014. Arbuscular mycorrhizal fungi in successional stages of Caatinga in the semi-arid region of Brazil. Ciência Florestal 24: 137-148. SANTOS NETO AP, BARRETO PAB, GAMA-RODRIGUES EF, NOVAES AB & PAULA A. 2015. Produção de serapilheira em Floresta Estacional Semidecidual e em plantios de Pterogyne nitens Tul. e Eucalyptus urophylla S. T. Blake no sudoeste da Bahia. Ciência Florestal 25: 633-643. SOUZA CA, GALLARDO ALCF, SILVA ÉD, MELLO YC, RIGHI CA & SOLERA ML. 2016. Serviços Ambientais Associados à Recuperação de Áreas degradadas por Mineração: Potencial para Pagamento de Serviços Ambientais. Ambient Soc 19: 139-166. SANTOS HG, JACOMINE PKT, ANJOS LHC, OLIVEIRA VA, LUMBRERAS JF, COELHO MR, ALMEIDA JÁ, ARAUJO FILHO JC, OLIVEIRA JB & CUNHA TJF. 2018a. Sistema Brasileiro de Classificação de Solos, 5ª ed., Brasília: Embrapa, 356 p. TEIXEIRA-RIOS T, SOUZA RGD, MAIA LC, OEHL F & LIMA CEP. 2013. Arbuscular mycorrhizal fungi in a semi-arid, limestone miningimpacted area of Brazil. Acta Bot Bras 27: 688-693. SANTOS RS, BARRETO PAB & SCORIZA RN. 2014. Efeito da sazonalidade na comunidade de fungos micorrízicos arbusculares em um fragmento de mata de cipó em Vitória da Conquista, Bahia. Rev Bras Biocienc 12: 46-51. THEODORO VCA, ALVARENGA MIN, GUIMARÃES RJ & MOURÃO- JÚNIOR M. MYCORRHIZAL FUNGI IN COFFEE PRODUCTION SYSTEMS richness and nestedness of arbuscular mycorrhizal fungi in agricultural soils. Mol Ecol 21: 2341-2353. Correspondence author: Patrícia Anjos Bittencourt Barreto-Garcia E-mail: patriciabarreto@uesb.edu.br Correspondence author: Patrícia Anjos Bittencourt Barreto-Garcia E-mail: patriciabarreto@uesb.edu.br VIEIRA LC, SILVA DKAD, ESCOBAR IEC, SILVA JMD, MOURA IAD, OEHL F & SILVA GAD. 2020. Changes in an Arbuscular Mycorrhizal Fungi Community Along an Environmental Gradient. Plants 9: 1-17. Author contributions WTB: conceptualization, data curation, formal analysis, investigation, methodology and writing – original draft; PABBG: conceptualization, formal analysis, methodology, supervision and writing – review & editing; OJSJ: methodology and resources; RNS: data curation, formal analysis and supervision; MSS: investigation and methodology. All authors critically reviewed the manuscript and approved the final version. YOUNG A. 1994. Agroforestry for soil conservation. Wallingford: CAB/ICRAF, 276 p. ZHU HH, YAO Q, SUN XT & HU YL. 2007. Colonization, ALP activity and plant growth promotion of native and exotic arbuscular mycorrhizal fungi at low pH. Soil Biol Biochem 39: 942-950. ZHU HH, YAO Q, SUN XT & HU YL. 2007. Colonization, ALP activity and plant growth promotion of native and exotic arbuscular mycorrhizal fungi at low pH. Soil Biol Biochem 39: 942-950. REFERENCES 2003. Carbono biomassa e micorriza em solo sob mata nativa e agroecossistemas cafeeiros. Acta Sci Agron 25: 147-153. SANTOS RS, BARRETO-GARCIA PAB & SCORIZA RN. 2018b. Fungos micorrízicos arbusculares e serapilheira como indicadores do efeito de borda em fragmento de floresta estacional. Ciência Florestal 28: 324-335. VENDRAMI JP, JURINITZ CF & CASTANHO CT. 2012. Litterfall and leaf decomposition in forest fragments under different successional phases on the Atlantic Plateau of the state of Sao Paulo, Brazil. Biota Neotrop 12: 136-143. SCHENCK NC & PÉREZ Y. 1988. Manual for the identification of VA mycorrhizal fungi, 2nd ed., Gainesville: INVAM, 245 p. SCORIZA RN, PEREIRA MG, PEREIRA GHA, MACHADO DL & SILVA EMR. 2012. Métodos para coleta e análise de serrapilheira VERBRUGGEN E, VAN DER HEIJDEN MG, WEEDON JT, KOWALCHUK GA & RÖLING WF. 2012. Community assembly, species An Acad Bras Cienc (2022) 94(3) e20201228 14 \| 15 MYCORRHIZAL FUNGI IN COFFEE PRODUCTION SYSTEMS WELLUMA T. BARROS et al. ORIVALDO JOSÉ SAGGIN JÚNIOR3 https://orcid.org/0000-0001-9209-9738 ORIVALDO JOSÉ SAGGIN JÚNIOR3 https://orcid.org/0000-0001-9209-9738 RAFAEL N. SCORIZA4 https://orcid.org/0000-0001-9173-9510 RAFAEL N. SCORIZA4 https://orcid.org/0000-0001-9173-9510 An Acad Bras Cienc (2022) 94(3) e20201228 15 \| 15 How to cite BARROS WT, BARRETO-GARCIA PAB, SAGGIN JÚNIOR OJ, SCORIZA RN & SILVA MS. 2022. Arbuscular mycorrhizal fungi community in coffee agroforestry, consortium and monoculture systems. An Acad Bras Cienc 94: e20201228. DOI 10.1590/0001-3765202220201228. Manuscript received on July 31, 2020; accep- ted for publication on December 7, 2020 WELLUMA T. BARROS1 https://orcid.org/0000-0003-3218-9349 WELLUMA T. BARROS1 https://orcid.org/0000-0003-3218-9349 PATRÍCIA A.B. BARRETO-GARCIA2 https://orcid.org/0000-0002-8559-2927 MAICON S. DA SILVA4 https://orcid.org/0000-0001-7361-4095 1Universidade Estadual do Sudoeste da Bahia - UESB, Programa de Pós-Graduação em Agronomia, Estrada do Bem Querer, Km 4, Caixa Postal 95, 45083-900 Vitória da Conquista, BA, Brazil 2Universidade Estadual do Sudoeste da Bahia – UESB, Departamento de Engenharia Agrícola e Solos, Estrada do Bem Querer, Km 4, Caixa Postal 95, 45083-900 Vitória da Conquista, BA, Brazil 3Empresa Brasileira de Pesquisa Agropecuária – Embrapa Agrobiologia, Rodovia BR-465, Km 7, Bairro Ecologia, 23891-000 Seropédica, RJ, Brazil 4Universidade Estadual do Sudoeste da Bahia – UESB, Programa de Pós-Graduação em Ciências Florestais, Estrada do Bem Querer, Km 4, Caixa Postal 95, 45083-900 Vitória da Conquista, BA, Brazil 4Universidade Estadual do Sudoeste da Bahia – UESB, Programa de Pós-Graduação em Ciências Florestais, Estrada do Bem Querer, Km 4, Caixa Postal 95, 45083-900 Vitória da Conquista, BA, Brazil An Acad Bras Cienc (2022) 94(3) e20201228 15 \| 15
https://openalex.org/W1980049367	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0095989&type=printable	English	null	Comparative Structural Modeling of Six Old Yellow Enzymes (OYEs) from the Necrotrophic Fungus Ascochyta rabiei : Insight into Novel OYE Classes with Differences in Cofactor Binding, Organization of Active Site Residues and Stereopreferences	PloS one	2,014	cc-by	9,525	Abstract Old Yellow Enzyme (OYE1) was the first flavin-dependent enzyme identified and characterized in detail by the entire range of physical techniques. Irrespective of this scrutiny, true physiological role of the enzyme remains a mystery. In a recent study, we systematically identified OYE proteins from various fungi and classified them into three classes viz. Class I, II and III. However, there is no information about the structural organization of Class III OYEs, eukaryotic Class II OYEs and Class I OYEs of filamentous fungi. Ascochyta rabiei, a filamentous phytopathogen which causes Ascochyta blight (AB) in chickpea possesses six OYEs (ArOYE1-6) belonging to the three OYE classes. Here we carried out comparative homology modeling of six ArOYEs representing all the three classes to get an in depth idea of structural and functional aspects of fungal OYEs. The predicted 3D structures of A. rabiei OYEs were refined and evaluated using various validation tools for their structural integrity. Analysis of FMN binding environment of Class III OYE revealed novel residues involved in interaction. The ligand para-hydroxybenzaldehyde (PHB) was docked into the active site of the enzymes and interacting residues were analyzed. We observed a unique active site organization of Class III OYE in comparison to Class I and II OYEs. Subsequently, analysis of stereopreference through structural features of ArOYEs was carried out, suggesting differences in R/S selectivity of these proteins. Therefore, our comparative modeling study provides insights into the FMN binding, active site organization and stereopreference of different classes of ArOYEs and indicates towards functional differences of these enzymes. This study provides the basis for future investigations towards the biochemical and functional characterization of these enigmatic enzymes. Citation: Nizam S, Gazara RK, Verma S, Singh K, Verma PK (2014) Comparative Structural Modeling of Six Old Yellow Enzymes (OYEs) from the Necrotrophic Fungus Ascochyta rabiei : Insight into Novel OYE Classes with Differences in Cofactor Binding, Organization of Active Site Residues and Stereopreferences. PLoS ONE 9(4): e95989. doi:10.1371/journal.pone.0095989 Editor: Valerie de Cre´cy-Lagard, University of Florida, United States of America Editor: Valerie de Cre´cy-Lagard, University of Florida, United States of America Received January 24, 2014; Accepted April 2, 2014; Published April 28, 2014 Copyright: 2014 Nizam et al. This is an open-access article distributed under the terms of the Creative Commons Attributi unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Abstract Funding: This work was supported by research grant from Department of Biotechnology, Government of India (File No: BT/PR10605/PBD/16/791/2008) and a core grant from National Institute of Plant Genome Research. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: pkv@nipgr.ac.in bacteria [11–13], oxidative stress response in yeasts [14–16], jasmonic acid biosynthesis in plants [17], and ergot alkaloid biosynthesis in filamentous fungi Aspergillus fumigatus and Claviceps purpuria [18]. Furthermore, biochemical characterizations of OYEs have revealed their potential to catalyze the stereoselective reduction of activated C = C bonds of structurally diverse a,b- unsaturated compounds [19]. These optically active reduced products include many commercially useful substrates for indus- trial applications [20]. Therefore, from the last few years, OYEs are being investigated as biocatalysts for the affordable production of a variety of biotechnological and pharmaceutical compounds. Several OYE homologs from yeasts, bacteria and plants have been crystallized and their structures have been resolved [21–23]. All of these OYEs were shown to fold into a (b/a)8 barrel (or TIM barrel) with the FMN binding within the barrel near the carboxy- terminus of the b-sheet. Despite having a conserved overall structure, mechanistic differences and variation in substrate bacteria [11–13], oxidative stress response in yeasts [14–16], jasmonic acid biosynthesis in plants [17], and ergot alkaloid biosynthesis in filamentous fungi Aspergillus fumigatus and Claviceps purpuria [18]. Furthermore, biochemical characterizations of OYEs have revealed their potential to catalyze the stereoselective reduction of activated C = C bonds of structurally diverse a,b- unsaturated compounds [19]. These optically active reduced products include many commercially useful substrates for indus- trial applications [20]. Therefore, from the last few years, OYEs are being investigated as biocatalysts for the affordable production of a variety of biotechnological and pharmaceutical compounds. S l O h l f b i d l h Comparative Structural Modeling of Six Old Yellow Enzymes (OYEs) from the Necrotrophic Fungus Ascochyta rabiei : Insight into Novel OYE Classes with Differences in Cofactor Binding, Organization of Active Site Residues and Stereopreferences Shadab Nizam, Rajesh Kumar Gazara, Sandhya Verma, Kunal Singh, Praveen Kumar Verma* Plant Immunity Laboratory, National Institute of Plant Genome Research, Aruna Asaf Ali Marg, New Delhi, India Phylogenetic analysis of ArOYEs y g y To investigate the evolutionary aspect of ArOYEs, phylogenetic analysis was carried out. It was conducted by the means of Bayesian inference (BI) using MrBayes (v3.2.2). Phylogenetic analysis indicated that six ArOYEs make two distinct clades, supported by robust posterior probabilities (100%) (Figure S3 in File S1). It was observed that ArOYE1, ArOYE2 and ArOYE3 make one clade, similarly ArOYE4, ArOYE5 and ArOYE6 made another clade. However, ArOYE4 and ArOYE5 were grouped together suggesting that they are more closely related and ArOYE6 is a distantly related member of the gene family (Figure S3 in File S1). To gain further insight regarding the evolution of ArOYEs, phylogenetic analysis was carried out with previously reported OYEs. This analysis indicated that six ArOYEs along with 34 previously known members make two distinct clades with robust branch support values (Figure 2). The first clade consists of Class I OYE from bacteria, yeasts, plants and filamentous fungi. The second clade consists of Class II OYEs along with ArOYE6. Further analysis of Class I OYEs revealed monophyletic origin of fungal and plant OYEs supported with high posterior probability (100%), indicating towards a common ancestor. Within the fungal subgroup, OYEs of yeasts are grouped together, whereas ArOYE1 and ArOYE2 are grouped along with OYEs of A. fumigatus and C. purpuria. Interestingly, ArOYE3 was found to be a distantly related member and wasn’t grouped with any of the yeast or fungal OYEs. In contrast to plant and fungal OYEs, bacterial OYEs appeared paraphyletic, indicating towards diverse ancestors. Analysis of Class II OYEs revealed two subgroups. The first subgroup consists of bacterial OYEs and the second subgroup consists of ArOYE4 In the present study, homology models of six A. rabiei OYEs were generated in order to get an in depth idea of their structural and functional aspects. The predicted structures were refined by taking advantages of MODELLER and energy minimization, and evaluated by PROCHECK, ProSA and QMEAN to analyze their structural integrity. Each 3D model was compared with the representative member of the respective OYE Class. Subsequent- ly, cofactor binding environment of each ArOYE was examined. The ligand para-hydroxybenzaldehyde (PHB) was docked into the models of each ArOYE and its interactions with the active site residues were analysed. Furthermore, structural features respon- sible for stereopreference regarding R/S selectivity of each ArOYE were analyzed. Sequence comparison and alignment of ArOYEs Sequence comparison and alignment of ArOYEs q p g To investigate the sequence conservation of ArOYEs, multiple sequence alignment was carried out. Full length sequences of ArOYEs were aligned using PROMALS3D program with default parameters. All the ArOYEs proteins varied moderately in their lengths (367–473 aa) as well as in positions of the conserved motifs. Sequence alignment suggested that there is less sequence conservation among the members (13–49% identity) and only the region containing the active site residues and the YGGS motif is well conserved among the six ArOYEs (Figure S1 in File S1). Comparing the sequences of members of same OYE class suggests more sequence similarity. Class I ArOYEs (ArOYE1-3) share 38– 49% amino acid identity and Class II ArOYEs (ArOYE4 and ArOYE5) share 42% amino acid identity. In contrast, sequence identity of OYE proteins is fairly low between the members of Class I and Class II (19–26%), Class II and Class III (13–18%), and Class I and Class III (15–18%). The deduced amino acid sequences of ArOYE1-6 showed predicted molecular mass in the range of 40.6–51.5 kDa and theoretical pI in the range of 5.6–6.28 (Table 1). The analysis of ArOYE1-6 sequences in the conserved domain database (CDD) available at NCBI (http://www.ncbi. nlm.nih.gov/Structure/cdd/wrpsb.cgi) revealed interesting re- sults. CDD predicted a conserved ‘OYE_like_FMN’ domain in ArOYE1-3 (Figure S2 in File S1). In contrast, it predicted ‘OYE_YqjM_FMN’ domain in ArOYE4 and ArOYE5, and ‘OYE_like_2_FMN’ domain in ArOYE6. To gain further insight, sequence alignment of ArOYEs was carried out with the previously known members. Sequence alignment suggested high sequence divergence among the homologs, sharing 11–91% of amino acid identity. The sequence conservation occurs among the core active site residues and the loop region containing the YGGS motif (Figure 1). Recently, to gain some insight regarding the distribution of OYEs and their physiological function in fungi, we carried out a comprehensive genome-wide identification of OYE proteins in 60 fungal species [32]. On the basis of active site residues and phylogeny, the identified OYEs were classified into three classes. Our study not only showed the existence of thermophilic-like OYEs in the genomes of several fungi but also suggested the presence of a novel OYE class, in addition to the classical OYEs. Therefore, we named the classical OYEs as Class I, thermophilic- like OYEs as Class II and novel class OYEs as Class III. Introduction Old Yellow Enzyme (OYE1) was initially isolated from the yeast Saccharomyces pastorianus by Warburg & Christian (1933) [1]. It was the first enzyme shown to possess a flavin cofactor, flavin mono- nucleotide (FMN). It has been well established that NAD(P)H serves as the physiological reductant for the enzyme-bound flavin [2], whereas several compounds such as quinines and many a/b- unsaturated aldehydes and ketones can act as oxidants [3]. Even though, majority of them are not naturally occurring. However, despite extensive characterization of this enzyme, the true physiological oxidant of OYE remains elusive till date. The gene encoding OYE1 was identified 58 years after the protein isolation [4]. Since then, a number of OYE homologs have been identified from other yeasts, bacteria, protists, plants and filamentous fungi [5–10]. Numerous metabolic functions for OYE homologs have been suggested including degradation of nitrate ester explosives in Several OYE homologs from yeasts, bacteria and plants have been crystallized and their structures have been resolved [21–23]. All of these OYEs were shown to fold into a (b/a)8 barrel (or TIM barrel) with the FMN binding within the barrel near the carboxy- terminus of the b-sheet. Despite having a conserved overall structure, mechanistic differences and variation in substrate April 2014 \| Volume 9 \| Issue 4 \| e95989 PLOS ONE \| www.plosone.org 1 Structural Modelling of Old Yellow Enzymes (OYEs) from Ascochya rabiei preference occur between the OYE family members. On the basis of distinct sequence and structural features, Toogood et al., divided OYEs into two classes [24]. The first class includes the well described and investigated members such as OYE1 from Saccharomyces pastorianus [25], 12-oxophytodienoate reductase from plants [26] and morphinone reductase from bacteria [27] and thus was named as classical OYEs. However, structures of only two fungal OYEs (from S. pastorianus and Pichia stipitis, members of Saccharomycetes), belonging to this class have been solved so far [25,28]. The second class was named as thermophilic-like OYEs and includes reductases such as YqjM from Bacillus subtilis [29], PpOYE (XenA) from Pseudomonas putida 86 [30] as well as TsOYE from Thermus scotoductus SA-01 [31]. Thermophilic-like OYEs are limited to bacteria only and structure of this OYE class has not been reported from eukaryotes. Members of classical and thermophilic-like OYEs show notable structural differences. Thermophilic-like OYEs posses a unique shared active site composition which is not observed in the active sites of classical OYEs. Introduction In thermophilic-like OYEs, an arginine [29] or tryptophan finger [30] protrudes from one monomer into the active site of the adjacent monomer. Therefore, thermophilic-like OYEs are mostly tetrameric with few dimeric proteins, whereas classical OYEs are mostly dimeric with certain monomeric OYEs [49]. Sequence comparison and alignment of ArOYEs Interestingly, it was observed that majority of fungal species (39 out of 60 species) analyzed in this study, posses all the three OYE classes in their genomes. One of such fungal species is Ascochyta rabiei, the causal agent of Ascochyta blight (AB) in chickpea worldwide. Recent studies related to genome sequencing of this phytopathogen in our laboratory revealed six OYEs (ArOYE1-6) of which ArOYE1-3 are Class I, ArOYE4 and ArOYE5 are Class II and ArOYE6 is Class III member. However, the structural organization regarding FMN binding and active site organization of Class III OYEs, eukaryotic Class II OYEs and Class I OYEs of filamentous fungi have not been studied yet. Homology modeling, refinement and validation of ArOYEs To study the structure of representative members of all the three OYE classes, homologs of OYE from A. rabiei were selected for homology modeling. The best templates of ArOYEs were selected through PSI-BLAST of each target protein against the PDB database. Single templates were used for each target protein except ArOYE5 and ArOYE6. Due to lack of crystal structure of Class III member from any organism, two proteins (TpOYE from Thermoanaerobacter pseudethanolicus and 12-oxophytodienoate reduc- tase, SlOPR1 from Solanum lycopersicum) having moderate sequence identity with ArOYE6 were taken as templates. TpOYE belongs to Class II while SlOPR1 is a member of Class I OYE. Table 1 shows the proteins used as templates for homology modeling along with PDB IDs, resolution and their identity with the respective target protein. OYE domains of all the target proteins were modeled using MODELLER 9v11. Best models for each ArOYEs were chosen on the basis of their DOPE score. Using loop refinement protocol of MODELLER, initial refinement of each 3D model was carried out. The PROCHECK, ProSA and Qmean analyses were performed to assess the quality of the final structural models (values shown in Table S1 in File S1). The Ramachandran plot showed the modeled domains of each ArOYE has more than 96% residues present in most favored and allowed regions (Figure S4a, Table S1 in File S1). Majority of the remaining residues (0.6–2.5%) were present in the generously allowed region, whereas only few residues (0–1.2%) were found in the disallowed region. The PROCHECK result summary showed the distribution of the main chain bond lengths (97.0–99.2%), bond angles (89.3–91.8) and planar groups (99.3–100%) present in all the modeled structures were within limits (Table S1 in File S1). Moreover, main-chain and side-chain parameters of each struc- ture were in the better region. For a 3D model to be reliable, its goodness factor (G-factor) should be above 20.50. G-factor predicts the quality of overall bond and angle distances. The G- factor overall scores observed in ArOYE models were in the range of 20.25 to 20.11, suggesting reliable models of all the ArOYEs. The z-score calculated from ProSA-web server indicates the overall model quality and measures the deviation of the total energy of the structure with respect to an energy distribution derived from all experimental structures deposited in the Protein Data Bank (PDB). Phylogenetic analysis of ArOYEs Our study for the first time provides new insights towards the structural organization of novel OYE classes and indicates differences in substrate specificity and possible function. PLOS ONE \| www.plosone.org April 2014 \| Volume 9 \| Issue 4 \| e95989 2 Structural Modelling of Old Yellow Enzymes (OYEs) from Ascochya rabiei Table 1. Proteins used as templates for homology modeling of A. rabiei OYEs. Target MW PI Template Template’s Resolution (A˚ ) % Sequence identity Description Organism ArOYE1 40.6 6.04 4K7Y 1.2 41 Old Yellow Enzyme (OYE1) Saccharomyces pastorianus ArOYE2 42.5 5.6 4K7Y 1.2 40 Old Yellow Enzyme (OYE1) Saccharomyces pastorianus ArOYE3 44.9 5.71 3P8I 1.19 43 Pentaerythritol tetranitrate reductase Enterobacter cloacae ArOYE4 46.5 6.28 3L5L 1.03 41 Xenobiotic reductase A (XenA) Pseudomonas putida ArOYE5 51.5 5.88 1Z41 1.3 35 Probable NADH-dependent flavin oxidoreductase (YqjM) Bacillus subtilis 3L5L 1.03 38 Xenobiotic reductase A (XenA) Pseudomonas putida ArOYE6 49.2 5.62 3KRU 1.6 29 NADH:flavin oxidoreductase/NADH oxidase Thermoanaerobacter pseudethanolicus 1ICP 1.9 31 12-oxophytodienoate reductase (OPR1) Solanum lycopersicum PLOS ONE \| www.plosone.org and ArOYE5 along with Pseudomonas putida OYE (PpOYE). This indicates a closer evolutionary relationship of ArOYE4 and ArOYE5 with PpOYE in comparison to other Class II OYE proteins. ArOYE6 was found as a distantly related member of all the OYEs analyzed further substantiating it to be a member of novel OYE class. Homology modeling, refinement and validation of ArOYEs The z-scores of combined energy for modeled ArOYEs were negative and in the range of 28.61 to 26.13, suggesting the overall good quality of 3D structures (Figure S4b in File S1). Final confirmation was done by performing QMEAN analysis of the 3D models. QMEAN is a composite scoring function for homology models which estimates the quality of single model on the basis of the geometrical analysis. The QMEANnorm score and QMEAN Z-score of all the six ArOYEs suggest good quality models (Figure S4c, Table S1 in File S1). All the validated models were subjected to energy minimization using the GROMOS96 force field of Deep View. Finally, all the validated models were aligned with their respective templates, and their RMSD and TM-score were calculated using TM-Align. RMSD values of ArOYEs were in the range of 0.27 to 1.02 (Table S2 in File S1), suggesting the structural feature of each model is very close to its respective template. April 2014 \| Volume 9 \| Issue 4 \| e95989 3 Structural Modelling of Old Yellow Enzymes (OYEs) from Ascochya rabiei Figure 1. Multiple sequence alignment of ArOYE1-6 along with previously reported OYEs. The alignment includes OYEs from bacteria [Pseudomonas syringae (PsNcr) AAD16106.1, Pseudomonas fluorescens (PfXenB) AAF02539.1, Shewanella oneidensis (SYE1) NP_718044.1, (SYE2) NP_718043.1, (SYE3) NP_719682.1, (SYE4) NP_718946.1, Agrobacterium radiobacter (ArNerA) CAA74280.1, Pseudomonas putida (PpmorB) AAC43569.1, (PpOYE) NP_743414.1, Enterobacter cloacae (EclOnr) AAB38683.1, Escherichia coli (EcNer) NP_416167.1, Geobacter metallireducens (GmOYE) YP_006721534.1, Thermus thermophilus (TtOYE) YP_143423.1, Thermus scotoductus (TsOYE) YP_004203660.1, Thermoanaerobacter pseudethanolicus (TpOYE) YP_001664021.1, Geobacillus kaustophilus (GkOYE) YP_148185.1 and Bacillus subtilis (BsYqjM) NP_390263.1], yeast [Saccharomyces cerevisiae (OYE2) NP_012049.1, (OYE3) NP_015154.1, Kluyveromyces lactis (KYE) AAA98815.1, Saccharomyces pastorianus (OYE1) Q02899.3, Hansenula polymorpha (HYE1) AAN09952.1, (HYE2) AAN09953.1, (HYE3) AAN09954.1, and Pichia stipitis (PsOYE) XP_001384055.1], filamentous fungi [Aspergillus fumigatus (AfEasA) Q4WZ70.1 and Claviceps purpurea (CpEasA) AET79178.1], land plants [Arabidopsis thaliana (AtOPR1) CAA71627.1, (AtOPR2) NP_177795.1, (AtOPR3) NP_178662.1 and Solanum lycopersicum (SlOPR1) NP_001234781.1, (SlOPR2) NP_001233868.1, (SlOPR3) NP_001233873.1] and protozoa [Trypanosoma cruzi (TcOYE) AAA74448.1]. The multiple sequence and structure alignment program PROMALS3D was used to generate the alignment using default parameters. The positions of the conserved active sites are highlighted with the rectangular boxes. The consensus sequence Figure 1. Multiple sequence alignment of ArOYE1-6 along with previously reported OYEs. Homology modeling, refinement and validation of ArOYEs The alignment includes OYEs from bacteria [Pseudomonas syringae (PsNcr) AAD16106.1, Pseudomonas fluorescens (PfXenB) AAF02539.1, Shewanella oneidensis (SYE1) NP_718044.1, (SYE2) NP_718043.1, (SYE3) NP_719682.1, (SYE4) NP_718946.1, Agrobacterium radiobacter (ArNerA) CAA74280.1, Pseudomonas putida (PpmorB) AAC43569.1, (PpOYE) NP_743414.1, Enterobacter cloacae (EclOnr) AAB38683.1, Escherichia coli (EcNer) NP_416167.1, Geobacter metallireducens (GmOYE) YP_006721534.1, Thermus thermophilus (TtOYE) YP_143423.1, Thermus scotoductus (TsOYE) YP_004203660.1, Thermoanaerobacter pseudethanolicus (TpOYE) YP_001664021.1, Geobacillus kaustophilus (GkOYE) YP_148185.1 and Bacillus subtilis (BsYqjM) NP_390263.1], yeast [Saccharomyces cerevisiae (OYE2) NP_012049.1, (OYE3) NP_015154.1, Kluyveromyces lactis (KYE) AAA98815.1, Saccharomyces pastorianus (OYE1) Q02899.3, Hansenula polymorpha (HYE1) AAN09952.1, (HYE2) AAN09953.1, (HYE3) AAN09954.1, and Pichia stipitis (PsOYE) XP_001384055.1], filamentous fungi [Aspergillus fumigatus (AfEasA) Q4WZ70.1 and Claviceps purpurea (CpEasA) AET79178.1], land plants [Arabidopsis thaliana (AtOPR1) CAA71627.1, (AtOPR2) NP_177795.1, (AtOPR3) NP_178662.1 and Solanum lycopersicum (SlOPR1) NP_001234781.1, (SlOPR2) NP_001233868.1, (SlOPR3) NP_001233873.1] and protozoa [Trypanosoma cruzi (TcOYE) AAA74448.1]. The multiple sequence and structure alignment program PROMALS3D was used to generate the alignment using default parameters. The positions of the conserved active sites are highlighted with the rectangular boxes. The consensus sequence is illustrated below the alignment. doi:10.1371/journal.pone.0095989.g001 Characterization of homology model of ArOYEs Comparing the predicted structures of ArOYE1-3 with OYE1, suggested their close structural resemblance (Figure 3). On the contrary, closer examination of the structures revealed differences. Although conformations of sheets and helices were in accordance with OYE1, the loops at the COOH-terminal end of the b-sheets of ArOYE1-3 adopted different conformations in the (b/a)8- barrel. The marked structural difference between ArOYE1-3 and OYE1 lies in the loop bL4. The loop bL4 contains the core active site residues viz. His191, Asn194, Tyr196 in case of OYE1. Although these residues are conserved in ArOYE1-3 as well but the length of the loop is different. The loop bL4 in OYE1 consists of 25 residues, whereas only 15 residues are present in ArOYE1-3. In order to get an insight about the structural conservation of ArOYE1-3, their 3D structures were superimposed upon each other (Figure S7a in File S1). All the three structures with eight a- helices and b-sheets along with secondary helices and sheets The 3D models of all the ArOYEs consist of eight b-sheets and eight a-helices (Figure S5 in File S1). All the 3D models comprised of one compact domain representing the frequently observed (b/ a)8-barrel or TIM barrel fold, where a cylindrical core of eight twisted b-strands is surrounded by eight helices. Additional secondary structural elements occur on loops formed between the alternating sheet and helix core elements. Similar to other OYE homologs, all of the turns at the NH2-terminal end of the barrel are composed of only three or four residues, while the loops at the COOH-terminal end are much longer and build up the active sites. All ArOYE modeled structures, exhibit a characteristic short b-hairpin prior to helix a1 that closes the barrel at the N terminus. The overall structures of ArOYEs strongly resemble the structures of other OYE homologs (Figure S6 in File S1). PLOS ONE \| www.plosone.org April 2014 \| Volume 9 \| Issue 4 \| e95989 4 Structural Modelling of Old Yellow Enzymes (OYEs) from Ascochya rabiei overlapped completely upon each other, suggesting these OYEs is in loop bL4, which consists of 25 residues in both ArOYE4 and Figure 2. Evolutionary relationships of OYE family proteins. The multiple sequence alignment generated by PROMALS3D server was used to build the phylogenetic tree by Bayesian inference in MrBayes. The OYE proteins were classified into three distinct classes, designated as Class I, II, and I. April 2014 \| Volume 9 \| Issue 4 \| e95989 Characterization of homology model of ArOYEs Different colour was assigned to each class. The numbers at the nodes indicates the Bayesian posterior probabilities. doi:10.1371/journal.pone.0095989.g002 Structural Modelling of Old Yellow Enzymes (OYEs) from Ascochya rabie Figure 2. Evolutionary relationships of OYE family proteins. The multiple sequence alignment generated by PROMALS3D server was used to build the phylogenetic tree by Bayesian inference in MrBayes. The OYE proteins were classified into three distinct classes, designated as Class I, II, and III. Different colour was assigned to each class. The numbers at the nodes indicates the Bayesian posterior probabilities. doi:10.1371/journal.pone.0095989.g002 overlapped completely upon each other, suggesting these OYEs have conserved structural organization. is in loop bL4, which consists of 25 residues in both ArOYE4 and ArOYE5. In contrast, 33 residues are present in YqjM. In addition, loop bL3 consists of 52 residues in ArOYE4, 73 residues in ArOYE5, and only 30 residues in YqjM. Another difference is in loop aL7 which is 4 and 5 residues in YqjM and ArOYE4, respectively. On the contrary, loop aL7 of ArOYE5 consists of 41 residues. This is evident from sequence alignment as well where a gap is introduced for proper alignment. However, this stretch is Since ArOYE4 and ArOYE5 are members of Class II, the predicted 3D structures were compared with the crystal structure of YqjM. As expected, the sheets and helices of both ArOYE4 and ArOYE5 were properly aligned with YqjM. In contrast, the loop regions of both ArOYEs showed different conformations from YqjM (Figure 3). The major structural difference in the monomers April 2014 \| Volume 9 \| Issue 4 \| e95989 PLOS ONE \| www.plosone.org 5 Structural Modelling of Old Yellow Enzymes (OYEs) from Ascochya rabiei Figure 3. Comparison of ArOYEs with yeast OYE1 and YqjM. ArOYE1-3 (red and yellow) were superimposed upon yeast OYE1 (sky blue and dark blue), ArOYE4 and ArOYE5 (red and yellow) were superimposed upon B. subtilis YqjM (sky blue and dark blue), and ArOYE6 was superimposed upon both OYE1 and YqjM. doi:10.1371/journal.pone.0095989.g003 g y y Figure 3. Comparison of ArOYEs with yeast OYE1 and YqjM. ArOYE1-3 (red and yellow) were superimposed upon yeast OYE1 (sky blue and dark blue), ArOYE4 and ArOYE5 (red and yellow) were superimposed upon B. subtilis YqjM (sky blue and dark blue), and ArOYE6 was superimposed upon both OYE1 and YqjM. doi:10.1371/journal.pone.0095989.g003 well conserved in corresponding OYE of closely related fungi. Characterization of homology model of ArOYEs These analyses suggest ArOYE4 and ArOYE5 are slightly different in their overall geometry and points towards different enzymatic and molecular functions (Figure S7b in File S1). 25 residues in ArOYE6, whereas it is 33 residues in YqjM. Taking together, structural comparisons indicate the overall structure of ArOYE6 resembling more with Class II proteins. In addition, it reveals the notable difference with Class II and thus indicates towards a novel structural organization of this class of fungal OYEs. Since Class I SlOPR1 and Class II TpOYE were used as templates for generating the 3D structure of ArOYE6, therefore we compared the modeled structure with both OYE1 and YqjM. Superimposing 3D structures of ArOYE6 with OYE1 suggested completely overlapping eight a-helices and b-sheets, however conformational differences were observed among the loop regions (Figure 3). The most notable difference was in the loop bL3. In ArOYE6, bL3 is 39 residues long with no secondary a-helices or b-sheets, whereas in OYE1 it is 48 residues long and contains two secondary a-helices and b-sheets. Superimposing the structure of ArOYE6 with YqjM suggested the proteins share more structural conservation (Figure 3). In contrast, we observed a notable difference between the two structures. The loop region bL4 is only FMN binding sites of ArOYEs The crystal structure of YqjM has revealed that the residues Ser23, Pro24, Cys26, Ala60, Gln102, His164, His167, Arg215, Ser249, Gln265, Gly284, Met285, Phe305, Gly307, Arg308, Glu309 and Arg312 are involved in FMN binding [29]. Out of these seventeen residues, eleven residues (Pro24, Cys26, Ala60, Gln102, His164, His167, Arg215, Ser249, Gln265, Gly284, and Arg308, numbering according to YqjM) are conserved in both ArOYE4 and ArOYE5. Additionally, residues Ser23, Phe305 and Gly307 are conserved in ArOYE4, whereas they are substituted with similar amino acids Ala, Leu and Ala in ArOYE5. Further confirmation of the FMN binding environment of these ArOYEs was carried out as mentioned above. LIGPLOT prediction analysis revealed that out of the 17 residues of YqjM, 13 residues of ArOYE4 and 12 residues of ArOYE5 form hydrogen bond and hydrophobic interaction with FMN (Figure 4, Table S3 in File S1). Thus, the requirements for a functional FMN-binding site are fulfilled by the ArOYE4 and ArOYE5 proteins. Therefore, our sequence and structural analyses suggests that FMN-binding environment in ArOYE4 and ArOYE5 is at par with Class II OYEs. p In the absence of any known structure of Class III OYEs, model of ArOYE6 was directly taken for LIGPLOT analysis. The analysis indicated eight residues (Ala38, Thr40, Asn80, His196, Lys249, Phe306, Phe328, and Lys329, numbering according to ArOYE6) that form hydrogen bond and six residues (Gly37, Met39, Arg42, His199, Phe305 and Arg355) that form hydrophobic interaction with FMN (Figure 4, Table S3 in File S1). All the residues that form hydrogen bond are well conserved among the top 20 hits (Figure S8c in File S1), which we got from PSI-BLAST of ArOYE6 against the non-redundant protein database at NCBI. In addition, residues involved in the formation of hydrophobic interaction with FMN are also well conserved or substituted with similar amino acid residues. Comparing the residues of ArOYE6 that interact with FMN to that of other ArOYEs reveal vast difference in FMN binding environment among different classes of OYE. Therefore, our structural analysis for the first time reveals the FMN binding environment of ArOYE6 in particular and Class III OYEs in general. In order to validate the active site residues of all ArOYEs, para- hydroxybenzyaldehyde (PHB) was docked into the active site pocket of each 3D model (Figure S9 in File S1). Interaction of PHB was analyzed with the predicted active site residues of each ArOYE (Figure 6). FMN binding sites of ArOYEs Comparing the active site residues side by side of ArOYE1-3 with OYE1 suggests similarities in the conformation of active site residues (Figure 5). Similarly, the crystal structure of YqjM has revealed that the residues Cys26, Tyr28, His164, His167, Tyr169 and Arg336 are involved in substrate binding [29]. All of these six residues are conserved in both ArOYE4 and ArOYE5 (Figure S8b in File S1). Comparing the catalytic region of ArOYE4 and ArOYE5 with YqjM suggested high similarities (Figure 5). This analysis authenticates that ArOYE4 and ArOYE5 are true eukaryotic homologs of YqjM. Due to lack of crystal structure of Class III OYE, the true active site composition of this class is not known. To get idea regarding the active site of Class III, sequence comparison of ArOYE6 was carried out with Class I and Class II ArOYEs. Sequence alignment of ArOYE6 with other ArOYEs suggested that the core catalytic residues of OYE family (His196, His199, Tyr201, numbering according to ArOYE6) are also conserved in this protein (Figure 1). Additionally, sequence alignment was performed using ArOYE6 and its top 20 hits, which were obtained from a PSI- BLAST against the non-redundant protein database of NCBI with an E-value threshold of 1025. Sequence alignment revealed that ArOYE6 shares high sequence identity (63–74%) with these proteins (Figure S8c in File S1). The core active site residues of ArOYE6 (His196, His199, Tyr201) are also well conserved among these proteins. However, alignment studies did not give any clue regarding the accessory residues involved in active site formation. Further analysis indicated the sequence conservation throughout the length of these proteins suggesting the possibilities of other conserved accessory residues involved in the formation of active site of Class III OYEs. Therefore, the 3D model of ArOYE6 was analyzed using active site prediction programs POOL and Q- SiteFinder. The predicted residues were validated through analyzing their position in the ArOYE6 structure (Figure 5). Only the residues making the active site pocket were selected. In this way following residues viz. Thr40, Arg42, Asn80, His196, His199, Tyr201 and Arg355 were selected as the predicted active site residues of ArOYE6. These residues are well conserved among the top 20 hits of ArOYE6 further demonstrating their role as active site residues (Figure S8c in File S1). FMN binding sites of ArOYEs Close proximity of these residues with PHB substantiates their role in catalytic functions. Stereopreferences of ArOYEs In recent years, it has been discovered that OYE family proteins carry out stereoselective reduction of activated C = C bonds [19]. In addition, to analyze the stereopreferences of OYEs, Oberdorfer et al. carried out extensive studies of structural features of exclusive R and S selective OYEs [33]. They observed a clear structure- specificity correlation and identified clusters on the basis of pseudo-atom distances. Their results clearly showed that in exclusive R-selective OYEs the pseudo-atom distance is .8 A˚ , whereas in exclusive S-selective OYEs the pseudo-atom distance is ,7 A˚ . However, some special cases of OYEs were also observed, FMN binding sites of ArOYEs To introduce FMN into the 3D structures of ArOYEs, MODELLER was used to supply restrains on the relative orientation of the FMN from respective templates to targets. FMN binding environment was then compared in a class-wise manner. From the crystal structure of OYE1, it has been deduced that Pro35, Thr37, Gly72, Gln114, Arg243, Gly324, Asn325, Phe326, Gly345, Gly347, and Arg348 contribute to the FMN binding sites [27]. Out of these eleven residues, seven residues (Pro35, Thr37, Gln114, Arg243, Gly324, Gly347 and Arg348, numbering according April 2014 \| Volume 9 \| Issue 4 \| e95989 PLOS ONE \| www.plosone.org 6 Structural Modelling of Old Yellow Enzymes (OYEs) from Ascochya rabiei Structural Modelling of Old Yellow Enzymes (OYEs) from Ascochya rabiei to OYE1) are conserved in ArOYE1-3. The residue, Phe326 is conserved in ArOYE1 and ArOYE3, whereas it is substituted with similar amino acid (Tyr) in ArOYE2. Similarly, Gly72 is conservatively substituted with (Ala) in all the three ArOYEs, and Gly345 is substituted with similar amino acid (Val) in ArOYE1 and (Ile) in ArOYE2 and ArOYE3. Only a single non-conservative amino acid substitution (Asn325 to Gly) was observed in all the three ArOYEs. In order to further confirm the FMN binding environment of these ArOYEs, LIGPLOT analysis was carried out. This analysis predicted the formation of hydrogen bond and/ or hydrophobic interactions between these conserved residues and FMN (Figure 4). All the conserved and substituted residues were predicted to form hydrogen bond and/or hydrophobic interaction with FMN in ArOYE1-3 (Table S3 in File S1). Although the FMN binding residues are conserved among ArOYE1-3, their confor- mation is different in these proteins. Thus our analysis reveals similarity in the residues involved in FMN binding at the same time indicates differences in conformation of the FMN and interacting residues, pointing towards different substrate specificity among ArOYE1-3. Phe250 and Tyr375, numbering according to OYE1) are conserved in ArOYE1-3, whereas a residue corresponding to Phe296 in OYE1 is absent in all the three ArOYEs (Figure S8a in File S1). Therefore, the major difference between OYE1 and ArOYE1-3 in the catalytic region is the bigger size of the active site pocket in ArOYEs because of the absence of Phe296. Thus active sites of ArOYE1-3 appear to be more accessible to bulky substrates than that of OYE1. Active Site organization of ArOYEs To analyze the catalytic environment of ArOYEs, detailed analysis of the active site residues was carried out. The crystal structure of OYE1 substantiates that His191, Asn194, Tyr196, Phe250, Phe296 and Tyr375 contribute towards substrate binding [27]. Out of these six residues, five residues (His191, Asn194, Tyr196, PLOS ONE \| www.plosone.org April 2014 \| Volume 9 \| Issue 4 \| e95989 7 Structural Modelling of Old Yellow Enzymes (OYEs) from Ascochya rabiei Figure 4. Analysis of FMN binding to the modeled ArOYEs. Ligplot diagrams of ArOYEs generated using PDBsum (44, 45), showing hydrogen bond as well as non bonded interactions. doi:10.1371/journal.pone.0095989.g004 Figure 4. Analysis of FMN binding to the modeled ArOYEs. Ligplot diagrams of ArOYEs generated using PDBsum (44, 45), showing hydrogen bond as well as non bonded interactions. doi:10.1371/journal.pone.0095989.g004 which showed intermediate distances (7.0–7.9 A˚ ) and thus exhibit moderate stereospecificity. indicating exclusive S-selectivity of these proteins (Figure 7). In the same way, the pseudo-atom distance of ArOYE6 was 6.4 A˚ , indicating this Class III OYE protein to be exclusive S-selective (Figure 7). Therefore, our results shows that ArOYEs have all the three types of stereopreferences from exclusive R-selective, moderate selective and exclusive S-selective. To analyze the stereopreferences of ArOYEs, we followed the structural features described by Oberdorfer et al. [33]. Corre- sponding residues involved in stereoselectivity were identified in each ArOYE through sequence and structure alignments. Pseudo- atoms were generated for the residue pair involved in stereoselec- tivity, in each ArOYEs. Analysis of pseudo-atom distances in ArOYEs revealed interesting results. The pseudo-atom distance is .8 A˚ in both ArOYE1 and ArOYE2, which indicates these proteins are exclusive R-selective (Figure 7). Interestingly, the pseudo-atom distance of ArOYE3 was 7.2 A˚ , suggesting moderate stereospecificity in this OYE. Similarly, in ArOYE4 and ArOYE5 the pseudo-atom distances were 6.5 A˚ and 5.8 A˚ , respectively thus Discussion Combinations of site-directed mutagenesis (SDM) and crystal structure studies have characterized the active site residues of OYE1 (OYE from S. pastorianus), which are well conserved across similar OYE proteins. A difference in active site organization was first observed when crystal structure of YqjM from Bacillus subtilis April 2014 \| Volume 9 \| Issue 4 \| e95989 PLOS ONE \| www.plosone.org 8 Structural Modelling of Old Yellow Enzymes (OYEs) from Ascochya rabiei Structural Modelling of Old Yellow Enzymes (OYEs) from Ascochya rabiei Figure 5. Characterization and comparison of the active sites of ArOYEs. The predicted active site residues were located in each ArOYE. Active site residue organization in ArOYE1-3 and their superimposition upon each other along with OYE1 (PDB ID: 1OYB, skyblue), in ArOYE4 and ArOYE5 and their superimposition upon each other along with YqjM (PDBID: 1Z42, sky blue), and in ArOYE6 and its superimposition upon OYE1 and YqjM. doi:10.1371/journal.pone.0095989.g005 Figure 5. Characterization and comparison of the active sites of ArOYEs. The predicted active site residues were located in each ArOYE. Active site residue organization in ArOYE1-3 and their superimposition upon each other along with OYE1 (PDB ID: 1OYB, skyblue), in ArOYE4 and ArOYE5 and their superimposition upon each other along with YqjM (PDBID: 1Z42, sky blue), and in ArOYE6 and its superimposition upon OYE1 and YqjM. doi:10.1371/journal.pone.0095989.g005 was resolved [29]. Thereafter, several homologs of YqjM have been isolated from other bacteria and their sequences were reported to posses the active site residues identical to YqjM. Few of these proteins were crystallized, which further confirmed the active site environment similar to that of YqjM. Thus a new class of OYE including YqjM and related bacterial proteins was discovered [24]. This class of OYE proteins contain an arginine or tryptophan finger, which protrudes from one monomer into the active site of the adjacent monomer. Thus displaying shared active site architecture. However, crystal structures of YqjM and related proteins have been reported only from bacteria and there is no structural information of eukaryotic homolog, till date. In order to gain some insight regarding the physiological function of OYEs in fungi, a comprehensive genome-wide identification of OYE proteins was carried out in 60 fungal species [32]. Active site residues and phylogeny were used to classify the identified OYEs into three classes viz. Class I, Class II and Class III. Discussion Class I OYEs posses the active site organization of OYE1, Class II OYEs contain the active site of YqjM, whereas Class III proteins appeared to have a unique active site organization. Thus our study showed the existence of YqjM like OYEs in the genomes of several fungi along with a novel OYE class, Class III. However, nothing is known about the active site organization or FMN binding environment of this class of proteins. In addition, there is no structural information about eukaryotic Class II OYEs and Class I OYEs of filamentous fungi. Therefore, due to lack of any experimental data, we decided to carry out in silico homology modeling of all the six OYEs of the chickpea blight fungus Ascochyta rabiei (ArOYE1-6), belonging to all the three OYE classes. In silico homology modeling clearly indicated the overall structure of ArOYE1-3 resembles that of yeast OYE with few differences. Similarly, the structures of ArOYE4 and ArOYE showed the typical shared active site composition of YqjM class of OYE proteins. However, structure of ArOYE6 was quite different from the structures of OYE1 and YqjM, suggesting that it is a novel class of OYE protein. After determining and characterizing the structure of all the six ArOYEs, our next aim was to predict the FMN binding environment in these OYEs. Previous reports suggested that the cofactor FMN is non-covalently bound in the active site with the si-side of the alloxazine ring facing the solvent [25–29]. It is bound at the C-terminal end of the b-barrel, where loops bL1-bL8 set up the active-site cavity above the FMN. This binding environment is observed in both OYE1 and YqjM like proteins, however the residues involved are different between the two classes. In accordance with the previous reports, our study suggests that in all ArOYE models, FMN binds within the barrel near the carboxy-terminal ends of the b-strands in an extended conformation that lies roughly perpendicular to the barrel axis. In contrast, the residues involved in binding are different among the three OYE classes (Figure 4). Additionally, we observed difference in conformation among the members of the same class indicating towards different biochemical properties of ArOYEs. Active site predictions confirmed this hypothesis. Except for the core active site residues (His196, His199, Tyr201, residue numbering according to ArOYE6), the accessory residues are different in all the six ArOYEs. Discussion Accessory residues of Class I ArOYEs are identical to PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org April 2014 \| Volume 9 \| Issue 4 \| e95989 9 Structural Modelling of Old Yellow Enzymes (OYEs) from Ascochya rabiei April 2014 \| Volume 9 \| Issue 4 \| e95989 April 2014 \| Volume 9 \| Issue 4 \| e95989 PLOS ONE \| www.plosone.org 10 Structural Modelling of Old Yellow Enzymes (OYEs) from Ascochya rabiei Structural Modelling of Old Yellow Enzymes (OYEs) from Ascochya rabiei Figure 6. Docking of para-hydroxybenzyaldehyde (PHB) in the active sites of ArOYEs. para-hydroxybenzyaldehyde (PHB) was docked in the respective active site pocket of each ArOYE. Active site residues interacting with the ligand were analyzed. Position of active site residues in respective ArOYE is indicated by numbers. doi:10.1371/journal.pone.0095989.g006 Figure 6. Docking of para-hydroxybenzyaldehyde (PHB) in the active sites of ArOYEs. para-hydroxybenzyaldehyde (PHB) was docked in the respective active site pocket of each ArOYE. Active site residues interacting with the ligand were analyzed. Position of active site residues in respective ArOYE is indicated by numbers. doi:10.1371/journal.pone.0095989.g006 organization of Class III OYEs. Furthermore, analysis of structural features involved in stereopreference of ArOYEs suggests differ- ences in R/S selectivity of these OYEs. Future studies involving biochemical and molecular characterizations may further improve our understanding of the diverse enzymatic and molecular functions of these OYEs. OYE1, whereas Class II ArOYEs posses accessory residues similar to YqjM. In contrast, the accessory residues of Class III OYE, ArOYE6 showed marked difference from Class I and Class II OYEs. On a broader way, the active site arrangement of ArOYE6 is somewhat related to Class II OYEs than Class I OYEs, suggesting that ArOYE6 is more closely related to Class II OYEs. On the other hand, accessory residues are different from members of both Class I and II proteins. The major differences with Class II proteins are substitution of Cys26 and Tyr28 of YqjM with Thr40 and Arg42 in ArOYE6 (Figure 5). Another important difference in ArOYE6 is the non-shared active site architecture. In Class II OYEs an arginine or tryptophan finger protrudes from one monomer into the active site of the adjacent monomer. In contrast, Arg355 comes from the same monomer in ArOYE6, therefore suggesting the monomeric nature of the enzyme. Materials and Methods Sequence alignment and phylogenetic analysis References 1. Warburg O, Christian W (1933) u¨ber das gelbeoxydationsferment. Biochem Z 263: 228–229. 9. Schaller F, Weiler EW (1997) Molecular cloning and characterization of 12- oxophytodienoate reductase, an enzyme of the octadecanoid signaling pathway from Arabidopsis thaliana. J Biol Chem 272: 28066–28072. 2. Massey V, Schopfer LM (1986) Reactivity of old yellow enzyme with alpha- NADPH and other pyridine nucleotide derivatives. J Biol Chem 261: 1215– 1222. 10. Cheng JZ, Coyle CM, Panaccione DG, O’Connor SE (2010) A role for old yellow enzyme in ergot alkaloid biosynthesis. J Am Chem Soc 132: 1776–1777. 3. Durchschein K, Hall M, Faber K (2013) Unusual reactions mediated by FMN- dependent ene- and nitro-reductases. Green Chem 15: 1764–1772. 3. Durchschein K, Hall M, Faber K (2013) Unusual reactions mediated by FMN- dependent ene- and nitro-reductases. Green Chem 15: 1764–1772. 11. White GF, Snape JR, Nicklin S (1996) Biodegradation of glycerol trinitrate and pentaerythritoltetranitrate by Agrobacterium radiobacter. Appl Environ Microbiol 62: 637–642. 4. Saito K, Thiele DJ, Davio M, Lockridge O, Massey V (1991) The cloning and expression of a gene encoding old yellow enzyme from Saccharomyces carlsbergensis. J Biol Chem 266: 20720–20724. 4. Saito K, Thiele DJ, Davio M, Lockridge O, Massey V (1991) The cloning and expression of a gene encoding old yellow enzyme from Saccharomyces carlsbergensis. J Biol Chem 266: 20720–20724. 12. French CE, Nicklin S, Bruce NC (1996) Sequence and properties of pentaerythritoltetranitratereductase from Enterobacter cloacae PB2. J Bacteriol 178: 6623–6627. 5. Stott K, Saito K, Thiele DJ, Massey V (1993) Old Yellow Enzyme: the discovery of multiple isozymes and a family of related proteins. J Biol Chem 268: 6097– 6106. 5. Stott K, Saito K, Thiele DJ, Massey V (1993) Old Yellow Enzyme: the discovery of multiple isozymes and a family of related proteins. J Biol Chem 268: 6097– 6106. 13. Nivinskas H, Sarlauskas J, Anusevicius Z, Toogood HS, Scrutton NS, et al. (2008) Reduction of aliphatic nitroesters and N -nitramines by Enterobacter- cloacae PB2 pentaerythritoltetranitratereductase. FEBS J 275: 6192–6203. 6. French CE, Bruce NC (1995) Bacterial morphinonereductase is related to Old- Yellow Enzyme. Biochem J 312: 671–678. 14. Komduur JA, Leao AN, Monastyrska I, Veenhuis M, Kiel JA (2002) Old yellow enzyme confers resistance of Hansenulapolymorpha towards allyl alcohol. Curr Genet 41:401–406. 7. Niino YS, Chakraborty S, Brown BJ, Massey V (1995) A new old yellow enzyme of Saccharomyces cerevisiae. J Biol Chem 270: 1983–1991. 15. In-silico homology modeling of ArOYEs gy g In order to carry out homology modeling of ArOYEs, best templates were selected through PSI BLAST of each target protein against the PDB database (http://www.rcsb.org/pdb/home/ home.do). The significant hits with .40% sequence identity and atomic resolution , 1.8 Angstrons, were selected as templates for each target protein except ArOYE5 and ArOYE6 where the templates taken were having ,40% sequence identity. Table 1 shows the proteins used as templates for homology modeling along with PDB IDs, atomic resolution and their identity with the target protein. The three-dimensional structures of the target proteins were generated using a restrained-based approach in MODEL- LER9v11 [37]. For each OYE, 10 models were created and the one with the best score in terms of the discrete optimized protein energy (DOPE) potential implemented in MODELLER was chosen. Initial refinement of the 3D models generated was carried out with the help of loop refinement protocol of MODELLER. The assessment of the final structural models was carried out with PROCHECK [38], ProSA [39,40] and QMEAN [41] analyses. The final deviation in the protein structure geometry was regularized by energy minimization with the GROMOS96 force field [42] using Deep View [43]. All the structures were visualized using PyMOL (http://www.pymol.org/). Sequence alignment and phylogenetic analysis Multiple sequence and structure alignment program PRO- MALS3D server [34] was used to carry out protein sequence alignment of the full-length OYEs using default parameters. Sequence alignments were visualized using Jalview [35]. The phylogenetic relationships among OYE family members were determined by the means of Bayesian Markov Chain Monte Carlo (MCMC) inference of phylogeny as employed in MrBayes (v3.2.2) [36]. Two independent runs were performed using mixed amino acid substitution model where each run comprised 3,000,000 iterations, four simultaneous Markov Chain Monte Carlo (MCMC) chains and a sampling frequency of every 300 iterations with MCMC left at default settings. Tracer software (v1.5) (http:// In conclusion, using comparative homology modeling of six ArOYEs our study provides the first report about the structural analysis of fungal OYEs. Novel residues of Class III OYE, involved in interaction with FMN were revealed. In addition, active site residues of ArOYE6 was predicted and validated through docking of para-hydroxybenzaldehyde (PHB). In compar- ison to Class I and II OYEs our study reveals a unique active site Figure 7. Stereopreference in ArOYEs. Pseudo-atoms were generated for the residues described by Oberdorfer et al. (33) involved in the stereoselectivity of ArOYEs. Bond distances between pseudo-atoms were analyzed for each ArOYE. Pseudo-atom distances indicate that ArOYE1 and ArOYE2 are exclusive R-selective, ArOYE3 is moderately selective and ArOYE4-6 are exclusive S-selective. doi:10.1371/journal.pone.0095989.g007 Figure 7. Stereopreference in ArOYEs. Pseudo-atoms were generated for the residues described by Oberdorfer et al. (33) involved in the stereoselectivity of ArOYEs. Bond distances between pseudo-atoms were analyzed for each ArOYE. Pseudo-atom distances indicate that ArOYE1 and ArOYE2 are exclusive R-selective, ArOYE3 is moderately selective and ArOYE4-6 are exclusive S-selective. doi:10.1371/journal.pone.0095989.g007 April 2014 \| Volume 9 \| Issue 4 \| e95989 PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org 11 Structural Modelling of Old Yellow Enzymes (OYEs) from Ascochya rabiei alignments. Active sites of ArOYE6 was predicted using the POOL server [46] (http://www.pool.neu.edu) and Q-SiteFinder [47] (http://www.bioinformatics.leeds.ac.uk/qsitefinder). tree.bio.ed.ac.uk/software/tracer/) was employed to inspect con- vergence of the runs by analyzing the trace files generated by the Bayesian MCMC runs. Finally, a single tree was generated from the sampled trees obtained from independent runs by using the Sumt function of MrBayes. FigTree (v1.4.) (http://tree.bio.ed.ac. uk/software/figtree/) was used to visualize the phylogenetic trees. Ligand (substrate) structure Structures of substrate para-hydroxybenzyaldehyde (PHB), which is known ligand of OYE proteins was obtained from NCBI PubChem. 2D structure was then sketched with ChemSketch tool (http://www.acdlabs.com/resources/freeware/chemsketch/) in MOL format then converted to PDB format using OpenBabel v2.3.1 (http://openbabel.org) for docking purpose. Acknowledgments FMN was introduced into 3D structures through the restrains on the relative orientation of the cofactor from respective templates using MODELLER 9v11. FMN binding sites, in each ArOYEs were analyzed by generating LIGPLOT diagram using PDBsum [44,45]. FMN interacting residues of each ArOYEs were categorized into two groups on the basis of type of interactions (Table S3 in File S1). We gratefully acknowledge a research grant from Department of Biotechnology, Government of India (File No: BT/PR10605/PBD/16/ 791/2008) and a core grant from National Institute of Plant Genome Research, New Delhi, India for funding this work. SN and SV acknowledge University Grants Commission and Council of Scientific and Industrial Research, Government of India, respectively for their fellowships. KS acknowledges NIPGR for fellowship. (Table S3 in File S1). Author Contributions For the prediction of active site residues of ArOYEs, sequences of ArOYE1-5 were aligned with sequences of their respective templates using PROMALS3D. Subsequently, active site residues of all targets were identified by selecting the residues from Conceived and designed the experiments: SN PKV. Performed the experiments: SN RKG SV KS. Analyzed the data: PKV SN. Wrote the paper: SN SV PKV. Conceived and designed the experiments: SN PKV. Performed the experiments: SN RKG SV KS. Analyzed the data: PKV SN. Wrote the paper: SN SV PKV. Ligand docking The best 3D modeled structures of ArOYEs were utilized for docking using AutoDock4 [48]. Input structures of ArOYEs and PHB ligand were prepared by adding gasteiger charges and merging non-polar hydrogens using AutoDockTools. Map files were generated with AutoGrid4 using grid points 38 A˚644 A˚656 A˚ for ArOYE1, 40 A˚640 A˚640 A˚ for Ar- OYE2-5, and 62 A˚648 A˚650 A˚ for ArOYE6 with 0.375 A˚ spacing. Lamarckian Genetic Algorithm was used for simulations. The best docked ligand was chosen on the basis of lowest binding energy and conformation. Distances of interactions of ligand molecule with active site residues were analyzed using PyMOL (http://www.pymol.org/). Structural Modelling of Old Yellow Enzymes (OYEs) from Ascochya rabiei 31. Opperman DJ, Sewell BT, Litthauer D, Isupov MN, Littlechild JA, et al. (2010) Crystal structure of a thermostable old yellow enzyme from Thermusscotoductus SA-01. Biochem Biophys Res Commun 393: 426–431. 16. Odat O, Matta S, Khalil H, Kampranis SC, Pfau R, et al. (2007) Old Yellow Enzymes, Highly Homologous FMN Oxidoreductases with Modulating Roles in Oxidative Stress and Programmed Cell Death in Yeast. J Biol Chem 282: 36010–36023. p y 32. Nizam S, Verma S, Borah NN, Gazara RK, Verma PK (2014) Comprehensive genome-wide analysis reveals different classes of enigmatic old yellow enzyme in fungi. Sci Rep 4, 4013; DOI:10.1038/srep04013. 17. Strassner J, Furholz A, Macheroux P, Amrhein N, Schaller A (1999) A homolog of Old Yellow Enzyme in tomato. Spectral properties and substrate specificity of the recombinant protein. J Biol Chem 274: 35067–35073. 33. Oberdorfer G, Steinkellner G, Stueckler C, Faber K, Gruber K (2011) Stereopreferences of old yellow enzymes: structure correlations and sequence patterns in enoate reductases. ChemCatChem 3: 1562–1566. 18. Coyle CM, Cheng JZ, O’Connor SE, Panaccione DG (2010) An old yellow enzyme gene controls the branch point between Aspergillusfumigatus and Clavicepspurpurea ergot alkaloid pathways. Appl Environ Microbiol 76: 3898– 3903. p 34. Pei J, Kim BH, Grishin NV (2008) PROMALS3D: a tool for multiple protein sequence and structure alignments. Nuc Acids Res 36: 2295–2300. 35. Waterhouse AM, Procter JB, Martin DMA, Clamp M, Barton GJ (2009) Jalview Version—2 a multiple sequence alignment editor and analysis workbench. Bioinformatics 25: 1189–1191. 19. Winkler CK, Tasnadi G, Clay D, Hall M, Faber K (2012) Asymmetric bioreduction of activated alkenes to industrially relevant optically active compounds. J Biotechnol 162: 381–389. 36. Ronquist F, Huelsenbeck JP (2003) Mrbayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19: 1572–1574. 20. Ernst H (2002) Recent advances in industrial carotenoid synthesis. Pure App Chem 74: 2213–2226. 37. Sali A, Blundell TL (1993) Comparative protein modelling by satisfaction of spatial restraints. J Mol Biol 234: 779–815. 21. Fox KM, Karplus PA (1994) Old yellow enzyme at 2 A˚ resolution: overall structure, ligand binding, and comparison with related flavoproteins. Structure 2: 1089–1105. p 38. Laskowski RA, Macarthur MW, Moss DS, Thornton JM (1993) PROCHECK: A program to check the stereochemical quality of protein structures. J Appl Crystallogr 26: 283–291. 22. Barna TM, Khan H, Bruce NC, Barsukov I, Scrutton NS, et al. Structural Modelling of Old Yellow Enzymes (OYEs) from Ascochya rabiei (2001) Crystal Structure of PentaerythritolTetranitrateReductase: ‘‘Flipped’’ Binding Geome- tries for Steroid Substrates in Different Redox States of the Enzyme. J Mol Biol 310: 433–447. y g 39. Sippl MJ (1993) Recognition of Errors in Three-Dimensional Structures of Proteins. Proteins 17: 355–362. 40. Wiederstein M, Sippl MJ (2007) ProSA-web: interactive web service for the recognition of errors in three-dimensional structures of proteins. Nuc Acids Res 35: 407–410. 23. van den Hemel D, Brige´ A, Savvides SN, Van Beeumen J (2006) Ligand-induced Conformational Changes in the Capping Subdomain of a Bacterial Old Yellow Enzyme Homologue and Conserved Sequence Fingerprints Provide New Insights into Substrate Binding. J Biol Chem 281: 28152–28161. 41. Benkert P, Ku¨nzli M, Schwede T (2009) QMEAN server for protein model quality estimation. Nucleic Acids Res 37: 510–514. 24. Toogood HS, Gardiner JM, Scrutton NS (2010) Biocatalytic reductions and chemical versatility of the old yellow enzyme family of flavoprotein oxidoreductases. ChemCatChem 2: 892–914. 42. Gunsteren VWF, Billeter SR, Eising A, Hunenberger PH, Kruger P, et al. (1996) Biomolecular Simulations: The GROMOS 96 Manual and User Guide. Zu¨rich: Verlag der FachvereineHochschulverlag AG an der ETH Zurich. Verlag der FachvereineHochschulverlag AG an der ETH Zurich 25. Karplus PA, Fox KM, Massey V (1995) Flavoprotein structure and mechanism. 8. Structure-function relations for old yellow enzyme. FASEB J 9: 1518–1526. 43. Guex N, Peitsch MC (1997) SWISS-MODEL and the Swiss-PdbViewer: an environment for comparative protein modeling. Electrophoresis 18: 2714–2723. 26. Breithaupt C, Strassner J, Breitinger U, Huber R, Macheroux P, et al. (2001) X- Ray Structure of 12-Oxophytodienoate Reductase 1 Provides Structural Insight into Substrate Binding and Specificity within the Family of OYE. Structure 9: 419–429. 44. Laskowski RA, Hutchinson EG, Michie AD, Wallace AC, Jones ML, et al.(1997) PDBsum: a Web-based database of summaries and analyses of all PDB structures. Trends Biochem Sci 22: 480–490. J ( ) PDBsum: a Web-based database of summaries and analyses of all PDB structures. Trends Biochem Sci 22: 480–490. 45. de Beer TA, Berka K, Thornton JM, Laskowski RA (2014) PDBsum additions. Nucleic Acids Res 42: 292–296. 27. Barna T, Messiha HL, Petosa C, Bruce NC, Scrutton NS, et al. (2002) Crystal structure of bacterial morphinone reductase and properties of the C191A mutant enzyme. J Biol Chem 277: 30976–30983. 46. Somarowthu S, Ondrechen MJ (2012) POOL server: machine learning application for functional site prediction in proteins. Bioinformatics 28: 2078– 2079. 28. References Trotter EW, Collinson EJ, Dawes IW, Grant CM (2006) Old yellow enzymes protect against acrolein toxicity in the yeast Saccharomyces cerevisiae. Appl Environ Microbiol 72: 4885–4892. 8. Miranda M, Ramirez J, Guevara S, Ongay-Larios L, Pena A, et al. (1995) Nucleotide sequence and chromosomal localization of the gene encoding the Old Yellow Enzyme from Kluyveromyceslactis. Yeast 11: 459–465. 8. Miranda M, Ramirez J, Guevara S, Ongay-Larios L, Pena A, et al. (1995) Nucleotide sequence and chromosomal localization of the gene encoding the Old Yellow Enzyme from Kluyveromyceslactis. Yeast 11: 459–465. April 2014 \| Volume 9 \| Issue 4 \| e95989 PLOS ONE \| www.plosone.org 12 Structural Modelling of Old Yellow Enzymes (OYEs) from Ascochya rabiei Structural Modelling of Old Yellow Enzymes (OYEs) from Ascochya rabiei Pompeu YA, Sullivan B, Walton AZ, Stewart JD (2012) Structural and catalytic characterization of Pichiastipitis OYE 2.6, a useful biocatalyst for asymmetric alkene reductions. Adv Synth Cat 354: 1949–1960. 47. Laurie AT, Jackson RM (2005) Q-SiteFinder: an energy-based method for the prediction of protein-ligand binding sites. Bioinformatics 21: 1908–1916. 29. Kitzing K, Fitzpatrick TB, Wilken C, Sawa J, Bourenkov GP, et al. (2005) The 1.3 A˚ crystal structure of the flavoproteinYqjM reveals a novel class of old yellow enzymes. J Biol Chem 280: 27904–27913. 48. Morris GM, Huey R, Lindstrom W, Sanner MF, Belew RK, et al. (2009) AutoDock4 and AutoDockTools4: Automated docking with selective receptor flexibility. J Comput Chem 30: 2785–2791. 30. Griese JJ, P Jakob R, Schwarzinger S, Dobbek H (2006) Xenobiotic reductase A in the degradation of quinoline by Pseudomonas putida 86: physiological function, structure and mechanism of 8-hydroxycoumarin reduction. J Mol Biol 361: 140– 152. 49. Murakami MT, Rodrigues NC, Gava LM, Honorato RV, Canduri F, et al. (2013) Structural studies of the Trypanosoma cruzi Old Yellow Enzyme: insights into enzyme dynamics and specificity. Biophys Chem. doi: 10.1016/ j.bpc.2013.08.004. PLOS ONE \| www.plosone.org April 2014 \| Volume 9 \| Issue 4 \| e95989 13
https://openalex.org/W2785909275	https://scholarlypublications.universiteitleiden.nl/access/item%3A3007645/view	English	null	Replication of Type 2 diabetes-associated variants in a Saudi Arabian population	Physiological genomics/Physiological genomics (Print)	2,018	cc-by	1,345	Replication of Type 2 diabetes-associated variants in a Saudi Arabian population Ruifang Li-Gao,1 Salma M. Wakil,3 Brian F. Meyer,3 Nduna Dzimiri,3 and Dennis O. Mook-Kanamori1,2 1Department of Clinical Epidemiology, Leiden University Medical Center, Leiden, the Netherlands; 2Department of Public Health and Primary Care, Leiden University Medical Center, Leiden, the Netherlands; and 3Genetics Department, King Faisal Specialist Hospital and Research Center, Riyadh, Kingdom of Saudi Arabia Submitted 28 September 2017; accepted in ﬁnal form 15 February 2018 Ruifang Li-Gao,1 Salma M. Wakil,3 Brian F. Meyer,3 Nduna Dzimiri,3 and Dennis O. Mook-Kanamori1,2 1Department of Clinical Epidemiology, Leiden University Medical Center, Leiden, the Netherlands; 2Department of Public Health and Primary Care, Leiden University Medical Center, Leiden, the Netherlands; and 3Genetics Department, King Faisal Specialist Hospital and Research Center, Riyadh, Kingdom of Saudi Arabia Submitted 28 September 2017; accepted in ﬁnal form 15 February 2018 Li-Gao R, Wakil SM, Meyer BF, Dzimiri N, Mook-Kanamori DO. Replication of Type 2 diabetes-associated variants in a Saudi Arabian population. Physiol Genomics 50: 296–297, 2018. First pub- lished February 16, 2018; doi:10.1152/physiolgenomics.00100.2017.— Over 120 Type 2 diabetes (T2D) loci have been identiﬁed from genome- wide association studies (GWAS), mainly from Caucasian populations. Very limited knowledge is available on the Saudi Arabian population. In this study, 122 previously reported T2D-related variants from 84 loci were examined in a Saudi Arabian cohort of 1,578 individuals (659 T2D cases and 919 controls). Eleven single nucleotide polymorphisms (SNPs) corresponding to nine independent loci had a P value 0.05. If a more stringent Bonferroni threshold of P 4.1 104 ( 0.05/122) were applied, none of the SNPs would have reached the signiﬁcance level. Nine of the SNPs with a P value 0.05 showed similar odds ratios as previously described, but rs11605924 (CRY2) and rs9470794 (ZFAND3) were in the opposite direction. This study demonstrates the importance of large-scale GWAS in the Saudi Arabian population to identify ethnicity-speciﬁc disease-associated variants. Cohort details. This study was performed in a population- based case-control study for coronary artery disease (CAD) and myocardial infarction (MI) in Saudi Arabia (2). The study population was composed of 5,668 Saudi Arabian individuals, with 2,668 CAD and MI patients cases and 3,000 controls. For the current analysis only controls were used. Type of study. Candidate SNPs. Type of study. Candidate SNPs. Type of study. Candidate SNPs. Details of SNPs studied. We examined 153 T2D-associated loci reported in Prasad and Groop (1). Replication of Type 2 diabetes-associated variants in a Saudi Arabian population After imputation by the 1000G reference panel, 149 out of 153 SNPs were available. Nine SNPs were removed due to low imputation quality (imputation info 0.4), and we dropped 18 SNPs because of a global minor allele frequency (MAF) 0.1, leaving 122 vari- ants belonging to 84 loci for analyses. Analysis model. In the current case-control analysis, additive genetic models were used to assess the associations to the risk of T2D by logistic regression, adjusted for age, sex, body mass index (BMI), and the ﬁrst four principal components. The power calculation was conducted under the assumptions of 20% T2D prevalence, MAF 0.1, genotype relative risk 1.3, and 5% type I error rate. Around 600 cases were needed to achieve 80% power. additive model; replication; Saudi Arabia; SNPs; Type 2 diabetes additive model; replication; Saudi Arabia; SNPs; Type 2 diabetes RESULTS With 659 T2D cases [mean (SD) age: 58 (12) yr, BMI: 31.0 (6.6) kg/m2, 53.7% men] and 919 controls [45 (16) yr, 28.3 (6.5) kg/m2, 54.1% men], 11 SNPs corresponding to nine independent loci had a P value 0.05. rs7901695, rs4506565, rs7903146 located in the TCF7L2 gene belong to the same loci, with linkage disequilibrium (LD) of 0.97, 0.89, and 0.90, respectively, between rs7901695 and rs4506565, rs7901695 and rs7903146, and rs4506565 and rs7903146. All three SNPs in this signal had pronounced P values (all 0.003) and obtained similar odds ratios (ORs) as reported in the European- ancestry GWAS (ORs 1.34, 1.32, 1.31) (see supplemental tables). (The online version of this article contains supplemen- tal material.) In contrast, rs11605924 located in the CRY2 gene and rs9470794 located in the ZFAND3 gene were found to have opposite effects on T2D by estimated ORs equal to 0.80 and 0.77, respectively. If a more stringent Bonferroni threshold of P 4.1 104 ( 0.05/122) were applied, none of the SNPs would have reached the signiﬁcance level. BACKGROUND/MOTIVATION FOR THE STUDY To date, over 120 Type 2 diabetes (T2D) loci have been identiﬁed through genome-wide association studies (GWAS) (1), mainly from Caucasian populations. The prevalence of T2D differs signiﬁcantly among ethnicities because of distinct environmental and genetic factors. In the past 5 yr, more studies have focused on multiancestry GWAS for T2D. De- spite a striking local diabetic epidemic in Saudi Arabia, there is limited knowledge on the genetic basis of T2D from Middle Eastern populations. Therefore, some initial evidence of over- lap in T2D susceptibility loci in the Saudi Arabian population is warranted to ﬁll in the void of genetic basis of T2D in ethnic Arabs. PHENOTYPE T2D was characterized by combinations of decreased insulin secretion and sensitivity (also deﬁned as insulin resistance). The study candidates for T2D fulﬁlled the World Health Organization criteria and the American Association for Diabe- tes Guidelines for the disease. Physiol Genomics 50: 296–297, 2018. First published February 16, 2018; doi:10.1152/physiolgenomics.00100.2017. Physiol Genomics 50: 296–297, 2018. First published February 16, 2018; doi:10.1152/physiolgenomics.00100.2017. Physiol Genomics 50: 296–297, 2018. Licensed under Creative Commons Attribution CC-BY 4.0: © the American Physiological Society. eISSN: 1531-2267. Downloaded from www.physiology.org/journal/physiolgenomics at Leids Univers Medisch Centrum (145.088.209.033) on August 9, 2019. Downloaded from www.physiology.org/journal/physiolgenomics at Leids Univers Medisch Centrum (145.088.209.033) on August 9, 2019. AUTHOR CONTRIBUTIONS R.L.-G. and D.O.M.K. analyzed data; R.L.-G. and D.O.M.K. interpreted results of experiments; R.L.-G. prepared ﬁgures; R.L.-G. drafted manuscript; R.L.-G., S.M.W., B.F.M., N.D., and D.O.M.K. edited and revised manuscript; R.L.-G., S.M.W., B.F.M., N.D., and D.O.M.K. approved ﬁnal version of manuscript; S.M.W. performed experiments; B.F.M. and N.D. conceived and designed research. ACKNOWLEDGMENTS 1. Prasad RB, Groop L. Genetics of type 2 diabetes-pitfalls and possibilities. Genes (Basel) 6: 87–123, 2015. doi:10.3390/genes6010087. 1. Prasad RB, Groop L. Genetics of type 2 diabetes-pitfalls and possibilities. Genes (Basel) 6: 87–123, 2015. doi:10.3390/genes6010087. The authors thank Editha Andres, Nejat Mazher, and Dr. Maie Alshaid for assistance in patient sample and clinical data collection. 2. Wakil SM, Ram R, Muiya NP, Mehta M, Andres E, Mazhar N, Baz B, Hagos S, Alshahid M, Meyer BF, Morahan G, Dzimiri N. A genome- wide association study reveals susceptibility loci for myocardial infarction/ coronary artery disease in Saudi Arabs. Atherosclerosis 245: 62–70, 2016. doi:10.1016/j.atherosclerosis.2015.11.019. Physiol Genomics • doi:10.1152/physiolgenomics.00100.2017 • www.physiolgenomics.org ww.physiology.org/journal/physiolgenomics at Leids Univers Medisch Centrum (145.088.209.033) on August 9, 2019. Physiol Genomics • doi:10.1152/physiolgenomics.00100.2017 • www.physiolgenomics.org Downloaded from www.physiology.org/journal/physiolgenomics at Leids Univers Medisch Centrum (145.088.209.033) on August 9, 2019. INTERPRETATION Address for reprint requests and other correspondence: R. Li-Gao, Dept. of Clinical Epidemiology, Leiden Univ. Medical Center, Albinusdreef 2, Leiden 2333 ZA, the Netherlands (e-mail: r.li@lumc.nl). The three variants located in the TCF7L2 gene showed similar ORs as in the European-ancestry GWAS, suggesting 296 ownloaded from www.physiology.org/journal/physiolgenomics at Leids Univers Medisch Centrum (145.088.209.033) on A 297 REPLICATION OF T2D-ASSOCIATED SNPs IN A SAUDI POPULATION Physiol Genomics • doi:10.1152/physiolgenomics.00100.2017 • www.physiolgenomics.org Downloaded from www.physiology.org/journal/physiolgenomics at Leids Univers Medisch Centrum (145.088.209.0 DISCLOSURES the validity of the current analysis. The two SNPs with oppo- site effects may indicate Saudi Arabian-speciﬁc genetic infor- mation on T2D. Due to the moderate effect sizes of these T2D-associated SNPs (median OR 1.12 in European ances- try), the current study is still underpowered, which partially explains the large amount of unreplicated SNPs (n 111/122). Additionally, controls were relatively younger than the T2D cases in the current case-control study, and this age discrep- ancy may confound the analysis, although we adjusted for age in the multivariable model. No conﬂicts of interest, ﬁnancial or otherwise, are declared by the authors. Physiol Genomics • doi:10.1152/physiolgenomics.00100.2017 • www.physiolgenomics.org GRANTS This study was funded through Royal Cardiovascular Research Grant RAC2030012 under the King Faisal Specialist Hospital and Research Centre.
https://openalex.org/W2164696253	https://link.springer.com/content/pdf/10.1007%2Fs10728-015-0300-4.pdf	English	null	Potential International Approaches to Ownership/Control of Human Genetic Resources	Health care analysis	2,015	cc-by	8,543	Health Care Anal (2016) 24:260–277 DOI 10.1007/s10728-015-0300-4 ORIGINAL ARTICLE & Catherine Rhodes yhtacrhodes@yahoo.co.uk; carhodes1@outlook.com 1 Institute for Science, Ethics and Innovation, 3.614 Stopford Building, Faculty of Life Sciences, University of Manchester, Oxford Road, Manchester M13 9PL, UK Introduction Scientiﬁc work utilising human genetic resources has great potential for contribut- ing to addressing major global health challenges. The approach adopted to their ownership in international governance will have signiﬁcant implications for how they are accessed and used, who participates in this work, the direction it takes and who beneﬁts from it. This paper outlines the different approaches to genetic resources ownership/control that have been taken by the international community, before assessing which will be the most appropriate approach to adopt toward governance of human genetic resources, which—in the absence of an agreed international approach—are likely to fall under free access, which tends to privilege the interests of private companies and scientiﬁcally and technologically advanced states, and is therefore inappropriate to serve global health needs. Genetic Resources Genetic resources have been deﬁned in international law as ‘‘genetic material of actual or potential value’’, with genetic material being ‘‘any material of plant, animal, microbial or other origin containing functional units of heredity’’ (Convention on Biological Diversity, Article 2 [3]). The term can also be understood to include associated data. There is still some uncertainty about whether the term genetic resources covers ‘derivatives’, deﬁned in Article 2 of the Nagoya Protocol to the Convention on BiologicalDiversityas‘‘anaturallyoccurringbiochemicalcompoundresultingfromthe genetic expression or metabolism of biological or genetic resources, even if it does not contain functional units of heredity’’ [4] (see for example section 2.4.1 in [15, 28]). Traditional knowledge associated with certain genetic resources also receives particular protection, but is not covered in any detail in this paper. Potential International Approaches to Ownership/ Control of Human Genetic Resources Catherine Rhodes1 Published online: 22 August 2015 The Author(s) 2015. This article is published with open access at Springerlink.com Published online: 22 August 2015 Published online: 22 August 2015 Published online: 22 August 2015 The Author(s) 2015. This article is published with open access at Springerlink.com Abstract In its governance activities for genetic resources, the international community has adopted various approaches to their ownership, including: free access; common heritage of mankind; intellectual property rights; and state sover- eign rights. They have also created systems which combine elements of these approaches. While governance of plant and animal genetic resources is well- established internationally, there has not yet been a clear approach selected for human genetic resources. Based on assessment of the goals which international governance of human genetic resources ought to serve, and the implications for how they will be accessed and utilised, it is argued that common heritage of mankind will be the most appropriate approach to adopt to their ownership/control. It does this with the aim of stimulating discussion in this area and providing a starting point for deeper consideration of how a common heritage of mankind, or similar, regime for human genetic resources would function and be implemented. Keywords International governance Human genetic resources Common heritage of mankind Approaches to genetic resources ownership Genetic resources governance Abbreviations CBD Convention on Biological Diversity CHM Common heritage of mankind FAO Food and Agriculture Organisation GISRS Global Inﬂuenza Surveillance and Response System 123 261 Health Care Anal (2016) 24:260–277 TRIPS Trade Related Aspects of Intellectual Property Rights Agreement UNCLOS United Nations Convention on the Law of the Sea UNESCO United Nations Educational, Scientiﬁc and Cultural Organisation UNGA United Nations General Assembly WHO World Health Organisation WIPO World Intellectual Property Organisation WTO World Trade Organisation WTO-TNC World Trade Organisation-Trade Negotiations Committee TRIPS Trade Related Aspects of Intellectual Property Rights Agreement UNCLOS United Nations Convention on the Law of the Sea UNESCO United Nations Educational, Scientiﬁc and Cultural Organisation UNGA United Nations General Assembly WHO World Health Organisation WIPO World Intellectual Property Organisation WTO World Trade Organisation WTO-TNC World Trade Organisation-Trade Negotiations Committee Human Genetic Resources In line with this deﬁnition, human genetic resources are understood to be human genetic material, containing functional units of heredity, with actual or potential value, and associated data. 12 123 123 262 Health Care Anal (2016) 24:260–277 International Community Where the term international community is used in this paper, it primarily refers to states and the international organisations of which they are members. 1 This is not the only way in which these approaches might be characterised and categorised. There are also other potential approaches which could be adopted by the international community (e.g. public trusteeship as outlined by Sand [20], however the focus in the paper is on those approaches already used by the international community in genetic resources governance. 2 The terms ownership and control are distinct. They are both used in this paper because some of the international approaches to genetic resources exclude ownership, while others blend elements of ownership and control. International Governance The term international governance refers to rules, norms, institutions, procedures and mechanisms which govern the behaviour of states and other international actors in the absence of supranational government. Signiﬁcant among these in the genetic resources area are international organisations, treaties, standards, guidelines and codes, and information exchange, reporting and surveillance mechanisms (examples are shown in Table 1). A useful, complementary deﬁnition of international governance is provided by Finkelstein [10, p. 368]: ‘‘any purposeful activity intended to ‘control’ or inﬂuence someone else that either occurs in the arena occupied by nations or, occurring at other levels, projects inﬂuence into that arena’’. This paper focuses on elements of governance that are potentially universal—that is open to all states to subscribe to, without any limitations on e.g. geographic or economic grounds. Table 1 Selected examples of international governance mechanisms for genetic resources International organisations Treaties Standards/ guidelines/codes/ declarations Reporting and surveillance mechanisms/expert networks Food and Agriculture Organisation United Nations Educational, Scientiﬁc and Cultural Organisation World Animal Health Organisation World Health Organisation World Intellectual Property Organisation World Trade Organisation Biological Weapons Convention Convention on Biodiversity International Plant Protection Convention International Treaty on Plant Genetic Resources for Food and Agriculture Nagoya Protocol on Access to Genetic Resources and the Fair and Equitable Sharing of the Beneﬁts Arising from their Utilisation Aquatic Animal Health Code Terrestrial Animal Health Code Laboratory Biosafety Manual Laboratory Biosecurity Guidance Universal Declaration on the Human Genome and Human Rights United Nations Declaration on the Rights of Indigenous Peoples Emergency Prevention and Response System—Food Safety Global Inﬂuenza Surveillance and Response System World Animal Health Information Service World Information and Early Warning System for Plant Genetic Resources for Food and Agriculture Table 1 Selected examples of international governance mechanisms for genetic resources 12 123 Health Care Anal (2016) 24:260–277 263 Governing Genetic Resources at the International Level There have been international efforts to govern genetic resources for over 60 years. At ﬁrst these focused on exchanges of plant genetic resources, and governance is most developed in this area. Many of the current rules on genetic resources—while having wider scope—are designed around historical patterns of exploitation and exchange associated with plants. Genetic resources governance is least developed for the areas of human and microbial genetic resources. For human genetic resources it is currently limited to three declarations of principles (not legally- binding on states) and concentrated within the United Nations Educational, Scientiﬁc and Cultural Organisation. Human genetic resources could justiﬁably fall within the remit of the World Health Organisation in regard to their (primary) utility in the understanding of human disease. The Organisation for Economic Cooperation and Development has also published some guidelines relating to human genetic databanks—I do not consider these to fall in the scope of international rules, which for the purposes of this analysis, I limit to those rules that are potentially universal— however, they provide a useful indicator of the direction the international community may take as it develops rules in this area. Genetic resources governance has expanded massively in scope from its original concern with facilitating exchange of plant genetic material. As well as covering collection, exchange and banking of genetic resources, it now covers several other issues including: conservation; protection of human rights; protection of human, animal and plant health; food security; climate change adaptation and mitigation; ﬁnance, funding and capacity building (particularly in science and technology); access and beneﬁt-sharing; and ownership and control rights. Approaches to genetic resources ownership and control are of central interest in this paper. 2 The terms ownership and control are distinct. They are both used in this paper because some of th international approaches to genetic resources exclude ownership, while others blend elements ownership and control. International Approaches to Ownership/Control of Genetic Resources There are, broadly, ﬁve approaches1 to ownership/control2 of genetic resources that are used by the international community: free access; state sovereign rights; intellectual property rights; common heritage of mankind; and mixed systems. Their general use is outlined next, before they are discussed in the more speciﬁc context of human genetic resources governance. 12 123 3 264 Health Care Anal (2016) 24:260–277 Free Access Where a free access approach is applied to genetic resources, anyone is free to access them, to use them as they choose, and to subsequently claim proprietary rights over them (excluding others from access/use if they choose to do so). This was the dominant international approach to genetic resources governance prior to the application of state sovereign rights in the early 1990s. It is a default position where no rules have been established, and still operates for certain genetic resources, such as those located in some areas deﬁned as ‘beyond national jurisdiction’—for example marine genetic resources in the high seas and seabed beyond state jurisdiction. Free access is controversial because it tends to favour those who have particular knowledge and expertise, and the ﬁnancial and technological means to access and exploit the resources [1]—which are primarily concentrated in developed countries. Free access therefore tends to have the effect of concentrating beneﬁts of research on and use of genetic resources in a limited number of individuals, groups and states, regardless of where they are sourced from. State Sovereign Rights Article 6—Access to Genetic Resources Article 6—Access to Genetic Resources Article 6—Access to Genetic Resources 1. In the exercise of sovereign rights over natural resources, and subject to domestic access and beneﬁt-sharing legislation or regulatory requirements, access to genetic resources for their utilization shall be subject to the prior informed consent of the [State] Party providing such resources… 3. Pursuant to paragraph 1 above, each Party requiring prior informed consent shall take the necessary legislative, administrative or policy measures, as appropriate, to:… (g) Establish clear rules and procedures for requiring and establishing mutually agreed terms. Such terms shall be set out in writing and may include, inter alia: … (ii) Terms on beneﬁt-sharing, including in relation to intellectual property rights; Use of a sovereign rights approach does not necessarily preclude subsequent claims of intellectual property rights being made by the user—it will depend on the terms agreed between the user and provider state. Access and beneﬁt-sharing arrangements based on state sovereignty are generally made through use of standard material transfer agreements which form a contract between the provider and the user.3 Recommendations and/or requirements for the content of these agreements are generally set out in the relevant treaty, and model contracts are usually provided by the relevant international organisation. Often, within provisions on access and beneﬁt-sharing, there is incorporation of respect for the rights of farmers, local and indigenous communities over genetic resources and associated knowledge, which they traditionally hold or have played a key role in developing. States are expected, for example, to ensure such groups are involved in consent processes and beneﬁt-sharing negotiations. 3 An explanation of such a contract is provided by the secretariat of the International Treaty on Plant Genetic Resources at http://www.planttreaty.org/content/what-standard-material-transfer-agreement- smta. State Sovereign Rights State sovereignty has long been accepted to include rights to territorial integrity and control over resources within that territory—particularly mineral resources. This is, for example, repeatedly stated in resolutions of the United Nations General Assembly, such as Resolution 3016(XXVII) ‘Permanent sovereignty over natural resources of developing countries’, which: ‘‘Reafﬁrms the right of States to permanent sovereignty over all their natural resources, on land within their international boundaries’’ [27]. State sovereign rights have been applied to genetic resources by the international community since the early 1990s, and are now the dominant international approach to genetic resources ownership/control. While not yet explicitly extended to human genetic resources, sovereign rights were extended to viral genetic resources in 2011 on quite spurious grounds [18], and so while their extension to human genetic resources seems inappropriate, it is not implausible. Statements of state sovereignty over genetic resources in international law generally use similar wording to that found in the Convention on Biological Diversity (CBD), but are often speciﬁc to particular sub-sets/types of genetic resources (such as plant genetic resources, or inﬂuenza genetic resources). Based on recognition of ‘‘the sovereign rights of States over their natural resources’’ Article 15.1 of the CBD states that: ‘‘the authority to determine access to genetic resources rests with the national governments and is subject to national legislation’’. Within international agreements that adopt a sovereign rights approach, access to genetic resources is made subject to the prior informed consent of the provider state, which can negotiate conditions on the sharing of any beneﬁts that arise from their subsequent exploitation. This is, for example, set out in Articles 5 and 6 of the Nagoya Protocol (to the Convention on Biological Diversity) on Access to Genetic Resources and the Fair and Equitable Sharing of the Beneﬁts Arising from their Utilisation: 123 265 Health Care Anal (2016) 24:260–277 Article 5—Fair and Equitable Beneﬁt-sharing …beneﬁts arising from the utilization of genetic resources as well as subsequent applications and commercialization shall be shared in a fair and equitable way with the [State] Party providing such resources… Such sharing shall be upon mutually agreed terms. …beneﬁts arising from the utilization of genetic resources as well as subsequent applications and commercialization shall be shared in a fair and equitable way with the [State] Party providing such resources… Such sharing shall be upon mutually agreed terms. 5 To illustrate how extensive these discussions are, they have received the following coverage in TRIPS Council meeting minutes within the last 2 years: paragraphs 9–65 in IP/C/M/76/Add.1 Minutes of Meeting 11 June 2014; paragraphs 22–114 in IP/C/M/75/Add.1 Minutes of Meeting 25–26 February 2014; paragraphs 8–66 in IP/C/M/74/Add.1 Minutes of Meeting 10–11 October 2013; paragraphs 1–72 in IP/C/ M/73/Add.1 Minutes of Meeting 11-12 June 2013; paragraphs 5.1–5.50 in IP/C/M/72 Minutes of Meeting 5–6 March 2013; and paragraphs 23–97 in IP/C/M/71 Minutes of Meeting 6–7 November 2012. All of these documents are available through WTO Documents Online Search Facility. http://www.wto.org/english/ res_e/res_e.htm. Intellectual Property Rights There are two main forms of intellectual property right which can be claimed over genetic resources—patents and plant variety rights. The latter obviously do not apply to human genetic resources; there is considerable controversy over whether patents should apply to them. Patents are increasingly being claimed over a range of genetic resources. The appropriateness of this is a subject of intense debate, both because of disputes over the extent to which genetic resources fulﬁl criteria of 12 3 266 Health Care Anal (2016) 24:260–277 novelty and inventiveness required for grant of patents,4 and also because it is seen as frequently being unfair to provider states and communities—their contributions are rarely acknowledged, their knowledge is often absent from searches for prior art, and they may be excluded from beneﬁts arising from commercial exploitation. This is for example a major source of contention in the World Trade Organisation’s TRIPS Council5 (the committee which oversees the review of the Trade Related Aspects of Intellectual Property Rights Agreement, which sets international minimum standards of intellectual property protection). The subject is considered under combined agenda items on: Review of the Provisions of Article 27.3 (b); Relationship between the TRIPS Agreement and the Convention on Biological Diversity; and Protection of Traditional Knowledge and Folklore, at each regular meeting of the TRIPS Council. South Africa, for example, summarised the problem areas in its statement to the Council in November 2012 [34, pgh. 33], noting ‘‘three fundamental conﬂicts between the Convention on Biological Diversity and TRIPS Agreement’’: ﬁrst, that TRIPS overlooks state sovereign rights over genetic resources as provided by the CBD; second, that TRIPS negates states’ legal authority to determine beneﬁt-sharing from commercial use of genetic resources; and third, that TRIPS does not incorporate requirements for prior informed consent within patent applications. Very limited progress has been made on the issues over the past 15 years, but discussions remain on-going. Reform suggestions made, for example in the World Trade Organisation’s Trade Negotiations Committee [35, pghs. 4–6, and 36] and through the World Intellectual Property Organisation’s Intergovernmental Com- mittee on Intellectual Property Rights, Genetic Resources, Traditional Knowledge and Folklore [29, 30], have included: incorporating traditional knowledge in prior art searches; requiring disclosure of origin of genetic resources or traditional knowledge used in patent applications; and requiring evidence of compliance with access and beneﬁt-sharing rules in order to be granted a patent. 4 Article 27—Patentable Subject Matter—of the Agreement on Trade Related Aspects of Intellectual Property Rights (TRIPS) states that ‘‘patents shall be available for any inventions, whether products or processes, in all ﬁelds of technology, provided that they are new, involve an inventive step and are capable of industrial application’’. Common Heritage of Mankind The concept of common heritage of mankind6 began to be articulated internationally during the late 1960s and has been used by developing states in efforts to shape 4 Article 27—Patentable Subject Matter—of the Agreement on Trade Related Aspects of Intellectual Property Rights (TRIPS) states that ‘‘patents shall be available for any inventions, whether products or processes, in all ﬁelds of technology, provided that they are new, involve an inventive step and are capable of industrial application’’. 6 It is worth noting that there is a distinction between a commons and a common heritage of mankind approach—which is one way of managing a commons area/commons resources. Commons may also be subject to free access approaches for example—which, as noted in the main text, is the case for marine genetic resources within the high seas. 123 7 There are several conceptions of CHM in the literature; these are generally consistent with Joyner’s model, although not all include the points about scientiﬁc research (see, for example, [20] and [16]). Literature on CHM mainly stems from the ﬁeld of environmental law. Taylor and Stroud’s [22] book Common Heritage of Mankind: A Bibliography of Legal Writing is an excellent starting point for those wanting to further explore the topic. 123 Health Care Anal (2016) 24:260–277 267 international law relating to common areas (there are four ‘global commons’ managed by the international community: Antarctica; the high seas and seabed beyond areas of national jurisdiction; the atmosphere; and outer space). While developed in relation to common areas, the common heritage of mankind approach can be adapted to common resources. Joyner [12, pp. 191–2] conceptualised common heritage of mankind7 for common areas as being based on ﬁve main elements: 1. not… subject to appropriation of any kind, either public or private, national or corporate… owned by no one, though hypothetically managed by everyone. Sovereignty would be absent, as would all its legal attributes and ramiﬁca- tions… legally the entire area would be administered by the international community. 2. all people would be expected to share in the management of a common space area… States or national governments would be precluded from this legal function, save as the representative agents of all mankind… universal popular interests would assume priority, and thereby supply the foundation for any administrative decisions made affecting the region. 3. if natural resources were exploited… any economic beneﬁts derived from those efforts would be shared internationally. Under a CHM [Common Heritage of Mankind] regime, agencies engaged in commercial proﬁt or private gain would be deemed inappropriate, unless they operated to enhance the common beneﬁt of all mankind. 4. use of the area must be limited to exclusively peaceful purposes 5. scientiﬁc research… would be freely and openly permissible, so long as the environment of the common space area was in no way physically threatened or ecologically impaired. All research results would be made available as soon as possible to anyone who genuinely expressed interest in them. Under a CHM regime, scientiﬁc research would be conducted to beneﬁt all peoples, not merely the State or government which sponsored the research. Furthermore, the scientiﬁc fruits of such research would be freely and publicly exchanged in the hope of fostering greater scientiﬁc co-operation and more extensive knowledge about the region. Joyner also noted that an international authority would be needed in order to effectively manage such areas as common heritage of mankind, and that the authority would need to fulﬁl various legal functions such as ‘‘distributing users’ rights and economic beneﬁts… and facilitating the settlement of disputes’’ [12, p. 194]. 8 Regardless of the approach taken to the ownership of genetic resources, this clause will apply because of existing legal obligations in the Biological Weapons Convention, which prohibit any non-peaceful use of biological materials. (The Chemical Weapons Convention will similarly apply where genetic resources are exploited in order to produce chemical agents.). 123 Applying these elements to genetic resources would mean that: resources would not be subject to appropriation and would be managed in line with universal interests; any economic (or other) beneﬁts arising from their exploitation would be 12 3 268 Health Care Anal (2016) 24:260–277 shared internationally; their use would be limited to exclusively peaceful purposes8; and scientiﬁc research using genetic resources would be conducted for the beneﬁt of all. Elements of the common heritage approach are covered in more detail below, where it is applied speciﬁcally to human genetic resources. shared internationally; their use would be limited to exclusively peaceful purposes8; and scientiﬁc research using genetic resources would be conducted for the beneﬁt of all. Elements of the common heritage approach are covered in more detail below, where it is applied speciﬁcally to human genetic resources. Common heritage of mankind has little current use as an approach to genetic resources governance, but it is promoted by many developing countries for marine genetic resources in areas beyond national jurisdiction. Currently, these resources are subject to free access, which is advantaging rich, technologically advanced states and commercial enterprises which are able to extract the resources and exploit them. The United Nations Convention on the Law of the Sea (UNCLOS) places the seabed and ocean ﬂoor and the mineral resources found there under the common heritage of mankind [26, Article 136], and while developing countries argue that this should apply to genetic resources as well, developed countries argue that they instead fall under the freedom of the high seas [2]. There was also an unsuccessful attempt to have common heritage of mankind applied to plant genetic resources in the early 1980s, within the Food and Agriculture Organisation’s International Undertaking on Plant Genetic Resources, which origi- nally stated that: ‘‘This Undertaking is based on the universally accepted principle that plant genetic resources are a heritage of mankind and consequently should be available without restriction’’ [6, Article 1]. The Undertaking received little support from developed states because of its inclusion of newly developed varieties and breeding lines within the scope of common heritage, and was amended in 1991 to give priority to the sovereign rights of states: ‘‘Recognizing that:—the concept of mankind’s heritage, as applied in the International Undertaking on Plant Genetic Resources, is subject to the sovereignty of the states over their plant genetic resources…’’ [7]. 9 Such acts are, of course, subject to limitations from other areas of law—this does not give the right to extract genetic material from an individual without their consent, for example. Mixed Systems The international community has established two systems for genetic resources which combine elements of state sovereignty and centralised access and/or beneﬁt-sharing mechanisms, with some allowance for claims to intellectual property rights. The ﬁrst of these is the Multilateral System of Access and Beneﬁt-Sharing created by the International Treaty on Plant Genetic Resources, which became operational in October 2007 [8]. It covers a list of food and forage crops determined to be particularly important to food security. States agree to exercise their sovereign rights through the System, using it to facilitate access to their listed plant genetic resources [9, Article 12.3 (d) and (f)]. They are encouraged to do so through use of standard material transfer agreements, which should include provisions on beneﬁt-sharing—including beneﬁts such as information exchange, technology transfer, capacity building, and monetary beneﬁts from commercialisation [9, Article 12.4 and Article 13]. The second example is the Pandemic Inﬂuenza Preparedness Framework adopted by the World Health Organisation in 2011. This focuses on inﬂuenza viruses with 12 123 269 Health Care Anal (2016) 24:260–277 human pandemic potential and centralises the sharing of inﬂuenza viral genetic resources for international collaborative research efforts within its Global Inﬂuenza Surveillance and Response System (GISRS) and between the System and external entities (generally pharmaceutical companies and vaccine manufacturers). It also provides centralised beneﬁt-sharing systems including vaccine and anti-viral stockpiles, which can be distributed during pandemics. State sovereign rights over their inﬂuenza viral genetic resources are recognised alongside the importance of sharing them for global public health efforts. Sharing of biological materials under the Framework is done using standard material transfer agreements in two forms: one between entities within GISRS—Standard Material Transfer Agreement 1; the other between the World Health Organisation and entities external to the GISRS—Standard Material Transfer Agreement 2 [32, Annex 1 and Annex 2]. The state affected by an inﬂuenza outbreak with human pandemic potential is expected to submit samples to the GISRS in a timely manner [32, pgh. 5.1.1]. The state may also provide samples directly to companies, provided GISRS access is prioritised [32, pgh. 5.1.4]. Subsequent claims of intellectual property rights by such companies does not seem to be precluded, however the implications of this for work within GISRS are unclear. 10 This point is not integral to Joyner’s outline of CHM. However, special attention to the needs of developing countries does appear in relation to the management of CHM resources (for example Article 140 of the UNCLOS states that where activities take place to the beneﬁt of mankind in the ‘area’, particular consideration should be given to the interests and needs of developing countries) and frequently appears in international rules on genetic resources. Governing Human Genetic Resources at the International Level Applying state sovereign rights to human genetic resources would be problematic and has not entered state practice. It would, for example, imply that the state had a right to determine access to the genetic material of people within its jurisdiction, and to be party to any beneﬁts accruing from their utilisation. Currently, there is no established international approach to the ownership of human genetic resources. On a practical basis this means that free access is—by default—likely to apply. As outlined above, this means that anyone can access such resources, utilise them as they choose,9 and claim proprietary rights over them. Free access situations tend to privilege those with advanced scientiﬁc and technological capacity, and may well lead to concentration of the beneﬁts of human genetic research in developed states and commercial entities. Claims of intellectual property rights can be detrimental to the achievement of important global goals—in this case advances in human health, and increase the costs of participation in scientiﬁc research. Concern about this situation was expressed at length in the World Health Organisation [31] report Genomics and World Health. It noted, for example, that trends in DNA patenting (of human and pathogenic genetic material) mean that ‘‘future proﬁts and resource ﬂows in genomics will be concentrated in the developed economies in general, and in the United States in particular’’ (p. 128) and concluded that ‘‘the current position regarding DNA patenting is retarding rather than stimulating both scientiﬁc and economic progress’’ (p. 138). These patenting trends have continued—a 2013 study found that approximately 41 % of human genes are covered by patents [19]. 12 3 270 Health Care Anal (2016) 24:260–277 There are ongoing disputes about whether proprietary rights over human genetic resources are valid or appropriate. As well as the broader arguments raised against the patenting of genetic resources in general (outlined earlier), additional arguments have been put forward in opposition to patenting of human genetic resources—for example in regard to the added expense this might bring to the cost of diagnostics— the Myriad case being an example of this (see [14] for an overview of the case and its implications), the impacts it can have on research with major public health beneﬁts, and concerns over the commodiﬁcation of life (see for example [11, 13]). Governing Human Genetic Resources at the International Level Given these problems with use of state sovereign rights, free access and intellectual property rights approaches to human genetic resources governance, a common heritage approach may be the most suitable alternative. Goals of Human Genetic Resources Governance Goal: achieving advances in human health (e.g. through better understanding, diagnosis, prevention and treatment of disease). The primary utility of human genetic resources is for research into human disease processes and their interactions with environmental factors. There is a common global interest in efforts for disease control and for scientiﬁc and medical work which supports these; within international governance a focus on global priority health needs is appropriate. A common heritage approach will direct work on human genetic resources towards universal interests (under point 2) and so ﬁts well with this goal. This is supported by the Universal Declaration on the Human Genome11 and Human Rights [23], the International Declaration on Human Genetic Data [24], and the Universal Declaration on Bioethics and Human Rights [25], which all incorporate the principle that research involving human genetic resources should beneﬁt humanity as a whole, and particularly in relation to health goals: ‘‘The applications of research… concerning the human genome, shall seek to offer relief from suffering and improve the health of individuals and humankind as a whole’’ [23, Article 12]. The importance of this has been recognised in scientists’ reﬂections on progress within the Human Genome Project—John Sulston, for example, noting that: Science is international, and its beneﬁts ought to be international as well—at least with regard to such a basic human need as healthcare. Yet this is far from the case. Most biomedical research is aimed at the rich markets, and most of the human disease burden is, so far, untouched by technological progress. We urgently need to do better—not just for the sake of our common humanity but for all our futures, as a divided world is unstable and dangerous [21, p. 14]. Goal: capacity building and reduction of inequalities in health and medical research and healthcare systems. There is also a common interest in the management and use of human genetic resources contributing to development and reduction in inequalities. To make responses to health threats sustainable, substantial capacity building is needed in order to reduce inequalities in e.g. health infrastructure, and access to medical care and medicines (this has been sharply illustrated during the current Ebola outbreak— see, for example, [33]). There also need to be reductions of inequalities in scientiﬁc and technological capacities between states. These currently have a major inﬂuence on what research gets done, how it is directed, who participates, and who beneﬁts from it. The Appropriateness of a Common Heritage Approach to Human Genetic Resources This section considers whether a common heritage approach to governance of human genetic resources is appropriate both in terms of the goals that such governance ought to be achieving and the principles articulated by the international community about the status and appropriate management of human genetic material. Joyner’s ﬁve elements of common heritage of mankind would apply to human genetic resources as follows: 1. Human genetic resources could not be owned and/or appropriated by anyone, nor could they be subject to sovereign rights. They would be administered by the international community on behalf of all mankind, with attention being given to the priority needs of developing countries where necessary.10 2. The international community would share responsibility for the resources and would manage them on the basis of universal interests. 2. The international community would share responsibility for the resources and would manage them on the basis of universal interests. 3. Beneﬁts from the exploitation of human genetic resources would be shared internationally on the basis of common global interests and with attention to the needs of developing countries, and ‘‘agencies engaged in commercial proﬁt or private gain would be deemed inappropriate, unless they operated to enhance the common beneﬁt of all mankind’’ [12, p. 192]. 4. The use of human genetic resources would be limited to peaceful purposes 5. Scientiﬁc research on human genetic resources would be freely and openly permitted (provided it is in compliance with other international and applicable domestic laws—for example on the protection of research subjects). The results of such research would rapidly be made freely available to anyone with a genuine interest. Scientiﬁc research would be directed to the beneﬁt of all peoples and the fruits of such research would be freely and publicly exchanged. (Adapted from [17], p. 235). 12 123 123 Health Care Anal (2016) 24:260–277 271 11 It is worth noting that the human genome and human genetic resources are distinct, and discussion on the human genome as a commons and application of CHM to the human genome have a narrower focus than human genetic resources. Goals of Human Genetic Resources Governance Measures to facilitate this will include extensive capacity building efforts of various kinds to increase participation in research using human genetic resources (e.g. in 12 3 272 Health Care Anal (2016) 24:260–277 infrastructure, education, science, technology, knowledge, expertise, research facilities and equipment, regulation and administration). A common heritage approach will require equitable sharing of economic and other beneﬁts of work with human genetic resources internationally, including both increased knowledge and the improved products that might result from it (ﬁtting with points 3 and 5 of Joyner’s approach). Such beneﬁt-sharing can support the needed capacity-building efforts and assist reduction of inequalities in the areas of health and scientiﬁc and technological capacities. The international authority that would be associated with management of human genetic resources under a common heritage approach, could help to direct funds (derived from centralised beneﬁt-sharing mechanisms) toward capacity-building, and take on other related functions, in a similar way to the governing body for the UN Convention on the Law of the Sea (known as ‘the Authority’). The Authority is assigned various responsibilities relating to the conduct, coordination and oversight of activities—such as marine scientiﬁc research—that take place within the ‘Area’ (which is beyond national jurisdiction and to which common heritage of mankind applies). These include: • Promoting scientiﬁc research for the beneﬁt of mankind as a whole, with particular consideration to the needs and interests of developing states (Articles 140 and 143). • Promoting international cooperation in such research including capacity- building for developing states (Article 143.3). • Facilitating the exchange and dissemination of scientiﬁc knowledge, data and information, and promoting provision of scientiﬁc and technical assistance and technology transfer, so that all can beneﬁt from research in the Area (Articles 144, 202, 244.2 and 266). • Promoting effective participation in activities by developing states (Article 148 Promoting effective participation in activities by developing states (Article 148). Such capacity-building efforts are promoted extensively in the three UNESCO declarations, for example: Such capacity-building efforts are promoted extensively in the three UNESCO declarations, for example: (a) … beneﬁts resulting from the use of human genetic data, human proteomic data or biological samples collected for medical and scientiﬁc research should be shared with the society as a whole and the international community. Goals of Human Genetic Resources Governance In giving effect to this principle, beneﬁts may take any of the following forms: (i) special assistance to the persons and groups that have taken part in the research; (ii) access to medical care; (iii) provision of new diagnostics, facilities for new treatments or drugs stemming from the research; (iv) support for health services; (v) capacity-building facilities for research purposes; (v) capacity building facilities for research purposes; (vi) development and strengthening of the capacity of developing countries to collect and process human genetic data, taking into consideration their speciﬁc problems [24, Article 19]. 12 3 273 Health Care Anal (2016) 24:260–277 (a) In the framework of international cooperation with developing countries, states should seek to encourage measures enabling: … (ii) the capacity of developing countries to carry out research on human biology and genetics, taking into consideration their speciﬁc problems, to be developed and strengthened; (iii) developing countries to beneﬁt from the achievements of scientiﬁc and technological research so that their use in favour of economic and social progress can be to the beneﬁt of all; (iv) the free exchange of scientiﬁc knowledge and information in the areas of biology, genetics and medicine to be promoted [23, Article 19]. However, the statement in Article 4 of the Universal Declaration on the Human Genome and Human Rights that ‘‘the human genome in its natural state shall not give rise to ﬁnancial gains’’ [23] is problematic: ‘natural state’ is subject to varying interpretations and this is one reason for controversies over patentability of genetic resources (i.e. whether they are inventions or discoveries); and to ﬁt with the common heritage approach it will need to extend beyond this to ‘worked’ resources, associated data and products and processes emerging from research. It would need to be amended, for example, to state that ﬁnancial gains from work on the human genome, even in its natural state, are acceptable as long as they are shared internationally and result from work which is for common beneﬁt. Such an amendment reﬂects Joyner’s approach at point 3 and is consistent with the extracts from the Universal Declaration on the Human Genome and Human Rights and the International Declaration on Human Genetic Data quoted above. q Goal: effective collection and exchange mechanisms and procedures to facilitate scientiﬁc research. Goal: interoperability between collections (e.g. 12 It does this, for example, in its International Code of Conduct for Plant Germplasm Collection and Transfer (1993), and Genebank Standards for Plant Genetic Resources for Food and Agriculture (2013). Goals of Human Genetic Resources Governance in the types of data collected and how it is recorded, in software systems, and in standards relating to e.g. conﬁdentiality and anonymity). As well as beneﬁt-sharing and capacity building, the international authority would have other administrative, regulatory and oversight functions in relation to the management of human genetic resources. These could include the development of standards for collection, storage and exchange (the FAO has undertaken similar tasks for plant genetic resources12). This would, for example, boost collaboration by opening ‘‘up a wider range of samples and improve applicability to a broad range of contexts’’ in health-related research on human genetic resources, and ‘‘provide clarity on roles and responsibilities and simplify transactions’’ between genebanks and research institutions, helping ‘‘to reduce restrictions on exchange that stem from uncertainty about whether the standards required by the provider… can or will be met by the recipient’’ [17, p. 68]. The beneﬁts of facilitating international collaborations in human genetic and genomic research projects were outlined by Collins in [5]: A second lesson from the HGP [Human Genome Project] was the importance A second lesson from the HGP [Human Genome Project] was the importance of international participation. Although the complexities of organizing of international participation. Although the complexities of organizing 12 3 274 Health Care Anal (2016) 24:260–277 collaborations and conference calls across multiple time zones can present challenges, the arguments in favour of maximum worldwide participation are strong… Furthermore, the human genome is our shared inheritance; it is therefore highly appropriate and desirable to have scientists of many different languages and cultures working together on such projects. Finally, the opportunity to include participation by scientists in countries where the infrastructure for biomedical research is not yet optimally developed can provide a valuable ‘on ramp’ for such individuals to acquire technology, funding and public recognition within their own countries. The UN Convention on the Law of the Sea’s provisions on the application of common heritage of mankind contain clauses regarding the conduct of scientiﬁc research that, for example, relate to protection of the environment and sustainable management. Common heritage applied to human genetic resources could include protective measures for human participants in research and some limitations on access to data and materials so that, e.g. conﬁdentiality and informed consent can be preserved. 13 These include the Human Genome Project, the 1000 Genomes Project, and the Encyclopaedia of DNA Elements Project. Conclusion The international community has a range of options which it could select for the governance of human genetic resources. Until it does so, it appears that they will, by default, fall under free access, privileging the interests of certain groups over others. This is not appropriate for resources which have a key role in combating global disease threats. Adoption of a common heritage of mankind approach to ownership/control of human genetic resources will align well with the key practical goals for their international governance, and with principles that have been outlined by the international community in relation to the management of the human genome and human genetic data. By excluding human genetic resources from state sovereign rights, intellectual property rights and other forms of appropriation, the common heritage approach will facilitate the dissemination of scientiﬁc knowledge and data, and broad international participation in research. It will also mean that decisions on which priorities to pursue will be based on universal interests rather than national interests and commercial potential. Acknowledgments The research conducted for this paper was supported by the Wellcome Trust Strategic Programme on the Human Body, Its Scope, Limits and Future (Grant Number WT087439/Z/08/Z). Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, dis- tribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Goals of Human Genetic Resources Governance This would be achieved through standard setting functions assigned to the associated international authority and this would enable interoperability to be maintained, alongside assurance that compatible protective measures are in place. Goal: facilitation of scientiﬁc research on human genetic resources. Scientiﬁc practice—for example in large-scale, international collaborative genome projects13—indicates that releasing information into the public domain contributes signiﬁcantly to rapid developments in the ﬁeld of human genomics (see for example [5, 21]). A common heritage approach would freely and openly permit scientiﬁc research on human genetic resources, with research results being made available as soon as possible to all with a genuine interest. Research on human genetic resources would be conducted for the beneﬁt of all peoples, and its ‘fruits’ would be freely and publicly exchanged. If the sharing in the ‘fruits of such research’ involves (as it seems to) more than the sharing of results, and extends for example to access to products and processes which utilise human genetic resources, then there will also need to be centralised funding schemes that enable some form of cost-sharing. This element of a common heritage approach is also supported by provisions within the three UNESCO Declarations. The Universal Declaration on the Human Genome and Human Rights, for example, states that: ‘‘states should take appropriate measures to foster the intellectual and material conditions favourable to freedom in the conduct of research on the human genome’’; and ‘‘States should make every effort… to continue fostering the international dissemination of scientiﬁc knowl- edge concerning the human genome, human diversity and genetic research and, in that regard, to foster scientiﬁc and cultural cooperation, particularly between industrialized and developing countries’’ [23, Articles 14 and 18]. Very similar provisions appear in the International Declaration on Human Genetic Data and the Universal Declaration on Bioethics and Human Rights. 123 275 Health Care Anal (2016) 24:260–277 Goal: an internationally agreed approach to their management, to facilitate these other activities. Given these goals, it is clear that taking the step of achieving an internationally agreed approach to the management of human genetic resources is necessary in order to optimise the contribution of scientiﬁc research to human health. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, dis- tribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Acknowledgments The research conducted for this paper was supported by the Wellcome Trust Strategic Programme on the Human Body, Its Scope, Limits and Future (Grant Number WT087439/Z/08/Z). References 1. Bonfanti, A., & Trevisanut, S. (2011). TRIPs on the high seas: Intellectual property rights on marin genetic resources. Brooklyn Journal of International Law, 37(1), 188–232. 2. Broggiato, A. (2011). Marine genetic resources beyond national jurisdiction: Coordination and harmonisation of governance regimes. Environmental Policy and Law, 41(1), 35–42. 3. CBD Secretariat. (1992). Convention on biological diversity. http://www.cbd.int/doc/legal/cbd-en. pdf. Accessed 03 Dec 2014. 3. CBD Secretariat. (1992). Convention on biological diversity. http://www.cbd.int/doc/legal/cbd-en. pdf. Accessed 03 Dec 2014. 4. CBD Secretariat. (2010). Nagoya protocol on access to genetic resources and the fair and equitable sharing of the beneﬁts arising from their utilisation. http://www.cbd.int/abs/doc/protocol/nagoya- protocol-en.pdf. Accessed 03 Dec 2014. 4. CBD Secretariat. (2010). Nagoya protocol on access to genetic resources and the fair and equitable sharing of the beneﬁts arising from their utilisation. http://www.cbd.int/abs/doc/protocol/nagoya- protocol-en.pdf. Accessed 03 Dec 2014. 5. Collins, F. S. (2006). The heritage of humanity (pp. 9–12). In Nature human genome. http://www. nature.com/nature/supplements/collections/humangenome/commentaries/0009hgc.pdf. Accessed 03 Dec 2014. 5. Collins, F. S. (2006). The heritage of humanity (pp. 9–12). In Nature human genome. http://www. nature.com/nature/supplements/collections/humangenome/commentaries/0009hgc.pdf. Accessed 03 Dec 2014. 12 3 276 Health Care Anal (2016) 24:260–277 6. FAO. (1983). Annex to resolution 8/83: International undertaking on plant genetic resources. In Report of the conference of the FAO—twenty-second session. http://www.fao.org/docrep/x5563E/ x5563e0a.htm. Accessed 28 Nov 2014. 7. FAO. (1991). Resolution 3/91: Annex 3 to the international undertaking on plant genetic resources. In Report of the conference of the FAO—Twenty-sixth session. http://www.fao.org/docrep/x5587E/ x5587e06.htm. Accessed 28 Nov 2014. 8. FAO. (2007). News story—Plant gene pool becomes operational: Multilateral system boosts the exchange of plant genetic material. http://www.fao.org/Newsroom/en/news/2007/1000690/index. html. Accessed 28 Nov 2014. 9. FAO. (2004). International treaty on plant genetic resources for food and agriculture. ftp://ftp.- fao.org/docrep/fao/011/i0510e/i0510e.pdf. Accessed 28 Nov 2014. g p p 10. Finkelstein, L. S. (1995). What is global governance? Global Governance, 1(3), 363–372. 11. Fowler, C. A. (2010). Comment—Ending genetic monopolies: How the TRIPS Agreement’s failure to exclude gene patents thwarts innovation and hurts consumers worldwide. American University International Law Review, 25(5), 1073–1105. 12. Joyner, C. (1986). Legal implications of the concept of common heritage of mankind. International and Comparative Law Quarterly, 35(1), 190–199. 13. Kers, J. G., Van Burg, E., Stoop, T., & Cornel, M. C. (2014). Trends in genetic patent applications: The commercialization of academic intellectual property. European Journal of Human Genetics, 22, 1155–1159. 14. Kesselheim, A. S., Cook-Deegan, R. M., Winickoff, D. E., & Mello, M. M. References (2013). Gene patenting: The Supreme Court ﬁnally speaks. New England Journal of Medicine, 369(9), 869–875. 15. Nijar, G. S. (2011). The Nagoya protocol on access and beneﬁt-sharing of genetic resources: Analysis and implementation options for developing countries, South Centre Research Paper No. 36, http://www.southcentre.int/wp-content/uploads/2013/08/Ev_130201_GNjar1.pdf. Accessed 30 Sept 2014. 16. Noyes, J. E. (2012). The common heritage of mankind: Past, present, and future. Denver Journal International Law and Policy, 40, 447–471. 17. Rhodes, C. (2013). Governance of genetic resources: A guide to navigating the complex glob landscape. Cheltenham: Edward Elgar. 18. Rhodes, C. (2013). Sovereign wrongs: Ethics in the governance of pathogenic genetic resources. Ethics in Biology, Engineering and Medicine, An International Journal, 3(103), 97–114. 19. Rosenfeld, J., & Mason, C. E. (2013). Pervasive sequence patents cover the entire human genom Genome medicine. doi:10.1186/gm431. 20. Sand, P. H. (2004). Sovereignty bounded: Public trusteeship for common pool resources? Global Environmental Politics, 4(1), 47–71. 21. Sulston, J. (2006). Common cause (p. 14). In Nature human genome. http://www.nature.com/nature/ supplements/collections/humangenome/commentaries/0014hgc.pdf. Accessed 03 Dec 2014. 21. Sulston, J. (2006). Common cause (p. 14). In Nature human genome. http://www.nature.com/nature/ supplements/collections/humangenome/commentaries/0014hgc.pdf. Accessed 03 Dec 2014. 22. Taylor, P., & Stroud, L. (2013). Common heritage of mankind: A bibliography of legal writin Malta: Fondation de Malte. 23. UNESCO. (1997). Universal declaration on the human genome and human rights. http://unesdo unesco.org/images/0011/001102/110220e.pdf#page=47. Accessed 03 Dec 2014. 24. UNESCO. (2003). International declaration on human genetic data. http://unesdoc.unesco.or images/0013/001331/133171e.pdf#page=45. Accessed 03 Dec 2014. g p p g 25. UNESCO. (2005). Universal declaration on bioethics and human rights. http://unesdoc.unesco.org/ images/0014/001428/142825e.pdf#page=80. Accessed 03 Dec 2014. 26. United Nations Division for Ocean Affairs and the Law of the Sea. (1982). United nations convention on the law of the sea. http://www.un.org/depts/los/convention_agreements/texts/unclos/unclos_e.pdf. Accessed 03 Dec 2014. 27. United Nations General Assembly. (1972). Resolution 3016(XXVII) Permanent sovereignty over natural resources of developing countries. Accessed through www.un.org/documents/ga/res/27/ ares27.htm, 27 Nov 2014. 28. Vogel, J. H., et al. (2011). The economics of information studiously ignored in the Nagoya protocol on access to genetic resources and beneﬁt-sharing. Law, Environment and Development Journal, 7(1): 54–65. http://www.abs-initiative.info/ﬁleadmin/media/ABS_Simply_explained/Nagoya_Protocol/Vogel- etal.pdf. Accessed 30 Sept 2014. etal.pdf. Accessed 30 Sept 2014. 29. WIPO—Intergovernmental Committee on Intellectual Property and Genetic Resources, Traditional Knowledge and Folklore. (2014a). WIPO/GRTKF/IC/28/4. Consolidated document relating to 123 277 Health Care Anal (2016) 24:260–277 intellectual property and genetic resources. http://www.wipo.int/edocs/mdocs/tk/en/wipo_grtkf_ic_ 28/wipo_grtkf_ic_28_4.pdf. Accessed 28 Nov 2014. intellectual property and genetic resources. http://www.wipo.int/edocs/mdocs/tk/en/wipo_grtkf_ic 28/wipo_grtkf_ic_28_4.pdf. References Accessed 28 Nov 2014. 30. WIPO—Intergovernmental Committee on Intellectual Property and Genetic Resources, Traditional Knowledge and Folklore. (2014b). WIPO/GRTKF/IC/28/5. The protection of traditional knowledge: Draft articles. http://www.wipo.int/edocs/mdocs/tk/en/wipo_grtkf_ic_28/wipo_grtkf_ic_28_5.pdf. Accessed 28 Nov 2014. 31. World Health Organisation. (2002). Genomics and world health: Report of the advisory commissi on health research. http://www.who.int/rpc/genomics_report.pdf. Accessed 28 Nov 2014. 32. World Health Organisation. (2011). Pandemic inﬂuenza preparedness framework. http://www.wh int/inﬂuenza/resources/pip_framework/en/. Accessed 28 Nov 2014. 33. World Health Organisation. (2014). Statement on the 1st meeting of the IHR emergency committee on the 2014 Ebola outbreak in West Africa. http://www.who.int/mediacentre/news/statements/2014/ ebola-20140808/en/. Accessed 28 Nov 2014. 34. WTO. TRIPS Council. (2013). IP/C/M/71 minutes of meeting 6–7 November 2012. Available through WTO D t O li S h F ilit htt // t / li h/ / ht 34. WTO. TRIPS Council. (2013). IP/C/M/71 minutes of meeting 6–7 November 2012. Available throug WTO Documents Online Search Facility. http://www.wto.org/english/res_e/res_e.htm 35. WTO-TNC. (2008). TN/C/W/52 draft modalities for TRIPS related issues: Communication from Albania, Brazil, China, Colombia, Ecuador, the European Communities, Iceland, India, Indonesia, the Kyrgyz Republic, Liechtenstein, the Former Yugoslav Republic of Macedonia, Pakistan, Peru, Sri Lanka, Switzerland, Thailand, Turkey, the ACP Group and the Africa Group. Available through WTO Documents Online Search Facility, http://www.wto.org/english/res_e/res_e.htm 36. WTO-TNC. (2011). TN/C/W/59 draft decision to enhance mutual supportiveness between the TRIPS agreement and the convention on biological diversity: Communication from Brazil, China, Colombia, Ecuador, India, Indonesia, Peru, Thailand, the ACP Group and the Africa Group. Available through WTO Documents Online Search Facility. http://www.wto.org/english/res_e/res_e.htm 123
https://openalex.org/W4324126554	https://zenodo.org/records/7728210/files/ZDIFT%200909.pdf	Javanese	null	ISH JOYLARIDAGI HOLATLARNI ERGONOMIKA ASOSIDA O'RGANISH	Zenodo (CERN European Organization for Nuclear Research)	2,023	cc-by	1,704	1. Toshkent Davlat transport universiteti.Assistent. 1. Toshkent Davlat transport universiteti.Assistent. 2. Toshkent Davlat transport universiteti.MMX-2 guruh talabasi. 3. Toshkent Davlat transport universiteti.MMX-2 guruh talabasi. JOYLARIDAGI HOLATLARNI ERGONOMIKA ASOSIDA O’RGANISH 1. Zuhriddinov Hayotbek Qaxramonjon o’g’li 1. Zuhriddinov Hayotbek Qaxramonjon o g 2. Davrenov Shuxrat Baurjan o‘g‘li 3 Seylbekov Yerlan Niyetbay o‘g‘li 3. Seylbekov Yerlan Niyetbay o‘g‘li References: References: 1. SanPiN 2.2.4.548-96 Sanoat binolarining mikroiqlimiga gigienik talablar. 2. Sulaymanov S., Kamilov K. M., Talipov M. M. TO THE PREVENTION OF FIRES RELATED TO ACCIDENTS OF MUNICIPAL-ENERGY NETWORKS OF THE DESTROYED PART OF THE CITY //Journal of Tashkent Institute of Railway Engineers. – 2020. – Т. 16. – №. 2. – С. 158-161. 3. Sulaymanov S., Kamilov K. M. ANALYSIS OF VIDEO MONITORING OF RESULTS OF LABOR ACTIVITIES OF TRAIN DISPATCHER (AS A TRAFFIC DISPATCHER OF THE SINGLE DISPATCH CENTER OF THE JOINT-STOCK COMPANY" UZBEKISTAN TEMIR YOLLARI".) //Journal of Tashkent Institute of Railway Engineers. – 2019. – Т. 15. – №. 2. – С. 198-201. 1. SanPiN 2.2.4.548-96 Sanoat binolarining mikroiqlimiga gigienik talablar. 2. Sulaymanov S., Kamilov K. M., Talipov M. M. TO THE PREVENTION OF FIRES RELATED TO ACCIDENTS OF MUNICIPAL-ENERGY NETWORKS OF THE DESTROYED PART OF THE CITY //Journal of Tashkent Institute of Railway Engineers. – 2020. – Т. 16. – №. 2. – С. 158-161. 3. Sulaymanov S., Kamilov K. M. ANALYSIS OF VIDEO MONITORING OF RESULTS OF LABOR ACTIVITIES OF TRAIN DISPATCHER (AS A TRAFFIC DISPATCHER OF THE SINGLE DISPATCH CENTER OF THE JOINT-STOCK COMPANY" UZBEKISTAN TEMIR YOLLARI" ) //Journal of 1. SanPiN 2.2.4.548-96 Sanoat binolarining mikroiqlimiga gigienik talablar. 2. Sulaymanov S., Kamilov K. M., Talipov M. M. TO THE PREVENTION OF FIRES RELATED TO ACCIDENTS OF MUNICIPAL-ENERGY NETWORKS OF THE DESTROYED PART OF THE CITY //Journal of Tashkent Institute of Railway Engineers. – 2020. – Т. 16. – №. 2. – С. 158-161. 3 Sulaymanov S Kamilov K M ANALYSIS OF VIDEO MONITORING OF RESULTS OF LABOR 3. Sulaymanov S., Kamilov K. M. ANALYSIS OF VIDEO MONITORING OF RESULTS OF LABOR ACTIVITIES OF TRAIN DISPATCHER (AS A TRAFFIC DISPATCHER OF THE SINGLE DISPATCH CENTER OF THE JOINT-STOCK COMPANY" UZBEKISTAN TEMIR YOLLARI".) //Journal of Tashkent Institute of Railway Engineers. – 2019. – Т. 15. – №. 2. – С. 198-201. 4. Kamilov X., Zuhriddinov H. CALCULATION MODEL OF THE EFFICIENCY OF THE MEANS OF PROTECTION AGAINST THE ELECTROMAGNETIC FIELD (BY THE EXAMPLE OF A TRAIN DISPATCH WORKSTATION) //Zamonaviy dunyoda ilm-fan va texnologiya. – 2022. – Т. 1. – №. 6. – С. 183-189. 4. Kamilov X., Zuhriddinov H. CALCULATION MODEL OF THE EFFICIENCY OF THE MEANS OF PROTECTION AGAINST THE ELECTROMAGNETIC FIELD (BY THE EXAMPLE OF A TRAIN DISPATCH WORKSTATION) //Zamonaviy dunyoda ilm-fan va texnologiya. – 2022. – Т. 1. – №. 6. – С. https://doi.org/10.5281/zenodo.7728210 Annotasiya: Ushbu maqolada ofis maydonining ergonomikasi, unumdorlikka ta'sir qiluvchi asosiy omillar va ish joyini loyihalashda muhim bo'lgan jihatlar tasvirlangan. Kalit so'zlar:Ergonomika, ish joyi, talablar, ish joylari, unumdorlik, mehnat sharoi Har qanday kompaniyaning muvaffaqiyatli ishlashi bevosita xodimlarning mehnatiga bog'liq, shuning uchun kompaniya ish jarayonini qulay sharoitlarda tashkil etishga alohida e'tibor qaratishi kerak[1]. Ish jarayonida qulaylik jamoadagi do'stona muhit va ofis maydonini oqilona rejalashtirishdan iborat. Ikkinchisining asosi har bir xodimning o'zi ham, jamoada ham imkon qadar samarali ishlashi uchun ish zonalariga bo'linishdir[2,3]. Har qanday joyda, hatto kichik ofisda ham qabulxona, xodimlar xonasi, menejerning kabineti, majlislar xonasi mavjud. Ushbu zonalarning har birining ish joylarining o'lchamiga qo'yiladigan talablarga rioya qilish psixologik noqulaylikdan qochish imkonini beradi[4,5]. Qabul qilish maydoni bevosita tashrif buyuruvchilar oqimiga bog'liq, ammo standartlarga ko'ra u 10 m2 dan kam bo'lishi mumkin emas[6]. Uchrashuv zali, to'g'ridan-to'g'ri maqsadiga qo'shimcha ravishda, tovarlar yoki xizmatlarni taqdim etish va namoyish qilish uchun joy sifatida ham foydalanish mumkin, shuning uchun unda barcha taqdimot jihozlarini joylashtirish oqilona joylashtirilishi kerak[7]. Menejerning to'liq va qulay ish jarayoni uchun 12 m2 ofisga ega bo'lish kifoya. Xodimlar uchun xonalar xodimlar soniga va ularning ofis atrofidagi harakatlariga qarab ishlab chiqilishi kerak[8,9]. Ba'zan ish joylarining ovozli va vizual izolyatsiyasini ham ta'minlash kerak. Shuningdek, xodimlar tomonidan ishlatiladigan asbob-uskunalarning joylashishini hisobga olish kerak[10,11]. Uni barcha ishlaydigan marshrutlar chorrahasida o'rnatish muhim hisoblanadi. Ish joylarining maydonlariga qo'yiladigan talablar GOSTR 50923-96 tomonidan tartibga solinadi. Interyerlarning rang uslubi har doim ham katta ahamiyatga ega emas. Biroq, rang sxemasi insonning ruhiy va fiziologik holatiga katta ta'sir qiladi, shuning uchun uning ahamiyatini e'tiborsiz qoldirib bo'lmaydi[12,13]. Insonning ishlashi va sog'lig'iga ta'sir qiluvchi bir xil darajada muhim omil bu yorug'likdir. Nur insonning fiziologik va psixologik holatiga ta'sir qiladi. Ofis binolarini yoritishga qo'yiladigan talablar SP 52.13330.2011. Ofisdagi harorat va namlik kabi omillar ham xodimlarning ishlashiga ta'sir qiladi[14,15]. Tadqiqotlar shuni ko'rsatdiki, agar mikroiqlimga qo'yiladigan talablar bajarilmasa, ish tezligi pasayadi va xatolar soni ortadi. Ish xonasida mikroiqlimga qo'yiladigan talablar SanPin 2.2.4.548-96. Ish joyi - bu xodim ish vaqtining ko'p qismini (50% dan ko'prog'ini yoki doimiy ravishda ikki soatdan ko'proq) o'tkazadigan joy. Agar bir vaqtning o'zida ish joyining turli nuqtalarida ish olib borilsa, butun ish maydoni doimiy ish joyi hisoblanadi[16]. Kengroq ma'noda, bu mehnat natijalarini olishning maqsadli funktsiyasiga muvofiq individual mehnat jarayonlarini amalga 55 55 oylashtirilgan vosi ` oshirish uchun ishchi joylashtirilgan vositalar va mehnat predmeti bilan o'zaro bog'langan atrofdagi elementar joyining tarkibiy qismidir[17,18]. Ish joyi ergonomikasining asosiy tamoyillari stress va qulaylikni minimallashtirishdir. Ofis xodimlari ish vaqtining ko'p qismini o'tirgan holatda o'tkazadilar va stul yoki oddiy stulda uzoq vaqt o'tirish tanaga zarar etkazishi mumkin. Ergonomik stul qo'l suyanchiqlari, bosh suyagi bilan jihozlangan bo'lishi kerak, sozlanishi, umurtqa pog'onasidagi yukni kamaytiradigan anatomik shakllarga ega bo'lishi kerak[19,20]. Ish joyining parametrlari va o'lchamlari GOSTR ISO 7250-2008 talablariga javob berishi kerak. To'liq ish uchun har xil turdagi qo'shimchalar, stendlar kerak bo'ladi. Biroq, ular bilan stolni chalkashtirib yubormaslik kerak[21,22]. G'ildiraklardagi osilgan javonlar yoki yotoqxona stollari eng yaxshi yechimdir. U ish joyini "hamma narsa qo'lida" tamoyili bo'yicha tashkil qila oladi. Ofis mebellarini to'g'ri joylashtirish keraksiz energiya xarajatlarini bartaraf etishga imkon beradi[23,24]. Ergonomika sohasidagi tadqiqotlar shuni ko'rsatdiki, ish joyini to'g'ri rejalashtirish ish vaqtini taxminan 30% tejash va buning natijasida mehnat unumdorligini oshirish imkonini beradi. References: 183-189. 5. Ogli, Z. K. Q. (2022). MA’LUMOTLARNI OPTIK DATCHIKLAR YORDAMIDA YETKAZISH VA O ‘LCHASH TIZIMLARINI ISHLAB CHIQISH. Трансформация моделей корпоративного управления в условиях цифровой экономики, 1(1), 237-241. 6. Zuhriddinov, H. (2022). ELIMINATION OF VARIOUS HAZARDS THROUGH THE USE OF OPTICAL SENSORS IN THE ENERGY, CIVILIAN AND TRANSPORT SECTORS. Academic research in modern science, 1(9), 433-441. 7. Qaxramonjon o’g’li, Z. H. MA’LUMOTLARNI OPTIK DATCHIKLAR YORDAMIDA YETKAZISH VA O ‘LCHASH TIZIMLARINI ISHLAB CHIQISH. Iqtisodiyotni raqamlashtirish sharoitida korporativ boshqaruv modellarining transformatsiyasi xalqaro ilmiy-amaliy anjumani, 10. 8. Qaxramonjon o’g’li, Z. H. HOZIRGI ZAMONAVIY RIVOJLANAGAN DAVRDA OPTIK DATCHIKLARDAN FOYDALANIB TURLI SOHALARDAGI HAVFLARNI OLDINI OLISHNI O’RGANISH. Iqtisodiyotni raqamlashtirish sharoitida korporativ boshqaruv modellarining transformatsiyasi xalqaro ilmiy-amaliy anjumani, 10. 8. Qaxramonjon o’g’li, Z. H. HOZIRGI ZAMONAVIY RIVOJLANAGAN DAVRDA OPTIK DATCHIKLARDAN FOYDALANIB TURLI SOHALARDAGI HAVFLARNI OLDINI OLISHNI O’RGANISH. Iqtisodiyotni raqamlashtirish sharoitida korporativ boshqaruv modellarining transformatsiyasi xalqaro ilmiy-amaliy anjumani, 10. 56 9. Alimovich, M. O., & Qaxramonjon o’g’li, Z. H. QISHLOQ XO’JALIGIDA NAMLIK DATCHIKLARIDAN OQILONA FOYDALANISH USULLARI. Journal of Advanced Research and Stability. 9. Alimovich, M. O., & Qaxramonjon o’g’li, Z. H. QISHLOQ XO’JALIGIDA NAMLIK DATCHIKLARIDAN OQILONA FOYDALANISH USULLARI. Journal of Advanced Research and Stability. 10. Qaxramonjon o’g’li, Z. H. OPTIK TOLALI DATCHIKLARNING BOSHQADATCHIKLARDAN FOYDALANISHDAGI AFZALLIKLARI. ОБРАЗОВАНИЕ И НАУКА В XXI ВЕКЕ, (25). 10. Qaxramonjon o’g’li, Z. H. OPTIK TOLALI DATCHIKLARNING BOSHQADATCHIKLARDAN FOYDALANISHDAGI AFZALLIKLARI. ОБРАЗОВАНИЕ И НАУКА В XXI ВЕКЕ, (25). ( ) 11. Qaxramonjon o’g’li, Z. H. (2022). ANALYSIS OF SAFETY IN CONSTRUCTION SITES USING OPTICAL SENSORS. Web of Scientist: International Scientific Research Journal, 3(6), 131-140. 12. O’G’li, Z. H. Q. (2022). Analysis of safety in construction sites using optical sensors. 11. Qaxramonjon o’g’li, Z. H. (2022). ANALYSIS OF SAFETY IN CONSTRUCTION SITES USING OPTICAL SENSORS. Web of Scientist: International Scientific Research Journal, 3(6), 131-140. 12. O’G’li, Z. H. Q. (2022). Analysis of safety in construction sites using optical sensors. 13. Ogli, Z. K. Q. (2022). HOZIRGI ZAMONAVIY RIVOJLANAGAN DAVRDA OPTIK DATCHIKLARDAN FOYDALANIB TURLI SOHALARDAGI HAVFLARNI OLDINI OLISHNI O’RGANISH. Трансформация моделей корпоративного управления в условиях цифровой экономики, 1(1), 231-236. 13. Ogli, Z. K. Q. (2022). HOZIRGI ZAMONAVIY RIVOJLANAGAN DAVRDA OPTIK DATCHIKLARDAN FOYDALANIB TURLI SOHALARDAGI HAVFLARNI OLDINI OLISHNI O’RGANISH. Трансформация моделей корпоративного управления в условиях цифровой экономики, 1(1), 231-236. 14. Xakimovich, A. S., & Qaxramonjon o’g’li, Z. H. (2022). Prediction of Situations That May Occur in Emergency Situations of Bridges by Means of Optical Sensors. Texas Journal of Engineering and Technology, 13, 55-59. 15. Qaxramonjon o’g’li, Z. References: H., & Xakimovich, A. S. Prediction of Situations That May Occur in Emergency Situations of Bridges by Means of Optical Sensors. 55-59 page. 16. Xakimovich, A. S., & Qaxramonjon o’g’li, Z. H. (2022). Analyzing the Results of Monitoring the Situations that May Occur in Emergency Situations of Bridges Through Various Optical Sensors. Global Scientific Review, 8, 80-88. 17. Abdazimov, S. X., & Zuhriddinov, H. (2022). CONTINUOUS MONITORING SYSTEM ON BRIDGES TO PREVENT EMERGENCIES. Journal of Integrated Education and Research, 1(6), 95- 99. 18. Abdazimov, S. X., & Zuhriddinov, H. (2022). REVIEW THE BRIDGE MONITORING SYSTEM ON A REGULAR BASIS TO PREVENT EMERGENCY SITUATIONS. Journal of Integrated Education and Research, 1(6), 90-94. 19. Musayev, S. G., & Zuhriddinov, H. (2022). BINOLARDA KELIB CHIQISHI MUMKIN BO’LGAN FAVQULODDAGI VAZIYATLARDA YONG’IN HODISALARINI OPTIK HARORAT DATCHIKI ORQALI ANIQLASH. Journal of Integrated Education and Research, 1(6), 85-89. 20. Kamilov, X., & Zuhriddinov, H. (2022). CALCULATION MODEL OF THE EFFICIENCY OF THE MEANS OF PROTECTION AGAINST THE ELECTROMAGNETIC FIELD (BY THE EXAMPLE OF A TRAIN DISPATCH WORKSTATION). Zamonaviy dunyoda ilm-fan va texnologiya, 1(6), 183- 20. Kamilov, X., & Zuhriddinov, H. (2022). CALCULATION MODEL OF THE EFFICIENCY OF THE MEANS OF PROTECTION AGAINST THE ELECTROMAGNETIC FIELD (BY THE EXAMPLE 20. Kamilov, X., & Zuhriddinov, H. (2022). CALCULATION MODEL OF THE EFFICIENCY OF THE MEANS OF PROTECTION AGAINST THE ELECTROMAGNETIC FIELD (BY THE EXAMPLE OF A TRAIN DISPATCH WORKSTATION). Zamonaviy dunyoda ilm-fan va texnologiya, 1(6), 183- 189. OF A TRAIN DISPATCH WORKSTATION). Zamonaviy dunyoda ilm-fan va texnologiya, 1(6), 183- 189. 21. Abdazimov, S., & Zuhriddinov, H. (2022). MONITORING USING FIBER BRAGG GRID SENSORS IN EMERGENCY PREVENTION OF BRIDGES. Eurasian Journal of Academic Research, 2(11), 1066-1075. 22. угли Зухриддинов, Х. Қ., & Амиров, М. У. (2022). АНАЛИЗ СИСТЕМ ИЗМЕРЕНИЯ ДАННЫХ С ПОМОЩЬЮ ВОЛОКОННО-ОПТИЧЕСКИХ ДАТЧИКОВ. INNOVATIVE DEVELOPMENT IN THE GLOBAL SCIENCE, 1(6), 150- 158. 23. Gulamovich, M. S., & O’G’Li, Z. H. Q. (2022). PEDAGOG XODIMLARDAGI ERGONOMIK BILIM VA KO’NIKMALARINI ZAMONONAVIY OLIY TA’LIM MUASSASALARIDAGI HOLATINI O’RGANISH. Ta’lim fidoyilari, 28, 21-29. 57
https://openalex.org/W3045555739	https://hal.archives-ouvertes.fr/hal-02943927/document	English	null	To-Do and Not-To-Do in Model Studies of the Uptake, Fate and Metabolism of Metal-Containing Nanoparticles in Plants	Nanomaterials	2,020	cc-by	9,919	To cite this version: Justyna Wojcieszek, Javier Jiménez-Lamana, Lena Ruzik, Joanna Szpunar, Maciej Jarosz. To-Do and Not-To-Do in Model Studies of the Uptake, Fate and Metabolism of Metal-Containing Nanoparticles in Plants. Nanomaterials, 2020, 10 (8), pp.1480. 10.3390/nano10081480. hal-02943927 To-Do and Not-To-Do in Model Studies of the Uptake, Fate and Metabolism of Metal-Containing Nanoparticles in Plants Justyna Wojcieszek, Javier Jiménez-Lamana, Lena Ruzik, Joanna Szpunar, Maciej Jarosz To cite this version: Justyna Wojcieszek, Javier Jiménez-Lamana, Lena Ruzik, Joanna Szpunar, Maciej Jarosz. To-Do and Not-To-Do in Model Studies of the Uptake, Fate and Metabolism of Metal-Containing Nanoparticles in Plants. Nanomaterials, 2020, 10 (8), pp.1480. 10.3390/nano10081480. hal-02943927 Received: 6 July 2020; Accepted: 23 July 2020; Published: 28 July 2020 Abstract: Due to the increasing release of metal-containing nanoparticles into the environment, the investigation of their interactions with plants has become a hot topic for many research ﬁelds. However, the obtention of reliable data requires a careful design of experimental model studies. The behavior of nanoparticles has to be comprehensively investigated; their stability in growth media, bioaccumulation and characterization of their physicochemical forms taken-up by plants, identiﬁcation of the species created following their dissolution/oxidation, and ﬁnally, their localization within plant tissues. On the basis of their strong expertise, the authors present guidelines for studies of interactions between metal-containing nanoparticles and plants. Keywords: metal-containing nanoparticles; model plants; nano-bio interactions; transformations; physico-chemical characterization; mass spectrometry HAL Id: hal-02943927 https://hal.science/hal-02943927v1 Submitted on 21 Sep 2020 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. nanomaterials nanomaterials Justyna Wojcieszek 1 , Javier Jiménez-Lamana 2,* , Lena Ruzik 1, Joanna Szpunar 2 and Maciej Jarosz 1 1 Chair of Analytical Chemistry, Faculty of Chemistry, Warsaw University of Technology, 3 Noakowskiego str., 00-664 Warsaw, Poland; jwojcieszek@ch.pw.edu.pl (J.W.); lenka@ch.pw.edu.pl (L.R.); mj@ch.pw.edu.pl (M.J.) 1 Chair of Analytical Chemistry, Faculty of Chemistry, Warsaw University of Technology, 3 Noakowskiego str., 00-664 Warsaw, Poland; jwojcieszek@ch.pw.edu.pl (J.W.); lenka@ch.pw.edu.pl (L.R.); mj@ch.pw.edu.pl (M.J.) 2 Universite de Pau et des Pays de l’Adour, E2S UPPA, CNRS, Institute of Analytical and Physical Chemistry for the Environment and Materials (IPREM), UMR 5254, 64053 Pau, France; joanna.szpunar@univ-pau.fr * Correspondence: j.jimenez-lamana@univ-pau.fr; Tel.: +33-540175090 1 Chair of Analytical Chemistry, Faculty of Chemistry, Warsaw University of Technology, 3 Noakowskiego str., 00-664 Warsaw, Poland; jwojcieszek@ch.pw.edu.pl (J.W.); lenka@ch.pw.edu.pl (L.R.); mj@ch.pw.edu.pl (M.J.) 2 Universite de Pau et des Pays de l’Adour, E2S UPPA, CNRS, Institute of Analytical and Physical Chemistry for the Environment and Materials (IPREM), UMR 5254, 64053 Pau, France; joanna.szpunar@univ-pau.fr * Correspondence: j.jimenez-lamana@univ-pau.fr; Tel.: +33-540175090 00 664 Warsaw, Poland; jwojcieszek@ch.pw.edu.pl (J.W.); lenka@ch.pw.edu.pl (L.R.); mj@ch.pw.edu.pl (M.J.) 2 Universite de Pau et des Pays de l’Adour, E2S UPPA, CNRS, Institute of Analytical and Physical Chemistry for the Environment and Materials (IPREM), UMR 5254, 64053 Pau, France; joanna.szpunar@univ-pau.fr * Correspondence: j.jimenez-lamana@univ-pau.fr; Tel.: +33-540175090 j * Correspondence: j.jimenez-lamana@univ-pau.fr; Tel.: +33-540175090 To-Do and Not-To-Do in Model Studies of the Uptake, Fate and Metabolism of Metal-Containing Nanoparticles in Plants Justyna Wojcieszek 1 , Javier Jiménez-Lamana 2,* , Lena Ruzik 1, Joanna Szpunar 2 and Maciej Jarosz 1 ustyna Wojcieszek 1 , Javier Jiménez-Lamana 2,* , Lena Ruzik 1, Joanna Szpunar 2 and 1. Introduction On the other hand, metal-containing NPs may promote the plant growth and seed germination. For example, exposure of tomato to strong irradiance and TiO2 NPs resulted in better ﬂower and fruit production, increased anthocyanin and carotenoids concentration and high enzyme activity although rapid chlorophyll content decrease was also observed [11]. The uptake and translocation of Fe3O4 NPs in Hordeum vulgare L. plants resulted in promoted gene expression and increase of some phenological parameters such as chlorophyll, total soluble protein and number of chloroplasts [9], although in Eichhornia crassipes plants a distinct decrease in chlorophyll content and catalase activity and an increase of malondialdehyde (MDA) content was observed after Fe3O4 NPs treatment at higher concentrations [19]. In this context, the comprehensive investigation of the behavior of metal-containing NPs throughout the whole process of interaction with plants—uptake, bioaccumulation, and translocation—is needed. However, such a challenge can only be accomplished through a careful design of experiments, where several factors must be taken into account, as well as through the use of several techniques that provide complementary information. The use of each technique will depend on the specific behavior of each metal-containing NPs (i.e., if they remain intact or they undergo dissolution and/or agglomeration) and thus on their chemical and physical nature. A number of analytical techniques is being currently used for the analysis and characterization of metal-containing NPs, such as transmission electron microscopy (TEM), scanning electron microscopy (SEM), atomic force microscopy (AFM), X-ray diﬀraction (XRD), energy dispersive X-ray spectroscopy (EDX), microbeam X-Ray Fluorescence (µ-XRF), microbeam X-ray absorption spectroscopy (µ-XAS) [3,20]. Microscopy based techniques are a commonly accepted characterization tool, TEM being the most widely used technique among them [4]. These techniques can provide information about particle size, shape, and agglomeration of metal-containing NPs accumulated by plants at the cellular and subcellular level [20]. However, the obtention of reliable data depends on statistical tools and a time consuming sample preparation based on a drying process, which in addition can lead to aggregation of NPs, especially in environmental samples [21]. On the other hand, synchrotron radiation (SR) based techniques have great potential to investigate localization and speciation of metal-containing NPs in plants. 1. Introduction The extensive use of metal-containing nanoparticles (NPs) in an increasing number of applications is leading to their release into the environment, where they can interact with plants with unknown eﬀects [1]. This interaction may lead to some impact on plant physiological processes and eventually to the bioaccumulation of NPs, and products of their metabolism, in the animal and human food chain. In the last years, the number of model studies focused on the investigation of interactions between plant and engineered nanoparticles, especially metal-containing ones, has increased [2–4]. For example, the bioaccumulation of silver nanoparticles (AgNPs) and aluminum oxide nanoparticles (Al2O3 NPs) in roots of Lactuca sativa L. followed by their translocation to shoots has been demonstrated [5,6]. A similar behavior has been observed during the study of the uptake and translocation of lead sulﬁde nanoparticles (PbS NPs), iron (III) oxide nanoparticles (Fe2O3 NPs) and magnetite nanoparticles (Fe3O4 NPs) in Zea mays L., Triticum aestivum L. and Hordeum vulgare L., respectively [7–9]. Contradictory results have also been observed for the same type of metal-containing nanoparticles in diﬀerent plants: titanium oxide (TiO2 NPs) were not taken up by Coriandrum sativum L. [10], although their accumulation and translocation in tomato [11] or radish plants [12] has been reported. On the other hand, NPs may undergo diﬀerent transformations after their accumulation in roots, followed by their translocation to above-ground organs as it has been observed for selenium nanoparticles (Se NPs) and AgNPs in garlic and Arabidopsis plants, respectively [13,14]. Finally, it is worth mentioning the accumulation of AgNPs in stems of three diﬀerent tree species was faster after foliar treatment compared to roots treatment [15]. www.mdpi.com/journal/nanomaterials Nanomaterials 2020, 10, 1480; doi:10.3390/nano10081480 www.mdpi.com/journal/nanomaterials 2 of 16 Nanomaterials 2020, 10, 1480 After uptake and accumulation, metal-containing NPs can interact with plants at the cellular and subcellular levels, facilitating changes to morphological and physiological states, which may be suppressive or stimulatory [2]. For instance, Ag NPs caused oxidative stress and exhibited toxicity when applied in higher concentrations to Allium cepa roots, regardless of surface coating used [16]. An increase of peroxidase, catalase, superoxide, dismutase activity, and inhibition in plant growth has been detected in Lemna minor after copper oxide nanoparticles (CuO NPs) treatment [17]. It has also been reported that the glutathione content and antioxidant power decreased signiﬁcantly after Trigonella foenum cultivation with Al2O3 NPs [18]. 1. Introduction The combination of high-resolution synchrotron x-ray ﬂuorescence microscopy (SR-XFM), oﬀering multi-elemental detection down to the tens of nm, and spatially resolved XAS is a powerful technique that can provide information about elemental composition, localization and chemical speciation with minimal sample preparation and non-destructive analysis [22]. For instance, it has been successfully applied to the study TiO2 NPs [23,24], AgNPs [23,25], zinc oxide nanoparticles (ZnO NPs) [26,27] or cerium oxide nanoparticles (CeO2 NPs) [27] in diﬀerent plants. The application of these techniques to the study of interactions between NPs and plants has been recently reviewed by Castillo-Michel et al. [22]. However, the majority of these techniques are inadequate for the characterization of NPs in complex matrices at low concentrations. In this context, the use of analytical methods based on the high-sensitive and element-speciﬁc technique of inductively coupled plasma mass spectrometry (ICP-MS) allows NPs detection at environmentally relevant concentrations. Indeed, an analytical tool that has become popular in these kinds of studies is Single Particle (SP) ICP-MS, thanks to a combination of the beneﬁts of ICP-MS, with those of a particle counting technique [28]. SP-ICP-MS can provide information about the particle size, particle size distribution as well as quantitative information about the metal in its dissolved and nanoparticulate form in a single analysis. Typically, the analysis of 3 of 16 Nanomaterials 2020, 10, 1480 a NP solution by SP-ICP-MS produces a time scan with two diﬀerent types of signal: a steady signal at low counts due to the background or the presence of the analyte in its dissolve form; and a number of pulses above the background due to the presence of the analyte in its nanoparticulated form [29]. Authors present here a step-by-step guideline in order to obtain reliable data in metal-containing NPs-plant interaction studies, including: the study of the stability of NPs in nutrient solutions used for plant cultivation; the optimization of a procedure able to extract NPs from the plant matrix without altering their properties; the analysis of NPs in plant tissues by monitoring their possible transformations; the identiﬁcation of new metal species created within the plant as a result of dissolution/oxidation processes; the study of the spatial distribution of NPs in plant tissues. A ﬂowchart with the necessary steps in these kinds of studies is shown in Figure 1. 2. Transformation of Metal-Containing Nanoparticles in Growth Media Model studies carried out in order to get deeper insight about metal-containing NPs interactions with plants include several steps. The ﬁrst one involves the growth of plants in the presence of metal/metal oxide NPs in a soil environment or in a hydroponic solution, being the latter the most used in literature. Diﬀerent media can be used for plant cultivation, for example, Murashige and Scoog, Hoagland or Knop nutrient solution [30,31]. The composition of the nutrient solution may have a negative impact on the interpretation of the results and may need to be adapted or modiﬁed. On the one hand, and due to the high sensitivity of the analytical techniques used in this kind of study, the presence of the metal of interest even at low concentration in the medium may lead to the occurrence of false positives when it comes to the analysis of plant tissues. For instance, salts usually present in growth media, like ZnSO4, CuSO4 or FeSO4, should be removed during the analysis of ZnO, CuO or Fe2O3 NPs, respectively. A possible alternative could be the use of isotopically labeled metal NPs, which would allow the discrimination from diﬀerent metal sources. Isotopic labeling of NPs enables their sensitive tracing in the presence of background elements in complex plant matrices [32]. On the other hand, special attention has to be paid if metal compounds with complexing agents such as ethylenediaminetetraacetic acid (EDTA), iminodiacetic acid (IMDA) or diethylenetriaminepentaacetic acid (DTPA) are added to a growth solution. For example, the presence of EDTA, a very strong ligand, can bind metal ions potentially released from the analyzed NPs and shift the equilibrium in the system, leading hence to biased results. In addition, the identiﬁcation of metal-EDTA complexes created as a result of metal releasing from NPs will not be useful information as this kind of compound cannot be formed inside plant tissues. In this context, Fe-EDTA present in growth media like Knop nutrient solution must be replaced by another salt, like FeSO4 [33]. Metal-containing NPs are reactive species that can interact with the surrounding environment. In this context, the stability of NPs in the growth medium used for plant cultivation needs to be investigated before the cultivation and analysis of plants. These studies are usually performed by spiking the nutrient solution with a suspension of metal-containing NPs followed by its analysis immediately upon addition of the NPs suspension and over time. 1. Introduction Authors use their extensive experience with the investigation of NPs of diﬀerent chemical nature and properties to explain the diﬀerent scenarios stressing some critical points where special attention needs to be paid. It must be stated that the guidelines described here apply to the experimental scheme shown in Figure 1, through the use of diﬀerent analytical techniques based on mass spectrometry, which has proven to give comprehensive information. However, it must be highlighted that, besides the general rules draw in this manuscript, each type of metal-containing NP has speciﬁc properties (size, shape, type of metal, coating, etc.) and will require an individual study. 4 of 16 Nanomaterials 2020, 10, 1480 Figure 1. Flowchart presenting possible scenarios and steps to be carried out during studies of nanoparticles (NPs)–plant interactions. Figure 1. Flowchart presenting possible scenarios and steps to be carried out during studies of nanoparticles (NPs)–plant interactions. Figure 1. Flowchart presenting possible scenarios and steps to be carried out during studies of nanoparticles (NPs)–plant interactions. Nanomaterials 2020, 10, 1480 5 of 16 2. Transformation of Metal-Containing Nanoparticles in Growth Media The last analysis should be performed at the endpoint time of the subsequent plant cultivation. This previous study is critical in model studies of NPs-plants interaction in order to elucidate if the possible transformations of NPs take place before or after their uptake by plants. In general, there are three possible scenarios according to the transformations that NPs can undergo: (i) NPs remain unchanged in medium; (ii) NPs are partially or totally dissolved; and (iii) NPs agglomerate over time. The occurrence (or not) and extent of these transformations will depend on the nature of the NPs used and requires an individual and speciﬁc study. For this purpose, SP-ICP-MS is a technique that can provide information about NP size distribution and the physico-chemical form of the metal of interest in a single analysis, which makes it an ideal tool to monitor possible NP transformation in growth medium. Examples of application of SP-ICP-MS to the characterization of metal-containing nanoparticles in hydroponic solution under the three mentioned scenarios are described below. The simplest situation that may happen during the study of the stability of NPs in a nutrient solution is that they are stable during the whole cultivation period. In that case, the obtained size distribution must be in good agreement with that obtained for the stock suspension [34]. Additionally, in SP-ICP-MS, the absence of a high background signal (corresponding to the metal in its dissolved from) and pulses of high intensity signal (corresponding to NPs of bigger sizes) will prove that NPs do not undergo any transformation such as dissolution or agglomeration in nutrient solution used for plant cultivation. As a consequence, any NPs transformation detected during the analysis of tissues of treated plants, must be attributed to processes taking place inside plant tissues, after the uptake and accumulation of NPs. However, growth medium can cause the dissolution of metal-containing NPs. It can happen especially in the case of microelements that are present in high abundance in natural environments, 6 of 16 Nanomaterials 2020, 10, 1480 such as zinc or copper, usually present in diﬀerent forms such as free ions, compounds with diﬀerent bioligands or as a component of rocks. This phenomenon can be easily identiﬁed thanks to the use of SP-ICP-MS. 2. Transformation of Metal-Containing Nanoparticles in Growth Media In this case, on the time scans obtained during SP-ICP-MS analysis of growth medium, a steady signal, i.e., characteristic of the dissolved form of the metal, is registered, whereas a signiﬁcant number of pulses proving the presence of NPs is observed after analysis of fresh NPs stock suspension. If only a steady signal is observed after analysis of growth medium, it leads to the conclusion that plants are mainly taking up metal in its dissolved form and therefore the accumulation of NPs is negligible. In the case of the presence of only the dissolved form of metal, speciation studies leading to the quantiﬁcation and identiﬁcation of metal complexes formed inside the plant tissues must be carried out, as it is explained in detail in Section 6 of this manuscript. The dissolution of NPs is time dependent and can be partial, with both dissolved and particulate form of metal present in the growth medium. In this case, if both forms of metal are taken up by plants, both NP characterization by SP-ICP-MS as well as speciation studies by the use of hyphenated techniques should be performed. If only metal ions are taken up by plants, then characterization of NPs is not possible and the direction of the study has to be changed. The dissolution rate of NPs strongly depends on media composition as well as on the surface coating of NPs. It has been shown in diﬀerent studies that bare nanoparticles are more susceptible to transformation than coated NPs [35,36]. In the last scenario, NPs can undergo agglomeration as it was observed for some nanoparticles based on metal oxides [12,37]. In this case, the extent of the agglomeration must be determined since it may have an inﬂuence on the potential uptake by the plant. For instance, it can happen that the median diameter of NPs shifts slightly toward bigger sizes after diﬀerent times of incubation or that NP size duplicate or triplicate, which leads to the conclusion that NPs can undergo agglomeration in a smaller or bigger degree after diﬀerent contact time with growth medium. In any case, the critical point is to determine whether agglomerates of NPs created in a nutrient solution are taken up by plant tissues. Typically, only NPs at smaller sizes than those observed in the growth medium are accumulated in plant leaves and roots [12,37,38]. 4. Extraction of Intact Nanoparticles Although analysis of total metal content provides a general idea of the metal bioaccumulation by plants, when it comes to studies with metal-containing NPs, important information like the physico-chemical form, the size distribution or the nanoparticle number concentration is lost after acid digestion. The correct interpretation of the data strongly depends on the extraction of NPs from the plant matrix preserving their native conditions. Therefore, the ﬁrst important step in the analytical procedure is to develop and optimize an eﬃcient extraction process of NPs from plant material. Plant tissue matrix is generally made of some or all of the following components: macro and micronutrients, vitamins, amino acids or other nitrogen supplements. In addition, the plant cell wall is composed primarily of polysaccharides, cellulose being its major component, and it is organized into paracrystalline structures inserted in a rich matrix of diverse polysaccharides, including hemicelluloses and pectins, structural glycoproteins and lignin in certain tissues [44]. NPs can penetrate through the cell wall, so the digestion of polysaccharides is needed (for example, pectin consists of four major polysaccharide domains: homogalacturonan (HGA), rhamnogalacturonan I (RGI), rhamnogalacturonan II (RGII) and xylogalacturonan (XGA)) with the preservation of the NPs at the same time. This can be done through the use of alkaline solutions or enzymes, for example, pectinase, hemicellulase, and cellulase, which are perfectly suited for breaking down the polysaccharides found in plant cell walls. Alkaline treatments with tetramethylammonium hydroxide (TMAH) have shown a high-eﬃciency extraction of diﬀerent NPs (Ag, Au, or carbon nanotubes) from tissues with a low amount of salts remaining in solution after digestion [45]. It should be mentioned that an alkaline treatment followed by SP-ICP-MS analysis could not be used for extracting AgNPs from tissue samples. This is important information regarding the changes in the state of AgNPs, most probably due to Ag+ precipitation and/or AgNPs aggregation [46]. Enzymatic digestion, which commonly works with mild conditions, i.e., at moderate temperatures and pH conditions, can be an appealing sample pre-treatment for isolating NPs without their degradation. The use of a multi-component enzyme mixture containing cellulase, hemicellulase, and pectinase (Macerozyme R-10) was proposed by Dan et al. for isolating gold nanoparticles (AuNPs) from roots of tomato plants [47]. In addition, a suitable extraction method should not only extract intact NPs but also a representative amount of them. For this purpose, the mentioned enzymatic digestion method was further developed by Jimenez-Lamana et al. [34]. 3. Sample Preparation and Total Content Determination After an investigation of NPs stability in nutrient solution, the cultivation of plants in the presence of NPs and control plants is the next step to be carried out. Afterward, plants are divided into diﬀerent organs/tissues and subsequently grounded. The use of a pestle and mortar is highly recommended. The translocation factor from roots to above-ground organs can be easily calculated at this stage, by determining the total content of metal within the diﬀerent plant organs. For this purpose, mineralization processes with oxidizing acids and heating systems or microwave-assisted techniques are commonly used for acid digestion of organic matrices [43], followed by quantitative analysis by standalone ICP-MS or inductively coupled plasma optical emission spectrometry (ICP-OES). The choice of the acid(s) used for the digestion of plant tissues will depend on the nature of the metal the NPs are made of. Some metal-containing nanoparticles can dissolve under acidic conditions, mainly by the use of concentrated nitric acid, but others will require diﬀerent or additional reagents: aqua regia for platinum nanoparticles (PtNPs) and palladium nanoparticles (PdNPs) [34,38]; hydrogen peroxide for ZnO NPs and CeO2 NPs [33,37]; or hydroﬂuoric acid for TiO2 NPs. 2. Transformation of Metal-Containing Nanoparticles in Growth Media The occurrence of agglomeration processes can be avoided by the use of coated-NPs and/or by the addition of some additional reagents such as organic acids or enzymes to the solution used for plant cultivation. The former option is clearly advised, since the latter may be problematic from the point of view of mimicking natural conditions and should be avoided as much as possible. As it has been described on the three scenarios, is it important to monitor the possible transformation processes of NPs in growth medium used during plant cultivation. The use of SP-ICP-MS can provide clear information on whether NPs are stable or if they undergo different transformations such as dissolution or agglomeration in a single analysis. Finally, it should be mentioned that the studies of the interaction between metal-containing NPs and plants can also be performed in solid media, in order to mimic natural conditions, where NPs are released into soil environments. Therefore, the stability of metal-containing NPs in a soil environment must be investigated, not only to investigate the uptake by plants but also to understand the terrestrial toxicity of NPs [39]. However, these studies are less convenient than those performed in a liquid medium, since they imply an additional sample preparation step: the extraction of NPs from the solid medium without changing their physico-chemical form. In this context, extractions with puriﬁed water and with tetrasodium pyrophosphate (TSPP), with sonication to enhance particle dispersion, followed by analysis by SP-ICP-MS have been proposed [40,41]. The type of media (liquid or solid) used for plant cultivation may have a signiﬁcant inﬂuence on NPs properties. For example, the bioavailability and the eﬀect of the silver ions released by AgNPs have been shown to be lower in a soil medium compared with an agar medium [42]. In any case, regardless of the medium used for plant cultivation, the fate and possible transformations of NPs must be monitored. Nanomaterials 2020, 10, 1480 7 of 16 7 of 16 4. Extraction of Intact Nanoparticles As it was demonstrated in this study, diﬀerent parameters (type of buﬀer used, amount of sample, amount of enzyme, sonication power, sonication time, incubation time) need to be studied and optimized to obtain the highest number of NPs from the diﬀerent plant tissues. A typical enzymatic digestion procedure for the extraction of metal-containing NPs from plant tissues is shown in Figure 2. It is important to highlight the use of well-grounded samples in order to 8 of 16 Nanomaterials 2020, 10, 1480 provide the maximum physical contact between sample and reagents. Ground samples are mixed with citrate buﬀer and the mixture is next homogenized using an ultrasonic probe while the tube is kept in ice. Milder procedures, like bath sonication or shaking, should be avoided, since they do not provide a successful extraction of NPs from plant tissues. On the other hand, nominal powers of a probe higher than 35% are not recommended, to avoid excessive heating of the sample. After the end of homogenization, the enzyme solution is added. The samples are shaken in a water bath at 37 ◦C for 24 h. After the incubation, the obtained suspensions are ﬁltered with a 0.45 µm syringe ﬁlter because of the presence of a remaining solid after homogenization and incubation. Figure 2. Steps to be performed in a typical enzymatic digestion procedure. Figure 2. Steps to be performed in a typical enzymatic digestion procedure. Important precautions must be taken depending on the nature of the metal-containing NPs object of the study. For instance, the sonication probe may leach signiﬁcant amounts of titanium into the suspension which, even at trace levels, may lead to the contamination of the plant samples and hence the occurrence of false positives in studies of TiO2 NPs interactions with plants [12]. To avoid that, a tissue grinder set can be used instead. The eﬀect that the digestion procedure may have on NPs (dissolution and/or aggregation) must be investigated. To do so, the same procedure must be performed on a suspension of the metal-containing NPs followed by its analysis. As indicated above, the characterization and monitoring of metal-containing NPs and the processes they may undergo can be easily carried out by SP-ICP-MS. In one single analysis, SP-ICP-MS can provide the necessary information to decide if the chosen digestion protocol is suitable for extracting NPs from the plant tissues without altering their properties. 4. Extraction of Intact Nanoparticles Finally, the inﬂuence of the plant matrix can be additionally investigated by submitting control plant tissues (i.e., cultivated in the absence of NPs) spiked with a suspension of metal-containing NPs to the digestion procedure, obtaining the corresponding size distributions by SP-ICP-MS and comparing with the original NP size distribution. It is important to mention that the use of a ﬁltration step after the digestion procedure could imply the loss of bigger NPs [12], leading to unreliable results. In that case, ﬁltration should be discarded and it is advisable to let the suspensions settle down after digestion for at least one hour and take the supernatants to analyze. The use of centrifugation at this step is not recommended since NPs will also settle down to the bottom of the suspension. 5. Uptake, Translocation and Biotransformation Once the stability of the metal-containing NPs suspension in the growth media and the suitability of the digestion procedure has been investigated, the plant cultivation in the presence of NPs must be carried out. During this process, a complementary evaluation by plant scientists should be carried out, i.e., the tolerance of the plant to the NP concentration in terms of phytotoxic eﬀects. Diﬀerent factors, such as color of plant tissues, biomass production or tissue hydration can be determined and compared with those obtained for control plant samples. If possible, detailed tests for eventual phytotoxicity on the cellular level should be undertaken. 9 of 16 Nanomaterials 2020, 10, 1480 As it was mentioned above, the total content of metal taken up by the plant does not provide information about the form of element accumulated in plant tissues or about possible transformations of NPs that can happen during uptake and transport. In this context, and in order to investigate the physico-chemical form of metal accumulated inside plant tissues, SP-ICP-MS can be a valuable tool. It is important to mention that in SP-ICP-MS, the dilution of the sample plays an important role, in order to be able to detect signals produced by individual NPs. Typically, samples with NP concentration around 1 × 108 NP L-1 are analyzed in SP-ICP-MS. This is especially important in the case of roots, where higher NPs concentrations are expected. Diﬀerent scenarios can be considered during SP-ICP-MS analysis, depending on several factors such as the chemical nature of the metal-containing NPs, type of plant or conditions of cultivation. Similarly to the investigation of metal-containing NPs stability in the growth media, unchanged NPs can be taken up and accumulated in plants, i.e., no transformations such as agglomeration or dissolution take place. This can be easily observed by a simple comparison of the size distributions obtained in plant tissues with the one obtained for a fresh suspension of the metal-containing NPs. The presence of NPs in both roots and leaves presenting the same size distribution and nominal diameter as the stock NPs suspension means that analyzed plant has an ability not only to uptake and accumulate NPs in roots but also to translocate them to above ground organs. A diﬀerent situation can be found when metal-containing NPs undergo agglomeration inside plant tissues [34]. This phenomenon can be clearly seen when observing the size distribution obtained by SP-ICP-MS. 5. Uptake, Translocation and Biotransformation If the agglomeration process occurs, two populations are observed on the size distribution: a main one at sizes close to the nominal diameter, corresponding hence to unchanged NPs; and a second population at larger sizes not observed during the analysis of stock suspension. In order to elucidate at which step of the plant cultivation the metal-containing NPs undergo agglomeration, the particle size distribution obtained in the roots must be compared with the one obtained previously in the stability study in growth media. If no agglomeration is observed in the nutrient solution spiked with NPs, the presence of second distribution at higher sizes will imply that NPs undergo agglomeration during their uptake. On the other hand, agglomeration can already occur in a nutrient solution used for plant cultivation. Again, a comparison between NP size distributions obtained in plant tissues and those obtained in the growth medium spiked by NPs over time must be performed. The key question here is whether agglomerates eventually created in a nutrient solution are taken up by plant tissues. For example, only smaller NPs may be taken up by plant roots, followed by the transport of intact NPs to stems and leaves [12], suggesting that agglomerates are not taken up by plant tissues. This can be easily recognized by a simple comparison of size distributions, since if only smaller NPs are taken up by plants, size distributions obtained in roots after plant cultivation will present particles with lower diameters in comparison with analysis of NPs suspension in growth medium. However, it can also happen, despite the fact that only smaller NPs are taken up by roots, that bigger NPs are found in the analysis of leaves and stems [37]. In this case, the results suggest that NPs can also undergo agglomeration at the endpoint of their transportation, where nanoparticles are more locally concentrated and hence agglomeration by contact is more likely to occur. The uptake of metal ions followed by re-precipitation can also be considered as a pathway of NPs accumulation in plants, which was already suggested during analysis of CeO2 NPs in three plant species by scanning transmission electron microscopy (STEM) [48]. Finally, the dissolution of NPs can also be observed after SP-ICP-MS analysis, which resulted in a steady signal on time scans, without pulses characteristic for particulate form of metal. Similarly to the case of agglomeration, dissolution can appear at diﬀerent stages of the experiment. 5. Uptake, Translocation and Biotransformation For instance, partial dissolution of metal-containing NPs may occur inside plant tissues after their uptake as intact NPs [38], which brings the opportunity to study both NPs characterization by SP-ICP-MS together with metal speciation in plant tissues by the use of separation techniques coupled to mass spectrometry detection [38]. 10 of 16 Nanomaterials 2020, 10, 1480 Figure 3 reﬂects how a simple comparison of size distributions obtained by SP-ICP-MS of metal-containing NPs in growth medium, roots and leaves can provide useful information. Example 1 shows the case of NPs that agglomerate in growth medium, only smaller ones are taken up by the plant and a re-agglomeration process occurs during transport to leaves. In example 2, agglomerates are not taken up from medium either but NPs remain intact during transport. Finally, in example 3, NPs remain unchanged in growth medium but they undergo agglomeration during uptake and/or transport. Figure 3. Comparison of size distributions obtained for metal-containing NPs in growth medium, roots and leaves in 3 diﬀerent scenarios. Figure 3. Comparison of size distributions obtained for metal-containing NPs in growth medium, roots and leaves in 3 diﬀerent scenarios. 6. Speciation Studies As it has been mentioned, metal-containing NPs may undergo partial or total dissolution in the nutrient solution, leading to the uptake of metal ions by roots [33]. The analysis of the total metal content in plant organs can provide preliminary conclusions about the physico-chemical form of NPs, as the translocation factor of analyzed metal from roots to leaves is usually signiﬁcantly higher if dissolved metal is transported within the plant. If the dissolution of the metal-containing NPs in growth medium is suspected, the cultivation of an additional set of plants treated with corresponding metal salt at the same metal concentration is recommended. In this case, the comparison of total metal content in plant tissues treated with metal salt and metal-containing NPs can give additional information about the uptake mechanisms. In any case, and regardless of whether a dissolution process takes place already in the growth medium used for plant cultivation or inside plant tissues after NPs uptake and accumulation, the presence of the dissolved form of metal redirects the investigation towards another direction. The key point is that the presence of the metal in its free form may lead to the formation of new species inside the plant and hence studies on metal speciation to determine and identify these new species are needed. For this purpose, the extraction of metal compounds formed in plant tissues must be carried out in order to release them from solid matrix. If one-step extraction turns out to be insuﬃcient, additional steps should be applied, for example, by the use of enzymes such as pectinase or cellulase. The use of these enzymes leads to an increase of extraction eﬃciency by degradation of the plant cell wall, which allows the release of metal accumulated in this part of the tissue. The eﬃciency of extraction can be determined by the digestion of each extract followed by metal content analysis and comparison with the total metal concentration. It should be highlighted that, due to its diﬀerent chemical nature, procedure of extraction of metal compounds must be diﬀerent than the digestion procedure used for extraction of intact NPs from plant tissues. 6. Speciation Studies 11 of 16 Nanomaterials 2020, 10, 1480 Speciation analysis can be successfully performed by the use of hyphenated techniques, based on the coupling of eﬀective separation technique such as chromatography or electrophoresis to ICP-MS for sensitive element-speciﬁc detection or/and to mass spectrometry with soft ionization (electrospray (ESI) MS, matrix-assisted laser desorption/ionization (MALDI) MS) for identiﬁcation of a compound structure. For analysis of complicated matrix such as plant samples, an initial fractionation of extracted compounds in terms of molecular mass can be performed by size exclusion chromatography (SEC) coupled to ICP-MS. SEC is used for simple fractionation of metallocompounds according to their molecular mass. In addition, SEC analyses are performed under mild conditions and without the use of organic solvents, which is important to maintain the stability of metal species. However, each peak obtained during SEC-ICP-MS analysis contains a signiﬁcant number of metal compounds, since resolution of SEC is rather low. Therefore, prior to the identiﬁcation of extracted complexes, a diﬀerent technique that allows better separation of initially fractionated species, should be applied. One of the possible options is that after SEC-ICP-MS analysis, fractions corresponding to each peak observed on the chromatograms can be collected, lyophilized and analyzed by hydrophilic interaction chromatography (HILIC) coupled to ICP-MS [7], which provides a more eﬃcient separation of metal compounds, in terms of their polarity. Results obtained by using ICP-MS as an elemental detector provide information about type and nature of extracted metal complexes, without information about the ligands bound to the analyzed metal. Therefore, after characterization of the extracted metal species by means of two-dimensional liquid chromatography coupled to ICP-MS, identiﬁcation of the compounds formed in plant tissues must be performed. For this purpose, tandem mass spectrometry (MS/MS) with soft ionization such as ESI or MALDI is usually used by fragmentation of molecular ions and detection of speciﬁc fragment ions. It can be performed by direct analysis of samples or after their separation using chromatographic techniques. However, in the case of matrices that contain many diﬀerent compounds, the results obtained by direct MS/MS analysis can be complex to interpret. In order to identify extracted metal complexes, HILIC chromatography coupled to high resolution electrospray tandem mass spectrometry (ESI-MS/MS) is highly recommended to use. HILIC chromatography is well suited to mass spectrometry detection, as typical eluents used in this separation technique contain a high percentage of organic solvent in water or volatile buﬀer. 6. Speciation Studies The high resolution of state-of-the-art ESI-MS together with high mass accuracy allow for automatic data mining in search for metal species and organic compounds, based on their isotopic patterns and species mass defects. The mass spectrum registered during analysis of extracted metal compounds must be searched for parent ions with a speciﬁc isotopic pattern corresponding to analyzed metal (unless it is monoisotopic) and chosen ions should be fragmented next for determination of species structure. Collision energy should be optimized during analysis since it may occur that fragmentation is too strong or not strong enough and contribution of the parent ion is too big. Thanks to this approach, the identiﬁcation of extracted complexes can be conﬁrmed in terms of molecular mass, MS/MS fragmentation and by matching of the registered isotopic patterns with the theoretical ones. If there is one major metal compound observed, the quantiﬁcation can be carried out by an external calibration curve and/or standard addition method. Finally, it should be highlighted that the use of hyphenated techniques for metal speciation i i t i ll th d t ti d id tiﬁ ti f i t l l t Finally, it should be highlighted that the use of hyphenated techniques for metal speciation in various matrices allows the detection and identiﬁcation of even minor metal complexes at environmentally relevant concentrations. 7. Spatial Distribution The detection and characterization of NPs in above ground tissues demonstrates the ability of plants to take up and translocate NPs. However, the exact pathways and mechanisms of uptake and transport of NPs are still unclear. For example, damage of the physiological barriers of roots [4] or penetration into the root epidermis and cortex through the apoplastic pathway have been suggested [49]. In this context, the study of the spatial distribution of metal-containing NPs in plant roots can help in ﬁlling this gap. 12 of 16 12 of 16 Nanomaterials 2020, 10, 1480 The analytical techniques described so far are not able to provide this information. However, Laser Ablation (LA) ICP-MS has proved to be a valuable analytical tool for bioimaging of metal species in plant tissues [50]. This technique is based on the vaporization of a solid sample by using pulses from a focused laser beam, the ablated material being transported to the ICP-MS in a gas ﬂow of Ar or He [51]. Even though LA-ICP-MS is not able to provide information about the physico-chemical form of the metal analyzed, it can be successfully applied when the information about the presence of the metal in its nanoparticulate form has been conﬁrmed by another technique [37]. Sample preparation is a critical step for the analysis of plant organs by LA-ICP-MS. Usually, thin layers of the plant tissue are prepared with a microtome, which are directly mounted into glass slides. However, the sample preparation procedure will depend on the shape and geometry of the analyzed organ. The analysis performed by LA-ICP-MS can provide useful information about the localization of NPs inside the tissue that will help to elucidate the uptake and transport mechanisms. For instance, the accumulation and transport of CeO2 NPs by secondary roots from the bottom towards the central part of radish roots was suggested after analysis by LA-ICP-MS [37]. LA-ICP-MS can also provide quantitative information but requires the use and availability of matrix matching standards to constitute calibration curves and appropriate internal standards (IS) to compensate signal variation during laser beam-sample interaction, transportation of the aerosol, and instrumental drifts [52,53], making the analyses more complex. In addition, the use of a single imaging technique like LA-ICP-MS may result insuﬃcient in studies at low concentrations, where the few observable NPs can be almost indistinguishable from naturally occurring NPs or other background signals [54]. 7. Spatial Distribution It is then advisable, when possible, to combine the information obtained about spatial distribution of metal nanoparticles by LA-ICP-MS, with the information obtained with other analytical techniques: total metal determination (ICP-MS) and characterization of physico-chemical form (SP-ICP-MS). References Eﬀects, uptake, and translocation of aluminum oxide nanoparticles in lettuce: A comparison study to phytotoxic aluminum ions. Sci. Total Environ. 2020, 719, 137393. [CrossRef] 6. Hayes, K.L.; Mui, J.; Song, B.; Shirzaei, E.; Eisenman, S.W.; Shef, J.B.; Kim, B. Eﬀects, uptake, and translocation of aluminum oxide nanoparticles in lettuce: A comparison study to phytotoxic aluminum ions. Sci. Total Environ. 2020, 719, 137393. [CrossRef] 7. Ullah, H.; Li, X.; Peng, L.; Cai, Y.; Mielke, H.W. In vivo phytotoxicity, uptake, and translocation of nanoparticles in maize (Zea mays L.) plants. Sci. Total Environ. 2020, 737, 139558. [CrossRef] 8. Al-amri, N.; Tombuloglu, H.; Slimani, Y.; Akhtar, S. Size eﬀect of iron (III) oxide nanomaterials on the growth, and their uptake and translocation in common wheat (Triticum aestivum L.). Ecotoxicol. Environ. Saf. 2020, 194, 110377. [CrossRef] 9. Tombuloglu, H.; Slimani, Y.; Tombuloglu, G.; Almessiere, M. Uptake and translocation of magnetite (Fe3O4) nanoparticles and its impact on photosynthetic genes in barley (Hordeum vulgare L.). Chemosphere 2019, 226, 110–122. [CrossRef] 10. Hu, J.; Wu, X.; Wu, F.; Chen, W.; White, J.C.; Yang, Y.; Wang, B.; Xing, B.; Tao, S.; Wang, X. Potential application of titanium dioxide nanoparticles to improve the nutritional quality of coriander (Coriandrum sativum L.). J. Hazard. Mater. 2020, 389, 121837. [CrossRef] 11. Ko, J.A.; Hwang, Y.S. Eﬀects of nanoTiO2 on tomato plants under diﬀerent irradiances. Environ. Pollut. 2019, 255, 113141. [CrossRef] 12 Wojcieszek J ; Jimenez Lamana J ; Ruzik L ; Asztemborska M ; Jarosz M ; Szpunar J Characterization of 255, 113141. [CrossRef] 12. Wojcieszek, J.; Jimenez-Lamana, J.; Ruzik, L.; Asztemborska, M.; Jarosz, M.; Szpunar, J. Characterization of TiO2 NPs in radish (Raphanus sativus L.) by Single Particle ICP-QQQ-MS. Front. Environ. Sci. 2020, 8, 100. 2. Wojcieszek, J.; Jimenez-Lamana, J.; Ruzik, L.; Asztemborska, M.; Jarosz, M.; Szpunar, J. Characterizatio TiO2 NPs in radish (Raphanus sativus L.) by Single Particle ICP-QQQ-MS. Front. Environ. Sci. 2020, 8, 10 13. Li, Y.; Zhu, N.; Liang, X.; Zheng, L.; Zhang, C.; Li, Y. A comparative study on the accumulation, translocation and transformation of selenite, selenate, and SeNPs in a hydroponic-plant system. Ecotoxicol. Environ. Saf. 2020, 189, 109955. [CrossRef] [PubMed] 14. Bao, D.; Zheng, G.O.; Chen, Z. Characterization of silver nanoparticles internalized by Arabidopsis plants using single particle ICP-MS analysis. Front. Plant Sci. 2016, 7, 1–8. [CrossRef] [PubMed] 15. obtained about the physico-chemical form of NPs inside plants, can provide new insights about the uptake and translocation pathways. obtained about the physico-chemical form of NPs inside plants, can provide new insights about the uptake and translocation pathways. Author Contributions: J.W. and J.J.-L. wrote and revised the original draft and prepared the ﬁnal version of the manuscript. L.R. contributed to the writing, revision and ﬁgure preparation. J.S. conceptualized, reviewed and advised the work. M.J. has supervised and managed the funding resources. All authors have read and agreed to the published version of the manuscript. Funding: This work was ﬁnancially supported by Warsaw University of Technology and the National Science Centre, Poland (grant No. 2015/18/M/ST4/00257) Conﬂicts of Interest: The authors declare no conﬂict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. References 1. Ma, X.; Geiser-lee, J.; Deng, Y.; Kolmakov, A. Interactions between engineered nanoparticles (ENPs) and plants: Phytotoxicity, uptake and accumulation. Sci. Total Environ. 2010, 408, 3053–3061. [CrossRef] [PubMed] 2 Khan M R ; Adam V; Rizvi T F ; Zhang B ; Ahamad F ; Josko I ; Zhu Y; Yang M ; Mao C 1. Ma, X.; Geiser-lee, J.; Deng, Y.; Kolmakov, A. Interactions between engineered nanoparticles (ENPs) and plants: Phytotoxicity, uptake and accumulation. Sci. Total Environ. 2010, 408, 3053–3061. [CrossRef] [PubMed] 1. Ma, X.; Geiser-lee, J.; Deng, Y.; Kolmakov, A. Interactions between engineered nanoparticles (ENPs) and plants: Phytotoxicity, uptake and accumulation. Sci. Total Environ. 2010, 408, 3053–3061. [CrossRef] [PubMed] 2. Khan, M.R.; Adam, V.; Rizvi, T.F.; Zhang, B.; Ahamad, F.; Josko, I.; Zhu, Y.; Yang, M.; Mao, C. Nanoparticle—Plant interactions: Two-way traﬃc. Small 2019. [CrossRef] [PubMed] 2. Khan, M.R.; Adam, V.; Rizvi, T.F.; Zhang, B.; Ahamad, F.; Josko, I.; Zhu, Y.; Yang, M.; M Nanoparticle—Plant interactions: Two-way traﬃc. Small 2019. [CrossRef] [PubMed] 3. Shrivastava, M.; Srivastav, A.; Gandhi, S.; Rao, S.; Roychoudhury, A.; Kumar, A.; Singhal, R.K.; Kumar, S.; Singh, S.D. Monitoring of engineered nanoparticles in soil-plant system: A review. Environ. Nanotechnol. Monit. Manag. 2019, 11, 100218. [CrossRef] 3. Shrivastava, M.; Srivastav, A.; Gandhi, S.; Rao, S.; Roychoudhury, A.; Kumar, A.; Singhal, R.K.; Kumar, S.; Singh, S.D. Monitoring of engineered nanoparticles in soil-plant system: A review. Environ. Nanotechnol. Monit. Manag. 2019, 11, 100218. [CrossRef] 4. Lv, J.; Christie, P.; Zhang, S. Uptake, translocation, and transformation of metal-based nanoparticles in plants: Recent advances and methodological challenges. Environ. Sci. Nano 2019, 6, 41–59. [CrossRef] 4. Lv, J.; Christie, P.; Zhang, S. Uptake, translocation, and transformation of metal-based nanoparticles in plants: Recent advances and methodological challenges. Environ. Sci. Nano 2019, 6, 41–59. [CrossRef] 5. Torrent, L.; Iglesias, M.; Marguí, E.; Hidalgo, M.; Verdaguer, D.; Llorens, L.; Kodre, A.; Kavˇciˇc, A.; Vogel-mikuš, K. Uptake, translocation and ligand of silver in Lactuca sativa exposed to silver nanoparticles of diﬀerent size, coatings and concentration. J. Hazard. Mater. 2020, 384, 121201. [CrossRef] 5. Torrent, L.; Iglesias, M.; Marguí, E.; Hidalgo, M.; Verdaguer, D.; Llorens, L.; Kodre, A.; Kavˇciˇc, A.; Vogel-mikuš, K. Uptake, translocation and ligand of silver in Lactuca sativa exposed to silver nanoparticles of diﬀerent size, coatings and concentration. J. Hazard. Mater. 2020, 384, 121201. [CrossRef] 6. Hayes, K.L.; Mui, J.; Song, B.; Shirzaei, E.; Eisenman, S.W.; Shef, J.B.; Kim, B. 8. Conclusions The investigation of interactions between metal-containing NPs and plants requires several steps that need to be carefully designed, each of them requiring a careful and individual evaluation depending on the plant used in a model study, the cultivation conditions and especially the type of metal NPs studied. In addition, for a correct characterization of metal-containing NPs at each step and/or identiﬁcation of new species created, a multi-technique approach is needed. Here we summarize the most important recommendations developed along the text: - The behavior of metal-containing NPs in the nutrient solution used for plant cultivation must be investigated before starting the cultivation experiment. The occurrence of a transformation process in the growth medium (agglomeration/dissolution) may inﬂuence the rest of the study. - The behavior of metal-containing NPs in the nutrient solution used for plant cultivation must be investigated before starting the cultivation experiment. The occurrence of a transformation process in the growth medium (agglomeration/dissolution) may inﬂuence the rest of the study. - NPs must be extracted from the plant material without altering their properties. The use of an enzymatic digestion with an enzyme capable of digesting plant cell walls, like pectinase, cellulase or hemicellulase is necessary. A cocktail of enzymes (e.g., Macerozyme R-10) is recommended. - SP-ICP-MS has proved to be a useful tool for size characterization of metal-containing NPs in plant tissues as well as to monitor possible NPs transformation inside the plants. - The comparison of NPs size distributions in the nutrient solution, roots and above-ground organs provides essential information about the mechanisms of uptake and translocation of metal-containing NPs by plants. - If NPs dissolution takes place, speciation studies leading to the identiﬁcation of new metal species created within plant tissues can be carried out by hyphenated techniques, which include separation of extracted compounds by chromatography or electrophoresis, followed by their characterization and/or identiﬁcation by mass spectrometry. - Additional information about the spatial location of metal following the uptake of NPs in tissues can be obtained by laser ablation coupled to ICP-MS. This, in combination with the information - Additional information about the spatial location of metal following the uptake of NPs in tissues can be obtained by laser ablation coupled to ICP-MS. This, in combination with the information 13 of 16 Nanomaterials 2020, 10, 1480 References Cocozza, C.; Perone, A.; Giordano, C.; Salvatici, M.C.; Pignattelli, S.; Raio, A.; Schaub, M.; Sever, K.; Innes, J.L.; Tognetti, R.; et al. Silver nanoparticles enter the tree stem faster through leaves than through roots. Tree Physiol. 2019, 39, 1251–1261. [CrossRef] [PubMed] 14 of 16 14 of 16 Nanomaterials 2020, 10, 1480 16. Souza, I.R.; Silva, L.R.; Fernandes, L.S.P.; Salgado, L.D.; Silva, H.C.; Assis, D.; Firak, D.S.; Bach, L.; Santos-ﬁlho, R.; Voigt, C.L.; et al. Visible-light reduced silver nanoparticles’ toxicity in Allium cepa test. Environ. Pollut. 2020, 257, 113551. [CrossRef] [PubMed] 7. Song, G.; Hou, W.; Gao, Y.; Wang, Y.; Lin, L.; Zhang, Z.; Niu, Q.; Ma, R.; Mu, L.; Wang, H. Eﬀects of C nanoparticles on Lemna minor. Bot. Stud. 2016, 57, 1–8. [CrossRef] 18. Owji, H.; Hemmati, S.; Heidari, R.; Hakimzadeh, M. Eﬀect of alumina ( Al2O3) nanoparticles and macroparticles on Trigonella foenum—Graceum L. In vitro cultures: Assessment of growth parameters and oxidative stress—Related responses. 3 Biotech 2019, 9, 1–12. [CrossRef] 19. Ding, Y.; Bai, X.; Ye, Z.; Ma, L.; Liang, L. Toxicological responses of Fe3O4 nanoparticles on Eichhornia crassipes and associated plant transportation. Sci. Total Environ. 2019, 671, 558–567. [CrossRef] 20. Cristina, S.; Arruda, C.; Luiz, A.; Silva, D.; Moretto, R.; Antunes, R.; Aurélio, M.; Arruda, Z. Nanoparticles applied to plant science: A review. Talanta 2015, 131, 693–705. [CrossRef] 21. Fabrega, J.; Luoma, S.N.; Tyler, C.R.; Galloway, T.S.; Lead, J.R. Silver nanoparticles: Behaviour and eﬀects in the aquatic environment. Environ. Int. 2011, 37, 517–531. [CrossRef] 22. Castillo-michel, H.A.; Larue, C.; Pradas, A.E.; Cotte, M.; Sarret, G. Practical review on the use of synchrotron based micro- and nano- X-ray ﬂuorescence mapping and X-ray absorption spectroscopy to investigate the interactions between plants and engineered nanomaterials. Plant Physiol. Biochem. 2017, 110, 13–32. [CrossRef] [PubMed] 23. Larue, C.; Castillo-michel, H.; Stein, R.J.; Fayard, B.; Pouyet, E.; Villanova, J.; Magnin, V.; Pradas, A.; Trcera, N.; Legros, S.; et al. Innovative combination of spectroscopic techniques to reveal nanoparticle fate in a crop plant. Spectrochim. Acta Part B At. Spectrosc. 2016, 119, 17–24. [CrossRef] 24. Servin, A.D.; Castillo-Michel, H.; Hernandez-Viezcas, J.A.; Diaz, B.C.; Peralta-videa, J.R.; Gardea-torresdey, J.L. Synchrotron Micro-XRF and Micro-XANES conﬁrmation of the uptake and translocation of TiO2 nanoparticles in cucumber (Cucumis sativus) plants. Environ. Sci. Technol. 2012, 46, 7637–7643. [CrossRef] 25. Yin, L.; Cheng, Y.; Espinasse, B.; Colman, B.P.; Au, M.; Wiesner, M.; Rose, J.; Liu, J.; Bernhardt, E.S. References More than the ions: The eﬀects of silver nanoparticles on Lolium multiﬂorum. Environ. Sci. Technol. 2011, 45, 2360–2367. [CrossRef] [PubMed] 26. Hernandez-viezcas, J.A.; Castillo-michel, H.; Servin, A.D.; Peralta-videa, J.R.; Gardea-torresdey, J.L. Spectroscopic veriﬁcation of zinc absorption and distribution in the desert plant Prosopis juliﬂora-velutina (velvet mesquite) treated with ZnO nanoparticles. Chem. Eng. J. 2011, 170, 346–352. [CrossRef] [PubMed] 27. Hernandez-viezcas, J.A.; Castillo-michel, H.; Andrews, J.C.; Cotte, M.; Rico, C.; Peralta-videa, J.R.; Ge, Y.; Priester, J.H.; Holden, A.; Gardea-torresdey, J.L. In situ synchrotron X-ray ﬂuorescence mapping and speciation of CeO2 and ZnO nanoparticles in soil cultivated soybean (Glycine max). ACS Nano 2013, 7, 1415–1423. [CrossRef] 28. Laborda, F.; Bolea, E.; Jiménez-Lamana, J. Single particle inductively coupled plasma mass spectrometry for the analysis of inorganic engineered nanoparticles in environmental samples. Trends Environ. Anal. Chem. 2016, 9, 15–23. [CrossRef] 29. Laborda, F.; Bolea, E.; Jiménez-Lamana, J. Single particle inductively coupled plasma mass spectrometry: A powerful tool for nanoanalysis. Anal. Chem. 2014, 86, 2270–2278. [CrossRef] 30. Murashige, T.; Skoog, F. A Revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol. Plant 1962, 15, 473–497. [CrossRef] 31. Hoagland, D.R.; Arnon, D.I. The water-culture method for growing plants without soil. Calif. Agric. Exp. Stn. Circ. 1949, 347, 32. 32. Nath, J.; Dror, I.; Landa, P.; Motkova, K.; Vanek, T.; Berkowitz, B. Isotopic labelling for sensitive detection of nanoparticle uptake and translocation in plants from hydroponic medium and soil. Environ. Chem. 2019, 16, 391–400. [CrossRef] 33. Wojcieszek, J.; Jimenez-Lamana, J.; Bierla, K.; Asztemborska, M.; Ruzik, L.; Jarosz, M.; Szpunar, J. Elucidation of the fate of zinc in model plants using single particle ICP-MS and ESI tandem MS. J. Anal. At. Spectrom. 2019, 34, 683–693. [CrossRef] 34. Jiménez-Lamana, J.; Wojcieszek, J.; Jakubiak, M.; Asztemborska, M.; Szpunar, J. Single particle ICP-MS characterization of platinum nanoparticles uptake and bioaccumulation by Lepidium sativum and Sinapis alba plants. J. Anal. At. Spectrom. 2016, 31, 2321–2329. [CrossRef] Nanomaterials 2020, 10, 1480 15 of 16 15 of 16 35. Knapen, D.; Bals, S.; Blust, R.; Adam, N. The uptake of ZnO and CuO nanoparticles in the water- ﬂea Daphnia magna under acute exposure scenarios. Environ. Pollut. 2014, 194, 130–137. [CrossRef] 36. Merdzan, V.; Domingos, R.F.; Monteiro, C.E.; Hadioui, M.; Wilkinson, K.J. The eﬀects of diﬀerent coatings on zinc oxide nanoparticles and their inﬂuence on dissolution and bioaccumulation by the green alga, C. reinhardtii. Sci. Total Environ. 2014, 488–489, 316–324. References [CrossRef] [PubMed] 37. Wojcieszek, J.; Jiménez-Lamana, J.; Bierła, K.; Ruzik, L.; Asztemborska, M.; Jarosz, M.; Szpunar, J. Uptake, translocation, size characterization and localization of cerium oxide nanoparticles in radish (Raphanus sativus L.). Sci. Total Environ. 2019, 683, 284–292. [CrossRef] 38. Ki´nska, K.; Jiménez-Lamana, J.; Kowalska, J.; Krasnod˛ebska-Ostr˛ega, B.; Szpunar, J. Study of the uptake and bioaccumulation of palladium nanoparticles by Sinapis alba using single particle ICP-MS. Sci. Total Environ. 2018, 615, 1078–1085. [CrossRef] 39. Yang, J.; Cao, W.; Rui, Y. Interactions between nanoparticles and plants: Phytotoxicity and defense mechanisms. J. Plant Interact. 2017, 12, 158–169. [CrossRef] 40. Schwertfeger, D.M.; Velicogna, J.R.; Jesmer, A.H.; Saatcioglu, S.; Mcshane, H.; Scroggins, R.P.; Princz, J.I. Extracting metallic nanoparticles from soils for quantitative analysis: method development using engineered silver nanoparticles and SP-ICP-MS. Anal. Chem. 2017, 89, 2505–2513. [CrossRef] 41. Mahdi, K.N.M.; Peters, R.J.B.; Klumpp, E.; Bohme, S.; Ploeg, M.; Van Der Ritsema, C.; Geissen, V. Silver nanoparticles in soil: Aqueous extraction combined with single-particle ICP-MS for detection and characterization. Environ. Nanotechnol. Monit. Manag. 2017, 7, 24–33. [CrossRef] 42. Lee, W.; Kwak, J.I; An, Y. Eﬀect of silver nanoparticles in crop plants Phaseolus radiatus and Sorghum bicolor: Media eﬀect on phytotoxicity. Chemosphere 2012, 86, 491–499. [CrossRef] [PubMed] 43. Laborda, F.; Bolea, E.; Cepriá, G.; Gómez, M.T.; Jiménez, M.S.; Pérez-Arantegui, J.; Castillo, J.R. Detection, characterization and quantification of inorganic engineered nanomaterials: A review of techniques and methodological approaches for the analysis of complex samples. Anal. Chim. Acta 2016, 904, 10–32. [CrossRef] [PubMed] 44. Stavolone, L.; Lionetti, V. Extracellular matrix in plants and animals: Hooks and locks for viruses. Front. Microbiol. 2017, 8, 1–8. [CrossRef] 45. Singh, G.; Stephan, C.; Westerhoﬀ, P.; Carlander, D.; Duncan, T.V. Measurement Methods to Detect, characterize, and quantify engineered nanomaterials in foods. Compr. Rev. Food Sci. Food Saf. 2014, 13, 693–704. [CrossRef] 46. Loeschner, K.; Navratilova, J.; Købler, C.; Mølhave, K.; Wagner, S.; Kammer, F. Von Der; Larsen, E.H. Detection and characterization of silver nanoparticles in chicken meat by asymmetric ﬂow ﬁeld ﬂow fractionation with detection by conventional or single particle ICP-MS. Anal. Bioanal. Chem. 2013, 405, 8185–8195. [CrossRef] 47. Dan, Y.; Zhang, W.; Xue, R.; Ma, X.; Stephan, C.; Shi, H. Characterization of gold nanoparticle uptake by tomato plants using enzymatic extraction followed by single-particle inductively coupled plasma-mass spectrometry analysis. Environ. Sci. Technol. 2015, 49, 3007–3014. [CrossRef] 48. © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). References Schwabe, F.; Tanner, S.; Schulin, R.; Rotzetter, A.; Stark, W.; Quadt, A.; Von Nowack, B. Dissolved cerium contributes to uptake of Ce in the three crop plants. Metallomics 2015, 7, 466–477. [CrossRef] 49. Zhao, L.; Peralta-videa, J.R.; Ren, M.; Varela-ramirez, A.; Li, C.; Hernandez-viezcas, J.A.; Aguilera, R.J.; Gardea-torresdey, J.L. Transport of Zn in a sandy loam soil treated with ZnO NPs and uptake by corn plants: Electron microprobe and confocal microscopy studies. Chem. Eng. J. 2012, 184, 1–8. [CrossRef] 50. Hansen, T.H.; de Ban, T.C.; Laursen, K.H.; Pedas, P.; Husted, S.; Schjoerring, J.K. Multielement plant tissue analysis using ICP spectrometry. In Plant Mineral Nutrients: Methods in Molecular Biology (Methods and Protocols); Maathuis, F., Ed.; Humana Press: Totowa, NJ, USA, 2013; p. 141. 51. Durrant, S.F. Laser ablation inductively coupled plasma mass spectrometry: Achievements, problems, prospects. J. Anal. At. Spectrom. 1999, 14, 1385–1403. [CrossRef] 52. Becker, J.S.; Dietrich, R.C.; Matusch, A.; Pozebon, D.; Dressler, V.L. Quantitative images of metals in plant tissues measured by laser ablation inductively coupled plasma mass spectrometry. Spectrochim. Acta Part B At. Spectrosc. 2008, 63, 1248–1252. [CrossRef] 52. Becker, J.S.; Dietrich, R.C.; Matusch, A.; Pozebon, D.; Dressler, V.L. Quantitative images of metals in plant tissues measured by laser ablation inductively coupled plasma mass spectrometry. Spectrochim. Acta Part B At. Spectrosc. 2008, 63, 1248–1252. [CrossRef] 16 of 16 Nanomaterials 2020, 10, 1480 16 of 16 53. Grijalba, N.; Legrand, A.; Holler, V.; Bouvier-capely, C. A novel calibration strategy based on internal standard—Spiked gelatine for quantitative bio-imaging by LA-ICP-MS: Application to renal localization and quantiﬁcation of uranium. Anal. Bioanal. Chem. 2020, 412, 3113–3122. [CrossRef] [PubMed] 53. Grijalba, N.; Legrand, A.; Holler, V.; Bouvier-capely, C. A novel calibration strategy based on internal standard—Spiked gelatine for quantitative bio-imaging by LA-ICP-MS: Application to renal localization and quantiﬁcation of uranium. Anal. Bioanal. Chem. 2020, 412, 3113–3122. [CrossRef] [PubMed] 54. Avellan, A.; Schwab, F.; Masion, A.; Chaurand, P.; Borschneck, D.; Vidal, V.; Rose, J.; Santaella, C.; Levard, C. Nanoparticle uptake in plants: Gold nanomaterial localized in roots of arabidopsis thaliana by X-Ray computed nanotomography and hyperspectral imaging. Environ. Sci. Technol. 2018, 51, 8682–8691. [CrossRef] [PubMed] 54. Avellan, A.; Schwab, F.; Masion, A.; Chaurand, P.; Borschneck, D.; Vidal, V.; Rose, J.; Santaella, C.; Levard, C. Nanoparticle uptake in plants: Gold nanomaterial localized in roots of arabidopsis thaliana by X-Ray computed nanotomography and hyperspectral imaging. Environ. Sci. Technol. 2018, 51, 8682–8691. References [CrossRef] [PubMed] © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
https://openalex.org/W2344403975	https://www.pure.ed.ac.uk/ws/files/60172454/BalmerEtalJRI2016FiveRulesOfThumb.pdf	English	null	Five rules of thumb for post-ELSI interdisciplinary collaborations	Journal of responsible innovation	2,016	cc-by	5,120	Edinburgh Research Explorer General rights C i h f h General rights Copyright for the publications made accessible via the Edinburgh Research Explorer is retained by the author(s) and / or other copyright owners and it is a condition of accessing these publications that users recognise and abide by the legal requirements associated with these rights. Five rules of thumb for Post-ELSI interdisciplinary collaborations Citation for published version: Balmer, A, Calvert, J, Marris, C, Molyneux-Hodgson, S, Frow, E, Kearnes, M, Bulpin, K, Schyfter Camacho, P, Mackenzie, A & Martin, P 2016, 'Five rules of thumb for Post-ELSI interdisciplinary collaborations', Journal of Responsible Innovation, vol. 3, no. 1, pp. 73-80. https://doi.org/10.1080/23299460.2016.1177867 Digital Object Identifier (DOI): 10.1080/23299460.2016.1177867 Link: Link to publication record in Edinburgh Research Explorer Document Version: Publisher's PDF, also known as Version of record Published In: Journal of Responsible Innovation Journal of Responsible Innovation ISSN: 2329-9460 (Print) 2329-9037 (Online) Journal homepage: http://www.tandfonline.com/loi/tjri20 Take down policy Take down policy The University of Edinburgh has made every reasonable effort to ensure that Edinburgh Research Explorer content complies with UK legislation. If you believe that the public display of this file breaches copyright please contact openaccess@ed.ac.uk providing details, and we will remove access to the work immediately and investigate your claim. Download date: 24. Oct. 2024 Five rules of thumb for post-ELSI interdisciplinary collaborations Andrew S. Balmer, Jane Calvert, Claire Marris, Susan Molyneux-Hodgson, Emma Frow, Matthew Kearnes, Kate Bulpin, Pablo Schyfter, Adrian Mackenzie & Paul Martin To cite this article: Andrew S. Balmer, Jane Calvert, Claire Marris, Susan Molyneux-Hodgson, Emma Frow, Matthew Kearnes, Kate Bulpin, Pablo Schyfter, Adrian Mackenzie & Paul Martin (2016) Five rules of thumb for post-ELSI interdisciplinary collaborations, Journal of Responsible Innovation, 3:1, 73-80, DOI: 10.1080/23299460.2016.1177867 To link to this article: https://doi.org/10.1080/23299460.2016.1177867 © 2016 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group Accepted author version posted online: 25 Apr 2016. Published online: 07 Jun 2016. Submit your article to this journal Article views: 1094 View related articles View Crossmark data Citing articles: 10 View citing articles © 2016 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/ licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. CONTACT Andrew S. Balmer andrew.balmer@manchester.ac.uk †Present afﬁliation: Centre for Food Policy, Sociology Department, City University, London, UK. ABSTRACT In this paper we identify ﬁve rules of thumb for interdisciplinary collaboration across the natural and social sciences. We link these to efforts to move away from the ‘ethical, legal and social issues’ framework of interdisciplinarity and towards a post-ELSI collaborative space. It is in trying to open up such a space that we identify the need for: collaborative experimentation, taking risks, collaborative reﬂexivity, opening-up discussions of unshared goals and neighbourliness. Five rules of thumb for post-ELSI interdisciplinary collaborations Andrew S. Balmera , Jane Calvertb, Claire Marrisc†, Susan Molyneux-Hodgsond, Emma Frowe,f, Matthew Kearnesg, Kate Bulpinh, Pablo Schyfterb, Adrian Mackenziei and Paul Martind Andrew S. Balmera , Jane Calvertb, Claire Marrisc†, Susan Molyneux-Hodgsond, Emma Frowe,f, Matthew Kearnesg, Kate Bulpinh, Pablo Schyfterb, Adrian Mackenziei and Paul Martind aSociology and the Morgan Centre for Research into Everyday Lives, University of Manchester, Manchester, UK; bScience, Technology and Innovation Studies, University of Edinburgh, Edinburgh, UK; cDepartment of Social Science, Health & Medicine, King’s College London, London, UK; dDepartment of Sociological Studies, University of Shefﬁeld, Shefﬁeld, UK; eSchool of Biological & Health Systems Engineering, Arizona State University, Tempe, AZ, USA; fThe Consortium for Science, Policy & Outcomes, Arizona State University, Tempe, AZ, USA; gEnvironmental Humanities, School of Humanities and Languages, University of New South Wales, Sydney, Australia; hSociology, University of Manchester, Manchester, UK; iCentre for Science Studies, Department of Sociology, Lancaster University, Lancaster, UK Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=tjri20 JOURNAL OF RESPONSIBLE INNOVATION, 2016 VOL. 3, NO. 1, 73–80 http://dx.doi.org/10.1080/23299460.2016.1177867 JOURNAL OF RESPONSIBLE INNOVATION, 2016 VOL. 3, NO. 1, 73–80 http://dx.doi.org/10.1080/23299460.2016.1177867 Introduction In recent years, dissatisfaction has grown with the ‘ethical, legal and social issues’ (ELSI) framework for interdisciplinary collaboration between natural and social scientists in sociotechnical knowledge production and innovation. In particular, critics from the ﬁeld of science and technology studies argue that such projects place too much emphasis on the promises surrounding sociotechnical innovation rather than on its practices, result- ing in ‘speculative ethics’ (Nordmann and Rip 2009) and that there is a danger of such work becoming little more than a box-ticking exercise (Jasanoff 2007). All of this can result in social scientists being positioned as ‘nay-sayers’, the voice of negative criticism (Fortun 2005). This is partly a product of how our critical apparatus is often integrated into scientiﬁc and governance practices through ELSI-style processes of engagement in order to address funders’ and government’s requirements to anticipate ‘negative conse- quences’, thereby placing us in the role of ‘foreteller’ (Balmer et al. 2015). This signiﬁcantly constrains opportunities for bringing about changes in practice and for productive relations between natural and social scientists. 74 74 A. S. BALMER ET AL. In trying to overcome such limitations, to move the integration (Fisher and Maricle 2015) of social science upstream, and attend to practices of research and innovation, science and technology studies (STS) researchers have sought to inhabit more collabora- tive, reﬂexive and, coproductive roles (Calvert and Martin 2009). Several forms of inte- gration have been developed to help accomplish such a shift, amongst them: upstream public engagement (Wilsdon and Willis 2004); contructive technology assessment (CTA) (Schot and Rip 1997); anticipatory governance and real time technology assess- ment (Barben et al. 2008); human practices (Rabinow and Bennett 2012); and responsible innovation (Owen, Bessant, and Heintz 2013). However, much less has been said regard- ing the everyday struggles to bring about changes in such relations as we seek to move towards more collaborative practices (Balmer et al. 2015; Balmer, Bulpin, and Moly- neux-Hodgson 2016; Fitzgerald and Callard 2014; Rabinow and Bennett 2012). In this article, we draw on our experiences of and reﬂections on interactions with natural scientists and engineers in the context of synthetic biology, which amount collec- tively to more than 48 researcher years of entanglement. We extend our previous com- ments on this topic (Balmer et al. 2012, 2015) to put forward ﬁve ‘rules of thumb’ for developing ‘post-ELSI forms’ of collaboration (Balmer and Bulpin 2013; Rabinow and Bennett 2012). Introduction Rules of thumb offer a general guide based on practice rather than theory – a ﬂexible and adaptable sense of how to approach a subject rather than a strict set of procedures to follow. Taking risks Experimenting with collaborative relationships often requires that social scientists move from role to role, sometimes shifting into more external positions, and at other times into more coproductive and collegial alignments (Balmer et al. 2015). These shifts of pos- ition come with shifts in the kinds of risks one is required to take and with what is put at risk. Collaborative positions involve more risk, professionally and personally, than do external positions (Balmer, Bulpin, and Molyneux-Hodgson 2016). For example, the risks taken with one’s career are more signiﬁcant, since to experiment with collaboration can cost time and effort. Interdisciplinary outputs are generally awarded less value than single discipline scholarly contributions in academic life (Klein 1990). Professional risks such as these are most acute for those whose jobs are funded by grants led by natural scien- tists, and most particularly for junior researchers in such positions. In this regard, the various roles one can take involve different levels and different kinds of vulnerability. These must be acknowledged by research funders, universities, policy-makers and princi- pal investigators on collaborative grants. Ensuring that there are practical protections for those at risk is an important part of ensuring that collaborative experiments can be pro- ductive for all those involved. Nonetheless, we believe it is vital that we take risks. For example, we must take more risks with how we represent our research and ﬁndings. Even in the most coproductive and collegiate collaborations, scientists and engineers do not invest as much time in understanding social science as do social scientists in understanding science and engineer- ing. Natural scientists and engineers often challenge our writing style, arguing that it is opaque and overly technical. Clearly there is a power differential at work in these chal- lenges: scientists expect our knowledge to be transparent, but are happy enough for their own literature to remain inaccessible to outsiders. Attempts to shift this, and to engage scientists and engineers in the academic literature generally end in frustration. Using art and design, creative and playful methods, as well as experimenting with different forms of textual representations is thus going to be an important part of how more sub- stantive integration is accomplished. This will involve experimenting with how our research is integrated and how critical commentary is developed and deployed. Collaborative experimentation Our ﬁrst rule of thumb is that experimentation with post-ELSI forms of integration should be developed collaboratively with scientists and engineers. In this regard, commitments to working together should be practical and rooted in the everyday situations in which they are to be implemented. They should be sensitive to the everyday nature of academic and industrial work. For example, they might have to take account of the time required and costs incurred in attempts to collaborate, ensuring that adequate resources are devoted to experiments with novel practices. Experiments should be sensitive to context: not all sociotechnical research and innovation practices can be integrated with social science practices in the same fashion. General approaches, as developed in CTA and so forth can be useful but may need to be refashioned within the speciﬁc set of social, technical, legal and political relations within a given project, ﬁeld, or discipline. Researchers should experiment with different approaches, bringing together different orientations into novel combinations in order to explore new ways of working together. The onus in collaborations tends to be on social scientists to work towards integrating themselves. However, it is important to emphasise the need for scientists and engineers themselves to experiment with how they conduct their everyday work, make knowledge and develop technical innovations as part of an interdisciplinary mix. If this commitment cannot be ensured then efforts on all parts are likely to fail. As such, clear commitments to experiment with working collaboratively should be made before grant applications are awarded and time devoted to these experimental processes. This has implications for research funders, who must ensure that social science is being integrated in a sensible and productive fashion, and that there are clear commitments to experimentation on both sides written into the structures of calls and funded grants. Social science cannot JOURNAL OF RESPONSIBLE INNOVATION 7 75 merely be an ‘add-on’, separated from the day-to-day work of innovation, and certainly should not be added to the grant at the last minute (Viseu 2015). g In order to fulﬁl our side of the bargain, social scientists have to take responsibility for how we enter into collaborations. Last-minute invitations to collaborate must often be refused. Moreover, we have to ﬁnd ways in which our expertise can form a part of mutually productive collaborative relationships rather than acting only as external critics. Collaborative experimentation This form of critique has its place as well, but alone will not be adequate to the task of achieving more substantive forms of integration and changes in practices on both sides. Much like experiments in science, we must be adventurous and playful, willing to explore the unknown, tinker with our methods and be resilient in the face of failure. In this regard, we have to take risks. Failures should not considered as disastrous but as lessons to be learned from. Taking risks There are some emerging examples of this kind of work in the context of synthetic biology (Balmer and Bulpin 2013; Ginsberg et al. 2014). 76 A. S. BALMER ET AL. 76 A. S. BALMER ET AL. A. S. BALMER ET AL. 76 Collaborative reﬂexivity Such experiments in form and practice can also help to bring about collaborative reﬂexivity. We view reﬂexive practice as vital to fruitful collaborations with scientists and engineers and to the possibility of making the move from instrumental and imposed roles to more copro- ductive and chosen ones. In this regard, helping to integrate reﬂexivity in science is an important outcome for collaborations, but we must also be reﬂexively attuned to how our collaborations themselves are enacted in day-to-day practice and to how they are awarded credibility. Collaborative reﬂexivity thus means engaging all collaborative partners in reﬂections on collaborative relationships, regarding how they are experienced; how this might be related to organisational, material, or social factors; how risks are being taken and vulnerabilities managed; and how contributions to collaborations are being valued. Furthermore, much like collaborative experimentation, collaborative reﬂexivity has to be situated and speciﬁc. What practicing reﬂexivity entails in one collaboration might be irrelevant in another and so it should be actively negotiated between practitioners, modi- ﬁed as collaborations develop, and evaluated based on conditions particular to the context. These encounters must be supported by all parties. Making reﬂexivity a collaborative enterprise might also help to free social scientists from being positioned either as foretel- lers or ‘yes men’ (Balmer et al. 2015). Indeed, regular engagement in collaborative reﬂex- ivity can help to talk about such issues, about the constraints faced by all parties, to discuss similarities and differences and to generally clear the air. On this note, it is important to be frank during collaborative reﬂections and to open-up discussions of unshared goals. Opening-up discussions of unshared goals We thus argue that as a rule of thumb it is generally worth the risk of being open and of opening-up such discussions, even if this sometimes comes at the cost of continued relationships. Opening-up discussions of unshared goals The integration of social sciences into science and engineering research is often under- stood to be important to ensuring national economic impact and successful innovation. But these might be less immediate goals for social scientists in such integrated positions, or at least those social scientists keen to bring about such impacts will generally hold a richer, more critical appreciation of the relations between science, innovation and the nation. As such, we think it is important to continue to negotiate expectations around what we hope to achieve from these collaborative experiments and what a successful impact might look like. Negotiating differences whilst maintaining relationships can often mean very frank discussions that – although they might not produce shared goals – can produce shared interests and more mutual understanding. Some have argued that, at least in some contexts, the answer lies in being comfortable with a degree of con- cealment about one’s aims in a collaboration (Fitzgerald et al. 2014). However, when working in long-term collaborations concealed goals and dispositions can become unbear- able, leading to signiﬁcant emotional burden and frustration. We thus argue that as a rule of thumb it is generally worth the risk of being open and of opening-up such discussions, even if this sometimes comes at the cost of continued relationships. The integration of social sciences into science and engineering research is often under- stood to be important to ensuring national economic impact and successful innovation. But these might be less immediate goals for social scientists in such integrated positions, or at least those social scientists keen to bring about such impacts will generally hold a richer, more critical appreciation of the relations between science, innovation and the nation. As such, we think it is important to continue to negotiate expectations around what we hope to achieve from these collaborative experiments and what a successful impact might look like. Negotiating differences whilst maintaining relationships can often mean very frank discussions that – although they might not produce shared goals – can produce shared interests and more mutual understanding. Some have argued that, at least in some contexts, the answer lies in being comfortable with a degree of con- cealment about one’s aims in a collaboration (Fitzgerald et al. 2014). However, when working in long-term collaborations concealed goals and dispositions can become unbear- able, leading to signiﬁcant emotional burden and frustration. Neighbourliness To develop these new ways of collaborating, and to stick with collaborations even when there are differences, the concept of neighbourliness might be a useful rule of thumb JOURNAL OF RESPONSIBLE INNOVATION 77 77 77 for ethical decision-making. The concept has a long tradition, which in the West is rooted in the Christian bible and epitomised in the story of the good Samaritan. The concept has long since been secularised and plays a role in English Common Law (van Rijswijk 2012). Moreover, in contemporary feminist and post-structuralist research, it has been explicitly connected to questions of power, vulnerability, community and difference (Derrida 2000). Neighbours in Christianity are not necessarily related through their physical or emotional connection to each other but rather through their commitment to God (Painter 2012), and in its feminist and post-structuralist manifestation they are related through a necessary commitment to a certain notion of justice; although the everyday meaning of being a neighbour does imply a geographical relation. For our purposes, the concept can bring some of these aspects together to serve as a rule of thumb, that reminds us to link questions of ethics in practices of collaboration to issues of power, vulnerability and proximity. This all relates to the question of difference between natural and social scientists, their paradigms of research, theories of life and so forth. Studies of ‘boundary work’ and ‘boundary objects’ (Gieryn 1983; Star and Griesemer 1989) have shown how differences between groups seeking to work together are managed through objects and practices which all parties can use in their own ways. Such boundaries can make interdisciplinary communication and to some degree an interdisciplinary community, possible. But good fences do not make good neighbours. Attempts to collaborate and to bring about the inte- gration of reﬂexivity demand more than these well-established ways of managing our differences. To be neighbourly, then, would mean to recognise our differences and to respect them, whilst seeking to welcome each other without losing our sense of ourselves and our own commitments, responsibilities and proclivities. It is fundamentally an ethical disposition, which does not mean shying away from conﬂict, but rather making conﬂicts and their causes part of how we collaborate. Neighbourliness In this regard, to be neighbourly to each other in an interdisciplinary collaboration would involve working together to identify our differences, to explore how we are differently vulnerable and how there might be different relations of power involved in our collaborative work. By doing so, we can make this relevant to the decisions that we make not only about how our collaborations are organised but also about the research and innovation itself. Neighbourliness can be a style of ethical engagement in collaborative experimentation that emphasises the need to remain close and to work together in the face of open differ- ences and contestation. It is also a general disposition, rather than a set of rules or pro- cedures, and so can be adaptable to shifting relations in moves towards collaborative post-ELSI dynamics. We have to work harder to ﬁnd commonalities, to identify interests, hopes and worries that we can share whilst attending to our differences in vulnerability and to power relations. To be neighbourly, therefore, means attending to the ethics of the collaboration itself, not simply to the ethical implications of different technologies or scientiﬁc practices. This orientation to the double movement of ethics, towards publics and other actors but also inwards into the collaboration is what will help to make collaborations more productive and enable us to move past the ELSI models of interdisciplinarity. Ultimately, however, there will be structural impediments to bringing about neigh- bourly relations, which have to do with how power is organised at a more institutional level, in universities, research funding structures and in government. This is because A. S. BALMER ET AL. 78 78 our collaborations involve not only social and ethical relations but also political ones. There are political impediments to moving towards integrated positions that continue to position social scientists in more impoverished roles, and which often require us to empathise with those in power but rarely demand that those with power empathise with us (Balmer et al. 2015). Being proximal to our colleagues in the natural sciences and engineering should not come with the requirement to ‘get on board’ with the aims of the project or to ‘be more positive’. As such, being neighbourly should not imply that we should accept inequalities and suffering (van Rijswijk 2012). Neighbourliness Rather we should use our proximity and pursuit of collaborative relations to try to overcome such inequal- ities to the extent that this work is tolerable. We do not need to suffer unendingly if things are not working, and should make sure that our participation in collaborations is fruitful for our ends too. Funding We are grateful to the research funders that made the work possible, including: Economic and Social Sciences Research Council [RES-451-26-0871], [RES-061-25-0208], [RES-145-28-0003]; Bio- technology and Biological Sciences Research Council [BB/M017702/1], [BB/M018040/1], [BB/ F018746/1]; Engineering and Physical Sciences Research Council [EP/J02175X/1], [EP/H01912X/ 1], [EP/G036004/1], [EP/K020781/1], [EP/H023488/1], [EP/F007388/1]; European Research Council [ERC 616510-ENLIFE]; the European Commission Framework Programme 7 [FP7- KBBE-2011-5]; the Australian Research Council [FT130101302], [CE140100036]; the White Rose Scholarship fund. Acknowledgements We would like to thank all of the participants of the ESRC Seminar Series on Synthetic Biology and the Social Sciences and all of those with whom we have sought to collaborate in the natural sciences and engineering. Disclosure statement No potential conﬂict of interest was reported by the authors. Notes on contributors Andrew S. Balmer is Lecturer in Sociology at the University of Manchester and a member of the Morgan Centre for Research into Everyday Lives. He conducts research in science and technology studies and sociology more broadly. He has written about several topics, including lie detection, synthetic biology and interdisciplinary collaboration. Jane Calvert is a Reader in Science, Technology and Innovation Studies at the University of Edin- burgh. Her current research, funded by a European Research Council Consolidator grant, focuses on attempts to engineer living things in the emerging ﬁeld of synthetic biology, which raises intri- guing questions about design, evolution and ‘life’. She is also interested in the governance of emer- ging technologies, and in interdisciplinary collaborations between scientists, engineers, social scientists, artists and designers. Claire Marris is Senior Lecturer in the Department of Sociology, City University London. She con- ducts research in social studies of science, with a focus on modern biotechnologies and genetic modiﬁcation. She is interested in the relationship between scientiﬁc evidence and policy making, 7 JOURNAL OF RESPONSIBLE INNOVATION 79 notably in the area of risk assessment for crops and foods; and in questions around science and democracy. Susan Molyneux-Hodgson co-founded and is currently Director of SATIS (Science and Technology in Society) research group. Her research is focused in three substantive areas: the sociology of scien- tiﬁc communities; science and society relations; and interdisciplinary collaboration. Emma Frow is an assistant professor at Arizona State University, holding a joint appointment with the School for the Future of Innovation in Society and the School of Biological & Health Systems Engineering. Her research interests center around governance, standardization and valuation prac- tices in engineering and the life sciences, with a focus on synthetic biology. She has participated in synthetic biology projects with science and engineering colleagues in Europe and the US since 2007. Matthew Kearnes is Associate Professor and an Australian Research Council Future Fellow in Environmental Humanities at the School of Humanities and Languages, University of New South Wales. Matthew’s research is situated between the ﬁelds of Science and Technology Studies (STS), environmental sociology and contemporary social theory. His current work is focused on the social and political dimensions of technological and environmental change, and he has published widely on the ways in which the development of novel and emerging technologies is entangled with profound social, ethical and normative questions. Andrew S. Balmer http://orcid.org/0000-0002-7146-0448 Andrew S. Balmer http://orcid.org/0000-0002-7146-0448 Notes on contributors Kate Bulpin is a PhD student in Sociology at the University of Shefﬁeld and Research Associate in Sociology at the University of Manchester. She has written various articles about synthetic biology, focusing on the training of novices in iGEM, the formation of new epistemic communities and the role of social scientists in interdisciplinary spaces. Pablo Schyfter is Lecturer in the Science, Technology and Innovation Studies (STIS) group at the University of Edinburgh. His research focuses on a number of topics, including the sociology of knowledge, science and technology studies, gender, feminist theory, engineering studies and phenomenology. Adrian Mackenzie is Professor in Technological Cultures, Department of Sociology, Lancaster Uni- versity, and has published work on technology: Transductions: bodies and machines at speed, (2002); Cutting code: software and sociality (2006), Wirelessness: Radical Empiricism in Network Cultures (2010), and Into the Data: An Archaeology of Machine Learning (2017). He is currently working on the circulation of data intensive methods across science, government, and business in network media. He currently co-directs the Centre for Science Studies, Lancaster University, UK. Paul Martin is Head of the Department of Sociological Studies, University of Shefﬁeld. He has two main areas of research interest. The ﬁrst is the ethical, legal and social issues associated with emer- ging medical technologies and the second focuses on the commercialization of biotechnology and expectation dynamics in medical innovation. His research has previously examined the develop- ment of gene therapy, genomics, pharmacogenetics, stem cells and regenerative medicine. y g p Balmer, A. S., K. Bulpin, J. Calvert, M. Kearnes, A. Mackenzie, C. Marris, P. Martin, S. Molyneux- Hodgson, and P. Schyfter. 2012. “Towards a Manifesto for Experimental Collaborations between Social and Natural Scientists.” Accessed https://experimentalcollaborations.wordpress.com/ about/. Balmer, A. S., and K. Bulpin. 2013. “Left to their Own Devices: Post-ELSI, Ethical Equipment and the International Genetically Engineered Machine (iGEM) Competition.” BioSocieties 8 (3): 311–335. References Balmer, A. S., and K. Bulpin. 2013. “Left to their Own Devices: Post-ELSI, Ethical Equipment and the International Genetically Engineered Machine (iGEM) Competition.” BioSocieties 8 (3): 311–335. Balmer, A. S., K. Bulpin, J. Calvert, M. Kearnes, A. Mackenzie, C. Marris, P. Martin, S. Molyneux- Hodgson, and P. Schyfter. 2012. “Towards a Manifesto for Experimental Collaborations between Social and Natural Scientists.” Accessed https://experimentalcollaborations.wordpress.com/ Balmer, A. S., and K. Bulpin. 2013. “Left to their Own Devices: Post-ELSI, Ethical Equipment and the International Genetically Engineered Machine (iGEM) Competition.” BioSocieties 8 (3): 311–335. B l A S K B l i J C l M K A M k i C M i P M i S M l Balmer, A. S., and K. Bulpin. 2013. “Left to their Own Devices: Post-ELSI, Ethical Equipment and the International Genetically Engineered Machine (iGEM) Competition.” BioSocieties 8 (3): 311–335. y g p Balmer, A. S., K. Bulpin, J. Calvert, M. Kearnes, A. Mackenzie, C. Marris, P. Martin, S. Molyneux- Hodgson, and P. Schyfter. 2012. “Towards a Manifesto for Experimental Collaborations between Social and Natural Scientists.” Accessed https://experimentalcollaborations.wordpress.com/ about/. A. S. BALMER ET AL. 80 80 Balmer, A. S., K. Bulpin, and S. Molyneux-Hodgson. 2016. Synthetic Biology: A Sociology of Changing Practices. Basingstoke: Palgrave Macmillan. Balmer, A. S., J. Calvert, C. Marris, S. Molyneux-Hodgson, E. Frow, M. Kearnes, K. Bulpin, P. Schyfter, A. Mackenzie, and P. Martin. 2015. “Taking Roles in Interdisciplinary Collaborations: Reﬂections on working in Post-ELSI Spaces.” Science and Technology Studies 28 (3): 3–25. Barben, D., E. Fisher, C. Selin, and D. H. Guston. 2008. “Anticipatory Goverance of Nanotechnology: Foresight, Engagement and Integration.” In The Handbook of Science and Technology Studies, edited by E. J. Hackett, O. Amsterdamska, M. Lynch, and J. Wacjman, 3rd ed., 979–1000. Cambridge: MIT Press. Calvert, J., and P. Martin. 2009. “The Role of Social Scientists in Synthetic Biology.” EMBO Reports 10 (3): 201–204. Derrida, J. 2000. Of Hospitality: Anne Dufourmantelle Invited Jacques Derrida to Respond. Translated by Rachel Bowlby. Stanford, CA: Stanford University Press. Fisher, E., and G. Maricle. 2015. “Higher-level Responsiveness? Socio-Technical Integration within US and UK Nanotechnology Research Priority Setting.” Science and Public Policy 42 (1): 72–85. Fitzgerald, D., and F. Callard. 2014. “Social Science and Neuroscience Beyond Interdisciplinarity: Experimental Entanglements.” Theory, Culture & Society 32 (1): 3–32. Fitzgerald, D., M. M. Littleﬁeld, K. J. Knudsen, J. Tonks, and M. J. Dietz. 2014. References “Ambivalence, Equivocation and the Politics of Experimental Knowledge: A Transdisciplinary Neuroscience Encounter.” Social Studies of Science 44 (5): 701–721. f Fortun, M. 2005. “For an Ethics of Promising, or: A Few Kind Words about James Watson.” New Genetics and Society 24 (2): 157–174. Gieryn, T. F. 1983. “Boundary-work and the Demarcation of Science from Non-science: Strains and Interests in Professional Ideologies of Scientists.” American Sociological Review 48 (6): 781–795. Ginsberg, A. D., J. Calvert, P. Schyfter, A. Elﬁck, and D. Endy. 2014. Synthetic Aesthetics: Investigating Synthetic Biology’s Designs on Nature. Cambridge: MIT Press. Jasanoff, S. 2007. Designs on Nature: Science and Democracy in Europe and the United States. Princeton, NJ: Princeton University Press. Klein, J. T. 1990. Interdisciplinarity: History, Theory, and Practice. Detroit, MI: Wayne State University Press. Nordmann, A., and A. Rip. 2009. “Mind the Gap Revisited.” Nature Nanotechnology 4 (5): 273–274. Owen, R., J. Bessant, and M. Heintz, eds. 2013. Responsible Innovation: Managing the Responsible Emergence of Science and Innovation in Society. Chichester: Wiley. Owen, R., J. Bessant, and M. Heintz, eds. 2013. Responsible Innovation: Managing the Responsible Emergence of Science and Innovation in Society. Chichester: Wiley. Painter, J. 2012. “The Politics of the Neighbour.” Environment and Planning D: Society and Space 30: 515–533. Rabinow, P., and G. Bennett. 2012. Designing Human Practices: An Experiment with Synthetic Biology. Chicago, IL: University of Chicago Press. van Rijswijk, H. 2012. “Neighbourly Injuries: Proximity in Tort Law and Virginia Woolf’s Theory of Suffering.” Feminist Legal Studies 20: 39–60. Schot, J., and A. Rip. 1997. “The Past and Future of Constructive Technology Assessment.” Technological Forecasting and Social Change 54 (2–3): 251–268. Star, S. L., and J. R. Griesemer. 1989. “Institutional Ecology,‘Translations’ and Boundary Objects: Amateurs and Professionals in Berkeley’s Museum of Vertebrate Zoology, 1907–39.” Social Studies of Science 19 (3): 387–420. f Viseu, A. 2015. “Integration of Social Science into Research is Crucial.” Nature 525: 291. Wil d n J nd R Willi 2004 S th h S i Wh P bli E t N d t Viseu, A. 2015. “Integration of Social Science into Research is Crucial.” Nature 525: 291. Wilsdon, J., and R. Willis. 2004. See-through Science. Why Public Engagement Needs to Move Upstream. London: DEMOS. Wilsdon, J., and R. Willis. 2004. See-through Science. Why Public Engagement Needs to Move Upstream. London: DEMOS.
https://openalex.org/W3038958603	https://translationalneurodegeneration.biomedcentral.com/track/pdf/10.1186/s40035-020-00203-4	English	null	Downregulated miR-18b-5p triggers apoptosis by inhibition of calcium signaling and neuronal cell differentiation in transgenic SOD1 (G93A) mice and SOD1 (G17S and G86S) ALS patients	Translational neurodegeneration	2,020	cc-by	15,051	RESEARCH Open Access © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. (2020) 9:23 (2020) 9:23 Kim et al. Translational Neurodegeneration https://doi.org/10.1186/s40035-020-00203-4 Downregulated miR-18b-5p triggers apoptosis by inhibition of calcium signaling and neuronal cell differentiation in transgenic SOD1 (G93A) mice and SOD1 (G17S and G86S) ALS patients Ki Yoon Kim1, Yu Ri Kim1, Kyung Won Choi1, Mijung Lee1, Somyung Lee1, Wooseok Im1, Je-Young Shin1, Jin Young Kim2, Yoon Ho Hong3, Manho Kim1, Jong-Il Kim4 and Jung-Joon Sung1* Abstract Background: MicroRNAs (miRNAs) are endogenous non-coding RNAs that regulate gene expression at the post- transcriptional level and are key modulators in neurodegenerative diseases. Overexpressed miRNAs play an important role in ALS; however, the pathogenic mechanisms of deregulated miRNAs are still unclear. Methods: We aimed to assess the dysfunction of RNAs or miRNAs in fALS (SOD1 mutations). We compared the RNA-seq of subcellular fractions in NSC-34 WT (hSOD1) and MT (hSOD1 (G93A)) cells to find altered RNAs or miRNAs. We identified that Hif1α and Mef2c were upregulated, and Mctp1 and Rarb were downregulated in the cytoplasm of NSC-34 MT cells. Results: SOD1 mutations decreased the level of miR-18b-5p. Induced Hif1α which is the target for miR-18b increased Mef2c expression as a transcription factor. Mef2c upregulated miR-206 as a transcription factor. Inhibition of Mctp1 and Rarb which are targets of miR-206 induces intracellular Ca2+ levels and reduces cell differentiation, respectively. We confirmed that miR-18b-5p pathway was also observed in G93A Tg, fALS (G86S) patient, and iPSC- derived motor neurons from fALS (G17S) patient. Conclusions: Our data indicate that SOD1 mutation decreases miR-18b-5p, which sequentially regulates Hif1α, Mef2c, miR-206, Mctp1 and Rarb in fALS-linked SOD1 mutation. These results provide new insights into the downregulation of miR-18b-5p dependent pathogenic mechanisms of ALS. Keywords: miRNAs, Hif1α, Mef2c, Mctp1 and Rarb * Correspondence: jjsaint@snu.ac.kr Correspondence: jjsaint@snu.ac.kr 1Department of Neurology, Seoul National University Hospital 28 yongon-Dong, Chongno-gu, Seoul 110-744, Republic of Korea Full list of author information is available at the end of the article © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Page 2 of 21 Kim et al. Translational Neurodegeneration (2020) 9:23 Kim et al. Translational Neurodegeneration (2020) 9:23 In this study, we performed whole transcriptome ana- lysis to explain the role of SOD1 mutation by studying the subcellular fractionation of NSC-34 hSOD1 (wtNSC- 34) and hSOD1 (G93A) (mtNSC-34) cells. With respect to our RNA-seq results, we found several altered RNAs [hypoxia inducible factor 1 alpha (Hif1α), myocyte spe- cific enhancer factor 2c (Mef2c), Mctp1, and Rarb] in mtNSC-34 cells. The RNA levels of Hif1α and Mef2c were upregulated in the nucleus and the cytoplasm of mtNSC-34 cells. Specifically, the cytoplasmic RNAs of Hif1α and Mef2c were higher in number than nuclear RNAs in mtNSC-34 cells. Furthermore, Mctp1 and Rarb transcripts were highly expressed in the nucleus, but were decreased in the cytoplasm of mtNSC-34 cells. For the reason that Hif1α, Mef2c, Mctp1, and Rarb were ob- served to be different in the cytoplasm of mtNSC-34 cells, we hypothesized that these genes were post- transcriptionally regulated in mtNSC-34 cells. To iden- tify the post-transcriptional regulation of Hif1α, Mef2c, Mctp1, and Rarb, we found that miR-18b-5p was in- volved in the regulation of Hif1α, and miR-206 regulated both Mctp1 and Rarb. To determine whether or not miR-18b-5p is related to SOD1 mutation in ALS, we val- idated the expression of miR-18b-5p, miR-206, Hif1α, Mef2c, Mctp1, and Rarb in vitro and in vivo. Background g Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder that causes the degeneration of upper and lower motor neurons (MNs) in the spinal cord, brainstem, and cerebral cortex [1–5]. The most pathogenic mechanisms of ALS are gene mutations in the following genes: Cu/Zn superoxide dismutase 1 (SOD1), Fused in Sarcoma (FUS), C9orf72 and TAR DNA-binding protein 43 (TDP-43) [5– 12]. These gene mutations are related to various RNA me- tabolisms [2, 5–12]. Current findings strikingly suggest that gene mutations regulate the biogenesis of microRNAs (miR- NAs); these miRNAs play a pivotal role in the pathogenesis of neurodegenerative diseases [9, 10]. However, the specific interactive mechanisms of gene mutations and miRNAs re- lated to ALS have not been fully elucidated [11, 12]. y RNA biogenesis, including mRNA transcription, spli- cing, export, stability, and microRNAs (miRNAS), is emerging as an important factor in the pathogenesis of ALS [1, 13]. Recently, differentially expressed miRNAs (miR-23a, miR-455, miRb1336 and miR-b2403) have been identified between normal and diseased tissue [9, 14], and miRNAs are presently emerging as key factors in ALS, as well as in Huntington’s disease (miR-9), Alz- heimer’s disease (miR29a/b-1), and Parkinson’s disease (miR-205) [9, 15–22]. miRNAs are small non-coding single-stranded RNA molecules that regulate protein synthesis via RNA-dependent post-transcriptional gene regulation [23–25]. According to recent reports, miR- NAs are associated with cellular processes, such as cal- cium signaling, and neuronal differentiation [26–30]. Our results indicate that a new molecular pathway for miR-18b-5p, which sequentially regulates Hif1α, Mef2c, miR-206, Mctp1, and Rarb is involved in the pathogenic mecha- nisms of ALS-linked SOD1 mutations. Annexin V and PI analysis by flowcytometry y y y y NSCs were seeded in 6-well tissue culture plates. For using Annexin-V-FITC and PI Apoptosis Detection Kit (556,547, BD Bioscience, Eugene, NJ, USA), the adherent NSCs were detached with TripleExpress (12605–010, GIBCO, NY 14072 USA). The culture medium was then added to inactivate trypsin. The supernatant was re- moved after centrifuging for 5 min at 1500×g. and cells were stained with Annexin V-FITC and PI according to the manufacturer’s instructions. The cells were analyzed immediately after staining using a FACSCalibur (BD Bio- sciences, San Jose, CA). For each measurement, at least 20,000 cells were counted. Fluorescence was evaluated using the green or red channel, and the data were ana- lyzed using Flowwing Software (Version 2.5.1, Unversity of Turku, Filand). Methods Animals Translational Neurodegeneration (EGF; Invitrogen, Carlsbad CA USA), and 10 ng/mL basic fibroblast growth factor (CTP0261, bFGF; Invitrogen, Carlsbad CA USA) for culturing NSCs. For inducing differ- entiation [42], when the cells formed neurospheres sized about 50–100 μm in diameter, they were resuspended and transferred into a sterile 15-ml tube. The neurosphere pel- let was obtained by centrifuging at 100×g for 5 min at room temperature, and resuspended with differentiation culture medium (DMEM/F12, 1% PSA, 2% B27, and 5% FBS (FBS;12,483–020, Gibco, Grand Island NY USA)). NOVUS, Abingdon OX14 3NB UK), rabbit anti-Mef2c antibody (LS-C31031, LSBio, Seattle WA USA), rabbit anti- Mctp1 antibody (NBP1–83604, Novus Biologicals, Oakville ON L6M 2 V5 Canada), rabbit anti-Rarb (ab53161, abcam, Cambridge CB2 0AX UK), rabbit anti-Bax (SC-493, Santa Cruz, Dallas Texas USA), rabbit anti-Bcl2 (SC-492, Santa Cruz, Dallas Texas USA), mouse anti-Flag (F3165, SIGMA, Burlington MA USA), goat anti-GFP antibody (600–101- 215, Rockland, Gilbertsville PA USA), rabbit anti-mCherry (M11217, Thermofisher, Waltham MA USA) and mouse anti β-actin (sc-47,778, SantaCruz, Dallas Texas USA), rabbit anti-SOD1 (97,959, abcam, Cambridge CB2 0AX UK). (EGF; Invitrogen, Carlsbad CA USA), and 10 ng/mL basic fibroblast growth factor (CTP0261, bFGF; Invitrogen, Carlsbad CA USA) for culturing NSCs. For inducing differ- entiation [42], when the cells formed neurospheres sized about 50–100 μm in diameter, they were resuspended and transferred into a sterile 15-ml tube. The neurosphere pel- let was obtained by centrifuging at 100×g for 5 min at room temperature, and resuspended with differentiation culture medium (DMEM/F12, 1% PSA, 2% B27, and 5% FBS (FBS;12,483–020, Gibco, Grand Island NY USA)). Methods Animals Calcium signaling is a ubiquitous system that is involved in the regulation of cellular processes such as cell prolifera- tion and apoptosis [29, 30]. Intracellular Ca2+ levels are tightly controlled by transporters, and binding proteins [30]. The multiple-C2 domain transmembrane protein 1 (Mctp1), which has Ca2+ binding-affinity C2 domains, is essential for neuronal calcium signaling [30, 31]. High cellular Ca2+ con- centration leads to apoptosis through mitochondrial dys- function [29]. According to a recent report, intracellular Ca2+ levels are not only increased, but Ca2+ buffering is also perturbed following SOD1 mutation in ALS [32]. Animal studies were carried out in accordance with the In- stitutional Animal Care and Use Committee (IACUC) guidelines of Seoul National University for the care and use of laboratory animals. Transgenic mice expressing the hu- man G93A-mutated SOD1 gene (B6SJL-Tg (SOD1-G93A) 1 Gur/J; Jackson Laboratory, Bar Harbor, Me, USA) were used in this study. WT and SOD1-G93A mice used for bio- chemical analyses were sacrificed 120 days after birth. Preparation of NSC for in vitro model cell Neural stem cells (NSC) were derived from the subventri- cular zone of 9-week-old mice. The culture method has been used previously [41]. Briefly, mice brain tissues were dissected and minced in a dish containing HBSS. The cells were trypsinized with TripleExpress (12604–013, Thermo Scientific, Waltham MA USA) and incubated for 15 min at 37 °C. And then cells were seeded in a 6-well plate after centrifuging and resuspending with DMEM/F12 (11,320, 033, Invitrogen, Waltham MA USA) supplied with 1% PSA (penicillin-streptomycin; 15,140,122, Invitrogen, Carlsbad CA USA), 2% B27 Supplement (17,504,044, Gibco, BRL, Carlsbad CA USA), 10 ng/mL epidermal growth factor Cell growth and differentiation are regulated by reti- noids (vitamin A derivatives) and play a prominent role in neuronal cells [33]. Retinoic acid (RA) is a biologically ac- tive form of vitamin A and it regulates cell proliferation and differentiation [33]. During this process, retinoic acid receptor beta (Rarb), a transcriptional co-regulator with retinoic X receptor (RXR) mediates RA response [34]. Overexpression of mutated human SOD1 in MNs, such as NSC-34 cells, proves impaired cell differentiation and induced apoptosis [35, 36]. Dysregulation of calcium signaling and neuronal cell differentiation are related to apoptosis and are representative events in ALS pathogen- esis [36–40]. Page 3 of 21 Kim et al. Translational Neurodegeneration (2020) 9:23 Kim et al. Lactate dehydrogenase (LDH) release assay Lactate dehydrogenase (LDH) release assay Cell culture medium was collected and briefly centrifuged. The supernatants were transferred into wells in 96-well plates. Equal amounts of lactate dehydrogenase assay sub- strate (MAK066, SIGMA, Burlington MA USA), enzyme and dye solution were mixed. A Half volume of the above mixture was added to one volume of medium supernatant. After incubating at room temperature for 30 min, the re- action was terminated by the addition of 1/10 volume of 1 N HCl to each well. Spectrophotometrical absorbance was measured at a wavelength of 490 nm and reference wavelength of 690 nm. RNA interference experiments and Western blot analysis 40 nM of siRNA duplex were transfected in NSC-34 cells with RNAiMax transfection reagent (13778–150, Invitro- gen, Carlsbad CA USA) according to the manufacturer’s in- structions. miR-18b-5p and miR-206 inhibitor (anit-18b and anti-206) were obtained from COSMO GENETECH. siRNAs were synthesis from COSMO GENETECH. Target sequences of mouse siHif1α, siMctp1 and siRarb were 5′- AAGCAUUUCUCUCAUUUCCUCAUGG-3′ 5′-GCCAC UAUAUAUCAAGGUATT-3′ and 5′-GGAGCCUUCAAA GCAGGAATT-3′. NSC-34 cells were collected at 48 h after siRNA transfection. NSC-34 cells were collected at 48 h after anti-18b, miR-206, mCherry-Mctp1 and eGFP-Rarb transfection with Lipofectamine 2000 (11,668,019, Invitro- gen, Carlsbad CA USA). The cells were dispersed by pipet- ting and 20–30 mg of frozen tissues were homogenized in lysis buffer (10 mM Tris at pH 7.4, 1 mM ethylenediamine- tetra acetic acid [EDTA] at pH 8.0500 mM NaCl, and 0.5% Triton X-100) and incubated for 30 min on ice. The sam- ples were centrifuged at 15000 rpm at 4 °C for 20 min, obtaining a supernatant (soluble proteins) and a pellet. The pellets was re-suspended in lysis buffer and sonicated 3 times for 10 s and shaken for 1 h at 4 °C. Samples were cen- trifuged at 15000 rpm at 4 °C for 20 min, obtaining a super- natant (insoluble proteins). (Primary antibodies used in this study are mouse anti-Hif1α antibody (NB100–449, Intracellular Ca2+ assay The day before the experiment, plate the cells overnight in growth medium using 4 × 104 to 8 × 104 cells per well at a plating volume of 100 μl per well for 96-well plates. After 48 h, FLUOFORTE Dye-Loading Solution added to each well and incubated the cell plates for 45 min at 37 °C and 15 min at room temperature. Then, fluores- cence was measured at 490/525 nm using a fluorescence plate reader. The changes in intracellular calcium levels of each group were measured by quantifying the fold change of the fluorescence level of Fluo-4 with respect to the basal level. RNA-seq Three sets of wt and mtNSC-34 cells were grown and harvested with each set from a separate passage of single cell line. Following subcellular fractionation, transcrip- tomes of 12 samples were analyzed by RNA-seq (Macro- gen Inc.), the Illumina standard kit was used according to the manufacturer’s protocol. Briefly, 3 μg of each total RNA sample was used for polyA mRNA selection using streptavidin-coated magnetic beads, followed by thermal mRNA fragmentation. The fragmented mRNA was Plasmid construction The 3’UTR of mouse Mctp1 was amplified by PCR from NSC-34 cDNA (forward primer, 5′- CCGCTCGAGAAAG CTTGAATAATAGAAAT-3′ and reverse primer, 5′- CTA GTCTAGAATACATGGGTTTTTTGTTTG-3′). The 3’UTR of mouse Rarb was amplified by PCR from NSC-34 cDNA (forward primer, 5′- CCGCTCGAGAACGTGTAA TTACCTTGAAA-3′ and reverse primer, 5′- CTAGTC TAGACAAAGTCTTCAGAAACTTAA-3′). The 3’UTR of mouse Hif1α was amplified by PCR from NSC-34 cDNA (forward primer, 5′- CCGCTCGAGTGTTGGTTATTTT TGGACACT-3′ and reverse primer, 5′-CTAGTCTAGA ATATTGCATGAGTAACTGCTGGT-3′) The PCR prod- uct was cloned into pmirGLO dual-Luciferase vector Page 4 of 21 Kim et al. Translational Neurodegeneration (2020) 9:23 Kim et al. Translational Neurodegeneration (2020) 9:23 Gibco, Grand Island NY USA),100 U/ml penicillin, 100 μg/ml streptomycin (15140–122, GIBCO Grand Is- land NY USA). NSC-34 cells were differentiated in DMEM with 1% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin and 20 μM all-trans-RA (R2625, Sigma, Bur- lington MA USA). Cells were fixed at room temperature using 4% paraformaldehyde washed with PBS. Non- specific proteins were blocked by incubation in PBS con- taining 0.05% Bovine Serum Albumin (82–100-6, Milli- pore, Kankakee illimois USA) and 0.03% Triton X-100 (T8787, SIGMA, St. Louis MO USA) and treated with pri- mary antibodies were anti-Oct4 (ab27985, abcam, Cam- bridge, CB2 0AX, UK), anti-Nanog (ab80892, abcam, Cambridge, CB2 0AX, UK), anti-Nestin (ab22035, abcam, Cambridge, CB2 0AX, UK), anti-Sox2 (ab97959, abcam, Cambridge, CB2 0AX, UK), anti-choline acetyltransferase (AB144P, Chemicon), HLXB9 polyclonal antibody (PA5– 23407, Thermo Fisher, Rockford IL USA), MAP2 (Santa Cruz, Dallas Texas USA), anti-SOD1 (abcam, Cambridge, CB2 0AX, UK), Proteostat Aggresome Detection kit (Enz- 51,035-k100, Enzo Life Science, Farmingdale NY USA). Cells were then incubated with fluorescence-labeled sec- ondary antibodies, which are Alexa Fluor 488, 555 and 594 (Life Technologies) and finally mounted on micro slides by using Aqueous/Dry Mounting Medium (MO1, biomeda, Foster City CA) with DAPI (D1306, Thermo, Eugene Oregon USA). Imaging was performed using a confocal microscope (LEICA STED CW). To measure MAP2 staining neurites, at least 30 neurons were analyzed from three different experiments. 20x magnification im- ages were acquired. ImageJ software was used to deter- mine the average neurite length. Gibco, Grand Island NY USA),100 U/ml penicillin, 100 μg/ml streptomycin (15140–122, GIBCO Grand Is- land NY USA). NSC-34 cells were differentiated in DMEM with 1% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin and 20 μM all-trans-RA (R2625, Sigma, Bur- lington MA USA). Cells were fixed at room temperature using 4% paraformaldehyde washed with PBS. Plasmid construction The mouse Mctp1 was amplified by PCR from NSC-34 cDNA (forward primer, 5′- CCCAAGCTTATGTACCAGTTGGATATCACACTA-3′ and reverse primer, 5′-CCCAAGCTTGCCAAGGTT GTTTTTTCTTCC-3′). The PCR product was cloned into mCherry C1 (632524, Clontech, Mountain View CA USA) with Hind III (R0104, NEW ENGLAND BioLabs, Ipswich MA USA) restriction enzyme sites. The mouse Rarb was amplified by PCR from NSC-34 cDNA (forward primer, 5′-CCGCTAGCATGAGC ACCAGCAGCCACGC-3′ and reverse primer, 5′- CCACCGGTCTGCAGCAGTGGTGACTGAC-3′) Table S1. The PCR product was cloned into eGFP N1 (PT3027–5, Clontech, Mountain View CA USA) with Nhe I and Age I (R0131, R0552, NEW ENGLAND Bio- Labs, Ipswich MA USA) restriction enzyme sites. The 3’UTR of Mctp1 and Rarb mutagenesis was performed by KOD-Plus-Mutagenesis Kit (F0936K, TOYOBO, Osaka Japan). Primer sequences are given in Table S1. Plasmid construction Non- specific proteins were blocked by incubation in PBS con- taining 0.05% Bovine Serum Albumin (82–100-6, Milli- pore, Kankakee illimois USA) and 0.03% Triton X-100 (T8787, SIGMA, St. Louis MO USA) and treated with pri- mary antibodies were anti-Oct4 (ab27985, abcam, Cam- bridge, CB2 0AX, UK), anti-Nanog (ab80892, abcam, Cambridge, CB2 0AX, UK), anti-Nestin (ab22035, abcam, Cambridge, CB2 0AX, UK), anti-Sox2 (ab97959, abcam, Cambridge, CB2 0AX, UK), anti-choline acetyltransferase (AB144P, Chemicon), HLXB9 polyclonal antibody (PA5– 23407, Thermo Fisher, Rockford IL USA), MAP2 (Santa Cruz, Dallas Texas USA), anti-SOD1 (abcam, Cambridge, CB2 0AX, UK), Proteostat Aggresome Detection kit (Enz- 51,035-k100, Enzo Life Science, Farmingdale NY USA). Cells were then incubated with fluorescence-labeled sec- ondary antibodies, which are Alexa Fluor 488, 555 and 594 (Life Technologies) and finally mounted on micro slides by using Aqueous/Dry Mounting Medium (MO1, biomeda, Foster City CA) with DAPI (D1306, Thermo, Eugene Oregon USA). Imaging was performed using a confocal microscope (LEICA STED CW). To measure MAP2 staining neurites, at least 30 neurons were analyzed from three different experiments. 20x magnification im- ages were acquired. ImageJ software was used to deter- mine the average neurite length. (E1980, Promega, Madison WI USA) with Xho I and Xba I (R0145, NEW ENGLAND BioLabs, Ipswich MA USA) re- striction enzyme sites. miR-18b-5p (forward primer, 5′- CGCGGATCCACCATGGTGATTTAATCAGA-3′ and reverse primer, 5′- CCGCTCGAGCCGTTCAAATCAT TTCTCAA-3′) and miR-206 (forward primer, 5′- CGCGGATCCATTCTTCACACTTCTCACTT-3′ and re- verse primer, 5′-CCGCTCGAG ACGAAGAAGTCAAC AGCATA-3′) were amplified from NSC-34 cDNA by PCR. The PCR product was cloned into pCDNA3 vector (V79020, Invitrogen, Carlsbad CA USA) with BamH I and Xho I (R0136,R0146,NEW ENGLAND BioLabs, Ipswich MA USA) restriction enzyme sites. The mouse Mctp1 was amplified by PCR from NSC-34 cDNA (forward primer, 5′- CCCAAGCTTATGTACCAGTTGGATATCACACTA-3′ and reverse primer, 5′-CCCAAGCTTGCCAAGGTT GTTTTTTCTTCC-3′). The PCR product was cloned into mCherry C1 (632524, Clontech, Mountain View CA USA) with Hind III (R0104, NEW ENGLAND BioLabs, Ipswich MA USA) restriction enzyme sites. (E1980, Promega, Madison WI USA) with Xho I and Xba I (R0145, NEW ENGLAND BioLabs, Ipswich MA USA) re- striction enzyme sites. miR-18b-5p (forward primer, 5′- CGCGGATCCACCATGGTGATTTAATCAGA-3′ and reverse primer, 5′- CCGCTCGAGCCGTTCAAATCAT TTCTCAA-3′) and miR-206 (forward primer, 5′- CGCGGATCCATTCTTCACACTTCTCACTT-3′ and re- verse primer, 5′-CCGCTCGAG ACGAAGAAGTCAAC AGCATA-3′) were amplified from NSC-34 cDNA by PCR. The PCR product was cloned into pCDNA3 vector (V79020, Invitrogen, Carlsbad CA USA) with BamH I and Xho I (R0136,R0146,NEW ENGLAND BioLabs, Ipswich MA USA) restriction enzyme sites. Subcellular fractionation The 3’UTR of Mctp1 and Rarb analysis was performed using (pmirGLO dual-luciferase vector (E1330, Promega, Madison WI USA)). pmirGLO-Mctp1 and Rarb reporter were transiently transfected in NSC-34 mouse motor- neuron-like cells (contNSC-34) with miR-206. The 3’UTR of HIF1α analysis was performed using (pmirGLO dual- luciferase vector (Promega)). pmirGLO- HIF1α reporter were transiently transfected in contNSC-34 cells with miR-18b-5p. The luciferase activity was measured 48 h after the transfection and normalized using Dual- luciferase Reporter System (E1980, Promega, Madison WI USA) according to the manufacturer’s instruction. wt and mtNSC-34 cells were grown in a 10 cm dish and they were harvested in 450 ul of ice-cold buffer A (10 mM HEPES at pH 7.9, 10 mM KCl, 1 mM dithiothreitol [DTT], and 0.1 mM EDTA at pH 8.0). NSC-34 WT and MT cells dispersed by pipetting and incubated for 25 min on ice. Then 5 μl of 10% NP-40 was added, and cells were incubated for 2 min on ice. The nuclei were precip- itated by centrifugation at 5000 rpm for 3 min at 4 °C. The supernatant was taken as the cytoplsamic fraction. Human spinal cord samples Post mortem spinal cord specimens from six normal controls and one with fALS (G86S) were used (supple- mentary Table S6). Control spinal cord samples were obtained from The Netherlands Brain Bank and the guidelines by The Netherlands Brain Bank were followed. Post mortem spinal cord specimens from fALS (G86S) spinal cord and blood samples from fALS (G17S) were analyzed with Institutional permission under Re- view Board in Seoul National University Hospital. Generation of iPS cell lines Peripheral blood mononuclear cells (PBMC) isolated from whole blood using the Ficoll-Paque (17–1440-03, GE Healthcare Life Sciences, Marlborough MA USA) were cultured and expanded in StemPro-34 medium(10, 639,011, GIBCO, Grand Island NY USA) supplemented with 1% penicillin-streptomycin (15,140,122, Life tech- nologies, Grand Island NY USA), hSCF 100 ng/mL, hFLT-3100 ng/mL, hIL-3 20 ng/mL, and hIL-6 20 ng/mL (all of them from Peprotech, Rocky Hill, NJ USA). 1 × 106 PBMC were transduced overnight with Sendai vi- ruses containing Oct3/4, Sox2, Klf4, and cMyc (A16517, CytoTune®-iPS Sendai Reprogramming Kit, Life tech- nologies, Grand Island NY USA) at multiplicity of infec- tion (MOI) of 5. After 3 days, the transduced cells were plated on the 20μg/ml mitomycin C (M4287, SIGMA, St. Louis MO USA) treated-Human scrotum foreskin fi- broblasts (HFFs) in Cell Start-coated 35 mm dishes in complete StemPro-34 medium without cytokines and were changed medium every day until iPSCs started transitioning. And then, colony-formed iPSCs were re- placed the mixture of the StemPro-34 medium without cytokines and DMEM F/12 with 15% Knockout SR, 40 ng/ml bFGF, 1% nonessential amino acids, 50 U/ml of penicillin, 50 μg/ml streptomycin and 0.1 mM 2- mercaptoethanol (all of them from GIBCO, Grand Island NY USA). At day 30 or later, colonies were mechanically picked and passaged onto freshly mitotically inactivated HFFs. The iPSC colonies were picked for expension. The expressed transcripts was quantified using Kallisto. DESeq2 and edgeR were used to identify transcripts that were differentially expressed between wt and mtNSC-34 cells. Different expression level of each transcript was defined as those that satisfied 2 criteria: \|log2(fold- change)\| > 1 and p < 0.01 after the Benjamini-Hochberg correction in DEseq2 and edgeR. Reverse transcription quantitative PCR (RT-qPCR) p q q Total RNA was extracted from wt and mtNSC-34 cells by TRIzol reagent (5741, MRC, Cincinnati OH USA). RNA was measured in a spectrophotometer at 260-nm absorbance. RNA analysis was conducted as follows. Fifty nanograms of RNA were used as a template for quantitative RT-PCR amplification, using SYBR Green Real-time PCR Master Mix (QPK201, Toyobo, Osaka Japan). Primers were standardized in the linear range of cycle before the onset of the plateau. Primer sequences are given in supplementary table 3 and 4. Mouse and human GAPDH was used as an internal control. Two- step PCR thermal cycling for DNA amplification and real-time data acquisition were performed with an ABI StepOnePlus™Real-Time PCR System using the follow- ing cycle conditions: 95 °C for 1 min × 1 cycle, and 95 °C for 15 s, followed by 62 °C for 1 min × 50 cycles. Fluores- cence data were analyzed by the ABI StepOnePlus soft- ware and expressed as Ct the number of cycles needed to generate a fluorescent signal above a predefined threshold. The ABI StepOnePlus software set baseline and threshold values. Expression of each gene was nor- malized to Gapdh and expression of each miRNA was normalized to U6. The fold change in mRNA and miR- NAs expression versus controls calculated using the 2(−ΔΔCT) method. For miRNAs RT-qPCR, 50 ng of total NSC-34 cell lines culture, cell differentiation with retinoic acid and immunofluorescence NSC-34 mouse motor neuron-like cell lines (contNSC-34, wtNSC-34 (human SOD1) and mtNSC-34 (human SOD1 (G93A)) kindly provided by H Ryu, Korea Institute of Sci- ence and Technology, Seoul, Korea) were grown in Dul- becco’s modified Eagle’s medium (SH30243, Hyclone, Logan UT USA) supplemented with 10% FBS (16,000,044, Page 5 of 21 Page 5 of 21 Kim et al. Translational Neurodegeneration (2020) 9:23 Kim et al. Translational Neurodegeneration (2020) 9:23 subjected to cDNA synthesis using reverse transcriptase (SuperScript II) and random primers. The cDNA was further converted into double stranded cDNA and, after an end repair process (Klenow fragment, T4 polynucleo- tide kinase and T4polymerase), was finally ligated to Illu- mina paired end (PE) adaptors. Size selection was performed using a 2% agarose gel, generating cDNA li- braries ranging in size from 200 to 250 bp. Finally, the li- braries were enriched using 10 cycles of PCR and purified by the QIAquick PCR purification kit (28,106, Qiagen, PL Venlo Netherlands). The enriched libraries were diluted with Elution Buffer to a final concentration of 10 nM. Each library was run at a concentration of 8 pM on one Genome Analyzer (GAIIx) lane using 53 bp sequencing. Reads were then processed and aligned to the mouse genome UCSC build mm9 using GSNAP. The unit of measurement is Reads Per Kilobase of exon per Million fragments mapped (RPKM). RNA was reverse transcribed, using GenoExplore micro- RNA RT-qPCR Kit (2001, Geno Sensor Corporation, Tempe, Arizona 85,282 USA), and subsequently quanti- fied using specific primers (GenoExplorer microRNA RT-qPCR primer sets (2003, Geno Sensor Corporation, Tempe, Arizona 85,282 USA)) for U6, miR-18b-5p and miR-206 (mouse and human). Generation of neural stem cell from iPSC Neural stem cells were cultured in DMEM/F12 supplemented with 1% nones- sential amino acids, 50 U/ml of penicillin, 50 μg/ml streptomycin and 0.1 mM 2-mercaptoethanol plus 0.5% N2 supplement and 40 ng/ml b-fibroblast growth factor onto the Cell Start-coated plates. USA). Control experiments were performed in the absence of primary antibody or in the presence of blocking peptide. Statistical analysis The data are presented as the mean ± standard error of the mean (SEM). Data analysis was performed by Stu- dent’s t test or one-way ANOVAs followed by Mann- Whitney and Kruskal-Wallis tests. Differences were con- sidered statistically significant when p < 0.05. Differentiation of neural stem cells into motor neurons Differentiation of neural stem cells into motor neurons Neural stem cells were cultured in DMEM/F12 with 1% nonessential amino acids, 50 U/ml of penicillin, 50 μg/ml streptomycin and 0.1 mM 2-mercaptoethanol, 0.5% N2 and 40 ng/ml b-FGF for 2 days onto the Cell Start with 1 μg/ml laminin and 5μg/ml heparin-coated plates and then chan- ged neural induction medium, which consists of the mix- ture of DMEM/F12 and neurobasal medium(A3582901, GIBCO, Grand Island NY USA) supplemented with 0.1 mM 2-mercaptoethanol, 0.5% N2 supplement (21985–023, 17,502–048, GIBCO, Grand Island NY USA) and 40 ng/ml b-fibroblast growth factor), 10 ng/ml neural growth factor (13,257,019, Invitrogen, Grand Island NY USA), 10 ng/ml sonic hedgehog (8908-SH, R&D Systems, Minneapolis MN USA), 10 μM forskolin and 1 μM retinoic acid (R2625, Sigma, Burlington MA USA), glial cell-derived neuro- trophic factor(GDNF; 212-GD), brain-derived neurotrophic factor (BDNF; 248-BDB), ciliary neurotrophic factor, insulin-like growth factor 1 and neurotrophin-3 (NT3) (267-N3, R&D Systems, Minneapolis MN USA), all at 10 ng/ml, every day or every other day for a week [44]. Generation of neural stem cell from iPSC Colonies were detached by 2 mg/ml dispase and trans- ferred in embryoid body (EB) medium that contains Es- sential 6 Medium supplemented with 15% knockout SR (10828–028, Gibco, Grand Island NY USA), 50 U/ml of Kim et al. Translational Neurodegeneration (2020) 9:23 Kim et al. Translational Neurodegeneration Page 6 of 21 penicillin, 50 μg/ml streptomycin (15140–122, GIBCO Grand Island NY USA) to 60-mm incoated bacterial plate at 37 for 5-7 days with change of medium every single day. And then, formed EBs transferred to Cell Start coated-35-mm culture dish. The culture method has been used previously [43]. When EBs attached to dish after 2–3 days, changed 0.5% N2 in DMEM/F12 supplemented with 1% nonessential amino acids, 50 U/ ml of penicillin, 50μg/ml streptomycin and 0.1 mM 2- mercaptoethanol to the same based-medium plus 1% N2 supplement and 40 ng/ml b-fibroblast growth factor every other day until neural structures appeared. These neural structures were mechanically isolated and cul- tured floating in the medium and then became spheres. These spheres were fragmented mechanically and cul- tured onto Cell start-coated culture dished for 1 day and treated accutase (A11105–01, Gibco, Grand Island NY USA) at 37 °C incubator or 1 h. Neural stem cells were cultured in DMEM/F12 supplemented with 1% nones- sential amino acids, 50 U/ml of penicillin, 50 μg/ml streptomycin and 0.1 mM 2-mercaptoethanol plus 0.5% N2 supplement and 40 ng/ml b-fibroblast growth factor onto the Cell Start-coated plates. penicillin, 50 μg/ml streptomycin (15140–122, GIBCO Grand Island NY USA) to 60-mm incoated bacterial plate at 37 for 5-7 days with change of medium every single day. And then, formed EBs transferred to Cell Start coated-35-mm culture dish. The culture method has been used previously [43]. When EBs attached to dish after 2–3 days, changed 0.5% N2 in DMEM/F12 supplemented with 1% nonessential amino acids, 50 U/ ml of penicillin, 50μg/ml streptomycin and 0.1 mM 2- mercaptoethanol to the same based-medium plus 1% N2 supplement and 40 ng/ml b-fibroblast growth factor every other day until neural structures appeared. These neural structures were mechanically isolated and cul- tured floating in the medium and then became spheres. These spheres were fragmented mechanically and cul- tured onto Cell start-coated culture dished for 1 day and treated accutase (A11105–01, Gibco, Grand Island NY USA) at 37 °C incubator or 1 h. SOD1 mutation (G93A) induces apoptosis by aberrant gene expression To study the mechanism of RNA biogenesis by SOD1 mutation, we performed transcriptome analysis to iden- tify RNA processing variation via subcellular fractionized RNAs in NSC-34 hSOD1 (wtNSC-34) and hSOD1 (G93A) (mtNSC-34) stable cell lines (Fig. 1a). By per- forming comparative RNA-seq analysis between the nu- cleus and cytoplasm of WT and mtNSC-34 cells, we identified significant changes in Mctp1 and Rarb expres- sion (Fig. 1a). The heat map shows that Mctp1 and Rarb were upregulated in the nucleus, but greatly downregu- lated in the cytoplasm of mtNSC-34 cells (Fig. 1a). To validate Mctp1and Rarb transcripts, we carried out re- verse transcriptase PCR (RT-PCR) analysis and con- firmed the presence of Mctp1 and Rarb mRNAs (Fig. 1b). To understand how Mctp1 and Rarb transcripts are regulated in mtNSC-34 cells, we performed revers tran- scription quantitative PCR (RT-qPCR) analysis in WT and mtNSC-34 cells (Fig. 1c). We found that Mctp1 and Rarb mRNAs were low in mtNSC-34 cells (Fig. 1c). These results led us to assume that Mctp1 and Rarb transcripts might be post-transcriptionally regulated or deficiently transported in the cytoplasm of mtNSC-34 cells. For the reason that miRNAs are one of the most representative the post-transcriptional regulators [45– 47], we focused on the post-transcriptional regulation and identified that Mctp1 and Rarb mRNAs are targeted by miR-206 (Additional file 1: Fig. S1A). Furthermore, we discovered that miR-206 was remarkably upregulated in mtNSC-34 cells (Fig. 1d). Moreover, we investigated whether or not Mctp1 and Rarb were related to calcium signaling and neuronal differentiation, respectively, and measured intracellular Ca2+ levels and total neurite length in WT and mtNSC-34 cells (Fig. 1e and f). As ex- pected, the intracellular Ca2+ levels increased, and the neurite outgrowth decreased significantly in mtNSC-34 cells with the aggregation of SOD1 (G93A) (Fig. 1e and f). These results showed that the downregulated Mctp1 and Rarb could stimulate the alteration of calcium sig- naling and cell differentiation in mtNSC-34 cells, re- spectively. From the RNA-seq results, we also identified that Hif1α and Mef2c, which are regulated by Hif1α [48], Confocal microscopy Immunofluorescence staining and confocal microscopy was used to determine mouse anti-Chat (AB144P, Chemicon, Tumecula CA USA). Images were analyzed using a spin- ning disk confocal microscope (Leica, Buffalo Grove IL USA). Deconvolution and 3-dimensional construction of the confocal image was performed by AQI-X-COMBO- CWF program (Media cybernetics Inc., Rockville, MD, Page 7 of 21 Kim et al. Translational Neurodegeneration (2020) 9:23 Fig. 1 SOD1 mutation (G93A) is related to altered gene expression and induces apoptosis. a The heat map of RNA-seq from subcellular fractionized wt and mtNSC-34 stable cells showed that relative expressions of Hif1α, Mef2c, Mctp1 and Rarb are altered by SOD1(G93A). The relative expression genes was displayed as colors: higher (red) or lower (green). The heat map represents the average of three samples. b RT-PCR analysis explained that Mctp1 and Rarb introns are upregulated in nucleus of mtNSC-34 cells and Mctp1 and Rarb exons are highly downregulated in cytoplasm of mtNSC-34 cells. c RT-qPCR analysis showed that Mctp1 and Rarb transcripts are decreased in mtNSC-34 cells. d miR-206 expression was induced in mtNSC-34 cells. e and f SOD1 aggregation (arrow) prevents neuronal differentiation (MAP2) and increases intracellular Ca2+ levels (WT (0.072) versus MT (0.108) in fluorescence intensities from baseline 490/525 ratio) in mtNSC-34 cells. Scale bar, 40 μm. Significantly different at , p < 0.05; , p < 0.005. The experiments were replicated 3 times Fig. 1 SOD1 mutation (G93A) is related to altered gene expression and induces apoptosis. a The heat map of RNA-seq from subcellular fractionized wt and mtNSC-34 stable cells showed that relative expressions of Hif1α, Mef2c, Mctp1 and Rarb are altered by SOD1(G93A). The relative expression genes was displayed as colors: higher (red) or lower (green). The heat map represents the average of three samples. b RT-PCR analysis explained that Mctp1 and Rarb introns are upregulated in nucleus of mtNSC-34 cells and Mctp1 and Rarb exons are highly downregulated in cytoplasm of mtNSC-34 cells. c RT-qPCR analysis showed that Mctp1 and Rarb transcripts are decreased in mtNSC-34 cells. d miR-206 expression was induced in mtNSC-34 cells. e and f SOD1 aggregation (arrow) prevents neuronal differentiation (MAP2) and increases intracellular Ca2+ levels (WT (0.072) versus MT (0.108) in fluorescence intensities from baseline 490/525 ratio) in mtNSC-34 cells. Scale bar, 40 μm. Significantly different at , p < 0.05; *, p < 0.005. Confocal microscopy The experiments were replicated 3 times increased in the nucleus and cytoplasm of mtNSC-34 cells (Fig. 1a). Interestingly, Mef2c has been reported to regulate miR-206 as a transcription factor [49]. We validated that Hif1α and Mef2c expression was significantly elevated in mtNSC-34 cells (Fig. 2a and b). From these results, we also hypothesized that an unknown target miRNA of Hif1α might be downregulated, and that the upregulated Hif1α could upregulate Mef2c expression serially. We also identified that the miR-18b-5p was target of Hif1α mRNAs (Additional file 1: Fig. S1B) [50]. RT-qPCR analysis con- firmed that miR-18b-5p was significantly reduced in mtNSC-34 cells (Fig. 2e). We also confirmed that only mtSOD1 (G93A) were associated with miR-18b-5p, Hif1α, Mef2c, Mctp1 and Rarb in NSC-34 (control, wtSOD1, and mtSOD1) stable cell lines (Fig. 2a-e). Owing to the fact that SOD1 mutations cause apoptosis [51], we measured the Page 8 of 21 Kim et al. Translational Neurodegeneration (2020) 9:23 Kim et al. Translational Neurodegeneration (2020) 9:23 Fig. 2 SOD1 mutation (G93A) induces apoptosis. a Western blot analysis verified the protein levels of Hif1α, Mef2c, Mctp1, Rarb, Bax, and Bcl2 in three different NSC-34 cell lines. b Quantification analysis of western blot. The data represent the average ± SEM of 3 separate experiments. Significantly different at , p < 0.5; *, p < 0.05. c Hif1α and Mef2c transcripts were increased in mtNSC-34 cells. d Mctp1 and Rarb transcripts were decreased in mtNSC-34 cells e miR-18b (miR-18b-5p) was reduced in mtNSC-34 cells. f LDH releases analysis explained that SOD1 mutation (G93A) induces apoptosis. g Bax mRNAs were upregulated and Bcl2 mRNAs were downregulated in mtNSC-34 cells. Significantly different at , p < 0.05; *, p < 0.005. The experiments were replicated 3 times Fig. 2 SOD1 mutation (G93A) induces apoptosis. a Western blot analysis verified the protein levels of Hif1α, Mef2c, Mctp1, Rarb, Bax, and Bcl2 in three different NSC-34 cell lines. b Quantification analysis of western blot. The data represent the average ± SEM of 3 separate experiments. Significantly different at , p < 0.5; *, p < 0.05. c Hif1α and Mef2c transcripts were increased in mtNSC-34 cells. d Mctp1 and Rarb transcripts were decreased in mtNSC-34 cells e miR-18b (miR-18b-5p) was reduced in mtNSC-34 cells. f LDH releases analysis explained that SOD1 mutation (G93A) induces apoptosis. g Bax mRNAs were upregulated and Bcl2 mRNAs were downregulated in mtNSC-34 cells. Confocal microscopy In order to confirm that the overexpression of miR-18b-5p was mediated by the regu- lation of Ca2+ signaling and cell differentiation, we mea- sured intracellular Ca2+ levels and total neurite length using the cell differentiation assay. We observed aggre- gated SOD1 in mtNSC-34 cells. Nevertheless, overex- pressed miR-18b-5p cells not only reduced intracellular Ca2+ levels, but also increased neurite outgrowth (Fig. 3i and j). These results proved that miR-18b-5p was related to Hif1α expression and apoptosis. S3H). In order to observe the reduced apoptosis, we ap- plied the LDH assay and, as we expected, LDH release was reduced by siHif1α in mtNSC-34 cells (Additional file 3: Fig. S3I). These results suggested that Hif1α directly regulated Mef2c and contributed to apoptosis. miR-206 not only controls post-transcriptional regulation of both Mctp1 and Rarb, but also induces apoptosis To further investigate the roles of miR-206, we car- ried out a luciferase reporter assay using the 3′ UTR of Mctp1 and Rarb. Mctp1 was significantly downreg- ulated and intracellular Ca2+ levels were increased by overexpressed miR-206 (Fig. 4a-d). Rarb levels were also decreased by miR-206 (Fig. 4d and e). The post- transcriptionally downregulation of Rarb by miR-206 also caused the neuronal differentiation (Fig. 4e). We then observed that overexpressed miR-206 induced apoptosis, because Bax expression was upregulated, while Bcl2 expression was downregulated by miR-206 (Fig. 4f and g). Overexpression of miR-206 was con- firmed by RT-qPCR analysis and miR-206 consider- ably induced LDH release (Fig. 4h and i). We opted to have it reconfirmed by performing flow cytometry analysis of mNSCs overexpressing miR-206 ectopi- cally. As a result, apoptosis was markedly induced (Fig. 4j). To identify whether or not miR-206 simul- taneously regulates Mctp1 and Rarb, we then per- formed anti-206 (anti-miR-206) experiments. Mctp1 and Rarb proteins were significantly increased by transfected anti-206 (anti-miR-206) in mtNSC-34 cells (Additional file 4: Fig. S4A and G). Anti-206 (anti- miR-206) also reduced Bax and induced Bcl2 levels in mtNSC-34 cells (Additional file 4: Fig. S4A, D, E, and G). Mctp1 and Rarb transcripts by anti-206 (anti- miR-206) showed the same results as western blot in the mtNSC-34 cells (Additional file 4: Fig. S4B and C). In addition, the LDH assay using anti-206 (anti- miR-206) in mtNSC-34 cells showed that anti-206 (anti-miR-206) restored cell death (Additional file 4: Fig. S4F). These findings suggested that miR-206 dir- ectly regulates Mctp1 and Rarb, and then induces apoptosis. Confocal microscopy Significantly different at , p < 0.05; *, p < 0.005. The experiments were replicated 3 times levels of Bax and Bcl2 as pro- and anti-apoptotic markers. Bax expression was increased, while Bcl2 expression was decreased in mtNSC-34 cells (Fig. 2a, b, and g). We then measured the amount of lactate dehydrogenase (LDH) re- leased to observe apoptosis in NSC-34 cell lines. As ex- pected, LDH release increased in mtNSC-34 cells (Fig. 2f). These results suggested that downregulated miR-18b-5p might sequentially control Hif1α, Mef2c, miR-206, Mctp1, and Rarb expression in SOD1 mutation. Downregulated miR-18b-5p directly upregulates Hif1α and enhances apoptosis in mtNSC-34 cells Downregulated miR-18b-5p directly upregulates Hif1α and enhances apoptosis in mtNSC-34 cells In order to examine whether or not sequential events of downregulated miR-18b-5p are associated with apop- tosis, we used RNAi method to reduce miR-18b-5p in contNSC-34 cells. Transfected Anti-18b (anti-miR-18b- 5p) elevated Hif1α and Mef2c proteins, while it de- creased Mctp1 and Rarb proteins (Additional file 2: Fig. S2A). RT-qPCR analysis also confirmed that mRNAs of In order to examine whether or not sequential events of downregulated miR-18b-5p are associated with apop- tosis, we used RNAi method to reduce miR-18b-5p in contNSC-34 cells. Transfected Anti-18b (anti-miR-18b- 5p) elevated Hif1α and Mef2c proteins, while it de- creased Mctp1 and Rarb proteins (Additional file 2: Fig. S2A). RT-qPCR analysis also confirmed that mRNAs of Page 9 of 21 Page 9 of 21 Kim et al. Translational Neurodegeneration (2020) 9:23 Kim et al. Translational Neurodegeneration Hif1α and Mef2c were decreased, and those of Mctp1 and Rarb were increased (Additional file 2: Fig. S2B-E). Downregulated miR-18b-5p was found to finally induce apoptosis, because expression of Bax increased, while ex- pression of Bcl2 decreased. In addition, LDH release was also increased by anti-18b (anti-miR-18b-5p) (Additional file 2: Fig. S2A, F, G, and H). Interestingly, RT-qPCR analysis results showed that miR-206 was rapidly in- duced by anti-18b (anti-miR-18b-5p) (Additional file 2: Fig. S2I and J). To identify apoptosis by downregulated miR-18b-5p, we isolated mouse neural stem cells (mNSCs) from the brains of mice and transfected anti- 18b (anti-miR-18b-5p). Flow cytometry analysis showed that anti-18b (anti-miR-18b-5p) elevates apoptosis in mNSCs (Additional file 2: Fig. S2K). Based on these re- sults, we naturally wondered whether transfected miR- 18b-5p could restored apoptosis. We ectopically expressed miR-18b-5p and measured the protein of Hif1α, Mef2c, Mctp1, Rarb, Bax, and Bcl2 in mtNSC-34 cells. Hif1α directly regulates Mef2c expression and apoptosis Hif1α directly regulates Mef2c expression and apoptosis As we had mentioned previously in this article that de- creased miR-18b-5p upregulated Hif1α, which subse- quently increased Mef2c, we confirm that Hif1α regulated Mef2c. We applied RNAi to decrease Hif1α, and observed the regulation of Mef2c, miR-206, Mctp1, and Rarb in mtNSC-34 cells (Additional file 3: Fig. S3). The expression of Mef2c was reduced by siHif1α in mtNSC-34 cells (Add- itional file 3: Fig. S3A-C). Mctp1 and Rarb expressions were also increased (Additional file 3: Fig. S3A, D, and E). To prove that Hif1α restored apoptosis, we observed the downregulation of Bax and upregulation of Bcl2 by siHif1α in mtNSC-34 cells (Additional file 3: Fig. S3A, F, and G). We also indirectly identified that Mef2c, inhibited by siHif1α, decreased miR-206 (Additional file 3: Fig. Post-transcriptionally downregulation of Mctp1 and Rarb inhibited calcium signaling and neuronal cell differentiation in mtNSC-34 cells Confocal microscopy Hif1α and Mef2c expressions decreased (Fig. 3a and b) while Mctp1 and Rarb expressions increased by increased miR-18b-5p (Fig. 3a and c). Ectopically overex- pressed miR-18b-5p induced the reversible changes in Bax and Bcl2 expressions in mtNSC-34 cells (Fig. 3a and d). miR-206 was also downregulated by overexpressed miR-18b-5p (Fig. 3e and f). The LDH release assay showed that induced miR-18b-5p prevented apoptosis (Fig. 3g). We also confirmed that Hif1α was target of miR-18b-5p in contNSC-34 cells (Fig. 3h). In order to confirm that the overexpression of miR-18b-5p was mediated by the regu- lation of Ca2+ signaling and cell differentiation, we mea- sured intracellular Ca2+ levels and total neurite length using the cell differentiation assay. We observed aggre- gated SOD1 in mtNSC-34 cells. Nevertheless, overex- pressed miR-18b-5p cells not only reduced intracellular Ca2+ levels, but also increased neurite outgrowth (Fig. 3i and j). These results proved that miR-18b-5p was related to Hif1α expression and apoptosis. Hif1α and Mef2c were decreased, and those of Mctp1 and Rarb were increased (Additional file 2: Fig. S2B-E). Downregulated miR-18b-5p was found to finally induce apoptosis, because expression of Bax increased, while ex- pression of Bcl2 decreased. In addition, LDH release was also increased by anti-18b (anti-miR-18b-5p) (Additional file 2: Fig. S2A, F, G, and H). Interestingly, RT-qPCR analysis results showed that miR-206 was rapidly in- duced by anti-18b (anti-miR-18b-5p) (Additional file 2: Fig. S2I and J). To identify apoptosis by downregulated miR-18b-5p, we isolated mouse neural stem cells (mNSCs) from the brains of mice and transfected anti- 18b (anti-miR-18b-5p). Flow cytometry analysis showed that anti-18b (anti-miR-18b-5p) elevates apoptosis in mNSCs (Additional file 2: Fig. S2K). Based on these re- sults, we naturally wondered whether transfected miR- 18b-5p could restored apoptosis. We ectopically expressed miR-18b-5p and measured the protein of Hif1α, Mef2c, Mctp1, Rarb, Bax, and Bcl2 in mtNSC-34 cells. Hif1α and Mef2c expressions decreased (Fig. 3a and b) while Mctp1 and Rarb expressions increased by increased miR-18b-5p (Fig. 3a and c). Ectopically overex- pressed miR-18b-5p induced the reversible changes in Bax and Bcl2 expressions in mtNSC-34 cells (Fig. 3a and d). miR-206 was also downregulated by overexpressed miR-18b-5p (Fig. 3e and f). The LDH release assay showed that induced miR-18b-5p prevented apoptosis (Fig. 3g). We also confirmed that Hif1α was target of miR-18b-5p in contNSC-34 cells (Fig. 3h). Confocal microscopy To confirm that miR-206 post- transcriptionally regulates Mctp1 and Rarb, we de- leted miR-206 binding sites from 3’UTR of Mctp1 and Rarb. We carried out a luciferase reporter assay using the mutation 3′ UTR of Mctp1 and Rarb. It did not show any significant change in miR-206 expres- sion (Additional file 4: Fig. S4H and I). differentiation in mtNSC-34 cells We have mentioned earlier in this article that downregu- lated miR-18b-5p regulated Hif1α which are related to Page 10 of 21 Kim et al. Translational Neurodegeneration (2020) 9:23 Kim et al. Translational Neurodegeneration (2020) 9:23 Fig. 3 miR-18b (miR-18b-5p) regulates Hif1α and reduces apoptosis in mtNSC-34 cells. a Overexpressed miR-18b (miR-18b-5p) decreased Hif1α and Mef2c proteins. Both Mctp1 and Rarb expression were increased by miR-18b (miR-18b-5p). Downregulated Bax and upregulated Bcl2 by miR- 18b (miR-18b-5p) diminished apoptosis in mtNSC-34 cells. b RT-qPCR analysis showed low expression of Hif1α and Mef2c mRNAs. c Mctp1 and Rarb transcripts were highly expressed by miR-18b (miR-18b-5p). d Bax mRNAs were decreased and Bcl2 mRNAs were increased by overexpressed miR-18b. e miR-18b (miR-18b-5p) was overexpressed in mtNSC-34 cells. f miR-206 was reduced by miR-18b (miR-18b-5p). g LDH release analysis explained that transfected miR-18b (miR-18b-5p) restores apoptosis. h Luciferases assay with 3′ UTR of Hif1α showed that Hif1α is target of miR-18b in contNSC-34 cells. i and j Overexpression of miR-18b (miR-18b-5p) enhanced neuronal differentiation (MAP2) and attenuated intracellular Ca2+ levels (Cont (0.098) versus miR-18b (miR-18b-5p) (0.051) in fluorescence intensities from baseline 490/525 ratio) in mtNSC-34 cells. Empty vector served as a negative control (Cont). Arrow represents SOD1 aggregation (green). Scale bar, 40 μm. Significantly different at , p < 0.05; *, p < 0.005. The experiments were replicated 5 times Fig. 3 miR-18b (miR-18b-5p) regulates Hif1α and reduces apoptosis in mtNSC-34 cells. a Overexpressed miR-18b (miR-18b-5p) decreased Hif1α and Mef2c proteins. Both Mctp1 and Rarb expression were increased by miR-18b (miR-18b-5p). Downregulated Bax and upregulated Bcl2 by miR- 18b (miR-18b-5p) diminished apoptosis in mtNSC-34 cells. b RT-qPCR analysis showed low expression of Hif1α and Mef2c mRNAs. c Mctp1 and Rarb transcripts were highly expressed by miR-18b (miR-18b-5p). d Bax mRNAs were decreased and Bcl2 mRNAs were increased by overexpressed miR-18b. e miR-18b (miR-18b-5p) was overexpressed in mtNSC-34 cells. f miR-206 was reduced by miR-18b (miR-18b-5p). g LDH release analysis explained that transfected miR-18b (miR-18b-5p) restores apoptosis. h Luciferases assay with 3′ UTR of Hif1α showed that Hif1α is target of miR-18b in contNSC-34 cells. i and j Overexpression of miR-18b (miR-18b-5p) enhanced neuronal differentiation (MAP2) and attenuated intracellular Ca2+ levels (Cont (0.098) versus miR-18b (miR-18b-5p) (0.051) in fluorescence intensities from baseline 490/525 ratio) in mtNSC-34 cells. Empty vector served as a negative control (Cont). Arrow represents SOD1 aggregation (green). Scale bar, 40 μm. differentiation in mtNSC-34 cells To investigate whether or not induced Mctp1 and Rarb had a direct thera- peutic effect on apoptosis, we transfected Mctp1 and Rarb in mtNSC-34 cells and measured Bax and Bcl2 expression. The overexpressed Mctp1 and Rarb did not induce significant changes, as in the loss-of- function studies, but co-transfected Mctp1 and Rarb reduced Bax and induced Bcl2 in mtNSC-34 cells (Additional file 5: Fig. S5A-C). The LDH assay also showed that simultaneous overexpression of Mctp1 and Rarb reduced apoptosis in mtNSC-34 cells (Add- itional file 5: Fig. S5D). To study whether or not Mctp1 and Rarb significantly affected intracellular Ca2+ levels and neurite outgrowth respectively, we ec- topically caused the occurrence of overexpression Mctp1 and Rarb. Mctp1 reduced intracellular Ca2+ levels, and Rarb enhanced neurite length in mtNSC- 34 cells (Additional file 5: Fig. S5E, F, and G). Downregulation of the miR-18b-5p signaling pathway is involved in diverse SOD1 mutations and in vivo studies To identify the pivotal role of the miR-18b-5p path- way in other SOD1 mutations, we ectopically caused the overexpression of SOD1 (G85R and D90A) in contNSC-34 cells. Transfected G85R and D90A dem- onstrated that Hif1α and Mef2c expression was in- creased and Mctp1 and Rarb expression was decreased (Additional file 6: Fig. S6A-C). G85R and D90A also enhanced apoptosis by difference the ex- pression of Bax and Bcl2 (Additional file 6: Fig. S6A and D). RT-qPCR analysis results showed that miR- 18b-5p was downregulated and miR-206 was upregu- lated by G85R and D90A (Additional file 6: Fig. S6E-G). Furthermore, we sought to verify the miR- 18b-5p pathway in G93A Tg and fALS (G86S) pa- tient. We first compared the miR-18b-5p pathway in the spinal cords of WT and G93A Tg. The expres- sion of Hif1α and Mef2c was highly increased, yet that of Mctp1 and Rarb was significantly decreased in G93A Tg (Fig. 6a and b). Increased Bax and de- creased Bcl2 indicated the apoptosis occurring in G93A Tg (Fig. 6a and b). RT-qPCR results also showed that miR-18b-5p was downregulated and miR-206 was upregulated in G93A Tg (Fig. 6c). Fur- ther, we confirmed the miR-18b-5p pathway in the spinal cord of the G86S patient. Hif1α and Mef2c expression significantly increased, while the expres- sion of Mctp1 and Rarb decreased in the G86S pa- tient (Fig. 6d and e). The expression of Bax and Bcl2 was induced and reduced respectively in the G86S patient (Fig. differentiation in mtNSC-34 cells Significantly different at , p < 0.05; *, p < 0.005. The experiments were replicated 5 times Kim et al. Translational Neurodegeneration (2020) 9:23 Page 11 of 21 Kim et al. Translational Neurodegeneration (2020) 9:23 Fig. 4 (See legend on next page.) Page 12 of 21 Kim et al. Translational Neurodegeneration (2020) 9:23 (See figure on previous page.) Fig. 4 miR-206 post-transcriptionally regulates Mctp1 and Rarb and elevates apoptotic cell death. a and b Luciferases assay with 3′ UTR of Mctp1 showed that Mctp1 is target of miR-206. Mctp1 transcripts was downregulated by increased miR-206. c Intracellular Ca2+ levels (Cont (0.069) versus miR-206 (0.122) in fluorescence intensities from baseline 490/525 ratio) was enhanced by miR-206. d miR-206 controlled Mctp1 and Rarb protein expression. e Luciferase assay with 3′ UTR of Rarb also verified that Rarb is target of miR-206. Rarb mRNAs was decreased by miR-206. Neuronal cell differentiation (MAP2) was reduced by miR-206. Scale bar, 40 μm. f and g Both Bax protein and mRNAs were increased by miR-206. Bcl2 proteins and mRNAs were decreased by miR-206. h miR-206 was overexpressed in contNSC-34 cells. i) LDH release assay showed that miR- 206 enhances apoptosis. j Flow cytometry analysis explained that overexpressed miR-206 activates apoptotic cell death in mouse NSCs. Empty vector served as a negative control (Cont). Significantly different at , p < 0.05; *, p < 0.005. The experiments were replicated 3 times multiple gene expression directly or indirectly (Mef2c, miR-206, Mctp1 and Rarb). These results in- dicated that Mctp1 and Rarb downregulated by miR- 206 induced apoptosis in mtNSC-34 cells, and down- regulation of Mctp1 and Rarb is related to the inhib- ition of calcium signaling and cell differentiation, respectively. To explore whether or not Mctp1 and Rarb deficiency directly induces apoptosis, we used RNAi methods in contNSC-34 cells. Interestingly, siMctp1 enhanced the intracellular Ca2+ concentra- tion, but did not affect Bax and Bcl2 expression (Fig. 5a-c and g-h). siRarb inhibited cell differentiation but did not induce a significant change in Bax and Bcl2 expression (Fig. 5a and d-h). Due to the fact that miR-206 simultaneously decreased Mctp1 and Rarb, we inhibited Mctp1 and Rarb simultaneously. Bax ex- pression was increased and Bxl2 was decreased by double-knockdown of Mctp1 and Rarb in contNSC-34 cells (Fig. 5a). RT-qPCR analysis also verified the up- regulated Bax transcripts and downregulated Bcl2 transcripts by co-transfected siMctp1 and siRarb (Fig. 5g and h). differentiation in mtNSC-34 cells LDH also was released more following the simultaneous reduction in Mctp1 and Rarb (Fig. 5i). Flow cytometry analysis also demonstrated enhanced apoptosis following ectopic downregulation of Mctp1 and Rarb in mNSCs (Fig. 5j). To investigate whether or not induced Mctp1 and Rarb had a direct thera- peutic effect on apoptosis, we transfected Mctp1 and Rarb in mtNSC-34 cells and measured Bax and Bcl2 expression. The overexpressed Mctp1 and Rarb did not induce significant changes, as in the loss-of- function studies, but co-transfected Mctp1 and Rarb reduced Bax and induced Bcl2 in mtNSC-34 cells (Additional file 5: Fig. S5A-C). The LDH assay also showed that simultaneous overexpression of Mctp1 and Rarb reduced apoptosis in mtNSC-34 cells (Add- itional file 5: Fig. S5D). To study whether or not Mctp1 and Rarb significantly affected intracellular Ca2+ levels and neurite outgrowth respectively, we ec- topically caused the occurrence of overexpression Mctp1 and Rarb. Mctp1 reduced intracellular Ca2+ levels, and Rarb enhanced neurite length in mtNSC- 34 cells (Additional file 5: Fig S5E F and G) Altogether, these gain- and loss-of-function studies concerning Mctp1 and Rarb imply that calcium sig- naling and neuronal cell differentiation are involved in important pathogenic mechanisms associated to SOD1 (G93A) mutations. multiple gene expression directly or indirectly (Mef2c, miR-206, Mctp1 and Rarb). These results in- dicated that Mctp1 and Rarb downregulated by miR- 206 induced apoptosis in mtNSC-34 cells, and down- regulation of Mctp1 and Rarb is related to the inhib- ition of calcium signaling and cell differentiation, respectively. To explore whether or not Mctp1 and Rarb deficiency directly induces apoptosis, we used RNAi methods in contNSC-34 cells. Interestingly, siMctp1 enhanced the intracellular Ca2+ concentra- tion, but did not affect Bax and Bcl2 expression (Fig. 5a-c and g-h). siRarb inhibited cell differentiation but did not induce a significant change in Bax and Bcl2 expression (Fig. 5a and d-h). Due to the fact that miR-206 simultaneously decreased Mctp1 and Rarb, we inhibited Mctp1 and Rarb simultaneously. Bax ex- pression was increased and Bxl2 was decreased by double-knockdown of Mctp1 and Rarb in contNSC-34 cells (Fig. 5a). RT-qPCR analysis also verified the up- regulated Bax transcripts and downregulated Bcl2 transcripts by co-transfected siMctp1 and siRarb (Fig. 5g and h). LDH also was released more following the simultaneous reduction in Mctp1 and Rarb (Fig. 5i). Flow cytometry analysis also demonstrated enhanced apoptosis following ectopic downregulation of Mctp1 and Rarb in mNSCs (Fig. 5j). Downregulated miR-18b-5p triggered apoptosis in differentiated MNs from hiPSC-derived NSCs of a SOD1 (G17S) fALS patient [52, 53]. Although SOD1, which is the first discovered ALS-causing mutated gene and is linked only with the ALS phenotype, does not have much functional correlation with FUS and TDP-43, many researchers have attempted to study dysregulated RNA biogenesis with regard to SOD1 mutations [9, 13]. However, pre- cise mechanisms of RNA metabolism in SOD1 muta- tions are still unclear. In the present study, we attempted to study abnormal RNA processing in SOD1 mutations in a different way compared to pre- vious efforts. To uncover abnormal RNA expression in the nucleus and cytoplasm, we performed RNA-seq in nuclear and cytoplasmic subcellular fractions from wtNSC-34 and mtNSC-34 cells. Then, we identified that mutated SOD1 (G93A) up or downregulated sev- eral gene expression in the nucleus and cytoplasm. Among them, we discovered that Hif1α and Mef2c were notably upregulated in the nucleus and cyto- plasm of mtNSC-34 cells, while Mctp1 and Rarb were upregulated in the nucleus, but were strongly down- regulated in the cytoplasm. These results led us to hypothesize that the altered expression of Mctp1 and Rarb might be affected by post-transcriptional regula- tion. We identified that Mctp1 and Rarb are target genes of miR-206 which is upregulated in mtNSC-34 cells. Previous functional studies on Mctp1 and Rarb have revealed that Mctp1 is related to calcium signal- ing [30, 31] and that Rarb is associated with cell dif- ferentiation [37]. Indeed, we found that the downregulation of Mctp1 and Rarb in mtNSC-34 cells and the intracellular Ca2+ levels increased and the neurite outgrowth was reduced. In the G86S patient study, we could not compare the normal lumber to G86S patient lumber tissues (Fig. 6d). However, we clearly confirmed miR-18b-5p pathway in cervical tissues. To support that miR-18b-5p pathway play a pivotal role in fALS patient motor neurons, we developed hiPSCs derived from WT and fALS SOD1 (G17S) patient blood. WT and G17S iPSCs were con- firmed using pluripotency markers and RT-PCR (Fig. 7a and Additional file 7: Fig. S7A). We also gen- erated NSCs, and the immunocytochemical staining demonstrated that SOX2 and Nestin expression were induced in WT and G17S NSCs (Fig. 7b). To validate the variation in miR-18b-5p, Hif1α, Mef2c, miR-206, Mctp1, and Rarb transcripts, we induced MNs from NSCs which were characterized by HB9 and ChAT (Fig. 7c). We confirmed that Hif1α and Mef2c tran- scripts significantly increased in G17S MNs (Fig. 7d). Downregulated miR-18b-5p triggered apoptosis in differentiated MNs from hiPSC-derived NSCs of a SOD1 (G17S) fALS patient Mctp1 and Rarb mRNAs remarkably decreased in G17S MNs (Fig. 7e). We also measured intracellular Ca2+ levels and neurite length. As we expected, Ca2+ was highly accumulated and neuronal cell differenti- ation was inhibited in G17S MNs (Fig. 7f and Add- itional file 7: Fig. S7B). We also verified that Bax transcripts were upregulated and Bcl2 transcripts were downregulated in G17S MNs (Additional file 7: Fig. S7C). We also measured downregulated miR-18b-5p and upregulated miR-206 in G17S MNs (Fig. 7g). LDH release was increased in G17S MNs (Additional file 7: Fig. S7D). Altogether, these findings show that the downregulated miR-18b-5p systematically controls Hif1α, Mef2c, miR-206, Mctp1, and Rarb expression and finally induces apoptosis in ALS. Previous research introduced us the idea that Mef2c could regulate miR-206 expression [49] and that Hif1α could also induce Mef2c as a transcription fac- tor [48]. In this research, we identified that both Hif1α and Mef2c were increased in mtNSC-34 cells. Earlier studies also shed some light on Hif1α regula- tion by miR-18b-5p [49]. Importantly, we first discov- ered that miR-18b-5p expression was significantly decreased in mtNSC-34 cells. Clues pertaining to al- tered miR-18b-5p, Hif1α, Mef2c, miR-206, Mctp1, and Rarb were perfectly correlated to each other and were related to apoptosis in mtNSC-34 cells. differentiation in mtNSC-34 cells 6d and e). RT-qPCR analysis showed decreased miR-18-5p and increased miR-206 in the G86S patient (Fig. 6f). These results strongly demon- strated that the miR-18b-5p pathway was generally involved in SOD1 mutation. Page 13 of 21 Kim et al. Translational Neurodegeneration (2020) 9:23 (2020) 9:23 Kim et al. Translational Neurodegeneration Fig. 5 (See legend on next page.) Fig. 5 (See legend on next page.) Page 14 of 21 Kim et al. Translational Neurodegeneration (2020) 9:23 (See figure on previous page.) Fig. 5 Downregulated Mctp1 and Rarb are associated with apoptotic cell death. a Bax proteins were increased by double knockdown of Mctp1 and Rarb. Bcl2 expression was decreased by siMctp1 and Rarb. b Mctp1 transcripts were downregulated. c Knockdown of Mctp1 enhanced Intracellular Ca2+ levels (siCont (0.063) versus siMctp1 (0.131) in fluorescence intensities from baseline 490/525 ratio). d Rarb transcripts were downregulated. e-f Transfected siRarb reduced neuronal cell differentiation (MAP2). Scale bar, 40 μm. g and h Double knockdown of Mctp1 and Rarb increased Bax transcripts and decreased Bcl2 transcripts. i Transfected siMctp1 and siRarb activated LDH release. j Flow cytometry verified that downregulated Mctp1 and Rarb significantly induce apoptotic cell death in mouse NSCs. Scrambled siRNA served as a negative control (siCont). Significantly different at , p < 0.05; *, p < 0.005. The experiments were replicated 3 times Downregulated miR-18b-5p triggered apoptosis in differentiated MNs from hiPSC-derived NSCs of a SOD1 (G17S) fALS patient Discussion Recently, various pathological pathways and mecha- nisms have gradually been discovered for ALS and FTLD [13]. SOD1, FUS, and TDP-43 are representa- tively associated with ALS pathogenesis [2–9]. Specif- ically, RNA processing studies are based on the pathogenic mechanisms of FUS and TDP-43 because they have several functional and structural similarities Kim et al. Translational Neurodegeneration (2020) 9:23 Page 15 of 21 Kim et al. Translational Neurodegeneration (2020) 9:23 Fig. 6 (See legend on next page.) Fig. 6 (See legend on next page.) Page 16 of 21 Kim et al. Translational Neurodegeneration (2020) 9:23 (See figure on previous page.) Fig. 6 Downregulated miR-18b (miR-18b-5p) by SOD1 mutations contributes apoptotic cell death in SOD1(G93A) Tg mice and fALS patient spinal cord tissues. a Hif1α, Mef2c and Bax expression were increased in G93A mice. Mctp1, Rarb and Bcl2 proteins were decreased in G93A mice. b mRNAs of Hif1α, Mef2c and Bax was highly expressed in G93A mice. Mctp1, Rarb and Bcl2 transcripts were significantly downregulated in G93A mice. c miR-18b (miR-18b-5p) was deeply reduced and miR-206 was dramatically induced in G93A mice (n = 5). d The protein levels of Hif1α, Mef2c and Bax was upregulated in fALS (G86S) patient (Cervical and lumber). Mctp1, Rarb and Bcl2 proteins were decreased in fALS (G86S) patient. Normal spinal cord tissues (Cervical (control 1 and 2) served as a negative control (Cont). e The transcripts of Hif1α, Mef2c and Bax was highly upregulated in fALS (G86S) patient (Cervicals). Mctp1, Rarb and Bcl2 transcripts were significantly downregulated in fALS (G86S) patient. f miR-18b (miR-18b-5p) expression was importantly decreased and miR-206 was highly expressed in fALS (G86S) patient (Cervial and Lumber). Normal spinal cord tissues (Cervicals (control 1 and 2) served as a negative control (Cont). hSOD1 immunoreactivity on western blots of the insoluble fraction of the G93A mice and fALS (G86S) patients tissues. Significantly different at , p < 0.05; *, p < 0.005 not clear. We also reconfirmed the downregulated miR-18b-5p pathway in the G93A Tg, G86S patient and G17S MNs. As observed in our in vitro studies, miR-18b-5p was incredibly downregulated and miR- 206 expression was upregulated in the G93A Tg, G86S patient and G17S MNs. The mRNAs of Hif1α, Mef2c, Mctp1 and Rarb were the same as those ob- served in vitro studies of the G93A Tg, G86S patient and G17S MNs. Discussion Intracellular Ca2+ levels were en- hanced and MN differentiation was significantly inhibited in G17S MNs. Increased Bax and decreased Bcl2 RNAs also indicated that apoptosis by downreg- ulated miR-18b-5p was elevated in the G93A Tg, G86S patient, and G17S MNs. Artificially increased miR-18b-5p directly downregu- lated Hif1α and Mef2c, restored intracellular Ca2+ levels, and induced neuronal differentiation in mtNSC-34 cells, because the decrease in miR-206 caused by reduced Mef2c upregulated both Mctp1 and Rarb in mtNSC-34 cells. In contrast, anti-18b (anti-miR-18b-5p) not only increased Hif1α and Mef2c levels, but also decreased Mctp1 and Rarb. Specifically, anti-18b (anti-miR-18b-5p) induced apop- tosis in mNSC and contNSC-34 cells. According to the hypothetical miR-18b-5p signal cascade, serial downregulated Hif1α directly reduced Mef2c, which also decreased miR-206 in mtNSC-34 cells. Mctp1 and Rarb indirectly upregulated by siHif1α decreased apoptosis in mtNSC-34 cells. Artificially increased miR-18b-5p directly downregu- lated Hif1α and Mef2c, restored intracellular Ca2+ levels, and induced neuronal differentiation in mtNSC-34 cells, because the decrease in miR-206 caused by reduced Mef2c upregulated both Mctp1 and Rarb in mtNSC-34 cells. In contrast, anti-18b (anti-miR-18b-5p) not only increased Hif1α and Mef2c levels, but also decreased Mctp1 and Rarb. Specifically, anti-18b (anti-miR-18b-5p) induced apop- tosis in mNSC and contNSC-34 cells. According to the hypothetical miR-18b-5p signal cascade, serial downregulated Hif1α directly reduced Mef2c, which also decreased miR-206 in mtNSC-34 cells. Mctp1 and Rarb indirectly upregulated by siHif1α decreased apoptosis in mtNSC-34 cells. According to the recent reports, apoptotic cell death of motor neurons (including SOD1, TDP-43, and FUS) [2–9, 54] and abnormal RNA metabolism (mRNA transcription and miRNAs) [9, 14, 55] in ALS are so controversial issues because non-apoptotic fea- tures have been found in ALS patients [54]. Besides, the crucial role of apoptotic cell death by abnormal RNA metabolism is still unclear. We, for the first time, have discovered that downregulated miR-18b-5p, which may be one of the important pathogenic mech- anisms in ALS associated SOD1 mutants (D90A, G17S, G85R, G86S and G93A) is associated with the sequential regulation of Hif1α, Mef2c, miR-206, Mctp1, and Rarb. Indeed, downregulated Mctp1 dir- ectly increased Ca2+ levels, and decreased Rarb signifi- cantly reduced cell differentiation in all investigated SOD1 mutations. The causes of down regulated miR- 18b-5p by SOD1 mutants need to be further exam- ined. Discussion It will provide novel insights into undescribed cellular processes and support to understand that miRNAs are related to important pathogenic mecha- nisms of sporadic and familial ALS. p p We first discovered that Mctp1 and Rarb were in- volved in the SOD1 mutation and were downregu- lated by miR-206. Our reporter assay showed that miR-206 significantly decreased both Mctp1 and Rarb, and then inhibited calcium signaling and neuronal differentiation. Increased miR-206 also induced apop- tosis in mNSCs and contNSC-34 cells. Indeed, anti- 206 (anti-miR-206) recovered apoptosis and increased Mctp1 and Rarb respectively in mtNSC-34 cells. To identify apoptosis by Mctp1 and Rarb, we reduced the levels of Mctp1 and Rarb. Intracellular Ca2+ was in- creased by siMctp1, and neurite outgrowth was de- creased by siRarb, but apoptosis was not enhanced. Notably, double-knockdown of Mctp1 and Rarb in- duced apoptosis in mNSCs and contNSC-34 cells. These results support the hypothesis that Mctp1 and Rarb are simultaneously downregulated by miR-206 and are correlated with apoptosis. This evidence ex- plains that the downregulated miR-18b-5p sequen- tially regulates Hif1α, Mef2c, miR-206, Mctp1, and Rarb. Other SOD1 mutations (G85R and D90A) also provoked the downregulated miR-18b-5p pathway as G93A does. Conclusions We proved that the miR-18b-5p pathway was func- tional in vitro, but whether or not the downregu- lated miR-18b-5p pathway could be revealed in the G93A Tg, G86S patient and G17S human MNs was We have provided strong evidence for the downregu- lated miR-18b-5p signaling pathway in ALS-related SOD1 mutations (Fig. 8). Our findings are as follows: (i) downregulated miR-18b-5p post-transcriptionally Kim et al. Translational Neurodegeneration (2020) 9:23 Page 17 of 21 Kim et al. Translational Neurodegeneration (2020) 9:23 Fig. 7 (See legend on next page.) Page 18 of 21 Kim et al. Translational Neurodegeneration (2020) 9:23 (See figure on previous page.) Fig. 7 Downregulated miR-18b (miR-18b-5p) by SOD1 mutations is associated with apoptotic cell death in motor neuron from hiPSC- derived NSCs fALS SOD1 (G17S). a Nanog (green) and Oct4 (red) iPSC marker are expressed in hiPSCs (normal and fALS SOD1 (G17S). Scale bar, 20 μm. b Neural stem cells (SOX2 and Nestin) was generated from hiPSCs (normal and fALS SOD1 (G17S)). Scale bar, 40 μm. c Differentiated motor neurons from hNSCs were double-stained with HB9 and ChAT antibodies. Scale bar, 20 μm. d Hif1α and Mef2c transcripts were upregulated in differentiated motor neurons SOD1 (G17S). e The mRNAs levels of Mctp1 and Rarb was downregulated in differentiated motor neurons SOD1 (G17S). f Intracellular Ca2+ levels (Normal (0.149) versus SOD1 (G17S) (0.215) in fluorescence intensities from baseline 490/525 ratio) were enhanced in differentiated motor neurons SOD1 (G17S). Total neurite length was reduced in differentiated motor neurons SOD1 (G17S). g miR-18b (miR-18b-5p) expression was deeply low and mir-206 was highly expression in differentiated motor neurons SOD1 (G17S). Significantly different at , p < 0.05; *, p < 0.005. The experiments were replicated 5 times controls Hif1α expression, (ii) Hif1α increases Mef2c as a transcription factor, (iii) Mef2c highly increases miR- 206 levels, (iv) miR-206 simultaneously degrades Mctp1 and Rarb, (v) decreased Mctp1 inhibits calcium signal- ing, (vi) reduced Rarb impedes neuronal cell differenti- ation, and (vii) downregulated miR-18b-5p enhances apoptotic cell death in ALS SOD1 mutations. This novel mechanism by which the downregulated miR-18b-5p is related to the regulation of several genes (Hif1α, Mef2c, miR-206, Mctp1, and Rarb) requires further investigation before it can be used for clinically meaningful applica- tion in ALS treatment. Fig. 8 Downregulated miR-18b (miR-18b-5p) signaling pathway in fALS linked SOD1 mutation. A schematic diagram illustrates that downregulated miR-18b (miR-18b-5p) by SOD1 mutation directly increases Hif1α. Supplementary information Additional file 6: Figure S6. The apoptotic cell death by SOD1 mutations (G85R and D90A) in fALS is related with miR-18b (miR-18b-5p) signaling pathway. (A) Immunoblot analysis showed that SOD1 (G85R and D90A) mutations increased both Hif1α and Mef2c. Mctp1 and Rarb were decreased by overexpressed SOD1 (G85R and D90A). Increased Bax and decreased Bcl1 proteins by SOD1 (G85R and D90A) were associated with apoptosis in NSC-34 cont cells. (B) RT-qPCR analysis explained that Hif1α and Mef2c were upregulated by SOD1 (G85R and D90A). (C) The mRNA levels of Mctp1 and Rarb was reduced by SOD1 (G85R and D90A). (D) Bax mRNA levels are increased and Bcl2 mRNA levels are decreased under overexpressed SOD1 (G85R and D90A) condition. (E) miR-18b (miR- 18b-5p) was reduced by SOD1 (G85R and D90A) (F) miR-206 was upregu- lated by SOD1 (G85R and D90A). (G) The mRNA levels of SOD1 (G85R and D90A) was increased in NSC-34 cont cells. Empty vector served as a negative control (Cont). The data represent the average ± SEM of 5 separ- ate experiments. Significantly different at , p < 0.05; *, p < 0.005. pp y Supplementary information accompanies this paper at https://doi.org/10. 1186/s40035-020-00203-4. Supplementary information accompanies this paper at https://doi.org/10. 1186/s40035-020-00203-4. Additional file 1 Figure S1. Mctp1 and Rarb are targeted by miR-206 and Hif1α is targeted by miR-18b (miR-18b-5p). (A) A schematic diagram explains consensus base pairing between miR-206 with the 3′ UTR se- quences of mouse Mctp1 and Rarb. (B) A schematic diagram shows con- sensus base pairing between miR-18b (miR-18b-5p) with the 3′ UTR sequences of mouse Hif1α. The identification of miR-18b (miR-18b-5p) target sequences was analyzed by TargetScan (http://www.targetscan. org). Additional file 2: Figure S2. Downregulated miR-18b (miR-18b-5p) by transfected anti-18b (anti-miR-18b-5p) controls alteration of several gene expressions and induces apoptotic cell death in NSC-34 cont cells. (A) anti-18b (anti-miR-18b-5p) increased Hif1α and Mef2c proteins. Both Mctp1 and Rarb proteins were decreased by anti-18b (anti-miR-18b-5p). Upregulated Bax and downregulated Bcl2 by anti-18b (anti-miR-18b-5p) induced apoptotic cell death. (B and C) anti-18b (anti-miR-18b-5p) in- creased Hif1α and Mef2c transcripts. (D and E) Mctp1 and Rarb mRNAs were decreased by anti-18b (anti-miR-18b-5p). (F and G) Bax mRNAs were upregulated and Bcl2 mRNAs were downregulated under knock down of miR-18b (miR-18b-5p) condition. (H) Lactate dehydrogenase (LDH) release analysis showed that anti-18b (anti-miR-18b-5p) induces cell death. Funding Th This research was supported by the Brain Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science and ICT (2017M3C7A102536521 and 2018R1A5A202596413). Availability of data and materials All raw data used and/or analyzed during the current study are available from the corresponding author on reasonable request. All raw data used and/or analyzed during the current study are available from the corresponding author on reasonable request. Acknowledgements We thank Dr. Kwang Woo Lee (Gachon University Gil Medical Center) for technical supports. Supplementary information Table S5 Human primer sequences that are used for reverse transcriptase PCR (RT-PCR). Table S6 Human spinal cord and blood samples Additional file 3: Figure S3. Knock down of Hif1α reduces apoptotic cell death in mtNSC-34 cells. (A) Transfected siHif1α decreased Mef2c pro- teins. Mctp1 and Rarb expressions were increased by knock down of Hif1α. siHif1α reduced Bax and induced Bcl2 protein levels. (B and C) RT- qPCR analysis showed downregulated Hif1α by siHif1α decreased Mef2c. (D and E) mRNA levels of Mctp1 and Rarb was increased by siHif1α. (F and G) siHif1α downregulated Bax and upregulated Bcl2 transcripts. (H) miR-206 expression was increased under knock down of Hif1α condition. (I) LDH release analysis showed that siHif1α restored apoptotic cell death. Scrambled siRNA served as a negative control (Cont). Significantly differ- ent at , p < 0.05; *, p < 0.005. The experiments were replicated 3 times. scription PCR (RT-qPCR). Table S5 Human primer sequences that are used for reverse transcriptase PCR (RT-PCR). Table S6 Human spinal cord and blood samples Authors’ contributions KKY designed the project, performed most of the experiments, analyzed the data and wrote the manuscript. YRK, KWC, SL, ML and WI performed experiments, analyzed data. JYS, JYK, YHH, MK and JIK analyzed data and reviewed the manuscript. JJS designed and supervised the project and wrote the manuscript. All authors read and approved the final manuscript. Additional file 5: Figure S5. Overexpressed Mctp1 and Rarb reduce apoptotic cell death in mtNSC-34 cells. (A) Co-transfected Mctp1 and Rarb decreased Bax proteins and increased Bcl2 proteins. (B) RT-qPCR analysis explained that mRNA levels of Bax were reduced by cotrans- fected Mctp1 and Rarb. (C) Bcl2 transcripts were induced by overex- pressed Mctp1 and Rarb. (D) LDH release showed that increased Mctp1 and Rarb reduced apoptosis. (E) Transfected Mctp1 reduced intracellular Ca2+ levels (Cont (0.025) versus Mctp1 (0.0078) in fluorescence intensities from baseline 490/525 ratio) and RT-qPCR analysis showed increased Mctp1 mRNAs. (F) Overexpressed Rarb enhanced neurite length. Signifi- cantly different at , p < 0.05; *, p < 0.005. (G) The confocal microscopy presented that overexpressed Rarb (GFP-Rarb) induced neurite outgrowth (MAP2). Empty vector served as a negative control (Cont). Scale bar, 20 μm. The experiments were replicated 5 times. Additional file 5: Figure S5. Overexpressed Mctp1 and Rarb reduce apoptotic cell death in mtNSC-34 cells. (A) Co-transfected Mctp1 and Rarb decreased Bax proteins and increased Bcl2 proteins. (B) RT-qPCR analysis explained that mRNA levels of Bax were reduced by cotrans- fected Mctp1 and Rarb. (C) Bcl2 transcripts were induced by overex- pressed Mctp1 and Rarb. (D) LDH release showed that increased Mctp1 and Rarb reduced apoptosis. (E) Transfected Mctp1 reduced intracellular Ca2+ levels (Cont (0.025) versus Mctp1 (0.0078) in fluorescence intensities from baseline 490/525 ratio) and RT-qPCR analysis showed increased Mctp1 mRNAs. (F) Overexpressed Rarb enhanced neurite length. Signifi- cantly different at , p < 0.05; *, p < 0.005. (G) The confocal microscopy presented that overexpressed Rarb (GFP-Rarb) induced neurite outgrowth (MAP2). Empty vector served as a negative control (Cont). Scale bar, 20 μm. The experiments were replicated 5 times. Abbreviations S h ALS: Amyotrophic later sclerosis; SOD1: Cu/Zn-superoxide dismutase; FUS: Fused in Sarcoma; Hif1α: Hypoxia inducible factor 1; iPSCs: induced pluripotent stem cells; NSCs: neural stem cells; miRNAs: microRNAs; miR- 18b: miR-18b-5p; Mctp1: Multiple C2 domains transmembrane protein 1; Mef2c: Myocyte specific enhancer factor 2c; MN: Motor neuron; Rarb: Retinoic acid receptor beta; TDP-43: TAR DNA-binding protein 43 Additional file 4: Figure S4. Reduced miR-206 in mtNSC-34 cells recov- ered apoptotic cell death. (A) Western blot analysis showed that trans- fected anti-206 (anti-miR-206) increased protein levels of Mctp1 and Rarb. Bax protein levels were reduced and Bcl2 protein levels were induced by anti-206 (anti-miR-206), respectively. (B and C) RT-qPCR results showed that Mctp1 and Rarb transcripts also were increased by anti-206 (anti- miR-206). (D and E) Bax mRNAs upregulated and Bcl2 mRNAs downregu- lated under transfected anti-206 (anti-miR-206) condition, respectively. (F) LDH release assay demonstrated that reduced miR-206 was associated apoptosis. (G) miR-206 was decreased by anti-206 (anti-miR-206). Scram- bled anti-mir served as a negative control (Cont). (H and I) Luciferase assay with mutation of miR-206 binding sites (3′ UTR of Mctp1 and Rarb) did not show any significant change. Significantly different at , p < 0.05; *, p < 0.005. The experiments were replicated 3 times. Conclusions Mef2c is controlled by overexpressed Hif1α. Increased Mef2c is related with miR-206 expression. Mctp1 and Rarb are downregulated by increased miR-206. Intracellular Ca2+ levels is increased by downregulated Mctp1 and Neuronal differentiation is reduced by downregulated Rarb. Apoptotic cell death is induced by prohibited Ca2+ signaling and Neuronal differentiation. As a result, downregulated miR-18b-5p by SOD1 mutation leads to apoptotic cell death in fALS linked SOD1 mutation Fig. 8 Downregulated miR-18b (miR-18b-5p) signaling pathway in fALS linked SOD1 mutation. A schematic diagram illustrates that downregulated miR-18b (miR-18b-5p) by SOD1 mutation directly increases Hif1α. Mef2c is controlled by overexpressed Hif1α. Increased Mef2c is related with miR-206 expression. Mctp1 and Rarb are downregulated by increased miR-206. Intracellular Ca2+ levels is increased by downregulated Mctp1 and Neuronal differentiation is reduced by downregulated Rarb. Apoptotic cell death is induced by prohibited Ca2+ signaling and Neuronal differentiation. As a result, downregulated miR-18b-5p by SOD1 mutation leads to apoptotic cell death in fALS linked SOD1 mutation Page 19 of 21 (2020) 9:23 Kim et al. Translational Neurodegeneration Supplementary information (I and J) RT-qPCR analysis demonstrated decreased miR-18b (miR-18b-5p) and increased miR-206 by anti-18b (anti-miR-18b-5p). (K) Flow cytometry ana- lysis explained that reduced miR-18b (miR-18b-5p) induces apoptotic cell death. Scrambled anti-mir served as a negative control (Cont). The data represent the average ± SEM of 3 separate experiments. Significantly dif- ferent at , p < 0.05; *, p < 0.005. Additional file 7: Figure S7. Downregulated miR-18b (miR-18b-5p) in iPSCs-derived motor neuron from SOD1 (G17S) ALS patient induce apop- totic cell death. (A) RT-PCR analysis verified that hiPSCs from normal and fALS SOD1 (G17S) were generated. (B) The immunoreactivity of ChAT (motor neuron) and MAP2 (neurite outgrowth) was expressed in differen- tiated motor neurons (normal vs SOD1 (G17S) patient). Scale bar, 20 μm. (C) Bax mRNAs were increased and Bcl2 mRNAs were decreased in iPSCs- derived motor neuron SOD1 (G17S) ALS patient. (D) LDH release analysis demonstrated that the apoptotic cell death was induced in iPSCs-derived motor neuron SOD1 (G17S) ALS patient. Significantly different at , p < 0.05; *, p < 0.005. The experiments were replicated 7 times. Additional file 8: Table S1 Mouse primer sequences that are used for cloning of 3’UTR of Hif1α, Rarb and Mctp1, miR-18b, miR-206, GFP-Rarb and mCherry-Mctp1. Table S2 Mouse primer sequences that are used for reverse transcriptase PCR (RT-PCR). Table S3 Mouse primer sequences that are used for quantitative reverse transcription PCR (RT-qPCR). Table S4 Human primer sequences that are used for quantitative reverse tran- scription PCR (RT-qPCR). Table S5 Human primer sequences that are used for reverse transcriptase PCR (RT-PCR). Table S6 Human spinal cord and blood samples Additional file 8: Table S1 Mouse primer sequences that are used for cloning of 3’UTR of Hif1α, Rarb and Mctp1, miR-18b, miR-206, GFP-Rarb and mCherry-Mctp1. Table S2 Mouse primer sequences that are used for reverse transcriptase PCR (RT-PCR). Table S3 Mouse primer sequences that are used for quantitative reverse transcription PCR (RT-qPCR). Table S4 Human primer sequences that are used for quantitative reverse tran- Additional file 8: Table S1 Mouse primer sequences that are used for cloning of 3’UTR of Hif1α, Rarb and Mctp1, miR-18b, miR-206, GFP-Rarb and mCherry-Mctp1. Table S2 Mouse primer sequences that are used for reverse transcriptase PCR (RT-PCR). Table S3 Mouse primer sequences that are used for quantitative reverse transcription PCR (RT-qPCR). Table S4 Human primer sequences that are used for quantitative reverse tran- scription PCR (RT-qPCR). References Couthouis J, Hart MP, Erion R, King OD, Diaz Z, Nakaya T, et al. Evaluating the role of the FUS/TLS-related gene EWSR1 in amyotrophic lateral sclerosis. Hum Mol Genet. 2012;21:2899–911. 6. Couthouis J, Hart MP, Erion R, King OD, Diaz Z, Nakaya T, et al. Evaluating the role of the FUS/TLS-related gene EWSR1 in amyotrophic lateral sclerosis. Hum Mol Genet. 2012;21:2899–911. 7. Kwiatkowski TJ Jr, Bosco DA, Leclerc AL, Tamrazian E, Vanderburg CR, Russ C, et al. Mutations in the FUS/TLS gene on chromosome 16 cause familial amyotrophic lateral sclerosis. Science. 2009;323:1205–8. 33. Mangelsdorf DJ, Thummel C, Beato M, Herrlich P, Schütz G, Umesono K, et al. The nuclear receptor superfamily: the second decade. Cell. 1995;83: 835–9. 8. Inukai Y, Nonaka T, Arai T, Yoshida M, Hashizume Y, Beach TG, et al. Abnormal phosphorylation of Ser409/410 of TDP-43 in FTLD-U and ALS. FEBS Lett. 2008;582:2899–904. 34. Li Y, Dawson MI, Agadir A, Lee MO, Jong L, Hobbs PD, et al. Regulation of RAR beta expression by RAR- and RXR-selective retinoids in human lung cancer cell lines: effect on growth inhibition and apoptosis induction. Int J Cancer. 1998;75:88–95. 9. Paez-Colasante X, Figueroa-Romero C, Sakowski SA, Goutman SA, Feldman EL. Amyotrophic lateral sclerosis: mechanisms and therapeutics in the epigenomic era. Nat Rev Neurol. 2015;11:266–79. 35. Pinto C, Cárdenas P, Osses N, Henríquez JP. Characterization of Wnt/β- catenin and BMP/Smad signaling pathways in an in vitro model of amyotrophic lateral sclerosis. Front Cell Neurosci. 2013;7:1–15. 10. van Zundert B, Brown RH Jr. Silencing strategies for therapy of SOD1- mediated ALS. Neurosci Lett. 2017;636:32–9. 36. Liang Y, Mirnics ZK, Yan C, Nylander KD, Schor NF. Bcl-2 mediates induction of neural differentiation. Oncogene. 2003;22:5515–8. 11. Narożna B, Langwiński W, Szczepankiewicz A. Non-coding RNAs in pediatric airway diseases. Genes. 2017;8:348. 37. Kruman II, Pedersen WA, Springer JE, Mattson MP. ALS-linked cu/Zn-SOD mutation increases vulnerability of motor neurons to excitotoxicity by a mechanism involving increased oxidative stress and perturbed calcium homeostasis. Exp Neurol. 1999;160:28–39. 12. Kosik KS. The neuronal microRNA system. Nat Rev Neurosci. 2006;7:911–20. 13. Bicker S, Schratt G. MicroRNAs in ALS: small pieces to the puzzle. EMBO J. 2015;34:2601–3. 14. Russell AP, Wada S, Vergani L, Hock MB, Lamon S, Léger B, et al. Disruption of skeletal muscle mitochondrial network genes and miRNAs in amyotrophic lateral sclerosis. Neurobiol Dis. 2013;49:107–17. 38. Pinton P, Ferrari D, Rapizzi E, Di Virgilio F, Pozzan T, Rizzuto R. Consent for publication Consent for publication Not applicable. 20. Cho HJ, Liu G, Jin SM, Parisiadou L, Xie C, Yu J, et al. MicroRNA-205 regulates the expression of Parkinson's disease-related leucine-rich repeat kinase 2 protein. Hum Mol Genet. 2013;22:608–20. Competing interests h h d l h 21. Butovsky O, Siddiqui S, Gabriely G, Lanser AJ, Dake B, Murugaiyan G, et al. Modulating inflammatory monocytes with a unique microRNA gene signature ameliorates murine ALS. J Clin Invest. 2012;122:3063–87. The authors declare that they have no competing interests. Author details 1 Author details 1Department of Neurology, Seoul National University Hospital 28 yongon-Dong, Chongno-gu, Seoul 110-744, Republic of Korea. 2Division of Mass Spectrometry Research, Korea Basic Science Institute, Daejun, South Korea. 3Department of Neurology, Seoul National University Seoul Metropolitan Government Boramae Medical Center, Seoul, South Korea. 4Department of Biochemistry and Molecular Biology, Seoul National University College of Medicine, Seoul, South Korea. 22. Koval ED, Shaner C, Zhang P, du Maine X, Fischer K, Tay J, et al. Method for widespread microRNA-155 inhibition prolongs survival in ALS-model mice. Hum Mol Genet. 2013;22:4127–35. 23. Han J, Pedersen JS, Kwon SC, Belair CD, Kim YK, Yeom KH, et al. Posttranscriptional crossregulation between Drosha and DGCR8. Cell. 2009; 136:75–84. 24. Lee Y, Ahn C, Han J, Choi H, Kim J, Yim J, et al. The nuclear RNase III Drosha initiates microRNA processing. Nature. 2003;425:415–9. 25. Diederichs S, Haber DA. Dual role for argonautes in microRNA processing and posttranscriptional regulation of microRNA expression. Cell. 2007;131: 1097–108. Received: 22 May 2019 Accepted: 1 June 2020 Received: 22 May 2019 Accepted: 1 June 2020 Received: 22 May 2019 Accepted: 1 June 2020 26. Chen Z, Li Y, Zhang H, Huang P, Luthra R. Hypoxia-regulated microRNA-210 modulates mitochondrial function and decreases ISCU and COX10 expression. Oncogene. 2010;29:4362–8. Ethics approval and consent to participate All animal experimental procedures were performed in compliance with the Institutional Animal Care and Use Committee guidelines at Seoul National University. Human samples were analyzed with Institutional permission under Review Board in Seoul National University Hospital. Control spinal cord Page 20 of 21 Page 20 of 21 Kim et al. Translational Neurodegeneration (2020) 9:23 Kim et al. Translational Neurodegeneration (2020) 9:23 samples were obtained from the Netherlands Brain Bank and the guidelines by The Netherlands Brain Bank were followed. 18. Wang WX, Huang Q, Hu Y, Stromberg AJ, Nelson PT. Patterns of microRNA expression in normal and early Alzheimer's disease human temporal cortex: white matter versus gray matter. Acta Neuropathol. 2011;121:193–205. 19. Gehrke S, Imai Y, Sokol N, Lu B. Pathogenic LRRK2 negatively regulates microRNA-mediated translational repression. Nature. 2010;466:637–41. 19. Gehrke S, Imai Y, Sokol N, Lu B. Pathogenic LRRK2 negatively regulates microRNA-mediated translational repression. Nature. 2010;466:637–41. References 1. Lagier-Tourenne C, Cleveland DW. Rethinking ALS: the FUS about TDP-43. Cell. 2009;136:1001–4. 1. Lagier-Tourenne C, Cleveland DW. Rethinking ALS: the FUS about TDP-43. Cell. 2009;136:1001–4. 1. Lagier-Tourenne C, Cleveland DW. Rethinking ALS: the FUS about TDP-43. Cell. 2009;136:1001–4. 27. Choi E, Cha MJ, Hwang KC. Roles of calcium regulating MicroRNAs in cardiac ischemia-reperfusion injury. Cells. 2014;3:899–913. 2. Ling SC, Polymenidou M, Cleveland DW. Converging mechanisms in ALS and FTD: disrupted RNA and protein homeostasis. Neuron. 2013;79:416–38. 2. Ling SC, Polymenidou M, Cleveland DW. Converging mechanisms in ALS and FTD: disrupted RNA and protein homeostasis. Neuron. 2013;79:416–38. 28. Makeyev EV, Zhang J, Carrasco MA, Maniatis T. The MicroRNA miR-124 promotes neuronal differentiation by triggering brain-specific alternative pre-mRNA splicing. Mol Cell. 2007;27:435–48. 3. Rosen DR, Siddique T, Patterson D, Figlewicz DA, Sapp P, Hentati A, et al. Mutations in cu/Zn superoxide dismutase gene are associated with familial amyotrophic lateral sclerosis. Nature. 1993;362:59–62. 3. Rosen DR, Siddique T, Patterson D, Figlewicz DA, Sapp P, Hentati A, et al. Mutations in cu/Zn superoxide dismutase gene are associated with familial amyotrophic lateral sclerosis. Nature. 1993;362:59–62. p p g 29. Berridge MJ, Bootman MD, Lipp P. Calcium--a life and death signal. Nature. 1998;395:645–8. 4. Gao FB, Almeida S, Lopez-Gonzalez R. Dysregulated molecular pathways in amyotrophic lateral sclerosis-frontotemporal dementia spectrum disorder. EMBO J. 2017;36:2931–50. 4. Gao FB, Almeida S, Lopez-Gonzalez R. Dysregulated molecular pathways in amyotrophic lateral sclerosis-frontotemporal dementia spectrum disorder. EMBO J. 2017;36:2931–50. 30. Qiu L, Yu H, Liang F. Multiple C2 domains transmembrane protein 1 is expressed in CNS neurons and possibly regulates cellular vesicle retrieval and oxidative stress. J Neurochem. 2015. https://doi.org/10.1111/jnc.13251. 5. DeJesus-Hernandez M, Mackenzie IR, Boeve BF, Boxer AL, Baker M, Rutherford NJ, et al. Expanded GGGGCC hexanucleotide repeat in noncoding region of C9ORF72 causes chromosome 9p-linked FTD and ALS. Neuron. 2011;72:245–56. 5. DeJesus-Hernandez M, Mackenzie IR, Boeve BF, Boxer AL, Baker M, Rutherford NJ, et al. Expanded GGGGCC hexanucleotide repeat in noncoding region of C9ORF72 causes chromosome 9p-linked FTD and ALS. Neuron. 2011;72:245–56. 31. Shin OH, Han W, Wang Y, Südhof TC. Evolutionarily conserved multiple C2 domain proteins with two transmembrane regions (MCTPs) and unusual Ca2+ binding properties. J Biol Chem. 2005;280:1641–51. 32. Tradewell ML, Cooper LA, Minotti S, Durham HD. Calcium dysregulation, mitochondrial pathology and protein aggregation in a culture model of amyotrophic lateral sclerosis: mechanistic relationship and differential sensitivity to intervention. Neurobiol Dis. 2011;42:265–75. 6. References The Ca2+ concentration of the endoplasmic reticulum is a key determinant of ceramide-induced apoptosis: significance for the molecular mechanism of Bcl-2 action. EMBO J. 2001;20:2690–701. 15. Martí E, Pantano L, Bañez-Coronel M, Llorens F, Miñones-Moyano E, Porta S, et al. A myriad of miRNA variants in control and Huntington's disease brain regions detected by massively parallel sequencing. Nucleic Acids Res. 2010; 38:7219–35. 39. Park JH, Park HS, Hong S, Kang S. Motor neurons derived from ALS-related mouse iPS cells recapitulate pathological features of ALS. Exp Mol Med. 2016;48:1–9. 16. Packer AN, Xing Y, Harper SQ, Jones L, Davidson BL. The bifunctional microRNA miR-9/miR-9 regulates REST and CoREST and is downregulated in Huntington's disease. J Neurosci. 2008;28:14341–6. 40. Chen H, Qian K, Du Z, Cao J, Petersen A, Liu H, et al. Modeling ALS with iPSCs reveals that mutant SOD1 misregulates neurofilament balance in motor neurons. Cell Stem Cell. 2014;14:796–809. 17. Hébert SS, Horré K, Nicolaï L, Papadopoulou AS, Mandemakers W, Silahtaroglu AN, et al. Loss of microRNA cluster miR-29a/b-1 in sporadic Alzheimer's disease correlates with increased BACE1/beta-secretase expression. Proc Natl Acad Sci U S A. 2008;105:6415–20. 41. Reynolds BA, Weiss S. Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system. Science. 1992;255: 1707–10. Page 21 of 21 Kim et al. Translational Neurodegeneration (2020) 9:23 Kim et al. Translational Neurodegeneration (2020) 9:23 42. Im W, Ban JJ, Chung JY, Lee ST, Chu K, Kim M. Multidrug resistance protein 1 reduces the aggregation of mutant huntingtin in neuronal cells derived from the Huntington's disease R6/2 model. Sci Rep. 2015;5:1–10. 43. Cho MS, Hwang DY, Kim DW. Efficient derivation of functional dopaminergic neurons from human embryonic stem cells on a large scale. Nat Protoc. 2008;3:1888–94. 44. Corti S, Nizzardo M, Nardini M, Donadoni C, Salani S, Ronchi D, et al. Embryonic stem cell-derived neural stem cells improve spinal muscular atrophy phenotype in mice. Brain. 2010;133:465–81. 45. Peters OM, Ghasemi M, Brown RH Jr. Emerging mechanisms of molecular pathology in ALS. J Clin Invest. 2015;125:1767–79. 46. Ha M, Kim VN. Regulation of microRNA biogenesis. Nat Rev Mol Cell Biol. 2014;15:509–24. 47. Kim KY, Hwang YJ, Jung MK, Choe J, Kim Y, Kim S, et al. A multifunctional protein EWS regulates the expression of Drosha and microRNAs. Cell Death Differ. 2014;21:136–45. 48. Krishnan J, Ahuja P, Bodenmann S, Knapik D, Perriard E, Krek W, et al. References Essential role of developmentally activated hypoxia-inducible factor 1alpha for cardiac morphogenesis and function. Circ Res. 2008;103:1139–46. 49. Rao PK, Kumar RM, Farkhondeh M, Baskerville S, Lodish HF. Myogenic factors that regulate expression of muscle-specific microRNAs. Proc Natl Acad Sci U S A. 2006;103:8721–6. 50. Dolt KS, Mishra MK, Karar J, Baig MA, Ahmed Z, Pasha MA. cDNA cloning, gene organization and variant specific expression of HIF-1 alpha in high altitude yak (Bos grunniens). Gene. 2007;386:73–80. 51. Pasinelli P, Brown RH. Molecular biology of amyotrophic lateral sclerosis: insights from genetics. Nat Rev Neurosci. 2006;7:710–23. insights from genetics. Nat Rev Neurosci. 2006;7:710–23. 52. Kim KY, Lee HW, Shim YM, Mook-Jung I, Jeon GS, Sung JJ. A phosphomimetic mutant TDP-43 (S409/410E) induces Drosha instability and cytotoxicity in Neuro 2A cells. Biochem Biophys Res Commun. 2015;464:236–43. 53. Mackenzie IR, Rademakers R, Neumann M. TDP-43 and FUS in amyotrophic lateralsclerosis and frontotemporal dementia. Lancet Neurol. 2010;9:995–1007. 54. Yamazaki M, Esumi E, Nakano I. Is motoneuronal cell death in amyotrophic lateral sclerosis apoptosis? Neuropathology. 2005;25:381–7. 55. Taylor JP, Brown RH Jr, Cleveland DW. Decoding ALS: from genes to mechanism. Nature. 2016;539:197–206.
https://openalex.org/W4323359545	https://aip.scitation.org/doi/pdf/10.1063/5.0123283	English	null	High-pressure studies of atomically thin van der Waals materials	Applied physics reviews	2,023	cc-by	32,174	REVIEW ARTICLE \| MARCH 07 2023 Articles You May Be Interested In In-plane and interlayer mechanical behaviors of diamane superlattice generated in twisted bilayer graphene J. Appl. Phys. (December 2022) Enhanced vertical piezoelectricity in nano-switch diamane structures by super-dipole-moment effect Appl. Phys. Lett. (April 2024) 24 October 2024 03:58:28 Interfacial thermal transport between graphene and diamane Interfacial thermal transport between graphene and diamane AFFILIATIONS AFFILIATIONS 1Physics Department, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA 2Departamento de Fısica, Universidade Federal de Ouro Preto, Ouro Preto MG 35400-000, Brazil 3Departamento de Fısica, Universidade Federal de Minas Gerais, Belo Horizonte MG 31270-901, Brazil 4Department of Physics, University of Washington, Seattle, Washington 98195, USA 5Department of Materials Science and Engineering, University of Washington, Seattle, Washington 98 Note: This paper is part of the special collection on Quantum Materials and 2D superlattices. a)Author to whom correspondence should be addressed: lmartins@mit.edu b)Electronic mail: rcomin@mit.edu c)Electronic mail: matheusmatos@ufop.edu.br d)Electronic mail: mazzoni@ﬁsica.ufmg.br e)Electronic mail: bernardo@ﬁsica.ufmg.br f)Electronic mail: myank@uw.edu 1Physics Department, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA 2Departamento de Fısica, Universidade Federal de Ouro Preto, Ouro Preto MG 35400-000, Brazil 3Departamento de Fısica, Universidade Federal de Minas Gerais, Belo Horizonte MG 31270-901, Brazil 4Department of Physics, University of Washington, Seattle, Washington 98195, USA 24 October 2024 03:58:28 High-pressure studies of atomically thin van der Waals materials Cite as: Appl. Phys. Rev. 10, 011313 (2023); doi: 10.1063/5.012328 Submitted: 29 August 2022 . Accepted: 13 February 2023 . Published Online: 7 March 2023 ABSTRACT Two-dimensional (2D) materials and their moire superlattices represent a new frontier for quantum matter research due to the emergent properties associated with their reduced dimensionality and extreme tunability. The properties of these atomically thin van der Waals (vdW) materials have been extensively studied by tuning a number of external parameters such as temperature, electrostatic doping, magnetic ﬁeld, and strain. However, so far pressure has been an under-explored tuning parameter in studies of these systems. The relative scarcity of high- pressure studies of atomically thin materials reﬂects the challenging nature of these experiments, but, concurrently, presents exciting oppor- tunities for discovering a plethora of unexplored new phenomena. Here, we review ongoing efforts to study atomically thin vdW materials and heterostructures using a variety of high-pressure techniques, including diamond anvil cells, piston cylinder cells, and local scanning probes. We further address issues unique to 2D materials such as the inﬂuence of the substrate and the pressure medium and overview efforts to theoretically model the application of pressure in atomically thin materials. V C 2023 Author(s). All article content, except where otherwise noted, is licensed under a Creative Commons Attribution (CC BY) license (http:// creativecommons.org/licenses/by/4.0/). https://doi.org/10.1063/5.0123283 TABLE OF CONTENTS I. INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 II. HIGH-PRESSURE EXPERIMENTS WITH DIAMOND ANVIL CELLS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 A. Instrumentation and experimental technique . . . . 3 1. DAC operation . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 2. The gasket . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 3. Determining the pressure: The ruby fluorescence method. . . . . . . . . . . . . . . . . . . . . . . 3 4. The choice of the pressure transmitting medium . . . . . . . . . ABSTRACT . . . . . . . . . . . . . . . . . . . . . . . . 4 B. The influence of the substrate and the PTM: Strain transfer, sample detachment, and detection of stress gradients . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 C. Evidence of pressure-induced formation of the 2D diamond from few-layer graphene . . . . . . . . . . 7 D. Pressure-tuning of the electronic band structure in monolayer TMDs. . . . . . . . . . . . . . . . . . . . . . . . . . 9 E. Pressure-tuning of the electronic structure in TMD heterostructures . . . . . . . . . . . . . . . . . . . . . . . . 10 III. HIGH-PRESSURE EXPERIMENTS WITH PISTON-CYLINDER PRESSURE CELLS . . . . . . 12 Applied Physics Reviews REVIEW scitation.org/journal/are High-pressure studies of atomically thin van der Waals materials Cite as: Appl. Phys. Rev. 10, 011313 (2023); doi: 10.1063/5.0123283 Submitted: 29 August 2022 . Accepted: 13 February 2023 . Published Online: 7 March 2023 Luiz G. Pimenta Martins,1,a) Riccardo Comin,1,b) Matheus J. S. Matos,2,c) Mario S. C. Mazzoni,3,d) Bernardo R. A. Neves,3,e) and Matthew Yankowitz4,5,f) AFFILIATIONS 1Physics Department, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA 2Departamento de Fısica, Universidade Federal de Ouro Preto, Ouro Preto MG 35400-000, Brazil 3Departamento de Fısica, Universidade Federal de Minas Gerais, Belo Horizonte MG 31270-901, Brazil 4Department of Physics, University of Washington, Seattle, Washington 98195, USA 5Department of Materials Science and Engineering, University of Washington, Seattle, Washington 98195, USA Note: This paper is part of the special collection on Quantum Materials and 2D superlattices. a)Author to whom correspondence should be addressed: lmartins@mit.edu b)Electronic mail: rcomin@mit.edu c)Electronic mail: matheusmatos@ufop.edu.br d)Electronic mail: mazzoni@ﬁsica.ufmg.br e)Electronic mail: bernardo@ﬁsica.ufmg.br f)Electronic mail: myank@uw.edu ABSTRACT Applied Physics Reviews REVIEW scitation.org/journal/are A. Instrumentation and experimental technique . . . . 12 1. Piston-cylinder cell assembly and operation . . 12 2. Electrical characterization of vdW samples . . . 13 3. Modifying the interlayer spacing between vdW crystals with pressure. . . . . . . . . . . . . . . . . 13 B. Moire materials under pressure . . . . . . . . . . . . . . . . 14 1. Angle-aligned graphene/BN . . . . . . . . . . . . . . . . 14 2. Twisted bilayer graphene. . . . . . . . . . . . . . . . . . . 14 3. Twisted double bilayer graphene . . . . . . . . . . . . 15 C. Controlling 2D magnetism with pressure. . . . . . . . 15 D. Pressure-tunable proximity effects. . . . . . . . . . . . . . 17 IV. HIGH PRESSURE EXPERIMENTS USING SCANNING PROBE MICROSCOPY. . . . . . . . . . . . . . . . 17 A. Instrumentation and experimental technique . . . . 17 B. Pressure-induced phase transition in 2D materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 C. Pressure-tuning of properties of 2D materials . . . . 20 V. MODELING ATOMICALLY THIN VAN DER WAALS MATERIALS AT HIGH PRESSURES . . . . . . . . 22 A. Methodology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 B. Structural phase transitions . . . . . . . . . . . . . . . . . . . . 22 C. Modulation of electronic properties. . . . . . . . . . . . . 23 VI. OUTLOOK . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I. INTRODUCTION Two-dimensional (2D) materials can be deﬁned as materials with a thickness of one or a few atoms.1 From the perspective of quantum matter research, the reduced dimensionality in 2D materials is linked to a reduction in the available phase space and the electronic screening of Coulomb forces, leading to enhanced quantum effects and increased interactions.2 For this reason, 2D materials often exhibit outstanding transport, optical and magnetic properties that have been intensively investigated over the past decades.3 The ability to stack dissimilar vdW crystals atop one another is another major advantage of these materi- als, forming the so-called van der Waals heterostructures featuring atomically sharp interfaces.3 Lattice mismatches and/or twist angles between neighboring crystals further lead to the emergence of moire patterns—geometric interference patterns that act as periodic poten- tials—which often result in a rich variety of physical phenomena not observed in the individual vdW layers alone. In order to glean insight into the physics of 2D vdW materials and moire heterostructures and unlock new materials functionalities, the properties of these systems have been extensively studied by tuning various external parameters including temperature,4–6 electric ﬁeld,7–9 electrostatic doping,10–13 magnetic ﬁeld,14–16 and in-plane strain.17–20 Pressure is another important thermodynamic variable that can be used to tune the prop- erties of materials, serving to reduce interatomic distances, strengthen- ing interatomic and magnetic interactions, and modifying the electronic density. Pressure, therefore, enables the realization of new physical phenomena as well as new material structures that are not accessible under ambient conditions. The relative scarcity of high- pressure studies involving 2D materials and heterostructures reveals that these are challenging experiments, but also present an opportunity Applied Physics Reviews . 24 A. High-pressure synthesis of novel 2D materials . . . 24 B. Pressure tuning of properties and many-body states of 2D materials and moire heterostructures 24 to study a relatively untapped research ﬁeld with a wealth of interest- ing new potential phenomena. Contrasting to other review articles on high-pressure studies of 2D systems,21,22 this review focuses solely on atomic-thin materials, which present their own experimental and theoretical challenges when compared to bulk 2D-layered van der Waals materials such as graphite (which sometimes are also referred to as 2D). For instance, it is well established that for atomically thin materials subjected to hydrostatic compression, the in-plane compression forces are exerted by the sub- strate via its adhesion to the 2D system, and not by the pressure medium, unlike for bulk materials.23–25 Also, unlike bulk materials, different compressibility between a solid pressure medium and the substrate can lead to stress gradients that can signiﬁcantly affect the interpretation of the experimental data. In this review, we address those issues in detail. Furthermore, previous reviews on the topic focused on high-pressure experiments using the diamond anvil cell (DAC) technique. In our work, apart from DACs, we review several high-pressure studies of atomically thin van der Waals materials using piston-cylinder cells and scanning probe microscopy techniques. An additional and important element of novelty is a dedicated session to the theoretical approach to model atomically thin systems at high pressures, including the theoretical formalism to simulate the applica- tion of pressure in those systems. This review is arranged as follows: Secs. II–IV describe high- pressure studies performed using different experimental techniques: diamond anvil cells (DACs), piston-cylinder cells, and scanning probe microscopy, respectively. A description of the instrumentation of each technique is reviewed at the beginning of each section. Section V dis- cusses the theoretical aspects of modeling atomic thin materials under compression. Section VI concludes with possible future research direc- tions in this ﬁeld. 24 October 2024 03:58:28 TABLE OF CONTENTS Appl. Phys. Rev. 10, 011313 (2023); doi: 10.1063/5.0123283 Applied Physics Reviews II. HIGH-PRESSURE EXPERIMENTS WITH DIAMOND ANVIL CELLS The ﬁeld of high-pressure research has continuously expanded since the pioneering work of Percy W. Bridgman, who won the Nobel Prize in Physics in 1946 for the invention of an instrument to produce extremely high pressures, and for his discoveries within the ﬁeld of high pressure physics.26 In the Bridgman era (1910–1950), high-pressure experiments were performed using the so-called Bridgman anvil and piston cylinder devices, which allowed to per- form electrical resistance and compressibility measurements up to 10 GPa,27 (as a comparison, the pressure at Earth’s core is estimated to be around 350 GPa). The diamond anvil cell (DAC) was later developed in 1958,28 revolutionizing high-pressure research since it allowed direct optical observation of the effects of static pressure on matter through different experimental techniques such as x-ray dif- fraction, optical absorption, reﬂectivity, and Raman spectroscopy.27 Nowadays, the typically accessible pressure achieved in DAC experi- ments ranges from 0.1 GPa to about 400 GPa- considered the pres- sure limit for conventional DACs29—however, improvements in DAC design can bring the maximum pressure to the terapascal range.30 That range of pressures provides a platform to study funda- mental physical phenomena, such as insulator–metal transitions,31,32 classical phase transitions,33,34 quantum phase transitions,35,36 and high-temperature superconductivity.37,38 10, 011313-2 10, 011313-2 Appl. Phys. Rev. 10, 011313 (2023); doi: 10.1063/5.0123283 V C Author(s) 2023 Appl. Phys. Rev. 10, 011313 (2023); doi: 10.1063/5.0123283 Applied Physics Reviews REVIEW scitation.org/journal/are Furthermore, given that the rules of chemistry can be quite differ- ent at high pressures, several new exotic materials have been obtained from high-pressure experiments39,40 such as materials with unprece- dented stoichiometries41 and with unusual chemical bonding.42 Therefore, high-pressure studies using DACs constitute a powerful experimental technique in condensed matter physics to synthesize new materials, tune materials’ properties, and observe novel many- body physics phenomena. the PTM as well as the sample/PTM volume ratio, as will be later dis- cussed. Since the compressed area is quite small (104 (lm)2), large pressures can be generated with the application of moderate forces. The pressure can be increased typically by tightening the screws that connect the two body parts of the DAC, with each body contain- ing one diamond, or by the use of a membrane that can be inﬂated by the injection of an inert gas, which will push one body part against the other. 2. The gasket The gasket is a crucial element for the operation of a diamond anvil cell. It (i) contains the PTM, allowing for the generation of hydrostatic pressures; (ii) prevents direct contact with the diamonds (since diamonds can break upon direct contact and applied force); and (iii) extrudes material around the diamonds, as illustrated in Fig. 1, forming a supporting ring which prevents the diamonds to crack due to the stress concentrated at the edges of the anvil faces.27 24 October 2024 03:58:28 The gasket preparation usually starts with the indentation of the metal foil by squeezing it between the two anvils, which will reduce the thickness of the compressed area. Ideally one should use the same dia- monds that will be used in the high-pressure experiment. The indenta- tion is performed in order to minimize the plastic deformation during the experiment, such that only the necessary degree of thinning is applied in order to pressurize the PTM.43 Typically the thinner the ﬁnal indentation thickness is, the higher the maximum pressure will be. FIG. 1. Schematic of a diamond anvil cell. Cross section view of a DAC in which the sample is compressed hydrostatically when two opposite diamonds are pushed together, squeezing the gasket between them and compressing the pressure medium, which transmits the forces to the sample. Ruby crystals are usually used as the pressure indicators due to the pressure-induced shifts in their ﬂuorescence energy. Reproduced with permission from Martins et al., Nat. Commun. 8, 1–9 (2017). Copyright 2017 Authors, licensed under a Creative Commons Attribution. After indentation, a hole is drilled at the center of the gasket with dimensions typically of 1/3–1/2 of the culet size, as previously men- tioned, which will work as the pressure chamber. Then, for the experi- ment, it is important that the gasket is seated on the lower diamond in the same orientation as it had during the indentation so that the indentation marks can align with those from the diamond facets. It is also important to choose the right material for the gasket for a given experiment. For instance, if the goal is to achieve higher pres- sures, one should choose hard yet malleable materials such as rhenium or stainless steel. It is also important to choose a material that is com- patible with the PTM/sample for the experimental conditions. 2. The gasket For instance, to avoid the formation of oxides originating from a chemical reaction between the PTM and the gasket material, stainless-steel gas- kets are a suitable option. FIG. 1. Schematic of a diamond anvil cell. Cross section view of a DAC in which the sample is compressed hydrostatically when two opposite diamonds are pushed together, squeezing the gasket between them and compressing the pressure medium, which transmits the forces to the sample. Ruby crystals are usually used as the pressure indicators due to the pressure-induced shifts in their ﬂuorescence energy. Reproduced with permission from Martins et al., Nat. Commun. 8, 1–9 (2017). Copyright 2017 Authors, licensed under a Creative Commons Attribution. 1. DAC operation Figure 1 shows schematics of a DAC. The principle of its opera- tion is quite simple. One initially places a thin metallic disk—the gas- ket—with a pre-drilled hole, on top of the ﬂat tip—the culet—of the bottom diamond. The dimensions of the gasket hole are typically 1/ 3–1/2 of the culet’s diameter on the order of 102 lm. Then, one adds the sample and a manometer—usually a few ruby crystals—inside of the gasket hole, together with the pressure transmitting medium (PTM). By bringing the top diamond as shown in Fig. 1, the pressure chamber will be sealed, with the sample, the pressure indicator, and the PTM contained by the gasket walls and the two culets of the dia- monds. High pressures are generated when the opposite diamonds are pushed together, squeezing the gasket and, therefore, the chamber, compressing the pressure medium, which transmits the forces to the sample. In this way, the uniaxial compression applied along the DAC axis is converted into hydrostatic pressure by the use of the PTM. However, the degree of hydrostaticity highly depends on the choice of II. HIGH-PRESSURE EXPERIMENTS WITH DIAMOND ANVIL CELLS Diamonds are chosen as anvils because of their superior hard- ness, being able to withstand pressures of GPa order without breaking, and the fact that they are transparent to x-rays and visible light. Thus, at each pressure, one can excite the system with a given light source, either a laser or a monochromatic x-ray beam, and collect the light emitted/scattered by the sample, therefore probing the modiﬁcations induced by pressure in the material. Different types of diamonds can be used, depending on the purpose. For instance, for Raman spectros- copy measurements, it is desirable to use diamonds with a very low defect density, typically type IIa diamonds, which will emit low- intensity ﬂuorescence signals (originated from optical transitions between defect levels). Applied Physics Reviews REVIEW scitation.org/journal/are method, small ruby crystals are inserted inside the gasket hole, as illus- trated in Fig. 1, and are used as manometers. measuring the spectral linewidth of the R1 ﬂuorescence peak from ruby, which can give information about the presence of local non- hydrostatic stress components. The R1 peak will broaden under the presence of local non-hydrostatic stress components because it is asso- ciated with an electronic transition within the Cr3þ impurities, which is highly sensitive to the local environment around the chromium ion. The ruby crystals are composed of a Al2O3 matrix with Cr3þ impurities. Upon excitation with visible light, those crystals emit two ﬂuorescence peaks termed as R1 and R2 at ambient temperature. The R1 and R2 lines are associated with electronic transitions within the Cr3þ impurity, and their energy will shift upon compression due to modiﬁcations in the crystal-ﬁeld-split energy levels induced by pres- sure.48 The calibration via ruby ﬂuorescence is based on the evolution of the R1-line energy with increasing pressure (P), and the connection between R1 energy and P was established via concomitant x-ray dif- fraction measurements of four metals (Mo, Cu, Pd, and Ag) and the ﬁtting of their speciﬁc volume with well-established equations of state for those metals.44,45 Therefore, one can determine the pressure (in GPa units) via the shift in the R1 line from ambient pressure condi- tions Dk ¼ ðkðPÞ kðP0ÞÞ, via the expression, This method remains popular and has still been used for instance, to determine the hydrostatic limits of several PTMs for low- temperature experiments,53 in which the solidiﬁcation of the PTM is unavoidable even at low pressures, due to the temperature drop. Importantly, some mediums can remain good quasi-hydrostatic PTMs even after their solidiﬁcation, if the presence of deviatoric stress is sufﬁciently small. One example is the water PTM. Even though water solidiﬁes at 1 GPa54 at ambient temperature, previous stud- ies52,54,55 show a lack of evidence of signiﬁcant pressure gradients or local non-hydrostatic stress components in the water medium up to approximately 8 GPa. P GPa ½ ¼ A B 1 þ Dk nm ½ k0 B 1 " # ; (1) Table I shows the solidiﬁcation pressure and quasi-hydrostatic limit at ambient temperature for several PTMs commonly used in high-pressure experiments. Applied Physics Reviews (1) where the A and B constants are 1904 and 7665, respectively, and k0 ¼ 694; 2 nm is the wavelength of the R1 ﬂuorescence line at a pres- sure of 1 bar.45 Another important factor to take into account when analyzing the degree of hydrostaticity in an HP experiment is the sample/PTM volume ratio. The higher this ratio is, the lower the degree of hydrosta- ticity will be. Other relevant aspect of choosing a PTM is the difﬁculty of its loading process into the DAC. Liquid PTMs are fairly simple to load, as long as they are not volatile, whereas loading gases is consider- ably more difﬁcult, often requiring equipment speciﬁcally designed for this purpose. Importantly, the positions of the R1 and R2 peaks also shift with temperature, and several works have investigated the pressure calibra- tion both in the low-temperature49 and high-temperature range.50 Alternative methods to determine the pressure inside of the chamber in DAC experiments are: keeping track of the ﬁrst-order Raman peak from the diamond window, which shows a linear shift with pressure,46 or measuring the pressure-dependence of the speciﬁc volume of a ref- erence material that has a well established-equation of state,47 such as Ag and Cu, via high-pressure x-ray diffraction. 24 October 2024 03:58:28 B. The influence of the substrate and the PTM: Strain transfer, sample detachment, and detection of stress gradients One of the main differences between high-pressure experiments involving bulk vs atomically thin materials is the inﬂuence of the sub- strate and the PTM on their mechanical responses. While for bulk materials, the compression is directly exerted by the PTM, for atomi- cally thin materials, it has been established that the in-plane compres- sion is exerted by the strain transfer from the substrate via its adhesion to the 2D material.23–25 That is, for a perfect adhesion, the extent to which the 2D material will be in-plane compressed will solely be 3. Determining the pressure: The ruby fluorescence method In high-pressure experiments using DACs, there are different methods that can be used to determine the pressure inside the cham- ber.44–47 A widely popular method is ruby ﬂuorescence.44,45 In this 10, 011313-3 Appl. Phys. Rev. 10, 011313 (2023); doi: 10.1063/5.0123283 V C Author(s) 2023 10, 011313-3 Applied Physics Reviews 4. The choice of the pressure transmitting medium If the adhesion is not ideal, the mechanical response of the 2D material will be deter- mined by the strain transfer from the substrate, while if the sample detaches from the substrate or if it is unsupported, its in-plane com- pression will be determined by the compressibility of the 2D material itself.24,58 The inﬂuence of the PTM should particularly be taken into account once the PTM solidiﬁes since eventual differential compress- ibility between the solid PTM and the substrate could lead to shear in- plane components on the 2D material25 that could lead to lattice dis- tortions and defects. In that context, Raman spectroscopy can provide direct information about the presence of stress/strain gradients across the compressed sample.56,59 It was also initially proposed that the PTM could chemically dope the 2D samples upon compression,60 which was later disproved24 for the graphene case. This section describes these works. of the N2 PTM above 1.2 GPa, consistent with the results from the fol- lowing work by Filintoglou et al.24 The effect of the number of gra- phene layers and the adhesion to the SiO2 substrate was studied in a subsequent work by Nicolle et al.60 via a calculation that took into account the competition between the adhesion and the elastic energies for graphene to conform to the rough SiO2 substrate. Their results showed that a debonding from the SiO2 substrate occurs for graphene layer thickness above two, which should result in a dxG/dP slope simi- lar to that of graphite. In the same work, the authors also proposed a giant doping effect from the PTM on their samples, which was later contested by Filintoglou et al.24 Filintoglou et al.24 investigated the contributions of both sub- strate and PTMs on the pressure response of graphene. The authors compressed monolayer graphene samples grown by chemical vapor deposition (CVD) on copper substrates using a polar (4:1 methanol/ ethanol, termed as alcohol by the authors) and non-polar (ﬂuorinert) PTM. In the 0–3 GPa range, they observed that the pressure slope for the G band is the same for both PTMs, suggesting that the pressure response of the G band frequency is mainly dictated by mechanical stress rather than the pressure doping from the PTM. 4. The choice of the pressure transmitting medium To further sup- port this claim and disentangle, the contributions from doping and strain, the authors plotted the frequencies of the G and 2D band (x2D) at increasing pressures, following the work of Lee et al.64 Their data show that the [xG(P), x2D(P)] points fall into a pure-strain line (d x2D dxG slope of 2.2 according to Lee et al.64) and constant electron- doping [Fig. 2(c)], conﬁrming that the PTM does not signiﬁcantly dope the samples as pressure increases. Importantly, the authors noticed a sudden drop in the frequency of the G band followed by an irreversible decrease in pressure slope from 9 cm–1/GPa to 5.6 cm–1/ GPa around 2 GPa for the ﬂuorinert PTM [Fig. 2(d)] without a signiﬁ- cant broadening of the G bandwidth. They attributed this behavior to the fact that the PTM solidiﬁes around that pressure, resulting in gra- phene being compressed between two solid surfaces, therefore, not being preferably adhered to the copper substrate, which should resem- ble the mechanical behavior of free-standing graphene. In fact, the pressure slope of 5.6cm–1/GPa is consistent with the recently reported slope of 5.4cm–1/GPa for unsupported graphene.58 Several groups have investigated the contributions from the sub- strate and the PTM in high-pressure DAC experiments via Raman spectroscopy by analyzing the pressure the evolution of the G band in graphene.23–25 The G band (approximately at 1580 cm1) is associated with the in-plane C–C bond-stretching mode in the graphene lattice and is extremely sensitive to the effects of strain and also doping.61 An important parameter to investigate the pressure evolution of Raman frequencies under strain is the Gr€uneisen parameter k. For a quasi- harmonic mode of frequency x, the Gr€uneisen parameter k can be deﬁned as62 xðPÞ xðP0Þ ¼ VðPÞ VðP0Þ k ; (2) 24 October 2024 03:58:28 (2) where P0 is the initial pressure. In the graphite case, given the consid- erable anisotropy between in-plane and out-of-plane compressibilities, one can deﬁne the Gr€uneisen parameter for the G band considering only the in-plane compressibility23 such that Eq. (2) can be rewritten as as xðPÞ xðP0Þ ¼ aðPÞ aðP0Þ 2kG ; (3) (3) where a is the lattice parameter of graphene.23 From Eq. (3), one can see that the G band frequency (xG) will blueshift upon compression. 4. The choice of the pressure transmitting medium Ideally, high-pressure experiments should be performed under hydrostatic conditions. However, every PTM solidiﬁes after a given pressure, and upon solidiﬁcation, the medium and consequently the sample can develop pressure gradients and local anisotropic stress components (also termed deviatoric stress) that can signiﬁcantly affect the interpretation of the experimental data depending on their magni- tude. A recent example is of the superconductor CaFe22As2 in which different high-pressure experiments reported conﬂicting conclusions about its PT phase diagram, with the contradiction being solved by the realization that the superconducting phase is extremely sensitive to non-hydrostatic conditions, being favored by uniaxial components while absent under hydrostatic conditions.51 Therefore, it is extremely important to identify the hydrostatic limits of the PTM for a given HP experiment, as well as the presence and strength of pressure gradients and local non-hydrostatic stress components. TABLE I. Solidiﬁcation pressure and hydrostatic/quasi-hydrostatic range for several PTMs commonly used in high pressure experiments. TABLE I. Solidiﬁcation pressure and hydrostatic/quasi-hydrostatic range for several PTMs commonly used in high pressure experiments. PTM Solidification pressure at room temperature (GPa) Hydrostatic/ quasi-hydrostatic range (GPa) References Water 1.0 8.0 52, 54–56. 4:1 methanol/ ethanol 10.5 10.5 57 Neon 4.8 15 57 Helium 12.1 >40 57 Argon 1.4 10 57 Nitrogen 2.4 12.0 57 In order to quantify the degree of hydrostaticity of a given PTM, a very popular technique was introduced by the seminal work of Piermarini et al.52 In their work, they investigated the hydrostatic limit of several PTMs in DAC experiments using two methods: (i) by mea- suring the pressure at several different locations from tiny ruby crystals spread across the chamber—which can give information about the presence of pressure gradients inside of the chamber, and (ii) by Appl. Phys. Rev. 10, 011313 (2023); doi: 10.1063/5.0123283 V C Author(s) 2023 10, 011313-4 Applied Physics Reviews REVIEW scitation.org/journal/are determined by the compressibility of the substrate. 4. The choice of the pressure transmitting medium In the light of the analysis by Filintoglou et al.,24 an important issue to be analyzed is how differences in the compressibility between the PTMs, once it solidiﬁes, and the substrate, can affect the mechani- cal response of a 2D material. This issue was investigated by Machon et al.25 In their work, the authors analyzed bi-layer graphene samples on SiO2/Si substrates compressed up to 8–10 GPa using three different PTMs:4:1 methanol/ethanol, water, and argon. Importantly, the 4:1 methanol/ethanol PTM remains liquid/hydrostatic up to 10.5GPa, while water turns into Ice VI and Ice VII at approximately 1 and 2 GPa, respectively, and argon solidiﬁes at 1.4 GPa- however, both water and argon PTMs remain quasi-hydrostatic up to higher pres- sures (see Table I). The authors observed a linear evolution of the G band frequency with pressure up to approximately 8 GPa for both the 4:1 methanol/ethanol and water PTMs, whereas the slope deviates from the linear behavior after the solidiﬁcation pressure for the argon PTM [Fig. 2(e)]. To understand those results, the authors plotted the pressure evolution of the solid surfaces and their pressure derivatives for water, N2, Ar, and SiO2, shown in Fig. 2(f). Notice that the surface compressibility of water and SiO2 are remarkably similar in the 0–8 GPa pressure range, resulting in an absence of in-plane shear The ﬁrst high-pressure work on graphene was carried out by Proctor et al.23 In their work, the authors compressed and exfoliated few layer graphene samples on a 300-nm SiO2-coated silicon substrate. For the mono, bi, and few-layer ﬂakes on the SiO2/Si substrate, they noticed that the thinner the sample is, the steeper its G band pressure slope (dxG/dP) is, as shown in Fig. 2(a). They proposed that the thin- ner the sample is, the greater the inﬂuence of the substrate will be on its in-plane compression. Since the SiO2/Si substrate is more com- pressible than graphene, dxG/dP will be higher for graphene on SiO2/ Si compared to unsupported graphene, the latter having similar dxG/ dP to graphite. To quantify this effect, the authors plotted the pressure evolution of the G band using Eq. (3) considering silicon’s compress- ibility for the aðPÞ=aðP0Þ term and graphene’s Gr€uneisen parameter kG ¼ 1:99 obtained from Ref. 63 [Fig. 2(b)]. For the G band, the agreement with the experimental data was good at low pressures (0–1.5 GPa) and started to deviate at higher pressures. 4. The choice of the pressure transmitting medium (e) G band frequency (black ﬁlled squares) and full width at half maximum (red empty squares) subtracted from their values at ambient pressure for bi-layer graphene on SiO2/Si as a function of pressure for 4:1 methanol/ethanol (left panel), water (center panel), and argon (right panel) PTMs. The verti- cal dotted lines correspond to different pressure-induced phase transitions of the PTMs to a solid phase, while the hatched region for argon corresponds to its pressure recrys- tallization domain. (f) Surface compression (let panel) and its derivative (right panel) as a function of pressure for different PTMs. [(e) and (f)] Reproduced with permission from Machon et al., J. Raman Spectrosc. 49 121 (2018). Copyright 2018 John Wiley and Sons. 24 October 2024 03:58:28 24 October 2024 03:58:28 FIG. 2. Inﬂuence of the substrate and the PTM via analysis of the pressure-evolution of the Raman G band in graphene. (a) Raman frequencies of the G band as a function of pressure for mono-, bi, and few-layer graphene on a SiO2-coated silicon substrate and of unsupported graphite using a N2 PTM. (b) G band frequencies as a function of pres- sure for monolayer graphene on SiO2/Si. The black solid line is the calculated xG using Eq. (3) considering silicon’s compressibility aðPÞ=aðP0Þ and using a Gr€uneisen param- eter of kG ¼ 1:99 obtained from Ref. 63. [(a) and (b)] Reprinted with permission from Proctor et al., Phys. Rev. B 80, 073408 (2009). Copyright 2009 the American Physical Society. (c) Correlation between the Raman frequencies of the G (xG) and 2D (x2D) bands for monolayer graphene at increasing pressures to extract information about doping and strain. The ﬁlled and empty squares correspond to two different pressure runs using a ﬂurorinert PTM, while the ﬁlled circles correspond to a pressure run using 4:1 meth- anol/ethanol PTM. The dotted straight lines correspond to pure-strain lines (constant doping) (d x2D/dxG slope of 2.264) (d). The star represents the (x2D,xG) values for undoped, unstrained monolayer graphene obtained from Ref. 64. The solid and doted lines starting from the star symbol correspond to pure electron and hole doping (no strain), respectively, as obtained from Ref. 65. (d) Pressure evolution of the G band frequency for monolayer graphene on the copper substrate. Empty and ﬁlled symbols cor- respond to increasing and decreasing pressures. 4. The choice of the pressure transmitting medium The authors attributed the deviation to graphene debonding at higher pressures. In fact, we point out that the debonding could be due to the solidiﬁcation 10, 011313-5 10, 011313-5 Appl. Phys. Rev. 10, 011313 (2023); doi: 10.1063/5.0123283 V C Author(s) 2023 Appl. Phys. Rev. 10, 011313 (2023); doi: 10.1063/5.0123283 Applied Physics Reviews scitation.org/journal/are FIG. 2. Inﬂuence of the substrate and the PTM via analysis of the pressure-evolution of the Raman G band in graphene. (a) Raman frequencies of the G band as a function of pressure for mono-, bi, and few-layer graphene on a SiO2-coated silicon substrate and of unsupported graphite using a N2 PTM. (b) G band frequencies as a function of pres- sure for monolayer graphene on SiO2/Si. The black solid line is the calculated xG using Eq. (3) considering silicon’s compressibility aðPÞ=aðP0Þ and using a Gr€uneisen param- eter of kG ¼ 1:99 obtained from Ref. 63. [(a) and (b)] Reprinted with permission from Proctor et al., Phys. Rev. B 80, 073408 (2009). Copyright 2009 the American Physical Society. (c) Correlation between the Raman frequencies of the G (xG) and 2D (x2D) bands for monolayer graphene at increasing pressures to extract information about doping and strain. The ﬁlled and empty squares correspond to two different pressure runs using a ﬂurorinert PTM, while the ﬁlled circles correspond to a pressure run using 4:1 meth- anol/ethanol PTM. The dotted straight lines correspond to pure-strain lines (constant doping) (d x2D/dxG slope of 2.264) (d). The star represents the (x2D,xG) values for undoped, unstrained monolayer graphene obtained from Ref. 64. The solid and doted lines starting from the star symbol correspond to pure electron and hole doping (no strain), respectively, as obtained from Ref. 65. (d) Pressure evolution of the G band frequency for monolayer graphene on the copper substrate. Empty and ﬁlled symbols cor- respond to increasing and decreasing pressures. The circle and square symbols correspond to compression with alcohol and fulorinert PTMs, respectively, while the triangle symbol corresponds to compression with ﬂuorinert after sample detachment. The dotted line is the calculated xG shift, considering the bulk modulus of copper and perfect adhesion of graphene. The numbers represent the dxG/dP pressure slopes. [(c) and (d)] Reprinted with permission from Filintoglou et al., Phys. Rev. B 88, 045418 (2013). Copyright 2013 the American Physical Society. 4. The choice of the pressure transmitting medium The circle and square symbols correspond to compression with alcohol and fulorinert PTMs, respectively, while the triangle symbol corresponds to compression with ﬂuorinert after sample detachment. The dotted line is the calculated xG shift, considering the bulk modulus of copper and perfect adhesion of graphene. The numbers represent the dxG/dP pressure slopes. [(c) and (d)] Reprinted with permission from Filintoglou et al., Phys. Rev. B 88, 045418 (2013). Copyright 2013 the American Physical Society. (e) G band frequency (black ﬁlled squares) and full width at half maximum (red empty squares) subtracted from their values at ambient pressure for bi-layer graphene on SiO2/Si as a function of pressure for 4:1 methanol/ethanol (left panel), water (center panel), and argon (right panel) PTMs. The verti- cal dotted lines correspond to different pressure-induced phase transitions of the PTMs to a solid phase, while the hatched region for argon corresponds to its pressure recrys- tallization domain. (f) Surface compression (let panel) and its derivative (right panel) as a function of pressure for different PTMs. [(e) and (f)] Reproduced with permission from Machon et al., J. Raman Spectrosc. 49 121 (2018). Copyright 2018 John Wiley and Sons. 10, 011313-6 10, 011313-6 Appl. Phys. Rev. 10, 011313 (2023); doi: 10.1063/5.0123283 V C Author(s) 2023 Applied Physics Reviews REVIEW scitation.org/journal/are an atomically thin diamond that could show additional unique fea- tures due to the quantum conﬁnement. Several theoretical models have predicted 2D diamond structures with different properties, how- ever, synthesizing stable and functional forms of these materials remains a current challenge. Different methods have been explored for its synthesis, including pressure-less chemical functionalization of bi- layer graphene69–71 as well as high-pressure compression of few-layer graphene using scanning probe microscopy tips72–75 or hydrostatic compression using DACs.56,76–78 This section focuses on the latter experimental method, while Sec. VB discusses different theoretical models of 2D-diamond structures. For a detailed review of all the experimental methods for 2D diamond synthesis, we refer the readers to Refs. 68 and 79. stress components, explaining the well-behaved linear evolution of G band frequency for the water case. On the other hand, the surface compressibility of solid Ar and SiO2 are considerably different, intro- ducing in-plane non-hydrostatic stress components, which would explain the deviations from the linear behavior of dxG/dP. 4. The choice of the pressure transmitting medium p We point out that a simple and direct method to determine the presence of pressure gradients generated by the PTM and/or substrate in an atomic-thin material is to acquire the Raman spectra at different locations across the sample. In the absence of considerably doping from the PTM (or if the doping can be considered to be homoge- neous), signiﬁcant variations in the Raman frequencies at different locations can be attributed to stress gradients. Using this method, Pimenta Martins et al.56 measured xG at three different locations in graphene powder compressed using water as the PTM. The powder consisted of a mixture of graphene ﬂakes with thicknesses ranging from mono-layer to graphite, with the majority of ﬂakes (86% in mass) with thickness below 20 layers. For each pressure, the authors calculated the difference between the maximum and the minimum values xG values (d xG). They observed that above 8 GPa, the d xG values start to increase, being a direct indication of the presence of pressure gradients, in qualitative agreement with the detection of pres- sure gradients in compressed water found by Piermarini et al.52 This method was also used by Pimenta Martins et al.,59 where the authors measured the Raman spectra of monolayer MoS2 compressed in a 4:1 methanol/ethanol PTM at 4.5 GPa and 10 K (the PTM is solid at that temperature). The authors observed no deviations of all Raman peaks under these conditions, in qualitative agreement with the absence of deviatoric stress for the 4:1 methanol/ethanol PTM at low tempera- tures (5 and 77 K) at that pressure, as investigated by Tateiwa and Haga53 and Feng et al.66 The conversion of graphite to the cubic diamond at high pres- sures and high temperatures is a canonical example of the use of pres- sure to synthesize novel materials and constitutes a common route of diamond production for practical applications. However, previous high-pressure studies carried out with graphite at room-temperature showed evidence of a structural phase transition to an insulating, transparent, and sp3 containing phase,33,80–82 with several proposed crystal structures.33,83–86 Similarly, recent DAC experiments carried out with few-layer graphene have obtained different evidence of a phase transition to a 2D-diamond like phase, with different structures being proposed. 4. The choice of the pressure transmitting medium g p p The ﬁrst experimental evidence for the existence of 2D diamond via hydrostatic compression was carried out by Pimenta Martins et al.76 In their work, the authors compressed two layers of CVD gra- phene transferred onto a Teﬂon substrate using water as the PTM and they observed a phase transition to a 2D diamond-like phase using Raman spectroscopy. This technique was chosen due to its sensitivity to different atomic hybridizations in carbon materials.87 Upon conver- sion of few-layer graphene to 2D diamond, as well as from graphite to bulk diamond, the carbon atoms change their hybridization from sp2 to sp3. The phase transition was detected by measuring the difference in frequency of the G band in the double-layer graphene system using two different laser excitation energies: 2.33 eV (green) and 2.54 eV (blue) (DxG ¼ xG;blue xG;green), as a function of pressure. According to Ferrari and Robertson.,87 for a pure sp2 system, one should expect that DxG ¼ 0, whereas for a mixed sp2–sp3 system, DxG 6¼ 0 (more precisely, the G band frequency should increase by increasing the laser excitation energy87) Thus, by plotting DxG as a function of pressure [see Fig. 3(a)], a clear discontinuity in DxG can be seen at approximately 5 and 7.5GPa for two different pressure runs, which can be associated with a structural sp2 to sp2–sp3 phase transition. According to the authors, upon phase transition, an sp3 matrix is formed, containing nanometer-sized sp2 clusters. Importantly, the behavior of DxG vs pressure cannot be affected by the solid PTM (water becomes Ice VI at 1 GPa and Ice VII at 2 GPa) since the Raman spectra using the 2.33 and 2.54 eV excitations were acquired at the same locations on the sample at each pressure. Furthermore, the authors found no evidence of phase transition (no signiﬁcant change in DxG) up to 13 GPa for monolayer graphene compressed in water as well as for double-layer graphene compressed in mineral-oil PTM [Fig. 3(b)]. 4. The choice of the pressure transmitting medium The latter results indicate that for the phase transition to occur, it is necessary at least two layers of graphene in the presence of water, in agreement with previous experimental results from tip-compression of few-layer graphene from Barboza 24 October 2024 03:58:28 From the combined information from those previous works, one can infer that whenever the compressed 2D material is under good hydrostatic or quasi-hydrostatic conditions, the Raman frequencies will shift linearly, without a signiﬁcant broadening of the peaks or changes in the Raman frequencies when measured at different loca- tions at a given pressure. To conclude this section, we highlight the recent work of Sun et al.,58 which investigated the mechanical response of unsupported graphene samples in an N,N-dimethylformamide (DMF) PTM at high pressures. They argued that it is physically meaningful to attribute a c33 elastic constant to graphene, that is, monolayer graphene gets squeezed upon out-of-plane compression as a result of the compres- sion of the out-of-plane pz orbitals. Their conclusions were based on the analysis of the sub-linear evolution of the G band in unsupported graphene, which in analogy to the graphite case, can give information about the graphene’s out-of-plane stiffness. C. Evidence of pressure-induced formation of the 2D diamond from few-layer graphene The isolation of graphene from the mechanical exfoliation of graphite67 motivated the search for 2D-versions of selected bulk mate- rials, since the reduced dimensionality could lead to novel and exotic properties, adding to those from their bulk counterparts. Diamond exhibits outstanding properties such as superior hardness and stiffness as well as the highest thermal conductivity at room temperature for bulk materials, apart from being transparent and chemically inert.68 Therefore, experimental efforts have been directed toward obtaining Appl. Phys. Rev. 10, 011313 (2023); doi: 10.1063/5.0123283 V C Author(s) 2023 10, 011313-7 10, 011313-7 Applied Physics Reviews FIG. 3. Evidence of 2D diamond formation from high-pressure hydrostatic compression of few-layer graphene. (a) Pressure evolution of DxG, deﬁned as the difference in the G band frequency measured with 2.54 and 2.33eV excitation, for two different pressure runs of CVD double-layer graphene samples on a Teﬂon substrate compressed in a water PTM. (b) Pressure evolution of DxG for monolayer graphene on Teﬂon compressed in water (top panel) and double-layer graphene on Teﬂon compressed in a mineral oil PTM. [(a) and (b)] Reproduced with permission from Martins et al., Nat. Commun. 8 1–9 (2017). Copyright 2017 Authors, licensed under a Creative Commons Attribution. (c) and (d) Sheet resistance (c) and optical absorbance measurements as a function of pressure for an exfoliated tri-layer graphene sample directly transferred onto the diamond culet. The ellipse in (d) highlights the presence of a weak absorption edge above approximately 30GPa. [(c) and (d)] Reproduced with permission from Ke et al., Proc. Natl. Acad. Sci. U. S. A. 116, 9186 (2019). Copyright 2019 APS. (e) Sheet resistance as a function of pressure for exfoliated bi-, tri-, tetra-, hexa-, and 12-layer graphene samples directly trans- ferred onto the diamond culet. Reprinted with permission from Ke et al., Nano Lett. 20, 5916–5921 (2020). Copyright 2020 American Chemical Society. (f)–(j) Evidence for the for- mation of a hard 2D phase for few-layer graphene compressed in water up to 8 GPa. (f)–(g) Optical images of a sample containing a four-layer graphene and a thin graphite ﬂake on a SiO2/Si substrate before (f) and after compression up to 8 GPa in water PTM (g). The red boxes indicate the regions of those sample where AFM measurements were car- ried out. C. Evidence of pressure-induced formation of the 2D diamond from few-layer graphene (h)–(i) Topographical AFM images of the four-layer (h) and graphite (i) ﬂakes where the blue lines correspond to height proﬁles shown on the on the right panels. Notice that samples and the substrate are leveled—an evidence for indentation. (j) Three-dimensional topographical AFM image of the graphite ﬂake showing that it penetrated into the harder SiO2/Si substrate. [(f)–(j)] Reprinted with permission from Pimenta Martins et al., Carbon 173, 744–747 (2021). Copyright 2021 Elsevier. 24 October 2024 03:58:28 FIG. 3. Evidence of 2D diamond formation from high-pressure hydrostatic compression of few-layer graphene. (a) Pressure evolution of DxG, deﬁned as the difference in the G band frequency measured with 2.54 and 2.33eV excitation, for two different pressure runs of CVD double-layer graphene samples on a Teﬂon substrate compressed in a water PTM. (b) Pressure evolution of DxG for monolayer graphene on Teﬂon compressed in water (top panel) and double-layer graphene on Teﬂon compressed in a mineral oil PTM. [(a) and (b)] Reproduced with permission from Martins et al., Nat. Commun. 8 1–9 (2017). Copyright 2017 Authors, licensed under a Creative Commons Attribution. (c) and (d) Sheet resistance (c) and optical absorbance measurements as a function of pressure for an exfoliated tri-layer graphene sample directly transferred onto the diamond culet. The ellipse in (d) highlights the presence of a weak absorption edge above approximately 30GPa. [(c) and (d)] Reproduced with permission from Ke et al., Proc. Natl. Acad. Sci. U. S. A. 116, 9186 (2019). Copyright 2019 APS. (e) Sheet resistance as a function of pressure for exfoliated bi-, tri-, tetra-, hexa-, and 12-layer graphene samples directly trans- ferred onto the diamond culet. Reprinted with permission from Ke et al., Nano Lett. 20, 5916–5921 (2020). Copyright 2020 American Chemical Society. (f)–(j) Evidence for the for- mation of a hard 2D phase for few-layer graphene compressed in water up to 8 GPa. (f)–(g) Optical images of a sample containing a four-layer graphene and a thin graphite ﬂake on a SiO2/Si substrate before (f) and after compression up to 8 GPa in water PTM (g). The red boxes indicate the regions of those sample where AFM measurements were car- ried out. (h)–(i) Topographical AFM images of the four-layer (h) and graphite (i) ﬂakes where the blue lines correspond to height proﬁles shown on the on the right panels. Notice that samples and the substrate are leveled—an evidence for indentation. C. Evidence of pressure-induced formation of the 2D diamond from few-layer graphene According to the authors, the pressure exerted by the van der Waals forces between the graphene sheets, which could be as high as 1 GPa, ionizes the trapped water and initiates the reaction that gives rise to the 2D sheets of metal oxides. g However, evidence of 2D diamond formation from compression of few-layer graphene has been reported even in the absence of chemi- cal functionalization. Ke et al.77 compressed an exfoliated tri-layer gra- phene sample using Daphne 7373 oil or argon PTMs (both chemically inert) and observed a bandgap opening at approximately 30 GPa via electrical and optical absorption measurements. For the transport measurements, the tri-layer graphene sample was transferred onto the diamond culet and the electrodes were directly patterned and depos- ited on top of it. The phase transition from a semi-metal to semicon- ductor was detected by monitoring the sheet resistance as a function of pressure and noticing a sudden increase in resistance at 33 GPa, fol- lowed by an increase by more than three orders of magnitude upon further compression to 59 GPa, as can be seen in Fig. 3(c). The sheet resistance showed a weak temperature-dependence below 34 GPa, signiﬁcantly increasing with decreasing temperature above that pressure—a signature of semiconducting behavior. A bandgap of 2.5eV was conﬁrmed by UV-vis absorption measurements as shown in Fig. 3(d), highlighting the absorption edge. The authors proposed that this phase transition could be associated with a diamond-like 2D phase. Using the same experimental methodology, Ke et al.78 com- pressed tri-, tetra-, hexa, and 12-layer graphene and from electrical resistance measurements, they observed a bandgap opening at 33.0, 27.1, 21.3, and 19.6GPa, respectively [Fig. 3(e)]. Notice that as the number of graphene layers increases, the critical pressure decreases. From optical absorption measurements, all samples showed an absorption edge at approximately 2.8 eV. The authors proposed via DFT calculations that the new phase consists of an atomically thin hexagonal diamond, formed with no need of chemical functionaliza- tion. High-pressure x-ray diffraction (XRD) data from a graphene powder was provided to indicate the formation of a powder of hexago- nal 2D diamonds; however, it is important to point out that their XRD signal could have contributions from thick graphite ﬂakes that are usu- ally present in graphene powders90 or bulk-like structures formed from the interlayer bonding of several ﬂakes. C. Evidence of pressure-induced formation of the 2D diamond from few-layer graphene y g p For the ﬁve-layer graphene and graphite ﬂakes, an additional evi- dence of phase transition was a change in the xG vs P slope, becoming steeper between 4 and 7 GPa when compared to the 0–4 GPa range. As discussed in Sec. II B, a change in d xG=dP had been previously attributed to sample detachment by Filintoglou et al., however in their work, d xG=dP reduced without signiﬁcant changes in G bandwidth and 2D band intensity, as expected for a sample detachment process, unlike the results of Pimenta Martins et al.,56 where d xG=dP increased with signiﬁcant changes in G bandwidth and quenching of 2D band intensity. Therefore, Pimenta Martins et al. attributed the change in d xG=dP, to changes in the mechanical properties of the system. For the evidence of transparency, the authors observed changes in the optical contrast images of the ﬂakes in the same 4–7 GPa pressure range of the changes in the Raman spectra, culmi- nating in the detection of a Raman peak from the SiO2/Si substrate underneath the graphite ﬂake after 7 GPa, indicated a gradual surface- to-bulk increase in transparency due to the phase transition process. Finally, for the evidence of hardness, the authors observed indentation marks on the SiO2/Si substrate from a four-layer graphene and graph- ite samples compressed up to 8 GPa, from AFM topographic measure- ments as shown in Figs. 3(f)–3(j). The fact that SiO2 is signiﬁcantly harder than graphene systems (Vickers hardness of 564.9 kgf/mm2 for amorphous SiO2 compared to 7–11 kgf/mm2 for graphite) is evidence that the indentation can only be explained by the formation of a hard phase upon compression. Furthermore, they reported the lowest criti- cal pressure (approximately 4 GPa) and pressure-induced transpar- ency (completed at approximately 7 GPa) in graphite, which was attributed to the role of water in facilitating the phase transition. Their theoretical models, based on molecular dynamics simulations and ﬁrst-principles calculations, described a novel phase transition mecha- nism for compressed graphene systems in the presence of passivation groups on the surface layers. This model shows a gradual surface-to- bulk phase transition process, which starts with the sp2–sp3 rehybrid- ization of the ﬁrst two layers, forming diamondene and then propagates along the c axis to the bottom with increasing pressure. C. Evidence of pressure-induced formation of the 2D diamond from few-layer graphene (j) Three-dimensional topographical AFM image of the graphite ﬂake showing that it penetrated into the harder SiO2/Si substrate. [(f)–(j)] Reprinted with permission from Pimenta Martins et al., Carbon 173, 744–747 (2021). Copyright 2021 Elsevier. et al.72 and with density functional theory calculations in these two works.72,76 According to these calculations, the resulting structure is diamondene: a 2D ferromagnetic semiconductor with spin-polarized bands. Importantly, in diamondene, the chemical functionalization occurs only at the top graphene layer, leaving half the carbon atoms at the bottom layer with a dangling bond, which is responsible for the magnetism and spin-polarization properties. The role of water is to provide the chemical species –H or –OH that will form covalent bonds with the top graphene layer at high pres- sures,56,72,76 facilitating and stabilizing the sp3 conversion. The exact Appl. Phys. Rev. 10, 011313 (2023); doi: 10.1063/5.0123283 V C Author(s) 2023 Appl. Phys. Rev. 10, 011313 (2023); doi: 10.1063/5.0123283 10, 011313-8 10, 011313-8 Applied Physics Reviews REVIEW scitation.org/journal/are graphite. For all these ﬂakes, the evidence for the presence of sp3 car- bon came from an abrupt broadening of the G bandwidth, considered a signature of the presence of sp3 carbon in compressed graphitic sys- tems,80,91,92 accompanied by a suppression of the 2D band intensity. Other sources of G band broadening such as due to inhomogeneous stress introduced by the PTM or to the differential compressibility between the solid PTM and the substrate were ruled out due to the lack of signiﬁcant deviatoric stress for the water PTM up to approxi- mately 8 GPa52 (see discussion in Sec. II A 4) and the similarity between the surface compressibility of water (Ice VII) and the SiO2 substrate25 (see discussion in Sec. II B). The evidence of phase transi- tion based on the suppression of the 2D band can be understood as follows. The 2D band (2 700 cm1 under ambient conditions) arises from two-phonon double resonance Raman processes and can be drastically suppressed upon changes in the electronic structure caused by structural modiﬁcations in the graphene lattice.93 mechanism for this chemical reaction remains elusive, but it is known that the reactivity of water molecules increases with pressure- for instance, the ionization constant of water increases by 100 from 0.1MPa to 1 GPa.88,89 In fact, Vasu et al.89 reported on the synthesis of 2D structures of metal oxides by trapping their corresponding salts with water between graphene sheets. C. Evidence of pressure-induced formation of the 2D diamond from few-layer graphene 24 October 2024 03:58:28 The works thus far described in this section revealed isolated properties expected from a 2D diamond, such as sp3 content,76 trans- parency,77 and superior hardness73 (in this latter work, the compres- sion was applied using an AFM tip, as discussed in Sec. IVB), providing different pieces for the 2D-diamond puzzle. However, to form a coherent picture and obtain robust evidence of its existence, one needs to probe these different properties in the same system under compression. Pimenta Martins et al.56 performed such measurements, obtaining experimental evidence for the formation of a sp3-containing, transparent and hard 2D phase by compressing few-layer graphene on a SiO2/Si substrate using water as the PTM. Raman spectroscopy pro- vided evidence of a phase transition from a sp2 to a phase containing sp2 and sp3 domains at approximately 6 GPa for bi-layer graphene and starting at 4 GPa and ending at 7 GPa for ﬁve-layer graphene and D. Pressure-tuning of the electronic band structure in monolayer TMDs The process is illustrated in Fig. 4(e). It starts with the absorption of a photon near the K valley and the creation of an electron–hole pair. Then, the excited electron is scattered either to K0 or to the Q valley by the crea- tion of a phonon with momentum K for K–K0 scattering, or with momentum M for K–Q scattering. From momentum conservation, the electron is scattered back to the K valley by the creation of a second phonon with opposite momentum, then the electron–hole pair recom- bines and a photon is emitted. The Raman spectra of MoS2 is shown in Fig. 4(f), where the 2LA modes arising from K–K0 (K–Q0) scattering are highlighted in red (blue). Pimenta Martins et al.59 compressed monolayer MoS2 and WS2 on a SiO2/Si substrate using 4:1 methano- l:ethanol PTM and observed an enhancement of the 2LA band inten- sity for both materials as shown in Fig. 4(g) for MoS2. They attributed the enhancement to the combined effects of a bandgap opening and a reduction of DKQ at high pressures, which would increase the prob- ability of K–K0 and K–Q scattering events. The bandgap opening blue- shifts the B exciton energy (2.08 eV under ambient conditions and 2.13 eV at 4.5GPa) closer to the laser excitation (2.33 eV), bringing the system closer to a resonant absorption/emission condition. The K–Q crossing would resonantly enhance the probability of K-Q scattering events since upon crossing, the scattering from K to Q would involve a real intermediate state instead of a virtual one as under ambient condi- tions, as represented by MoS2 in Fig. 4(h). The K–Q crossing occurred near 3 and 2 GPa for MoS2 and WSe2, respectively, as evidenced by monitoring the evolution of their PL spectra. The authors pointed out that being able to probe multivalley scattering as a function of strain in monolayer TMDs should elucidate several phenomena including val- ley coherence and multivalley superconductivity.59 g y p y p We will start the discussion with the bandgap opening effect induced by pressure. Nayak et al.95 compressed monolayer semicon- ducting MoS2 on SiO2/Si up to 30 GPa using a neon PTM at ambient temperature and observed a blueshift of the PL from 1.85 eV at ambi- ent pressure to 2.08 eV at 16 GPa—an increase in approximately 12% of the bang gap, assuming a constant exciton binding energy. D. Pressure-tuning of the electronic band structure in monolayer TMDs The electronic band structure of monolayer semiconducting TMDs is highly sensitive to the effects of strain, therefore the applica- tion of hydrostatic pressures can induce profound changes in their Appl. Phys. Rev. 10, 011313 (2023); doi: 10.1063/5.0123283 V C Author(s) 2023 Appl. Phys. Rev. 10, 011313 (2023); doi: 10.1063/5.0123283 10, 011313-9 10, 011313-9 Applied Physics Reviews REVIEW scitation.org/journal/are band structure, and those changes can then be probed via Raman and photoluminescence spectroscopy. In the monolayer limit, these mate- rials are direct-gap semiconductors forming two spin-valley locked and spin-split valleys at the K/K0 points in the BZ as shown in Fig. 4(a). Apart from the K/K0 valleys, semiconducting TMDs also pos- ses six Q/Q0 valleys in the conduction band located roughly halfway between the C and the K points as shown in Fig. 4(a), which are also spin-valley locked and spin-split. However, phenomena involving K and Q scattering have been relatively unexplored. An important exam- ple of the potential of multivalley physics in TMDs is the observation of superconductivity in ion-gated few-layer semiconducting MoS2. 94 The origin of this multivalley superconductivity was attributed to an increase in the electron–phonon coupling upon gating due to the appearance of new scattering channels, such as K–Q and Q–Q scatter- ing, formed upon Fermi level crossing of K and Q valleys.94 However, those multivalley effects are usually absent in monolayer TMDs due to the large energy difference between K and Q valleys (DKQ). Pressure has the effect of shifting the energies of the K/K0 and Q/Q0 valleys, causing a bandgap opening and a reduction in DKQ, possibly unlocking new multivalley physics phenomena.59 band structure, and those changes can then be probed via Raman and photoluminescence spectroscopy. In the monolayer limit, these mate- rials are direct-gap semiconductors forming two spin-valley locked and spin-split valleys at the K/K0 points in the BZ as shown in Fig. 4(a). Apart from the K/K0 valleys, semiconducting TMDs also pos- ses six Q/Q0 valleys in the conduction band located roughly halfway between the C and the K points as shown in Fig. 4(a), which are also spin-valley locked and spin-split. However, phenomena involving K and Q scattering have been relatively unexplored. An important exam- ple of the potential of multivalley physics in TMDs is the observation of superconductivity in ion-gated few-layer semiconducting MoS2. 94 scattering between K and Q as well as K and K0 valleys. D. Pressure-tuning of the electronic band structure in monolayer TMDs Above 16 GPa the authors observed a quenching of the PL signal which they attributed to a crossover of the bandgap from direct to indirect due to a change of the valence band minima (VBM) from K to C, as indicated by their DFT calculations. Fu et al.96 also reported a bang gap opening in compressed monolayer MoS2 but the PL quenching was observed at a much lower pressure, around 5 GPa as shown in Fig. 4(b). The authors also attributed the quenching to a direct to indirect transition, however, due to a change of the conduction band minima (CBM) from K to Q at a pressure of 1.9 GPa. The crossing of the K and Q val- leys was supported by DFT calculations and by monitoring the energy of the predominant peak in the PL, with pressure. A mixing of the energies of the K and Q valleys upon reduction of DKQ would cause a deviation from the linear evolution of the predominant PL peak. A banggap opening and PL quenching was also observed in compressed WSe2 97 [see Fig. 4(c)]. The authors also reported a K–Q crossing at approximately 2.2 GPa by monitoring the negatively charged exciton (trion X) peak of WSe2 with increasing pressures and noticing its deviation from the linear behavior as seen in Fig. 4(d). Such deviation could be explained by the joint contributions to the PL signal from direct K–K and indirect K–Q transitions of the trions populating both K and Q valleys near K–Q crossing. Similar to the work of Fu et al.,96 the pressure evolution of X could be ﬁt by a model considering mix- ing of K and Q valleys with increasing pressure upon reduction of DKQ, as done for the C and X sate mixing upon C–X crossing in compressed quantum dots.98 24 October 2024 03:58:28 E. Pressure-tuning of the electronic structure in TMD heterostructures Heterostructures of MX2 TMDs typically form type II band alignment systems where the conduction band minina and the valence band maxima reside in opposite layers. Thus, upon photo excitation, a charge transfer process will occur where electrons and holes will accu- mulate on different layers as shown in Fig. 5(a). Due to the reduced dielectric screening and short interlayer distance, they will form bound electron–hole pairs where the carriers reside in opposite layers, these states are known as interlayer excitons (IXs). Their optical signature is a photoluminescence peak formed upon recombination of those elec- tron–hole pairs, with the peak energy being smaller than the energies of the intralayer excitons from the individual layers. Pressure is a par- ticularly important external tuning parameter to investigate those exci- tations since it reduces the interlayer distance directly modulating the IX energy. Xia et al.100 compressed a 2H-stacked CVD grown MoSe2/ WSe2 heterostructure and monitored changes in its electronic struc- ture by analyzing the evolution with pressure of the IX photolumines- cence peaks. The MoSe2/WSe2 heterostructure was directly transferred to the diamond’s culet and they used silicone oil as the PTM. Figure 5(b) shows the evolution of the IX PL spectra. Below 1 GPa, it con- sisted in two peaks termed as IA and IB, which according to their DFT calculations would correspond to a direct IX electron–hole pair recom- bination at K and an indirect IX recombination from Y to K, as indi- cated in Fig. 5(c). Absorption measurements conﬁrmed this scenario by the detection of an exciton peak with the same energy as IA and the absence of a peak with the IB energy. Above 1 GPa, the PL spectra Recently, it has been demonstrated that the modiﬁcations induced by pressure in the band structure of TMDs can be probed by monitoring the changes in the intensity and shape of the 2LA Raman band.59 The 2LA band is present in the Raman spectra of all semicon- ducting TMDs, and it is composed of several modes arising from two- phonon double-resonance Raman process involving electronic Appl. Phys. Rev. 10, 011313 (2023); doi: 10.1063/5.0123283 V C Author(s) 2023 10, 011313-10 10, 011313-10 Applied Physics Reviews FIG. 4. Pressure-tuning of the electronic band structure in monolayer TMDs. (a) Electronic band structure of monolayer MX2 TMDs under ambient conditions, where M is either Mo or W and X is either S or Se. E. Pressure-tuning of the electronic structure in TMD heterostructures The two valleys at K/K0 and the six valleys at Q/Q0 are spin-split and exhibit spin-valley locking. The energy difference between K and Q valleys (DEKQ) ranges from tens to hundreds of meV depending on the TMD.99 (b) Photoluminescence spectra of monolayer MoS2 at increasing pressures. Reprinted with permission from Fu et al. Sci., Adv. 3, e1700162 (2017). Copyright 2017 Authors, licensed under a Creative Commons Attribution 4.0 License. (c) Photoluminescence spectra of monolayer WSe2 at increasing pressures with the dashed lines indicating the exciton (X) and trion (X) peaks. (d) Pressure evolution of the X and X energy for monolayer WSe2. [(c) and (d)] Reproduced with permission from Ye et al., Nanoscale 8, 10843–10848 (2016). Copyright 2016 Royal Society of Chemistry. (e) Schematics of the two-phonon double resonance Raman processes that gives rise to the modes composing the 2LA band for MoS2 and WSe2. Upon absorption of a photon and creation of an electron–hole pair near K, the excited electron is scattered to K0 (Q) valley by the emission of a phonon with momentum K (M). The excited electron is then scattered back to K by the creation of a second phonon with opposite momentum and the electron–hole pair recombines emitting a photon. (f) Raman spectra of monolayer MoS2 highlighting in red (blue), modes arising from two-phonon K–K0 (K–Q) scattering processes. (g) Raman spectra of monolayer MoS2 with increasing pressures at 10K. The red arrows indicate an enhancement of the intensity of the 2LA and LA bands. (h) DFT calculations for the band structure of monolayer MoS2 with increas- ing pressures. Red and blue represent the different spin-polarized bands. The arrows represent the Raman processes associated with K–Q scattering at different pressures. Red and yellow arrows represent absorption and emission of a photon near the B exciton energy, while blue arrows represent spin-conserving phonon-assisted electronic scattering between K and Q valleys. [(a) and (e)–(h)] Reprinted with permission from Pimenta Martins et al., ACS Nano 16, 8064–8075 (2022). Copyright 2022 American Chemical Society. changes drastically, giving rise to two features termed as IC and ID, which according to DFT calculations would correspond to an indirect IX electron–hole pair recombination from K and Y to C, respectively, as indicated in Fig. 5(d). Notice that the effect of pressure was to change the VBM from K to C, which could be directly probed by the changes in the PL features. E. Pressure-tuning of the electronic structure in TMD heterostructures The features shown here are common to all monolayer MX2 TMDs. The two valleys at K/K0 and the six valleys at Q/Q0 are spin-split and exhibit spin-valley locking. The energy difference between K and Q valleys (DEKQ) ranges from tens to hundreds of meV depending on the TMD.99 (b) Photoluminescence spectra of monolayer MoS2 at increasing pressures. Reprinted with permission from Fu et al. Sci., Adv. 3, e1700162 (2017). Copyright 2017 Authors, licensed under a Creative Commons Attribution 4.0 License. (c) Photoluminescence spectra of monolayer WSe2 at increasing pressures with the dashed lines indicating the exciton (X) and trion (X) peaks. (d) Pressure evolution of the X and X energy for monolayer WSe2. [(c) and (d)] Reproduced with permission from Ye et al., Nanoscale 8, 10843–10848 (2016). Copyright 2016 Royal Society of Chemistry. (e) Schematics of the two-phonon double resonance Raman processes that gives rise to the modes composing the 2LA band for MoS2 and WSe2. Upon absorption of a photon and creation of an electron–hole pair near K, the excited electron is scattered to K0 (Q) valley by the emission of a phonon with momentum K (M). The excited electron is then scattered back to K by the creation of a second phonon with opposite momentum and the electron–hole pair recombines emitting a photon. (f) Raman spectra of monolayer MoS2 highlighting in red (blue), modes arising from two-phonon K–K0 (K–Q) scattering processes. (g) Raman spectra of monolayer MoS2 with increasing pressures at 10K. The red arrows indicate an enhancement of the intensity of the 2LA and LA bands. (h) DFT calculations for the band structure of monolayer MoS2 with increas- ing pressures. Red and blue represent the different spin-polarized bands. The arrows represent the Raman processes associated with K–Q scattering at different pressures. Red and yellow arrows represent absorption and emission of a photon near the B exciton energy, while blue arrows represent spin-conserving phonon-assisted electronic scattering between K and Q valleys. [(a) and (e)–(h)] Reprinted with permission from Pimenta Martins et al., ACS Nano 16, 8064–8075 (2022). Copyright 2022 American Chemical Society. 24 October 2024 03:58:28 FIG. 4. Pressure-tuning of the electronic band structure in monolayer TMDs. (a) Electronic band structure of monolayer MX2 TMDs under ambient conditions, where M is either Mo or W and X is either S or Se. The features shown here are common to all monolayer MX2 TMDs. A. Instrumentation and experimental technique 1. Piston-cylinder cell assembly and operation p y g The sample of interest is mounted atop a CuBe stage, which is initially detached from the main cell body. A Teﬂon cup ﬁlled with the desired PTM is positioned onto the sample stage, thereby sealing the sample completely within the PTM. The entire sample assembly ﬁts tightly into the bore of the pressure cell body, such that the sample resides roughly in the geometric center of the cell. A metal piston, typi- cally made from WC, sits atop the Teﬂon cup. Locking nuts enclose the assembly on each end of the cell. The pressure is controlled by applying force to the piston with a hydraulic press, thereby compress- ing the Teﬂon cup and reducing the volume of the sample space. The locking nut holding the piston in place is tightened as the Teﬂon cup is compressed, maintaining the pressure in cell even upon removing the load from the hydraulic press. After achieving the desired pressure at room temperature, the entire pressure cell can be afﬁxed to an insert for cryogenic measurements. Piston-cylinder pressure cells can be operated down to millikelvin temperatures and in magnetic ﬁelds in excess of 40 T. However, changing the pressure requires warming the cell to room temperature and temporarily reloading it into the hydrau- lic press. The piston-cylinder pressure cell was invented in the late 1800s101 and has since been employed across multiple ﬁelds of funda- mental science including condensed matter physics and geology. Although many variations of the piston-cylinder cell geometry have been developed over the years, all involve positioning a sample within a bath of a PTM and systematically compressing the sample enclosure with a metal piston to raise the pressure. Most piston-cylinder cells can support the application of pressure 3 GPa, although somewhat higher pressure is possible with great care. Although this is orders of magnitude smaller than the maximum achievable pressures in dia- mond anvil pressure cells (discussed in detail in Sec. II), piston- cylinder cells afford orders-of-magnitude larger sample volume and are generally much easier to operate. Figure 6(a) shows a schematic illustration of a piston-cylinder cell. The main body of the cell comprises a metal cylinder with a small- diameter hole bored axially through its center. III. HIGH-PRESSURE EXPERIMENTS WITH PISTON-CYLINDER PRESSURE CELLS double-wall design in which an inner metallic cylinder is tightly ﬁt within an outer metallic sleeve. Hard non-magnetic metals are generally desir- able for condensed matter experiments; cells are typically constructed with a CuBe outer sleeve and a superalloy inner shell (e.g., NiCrAl). E. Pressure-tuning of the electronic structure in TMD heterostructures Above 3 GPa, the authors observed a quenching of the PL signal, being consistent with the indirect nature of the IC and ID IXs PL peaks. 10, 011313-11 10, 011313-11 Appl. Phys. Rev. 10, 011313 (2023); doi: 10.1063/5.0123283 V C Author(s) 2023 Appl. Phys. Rev. 10, 011313 (2023); doi: 10.1063/5.0123283 Applied Physics Reviews scitation.org/journal/are FIG. 5. (a) Schematics of a type-II band alignment between MoSe2 and WSe2, which is typical of MX2 TMD heterostructures. Upon photoexcitation, electrons will accumulate in MoSe2 and holes will accumulate in WSe2, forming interlayer excitons (IXs). (b) Photoluminescence spectra of IXs of MoSe2/WSe2 with increasing pressures. (c) and (d) DFT calculations of the MoSe2/WSe2 band structure at ambient (c) and 2.8 GPa (d), where the origin of the PL peaks Ii (i ¼ A, B, C, and D) are indicated. Reprinted with per- mission from Xia et al., Nat. Phys. 17, 92–98 (2021). Copyright 2021 Springer Nature. 24 October 2024 03:58:28 24 October 2024 03:58:28 FIG. 5. (a) Schematics of a type-II band alignment between MoSe2 and WSe2, which is typical of MX2 TMD heterostructures. Upon photoexcitation, electrons will accumulate in MoSe2 and holes will accumulate in WSe2, forming interlayer excitons (IXs). (b) Photoluminescence spectra of IXs of MoSe2/WSe2 with increasing pressures. (c) and (d) DFT calculations of the MoSe2/WSe2 band structure at ambient (c) and 2.8 GPa (d), where the origin of the PL peaks Ii (i ¼ A, B, C, and D) are indicated. Reprinted with per- mission from Xia et al., Nat. Phys. 17, 92–98 (2021). Copyright 2021 Springer Nature. 2. Electrical characterization of vdW samples superconducting transition temperature of tin is measured for low- temperature calibration. In order to perform electrical transport measurements of samples under pressure, wires must penetrate into the sample space without allowing the PTM to leak out of the cell. To achieve this, a thin hole is drilled through the center of the sample stage prior to mounting the sample. A handful of insulated wires are threaded through this hole and epoxied into place (e.g., using Stycast), forming a leak-tight seal that can withstand at least one pressure campaign. The sample is then mounted atop the epoxy, and the wires are attached to the sample as desired. The pressure inside the sample space can be determined either optically or electrically. In the former case, an optical ﬁber is also inserted into the sample space through the stage, and ruby crystals are mounted near the end of the ﬁber. The ﬂuorescence of the ruby crystal is used to measure the pressure. In the latter case, additional wires can be used to measure the resistance of pressure-sensitive metals in a four-terminal conﬁguration. Typically, a coil of manganin wire is used to calibrate the pressure at room temperature, whereas the Creating electrical contacts to bulk materials is relatively straight- forward, as the ends of the wires are simply pasted onto the sample. However, establishing electrical contact to vdW devices presents addi- tional challenges since they typically reside on planar Si/SiO2 wafers and are highly sensitive to electrostatic discharge. Various schemes have been employed recently to overcome these challenges, including carefully afﬁxing the wafer to the epoxy and hand-pasting wires one- by-one onto the sample electrodes,102 and using designer printed cir- cuit boards compatible with wire bonding for sample mounting.103 A. Instrumentation and experimental technique 1. Piston-cylinder cell assembly and operation Although the body of the cell can be machined from a single piece of ultra-hard metal, modern piston-cylinder cells achieve the highest pressures by utilizing a hybrid 10, 011313-12 10, 011313-12 Appl. Phys. Rev. 10, 011313 (2023); doi: 10.1063/5.0123283 V C Author(s) 2023 Applied Physics Reviews scitation.org/journal/are FIG. 6. (a) Schematic of a vdW heterostructure device residing inside a piston-cylinder pressure cell. (b) Schematic of a monolayer graphene sheet encapsulated between boron nitride crystals and resting on a ﬂake of graphite. All of the layers compress toward each other under pressure. (c) Measurement of the gate capacitance, Cg, as a func- tion of pressure in devices of graphene encapsulated by BN with a graphite back gate. Square (triangle) markers denote quantities extracted from measurements of the low- ﬁeld Hall resistance (the integer quantum Hall gap with ﬁlling factor IQH ¼ þ14). Colors represent measurements from different devices. (d) Bandgap of the primary Dirac point (black squares) and the secondary Dirac point in the valence band (blue circles) as a function of pressure in an aligned graphene/BN device. The insets show cartoon illustrations of the graphene band structure at ambient and high pressure. Adapted with permission from Yankowitz et al., Nature 557, 404–408 (2018). Copyright 2018 Springer Nature. 24 October 2024 03:58:28 FIG. 6. (a) Schematic of a vdW heterostructure device residing inside a piston-cylinder pressure cell. (b) Schematic of a monolayer graphene sheet encapsulated between boron nitride crystals and resting on a ﬂake of graphite. All of the layers compress toward each other under pressure. (c) Measurement of the gate capacitance, Cg, as a func- tion of pressure in devices of graphene encapsulated by BN with a graphite back gate. Square (triangle) markers denote quantities extracted from measurements of the low- ﬁeld Hall resistance (the integer quantum Hall gap with ﬁlling factor IQH ¼ þ14). Colors represent measurements from different devices. (d) Bandgap of the primary Dirac point (black squares) and the secondary Dirac point in the valence band (blue circles) as a function of pressure in an aligned graphene/BN device. The insets show cartoon illustrations of the graphene band structure at ambient and high pressure. Adapted with permission from Yankowitz et al., Nature 557, 404–408 (2018). Copyright 2018 Springer Nature. B. Moire materials under pressure Heterostructures of vdW materials can be assembled by mechan- ically stacking various atomically thin crystals atop one another. A geometric interference effect known as a moire pattern emerges when neighboring vdW crystals are rotationally faulted and/or lattice mis- matched. The moire pattern acts as a periodic potential for charge car- riers in the heterostructure, and can have a wavelength many times larger than the atomic unit cells of the constituent crystals. Moire pat- terns were imaged by scanning tunneling microscopy in graphene rotated on graphite105 and graphene aligned with BN106–108 over a decade ago, but the study of moire materials expanded rapidly follow- ing the 2018 discoveries of superconductivity and strongly correlated states in magic-angle twisted bilayer graphene (tBLG).12,109 Numerous techniques have been developed to control the moire wavelength, including the “brute-force” method of simply fabricating many sam- ples with different twist angles, aligning straight crystalline edges,110 or by dynamically rotating one crystal atop another using an atomic force microscope tip.111,112 Equally important is the amplitude of the moire potential, which is dictated by the strength of the interlayer electronic coupling between the constituent vdW crystals. This coupling is set by the interlayer spacing between the crystals. Pressure therefore repre- sents a distinct experimental tuning knob to modify the properties of moire materials, as it directly controls the strength of the moire poten- tial by enhancing the interlayer electronic coupling. Pressure provides an alternative pathway toward tuning U/K within a single tBLG device, stemming from the similar roles of twist angle and interlayer coupling strength in determining the band struc- ture. A “magic angle” is possible for a wide range of angles just above 1, simply requiring the application of pressure to enhance the inter- layer coupling and ﬂatten the band. Figure 7(a) shows transport mea- surements of a tBLG device with a twist angle of 1.27 both at 0 GPa and at 2.21 GPa. Correlated states at integer band ﬁlling factors (, where ¼ 64 corresponds to full ﬁlling of the ﬂat bands, and is alter- natively referred to as 6ns) are nearly absent at 0 GPa, and no signa- tures of superconductivity are observed down to a base temperature of 300 mK. 1. Angle-aligned graphene/BN Young’s modulus of typical vdW crystals is orders of magnitude larger in the 2D plane than in the out-of-plane stacking direction,104 and the sample rests on a Si/SiO2 wafer on one side but is exposed directly to the PTM on the other. The latter complicates the relationship between the applied pressure and the lattice compression of the vdW crystals in the 2D plane. However, since the sample is much more deformable in along the vdW-stacked axis, the dominant effect of pressure is to reduce the interlayer spacing between all neighboring vdW crystals [see schematic in Fig. 6(b)]. The low-energy band structure of graphene is strongly modiﬁed when it is in close rotational alignment with BN. The moire potential creates secondary Dirac points at ﬁnite energy in the graphene band structure,108 opens a gap at the Dirac point by breaking the sublattice symmetry of the graphene,113 and generates a recursive Hofstadter butterﬂy spectrum in a magnetic ﬁeld.110,113,114 These features depend both on the moire wavelength and its amplitude. Under pressure, the energy gap of the Dirac point grows superlinearly owing to an enhancement of the moire amplitude [Fig. 6(d)]. Surprisingly, the gap at the secondary Dirac point in the valence band appears to be roughly insensitive to pressure. The graphene lattice is known to strain on the moire scale both in- and out-of-plane, and the details of this strain ﬁeld also depend on pressure. Theoretical modeling suggests that the modiﬁcations of the graphene strain proﬁle may reduce the gap at the secondary Dirac point in nearly equal magnitude to the amount it is enhanced by the larger moire amplitude, resulting in an approximate compensation.102 Yankowitz et al.102 established the relationship between pressure and interlayer spacing by investigating transport in heterostructures of graphene encapsulated by boron nitride (BN) crystals. A ﬂake of graphite positioned beneath the bottom BN dielectric acts as a back gate electrode. The graphene charge carrier density, n, is directly related to the applied gate voltage, Vg, by the gate capacitance per unit area, Cg, following the relationship Cg ¼ en=Vg, where e is the charge of the electron. Figure 6(c) shows measurements of Cg for a handful of such devices, normalized to its value at ambient pressure. 1. Angle-aligned graphene/BN For each data point, Cg is extracted by determining the value of n at a given Vg from the low-ﬁeld Hall effect or by tracking the position of a certain integer quantum Hall state. The gate capacitance can be written equiv- alently as Cg ¼ 0=t, where is dielectric constant of BN, 0 is the vacuum permittivity, and t is the thickness of the BN; thus, changes in both and t contribute to its evolution with pressure. Although these two quantities cannot be independently determined in the transport experiments, comparison to ab initio calculations reveal that the inter- layer spacing of the BN decreases by approximately 2.5% per gigapas- cal of applied pressure. Roughly similar compression is also likely for other vdW materials, as well as for interfaces between dissimilar crys- tals, since all such layers are held together by a similar vdW adhesion. 2. Twisted bilayer graphene The low energy bands of tBLG become extremely ﬂat when the two graphene sheets are twisted very near the “magic angle” of 1.1. Coulomb interactions between electrons become dominant in such samples, leading to the emergence of superconductivity and a variety of strongly correlated and topological states.115,116 The ratio of Coulomb to kinetic energy (U/K) grows continuously as the twist angle is tuned away from the magic angle, in principle enabling a care- ful mapping of the correlated phase diagram as the strength of the many-body interactions is varied. Although impressive progress has been made in this direction, a full understanding has been complicated by random inhomogeneous strains that form during sample fabrica- tion, uncontrolled and unknown alignment of the encapsulating BN crystals, and the requirement of fabricating and studying many sam- ples with slightly different twist angles. 24 October 2024 03:58:28 3. Modifying the interlayer spacing between vdW crystals with pressure The pressure inside the sample space of the piston-cylinder cell is very nearly hydrostatic, assuming a suitable choice of PTM. However, substrate-supported vdW heterostructures are highly anisotropic; the Appl. Phys. Rev. 10, 011313 (2023); doi: 10.1063/5.0123283 V C Author(s) 2023 Appl. Phys. Rev. 10, 011313 (2023); doi: 10.1063/5.0123283 10, 011313-13 10, 011313-13 Applied Physics Reviews REVIEW scitation.org/journal/are B. Moire materials under pressure In contrast, at 2.21 GPa correlated insulating states are clearly seen at ¼ 2; þ1; þ2 and þ 3, and superconductivity is observed for a region of hole doping just beyond ¼ 2 [Fig. 7(b)]. Additional measurements at 1.33 GPa show that the energy gaps of the correlated insulators and the critical temperature of superconductivity evolve non-monotonically with pressure [Figs. 7(c)–7(d)]. The non- monotonic evolution of the apparent strength of correlations follows the theoretical expectation that the band should be maximally ﬂat at a pressure of approximately 1.5 GPa for a sample at this twist angle.117,118 Future experiments mapping out a higher density of Appl. Phys. Rev. 10, 011313 (2023); doi: 10.1063/5.0123283 V C Author(s) 2023 10, 011313-14 10, 011313-14 Appl. Phys. Rev. 10, 011313 (2023); doi: 10.1063/5.0123283 Applied Physics Reviews scitation.org/journal/are FIG. 7. (a) Conductance of a tBLG device with a twist angle of 1.27 measured at 0 (gray) and 2.21 GPa (blue), acquired at a temperature of 300 mK. (b) Temperature- dependence measurements of the device resistance at various pressures acquired at doping indicated by the blue arrow in (a). The sample is metallic at 0 GPa and supercon- ducting at higher pressure. (c) Thermally activated energy gaps of the correlated insulating states at half and three-quarters ﬁlling of the ﬂatband as a function of pressure. (d) Pressure-dependence of the superconducting critical temperature, Tc. [(a)–(d)] Reprinted with permission from Yankowitz et al., Science 363, 1059 (2019). Copyright 2019 AAAS. (e) Measurements of the energy gaps in a tDBG device with a twist angle of 1.07 measured as a function of displacement ﬁeld and pressure. (f) Theoretical estimates of the gaps calculated from the single-particle band structure. [(e) and (f)] Reproduced with permission from Szentpeteri et al., Nano Lett. 21, 8777 (2021). Copyright 2021 Authors, licensed under a Creative Commons Attribution License. 24 October 2024 03:58:28 24 October 2024 03:58:28 FIG. 7. (a) Conductance of a tBLG device with a twist angle of 1.27 measured at 0 (gray) and 2.21 GPa (blue), acquired at a temperature of 300 mK. (b) Temperature- dependence measurements of the device resistance at various pressures acquired at doping indicated by the blue arrow in (a). The sample is metallic at 0 GPa and supercon- ducting at higher pressure. (c) Thermally activated energy gaps of the correlated insulating states at half and three-quarters ﬁlling of the ﬂatband as a function of pressure. B. Moire materials under pressure (d) Pressure-dependence of the superconducting critical temperature, Tc. [(a)–(d)] Reprinted with permission from Yankowitz et al., Science 363, 1059 (2019). Copyright 2019 AAAS. (e) Measurements of the energy gaps in a tDBG device with a twist angle of 1.07 measured as a function of displacement ﬁeld and pressure. (f) Theoretical estimates of the gaps calculated from the single-particle band structure. [(e) and (f)] Reproduced with permission from Szentpeteri et al., Nano Lett. 21, 8777 (2021). Copyright 2021 Authors, licensed under a Creative Commons Attribution License. pressure points and in samples with different twist angles will help to further elucidate this physics. ( ¼ 64). These gaps are anticipated by calculations of the single- particle band structure of tDBG and can be opened and closed depending on both the displacement ﬁeld and pressure. Szentpeteri et al.124 measured the pressure dependence of these gaps in a tDBG sample with a twist angle of 1.07 [Fig. 7(e)] and found that, in gen- eral, they were either weakly tuned ( ¼ 0) or substantially decreased ( ¼ 64) as the pressure is raised. The evolution of these gaps with pressure is reasonably well explained by the single-particle band struc- ture calculated with an enhancement in the interlayer coupling, shown in Fig. 7(f). Symmetry-broken states are only very weakly developed at this twist angle, so additional work remains necessary to map out the full evolution of the correlated and topological states in tDBG tuned by the combination of twist angle, doping, displacement ﬁeld, and pressure. 3. Twisted double bilayer graphene Many moire materials beyond tBLG can develop ﬂat bands driven by the superlattice potential, and in principle the correlated and topological properties of all such systems should also depend on pressure. Twisted double bilayer graphene (tDBG)—constructed by stacking and rotating two sheets of Bernal-stacked bilayer gra- phene119–123—has also been investigated under pressure.124 At ambi- ent pressure, tDBG exhibits relatively ﬂat moire bands over a wider range of twist angles than tBLG owing to additional band tunability in a perpendicular displacement ﬁeld. Correlated insulating states and associated isospin symmetry breaking have been observed in devices with twist angles just above 1. These correlated states exist roughly within a window of displacement ﬁeld in which band gaps are open at both the charge neutrality point ( ¼ 0) and full band ﬁlling C. Controlling 2D magnetism with pressure The pressure-dependence of the interlayer magnetic ordering is further it was quickly recognized that the interlayer stacking conﬁguration of the CrI3 is likely connected to its interlayer magnetic ordering. The bulk CrI3 crystal is known to undergo a structural transition from a monoclinic to rhomobohedral stacking conﬁguration upon reducing the temperature below 220 K,129 whereas Raman spectroscopy and second-harmonic generation measurements indicate that few-layer CrI3 remains stacked in the monolinic conﬁguration down to cryo- genic temperatures130 [see schematics in Fig. 8(a)]. Bulk crystals FIG. 8. (a) Schematic of the rhombohedral (top) and monoclinic (bottom) stacking conﬁgurations of bilayer CrI3. The green (purple) atoms represent the Cr atoms in the top (bottom) layer, while the brown ones represent the I atoms. The top (side) view is shown on the left (right). The black arrows represent the magnetic ordering. (b) Tunneling current, It, vs magnetic ﬁeld in a bilayer CrI3 tunnel junction device acquired at a series of pressures. (c) RMCD measured at three different points on a trilayer CrI3 ﬂake, as indicated by the inset illustrations in the bottom right of each panel. The measurements are performed at 0 GPa after a pressure of 2.45 GPa was applied to the sample. The cartoon insets in (b) and (c) depict the magnetic states at each applied magnetic ﬁeld. Adapted with permission from Song et al., Nat. Mater. 18, 1298–1302 (2019). Copyright 2019 Springer Nature. 24 October 2024 03:58:28 FIG. 8. (a) Schematic of the rhombohedral (top) and monoclinic (bottom) stacking conﬁgurations of bilayer CrI3. The green (purple) atoms represent the Cr atoms in the top (bottom) layer, while the brown ones represent the I atoms. The top (side) view is shown on the left (right). The black arrows represent the magnetic ordering. (b) Tunneling current, It, vs magnetic ﬁeld in a bilayer CrI3 tunnel junction device acquired at a series of pressures. (c) RMCD measured at three different points on a trilayer CrI3 ﬂake, as indicated b the inset ill strations in the bottom right of each panel The meas rements are performed at 0 GPa after a press re of 2 45 GPa as applied to the sample The FIG. 8. (a) Schematic of the rhombohedral (top) and monoclinic (bottom) stacking conﬁgurations of bilayer CrI3. The green (purple) atoms represent the Cr atoms in the top (bottom) layer, while the brown ones represent the I atoms. C. Controlling 2D magnetism with pressure A rapidly growing family of vdW magnets have been identiﬁed following the discovery of 2D magnetism in 2017.125,126 Chromium Appl. Phys. Rev. 10, 011313 (2023); doi: 10.1063/5.0123283 V C Author(s) 2023 Appl. Phys. Rev. 10, 011313 (2023); doi: 10.1063/5.0123283 10, 011313-15 Applied Physics Reviews REVIEW scitation.org/journal/are triiodide (CrI3) is a prototypical example of a 2D Ising ferromagnetic insulator, exhibiting out-of-plane ferromagnetism within each mono- layer and antiferromagnetic interlayer coupling.126 The layer- dependent magnetism of few-layer CrI3 crystals has been probed in a number of distinct ways, including optically using reﬂective magnetic circular dichroism (RMCD)126 and electrically in magnetic tunnel junction geometries.127,128 Following the isolation of monolayer CrI3, it was quickly recognized that the interlayer stacking conﬁguration of the CrI3 is likely connected to its interlayer magnetic ordering. The bulk CrI3 crystal is known to undergo a structural transition from a monoclinic to rhomobohedral stacking conﬁguration upon reducing the temperature below 220 K,129 whereas Raman spectroscopy and second-harmonic generation measurements indicate that few-layer CrI3 remains stacked in the monolinic conﬁguration down to cryo- genic temperatures130 [see schematics in Fig. 8(a)]. Bulk crystals further exhibit interlayer ferromagnet ordering below the Curie tem- perature, in contrast to the interlayer antiferromagnetism found in few-layer CrI3. triiodide (CrI3) is a prototypical example of a 2D Ising ferromagnetic insulator, exhibiting out-of-plane ferromagnetism within each mono- layer and antiferromagnetic interlayer coupling.126 The layer- dependent magnetism of few-layer CrI3 crystals has been probed in a number of distinct ways, including optically using reﬂective magnetic circular dichroism (RMCD)126 and electrically in magnetic tunnel junction geometries.127,128 Following the isolation of monolayer CrI3, Song et al.131 and Li et al.132 simultaneously reported that pres- sure could be used to directly manipulate the magnetic ordering of few-layer CrI3 by irreversibly altering its interlayer stacking conﬁgura- tion from monoclinic to rhombohedral. Figure 8(b) shows bulk tunneling measurements across a bilayer CrI3 surrounded by graphite electrodes at a few different pressures, taken from Ref. 131. Hysteresis in the tunneling current, indicating the magnetic ﬁeld at which the bilayer switches from interlayer antiferromagnetic to ferromagnetic, is found to persist to a pressure of at least 1.46 GPa. However, the hyster- esis vanishes in the measurement performed at 2.70 GPa, indicating that the CrI3 bilayer is a layer ferromagnet at all magnetic ﬁelds. A. Instrumentation and experimental technique conﬁrmed by optical spectroscopy performed on the samples before and after the pressure cycle. Figure 9 schematizes the process of pressure application onto vdW materials via SPM. A sample, consisting of mono- to few-layered ﬂakes of the chosen vdW material is placed (or grown) atop a hard and smooth substrate (a piece of doped Si wafer with a 300 nm-thick layer of Si oxide is a common choice) and, then, transferred onto any conventional SPM apparatus. The cantilever-tip system, an integral part of any SPM setup, provides the application of force (or pressure) onto the sample when it is brought into physical contact with the sam- ple surface by piezoelectric actuators. The cantilever acts as a spring with a characteristic constant k, enabling the application of controlled forces upon its deformation against the sample surface. Through the variation of the cantilever material, geometry, dimensions and defor- mation, the applied force may range from picoNewtons to microNewtons. A tip, at the end of the cantilever, transfers the cantile- ver deformation force onto the sample, creating an effective sample deformation area S. Due to the nanometric dimensions of the tip end and the range of the applied forces, such tip-sample interaction area may range from few nm2 up to hundreds of nm2. Therefore, the effec- tive applied pressure may range from few kPa up to tens of GPa. It is important to stress here that, different from DACs, where the applied pressure is isotropic, the SPM applied pressure has an axial nature and its value may vary across the effective deformation area S (being larger at the center and smaller at the border). Moreover, it is a localized effect. In other words, only the region of the sample directly beneath the SPM tip is eventually modiﬁed, with the remaining vdW material remaining unchanged. Figure 8(c) show RMCD measurements of a trilayer CrI3 sam- ple,131 acquired after applying a pressure of 2.45 GPa to the sample and then unloading the pressure back to ambient. Three different types of magnetic ordering are observed, corresponding to different regions of the sample. Region R exhibits a single hysteresis loop sur- rounding B ¼ 0, indicative of interlayer ferromagnetism. Regions P and Q are more complicated, with numerous step-like jumps in the RMCD and hysteresis surrounding numerous values of B. D. Pressure-tunable proximity effects When dissimilar vdW crystals are stacked atop one another, proximity effects between them can modify the properties of each of the constituent crystals.133 A well-studied example is the case of graphene on transition metal dichalcogenide (TMD) substrates. Although freestanding graphene has extraordinarily weak spin– orbit coupling (SOC) on the order of tens of leV, the proximity effect from the TMD substrate can enhance it to values of a few meV.134–139 Theoretical modeling, unsurprisingly, predicts that the value of the proximity-induced SOC grows rapidly as the inter- layer spacing between the graphene and TMD is reduced.135 F€ul€op et al.140 study the SOC in a graphene/WSe2 heterostructure up to a pressure of 1.8 GPa. They extract the strength of the Rashba SOC from measurements weak antilocalization and estimate that it is enhanced by approximately a factor of two over the accessible range of pressure. These measurements highlight the great promise of using pressure to directly control proximity effects in vdW heterostructures. 24 October 2024 03:58:28 As a consequence of its working mechanism and considering the relation between applied force and applied pressure in typical SPM experiments, the exact estimation of pressure values is not trivial. It requires the use of an approximate contact mechanics model (there are several: Hertz, JKR, DMT, Maugis, and others), the precise value of the tip radius R and the precise value of the sample deformation d under the applied force to estimate S (and, therefore, P).141,142 For example, using the simplest model—Hertz model for non-adhesive elastic deformations—the radius a of the contact area can be estimated as a ¼ (R.d)1=2, where R is the tip radius and d is the deformation of the vdW material.141,142 In most cases, the value of R is typically within the range between 10 and 30 nm and d is in the range of 0.1–1 nm. FIG. 9. Schematic illustration of SPM-controlled modiﬁcation of vdW materials via pressure application. The tip at the end of a cantilever (in yellow-green shades) compresses the vdW layers (blue-green shades) placed (or grown) atop a hard substrate (e.g., doped Si covered with a thin Si oxide layer). In addition to the mechanical characterization, the SPM apparatus also enables electrical characteri- zation via application of a bias V between tip and sample. C. Controlling 2D magnetism with pressure The top (side) view is shown on the left (right). The black arrows represent the magnetic ordering. (b) Tunneling current, It, vs magnetic ﬁeld in a bilayer CrI3 tunnel junction device acquired at a series of pressures. (c) RMCD measured at three different points on a trilayer CrI3 ﬂake, as indicated by the inset illustrations in the bottom right of each panel. The measurements are performed at 0 GPa after a pressure of 2.45 GPa was applied to the sample. The cartoon insets in (b) and (c) depict the magnetic states at each applied magnetic ﬁeld. Adapted with permission from Song et al., Nat. Mater. 18, 1298–1302 (2019). Copyright 2019 Springer Nature. ppl. Phys. Rev. 10, 011313 (2023); doi: 10.1063/5.0123283 10, 011313-16 A th ( ) 2023 Appl. Phys. Rev. 10, 011313 (2023); doi: 10.1063/5.0123283 V C Author(s) 2023 10, 011313-16 Applied Physics Reviews REVIEW scitation.org/journal/are A. Instrumentation and experimental technique These regions correspond to two different forms of interlayer antiferromag- netism, as indicated by the schematic insets surrounding the data. The results indicate that sufﬁciently high pressure drives an irreversible monoclinic-to-rhombohedral conversion of the stacking order of the CrI3, which persists even upon removing the pressure. Measurements of the polarization angle dependence of the Raman spectroscopy fur- ther corroborate this conclusion,131,132 showing a fourfold polarization dependence indicative of monoclinic stacking prior to applying pres- sure, and an absence of polarization dependence indicative of rhombo- hedral stacking subsequent to applying pressure. IV. HIGH PRESSURE EXPERIMENTS USING SCANNING PROBE MICROSCOPY One should note that the above methodology does enable the estimation of the injected charge density r in units of charge per area. However, as Eq. (6) shows, in order to calculate r, one needs to know the value of R, which is the radius of the effective disk of charges interacting signiﬁcantly with the EFM probe. Such an estimation of R should take into account the tip- ﬂake distance, tip radius r and even dielectric/screening properties of the ﬂake material. Thus, the estimation of R may become quite arbitrary and introduce an unnecessary source of quantitative error in the mea- sured charge values. Therefore, in many cases, the value of b is left non- estimated and the results are expressed in terms of the EFM signal Dx, which are surely proportional to the real quantitative charge values. where r is the EFM tip radius. In other words, Eq. (6) states that the amount of charges q is directly proportional to the square-root of the EFM response: q ¼ b(Dx)1=2, where b is a constant. One should note that the above methodology does enable the estimation of the injected charge density r in units of charge per area. However, as Eq. (6) shows, in order to calculate r, one needs to know the value of R, which is the radius of the effective disk of charges interacting signiﬁcantly with the EFM probe. Such an estimation of R should take into account the tip- ﬂake distance, tip radius r and even dielectric/screening properties of the ﬂake material. Thus, the estimation of R may become quite arbitrary and introduce an unnecessary source of quantitative error in the mea- sured charge values. Therefore, in many cases, the value of b is left non- estimated and the results are expressed in terms of the EFM signal Dx, which are surely proportional to the real quantitative charge values. The role of SPM in pressure-modiﬁcation of vdW materials goes beyond the modiﬁcation itself and includes some characterization possi- bilities as well. Since the pressure-induced modiﬁcations of vdW materi- als involve mechanical and/or electrical changes in the parent material, different SPM techniques, such as force spectroscopy, conductive AFM, electrostatic force microscopy (EFM), and scanning Kelvin force micros- copy (SKPM), can also be used to probe such electromechanical effects. IV. HIGH PRESSURE EXPERIMENTS USING SCANNING PROBE MICROSCOPY In addition to diamond anvil cells and piston-cylinder cells, scan- ning probe microscopy (SPM) constitutes a family of techniques that enables the application of large axial pressures onto thin vdW materi- als. Using a hard tip at the end of cantilever, it is possible to apply pres- sure in the range of several GPa under a chosen and controlled environment (ambient, vacuum, or different atmospheres and temper- atures). Through this approach, a variety of experiments have demon- strated the transformation of the parent vdW materials into novel phases with exquisite properties. In the following sections, we will describe some aspects of the required instrumentation (Sec. IV A), some representative studies of SPM-induced modiﬁcation of several vdW materials (Sec. IV B) and a few other SPM-enabled tweaking of their electromechanical properties (Sec. IVC). FIG. 9. Schematic illustration of SPM-controlled modiﬁcation of vdW materials via pressure application. The tip at the end of a cantilever (in yellow-green shades) compresses the vdW layers (blue-green shades) placed (or grown) atop a hard substrate (e.g., doped Si covered with a thin Si oxide layer). In addition to the mechanical characterization, the SPM apparatus also enables electrical characteri- zation via application of a bias V between tip and sample. FIG. 9. Schematic illustration of SPM-controlled modiﬁcation of vdW materials via pressure application. The tip at the end of a cantilever (in yellow-green shades) compresses the vdW layers (blue-green shades) placed (or grown) atop a hard substrate (e.g., doped Si covered with a thin Si oxide layer). In addition to the mechanical characterization, the SPM apparatus also enables electrical characteri- zation via application of a bias V between tip and sample. Appl. Phys. Rev. 10, 011313 (2023); doi: 10.1063/5.0123283 V C Author(s) 2023 Appl. Phys. Rev. 10, 011313 (2023); doi: 10.1063/5.0123283 10, 011313-17 10, 011313-17 Applied Physics Reviews REVIEW scitation.org/journal/are proportional to the gradient of the electrostatic tip-sample force dF/dz due to the electric ﬁeld E by proportional to the gradient of the electrostatic tip-sample force dF/dz due to the electric ﬁeld E by This gives a contact radius a in the range between 1 and 6nm. Using P¼ F/S, where P (F) is the applied pressure (force) and S is the area of contact S ¼ pa2, one gets a typical pressure of 10GPa for an applied force around 300 nN and a contact radius of around 3nm. IV. HIGH PRESSURE EXPERIMENTS USING SCANNING PROBE MICROSCOPY In force spectroscopy, the same procedure used to apply pressure onto the vdW material is used to record force-displacement curves, which can be analyzed based on some contact mechanics models and yield information about hardness and/or elastic modulus of the modiﬁed material.141,142 Regarding electrical characterization, a common tech- nique is conductive AFM (c-AFM), where a bias is applied between the AFM tip and sample and the ﬂowing electrical current is monitored. Albeit being an efﬁcient characterization method, c-AFM requires either a conducting substrate where the vdW is placed on or a direct electrical contact to the ﬂake (which requires sophisticated lithography processes). Nevertheless, there are two other electrical SPM techniques that do not require direct electric contact with the vdW ﬂake: EFM and SKPM, which characterize free charges, dielectric properties and work function variations.141,144 A common approach involves the use of a biased AFM tip to transfer electric charges to the vdW material, while applying the desired pressure, and the efﬁciency of this process is analyzed via EFM imaging.145–147 Speciﬁcally, in the charging process, a biased AFM tip is kept in contact with a given region (a single point—without any scan- ning) of the vdW material during a constant contact time (typically, t¼ 0.1 s145–147) transferring charges to it. After injection, the extra charges on the sample induce image charges of opposite sign in the EFM tip during the scan, where the tip is kept at a distance z from the surface, leading to an attractive tip-sample interaction which shifts the cantilever oscillation frequency to lower values.145–147 24 October 2024 03:58:28 Another important experimental issue for the correct investiga- tion of the charging process is the relative humidity of the environ- ment around a charged sample. Since the EFM methodology described above is relatively slow (it takes several minutes to acquire an EFM image), one needs to avoid any spontaneous discharging pro- cesses at this timescale if the correct values of q are to be estimated. Some works reported that water vapor molecules under ambient RH conditions are the main source for spontaneous discharge of electriﬁed surfaces.72,146 Therefore, for the correct estimation of the injected charge q, a dry environment (usually dry N2 atmosphere) surrounding the sample is employed. IV. HIGH PRESSURE EXPERIMENTS USING SCANNING PROBE MICROSCOPY Theoretically and within the Hertz model, the deformation d increases with applied force F as d ¼ {9F2/[16(RE0)2]}1=3, where E0 is the reduced elastic modulus given by 1/E0 ¼ (1-2 1)/E1 þ(1-2 2)/E2 and E1 (E2) are the elastic moduli and 1 (2) are the Poisson’s ratios associated with SPM tip and vdW ﬂake, respectively.142 Hence, the radius a of the con- tact area grows with applied force F as a ¼ (3RF/4E0)1=3.141,142 Even though this equation shows a simple relation between applied force and contact area, it depends on the reduced elastic modulus E0, which has a complicated convolution between the elastic properties of the ultrathin vdW ﬂake and its supporting substrate. In other words, the experimen- tal determination of E0 is non-trivial and, thus, the experimental deter- mination of the contact area is non-trivial (and prone to signiﬁcant approximation errors). Therefore, due to these large uncertainties in the quantitative values of the contact area, most SPM pressure-related stud- ies prefer to use experimental force values, which are much more precise and much less model-dependent than experimental pressure values. Nevertheless, there are sub-nanometer indentation techniques, where the substrate effect is well modeled and, thus, the tip pressure determi- nation is more reliable.143 Dx ¼ x0 2k @F @z ; (4) (4) where x0 and k are the cantilever resonant frequency and spring con- stant, respectively.141 The surface charge density r (r ¼ q/A) in a given ﬂake can be estimated from EFM images considering the electric ﬁeld E of a disk of effective radius R. It is known that the electric ﬁeld E at a distance z from the disk is E ¼ r 2 1 z ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ z2 þ R2 p ; (5) (5) where is the electrical permittivity of the air. After some manipula- tion of Eqs. (4) and (5), it results that the charge density r can be esti- mated by where is the electrical permittivity of the air. After some manipula- tion of Eqs. (4) and (5), it results that the charge density r can be esti- mated by r ¼ ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ 4kDxðz2 þ R2Þ3=2 pr2R2x0 s ; (6) (6) where r is the EFM tip radius. In other words, Eq. (6) states that the amount of charges q is directly proportional to the square-root of the EFM response: q ¼ b(Dx)1=2, where b is a constant. IV. HIGH PRESSURE EXPERIMENTS USING SCANNING PROBE MICROSCOPY SKPM characterization requires the applica- tion of both AC and DC biases between tip and sample (without any mechanical stimulation applied to the cantilever).141 The presence of free charges on the sample and/or work function differences induce an oscillatory force between tip and sample at the same frequency of the applied AC bias.141 Using appropriate DC bias applied to the tip and a feedback mechanism, it is possible to determine the sample’s surface potential and eventual work function differences.141,146 B. Pressure-induced phase transition in 2D materials Therefore, this EFM response Dx can be directly correlated with the amount of injected charges q as follows: the general theory of EFM shows that the frequency shift Dx measured in EFM images is As described above, SPM techniques enable simultaneous pressure-related modiﬁcation and subsequent characterization of Appl. Phys. Rev. 10, 011313 (2023); doi: 10.1063/5.0123283 V C Author(s) 2023 10, 011313-18 10, 011313-18 Applied Physics Reviews REVIEW scitation.org/journal/are force (by the SPM tip) for monolayer and bilayer graphene. While the charging efﬁciency is constant for monolayer graphene (black sym- bols), it decreases signiﬁcantly for bilayer (green symbols) and few- layer (not shown in this panel) graphene, which is a signature of gap opening and, thus, electrical evidence of the diamondization transi- tion.72,145 Structural evidence for such process [illustrated in the car- toon in Fig. 10(b)] were shown in a subsequent work using Raman spectroscopy at high pressures [see Fig. 3(a)].9 The same process was also observed for bilayer graphene grown on SiC using force materials. This is exempliﬁed in earlier studies with carbon nanotubes, where deformation-induced electronic transition is shown to occur in semiconducting nanotubes145 or where the universal behavior of car- bon nanotube deformation is demonstrated.148 The ﬁrst study of SPM-controlled pressure-induced phase transition in 2D materials was published in 2011,72 where the room-temperature diamondization of few-layer graphene was evidenced via electrical measurements [see Figs. 10(a) and 10(b)]. Panel (a) shows the charging efﬁciency, which is proportional to the EFM frequency shift Dx, as a function of applied FIG. 10. SPM-controlled pressure-induced modiﬁcation of vdW materials. (a) and (b) illustrate the process of graphene diamondization via pressure application [see the car- toon in (b)]. The graph in (a) shows a signiﬁcant decrease in the charge transfer efﬁciency (measured as the EFM frequency shift Dx) as the applied tip force increases for bilayer graphene, indicative of gap opening. [(a) and (b)] Reproduced with permission from Barboza et al., Adv. Mater. 23, 3014–3017 (2011). Copyright 2011 John Wiley and Sons. A similar diamondization process is evidenced in mechanical measurements shown in (c), where bilayer graphene becomes harder as it is compressed by the SPM tip (c) Reproduced from Gao et al., Nat. Nanotechnol. 13, 133 (2018). Copyright 2018 Springer Nature. (d) and (f) illustrate the pressure-induced modiﬁcation of h-BN, either via electrical characterization [(d) and (e)] or mechanical characterization (f). B. Pressure-induced phase transition in 2D materials The “diamondization” of h-BN leads to gap closure [(d) and (e))] and material hardening (f). [(d) and (e)] Reprinted with permission from Barboza et al., ACS Nano 12 5866 (2018). Copyright 2018 American Chemical Society. (f) Reproduced with permission from Cellini et al. Adv. Sci. 8 2002541 (2021). Copyright 2021 Authors, licensed under a Creative Commons Attribution License. The pressure effect on graphene/h-BN heterostructures is shown in panels (g) and (h), which show different electrical behaviors depending on the number of graphene layers atop a h-BN ﬂake. The SKPM image in (h) readily differentiates graphene and h-BN layers and the substrate. [(g) and(h)] Reprinted with permission from Barboza et al., Carbon 155, 108–113 (2021). Copyright 2021 Elsevier. (i) Pressure- induced transitions in TMDs were also investigated via electrical SPM characterization, showing two distinct mechanisms of gap closure. Reproduced with permission from Bessa et al., ACS Appl. Nano Mater. 4, 11305 (2021). Copyright 2021 American Chemical Society. 24 October 2024 03:58:28 FIG. 10. SPM-controlled pressure-induced modiﬁcation of vdW materials. (a) and (b) illustrate the process of graphene diamondization via pressure application [see the car- toon in (b)]. The graph in (a) shows a signiﬁcant decrease in the charge transfer efﬁciency (measured as the EFM frequency shift Dx) as the applied tip force increases for bilayer graphene, indicative of gap opening. [(a) and (b)] Reproduced with permission from Barboza et al., Adv. Mater. 23, 3014–3017 (2011). Copyright 2011 John Wiley and Sons. A similar diamondization process is evidenced in mechanical measurements shown in (c), where bilayer graphene becomes harder as it is compressed by the SPM tip. (c) Reproduced from Gao et al., Nat. Nanotechnol. 13, 133 (2018). Copyright 2018 Springer Nature. (d) and (f) illustrate the pressure-induced modiﬁcation of h-BN, either via electrical characterization [(d) and (e)] or mechanical characterization (f). The “diamondization” of h-BN leads to gap closure [(d) and (e))] and material hardening (f). [(d) and (e)] Reprinted with permission from Barboza et al., ACS Nano 12 5866 (2018). Copyright 2018 American Chemical Society. (f) Reproduced with permission from Cellini et al., Adv. Sci. 8 2002541 (2021). Copyright 2021 Authors, licensed under a Creative Commons Attribution License. The pressure effect on graphene/h-BN heterostructures is shown in panels (g) and (h), which show different electrical behaviors depending on the number of graphene layers atop a h-BN ﬂake. B. Pressure-induced phase transition in 2D materials 10(i)].159 The graph of charging efﬁ- ciency as a function of applied tip force shows an initial increase in charge efﬁciency at low forces (pressure) for all TMDs (MoS2, WS2, MoSe2, and WSe2), which is associated with surface functionaliza- tion.159 After well-marked plateaus, the charge efﬁciency increases again at higher applied forces (pressure), which is associated with structural changes in the TMDs (interlayer approximation and intra- layer deformation).153–159 spectroscopy [panel (c)] and conductive AFM.73 The graph in Fig. 10(c) indicates signiﬁcantly smaller indentation depths for bilayer gra- phene in comparison to the SiC substrate for the same applied tip force, evidencing a hardening effect of bilayer graphene upon pressure application.73 This hardening effect was further conﬁrmed via hydro- static high-pressure experiments, where bi-, four-, and ﬁve-layer gra- phene samples were compressed in a water medium. After the onset of the phase transition, revealed by Raman spectroscopy and optical images, the SiO2/Si substrate showed indentation marks along the edge of the samples56 (see discussion in Sec. II C). Additional conduc- tive AFM data under high pressure (data not shown) also show a decrease in electrical conductivity, consistent with the gap opening hypothesis.73 yp After the initial SPM studies with graphene systems, an immedi- ate question arose regarding whether the same effect could be observed in other systems. The most natural candidate would be hexagonal boron nitride (h-BN), also dubbed as “white graphene,” which has strikingly similar structure to graphene. However, opposite to gra- phene, h-BN is a wide bandgap material (6 eV) which may be used as a deep UV source.149 Due to its structural similarities with graphene, monolayers and up to few-layers h-BN were investigated using the same SPM methodology initially applied to graphene as shown in Figs. 10(d) and 10(e). The graph in Fig. 10(d) shows that the charging efﬁ- ciency of h-BN few-layers actually increase upon force (pressure) application by the SPM tip (green symbols), while the charging efﬁ- ciency remains constant for monolayer h-BN and the Si oxide sub- strate (orange and red symbols, respectively).146 Such increase in charging efﬁciency is indicative of gap closure,145 suggesting that a 3D-rehybridized BN material would be a conducting material. This was indeed conﬁrmed in an ensuing SPM experiment [summarized in panel, Fig. 10(e)]. The left side of Fig. B. Pressure-induced phase transition in 2D materials The SKPM image in (h) readily differentiates graphene and h-BN layers and the substrate. [(g) and(h)] Reprinted with permission from Barboza et al., Carbon 155, 108–113 (2021). Copyright 2021 Elsevier. (i) Pressure- induced transitions in TMDs were also investigated via electrical SPM characterization, showing two distinct mechanisms of gap closure. Reproduced with permission from Bessa et al., ACS Appl. Nano Mater. 4, 11305 (2021). Copyright 2021 American Chemical Society. 10, 011313-19 10, 011313-19 Appl. Phys. Rev. 10, 011313 (2023); doi: 10.1063/5.0123283 V C Author(s) 2023 Applied Physics Reviews REVIEW scitation.org/journal/are the formed heterostructures were compressed and their electrical response analyzed.147 Figures 10(g) and 10(h) illustrate the richness of phenomena occurring in such heterostructures under compression. The SKPM image in Fig. 10(h) shows the surface potential difference between a multilayered h-BN ﬂake (in blue-green shades) and a tri- layer graphene ﬂake (in yellow) placed atop it. Figure 10(g) summa- rizes the results for all investigated heterostructures in a graph of charging efﬁciency as a function of applied tip force. For the case of a monolayer graphene atop h-BN (red symbols), a signiﬁcant decrease in the charging efﬁciency indicates gap opening with covalent bonding between graphene and h-BN layers (a process similar to the diamond- ization of graphene).72,73,76,147 The phenomenology changes for the cases of bilayer (green symbols) and tri-layer (blue symbols) graphene atop h-BN. After an initial decrease in charge efﬁciency (indicative of gap opening) at small applied forces (pressure), both systems evolve to a ﬁnal conducting state (increase in charging efﬁciency), similar to the case of h-BN under pressure,146 as the applied force (pressure) increases.147 Transition metal dichalcogenides (TMDs) constitute another important class of vdW materials with attractive optoelec- tronic properties.1,151,152 Most TMDs are semiconducting materials with energy gaps in the 0.5–2 eV range1,151,152 and some theoretical studies have proposed a pressure-induced gap closure for these materi- als,153–155 which were indeed observed using different experimental techniques.156–158 A recent SPM-based study has investigated four dif- ferent TMDs and has shown two distinct pressure-induced gap closure process for each of them [see Figs. B. Pressure-induced phase transition in 2D materials 10(e) shows two structural mod- els for bilayer h-BN: uncompressed 2D h-BN (upper model) and the proposed 3D-rehybridized h-BN structure (lower model).146 The background image in Fig. 10(e) is an EFM image of a large h-BN few- layer ﬂake [green-yellow shades in Fig. 10(e)]. A small square region (2 lm2)—on the low-right part of Fig. 10(e)—was continuously compressed (F ¼ 300 nN), creating a rehybridized region.145 Then, single point charging procedures were carried out on a pristine region of the h-BN ﬂake [upper violet circle in Fig. 10(e)] and on the center of the rehybridized square region. Subsequent EFM imaging allowed the analysis of the charge spread in both pristine and modiﬁed regions. Being an insulator, electrical charges do not spread across the pristine h-BN and a small circular region is observed [upper violet circle in Fig. 10(e)]. However, the EFM image shows that the injected charge is homogeneously spread across the entire modiﬁed square region, evidencing the conducting nature of this new pressure-modiﬁed h-BN compound.146 Another recent study indicates a similar 3D- rehybridization of h-BN using SPM-based compression, as illustrated in Fig. 10(f)150. Similar to the graphene case in panel (c), the graph in panel f indicates a noticeable hardening of 2–3 layer h-BN upon com- pression when compared to the Si oxide substrate, suggesting a similar “diamondization” effect of h-BN.74,150 24 October 2024 03:58:28 C. Pressure-tuning of properties of 2D materials The use of SPM techniques to modify vdW materials via pressure application goes beyond the structural “diamondization” processes, including covalent bond formation, described above. There are also a few other examples of SPM-controlled pressure-modiﬁcation of mate- rials that lead to some interesting phenomena, as illustrated in Fig. 11. One such case uses a combination of pressure application and lateral tip movement to create a giant negative dynamic compressibility of some vdW materials (graphene, h-BN, MoS2, and talc)160,161—see Figs. 11(a)–11(d). The contact mode AFM images in panels (a)–(c) show the apparent height (thickness) evolution of a bilayer graphene ﬂake with the applied tip force (pressure) during the imaging scan: as the force (compression) increases, the bilayer graphene becomes apparently thicker. These seemingly counterintuitive results are sum- marized in the graph in panel (d), which shows the measured height proﬁle of the ﬂake for each applied force.160 Such giant negative com- pressibility effect is actually the result of a pressure-dependent shear effect which induces ﬂake corrugations as the tip is scanned across its surface.160 The effect of tip-induced deformation was also observed for epitaxial graphene grown on SiC162,163 and graphene on h-BN.164 As illustrated in Fig. 11(e), there is a measurable deformation of the epi- taxial graphene as the AFM tip-sample interaction increases.162 A sim- ilar study explores the possibility of pressure-induced commensurate Following the 3D-rehybridization processes shown to occur in graphene and h-BN,72,73,76,146,150 it is only natural to wonder what would happen to a graphene/h-BN heterostructure under compres- sion. Such question was answered in a SPM-based study where mono- layer, bilayer and tri-layer graphene were placed atop h-BN ﬂakes and Appl. Phys. Rev. 10, 011313 (2023); doi: 10.1063/5.0123283 V C Author(s) 2023 10, 011313-20 10, 011313-20 Applied Physics Reviews scitation.org/journal/are ki f h h BN [Fi 11(f) 11( )] 164 A ill d i h h ibili f i hi h f l l i i f FIG. 11. SPM-based pressure-tuned properties of vdW materials. (a)–(c) Contact mode AFM images of the same graphene bilayer ﬂake with increasing applied tip forces showing an apparent thickening (expansion) of graphene upon compression. Such giant negative compressibility is evidenced in the graph in panel (d). [(a)–(d)] Reprinted with permission from Barboza et al., Nano Lett. 12, 2313 (2012). Copyright 2012 American Chemical Society. (e) Effect of the AFM tip pressure on SiC-grown graphene morphol- ogy. A. Methodology From the methodological point of view, the problem of 2D nano- structures under pressure has been addressed by standard techniques, such as the Pseudopotential Density Functional Theory (DFT) formal- ism, the tight-binding method and molecular dynamics simulations. More subtle is the scheme adopted to simulate the application of pres- sure in a ﬁrst principles approach. The vacuum region inherent to 2D models prevents, in principle, a straightforward implementation of pressure effects with constraints imposed on the components of the stress tensor. Fixing position of selected atoms may introduce unphysi- cal ingredients to the problem. The issue has been circunvent with dis- tinct approaches. To mimic the effect of uniaxial pressure in silicene layers, Tantardini et al.167 has ﬁlled the vacuum region with helium layers. This scheme allows for a full relaxation of in-plane lattice vec- tors and do not involve any position constraint in the system. In another approach, the uniaxial pressure problem has been overcome by the adoption of the so-called “hard wall” constraints. As before, the method does not require any atom to be ﬁxed. However, limits are imposed on the maximum and minimum values of the vertical com- ponents (along the z-axis) of atomic positions—the force component Fz is set to zero if it is positive and the z-component of the position is larger than a predeﬁned upper limit, with analogous constraints imposed in the bottom region. The pressure is estimated by the remanent (or constrained) forces in one of the surfaces. The case of hydrostatic pressure can be addressed by a combination of schemes: in-plane pressure controlled by the constrained stress tensor, while vertical forces adjusted with the above method. C. Pressure-tuning of properties of 2D materials Reproduced with permission from Meza et al., Nanotechnology 26, 255704 (2015). Copyright 2015 IOP Publishing. (f)–(g) A Similar effect of graphene tip-induced defor- mation is observed when graphene is placed atop h-BN, leading to different topographic patterns. Reproduced with permission from Yankowitz et al., Nat. Commun. 7, 13168 (2016). Copyright 2016 Authors, licensed under a Creative Commons Attribution. (h) Cartoon illustrating a piezoelectric domain switch of MoS2 monolayers induced and con- trolled via the SPM tip. Reproduced with permission from Lipatov et al., npj 2D Mater. Appl. 6, 18 (2022). Copyright 2022 Authors, licensed under a Creative Commons Attribution. Applied Physics Reviews REVIEW scitation.org/journal/are 24 October 2024 03:58:28 24 October 2024 03:58:28 FIG. 11. SPM-based pressure-tuned properties of vdW materials. (a)–(c) Contact mode AFM images of the same graphene bilayer ﬂake with increasing applied tip forces showing an apparent thickening (expansion) of graphene upon compression. Such giant negative compressibility is evidenced in the graph in panel (d). [(a)–(d)] Reprinted with permission from Barboza et al., Nano Lett. 12, 2313 (2012). Copyright 2012 American Chemical Society. (e) Effect of the AFM tip pressure on SiC-grown graphene morphol- ogy. Reproduced with permission from Meza et al., Nanotechnology 26, 255704 (2015). Copyright 2015 IOP Publishing. (f)–(g) A Similar effect of graphene tip-induced defor- mation is observed when graphene is placed atop h-BN, leading to different topographic patterns. Reproduced with permission from Yankowitz et al., Nat. Commun. 7, 13168 (2016). Copyright 2016 Authors, licensed under a Creative Commons Attribution. (h) Cartoon illustrating a piezoelectric domain switch of MoS2 monolayers induced and con- trolled via the SPM tip. Reproduced with permission from Lipatov et al., npj 2D Mater. Appl. 6, 18 (2022). Copyright 2022 Authors, licensed under a Creative Commons Attribution. shown the possibility of switching the out-of-plane polarization of MoS2 via pressure application by an AFM tip.165 As illustrated by the cartoon in Fig. 11(h), some piezoelectric properties can be tuned in desired patterns on a MoS2 ﬂake.165 stacking of graphene on h-BN [Figs. 11(f)–11(g)].164 As illustrated in panel g, the pressure exerted by the STM tip deforms the monolayer graphene causing a signiﬁcant variation on the observed topography [left and right images in Fig. 11(f)].164 Finally, a recent study has 10, 011313-21 10, 011313-21 Appl. Phys. Rev. 10, 011313 (2023); doi: 10.1063/5.0123283 V C Author(s) 2023 Appl. Phys. Rev. 10, 011313 (2023); doi: 10.1063/5.0123283 Applied Physics Reviews REVIEW scitation.org/journal/are A. Methodology Alternatively, in the case of ﬂat surfaces, the vertical forces may be predeﬁned as con- straints in the upper atoms during the relaxation, while the hard wall scheme is kept only in the bottom surface.76 24 October 2024 03:58:28 The former has been observed and characterized in scanning probe microscopy (SPM) experiments72,79 and in pressure vessels cou- pled to Raman spectroscopy apparatus,76 while the latter have been extensively investigated68,79,169 since ﬁrst theoretical proposals of ana- logue structures based on ﬂuorinated graphene bilayers.170 In fact, the role of surface chemistry is so important in such thin material that some authors report the synthesis of diamane under ambient condi- tions in experiments in which a hot ﬁlament provides -H radicals to interact with graphene.171 Figures 12(a) and 12(b) show the relaxed DFT structures for both compounds. Both, diamondol and diamane have been theoretically character- ized by DFT calculations, which shows that the phase transition, esti- mated in the diamondol case to take place at P 10:0 GPa, is accompanied by strong modulation of electronic and mechanical properties. Diamane is a wide gap direct semiconductor. Diamondol— its single-sided counterpart—is a ferromagnetic semicondutor. This behavior is explained in terms of an array of unsaturated bonds left in the lower part of the structure. Each dangling bond possess a magnetic moment of 1 lB, within the DFT-GGA description, and a dispersive and spin polarized band shows up as the ﬁrst conduction band. The energy gap assumes the value of 1.1eV, considerably reduced com- pared to the diamane case. Figures 12(d) and 12(e) show band struc- tures for diamane and diamondol, highlighting the low energy bands, which deﬁnes the gap region. Temperature and layer thickness are also important variables. Kvashnin et al.,166 for instance, determine the Gibbs free energy from DFT calculations, which allows for the construction of the phase dia- gram including pressure, temperature, and layer thickness. In this approach, the Gibbs potential, following the work of Kern et al.,168 is given by The diamondization of a bilayer graphene could be probed by the assessment of its mechanical properties: Does the transformed structure exhibits stiffness and hardness compatible with a diamond- like ﬁlm? That was exactly the approach carried out by Gao et al.73 to prove the sp2 to sp3 rehybridization. With calculations based on (7) GðP; TÞ ¼ E0ðVÞ þ PV þ U0ðVÞ þ FvibðT; VÞ; (7) Appl. Phys. Rev. B. Structural phase transitions Few-layer graphene furnishes a good example of phase transition driven by pressure application. Indeed, if pressure is applied to a bilayer graphene in the presence of adsorbates, such as water vapor molecules, a concerted action may take place: (i) as a response to the pressure, the layers approach each other, which increases the sp3char- acter of the chemical bonding in the carbon system; (ii) the surface reactivity increases and the formation of –H and/or –OH radicals are facilitated in such scenario, resulting in the binding of chemical species in those atoms which would be left with unsaturated bonds in an all- sp3 conﬁguration; and (iii) a cooperative effect, driven by the geomet- ric corrugation characteristic of the tetrahedral sp3 bonds, propagates the reaction throughout the surface. The result may be a single-sided - H and/or –OH functionalized two-layer diamond-like structure, named diamondol (or graphone-like structure or diamondene), or a complete functionalized 2D-diamond, the diamane. V. MODELING ATOMICALLY THIN VAN DER WAALS MATERIALS AT HIGH PRESSURES in which E0 is the total energy, Fvib and U0 are the vibrational and zero-point energies, respectively, and are determined in terms of the phonon density of states gðV; xÞ, What makes a 2D pressure-induced phase transition and/or modulation of electronic properties special relative to its bulk counter- part? A ﬁrst aspect, as pointed out by Kvashinin et al.,166 concerns the central role played by surface chemistry in the 2D case, which may trigger structural phase transitions in relative smaller values of pres- sure. The covalent incorporation of other chemical species at the sur- face is, by itself, an important ingredient in the structural and electronic characterization of the resulting structure. A second aspect concerns the idiosyncrasies of layered compounds, which allows for electronic modulation by application of an in-plane strain or by a pressure-induced control of interlayer interactions. In the following, we shall use carbon and related materials to illustrate the ﬁrst point and transition metal dichalcogenides and twisted layers to address the second. Prior to this discussion, we shall present the main theoretical approaches that have been employed to address the problem of the characterization of 2D nanostructures under pressure. Fvib ¼ kBT ð X gðV; xÞln 1 e hx kBT dx; (8) U0ðVÞ ¼ 1 2 ð gðV; xÞhxdx: (9) (8) (9) Once the Gibbs potential is calculated, the temperature depen- dent phase transition pressure value can be determined. A. Methodology 10, 011313 (2023); doi: 10.1063/5.0123283 V C Author(s) 2023 10, 011313-22 10, 011313-22 Applied Physics Reviews REVIEW scitation.org/journal/are FIG. 12. Relaxed structures (a)–(c) and DFT-GGA band structures (d)–(e) for dia- mane, diamondol, and bonitrol, respectively, were calculated using the SIESTA soft- ware.172–176 The Fermi level is set to zero in all plots. Diamane is a direct bandgap semiconductor (d), while diamondol is a ferromagnetic semiconductor (e). An array of unsaturated bonds left in the lower part of the structure confers two dispersive and spin-polarized bands to diamondol. Bonitrol, on the other hand, has a metallic behavior (f), with the defective band crossing the Fermi level. Both diamondol and bonitrol possess a magnetic moment of 1 B. Light cyan, red, gray, blue, and pink spheres represent H, O, C, N, and B atoms, respectively. unsatured bonds, the defective band is relatively dispersive and crosses the Fermi level, rendering metallic behavior to the resulting com- pound. The threshold pressure required to trigger the process has been estimated to be 7.0 GPa. In few-layer systems (up to seven layers), cal- culations suggest that pressure values of this order keeps the rehybridi- zaton restricted to the two top layers.On the top surface, N–H bonds may be formed instead of B–OH bonds, leaving the dangling bonds at the bottom side centered at boron sites. This conﬁguration is thermo- dinamically less stable than the previous one, though. A complete dia- mondization of atomically thin hBN on top of SiO2 substrate—a BN diamane analogue—has also been observed and characterized with spectroscopic and theoretical techniques150 based on molecular dynamics simulations. The authors described its formation as induced by pressure with no need of chemical functionalizaton. The sp2 to sp3 conversion is accompanied by a stiffening phenomenon in the pres- sure range of 2.0–4.0 GPa for bi- and trilayers. The question on reversibility has also been addressed. The experi- mental reports72,150 suggests a reversible transformation. The theoreti- cal treatments rationalize this trend in terms of functionalization coverage: above a critical coverage, ultra-thin diamond-like structures may become stable.72 How would be the behavior of 2D few-layer structures based on heavier elements? A systematic study conducted by Tantardini et al. on silicene layers shed light on this issue.167 At zero pressure, silicene layers are corrugated, reﬂecting the predominance of a sp3 hybrid orbi- tals in the chemical bonds. A. Methodology The availability of d orbitals in the third electronic shell is a second important distinction relative to the carbon case. Upon uniaxial pressure, it is energetically more favorable for the hybridization in bi- and trilayers in AA and AAA stackings to pass from sp3 to sp3d, rather than to sp2. The characteristic geometry of sp3 hybrids is a trigonal bipyramidal arrangement, which confers a ﬂat conﬁguration to the layers. Therefore, the geometry does not evolve following a diamondization process, as in the carbon case. A charge redistribution does occur, though, and suggests the use of silicene layers as ﬁeld effect transistor pressure sensors. Flattening is estimated to take place at 15.0 and 2.0GPa for tri- and bilayers, respectively. The monolayer also ﬂattens under a 15.0 GPa uniaxial pressure but as a consequence of a sp3 to sp2 rehybridization. FIG. 12. Relaxed structures (a)–(c) and DFT-GGA band structures (d)–(e) for dia- mane, diamondol, and bonitrol, respectively, were calculated using the SIESTA soft- ware.172–176 The Fermi level is set to zero in all plots. Diamane is a direct bandgap semiconductor (d), while diamondol is a ferromagnetic semiconductor (e). An array of unsaturated bonds left in the lower part of the structure confers two dispersive and spin-polarized bands to diamondol. Bonitrol, on the other hand, has a metallic behavior (f), with the defective band crossing the Fermi level. Both diamondol and bonitrol possess a magnetic moment of 1 B. Light cyan, red, gray, blue, and pink spheres represent H, O, C, N, and B atoms, respectively. 24 October 2024 03:58:28 atomistic models combined with Hookean force ﬁelds, they were able to estimate force vs indentation depth curves in distinct scenarios. The comparison with experimental indentation curves strongly supported the phase transition hypothesis. The sp2-sp3 rehybridization may propagate throughout the struc- ture if more layers are available. In the diamondol case, it represents a top-bottom process, which requires increasing values of pressure. A. Methodology Here, the stacking order shows up as a new variable: in a few-layer gra- phene with AB stacking, the diamondization eventually requires layer sliding, which, interestingly, may lead to the stabilization (from the third layer, counting from the top surface, to the bottom of the struc- ture) of a hexagonal diamond lonsdaleite-type stacking.56 In principle, this agrees with calculations in graphite to diamond transitions, which indicates the important role of kinetics effects in favoring an initial nucleation mechanism for hexagonal diamond and its faster propaga- tion growth.177 B. Pressure tuning of properties and many-body states of 2D materials and moire heterostructures 185 These experiments can help unravel the interplay between stacking order, interlayer distance, and the number of layers in determining the magnetic ground state in these materials. Furthermore, NiI2 is an example of a multiferroic, a class of materials featuring coupled ferro- electric and magnetic order that have garnered wide interest for their exceptional static and dynamical magnetoelectric properties.186 Pressure may be an invaluable tool to tune and investigate the cou- pling between the magnetic and ferroelectric order in such atomically thin systems. The magnetic properties of vdW heterostructures such as CrBr3/CrI3 or WSe2/CrI3 187 can also be explored under pressure, enabling direct control of the exchange bias and other forms of mag- netic proximity effects that depend strongly on the interlayer distance. There is a vast range of physical properties of 2D materials and moire heterostructures (electronic, vibrational, magnetic, etc.) that can be investigated via pressure tuning. One interesting direction is to investigate the layer-dependent magnetism as a function of pressure and temperature in novel 2D van der Waals magnets such as NiI2. 185 p g p gy Pressure may also be an important variable in the growing ﬁeld of twistronics. The large cells required to represent small-twisted angles—as the 1.1 magic angle, which leads to ﬂatband phenomenol- ogy in bilayer graphene12,109—prevents the use of a ﬁrst principles for- malism. However, tight binding calculations117 have been employed in this context, particularly in addressing the effects of pressure in modu- lating the emergence and energy position of ﬂat bands. Carr et al.,117 for instance, have used interlayer coupling parameters determined, as a function of pressure, by a quadratic ﬁt; the pressure, in its turn, is given by a functional form of the type P ¼ AðeB 1Þ, in which is related to the interlayer distance d ( ¼ 1 d=d0), and A and B are adjustable parameters. Within this scheme, the pressure is shown to drive the emergence of ﬂat bands in larger twist angles: under 9.2 GPa (10% compression), the magic angle increases to 2.0 . The results indi- cates that pressure is a fundamental ingredient in describing correlated states, which may have important consequences in the ﬁeld of superconductivity. Regarding pressure tuning of many-body states, an exciting research avenue is the investigation of moire excitons at high pres- sures. B. Pressure tuning of properties and many-body states of 2D materials and moire heterostructures y y The changes in the electronic structure of TMDs due to varia- tions in the interlayer distance is another example of the role of pres- sure in modulating the electronic behavior. The top of the valence band at the C point in TMDs [see Fig. 4(h) for the MoS2 case] is domi- nated by Mo 4d 2 z and S 3pz orbitals, which strongly respond to vertical compressive forces159,180 moving up in energy, toward the Fermi level. Pressure may also be an important variable in the growing ﬁeld of twistronics. The large cells required to represent small-twisted angles—as the 1.1 magic angle, which leads to ﬂatband phenomenol- ogy in bilayer graphene12,109—prevents the use of a ﬁrst principles for- malism. However, tight binding calculations117 have been employed in this context, particularly in addressing the effects of pressure in modu- lating the emergence and energy position of ﬂat bands. Carr et al.,117 for instance, have used interlayer coupling parameters determined, as a function of pressure, by a quadratic ﬁt; the pressure, in its turn, is given by a functional form of the type P ¼ AðeB 1Þ, in which is related to the interlayer distance d ( ¼ 1 d=d0), and A and B are adjustable parameters. Within this scheme, the pressure is shown to drive the emergence of ﬂat bands in larger twist angles: under 9.2 GPa (10% compression), the magic angle increases to 2.0 . The results indi- cates that pressure is a fundamental ingredient in describing correlated states, which may have important consequences in the ﬁeld of The changes in the electronic structure of TMDs due to varia- tions in the interlayer distance is another example of the role of pres- sure in modulating the electronic behavior. The top of the valence band at the C point in TMDs [see Fig. 4(h) for the MoS2 case] is domi- nated by Mo 4d 2 z and S 3pz orbitals, which strongly respond to vertical compressive forces159,180 moving up in energy, toward the Fermi level. There is a vast range of physical properties of 2D materials and moire heterostructures (electronic, vibrational, magnetic, etc.) that can be investigated via pressure tuning. One interesting direction is to investigate the layer-dependent magnetism as a function of pressure and temperature in novel 2D van der Waals magnets such as NiI2. Applied Physics Reviews REVIEW scitation.org/journal/are der Waals materials from elemental compounds. Examples are the synthesis of pentagonal 2D NiN2 183 and the Dirac material BeN4 184 from high-pressure high-temperature (via laser heating) reaction between nickel and nitrogen, and beryllium and nitrogen, respectively. state in the K point. The prediction is, therefore, a direct to indirect bandgap transition and, eventually, a decrease in the gap value or even a semiconductor-metal transition. The K–Q crossover is indeed observed in photoluminesce measurements at a critical pressure of 1.9GPa96 and also in double-resonance Raman measurements.59 Similar analyses have been reported for distinct types of tensile and shear strains, for both few-layer and monolayer TMDs.178–180 A. High-pressure synthesis of novel 2D materials As discussed in Secs. II C and IVB, there has been compelling evidence for the high-pressure conversion of few-layer graphene to 2D diamond obtained either from SPM72,73 or DAC experiments,56,76–78 as well as evidence for the diamondization of hBN146,150 and the for- mation of covalent bonds between graphene and hBN from SPM experiments.147 Therefore, it would be highly insightful to obtain information about the new “hBN diamond” and hybrid “graphene- hBN” phases via spectroscopic or transport methods using DACs. For that purpose, the use of water PTM could be beneﬁcial to drive and reduce the critical pressures necessary to start the phase transi- tion.56,76,181 Furthermore, water should also functionalize monolayer TMDs under compression,159 leading to a metallic phase that could be characterized by a PL quenching and/or a change in the electrical resis- tance. Compression of twisted bilayer graphene could potentially lead to the formation of moire 2D diamonds with different spin- localization conﬁgurations depending on the twist-angle.182 In summary, the combination of high-pressure experiments and atomically thin van der Waals materials is a powerful one for both its scientiﬁc aspects, with a myriad of interesting phenomena, and its pos- sible technological outcomes. There is a vast range of possibilities to be explored, and we hope this review may help the advancement of this research ﬁeld. VI. OUTLOOK In this section, we discuss possible research directions in the ﬁeld of high-pressure studies of atomically thin van der Waals systems within two different approaches: (i) high-pressure synthesis of novel 2D materials and (ii) pressure tuning of properties and many-body states of 2D materials and moire heterostructures. B. Pressure tuning of properties and many-body states of 2D materials and moire heterostructures Moire excitons consist in excitons conﬁned to the local minima of the supperlattice potential in moire system, working as an artiﬁcial lattice of interacting quasiparticles.188 These excitations have been observed in several recent studies involving TMD heterostructures such as MoSe2/WSe2 189,190 and WSe2/WS2, 191 exhibiting an ultra- sharp photoluminescence signal as a characteristic signature. High pressure is a powerful tool to tune and investigate the interactions between moire excitons by reducing the average quasiparticle distance and increasing the strength of the moire potential, due to the in-plane compression and reduction of interlayer distance, respectively. The system could then be probed by photoluminescence spectroscopy and reﬂection contrast using DACs. There are also exciting open chal- lenges in performing transport measurements of strongly correlated, superconducting, and topological moire materials in DACs, affording the ability to apply substantially higher pressure than has been achieved in piston-cylinder cells so far. These experiments may help to uncover fundamentally new ground states that are inaccessible at ambient pressure. 24 October 2024 03:58:28 C. Modulation of electronic properties The case of transition metal dichalcogenides is illustrative of the role of pressure in changing electronic properties while keeping the overall crystal structure. The effect has been reported by several groups,59,96,178–180 and an intuitive grasp of the phenomenon may be pursued by analyzing orbital contributions for the bands close to the gap region.96 Indeed, the lowest conduction state in TMDs, such as MoS2 and MoSe2, is predominantly built upon contributions of Mo 4d z2 orbitals in the K point; From K to Q point, the band goes up in energy, reaches a local maximum and a second minimum at the Q point (a local one, 0.2 eV above that in the K point). At the Q point, the most important contributions come from the in-plane Mo orbitals 4d x2y2 and 4dxy. This is illustrated in Fig. 4(h) for the MoS2 case. The phenomenology has also been observed in other 2D materi- als—hexagonal boron nitride (hBN) is the immediate example due to the chemical similarities between C–C and B–N bonds. In fact, it leads to similar structural features, but, obviously, with marked differences in the electronic aspects. In the hBN case, –OH radicals may bind to the upper boron atoms as the layers approach each other, promoting the restructuring and leaving an array of dangling bonds in the nitro- gen atoms of the bottommost layer,146 as shown in Fig. 12(c). Again, localized states are responsible for magnetic ordering and spin polar- ized bands, as it can be seen in Fig. 12(f). Due to the proximity of these y y How are these features related to pressure effects in TMDs? An in-plane compression should have a larger effect in the states built upon the in-plane orbitals, those with predominant contributions in the Q point, and the net result would be a decrease in the energy of this state, which, eventually, would become lower in energy than the Appl. Phys. Rev. 10, 011313 (2023); doi: 10.1063/5.0123283 V C Author(s) 2023 10, 011313-23 10, 011313-23 Applied Physics Reviews REFERENCES pressure transmitting media,” Phys. Rev. B 88, 045418 (2013). 25 25D. Machon, C. Bousige, R. Alencar, A. Torres-Dias, F. Balima, J. Nicolle, G. de Sousa Pinheiro, A. G. Souza Filho, and A. San-Miguel, “Raman scattering studies of graphene under high pressure,” J. Raman Spectrosc. 49, 121–129 (2018). 1K. S. Novoselov, D. Jiang, F. Schedin, T. Booth, V. Khotkevich, S. Morozov, and A. K. Geim, “Two-dimensional atomic crystals,” Proc. Natl. Acad. Sci. U.S.A. 102, 10451–10453 (2005). 2P. Ajayan, P. Kim, and K. Banerjee, “Two-dimensional van der Waals materi- als,” Phys. Today 69(9), 38 (2016). 26See https://www.nobelprize.org/prizes/physics/1946/summary for “Nobel Prize Outreach AB” (accessed January 24, 2022). 2 3K. Novoselov, O. A. Mishchenko, O. A. Carvalho, and A. C. Neto, “2D mate- rials and van der Waals heterostructures,” Science 353, aac9439 (2016). y 27A. Jayaraman, “Diamond anvil cell and high-pressure physical investigations,” Rev. Mod. Phys. 55, 65 (1983). 4K. I. Bolotin, K. J. Sikes, J. Hone, H. Stormer, and P. Kim, “Temperature- dependent transport in suspended graphene,” Phys. Rev. Lett. 101, 096802 (2008). 4K. I. Bolotin, K. J. Sikes, J. Hone, H. Stormer, and P. Kim, “Temperature- dependent transport in suspended graphene ” Phys Rev Lett 101 096802 I. Bolotin, K. J. Sikes, J. Hone, H. Stormer, and P. Kim, “Tempe y 28W. A. Bassett, “Diamond anvil cell, 50th birthday,” High Pressure Res. 29, 163–186 (2009). 29 29B. Li, C. Ji, W. Yang, J. Wang, K. Yang, R. Xu, W. Liu, Z. Cai, J. Chen, and H.-k. Mao, “Diamond anvil cell behavior up to 4 mbar,” Proc. Natl. Acad. Sci. U. S. A. 115, 1713–1717 (2018). 0 5A. Taube, J. Judek, A. Łapinska, and M. Zdrojek, “Temperature-dependent thermal properties of supported MoS2 monolayers,” ACS Appl. Mater. Interfaces 7, 5061–5065 (2015). 30A. Dewaele, P. Loubeyre, F. Occelli, O. Marie, and M. Mezouar, “Toroidal diamond anvil cell for detailed measurements under extreme static pressur- es,” Nat. Commun. 9, 2913 (2018). 6A. Allain and A. Kis, “Electron and hole mobilities in single-layer WSe2,” ACS Nano 8, 7180–7185 (2014). 7 7Y. Zhang, T.-T. Tang, C. Girit, Z. Hao, M. C. Martin, A. Zettl, M. F. Crommie, Y. R. Shen, and F. Wang, “Direct observation of a widely tunable bandgap in bilayer graphene,” Nature 459, 820–823 (2009). 8 31M. P. Pasternak, R. D. Taylor, A. Chen, C. Meade, L. Falicov, A. Giesekus, R. Jeanloz, and Y. Y. Author Contributions 19A. Castellanos-Gomez, R. Roldan, E. Cappelluti, M. Buscema, F. Guinea, H. S. van der Zant, and G. A. Steele, “Local strain engineering in atomically thin MoS2,” Nano Lett. 13, 5361–5366 (2013). 20 Luiz Gustavo Pimenta Martins: Conceptualization (lead); Writing – original draft (equal); Writing – review & editing (equal). Riccardo Comin: Writing – original draft (equal); Writing – review & editing (equal). Matheus J. S. Matos: Writing – original draft (equal); Writing – review & editing (equal). Mario S. C. Mazzoni: Writing – original draft (equal); Writing – review & editing (equal). Bernardo Ruegger Almeida Neves: Writing – original draft (equal); Writing – review & editing (equal). Matthew Yankowitz: Writing – original draft (equal); Writing – review & editing (equal). 20Y. Bai, L. Zhou, J. Wang, W. Wu, L. J. McGilly, D. Halbertal, C. F. B. Lo, F. Liu, J. Ardelean, P. Rivera et al., “Excitons in strain-induced one-dimensional moire potentials at transition metal dichalcogenide heterojunctions,” Nat. Mater. 19, 1068–1073 (2020). 21L. Zhang, Y. Tang, A. R. Khan, M. M. Hasan, P. Wang, H. Yan, T. Yildirim, J. F. Torres, G. P. Neupane, Y. Zhang et al., “2D materials and heterostructures at extreme pressure,” Adv. Sci. 7, 2002697 (2020). F. Torres, G. P. Neupane, Y. Zhang et al., 2D materials and heterostructures at extreme pressure,” Adv. Sci. 7, 2002697 (2020). 22 at extreme pressure,” Adv. Sci. 7, 2002697 (2020). 22 22S. Pei, Z. Wang, and J. Xia, “High pressure studies of 2D materials and hetero- structures: A review,” Mater. Des. 213, 110363 (2021). 23J. E. Proctor, E. Gregoryanz, K. S. Novoselov, M. Lotya, J. N. Coleman, and M. P. Halsall, “High-pressure raman spectroscopy of graphene,” Phys. Rev. B 80, 073408 (2009). DATA AVAILABILITY Data sharing is not applicable to this article as no new data were created or analyzed in this study. 24K. Filintoglou, N. Papadopoulos, J. Arvanitidis, D. Christoﬁlos, O. Frank, M. Kalbac, J. Parthenios, G. Kalosakas, C. Galiotis, and K. Papagelis, “Raman spectroscopy of graphene at high pressure: Effects of the substrate and the pressure transmitting media,” Phys. Rev. B 88, 045418 (2013). 24 October 2024 03:58:28 The authors have no conﬂicts to disclose. The authors have no conﬂicts to disclose. 18C. Si, Z. Sun, and F. Liu, “Strain engineering of graphene: A review,” Nanoscale 8, 3207–3217 (2016). 19 Conflict of Interest 17N. Levy, S. Burke, K. Meaker, M. Panlasigui, A. Zettl, F. Guinea, A. C. Neto, and M. F. Crommie, “Strain-induced pseudo–magnetic ﬁelds greater than 300 tesla in graphene nanobubbles,” Science 329, 544–547 (2010). 18 AUTHOR DECLARATIONS Conflict of Interest y g y 16Y. Wang, Y. Huang, Y. Song, X. Zhang, Y. Ma, J. Liang, and Y. Chen, “Room- 16Y. Wang, Y. Huang, Y. Song, X. Zhang, Y. Ma, J. Liang, and Y. Chen, “Room- temperature ferromagnetism of graphene,” Nano Lett. 9, 220–224 (2009). g g g g g temperature ferromagnetism of graphene,” Nano Lett. 9, 220–224 (2009). 17 ACKNOWLEDGMENTS Work at MIT (L.G.P.M. and R.C.) was supported by the STC Center for Integrated Quantum Materials, NSF Grant No. DMR- 1231319. M.Y. acknowledges the support of the Army Research Ofﬁce under Grant No. W911NF-20–1-0211. M.J.S.M., M.S.C.M., Another interesting approach that has been recently demon- strated is the high-pressure synthesis of new classes of 2D layered van Appl. Phys. Rev. 10, 011313 (2023); doi: 10.1063/5.0123283 V C Author(s) 2023 10, 011313-24 Applied Physics Reviews REVIEW scitation.org/journal/are 13Y. Tang, L. Li, T. Li, Y. Xu, S. Liu, K. Barmak, K. Watanabe, T. Taniguchi, A. and B.R.A.N. acknowledge ﬁnancial support from CNPq, CAPES, FAPEMIG, and Brazilian Institute of Science and Technology (INCT) in Carbon Nanomaterials, Rede Mineira de Materiais Bidimensionais (FAPEMIG). M.J.S.M. acknowledges the support from Universidade Federal de Ouro Preto (UFOP). 13Y. Tang, L. Li, T. Li, Y. Xu, S. Liu, K. Barmak, K. Watanabe, T. Taniguchi, A. H. MacDonald, J. Shan et al., “Simulation of hubbard model physics in WSe2/ S l ” ( ) H. MacDonald, J. Shan et al., “Simulation of hubbard model physics in WSe2/ WS2 Moire superlattices,” Nature 579, 353–358 (2020). , J , p y 2 WS2 Moire superlattices,” Nature 579, 353–358 (2020). 14 WS2 Moire superlattices,” Nature 579, 353–358 (2020). 14 p 14K. S. Burch, D. Mandrus, and J.-G. Park, “Magnetism in two-dimensional van der Waals materials,” Nature 563, 47–52 (2018). 15K. F. Mak, J. Shan, and D. C. Ralph, “Probing and controlling magnetic states in 2D layered magnetic materials,” Nat. Rev. Phys. 1, 646–661 (2019). 16 REFERENCES Peter, “Pressure-induced metallization and the collapse of the magnetic state in the antiferromagnetic insulator NiI2,” Phys. Rev. Lett. 65, 790 (1990). 8L. A. Jauregui, A. Y. Joe, K. Pistunova, D. S. Wild, A. A. High, Y. Zhou, G. Scuri, K. De Greve, A. Sushko, C.-H. Yu et al., “Electrical control of interlayer exciton dynamics in atomically thin heterostructures,” Science 366, 870–875 (2019). 32R. Reichlin, K. E. Brister, A. K. McMahan, M. Ross, S. Martin, Y. K. Vohra, and A. L. Ruoff, “Evidence for the insulator-metal transition in xenon from optical, x-ray, and band-structure studies to 170 GPa,” Phys. Rev. Lett. 62, 669 (1989). 9Y. Tang, J. Gu, S. Liu, K. Watanabe, T. Taniguchi, J. Hone, K. F. Mak, and J. Shan, “Tuning layer-hybridized Moire excitons by the quantum-conﬁned stark effect,” Nat. Nanotechnol. 16, 52–57 (2021). ( ) 33T. Yagi, W. Utsumi, M-a Yamakata, T. Kikegawa, and O. Shimomura, “High- pressure in situ x-ray-diffraction study of the phase transformation from graphite to hexagonal diamond at room temperature,” Phys. Rev. B 46, 6031 (1992). 10S. Pisana, M. Lazzeri, C. Casiraghi, K. S. Novoselov, A. K. Geim, A. C. Ferrari, and F. Mauri, “Breakdown of the adiabatic Born–Oppenheimer approxima- tion in graphene,” Nat. Mater. 6, 198–201 (2007). 34H. Xia, S. J. Duclos, A. L. Ruoff, and Y. K. Vohra, “New high-pressure phase transition in zirconium metal,” Phys. Rev. Lett. 64, 204 (1990). 35 g 11S. Mouri, Y. Miyauchi, and K. Matsuda, “Tunable photoluminescence of monolayer mos2 via chemical doping,” Nano Lett. 13, 5944–5948 (2013). 12 35R. Jaramillo, Y. Feng, J. Lang, Z. Islam, G. Srajer, P. Littlewood, D. McWhan, and T. Rosenbaum, “Breakdown of the Bardeen–Cooper–Schrieffer ground state at a quantum phase transition,” Nature 459, 405–409 (2009). 12Y. Cao, V. Fatemi, S. Fang, K. Watanabe, T. Taniguchi, E. Kaxiras, and P. Jarillo-Herrero, “Unconventional superconductivity in magic-angle graphene superlattices,” Nature 556, 43–50 (2018). 10, 011313-25 Appl. Phys. Rev. 10, 011313 (2023); doi: 10.1063/5.0123283 V C Author(s) 2023 10, 011313-25 Appl. Phys. Rev. 10, 011313 (2023); doi: 10.1063/5.0123283 Applied Physics Reviews REVIEW scitation.org/journal/are 36Y. Wang, T. Rosenbaum, A. Palmer, Y. Ren, J.-W. Kim, D. Mandrus, and Y. Feng, “Strongly-coupled quantum critical point in an all-in-all-out anti- ferromagnet,” Nat. Commun. 9, 2953 (2018). transition metal dichalcogenides at high pressures,” ACS Nano 16, 8064–8075 (2022). 16, 60J. Nicolle, D. Machon, P. Poncharal, O. Pierre-Louis, and A. San-Miguel, “Pressure-mediated doping in graphene,” Nano Lett. 11, 3564–3568 (2011). REFERENCES g 37A. Drozdov, M. Eremets, I. Troyan, V. Ksenofontov, and S. I. Shylin, “Conventional superconductivity at 203 kelvin at high pressures in the sulfur hydride system,” Nature 525, 73–76 (2015). 38 61A. Jorio, M. S. Dresselhaus, R. Saito, and G. Dresselhaus, Raman Spectroscopy in Graphene Related Systems (John Wiley & Sons, 2010). in Graphene Related Systems (John Wiley & Sons, 2010). 62 y y 38J. A. Flores-Livas, L. Boeri, A. Sanna, G. Profeta, R. Arita, and M. Eremets, “A perspective on conventional high-temperature superconductors at high pres- sure: Methods and materials,” Phys. Rep. 856, 1–78 (2020). 62M. M. Cardona and G. G€untherodt, “Light scattering in solids IV,” Top. Appl. Phys. 54, 467 (1984). 63T. Mohiuddin, A. Lombardo, R. Nair, A. Bonetti, G. Savini, R. Jalil, N. Bonini, D. Basko, C. Galiotis, N. Marzari et al., “Uniaxial strain in graphene by Raman spectroscopy: G peak splitting, gr€uneisen parameters, and sample ori- entation,” Phys. Rev. B 79, 205433 (2009). g sure: Methods and materials,” Phys. Rep. 856, 1–78 (2020). 9 y p 39L. Zhang, Y. Wang, J. Lv, and Y. Ma, “Materials discovery at high pressures,” Nat. Rev. Mater. 2, 1–16 (2017). 40 40P. F. McMillan, “New materials from high-pressure experiments,” Nat. Mater. 1, 19–25 (2002). 64J. E. Lee, G. Ahn, J. Shim, Y. S. Lee, and S. Ryu, “Optical separation of mechanical strain from charge doping in graphene,” Nat. Commun. 3, 1024 (2012). 41W. Zhang, A. R. Oganov, A. F. Goncharov, Q. Zhu, S. E. Boulfelfel, A. O. Lyakhov, E. Stavrou, M. Somayazulu, V. B. Prakapenka, and Z. Kon^opkova, “Unexpected stable stoichiometries of sodium chlorides,” Science 342, 1502–1505 (2013). 65A. Das, S. Pisana, B. Chakraborty, S. Piscanec, S. K. Saha, U. V. Waghmare, K. S. Novoselov, H. R. Krishnamurthy, A. K. Geim, A. C. Ferrari et al., “Monitoring dopants by raman scattering in an electrochemically top-gated graphene transistor,” Nat. Nanotechnol. 3, 210–215 (2008). 66 42E. Horvath-Bordon, R. Riedel, A. Zerr, P. F. McMillan, G. Auffermann, Y. Prots, W. Bronger, R. Kniep, and P. Kroll, “High-pressure chemistry of nitride-based materials,” Chem. Soc. Rev. 35, 987–1014 (2006). 43 g 66Y. Feng, R. Jaramillo, J. Wang, Y. Ren, and T. Rosenbaum, “Invited article: High-pressure techniques for condensed matter physics at low temperature,” Rev. Sci. Instrum. 81, 041301 (2010). 67 43I. Spain and D. Dunstan, “The technology of diamond anvil high-pressure cells: II. Operation and use,” J. Phys. E 22, 923 (1989). 67K. S. REFERENCES Mizuki, “Ruby pressure scale in a low-temperature diamond anvil cell,” J. Appl. Phys. 112, 124503 (2012). ﬂuorinated single-layer diamond,” Nat. Nanotechnol. 15, 59–66 ( pp y ( ) 50D. D. Ragan, R. Gustavsen, and D. Schiferl, “Calibration of the ruby R1 and R2 ﬂuorescence shifts as a function of temperature from 0 to 600 k,” J. Appl. Phys. 72, 5539–5544 (1992). 72A. P. Barboza, M. H. Guimaraes, D. V. Massote, L. C. Campos, N. M. Barbosa Neto, L. G. Cancado, R. G. Lacerda, H. Chacham, M. S. Mazzoni, and B. R. Neves, “Room-temperature compression-induced diamondization of few- layer graphene,” Adv. Mater. 23, 3014–3017 (2011). 73 y 51H. Lee, E. Park, T. Park, V. Sidorov, F. Ronning, E. Bauer, and J. Thompson, y 51H. Lee, E. Park, T. Park, V. Sidorov, F. Ronning, E. Bauer, and J. Thompson, “Pressure-induced superconducting state of antiferromagnetic CaFe2 As2,” Phys. Rev. B 80, 024519 (2009). g J p “Pressure-induced superconducting state of antiferromagnetic CaFe2 As2,” Phys. Rev. B 80, 024519 (2009). 73Y. Gao, T. Cao, F. Cellini, C. Berger, W. A. De Heer, E. Tosatti, E. Riedo, and A. Bongiorno, “Ultrahard carbon ﬁlm from epitaxial two-layer graphene,” Nat. Nanotechnol. 13, 133 (2018). 74 y 52G. Piermarini, S. Block, and J. Barnett, “Hydrostatic limits in liquids and sol- ids to 100 kbar,” J. Appl. Phys. 44, 5377–5382 (1973). 53 74F. Cellini, F. Lavini, T. Cao, W. de Heer, C. Berger, A. Bongiorno, and E. Riedo, “Epitaxial two-layer graphene under pressure: Diamene stiffer than diamond,” FlatChem 10, 8–13 (2018). 75 53N. Tateiwa and Y. Haga, “Evaluations of pressure-transmitting media for cryogenic experiments with diamond anvil cell,” Rev. Sci. Instrum. 80, 123901 (2009). diamond,” FlatChem 10, 8–13 (2018). 75 75F. Cellini, F. Lavini, C. Berger, W. De Heer, and E. Riedo, “Layer dependence of graphene-diamene phase transition in epitaxial and exfoliated few-layer graphene using machine learning,” 2D Mater. 6, 035043 (2019). 54B. Olinger and P. M. Halleck, “Compression and bonding of ice VII and an empirical linear expression for the isothermal compression of solids,” J. Chem. Phys 62, 94–99 (1975). graphene using machine learning,” 2D Mater. 6, 035043 (2019). 76 76L. G. Pimenta, M. J. S. Matos, A. R. Paschoal, P. T. C. Freire, N. F. Andrade, A. L. Aguiar, J. Kong, B. R. A. Neves, A. B. de Oliveira, M. S. C. Mazzoni, A. REFERENCES Novoselov, A. K. Geim, S. Morozov, D. Jiang, Y. Zhang, S. Dubonos, I. Grigorieva, and A. Firsov, “Electric ﬁeld effect in atomically thin carbon ﬁlms,” Science 306, 666–669 (2004). 44H. Mao, P. Bell, J. t Shaner, and D. Steinberg, “Speciﬁc volume measurements of Cu, Mo, Pd, and Ag and calibration of the ruby R1 ﬂuorescence pressure Gauge from 0.06 to 1 mbar,” J. Appl. Phys. 49, 3276–3283 (1978). 45 68P. B. Sorokin and B. I. Yakobson, “Two-dimensional diamond–diamane: Current state and further prospects,” Nano Lett. 21, 5475–5484 (2021). 69 45H. Mao, J.-A. Xu, and P. Bell, “Calibration of the ruby pressure gauge to 800 kbar under quasi-hydrostatic conditions,” J. Geophys. Res. 91, 4673–4676, https://doi.org/10.1029/JB091iB05p04673 (1986). 46 69F. Piazza, K. Gough, M. Monthioux, P. Puech, I. Gerber, R. Wiens, G. Paredes, and C. Ozoria, “Low temperature, pressureless sp2 to sp3 transforma- f l h ll b ﬁl ” b ( ) , G g , , , G , Paredes, and C. Ozoria, “Low temperature, pressureless sp2 to sp3 trans tion of ultrathin, crystalline carbon ﬁlms,” Carbon 145, 10–22 (2019). 70 46M. Hanﬂand and K. Syassen, “A Raman study of diamond anvils under stress,” J. Appl. Phys. 57, 2752–2756 (1985). 47 tion of ultrathin, crystalline carbon ﬁlms,” Carbon 145, 10–22 (2019). 70 pp y 47W. Holzapfel, M. Hartwig, and W. Sievers, “Equations of state for Cu, Ag, and Au for wide ranges in temperature and pressure up to 500 GPa and above,” J. Phys. Chem. Ref. Data 30, 515–529 (2001). 70F. Piazza, M. Monthioux, P. Puech, and I. C. Gerber, “Towards a better understanding of the structure of diamano€ıds and diamano€ıd/graphene hybrids,” Carbon 156, 234–241 (2020). 71P. V. Bakharev, M. Huang, M. Saxena, S. W. Lee, S. H. Joo, S. O. Park, J. Dong, D. C. Camacho-Mojica, S. Jin, Y. Kwon et al., “Chemically induced transformation of chemical vapour deposition grown bilayer graphene into ﬂuorinated single-layer diamond,” Nat. Nanotechnol. 15, 59–66 (2020). 2 48K. Syassen, “Ruby under pressure,” High Pressure Res. 28, 75–126 (2008). 49H. Yamaoka, Y. Zekko, I. Jarrige, J.-F. Lin, N. Hiraoka, H. Ishii, K.-D. Tsuei, K. Syassen, Ruby under pressure, High Pressure Res. 28, 75 126 (2008). 49H. Yamaoka, Y. Zekko, I. Jarrige, J.-F. Lin, N. Hiraoka, H. Ishii, K.-D. Tsuei, and J. Mizuki, “Ruby pressure scale in a low-temperature diamond anvil cell,” J. Appl. Phys. 112, 124503 (2012). 50 and J. REFERENCES G S Filho and L G Cancado “Raman evidence for pressure induced forma 76L. G. Pimenta, M. J. S. Matos, A. R. Paschoal, P. T. C. Freire, N. F. Andrade, A. L. Aguiar, J. Kong, B. R. A. Neves, A. B. de Oliveira, M. S. C. Mazzoni, A. G. S. Filho, and L. G. Canc¸ado, “Raman evidence for pressure-induced forma- tion of diamondene,” Nat. Commun. 8, 96 (2017). 77 y 55E. Wolanin, P. Pruzan, J. Chervin, B. Canny, M. Gauthier, D. H€ausermann, and M. Hanﬂand, “Equation of state of ice VII up to 106 GPa,” Phys. Rev. B 56, 5781 (1997). G. S. Filho, and L. G. Canc¸ado, “Raman evidence for pressure-induced forma- tion of diamondene,” Nat. Commun. 8, 96 (2017). 56L. G. Pimenta Martins, D. L. Silva, J. S. Smith, A.-Y. Lu, C. Su, M. Hempel, C. Occhialini, X. Ji, R. Pablo, R. S. Alencar et al., “Hard, transparent, sp3-con- taining 2D phase formed from few-layer graphene under compression,” Carbon 173, 744–757 (2021). 77F. Ke, Y. Chen, K. Yin, J. Yan, H. Zhang, Z. Liu, J. S. Tse, J. Wu, H-k Mao, and B. Chen, “Large bandgap of pressurized trilayer graphene,” Proc. Natl. Acad. Sci. U. S. A. 116, 9186–9190 (2019). 8 78F. Ke, L. Zhang, Y. Chen, K. Yin, C. Wang, Y.-K. Tzeng, Y. Lin, H. Dong, Z. Liu, J. S. Tse et al., “Synthesis of atomically thin hexagonal diamond with compression,” Nano Lett. 20, 5916–5921 (2020). 9 57S. Klotz, J. Chervin, P. Munsch, and G. Le Marchand, “Hydrostatic limits of 11 pressure transmitting media,” J. Phys. D 42, 075413 (2009). 58 58Y. Sun, W. Liu, I. Hernandez, J. Gonzalez, F. Rodriguez, D. Dunstan, and C. Humphreys, “3D strain in 2D materials: To what extent is monolayer gra- phene graphite?,” Phys. Rev. Lett. 123, 135501 (2019). 79F. Lavini, M. Rejhon, and E. Riedo, “Two-dimensional diamonds from sp2-to- sp3 phase transitions,” Nat. Rev. Mater. 7, 814–832 (2022). 80M. Hanﬂand, H. Beister, and K. Syassen, “Graphite under pressure: Equation of state and ﬁrst-order raman modes,” Phys. Rev. B 39, 12598 (1989). 80M. Hanﬂand, H. Beister, and K. Syassen, “Graphite unde 80M. Hanﬂand, H. Beister, and K. Syassen, Graphite under pressure: Equation of state and ﬁrst-order raman modes,” Phys. Rev. B 39, 12598 (1989). 81 , , y , p p q of state and ﬁrst-order raman modes,” Phys. Rev. B 39, 12598 (1989). 81M. Hanﬂand, K. Syassen, and R. REFERENCES Adv. 3, e1700162 (2017). 97 116E. Y. Andrei and A. H. MacDonald, “Graphene bilayers with a twist,” Nat. Mater. 19, 1265–1275 (2020). 117 y y p 97Y. Ye, X. Dou, K. Ding, D. Jiang, F. Yang, and B. Sun, “Pressure-induced k–k crossing in monolayer WSe2,” Nanoscale 8, 10843–10848 (2016). 98 117S. Carr, S. Fang, P. Jarillo-Herrero, and E. Kaxiras, “Pressure dependence of the magic twist angle in graphene superlattices,” Phys. Rev. B 98, 085144 (2018). 98G. Li, A. Goni, K. Syassen, O. Brandt, and K. Ploog, “State mixing in InAs/ GaAs quantum dots at the pressure-induced c-x crossing,” Phys. Rev. B 50, 18420 (1994). 118B. L. Chittari, N. Leconte, S. Javvaji, and J. Jung, “Pressure induced compres- sion of ﬂatbands in twisted bilayer graphene,” Electron. Struct. 1, 015001 (2019). ( ) 99A. Kormanyos, G. Burkard, M. Gmitra, J. Fabian, V. Zolyomi, N. D. Drummond, and V. Fal’ko, “k p theory for two-dimensional transition metal dichalcogenide semiconductors,” 2D Mater. 2, 022001 (2015). 100 119C. Shen, Y. Chu, Q. Wu, N. Li, S. Wang, Y. Zhao, J. Tang, J. Liu, J. Tian, K. Watanabe, T. Taniguchi, R. Yang, Z. Y. Meng, D. Shi, O. V. Yazyev, and G. Zhang, “Correlated states in twisted double bilayer graphene,” Nat. Phys. 16, 520–525 (2020). 119C. Shen, Y. Chu, Q. Wu, N. Li, S. Wang, Y. Zhao, J. Tang, J. Liu, J. Tian, K. Watanabe, T. Taniguchi, R. Yang, Z. Y. Meng, D. Shi, O. V. Yazyev, and G. h “ l d d d bl b l h ” h 100J. Xia, J. Yan, Z. Wang, Y. He, Y. Gong, W. Chen, T. C. Sum, Z. Liu, P. M. Ajayan, and Z. Shen, “Strong coupling and pressure engineering in WSe2–MoSe2 heterobilayers,” Nat. Phys. 17, 92–98 (2021). 0 Zhang, “Correlated states in twisted double bilayer graphene,” Nat. Phys. 16, 520–525 (2020). 120 y y 101C. A. Parsons, “VIII. Experiments on carbon at high temperatures and under great pressures, and in contact with other substances,” Proc. R. Soc. London 44, 320–323 (1888). 120X. Liu, Z. Hao, E. Khalaf, J. Y. Lee, K. Watanabe, T. Taniguchi, A. Vishwanath, and P. Kim, “Tunable spin-polarized correlated states in twisted double bilayer graphene,” Nature 583, 221–225 (2020). 121 102M. Yankowitz, J. Jung, E. Laksono, N. Leconte, B. L. Chittari, K. Watanabe, T. Taniguchi, S. Adam, D. Graf, and C. R. REFERENCES Ferrari and J. Robertson, “Resonant Raman spectroscopy of disordered, amorphous, and diamondlike carbon,” Phys. Rev. B 64, 075414 (2001). 88 108M. Yankowitz, J. Xue, D. Cormode, J. D. Sanchez-Yamagishi, K. Watanabe, T. Taniguchi, P. Jarillo-Herrero, P. Jacquod, and B. J. LeRoy, “Emergence of superlattice Dirac points in graphene on hexagonal boron nitride,” Nat. Phys. 8, 382–386 (2012). 88W. L. Marshall and E. Franck, “Ion product of water substance, 0–1000 C, 1–10,000 bars new international formulation and its background,” J. Phys. Chem. Ref. data 10, 295–304 (1981). 89 89K. Vasu, E. Prestat, J. Abraham, J. Dix, R. Kashtiban, J. Beheshtian, J. Sloan, P. Carbone, M. Neek-Amal, S. Haigh et al., “Van der waals pressure and its effect on trapped interlayer molecules,” Nat. Commun. 7, 12168 (2016). 90 109Y. Cao, V. Fatemi, A. Demir, S. Fang, S. L. Tomarken, J. Y. Luo, J. D. Sanchez- Yamagishi, K. Watanabe, T. Taniguchi, E. Kaxiras, R. C. Ashoori, and P. Jarillo-Herrero, “Correlated insulator behaviour at half-ﬁlling in magic-angle graphene superlattices,” Nature 556, 80–84 (2018). 110 on trapped interlayer molecules,” Nat. Commun. 7, 12168 (2016). 90D. L. Silva, J. L. E. Campos, T. F. Fernandes, J. N. Rocha, L. R. Machado, E. M. Soares, D. R. Miquita, H. Miranda, C. Rabelo, O. P. V. Neto et al., “Raman spectroscopy analysis of number of layers in mass-produced graphene ﬂakes,” Carbon 161, 181–189 (2020). 91 graphene superlattices,” Nature 556, 80–84 (2018) 110 110L. A. Ponomarenko, R. V. Gorbachev, G. L. Yu, D. C. Elias, R. Jalil, A. A. Patel, A. Mishchenko, A. S. Mayorov, C. R. Woods, J. R. Wallbank, M. Mucha- Kruczynski, B. A. Pio, M. Potemski, I. V. Grigorieva, K. S. Novoselov, F. Guinea, V. I. Fal’ko, and A. K. Geim, “Cloning of Dirac fermions in graphene superlattices,” Nature 497, 594–597 (2013). 111 91M. Amsler, J. A. Flores-Livas, L. Lehtovaara, F. Balima, S. A. Ghasemi, D. Machon, S. Pailhe`s, A. Willand, D. Caliste, S. Botti et al., “Crystal structure of 91M. Amsler, J. A. Flores-Livas, L. Lehtovaara, F. Balima, S. A. Ghasemi, D. Machon, S. Pailhe`s, A. Willand, D. Caliste, S. Botti et al., “Crystal structure of cold compressed graphite,” Phys. Rev. Lett. 108, 065501 (2012). Machon, S. Pailhe`s, A. Willand, D. Caliste, S. Botti et al., “Crystal structure of cold compressed graphite,” Phys. Rev. Lett. 108, 065501 (2012). 92 y cold compressed graphite,” Phys. Rev. Lett. 108, 065501 (2012). 92 111R. Ribeiro-Palau, C. Zhang, K. REFERENCES Sonnenschein, “Optical reﬂectivity of graph- ite under pressure,” Phys. Rev. B 40, 1951 (1989). g y 59L. G. Pimenta Martins, B. R. Carvalho, C. A. Occhialini, N. P. Neme, J.-H. Park, Q. Song, P. Venezuela, M. S. Mazzoni, M. J. Matos, J. Kong et al., “Electronic band tuning and multivalley raman scattering in monolayer y 81M. Hanﬂand, K. Syassen, and R. Sonnenschein, “Optical reﬂectivity of graph- ite under pressure,” Phys. Rev. B 40, 1951 (1989). Appl. Phys. Rev. 10, 011313 (2023); doi: 10.1063/5.0123283 V C Author(s) 2023 10, 011313-26 10, 011313-26 Appl. Phys. Rev. 10, 011313 (2023); doi: 10.1063/5.0123283 Applied Physics Reviews REVIEW scitation.org/journal/are 82R. Aust and H. Drickamer, “Carbon: A new crystalline phase,” Science 140, 817–819 (1963). P. Makk, and S. Csonka, “New method of transport measurements on van der Waals heterostructures under pressure,” J. Appl. Phys. 130, 064303 (2021). 104 104O. L. Blakslee, D. G. Proctor, E. J. Seldin, G. B. Spence, and T. Weng, “Elastic constants of compression–annealed pyrolytic graphite,” J. Appl. Phys. 41, 3373 (1970). 83W. L. Mao, H.-K. Mao, P. J. Eng, T. P. Trainor, M. Newville, C.-C. Kao, D. L. Heinz, J. Shu, Y. Meng, and R. J. Hemley, “Bonding changes in compressed superhard graphite,” Science 302, 425–427 (2003). 105G. Li, A. Luican, J. M. B. Lopes dos Santos, A. H. Castro Neto, A. Reina, J. Kong, and E. Y. Andrei, “Observation of van Hove singularities in twisted gra- phene layers,” Nat. Phys. 6, 109–113 (2010). g 84Q. Li, Y. Ma, A. R. Oganov, H. Wang, H. Wang, Y. Xu, T. Cui, H.-K. Mao, and G. Zou, “Superhard monoclinic polymorph of carbon,” Phys. Rev. Lett. 102, 175506 (2009). 106J. Xue, J. Sanchez-Yamagishi, D. Bulmash, P. Jacquod, A. Deshpande, K. Watanabe, T. Taniguchi, P. Jarillo-Herrero, and B. J. LeRoy, “Scanning tunnelling microscopy and spectroscopy of ultra-ﬂat graphene on hexagonal boron nitride,” Nat. Mater. 10, 282–285 (2011). 107 85K. Umemoto, R. M. Wentzcovitch, S. Saito, and T. Miyake, “Body-centered tetragonal C4: A viable sp3 carbon allotrope,” Phys. Rev. Lett. 104, 125504 (2010). 86J.-T. Wang, C. Chen, and Y. Kawazoe, “Low-temperature phase transforma- tion from graphite to sp3 orthorhombic carbon,” Phys. Rev. Lett. 106, 075501 (2011). 107R. Decker, Y. Wang, V. W. Brar, W. Regan, H.-Z. Tsai, Q. Wu, W. Gannett, A. Zettl, and M. F. Crommie, “Local electronic properties of graphene on a bn substrate via scanning tunneling microscopy,” Nano Lett. 11, 2291–2295 (2011). 87A. C. REFERENCES Watanabe, T. Taniguchi, J. Hone, and C. R. Dean, “Twistable electronics with dynamically rotatable heterostructures,” Science 361, 690–693 (2018). 112 92Y. Wang, J. E. Panzik, B. Kiefer, and K. K. Lee, “Crystal structure of graphite under room-temperature compression and decompression,” Sci. Rep. 2, 520 (2012). 24 October 2024 03:58:28 112N. R. Finney, M. Yankowitz, L. Muraleetharan, K. Watanabe, T. Taniguchi, C. R. Dean, and J. Hone, “Tunable crystal symmetry in graphene–boron nitride heterostructures with coexisting Moire superlattices,” Nat. Nanotechnol. 14, 1029–1034 (2019). 113 93E. M. Ferreira, M. V. Moutinho, F. Stavale, M. Lucchese, R. B. Capaz, C. Achete, and A. Jorio, “Evolution of the raman spectra from single-, few-, and many-layer graphene with increasing disorder,” Phys. Rev. B 82, 125429 (2010). 113B. Hunt, J. D. Sanchez-Yamagishi, A. F. Young, M. Yankowitz, B. J. LeRoy, K. Watanabe, T. Taniguchi, P. Moon, M. Koshino, P. Jarillo-Herrero, and R. C. Ashoori, “Massive Dirac fermions and Hofstadter butterﬂy in a van der Waals heterostructure,” Science 340, 1427–1430 (2013). ( ) 94E. Piatti, D. De Fazio, D. Daghero, S. R. Tamalampudi, D. Yoon, A. C. Ferrari, and R. S. Gonnelli, “Multi-valley superconductivity in ion-gated MoS2 layers,” Nano Lett. 18, 4821–4830 (2018). y 95A. P. Nayak, T. Pandey, D. Voiry, J. Liu, S. T. Moran, A. Sharma, C. Tan, C.-H. Chen, L.-J. Li, M. Chhowalla et al., “Pressure-dependent optical and vibrational properties of monolayer molybdenum disulﬁde,” Nano Lett. 15, 346–353 (2015). 114C. R. Dean, L. Wang, P. Maher, C. Forsythe, F. Ghahari, Y. Gao, J. Katoch, M. Ishigami, P. Moon, M. Koshino, T. Taniguchi, K. Watanabe, K. L. Shepard, J. Hone, and P. Kim, “Hofstadter’s butterﬂy and the fractal quantum Hall effect in Moire superlattices,” Nature 497, 598–602 (2013). Hone, and P. Kim, “Hofstadter’s butterﬂy and the fractal quantum Hall effect in Moire superlattices,” Nature 497, 598–602 (2013). 96L. Fu, Y. Wan, N. Tang, Y.-M. Ding, J. Gao, J. Yu, H. Guan, K. Zhang, W. Wang, C. Zhang et al., “K-k crossover transition in the conduction band of monolayer MoS2 under hydrostatic pressure,” Sci. Adv. 3, e1700162 (2017). 97 p 115L. Balents, C. R. Dean, D. K. Efetov, and A. F. Young, “Superconductivity and strong correlations in Moire ﬂat bands,” Nat. Phys. 16, 725–733 (2020). 116 Wang, C. Zhang et al., K-k crossover transition in the conduction band of monolayer MoS2 under hydrostatic pressure,” Sci. Adv. 3, e1700162 (2017). 97 monolayer MoS2 under hydrostatic pressure,” Sci. REFERENCES Yankowitz, Z. Lin, Q. Jiang, K. Hwangbo, Q. Zhang, B. Sun, T. Taniguchi, K. Watanabe, M. A. McGuire, D. Graf, T. Cao, J.-H. Chu, D. H. Cobden, C. R. Dean, D. Xiao, and X. Xu, “Switching 2D magnetic states via pressure tuning of layer stacking,” Nat. Mater. 18, 1298–1302 (2019). g 152W. Choi, N. Choudhary, G. H. Han, J. Park, D. Akinwande, and Y. H. Lee, “Recent development of two-dimensional transition metal dichalcogenides and their applications,” Mater. Today 20, 116–130 (2017). 153 pressure tuning of layer stacking,” Nat. Mater. 18, 1298–1302 (2019 2 132T. Li, S. Jiang, N. Sivadas, Z. Wang, Y. Xu, D. Weber, J. E. Goldberger, K. Watanabe, T. Taniguchi, C. J. Fennie, K. F. Mak, and J. Shan, “Pressure- controlled interlayer magnetism in atomically thin CrI3,” Nat. Mater. 18, 1303–1308 (2019). pp y 153S. Bhattacharyya and A. K. Singh, “Semiconductor-metal transition in semi- conducting bilayer sheets of transition-metal dichalcogenides,” Phys. Rev. B 86, 075454 (2012). 133A. K. Geim and I. V. Grigorieva, “Van der waals heterostructures,” Nature 499, 419–425 (2013). 154A. Kumar and P. Ahluwalia, “Electronic structure of transition metal dichalco- genides monolayers 1H-MX2 (M ¼ Mo, W; X ¼ S, Se, Te) from ab-initio the- ory: New direct band gap semiconductors,” Eur. Phys. J. B 85, 1–7 (2012). 155 134A. Avsar, J. Y. Tan, T. Taychatanapat, J. Balakrishnan, G. K. W. Koon, Y. Yeo, J. Lahiri, A. Carvalho, A. S. Rodin, E. C. T. O’Farrell, G. Eda, A. H. Castro Neto, and B. €Ozyilmaz, “Spin–orbit proximity effect in graphene,” Nat. Commun. 5, 4875 (2014). 135 155X. Su, W. Ju, R. Zhang, C. Guo, Y. Yong, H. Cui, and X. Li, “Band gap modu- lation of transition-metal dichalcogenide MX2 nanosheets by in-plane strain,” Physica E 84, 216–222 (2016). 135Z. Wang, D.-K. Ki, H. Chen, H. Berger, A. H. MacDonald, and A. F. Morpurgo, “Strong interface-induced spin–orbit interaction in graphene on WS2,” Nat. Commun. 6, 8339 (2015). 136 y 156D. Fu, J. Zhou, S. Tongay, K. Liu, W. Fan, T.-J. King Liu, and J. Wu, “Mechanically modulated tunneling resistance in monolayer MoS2,” Appl. Phys. Lett. 103, 183105 (2013). 136Z. Wang, D.-K. Ki, J. Y. Khoo, D. Mauro, H. Berger, L. S. Levitov, and A. F. Morpurgo, “Origin and magnitude of ‘designer’ spin-orbit interaction in gra- phene on semiconducting transition metal dichalcogenides,” Phys. Rev. X 6, 041020 (2016). y 157A. P. Nayak, S. Bhattacharyya, J. Zhu, J. Liu, X. REFERENCES Dean, “Dynamic band-structure tun- ing of graphene Moire superlattices with pressure,” Nature 557, 404–408 (2018). 121Y. Cao, D. Rodan-Legrain, O. Rubies-Bigorda, J. M. Park, K. Watanabe, T. Taniguchi, and P. Jarillo-Herrero, “Tunable correlated states and spin-polarized phases in twisted bilayer–bilayer graphene,” Nature 583, 215–220 (2020). 122 122G. W. Burg, J. Zhu, T. Taniguchi, K. Watanabe, A. H. MacDoinald, and E. Tutuc, “Correlated insulating states in twisted double bilayer graphene,” Phys. Rev. Lett. 123, 197702 (2019). 103B. F€ul€op, A. Marffy, E. Tovari, M. Kedves, S. Zihlmann, D. Indolese, Z. Kovacs-Krausz, K. Watanabe, T. Taniguchi, C. Sch€onenberger, I. Kezsmarki, Appl. Phys. Rev. 10, 011313 (2023); doi: 10.1063/5.0123283 V C Author(s) 2023 10, 011313-27 10, 011313-27 Appl. Phys. Rev. 10, 011313 (2023); doi: 10.1063/5.0123283 Applied Physics Reviews REVIEW scitation.org/journal/are 141D. Bonnell, Scanning Probe Microscopy and Spectroscopy (Wiley-VCH, New York, NY, 2001). 123M. He, Y. Li, J. Cai, Y. Liu, K. Watanabe, T. Taniguchi, X. Xu, and M. Yankowitz, “Symmetry breaking in twisted double bilayer graphene,” Nat. Phys. 17, 26–30 (2021). 124 123M. He, Y. Li, J. Cai, Y. Liu, K. Watanabe, T. Taniguchi, X. Xu, and M. 142A. Renger and K. Johnson, Contact Mechanics (Cambridge University Press, Cambridge, UK, 1985). 124B. Szentpeeri, P. Rickhaus, F. K. de Vries, A. Marffy, B. F€ul€op, E. Tovari, K. Watanabe, T. Taniguchi, A. Kormanyos, S. Csonka, and P. Makk, “Tailoring the band structure of twisted double bilayer graphene with pressure,” Nano Lett. 21, 8777 (2021). g ) 143F. Cellini, Y. Gao, and E. Riedo, “A˚-indentation for non-destructive elastic moduli measurements of supported ultra-hard ultra-thin ﬁlms and nano- structures,” Sci. Rep. 9, 4075 (2019). p 144J. Stern, B. Terris, H. Mamin, and D. Rugar, “Deposition and imaging of local- ized charge on insulator surfaces using a force microscope,” Appl. Phys. Lett. 53, 2717–2719 (1988). 125C. Gong, L. Li, Z. Li, H. Ji, A. Stern, Y. Xia, T. Cao, W. Bao, C. Wang, Y. Wang, Z. Q. Qiu, R. J. Cava, S. G. Louie, J. Xia, and X. Zhang, “Discovery of intrinsic ferromagnetism in two-dimensional van der Waals crystals,” Nature 546, 265–269 (2017). 145A. Barboza, A. Gomes, B. Archanjo, P. Araujo, A. Jorio, A. Ferlauto, H. Chacham, B. Neves et al., “Deformation induced semiconductor-metal transi- tion in single wall carbon nanotubes probed by electric force microscopy,” Phys. Rev. Lett. 100, 256804 (2008). 126B. Huang, G. Clark, E. Navarro-Moratalla, D. R. Klein, R. Cheng, K. L. Seyler, D. Zhong, E. REFERENCES Schmidgall, M. A. McGuire, D. H. Cobden, W. Yao, D. Xiao, P. Jarillo-Herrero, and X. Xu, “Layer-dependent ferromagnetism in a van der Waals crystal down to the monolayer limit,” Nature 546, 270–273 (2017). 127 y 146A. P. Barboza, M. J. Matos, H. Chacham, R. J. Batista, A. B. de Oliveira, M. S. Mazzoni, and B. R. Neves, “Compression-induced modiﬁcation of boron nitride layers: A conductive two-dimensional bn compound,” ACS Nano 12, 5866–5872 (2018). 127T. Song, X. Cai, M. W.-Y. Tu, X. Zhang, B. Huang, N. P. Wilson, K. L. Seyler, L. Zhu, T. Taniguchi, K. Watanabe, M. A. McGuire, D. H. Cobden, D. Xiao, W. Yao, and X. Xu, “Giant tunneling magnetoresistance in spin-ﬁlter van der Waals heterostructures,” Science 360, 1214–1218 (2018). 128 147A. P. Barboza, A. C. Souza, M. J. Matos, J. C. Brant, T. C. Barbosa, H. Chacham, M. S. Mazzoni, and B. R. Neves, “Graphene/h-BN heterostructures under pressure: From van der Waals to covalent,” Carbon 155, 108–113 (2019). 128D. R. Klein, D. MacNeill, J. L. Lado, D. Soriano, E. Navarro-Moratalla, K. Watanabe, T. Taniguchi, S. Manni, P. Canﬁeld, J. Fernandez-Rossier, and P. Jarillo-Herrero, “Probing magnetism in 2D van der Waals crystalline insula- tors via electron tunneling,” Science 360, 1218–1222 (2018). 148A. Barboza, H. Chacham, and B. Neves, “Universal response of single-wall car- bon nanotubes to radial compression,” Phys. Rev. Lett. 102, 025501 (2009). 149 129M. A. McGuire, H. Dixit, V. R. Cooper, and B. C. Sales, “Coupling of crystal structure and magnetism in the layered, ferromagnetic insulator CrI3,” Chem. Mater. 27, 612–620 (2014). 149K. Watanabe, T. Taniguchi, and H. Kanda, “Direct-bandgap properties and evidence for ultraviolet lasing of hexagonal boron nitride single crystal,” Nat. Mater. 3, 404–409 (2004). 130Z. Sun, Y. Yi, T. Song, G. Clark, B. Huang, Y. Shan, S. Wu, D. Huang, C. Gao, Z. Chen, M. McGuire, T. Cao, D. Xiao, W.-T. Liu, W. Yao, X. Xu, and S. Wu, “Giant nonreciprocal second-harmonic generation from antiferromagnetic bilayer CrI3,” Nature 572, 497–501 (2019). 150F. Cellini, F. Lavini, E. Chen, A. Bongiorno, F. Popovic, R. L. Hartman, R. Dingreville, and E. Riedo, “Pressure-induced formation and mechanical prop- erties of 2D diamond boron nitride,” Adv. Sci. 8, 2002541 (2021). 151 24 October 2024 03:58:28 151Q. H. Wang, K. Kalantar-Zadeh, A. Kis, J. N. Coleman, and M. S. Strano, “Electronics and optoelectronics of two-dimensional transition metal dichalcogenides,” Nat. Nanotechnol. 7, 699–712 (2012). 131T. Song, Z. Fei, M. REFERENCES Wu, T. Pandey, C. Jin, A. K. Singh, D. Akinwande, and J.-F. Lin, “Pressure-induced semiconducting to metallic transition in multilayered molybdenum disulphide,” Nat. Commun. 5, 1–9 (2014). 137B. Yang, M.-F. Tu, J. Kim, Y. Wu, H. Wang, J. Alicea, R. Wu, M. Bockrath, and J. Shi, “Tunable spin–orbit coupling and symmetry-protected edge states in graphene/WS2,” 2D Mater. 3, 031012 (2016). 138 158Z.-H. Chi, X.-M. Zhao, H. Zhang, A. F. Goncharov, S. S. Lobanov, T. Kagayama, M. Sakata, and X.-J. Chen, “Pressure-induced metallization of molybdenum disulﬁde,” Phys. Rev. Lett. 113, 036802 (2014). 159 138B. Yang, M. Lohmann, D. Barroso, I. Liao, Z. Lin, Y. Liu, L. Bartels, K. Watanabe, T. Taniguchi, and J. Shi, “Strong electron-hole symmetric Rashba spin-orbit coupling in graphene/monolayer transition metal dichalcogenide heterostructures,” Phys. Rev. B 96, 041409(R) (2017). 159M. V. Bessa, W. D. Freitas, N. P. Neme, L. G. Martins, A. P. Barboza, M. J. Matos, M. S. Mazzoni, and B. R. Neves, “Electromechanical modulations in transition metal dichalcogenide nanosheets: Implications for environmental ” ACS A l N M t 4 11305 11311 (2021) g p sensors,” ACS Appl. Nano Mater. 4, 11305–11311 (2021). 160 139J. O. Island, X. Cui, C. Lewandowski, J. Y. Khoo, E. M. Spanton, H. Zhou, D. Rhodes, J. C. Hone, T. Taniguchi, K. Watanabe, L. S. Levitov, M. P. Zaletel, and A. F. Young, “Spin–orbit-driven band inversion in bilayer graphene by the van der Waals proximity effect,” Nature 571, 85–89 (2019). 140 sensors,” ACS Appl. Nano Mater. 4, 11305–11311 (2021). 160 160A. P. M. Barboza, H. Chacham, C. K. Oliveira, T. F. Fernandes, E. H. M. Ferreira, B. S. Archanjo, R. J. Batista, A. B. de Oliveira, and B. R. Neves, “Dynamic negative compressibility of few-layer graphene, h-BN, and MoS2,” Nano Lett. 12, 2313–2317 (2012). the van der Waals proximity effect,” Nature 571, 85–89 (2019). 140 140B. F€ul€op, A. Marffy, S. Zihlmann, M. Gmitra, E. Tovari, B. Szentpeeri, M. Kedves, K. Watanabe, T. Taniguchi, J. Fabian, C. Sch€onenberger, P. Makk, and S. Csonka, “Boosting proximity spin–orbit coupling in graphene/wse2 het- erostructures via hydrostatic pressure,” npj 2D Mater. Appl. 5, 82 (2021). 161A. B. Alencar, A. P. M. Barboza, B. S. Archanjo, H. Chacham, and B. R. Neves, “Experimental and theoretical investigations of monolayer and few-layer talc,” 2D Mater. 2, 015004 (2015). Appl. Phys. Rev. 10, 011313 (2023); doi: 10.1063/5.0123283 V C Author(s) 2023 10, 011313-28 10, 011313-28 Appl. Phys. Rev. REFERENCES 10, 011313 (2023); doi: 10.1063/5.0123283 Applied Physics Reviews REVIEW scitation.org/journal/are 162J. A. M. Meza, C. Lubin, F. Thoyer, and J. Cousty, “Tip induced mechanical deformation of epitaxial graphene grown on reconstructed 6H–SiC (0001) sur- face during scanning tunneling and atomic force microscopy studies,” Nanotechnology 26, 255704 (2015). 178P. Johari and V. B. Shenoy, “Tuning the electronic properties of semiconduct- ing transition metal dichalcogenides by applying mechanical strains,” ACS Nano 6, 5449–5456 (2012). 179E. Scalise, M. Houssa, G. Pourtois, V. Afanas’ev, and A. Stesmans, “Strain- induced semiconductor to metal transition in the two-dimensional honey- comb structure of MoS2,” Nano Res. 5, 43–48 (2012). 180 gy 163M. Rejhon, F. Lavini, A. Khosravi, M. Shestopalov, J. Kunc, E. Tosatti, and E. Riedo, “Relation between interfacial shear and friction force in 2D materials,” Nat. Nanotechnol. 17, 1280–1287 (2022). 180A. Kumar and P. K. Ahluwalia, “Semiconductor to metal transition in bilayer transition metals dichalcogenides MX2 ðM ¼ Mo; W; X ¼ S; Se; TeÞ,” Modell. Simul. Mater. Sci. Eng. 21, 065015 (2013). Nat. Nanotechnol. 17, 1280–1287 (2022). 164M. Yankowitz, K. Watanabe, T. Taniguchi, P. San-Jose, and B. J. LeRoy, “Pressure-induced commensurate stacking of graphene on boron nitride,” Nat. Commun. 7, 13168 (2016). g 181Z. Tao, J. Du, Z. Qi, K. Ni, S. Jiang, and Y. Zhu, “Raman spectroscopy study of sp2 to sp3 transition in bilayer graphene under high pressures,” Appl. Phys. Lett. 116, 133101 (2020). 165A. Lipatov, P. Chaudhary, Z. Guan, H. Lu, G. Li, O. Cregut, K. D. Dorkenoo, R. Proksch, S. Cheriﬁ-Hertel, D.-F. Shao et al., “Direct obser- vation of ferroelectricity in two-dimensional MoS2,” npj 2D Mater. Appl. 6, 18 (2022). 182L. A. Chernozatonskii, V. A. Demin, and D. G. Kvashnin, “Moire diamones: New diamond-like ﬁlms of semifunctionalized twisted graphene layers,” J. Phys. Chem. Lett. 13, 5399–5404 (2022). 166A. G. Kvashnin, L. A. Chernozatonskii, B. I. Yakobson, and P. B. Sorokin, “Phase diagram of quasi-two-dimensional carbon, from graphene to dia- mond,” Nano Lett. 14, 676–681 (2014). 183M. Bykov, E. Bykova, A. V. Ponomareva, F. Tasnadi, S. Chariton, V. B. Prakapenka, K. Glazyrin, J. S. Smith, M. F. Mahmood, I. A. Abrikosov et al., “Realization of an ideal Cairo tessellation in nickel diazenide NiN2: High-pressure route to pentagonal 2D materials,” ACS Nano 15, 13539–13546 (2021). 184 167C. Tantardini, A. G. Kvashnin, C. Gatti, B. I. Yakobson, and X. REFERENCES Gonze, “Computational modeling of 2D materials under high pressure and their chemical bonding: Silicene as possible ﬁeld-effect transistor,” ACS Nano 15, 6861–6871 (2021). 184M. Bykov, T. Fedotenko, S. Chariton, D. Laniel, K. Glazyrin, M. Hanﬂand, J. S. Smith, V. B. Prakapenka, M. F. Mahmood, A. F. Goncharov et al., “High-pres- sure synthesis of Dirac materials: Layered van der Waals bonded BeN4 poly- morph,” Phys. Rev. Lett. 126, 175501 (2021). 168G. Kern, G. Kresse, and J. Hafner, “Ab initio calculation of the lattice dynamics and phase diagram of boron nitride,” Phys. Rev. B 59, 8551–8559 (1999). p y 185Q. Song, C. A. Occhialini, E. Ergec¸en, B. Ilyas, D. Amoroso, P. Barone, J. Kapeghian, K. Watanabe, T. Taniguchi, A. S. Botana et al., “Evidence for a single-layer van der Waals multiferroic,” Nature 602, 601–605 (2022). 186 169L. A. Chernozatonskii, P. B. Sorokin, A. G. Kvashnin, and D. G. Kvashnin, “Diamond-like c2h nanolayer, diamane: Simulation of the structure and prop- erties,” JETP Lett. 90, 134–138 (2009). 186Y. Tokura, S. Seki, and N. Nagaosa, “Multiferroics of spin origin,” Rep. Prog. Phys. 77, 076501 (2014). 187 170Y. Takagi and K. Kusakabe, “Transition from direct band gap to indirect band gap in ﬂuorinated carbon,” Phys. Rev. B 65, 121103 (2002). 187D. Zhong, K. L. Seyler, X. Linpeng, N. P. Wilson, T. Taniguchi, K. Watanabe, M. A. McGuire, K.-M. C. Fu, D. Xiao, W. Yao et al., “Layer-resolved magnetic proximity effect in van der Waals heterostructures,” Nat. Nanotechnol. 15, 187–191 (2020). 187D. Zhong, K. L. Seyler, X. Linpeng, N. P. Wilson, T. Taniguchi, K. Watanabe, M. A. McGuire, K.-M. C. Fu, D. Xiao, W. Yao et al., “Layer-resolved magnetic 171F. Piazza, K. Cruz, M. Monthioux, P. Puech, and I. Gerber, “Raman evidence for the successful synthesis of diamane,” Carbon 169, 129–133 (2020). 172 24 October 2024 03:58:28 172J. M. Soler, E. Artacho, J. D. Gale, A. Garcıa, J. Junquera, P. Ordejon, and D. Sanchez-Portal, “The SIESTA method for ab initio order-N materials simu- lation,” J. Phys.: Condens. Matter 14, 2745 (2002). 173 188H. Yu, G.-B. Liu, J. Tang, X. Xu, and W. Yao, “Moire excitons: From program- mable quantum emitter arrays to spin-orbit–coupled artiﬁcial lattices,” Sci. Adv. 3, e1701696 (2017). 189 173P. Hohenberg and W. Kohn, “Inhomogeneous electron gas,” Phys. Rev. 136, B864–B871 (1964). 189K. Tran, G. Moody, F. Wu, X. Lu, J. Choi, K. Kim, A. Rai, D. A. Sanchez, J. Quan, A. REFERENCES Singh et al., “Evidence for moire excitons in van der Waals hetero- structures,” Nature 567, 71–75 (2019). 190 174W. Kohn and L. J. Sham, “Self-consistent equations including exchange and correlation effects,” Phys. Rev. 140, A1133–A1138 (1965). 175N. Troullier and J. L. Martins, “Efﬁcient pseudopotentials for plane-wave cal- culations,” Phys. Rev. B 43, 1993–2006 (1991). 6 190K. L. Seyler, P. Rivera, H. Yu, N. P. Wilson, E. L. Ray, D. G. Mandrus, J. Yan, W. Yao, and X. Xu, “Signatures of moire-trapped valley excitons in MoSe2/ WSe2 heterobilayers,” Nature 567, 66–70 (2019). 9 176L. Kleinman and D. M. Bylander, “Efﬁcacious form for model pseudopotentials,” Phys. Rev. Lett. 48, 1425–1428 (1982). 177 191C. Jin, E. C. Regan, A. Yan, M. I. B. Utama, D. Wang, S. Zhao, Y. Qin, S. Yang, Z. Zheng, S. Shi et al., “Observation of moire excitons in WSe2/WS2 hetero- structure superlattices,” Nature 567, 76–80 (2019). 177Y.-P. Xie, X.-J. Zhang, and Z.-P. Liu, “Graphite to diamond: Origin for kinetics selectivity,” J. Am. Chem. Soc. 139, 2545–2548 (2017). 10, 011313-29 10, 011313-29 Appl. Phys. Rev. 10, 011313 (2023); doi: 10.1063/5.0123283 V C Author(s) 2023 Appl. Phys. Rev. 10, 011313 (2023); doi: 10.1063/5.0123283 V C Author(s) 2023
https://openalex.org/W4283214364	https://www.cambridge.org/core/services/aop-cambridge-core/content/view/6745C33068868D3EAE2284BAB6A22284/S0829320122000084a.pdf/div-class-title-when-prisoners-right-to-die-goes-online-a-case-study-of-legal-and-penal-sensibilities-div.pdf	English	null	When Prisoners’ “Right to Die” Goes Online: A Case-Study of Legal and Penal Sensibilities	Canadian journal of law and society	2,022	cc-by	11,239	* Our research was possible thanks to the generous and knowledgeable tutorship of Alexander Luscombe, an exceptional and caring colleague of ours whose encouragement focused the idea for the paper and brought us together as collaborators. One of us (Joshua) would also like to acknowledge the Centre for Criminology and Sociolegal Studies for an appointment as Visiting Junior Fellow in 2019–2021 that enabled our collaboration. Abstract Prisoners in Canadian federal penitentiaries can obtain medical assistance in dying (MAiD). This raises questions about the nature and legitimacy of pain and death in incarceration. The authors analyze responses to a Canadian Broadcasting Corpo- ration online news article discussing the provision of MAiD to prisoners. The comments exemplify diﬀerent sensibilities about the state’s lethality with respect to prisoners. These sensibilities—both legal and penal—draw on an array of cultural referents to orient to prisoners’ deaths generally, but also MAiD speciﬁcally. The authors explore how certain referents factor in these legal and penal sensibilities and appear to mediate commenters’ judgements. For example, capital punishment factors signiﬁcantly in conversations about MAiD for prisoners, as well as imag- inations of prisoners’ bodies in pain. As a result, there is a spectacularization of prisoners’ carceral death, despite the humane, “civilized” death MAiD provides, which circumscribes how some commenters imagine the procedure and prisoners’ deaths. Keywords: Comment sections, capital punishment, law and pain, medical assis- tance in dying, punishment When Prisoners’ “Right to Die” Goes Online: A Case-Study of Legal and Penal Sensibilities Joshua D. M. Shaw and Daniel Konikoﬀ* Canadian Journal of Law and Society / Revue Canadienne Droit et Société, 2022, Volume 37, no. 3, pp. 451–471. doi:10.1017/cls.2022.8 © The Author(s), 2022. Published by Cambridge University Press on behalf of the Canadian Law and Society Association. This is an Open Access article, distributed under the terms of the Creative Commons Attribution licence (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted re-use, distribution, and reproduction in any medium, provided the original work is properly cited. 451 https://doi.org/10.1017/cls.2022.8 Published online by Cambridge University Press Introduction Prisoners in Canadian penitentiaries can request that a physician or nurse practi- tioner administer or prescribe a medical substance that causes their death (CSC 2017; Downie, Iftene, and Steeves 2019). Such requests are possible because the Criminal Code allows every competent, autonomous Canadian patient to obtain medical assistance in dying (MAiD) if they seek “death as a response to a grievous Joshua D. M. Shaw and Daniel Konikoﬀ 452 and irremediable medical condition.”1 The legal right to MAiD follows from the 2015 decision, Carter v Canada, where the Supreme Court of Canada declared that the criminal prohibition of MAiD constituted an unconstitutional limit on one’s right to life, liberty, and security of the person. Canada’s Criminal Code was amended in 2016 to provide a legal framework for MAiD, and in 2017 the Correctional Service of Canada (CSC) issued a policy—Guideline 800-9—which set out the process by which prisoners in federal penitentiaries could request and obtain MAiD (including the procedures of euthanasia and self-administered suicide) (CSC 2017). g p Important to the Supreme Court’s reasoning in Carter v Canada was the experience of the ideal candidate for MAiD: a competent, self-actualizing person, whose independence is failed by an ailing body. The ideal candidate for MAiD was a patient “imprisoned” in their own body due to their condition and the criminal law that prevented them from medically assisted death. But actual incarceration complicates this image. The vulnerability of incarceration places the state in an intimate relationship with the prisoner’s death, because the state acts not only as the custodian of prison conditions, admission, and release, but also as the custodian of prisoners’ care and access to MAiD. The relationship of the state to a prisoner’s MAiD thereby revives concerns about the state’s lethality approximately forty years after Canada’s Bill C-84 abolished capital punishment in 19762 (lethality in the sense that the state appears to be involved in the killing or “letting die” of its population). Concerns with respect to the state’s role are also magniﬁed by recent studies on the poor health outcomes of Canadian prisoners (e.g., Iftene 2019; Iftene and Downie 2020), which may compound a prisoner’s experience of their body and the desire for MAiD, bringing into doubt the quality of consent even when it is obtained in accordance with law and policy. 1 Carter v Canada, 2015 SCC 5, para 2. 2 See BillC-84,AnAct toamendthe CriminalCodein relation tothe punishmentfor murderand certain other serious oﬀences, 1st Sess, 30th Parl; However, capital punishment was still permitted for certain military oﬀences until 1999, see Bill C-25, An Act to amend the National Defence Act and to make consequential amendments to other Acts, 1st Sess, 36th Parl, 46-47 Elizabeth II, 1997-98. 3 Dalhousie Health Law Institute, A Prison-Focused Satellite Meeting After the Second International Conference on End-of-Life Law, Ethics, Policy and Practice, Medical Assistance in Dying for Canadian Prisoners (September 2017) http://www.dementiajustice.com/uploads/1/0/2/4/ 102466336/prison_meeting_report_sept_2017.pdf. https://doi.org/10.1017/cls.2022.8 Published online by Cambridge University Press 1 Carter v Canada, 2015 SCC 5, para 2. 2 Introduction Prisoners’ “right to die” may thereby confront foundational assumptions surrounding death behind bars, as well as MAiD,3 aﬀecting how individuals orient to and evaluate these public policies and make meanings about law and punishment. Beyond the content of Guideline 800-9 and the other directives and authorities cited in that guideline (see e.g., Downie, Iftene, and Steeves, 2019), as well as a recent study of how inmates in a Canadian penitentiary navigate end-of-life care and view MAiD (Shaw and Driftmier 2021), little is publicly known about MAiD in prisons (also see Shaw and Elger, 2016; Stensland and Sanders, 2016). The Oﬃce of the Correctional Investigator released an annual report in 2019–2020 that identi- ﬁed “three known cases of MAiD in federal corrections, two carried out in the community” (OSC 2020), but the report does not say much more than the Correctional Investigator’s recommendations to expand access to compassionate https://doi.org/10.1017/cls.2022.8 Published online by Cambridge University Press When Prisoners’ “Right to Die” Goes Online 453 release. There is also a dearth of cultural or popular critique. Apart from an online news article published by the Canadian Broadcasting Corporation (CBC), MAiD in prisons is rarely mentioned in public discourse. The lack of public discourse, in part, accounts for our study’s basis in the comment section of that CBC article. This comment section comprises a rare space in which the under-discussed subject of MAiD in prisons is considered. Further, the content of those comments demon- strates the charged nature of that discussion when brought into focus, with commenters pulling from a number of referents to make normative judgements of MAiD in prisons. Though freighted with its own methodological limits, using the comment section as our sample provides us an access point to an understudied dimension of punishment, namely the way individuals make meanings of medi- calized death in a punitive setting. For example, commenters engage with ideas about capital punishment, notions of legitimate pain for the prisoner’s body, and the state’s role in punitive practices. In other words, the comment section—as a space of dialogue and dissent—allows us to explore how individuals, when con- fronted by the idea of MAiD in prisons, make sense of their foundational assump- tions about MAiD and punishment. Foundational assumptions—of MAiD or of punishment—are inextricably cultural. https://doi.org/10.1017/cls.2022.8 Published online by Cambridge University Press Introduction As David Garland (2006, 421) writes, punishment and “penal institutions” are “grounded in cultural values and perceptions,” draw “upon speciﬁc sensibilities” and express “particular emotions.” Punishment and penal institutions are “sites of ritual performance and cultural production” and “produc[e] diﬀuse cultural eﬀects as well as crime control” (Garland 2006, 421). Penal sensibilities involve the everyday constructions and “structures of aﬀect” that shape experience and understanding, including that which emerges in conversation (Smith, Sparks, and Girling 2001). Penal sensibilities are involved in the formation and reproduc- tion of institutions of punishment and are products of punishment themselves, as these sensibilities “communicate values, moralities, and political understandings” surrounding punishment (Smith, Sparks, and Girling 2001, 397). Similarly, as Roger Cotterrell (2018, 527) writes, law and legal institutions inhabit culture. This requires the legal theorist to pay “more attention to the nature of law itself as not only an instrument of state regulation but also an aspect of culture,” including “aﬀective elements [like] emotional attachments, allegiances, resistances and rejections” (emphasis in original). These cultural dimensions include people’s everyday perceptions or understandings of, and attitudes toward, law in social context. These are popular forms of legal consciousness where detailed narratives, cultural referents, and “structures of feeling” or embodied experience constitute what law is, does, and means in social situations (Ewick and Silbey 1998; Silbey 2018). Given that penal and legal sensibilities share a cultural basis or form, we discuss them together without distinction. MAiD and punishment are no diﬀerent from these sensibilities generally; they cannot be understood by reading statutes, decisions, or policies alone—the “oﬃcial” narratives told by state documents are inevitably incomplete without the admixture of penal and legal sensibilities that arise in their wake (Ewick and Silbey 1998). Thus, the signiﬁcance of culture to law and punishment, and its incorporation in everyday life, suggests that quotidian sites of cultural exchange and production may also be sites where people potentially reevaluate or solidify, Joshua D. M. Shaw and Daniel Konikoﬀ 454 reinforce, demonstrate, and contest foundational assumptions of MAiD and punishment. p One such place in which penal and legal sensibilities now emerge is online, as internet users are able to express penal and legal sensibilities in discussion boards, on social media sites, and in comment sections. These online fora allow immediate or near-immediate engagement across vast distances, potentially in a more candid and regular manner than is possible through other media. Introduction Oftentimes, these anonymously authored posts are of questionable character, as comments do not need to meet any literary or intellectual standard. Nevertheless, the ideas that emerge from this online discourse can reveal compelling, honest, and often unexpected perspectives on punishment, generated by people outside the oﬃcial penal system. Comment sections can also reveal uninformed, disingenuous, and callous perspectives, but have intrinsic quality to the extent that they display real human sentiments or beliefs about MAiD in prisons (messy though these senti- ments may be). In this paper, we look at how these sensibilities emerge at the conversational site of a comment section of an online news article published by CBC on February 25, 2018 (Harris 2018). The online news article—“Watchdog calls for ‘compassionate’ parole as prison system adopts new assisted death policy”—reports on the CSC policy that allows Canadian penitentiaries to act, as the author characterizes it, as the “facilitator or enabler” of MAiD for prisoners. In our study of these comments, we trace how MAiD for prisoners, and its founda- tional assumptions, are constructed in commenters’ everyday conversation having regard to a cultural approach to law and punishment. We focus on the comment section as a particular site of sensibilities pertaining to state lethality. While a range of ethical judgements are made by commenters, with some in favour of the CSC policy and others against, commenters tend to draw on certain narratives, referents and symbols in their construction of state lethality. We foreground the comments in a discussion of how capital punishment is diﬀerentially understood in relation to MAiD, foreground the comments again in a section that looks at how conceptions of corporeality mediate diverging views, and then pull these strands together in a discussion of how the spectacularization of prisoner death through MAiD aﬀects ethical orientations to death in incarceration. In doing so, we undertake a critical analysis of paradigms that matter in the commenters’ framing of the issue, which shapes their relations to foundational assumptions about MAiD and punishment. Importantly, we do not assume a positivist relationship to identifying and analyzing discrete facts about the sensi- bilities observed in the comment section. https://doi.org/10.1017/cls.2022.8 Published online by Cambridge University Press Introduction Instead, we take a post-positivist orien- tation to qualitative analysis and theory, which assumes knowledge is provisional, inextricable from the act-situation of observation (including methods of collection and analysis), and approached “abductively” oﬀering plausible explanations from an array of theories and experiences available to the scholar (Brinkmann 2014). 4 Since the enactment of Guideline 800-9, Bill C-7, An Act to amend the Criminal Code (medical assistance in dying), was passed by Parliament and received Royal Assent in March 2021. The Act amends the Criminal Code to establish two streams for accessing MAiD: one set of safeguards for MAiD where an individual’s “natural death” is reasonably foreseeable, and another set of safe- guards where death is not foreseeable, but the individual otherwise qualiﬁes for MAiD. As of February 2022, Guideline 800-9 has not been amended in response to this legislative change; it is unclear how the legislative change has aﬀected the provision of MAiD to prisoners. Furthermore, these amendments were formulated and enacted years after the publication of the CBC article and the comment section. Accordingly, our analysis does not consider these recent amendments. Medical Assistance in Dying for Prisoners in Canada The CSC policy on MAiD for prisoners—Guideline 800-9—sets out the process by which prisoners in federal penitentiaries in Canada can request and obtain MAiD (which includes physician/nurse practitioner-administered and self-administered 455 When Prisoners’ “Right to Die” Goes Online medical assistance in dying) (CSC 2017). Prisoners are eligible for MAiD according to the same criteria set out for the public; however, Guideline 800-9, in tandem with other directives and authorities, indicates a process unique for those in federal custody. Guideline 800-9 adds additional steps to the making of a request and its evaluation (CSC 2017). It also presupposes that the prisoner requesting MAiD will be transferred out of the federal penitentiary into the community for the second of two eligibility assessments and, if eligible, the completion of the procedure. To facilitate such transfers, CSC will consider “all release options,” including parole, parole-by-exception, and temporary absence, and inmates can also write to the Governor General of Canada who may grant the inmate’s release under the Royal Prerogative of Mercy (Downie, Iftene, and Steeves 2019). Guideline 800-9 speciﬁes that the “external [environment] to CSC [is], namely, […] a community hospital or health care facility” (CSC 2017), where policies and procedures of the hospital would also apply. In “exceptional circumstances,” the CSC will permit a prisoner to obtain MAiD within the penitentiary institution or a regional hospital operated by CSC, but it must be at the request of the prisoner, approved by the CSC’s Assistant Commissioner of Health Services, and the procedure must involve a practitioner external to CSC.4 The Case Study: The CBC Article and Comment Section https://doi.org/10.1017/cls.2022.8 Published online by Cambridge University Press The Case Study: The CBC Article and Comment Section On February 25, 2018, CBC published an online news article by Kathleen Harris entitled “Watchdog calls for ‘compassionate’ parole as prison system adopts new assisted death policy.” In this article, Harris outlined a policy that the CSC adopted in late 2017, in which prisons are allowed to serve as “a facilitator or enabler” of an inmate’s medically assisted death or MAiD (CSC 2017). Harris further described a letter to acting CSC Commissioner Anne Kelly from the Correctional Investigator Ivan Zinger, which decried the new policy, instead urging the CSC to grant terminally ill inmates a more “humanitarian and compassionate” conditional release. The comment section for this article included 812 distinct comments posted by 163 separate users between February 25, 2018, at 4:23 AM EST and February 26, 2018, at 3:30 AM EST. We trawled comments manually, copying-and-pasting each comment into an Excel ﬁle along with its associated metadata. Each entry in our database was comprised of the commenter’s name, the comment’s date and timestamp, the content of the comment, and the number of “Likes” or “upvotes” their comment received by other readers. We also indicated whether a comment https://doi.org/10.1017/cls.2022.8 Published online by Cambridge University Press Joshua D. M. Shaw and Daniel Konikoﬀ 456 was an original post or a reply to another comment. Reply comments were further ﬂagged with information that identiﬁed the comment to which they were replying, to keep track of conversations in the comment section. was an original post or a reply to another comment. Reply comments were further ﬂagged with information that identiﬁed the comment to which they were replying, to keep track of conversations in the comment section. p Once we gathered all comments, we began conducting exploratory research, using the ﬁrst ﬁfty comments as a test sample from which to develop a codebook of relevant themes and categories present within the comments. The themes and categories we developed clustered around a number of aspects pertaining to punishment, such as punishment’s purposes (e.g., retribution, incapacitation, deterrence, rehabilitation, reintegration), critiques of the justice system, critiques of the administration of MAiD, references to the death penalty, as well as com- passion or animosity toward inmates. We expanded our selection to 100 comments and adopted processes of iterative categorization (Neale 2016), adding more themes as we read and re-read our dataset. https://doi.org/10.1017/cls.2022.8 Published online by Cambridge University Press The Case Study: The CBC Article and Comment Section 457 When Prisoners’ “Right to Die” Goes Online On this point, though, we remain convinced of the comment section as an analytically rich venue for our case study. For one, our comment section is a lightly moderated cultural space where people self-identiﬁed by name come to converse about the content produced by the CBC. We say lightly moderated because, while comments are screened by site editors before publication, CBC has previously stated that 85% to 90% of comments submitted to CBC.ca are published.5 This can be contrasted with platforms like Twitter, which engage in far more complex forms of gatekeeping and content moderation (see e.g., Konikoﬀ2021). The comment section’s innate porosity is, in fact, a beneﬁt to our method, in that the conversa- tional architecture of the comment section allows for varied cultural ideas and references to ﬁnd expression in the comments, as opposed to a debate forum that closely regulates the parameters of speech. And though they are by no means a common data source in sociolegal research, comment sections can oﬀer revealing insights into penal and legal sensibilities. g g Public opinion data is an often-tapped resource in criminal justice research. A number of these issues—such as attitudes toward policing, the courts, prisons, neighbourhood safety, and so on—lend themselves particularly well to public opinion research, given the public facing nature of criminality and criminal justice processes. Historically, the dominant ways to gauge public opinion, particularly on criminal justice phenomena, have been opinion polls and surveys (Berinsky 2017; Frost 2010). However, in our current digital era, scholars have argued that the digital footprint that individuals leave behind on social media and in comment sections can be used to measure public opinion and reﬂect their individual preferences (see e.g., Bond and Messing 2015; Lee and Nerghes 2018; Prichard et al. 2015). While we do not claim that comment sections represent public opinion (e.g., it was exceedingly rare for anyone in the comment section to oppose MAiD generally even though an Ipsos poll (n = 3500) conducted on behalf of Dying with Dignity suggests 13% of Canadians opposed the policy in 2021),6 comment sections overall are a site in which cultural attitudes ﬁnd expression, which may inform place-deﬁned understandings (Davies 2015) of state lethality and punishment. The Case Study: The CBC Article and Comment Section In developing these themes, we approached our comment section case study with a sense of abductive inquiry (Brinkmann 2014). A helpful mode of analysis for the “creative crafting of theory,” abduction requires researchers to approach their study with “a wide array of theorizations” to “render surprising situations understandable” (Tavory and Tim- mermans 2019, 536–541). Throughout coding, we abductively situated our qual- itative observations in conversation with sociolegal studies, jurisprudence, criminology and communication studies, as well as Canada’s abolition of capital punishment and legalization of MAiD. This emergent form of analysis allowed us to make sense of surprising comments and develop novel coding categories, challenging our conceptualizations of our comment section data while allowing us to engage with our case study more creatively. In this way, coding was only ever provisional, mediating our encounter with theories that might help make plausible sense of the sensibilities observed. After coding each comment and discussing discrepancies, we ﬂagged 456 com- ments as irrelevant due to their lack of substantive engagement with the article’s content and removed them from the dataset. Discrepancies in coding were resolved in discussion with reference to theories we each saw resonate with the comments. This left 356 relevant comments from which we were to conduct our analysis. Deeming this many comments irrelevant and excising them from our sample attests to the relatively unplanned, unsystematic nature of comment sections. In general, comment sections allow for plural, unrelated dialogues to start, stop, and change direction. Many commenters often engage directly with the topic of the relevant article, but others use the space as a jumping-oﬀpoint for discussions of other issues. Oﬀ-topic conversations appear to be generally tolerated by commen- ters, perhaps because CBC’s editorial staﬀdo not activate comment sections for all CBC articles, which shifts conversations to comment sections on unrelated stories. The mode of conversation in our case study was an open one; even when com- menters’ commentary dealt with MAiD for prisoners, it could also traﬃc in other events, concerns, and identities, whether related to punishment or not. While our abductive approach allowed for surprise and breakdown in our qualitative obser- vations, some comments were regarded as perhaps more outside the study’s parameters than they were empirically useful to understanding sensibilities that inform judgements of prisoners’ deaths. 5 https://www.cbc.ca/newsblogs/community/editorsblog/2016/03/reviewing-our-commenting- policy.html. 6 https://www.ipsos.com/en-ca/majority-canadians-support-access-medical-assistance-dying-maid. p y 6 https://www.ipsos.com/en-ca/majority-canadians-support-access-medical-assistance-dying-maid. 5 https://www.cbc.ca/newsblogs/community/editorsblog/2016/03/reviewing-our-commenting- policy.html. policy.html. 6 h // https://doi.org/10.1017/cls.2022.8 Published online by Cambridge University Press 5 https://www.cbc.ca/newsblogs/community/ li h l The Case Study: The CBC Article and Comment Section We consider place-based understandings of state lethality and punishment to be “[p]erformative ‘truths’ […] produced by actions that connect humans to the material (and ideational) world, constituting the subjects and objects” (Davies 2015, 221) that come to deﬁne social experience in a speciﬁc place. The comments here express sensibilities informed by the place and time of the comment section, and the experiences they bring to that virtual space, which together shapes foundational assumptions about MAiD and punishment, and commenters’ ethical judgements. Using comment sections as the basis of qualitative inquiry aligns with Garland’s (2006) call for the use of the tools of cultural analysis for making sense of punishment and penal techniques. Exploring the discourse of comment sections also aligns with Garland’s (2006, 428) concomitant call for an exploration of https://doi.org/10.1017/cls.2022.8 Published online by Cambridge University Press Joshua D. M. Shaw and Daniel Konikoﬀ 458 “audience and interpretation” when thinking through the meanings and sensibil- ities wrapped up in punishment. Comment sections, then, can serve as a sort of venue for informative “societal conversations” surrounding penality (Smith et al. 2001, 400). We have by no means “tapped into the essential inner world” of our commenters, nor can we say with certainty who these people really are, but their potential anonymity, as well as the occasional discursive haphazardness of their contributions, in no way undercut the surprising analytic insights their comments stand to reveal. The monologues and dialogues in the comment section neverthe- less represent cultural expressions of penal and legal sensibilities around MAiD, further reﬂecting what Garland (1990, 253) describes as punishment’s “positive capacity to create meaning.” https://doi.org/10.1017/cls.2022.8 Published online by Cambridge University Press The Spectre of Capital Punishment The causal agent coincides in MAiD and capital punishment through the use of the phrase “lethal injection,” relating the two procedures through their shared transgression of the prisoner’s living body: “ah I When Prisoners’ “Right to Die” Goes Online 459 love it, lethal injection by request.” But Brian’s reference to lethal injection, especially paired with a ﬂat, caustic aﬀect (“ah I love it”), while not a spectacular method of execution, is not devoid of violence (Garland 2011). Lethal injection was rejected by a Joint Committee’s ﬁnal report in 1956, because the method fused medicine and execution, and as Strange (2001, 380) put it, “required close and intimate connection between the prisoner and the person who injected the needle.” This close and intimate connection between executor and prisoner put the executor love it, lethal injection by request.” But Brian’s reference to lethal injection, especially paired with a ﬂat, caustic aﬀect (“ah I love it”), while not a spectacular method of execution, is not devoid of violence (Garland 2011). Lethal injection was rejected by a Joint Committee’s ﬁnal report in 1956, because the method fused medicine and execution, and as Strange (2001, 380) put it, “required close and intimate connection between the prisoner and the person who injected the needle.” This close and intimate connection between executor and prisoner put the executor in a qualitatively diﬀerent relation to the death caused than with the dispassionate execution by hanging, contrary to mid-twentieth-century sensibilities about killing (Strange 2001). This close and intimate connection is also drawn out when contrasting the lexical construction, “lethal injection,” with that particular to MAiD. Section 241.1 of Canada’s Criminal Code, in deﬁning MAiD, describes the method, in part, as “a substance […] that causes their death,” and is described in protocols of the Canadian Association of MAiD Assessors and Providers (CAMAP)—a voluntary association involved in the drafting of guidelines—as either a “lethal dose” or “lethal eﬀect.”7 Whereas the action-noun, injection, implies active, human intervention in extinguishing life, the passive construction of MAiD’s legislative deﬁnition—“a substance […] that causes”—places the prescribing or administering physician or nurse practitioner at some remove from death. Brian, in less than two words, compresses this distance relating MAiD to an active form of lethality that was repugnant to politicians in the twilight of capital punishment in Canada. 7 See e.g., https://camapcanada.ca/wp-content/uploads/2019/01/OralMAiD-Med.pdf; https:// camapcanada.ca/wp-content/uploads/2020/05/IV-protocol-ﬁnal.pdf. 8 Hansard (Commons Debates), May 6, 1976 on p 13243. The Spectre of Capital Punishment Capital punishment is a touchstone for many commenters, with several of them drawing on cultural images of executions to evaluate MAiD for prisoners. These cultural images of capital punishment occur despite its abolition in 1976 and the last executions in Canada taking place in 1962. Commenters associate death caused by MAiD with capital punishment, often with punitive emphasis. In other instances, capital punishment is not referred to explicitly, but commenters’ ratio- nales for MAiD coincide with those expressed historically in Canada. For example, Lucia invokes the violence of an “electric chair” to describe MAiD, connecting MAiD to execution: “The electric chair sounds like a great option for assisted suicide of prisoners. This sounds like we should bring back the death penalty under the guise of compassion for the most heinous inmates.” p y g p In invoking the electric chair, Lucia appears to imagine the prisoner in pain, in opposition to the humane death so important to the American state’s absolution of capital punishment (Garland 2011; Kaufman-Osborn 2001; Sarat 2001). The electric chair is a comparatively corporeal and spectacular reference. The electric chair’s violence has required US courts, in contexts of judicial review, to obscure and diminish signs that the prisoner suﬀered unnecessarily to preserve the method’s legitimacy (Kaufman-Osborn 2001) and necessitated additional means to “minimize the exposure of bodily ﬂuids and ﬂows” (Garland 2011, 778). Whether intentional or not, Lucia’s reference to the electric chair strongly associ- ates MAiD with such gore. Further, the physician or nurse practitioner, and their medicalized, therapeutic means of causing death, disappear in Lucia’s image, replaced by technology that re-aligns MAiD with the commission of punishment through execution. And perhaps most interestingly, Canada has no historical precedent of using the electric chair, having only executed prisoners by hanging (Strange 1995). Lucia sees MAiD for prisoners as an opportunity to restore capital punishment—a kind that never existed in Canada—even if it appears outwardly, through procedures attendant to MAiD, like an act of compassion. For Brian, the causal agent of death need not be spectacular, like an electric chair, nor risk the same pains. This is a way to save money and the end results are the same. The inmate who is going to die in prison dies. The Spectre of Capital Punishment Further, the outward appearance of MAiD procedures—such as Brian’s reference to “by request”—is overtaken by the imagined connection of capital punishment to MAiD through the mechanism of causing death. The Criminal Code establishes an array of procedural safeguards, which require a patient’s capacity to consent, the absence of coercion or pressure, intolerable suﬀering of a “grievous and irremediable medical condition,” periods for reﬂection, among other safeguards, dissimulated in Brian’s characterization, “lethal injection by request.” Death, and the state’s role in bringing about death, are also discussed in much more abstracted terms. Commenters invoke the argument, similarly raised in 1976 in favour of capital punishment, that supporting prisoners costs the Canadian government—and by extension, taxpayers—a lot of money,8 so prisoners’ prema- ture death, through MAiD, would limit government spending. Humphrey writes: “Why not. It’ll save us money.” Joe writes: “We should thank them for saving US money!” Lucia, again playing with terminology, writes: “The death penalty is a form of assisted suicide and a good cost saver.” Susan writes: Cons whine daily about money the Liberals spend but are OK with it costing $150,000[tokeep]aninmatethathasmajorhealthissuesaliveafewmoreyears. This is a way to save money and the end results are the same. The inmate who is going to die in prison dies. This is a way to save money and the end results are the same. The inmate who is going to die in prison dies. p / p / p / / / p 8 Hansard (Commons Debates), May 6, 1976 on p 13243. https://doi.org/10.1017/cls.2022.8 Published online by Cambridge University Press Joshua D. M. Shaw and Daniel Konikoﬀ 460 For Jason, the provision of care to prisoners is too expensive and could be avoided by facilitating MAiD, where MAiD is the prisoner’s choice: “If this is asked for by the inmates. All the power to them. Let THEM make that choice. Especially if it can save 150G/year per inmate.” In these examples, rationales for MAiD coincide with rationales previously used for capital punishment. These comments use prisoners’ deaths as a tool to critique government largesse, where largesse not only signals the unscrupulous use of the public purse but also something deeper about the nature of law and society. The Spectre of Capital Punishment In the case of capital punishment, as exempliﬁed in the 1976 speech of Kenneth Hurlburt, MP, in Parliament, prisoners’ deaths reinforce law’s hold over the hearts and minds of people, maintaining good order in awe of law’s authority. According to Hurlburt, abolishing the state’s “right to protect […] life by killing” and replacing it with a carceral-welfare state would lead Canada further into the excess and disorder of a criminogenic society. The “particular [historical and cultural] conjuncture” that lent support to capital punishment in the 1960s and 1970s for retentionists like Hurlburt was, as David Garland (2005, 357–58) notes, “shaped by fears about rising rates of crime and violence,” “urban breakdown,” and “moral decay.” Joe, for example, who, as noted above, celebrated that MAiD for prisoners saved taxpayers’ money, was also critical of desires of some to ensure that MAiD was carried out compassionately. “Certain members of society” are seen as being too soft on crime and prisoners, which, it is implied, feeds criminal activity: When convicts who CHOOSE to break our laws and threaten, injure and in many cases kill (mostly) innocent people, they give up not only their privilege to “compassion”; but their rights as Canadians and even human beings as well. Certain members of society waste far too much energy on those who CHOOSE to break OUR laws. Quite simply, convicted criminal should pay the SAME penalty, plus costs and inconvenience, as they have “charged” their victims. They, or their estate, should also pay all court, policing and jail costs as well. THAT would be justice—and it would lead to a decrease in criminal activity! Burt, also taking issue with parole as a means to ensure a more compassionate death, draws out concern for “moral decay” in more explicit terms: “Sorry…no ‘compassionate’ parole…the people they’ve hurt or killed do not get any compas- sion…So sick and tired of this country coddling criminals…They’re criminals—we should be coddling the victims whose lives they’ve ripped apart…This country is going to hell in a hand basket pretty darn fast…” Overall, where comments allude to or reference capital punishment, it is generally to imbed MAiD in a regime of punishment, bringing out its punitive or violent features or to encourage the movement to restoring the death penalty. https://doi.org/10.1017/cls.2022.8 Published online by Cambridge University Press Pain as Punishment Both where capital punishment is raised and where it is not, prisoners’ bodies tend to occupy a central place in commenters’ conversation. The centrality of the body is consistent with Alan Hyde’s (1997) argument that the human body ﬁgures signif- icantly in legal imagination. The body is simultaneously an object of regulation and a site of experience, which together mediate legal sensibilities (Hyde 1997). Further, as Roxanne Mykitiuk (1994, 84) argues, the body and its corporeal matters possess a certain “recalcitrance”—a capacity for agency or aﬀection of its own—that shapes, while it is concurrently reconstructed in, law (also see Shaw 2021; Shaw and Mykitiuk 2022). Insights about law and the body have aided scholars like Sarat (2001) in attending to the sensibilities that shape punishment and the regulation of death. Following Robert Cover’s (1986, 1601) observation that “[l]egal interpreta- tion plays on a ﬁeld of pain and death,” Sarat (2001) and colleagues (e.g., Kaufman- Osborn 2001) argue pain, speciﬁcally the body in pain, is uniquely generative of sensibilities that sustain (or, potentially, challenge) diﬀerent forms of state lethality. For example, the body in pain can be thought of as a record of state violence, whose existence can channel and magnify pleasure among those spectating and those invested in the continuation of these violent practices. Alternatively, pain signals an excess of state violence that demands others’ empathy (Hyde 1997); or pain, like death, is a (ontological) limit to law’s dominion that necessarily engenders resis- tance, to desperately seek escape from under the weight of the state’s preferred nomos (Cheah and Grosz 1996; Cover 1986). Pain and death are the body’s recalcitrance, which “live in and through various institutions and their linguistic practices, institutions and practices that are historically and culturally situated” (Sarat 2001, 7; also see Garland 2011). The body in pain starts to emerge in the comments where MAiD is constructed as an escape of punishment. Anything less than the complete duration of the sentence is an erosion of justice; the prisoner must stay in custody, ideally suﬀering the deprivation of comforts and rights that protect the body from pain. Further, the sentence must be carried out to its end despite a pain-wracked prisoner’s MAiD request, so even where pain is not explicitly acknowledged, it is implied. The Spectre of Capital Punishment Spectres of capital punishment thereby haunt conversation, cultural traces appear- ing in and shaping everyday sensibilities as commenters reconcile MAiD and punishment, and their image of the government, society and the CSC policy that operationalizes this “civilized” form of prisoner death. 461 When Prisoners’ “Right to Die” Goes Online Pain as Punishment For example, Angela characterizes MAiD and capital punishment as a metaphorical escape: “If it is a murderer who showed no compassion to their victims you believe they should be allowed to escape by a compassionate comfy death? I’m against the death penalty for that reason.” Likewise, Jeﬀis dismayed by MAiD cutting sentences short; Hugh considers MAiD tantamount to freedom, to which prisoners should not be entitled; and Teddy refers to MAiD as an escape from punishment, although his opposition to state lethality, generally, creates additional discomfort: I think that’s an escape route that should be banned. The prisoners lost the right to make that kind of determination when they were sentenced for the crime. It’s also a step along a very nasty slippery slope. I don’t want the state involved in killing prisoners, even letting them die is somewhat disturbing… https://doi.org/10.1017/cls.2022.8 Published online by Cambridge University Press Joshua D. M. Shaw and Daniel Konikoﬀ 462 Jack thinks punishment is best achieved through a “natural” death in prison, with which MAiD would interfere: “Why would anyone support assisted death for prisoners? They are in for life and need go by natural causes. They will get oﬀto [sic] easy with assisted death. We should have a referendum on this issue with the next federal election by putting this on a ballot to check yes or no.” y g y Jack’s invocation of referendum echoes death-penalty retentionists’ arguments during the parliamentary debate of Bill C-84, conﬁdent that a popular vote mirrors his desire. Elsewhere, his desire is expressed nakedly when he writes, “Leave these criminals alive so they can suﬀer their sentence until they die.” The presence of pain seems meaningful to such commenters “via its incorpo- ration within a cosmological narrative,” as Timothy Kaufman-Osborn (2001, 78– 79) describes it, with respect to the commenters’ proper place on earth: ordered, happy, and sovereign above an invasive and depraved Other. This is suggested by comments, like Beverly’s, that portray prisoners accessing MAiD outside the prison as a compromise to their duly deserved sentence: a “bending” and “changing” of punishment by allowing the prisoner Other to move outside the penitentiary among a non-criminal population. “This option needs to be preformed [sic] in the prison…enough bending, changing the sentencing. https://doi.org/10.1017/cls.2022.8 Published online by Cambridge University Press Pain as Punishment There are medical people in prisons…use them.” As noted earlier, Burt connects parole, which is to facilitate some prisoners with obtaining MAiD, to aﬄiction and decay: “[t]his country is going to hell in a hand basket pretty darn fast.” Terri—who elsewhere expresses anxiety about murderous criminals causing disorder and violating Judeo-Christian values—lets slip the role of race into these binaries. She also sees MAiD as another right aﬀorded to prisoners to the diminishment of incarceration: “They have even more rights than people on the outside paying for the service. Karla Homolka got free tuition and free language training in jail. Students on the outside graduate with huge debts. In the US, many inmates get a free tuition law degree so that they can challenge those who put them in jail for murder, etc.” j Further, dismayed with the state of society, Terri implies that punishment of the Other is not painful enough: “Palliative care, limited as it is, should be prioritised for non-criminals. Otherwise, why does anyone even bother to go to work and pay taxes if those at Club Fed get all the beneﬁts? We are the fools. I have already dropped out of this evil society and stick to my own demographic and will continue to do so.” In her many comments, Terri delineates between herself and others like her (“my own demographic”) and the “evil society” deﬁned by crime, disorder, and the transgression of Judeo-Christian values; her comments are suggestive of a racial- ized Other as distinct from and dangerous to law and order (Ahmed 1995). Terri’s desire for incarceration is inextricable from, as Henrique Carvalho and Anastasia Chamberlen (2018, 218) describe, the “pleasure of punishment […] directly linked to the speciﬁc kind of solidarity that punishment produces”: a white, lawful, and ordered society mediated through pain and punishment of the Other. The pain of prisoners is appropriated in these images as justice, which stands in contrast to law’s oﬃcial narratives historically in Canada (Strange 2001) and presently in the US, according to which capital punishment fulﬁlls “the sentimental When Prisoners’ “Right to Die” Goes Online 463 ideal of death that involves no pain” (Kaufman-Osborn 2001, 80; also see LaChance 2017). It stands in contrast to the abolition of corporal punishment as a necessary part in a civilized and modern Canada (Strange 2001), even if punishment continues to be experienced corporeally (Chamberlen 2018; Garland 2011). Pain as Punishment And it stands in contrast to how sentencing law, which treats the prison as a “black box,” renders prisoners’ corporeal experience unknowable, deferred to the remit of administrators (Kerr 2019). As Sarat (2001) notes, the retention of violence in punishment relies on its reconstruction as humane and controlled; the sentimental ideal emerges as a “civilizing process” to make punishment, including death, more civilized, painless, and implicated in projects of reform (Strange 2001). Pain, in modern medicalized discourse, is no longer anything but neuro-physical activity perceived by the mind; pain is not, as Christian theologies at times insisted, expressive of any moral economy or fundamental justice (Kaufman-Osborn 2001). The sentimental ideal exists in tandem with the disembodiment of the prisoner, so that punishment is understood to act principally on the body-less legal person, an immaterial subject of law, rather than through the body itself (Garland 2011; Kaufman-Osborn 2001). But the commenters turn the law’s oﬃcial narratives on their head. Pain obtains meaning again as punishment. And MAiD, to the extent it cannot be reconciled in commenters’ sensibilities as a means to eﬀect punishment, reﬂects an intrusion upon that pain. Refracting Sensibilities through the Body in Pain Alan Hyde (1997, 193) describes the “sentimentalized body” as a body that invites people to relate to each other, speciﬁcally by inviting someone to experience another’s suﬀering and pain as “a ﬁgure of empathy.” The “sentimentalized, empathized[-with] body” in pain thereby compels one to treat another as more than a disembodied or abstracted legal person (Hyde 1997, 195), potentiating diﬀerent relationships between people. In the alternative to the hostile comments so far documented, some commenters identify prisoners’ pain as similar to their own because of old age or illness, in this form of sentimentalized or empathetic relating. For example, Pete emphasizes that the prisoner is an individual in need of care, comparable to any individual outside the prison: “If an individual has a terminal disease and chooses medically assisted end of life care, who cares if the individual is in prison or not. The government should not interfere in the right of an individual to decide when to end their life when facing a terminal disease.” Glen similarly notes that prisoners should be entitled to a digniﬁed, painless death like anyone else: Hard not to agree to ANYONE’s wish for medical assistance in death (MAID) to end an agonized existence. Those with strong religious or moral beliefs are free to eliminate MAID from their own end of life situation…but, just as they are free to choose the option of their choice, they cannot impose their limitations on the choices available to others. https://doi.org/10.1017/cls.2022.8 Published online by Cambridge University Press For those who suggest palliative care can make MAID less desirable, while I (somewhat) agree with their view, it has to be recognized that the powerful drugs associated with palliative care cannot possibly be administered in a prison environment. I mean really, it would be like storing fresh meat in a tiger’s cage. 464 Joshua D. M. Shaw and Daniel Konikoﬀ For those who suggest palliative care can make MAID less desirable, while I (somewhat) agree with their view, it has to be recognized that the powerful drugs associated with palliative care cannot possibly be administered in a prison environment. I mean really, it would be like storing fresh meat in a tiger’s cage. 464 Joshua D. M. Shaw and Daniel Konikoﬀ 464 Joshua D. M. Refracting Sensibilities through the Body in Pain Shaw and Daniel Konikoﬀ Others, like Danny, frame the idea of prohibiting MAiD for prisoners as an extension of punishment: “There is no reason to stand in their way especially at this point in a prisoner’s life. If they are that sick, why prolong their punishment? It seems a tad extra cruel to me, we’re not that kind of society… are we?” And another: “Does one kind of pain cancel out another? The sentence itself is the punishment; anything beyond it is gratuitously cruel. I am not pleading leniency or clemency for the vast majority of criminals who, being healthy, are still a threat.” Keith writes: I don’t see any reason why inmates should not receive medical assistance to die if they meet all the criteria. To suggest otherwise is just unnecessarily cruel. And if necessary perform the procedure outside of prison under the hands of qualiﬁed medical professionals. You wouldn’t allow an inmate to die of appendicitis by denying treatment, so why would you deny one MAID? I don’t see any reason why inmates should not receive medical assistance to die if they meet all the criteria. To suggest otherwise is just unnecessarily cruel. And if necessary perform the procedure outside of prison under the hands of qualiﬁed medical professionals. You wouldn’t allow an inmate to die of appendicitis by denying treatment, so why would you deny one MAID? While the prisoner’s body in pain demands interrelation qua their experience as a patient, some commenters nonetheless construe prisoners as abstracted legal personalities. The recipient of MAiD is split into an embodied patient entitled to medical care and an imprisoned person who must be deprived of abstracted rights. This might necessitate for commenters that MAiD be provided in the prison, without parole. For example, one person writes, “I agree that the care be provided by a doctor and not prison staﬀand it should be carried out in a hospital or care center but I disagree it is a reason for parole. Let’s not forget these individuals in prisons carried out crimes, many violent, against innocent victims.” Jason writes: However we are not talking [about] the diﬀerence between suicide and MAID. MAID is only given to a terminally ill patient. It is not like the other inmates can talk a regular person into going to ask for MAID. https://doi.org/10.1017/cls.2022.8 Published online by Cambridge University Press Refracting Sensibilities through the Body in Pain It has to go through the same screening process style that anyone other person in normal public would have to go through. However I don’t believe the terminally ill person requesting the MAID having a life sentence should get the beneﬁt of tasting freedom. Even for a short time period. IMO the public hospital beds should be kept for those who have not broke[n] the law. In other cases, parole is understood as a legitimate option for a prisoner, where the prisoner’s release is appropriate. This is especially so where the prisoner is understood as frail or weak due to their intolerable suﬀering, thereby posing minimal risk to the public. For example, Philip writes: If these inmates are no threat to society I do not see why the Correctional Services do not have their own palliative care facility where the oﬀender and their family can be together to ease the death for everyone. https://doi.org/10.1017/cls.2022.8 Published online by Cambridge University Press 465 When Prisoners’ “Right to Die” Goes Online There would be little physical security required, it would be more like a remote hospices with facilities for family or friends to stay and be with the person. There would be little physical security required, it would be more like a remote hospices with facilities for family or friends to stay and be with the person. There would need to be an assessment of risk in each case, as anyone can cause harm and act violently regardless how ill they appear to be. Also this facility would not be an option to prison where the prisoner may live for several months, potentially years, though it could be a step to a parole to community care if it is available. Assisted dying should not be a protracted issue for anyone, when a person asks for help in dying and meets all the acceptable conditions to be allowed to do so, the entire process should take no more than days or hours from that point. One assumes the prisoner’s relatives have been involved in the process as soon as the person asks for help in dying. Elsewhere, in reply to someone else, Vern writes, “Wow! It’s like you didn’t even read the article. https://doi.org/10.1017/cls.2022.8 Published online by Cambridge University Press Refracting Sensibilities through the Body in Pain They’re talking about inmates who are too sick and frail to get out of bed, They’re talking about releasing them to palliative care centres where they would be heavily sedated to manage their pain. So you REALLY think they could pose any risk?” These commenters’ sensibilities perhaps share most with law’s oﬃcial narratives of the legal subject, incarceration, and the ideal candidate for MAiD, even though their sensibilities involve relating to the prisoner’s empathized- with body. For example, these commenters treat pain as irreconcilable with punishment; pain exceeds what these commenters expect of the prisoner’s sentence, which is instead thought to reﬂect an archaic form of retributive justice. Further, prisoners, like others who age or become ill, are seen as entitled to a painless, digniﬁed death. As Victoria states, inasmuch as the person is an inmate, punishment should be exacted against the prisoner as a legal subject through the deprivation of rights like freedom of movement. But as a patient, the prisoner’s pain is intelligible as a terminal or otherwise grievous and irremedi- able condition, which entitles the prisoner to MAiD like any other patient. As a patient, the prisoner becomes, as Hyde (1997, 199) described, a “sympathetic body” “that is the uniquely diﬀerentiated home of a unique human person, the body that is the sole medium through which that person has a world, relates to others, others who can enter relations with that person precisely because they feel that body’s pain.” These commenters appear to split the prisoner into diﬀerent legal persons (i.e. the inmate and the patient) and engage concurrently in a selective presencing and absencing of the body (Leder 1990) with respect to these diﬀerent personalities. Spatially and temporally, the body in pain is presenced within the immediate bounds of the patient entitled to MAiD, whose mental decision-making about bodily integrity is prioritized unless they are a class of person for whom this legal personality cannot be convincingly maintained. The prisoner’s pain as an inmate, which potentially underlies or contributes to the prisoner’s desire for MAiD, is absenced when the commenter identiﬁes the prisoner as a patient. In other words, by disembodying the prisoner qua their status as an inmate, and attributing pain to Joshua D. M. Refracting Sensibilities through the Body in Pain Shaw and Daniel Konikoﬀ 466 the medical condition for which MAiD is sought, these commenters selectively relate to the prisoner as a patient, averting their eyes from the active involvement of incarceration in acting on the body (Chamberlen 2018). A “civilized,” “non- carceral” death for prisoners by MAiD thereby becomes permissible through the alternation of embodiment and disembodiment in discourse, aﬀecting how com- menters relate to the prisoner’s body. Commenters can thereby maintain the notion of legal personality vital to liberal legal thought on the subject of rights and incarceration generally, as well as with respect to their perception of the ideal candidate for MAiD. https://doi.org/10.1017/cls.2022.8 Published online by Cambridge University Press Conclusion: Aesthetic Mediations Contemporary capital punishment has generally become like MAiD, according to Girling (2016, 356), in that both are emblematic of “[the] search for digniﬁed and painless death,” “in which the taking of life ‘assume[s] the character of a depoliticized humanitarian (non) event.’” As a result of decades of civilizing processes, the death penalty has become a form of death like the slow deaths of prisoners generally, and so to When Prisoners’ “Right to Die” Goes Online 467 spectacularize the execution focuses attention on the botched death, the means of execution, or ﬁdelity to procedure, as opposed to the lethal structure of carceral institutions. From Girling’s (2016) perspective, the non-spectacular death of contemporary capital punishment and slow death of incarceration generally require diﬀerent sensibilities and diﬀerent strategies than those enabled by these deaths’ ﬂeeting spectacularization (e.g., legal challenges of capital punishment) if abolitionists are to succeed in putting a stop to prisoners “doing life” (and “doing death”) behind bars (Girling 2016, 358). Our case study similarly demonstrates the aesthetic mediation of ethical judgement: a non-propositional form of ethics emerging in a place and time inﬂected by certain penal and legal sensibilities. But our observation of MAiD in the context of the comment section diﬀers from what Girling takes for granted in her comparison of capital punishment and MAiD. Girling treats MAiD as a non- event, which serves as an analogue to contemporary capital punishment. But for commenters, the provision of MAiD to prisoners is not a non-event. Discussing MAiD for prisoners appears to implicate a speciﬁc set of relations between the commenter and prisoner, which reposition the commenter in relation to the lethality of the prison. Generally, prisoners, old and young, die in prison without stirring aﬀections among the public. These deaths go unnoticed, uncared for and un-mourned. Prisoners die “slowly.” But by discussing the CSC’s policy, dying and pain in the prison take on diﬀerent meanings, exempliﬁed by the range of sensi- bilities expressed in the comment section. The deaths become noticed, cared for and mourn-able, or in place of mourning, become desirable. Commenters tend to relate with, or lean into, the prisoner they imagine in pain or death, which appears to spectacularize the prisoner’s death by MAiD. p p y In the case of the comment section, the bodies of prisoners (and of antici- pated or actual victims) play a fundamental part in staging MAiD. Conclusion: Aesthetic Mediations Evi Girling and Lizzie Seal (2016, 269) characterize prisoners experiencing “life without parole” as “sentenced to slow death by imprisonment, with no one ‘deciding death,’ no technologies of death, no rituals of execution.” Slow death is “the physical wearing out of a population and the deterioration of people in that population that is very nearly a deﬁning condition of their experience and historical existence” (Berlant 2007, 754). The slow death of incarceration can be contrasted with capital punishment, which often entails—especially historically—a diﬀerent approach of the state to the death caused. Historically, capital punishment entailed the spectacularization of death, where the state-power that eﬀected death was conﬁgured through the sovereign who made highly visible life-and-death decisions over subjects (Foucault 1977; Povinelli 2009). To some extent, this spectacular death persists in contemporary examples of capital punishment, namely in the United States, even though death has been “humanized” through the “medicalised aesthetics” of lethal injection (Girling 2016, 355). Namely, legal challenges to capital punishment, and the “ensuing spectacle of mitigation, delay, mercy (and its denial)” (Girling 2016, 354) can re-focus penal sensibilities among the US public on capital punishment. The possibility of death being slow or spectacular (or becoming one or the other) matters in the formation of ethical judgements, in part because the character or quality of a particular death has consequences for the framing of responses to an injustice. The manner in which a person is made or allowed to die ﬂows from the condensation of power relations implied aesthetically in the character or quality of death. For example, Girling (2016, 356) argues that the anaesthetization and medicalization of death in the “staging of modern executions” renders the spectacular appearance of prisoners’ pain in discourse “meaningless.” By meaningless, Girling is describing how a popular discourse intermittently consumed by the pains of capital punishment is ill equipped to conceptualize the slow violence of the state that an anaesthetized and medical- ized execution has become part of. https://doi.org/10.1017/cls.2022.8 Published online by Cambridge University Press Conclusion: Aesthetic Mediations Prisoners’ bodies exist in commenters’ imaginations as overﬂowing in pain that invites culturally formative relations between commenters and prisoners, commenters and victims, and commenters and prisoners’ deaths. Such relations enable a range of interpretations and ethical judgements for commenters; but in the time and place of this comment section, this tended to be realized in at least two diﬀerent ways. On the one hand, relating fomented sensibilities amenable to hate, revulsion, and a desire to kill, evidenced in the hostility of some comments. Signiﬁcantly, the spectre of capital punishment and its spectacularized image of the body in pain materialized in these kinds of comments. Commenters’ sensi- bilities could alternatively be redirected through empathetic relations, rendering prisoners’ deaths and state lethality more humanized to commenters. These generally operated through a mode of presencing and absencing the body, prioritizing the prisoners’ sentimentalized, empathetic body in pain as a patient over pains of imprisonment. Both modes of relating to prisoners enabled commenters to take notice, in their imagination, of prisoners’ dying by MAiD. What might otherwise be a non-event— a medicalized procedure undertaken to eliminate meaningless, debilitating pain— was transformed into a distinctive event in the process of its spectacularization. Whether MAiD was viewed as advancing capital punishment, diminishing the pain Joshua D. M. Shaw and Daniel Konikoﬀ 468 that should be suﬀered in punishment, or responding to the pains delimited as satisfying the patient’s status as the ideal patient, the prisoner’s body formed part of commenters’ ethical judgements, aﬀecting how the prisoner, their death, and the circumstances of their incarceration came to matter to the question of justice at stake. Commenters seemed to dismiss other prisoners, pains, or deaths that were incompatible with the bodies they imagined were eligible for MAiD. Further, although we can only oﬀer this comment speculatively in the absence of any comparative data, the medium of the comment section and its placement under a news article seemed to amplify its spectacularization. The reductive framing of the policy in the article (as opposed to structural histories of incarceration), the dramatization of polemic discourse between commenters (e.g., Liberals versus Conservatives), and ephemeral signiﬁcance (e.g., the attention of commenters is short as novel news stories come out and shift discourse) likely reinforced shallow engagement ill-suited to the challenge of identifying and responding ethically to prisoners’ deaths. https://doi.org/10.1017/cls.2022.8 Published online by Cambridge University Press Conclusion: Aesthetic Mediations Accordingly, irrespective of how commenters imagined pris- oners eligible for MAiD, slow deaths under incarceration were left unnoticed, uncared for and un-mourned in their discussion. The “civilized,” humanitarian form of death oﬀered by MAiD deﬂects from the pains and deaths that prisoners otherwise endure, leaving intact incarceration’s lethality. It is conceivable that the discussion of MAiD for prisoners could take place without the spectacularization of death, allowing for commenters to consider both the pain caused by prisoners’ medical conditions and that eﬀected by the slow death of incarceration, and frame their commentary on legal rights and entitlements accordingly. The presencing of the prisoner, as both an inmate and a patient, in addition to their other relations, could open the commenter to multiple deaths caused: the death which is slow according to structural conditions of incarceration, life course and medical illness, and that which occurs acutely. This concurrent presencing would seem to complicate the sensibilities by which commenters approached the question of MAiD for prisoners, inviting critiques of incarceration itself and consideration of less harmful alternatives, as well as attention to those structures that take eﬀect over a longer durée. g Deeper interrogation of foundational assumptions might thereby enable a more equitable study of MAiD for prisoners, as well as incarceration. Consistent with the cultural approach to penality and legality we have undertaken, such an interroga- tion should attend to how referents and aﬀects are made in a given place and shape discourse and social action, allowing the commenter to take careful, slow and stumbling steps with others in the deliberation over foundational assumptions. But penologists and legal scholars ought to take online sites like comment sections seriously, and not wish them away simply because their content may be discomﬁting or toxic. Comment sections are venues for meaning making, which have been known to aﬀect the formation of public opinion, and individual and collective behaviour. Further, commenters in online fora take form within our cultural milieu and so the conditions under which their sensibilities emerge must have some connection to our oﬄine worlds, even if digital infrastructures allow for and facilitate diﬀerences in the expression of penal and legal sensibilities. Conclusion: Aesthetic Mediations To When Prisoners’ “Right to Die” Goes Online 469 examine foundational assumptions without regard for the contribution of online fora, where we increasingly spend more time and where so many of our interactions are mediated, risks excluding formative dimensions of social life. For that reason, examining commenters’ discussion of MAiD for prisoners serves as an important case study of legal and penal sensibilities, bringing us closer to appreciating how individuals can orient to prisoners’ deaths generally, and with respect to MAiD speciﬁcally. The comment section demonstrates how penal and legal meaning can be made, as well as the eﬀects of these meanings upon ethical judgement. https://doi.org/10.1017/cls.2022.8 Published online by Cambridge University Press References Ahmed, S. 1995. Deconstruction and law’s other: Towards a feminist theory of embodied legal rights. Social and Legal Studies 4 (1): 55–73. Berlant, L. 2007. Slow death (sovereignty, obesity, lateral agency). Critical Inquiry 33 (4): 754–780. Berinsky, A. J. 2017. Measuring public opinion with surveys. Annual Review of Political Science 20:309–29. Brinkmann, S. 2014. Doing without data. Qualitative Inquiry 20 (6): 720–25. Bond, R., and S. Messing. 2015. Quantifying social media’s political space: Estimating ideology from revealed preferences on Facebook. American Political Science Review 109:62–78. Carvalho, H., and A. Chamberlen. 2018. Why punishment pleases: Punitive feelings in a world of hostile solidarity. Punishment and Society 20 (2): 217–34. Chamberlen, A. 2018. Embodying Punishment: Emotions, Identities, and Lived Experiences in Women’s Prisons. Oxford University Press. Cheah, P., and E. Grosz. 1996. The body of the law: Notes toward a theory of corporeal justice. In Thinking Through the Body of the Law, ed. P. Cheah et al., 1–26. NYU Press. CSC (Correctional Service of Canada), Guideline 800-9, Medical Assistance in Dying (11 November 2017), online: https://www.csc-scc.gc.ca/acts-and-regulations/800-9- gl-en.shtml. Cotterrell, R. 2018. Theory and values in socio-legal studies. Journal of Law and Society 44 519–36. Cover, R. 1986. Violence and the Word. The Yale Law Journal 95 (8): 1601–29. Davies, M. 2015. The Consciousness of Trees. Law and Literature 27 (2): 217–35 Downie, J., A. Iftene, and M. Steeves. 2019. Assisted dying for prison populations – Lessons from and for abroad. Medical Law International 19 (2–3): 207–25. Ewick, P., and S. Silbey. 1998. The common place of law: Stories from everyday life. Chicago: University of Chicago Press. Foucault, M. 1977. Discipline and punish: The birth of the prison. New York City: Vintage Books. Frost, N. A. 2010. Beyond public opinion polls: Punitive public sentiment and criminal justice policy. Sociology Compass 3/4:156–68. Garland, D. 2011. The problem of the body in modern state punishment. Social Research 78 (3): 767–98. Garland, D. 2006. Concepts as culture in the sociology of punishment. Theoretical Crimi- nology 10 (4): 419–47. Garland, D. 2005. Capital punishment and American culture. Punishment and Society 7 (4): 347–76. https://doi.org/10.1017/cls.2022.8 Published online by Cambridge University Press 470 Joshua D. M. Shaw and Daniel Konikoﬀ Garland, D. 1990. Punishment and modern society: A study in social theory. Chicago: University of Chicago Press. Girling, E. 2016. References Sites of crossing and death in punishment: The parallel lives, trade-oﬀs and equivalences of the death penalty and life without parole in the US. John Howard Journal of Crime and Justice 55 (3): 345–61. Girling, E., and Lizzie Seal. 2016. Introduction: The deathscapes of incarceration. John Howard Journal of Crime and Justice 55 (3): 267–77. Harris, Kathleen. 2018. Watchdog calls for ‘compassionate’ parole as prison system adopts new assisted death policy. CBC News, 28 February. https://www.cbc.ca/news/politics/ terminally-ill-inmates-csc-zinger-maid-1.4546773 y g Hyde, A. 1997. Bodies of Law. Princeton University Press. Iftene, A. 2018. Punishment for aging: Older federal prisoners and the challenge to the Canadian correctional and legal landscape. University of Toronto Press. Iftene, A., and J. Downie. 2020. End-of-life care for federally incarcerated individuals. McGill Journal of Law and Health 14 (1): 1–50. Kaufman-Osborn, T. V. 2001. What the law must not hear: On capital punishment and the voice of pain. In Pain, Death and the Law, ed. Sarat, A, 71–102. University of Michigan Press. Kerr, L. 2019. How the prison is a black box in punishment theory. University of Toronto Law Journal 69 (1): 85–116. Konikoﬀ, D. 2021. Gatekeepers of toxicity: Reconceptualizing twitter’s abuse and hate speech policies. Policy and Internet 13:502–21. LaChance, D. 2017. Executing humanity: Legal consciousness and capital punishment in the United States, 1915–1940. Law and History Review 35 (4): 929–76. Leder, D. 1990. The absent body. Chicago: University of Chicago Press. Lee J., and A. Nerghes. 2018. Refugee or migrant crisis? Labels, perceived agency, an sentiment polarity in online discussions. Social Media þ Society 4 (3): 1–22. Mykitiuk, R. 1994. Fragmenting the body. Australian Feminist Legal Studies 2 (1): 63–98 Neale, J. 2016. Iterative Categorization (IC): A systematic technique for analysing qualitativ data. Addiction 111 (6): 1096–1106. OSC (Oﬃce of the Correctional Investigator), Annual Report 2019–2020 (26 June 2020). https://www.oci-bec.gc.ca/cnt/rpt/annrpt/annrpt20192020-eng.aspx. Povinelli, E. A. 2009. The child in the broom closet: States of killing and letting die. In States of Violence: War, Capital Punishment and Letting Die, ed. A. Sarat and J. Culbert, 169– 91. University of Cambridge Press. Prichard, J., P. Watters, T. Krone, C. Spiranovic, and H. Cockburn. 2015. Social media sentiment analysis: A new empirical tool for assessing public opinion on crime?. Current Issues in Criminal Justice 27 (2): 217–36. Sarat, A. 2001. Introduction: On pain and death as facts of legal life. In Pain, Death and the Law, ed. A. Sarat, 1–14. References University of Michigan Press. Shaw, D. M., and B. S. Elger. 2016. Assisted suicide for prisoners? Stakeholder and prisoner perspectives. Death Studies 40 (8): 479–85. Shaw, J., and P. Driftmier. 2021. Dying with a smile, just knowing that somebody’s listened to me: End-of-life care and medical assistance in dying in Canadian prisons. OMEGA – Journal of Death and Dying. https://doi.org/10.1177/00302228211052341. Shaw, J. D. M. 2021 The spatio-legal production of bodies through the legal ﬁction of death. Law and Critique 32 (1): 69–90. Shaw, J. D. M., and R. Mykitiuk. 2022. Jurisgenerative tissues: Sociotechnical imaginaries and the legal secretions of 3D bioprinting. Law and Critique. https://doi.org/10.1007/ s10978-022-09319-0 https://doi.org/10.1017/cls.2022.8 Published online by Cambridge University Press When Prisoners’ “Right to Die” Goes Online 471 Silbey, S. 2018. Studying legal consciousness: Building institutional theory from micro data. Droit et société 100 (3): 685–731. Smith, M., R. Sparks, and E. Girling. 2001. Educating sensibilities: The image of “the lesson” in children’s talk about punishment. Punishment and Society 2 (4): 395–415. Stensland, M., and S. Sanders. 2016. Detained and dying: Ethical issues surrounding end-of- life care in prison. Journal of Social Work in End-of-Life and Palliative Care 12 (3): 259–76. Strange, C. 2001. The undercurrents of penal culture: Punishment of the body in mid- twentieth century Canada. Law and History Review 19 (2): 343–85. Strange, C. 1995. “The lottery of death”: Capital punishment, 1867–1976. Manitoba Law Journal 23:594–619. Tavory, I., and S. Timmermans. 2019. Abductive analysis and grounded theory. In The SAGE Handbook of Current Developments in Grounded Theory, ed. A. Bryant and K. Char- maz, 532–46. SAGE. Joshua D. M. Shaw Osgoode Hall Law School, York University joshuashaw@osgoode.yorku.ca Daniel Konikoff Centre for Criminology and Sociolegal Studies, University of Toronto daniel.konikoff@mail.utoronto.ca Joshua D. M. Shaw Osgoode Hall Law School, York University joshuashaw@osgoode.yorku.ca Daniel Konikoff Centre for Criminology and Sociolegal Studies, University of Toronto daniel.konikoff@mail.utoronto.ca Daniel Konikoff Centre for Criminology and Sociolegal Studies, University of Toronto daniel.konikoff@mail.utoronto.ca https://doi.org/10.1017/cls.2022.8 Published online by Cambridge University Press
https://openalex.org/W4286579690	https://gfzpublic.gfz-potsdam.de/pubman/item/item_5013448_2/component/file_5013503/5013448.pdf	English	null	An SVM-Based Snow Detection Algorithm for GNSS-R Snow Depth Retrievals	IEEE journal of selected topics in applied earth observations and remote sensing	2,022	cc-by	6,853	I. INTRODUCTION Abstract—The signal-to-noise ratio (SNR) is important observa- tions in global navigation satellite system-reﬂectometry (GNSS-R) technology. The oscillation frequency in the SNR arc is sensitive to different reﬂecting surfaces and can be used to build height model to track the variation of snow depth. However, it is difﬁcult to obtain retrieval results with snow depth of zero in the actual snow depth re- trieval experiments based on GNSS-R technology, which indicates that the classical model has nonnegligible retrieval errors in the snow-free state. This study aims to realize the detection of ground truth information before snow depth retrieval, i.e., classiﬁcation of snow-free state and snow-covered state. Machine learning was introduced to achieve the aforementioned purpose and the SNR arc was used as the input data. Compared with the current mainstream topography correction algorithms, the algorithm proposed in this study does not rely on any priori ground measured data and has theoretical universality. The detection results can constrain the retrieval snow depth in the snow-free state and, thus, improve the retrieval accuracy. The experimental results for the 2014 seasonal snowpack at P351 station in Idaho, USA, show that the detection results obtained based on support vector machines agree well with the measured snow depth provided by the SNOTEL network, and the overall detection accuracy can reach about 96%. The daily snowpack state is determined by the majority of SNR arcs detected during the day and is only considered reliable if the percentage exceeds 75%. Only one day of the detection results was inaccurate and only 8 days (8/365) did not reach the set threshold of 75%. With the help of the detection results, the root-mean-square error of snow depth retrieval can be reduced from 20 cm in the classical algorithmto15cm,whichresultsina25%improvementinretrieval accuracy. Moreover, this study broadens the application value of GNSS signals and provides a reference for the application of SNR in the detection ﬁeld. S S NOW is one of the important sources of water resources and a sensitive and important factor in climate change. Therefore, monitoring snowpack is of great signiﬁcance to climate prediction, hydrological research, and snow disaster prevention. Affected by topography, snowpack also exhibits great spatial variability. In addition, snowpack ablation is also nonuniform due to temperature, wind, and radiation [1]. Usually, snow data can be obtained from ground stations and satellite remote sensing. 6046 6046 IEEE JOURNAL OF SELECTED TOPICS IN APPLIED EARTH OBSERVATIONS AND REMOTE SENSING, VOL. 15, 2022 Yuan Hu, Xintai Yuan , Wei Liu , Qingsong Hu, Jens Wickert , and Zhihao Jiang Yuan Hu, Xintai Yuan , Wei Liu , Qingsong Hu, Jens Wickert , and Zhihao Jiang I. INTRODUCTION The snowdrift telemetry (SNOTEL) network [2], with nearly a thousand stations distributed in the United States, can provide data, such as snow depth, snowfall, and snow water equivalent (SWE). However, the measurement area of each site is only about 9 m2, which cannot adequately express the current status of in-situ snowpack. Besides, other classical snow monitoring methods, such as satellite remote sensing [3] and synthetic aperture radar [3], have certain limitations, such as spatial and temporal resolution, high cost, and susceptibility to external environmental inﬂuences. Under these conditions, there is a demand for a method that can measure large areas of snowpack at low cost to complement snow monitoring. Global navigation satellite system-reﬂectometry (GNSS-R) technology is a new branch of GNSS that has been gradually developed since the 1990’s and is also one of the research hot spots in the ﬁeld of remote sensing. Multipath signals are often suppressed as a source of error in high accuracy applications [4]. However, multipath signals actually carry much geometric and physical information from the reﬂecting surface that can be used in GNSS-R technology. The signal-to-noise ratio (SNR) data received by the GNSS antenna are formed by the interference of the direct and reﬂected signals and is a measure of the signal strength.Meanwhile,theSNRdataaretheimportantobservation for GNSS-R technology. Martin-Neira [5] revealed the inter- ference phenomenon between the direct satellite signal and the reﬂected signal. Bilich and Larson [6] further proposed the SNR power spectral maps for multipath evaluation and, thus, found a mapping relationship between SNR and multipath environment. It was also shown that the oscillation frequency in a robust SNR time series is a function of the distance to the reﬂected object. Jacobson [7] also demonstrated that GPS signal power correlates well with snow depth and can be used to track the variability of snow depth. Larson et al. [8] tried to retrieve the snow depth throughtheSNRdataofthestandardGPSreceiveronthebasisof the previous research, in preparation for the SWE measurement. Since then, the GNSS-R snow depth retrieval technology based Index Terms—Detection, global navigation satellite system- reﬂectometry (GNSS-R), signal-to-noise ratio (SNR), snow depth, support vector machine (SVM). Manuscript received 21 May 2022; revised 10 July 2022; accepted 19 July 2022. Date of publication 22 July 2022; date of current version 3 August 2022. Manuscript received 21 May 2022; revised 10 July 2022; accepted 19 July 2022. Date of publication 22 July 2022; date of current version 3 August 2022. This work was supported in part by the National Natural Science Foundation of China under Grant 52071199, in part by the Shanghai Natural Science FoundationunderGrant19ZR1422800,andinpartbytheNationalKeyResearch and Development Plan under Grant 2019YFD0901303. (Yuan Hu and Xintai Yuan are co-ﬁrst authors.) (Corresponding author: Wei Liu.) This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ Yuan Hu, Xintai Yuan, Qingsong Hu, and Zhihao Jiang are with the Shang- hai Ocean University, Shanghai 201306, China (e-mail: y-hu@shou.edu.cn; xt-yuan@foxmail.com; qshu@shou.edu.cn; 1512330397@qq.com). Wei Liu is with the Merchant Marine College, Shanghai Maritime University, Shanghai 201306, China (e-mail: liu@sreil.com). HU et al.: SVM-BASED SNOW DETECTION ALGORITHM FOR GNSS-R SNOW DEPTH RETRIEVALS 6047 Fig. 1. Classical GNSS-R snow depth retrieval model. (a) Diagram of the geometric relationship of GNSS-R altimetry. (b) Model predictions for GPS S1C multipath from snow-free state (black) and snow-covered state (red). on SNR data has received wide attention [9]–[13]. The existing classical snow depth retrieval model based on SNR data is a height model obtained by empirical derivation. In principle, the height model uses the oscillation frequency to solve for the reﬂector height, i.e., the distance from the antenna to the reﬂecting surface. However, it is difﬁcult to see retrieval results with a snow depth of zero in the actual snow depth retrieval experiment. In other words, the classical model cannot or is difﬁcult to detect snow-free state, and even the retrieval results will be affected by the topography and produce immeasurable errors between the measured snow depth [14]–[17]. Currently, most topography correction methods are based on known a priori information. Tabibi et al. [18] proposed to attenuate the effect of partial topography bias by changing the ﬁxed reﬂector height reference to improve the overall accuracy. Li et al. [19] used the antenna phase center correction model to redeﬁne the antenna height to reduce the effect of topography slope on the retrieval results. Although modifying the reﬂector height reference can improve the retrieval accuracy, the effect of topography correction is limited. The correction of snow-free states does not seem to be suitable for snow-covered states, and topographycorrectionreliesonknowntopographymeasurement data [20]. In this study, we still focus on the retrieval error in the snow- free state, but the focus is not on the antenna height instead on detecting the snow state on the ground. As the existing studies indicate, there is a difference between the SNR arcs collected in snow-free state and snow state. We propose to introduce support vector machine (SVM) and use SNR arcs as input samples to detect the ground truth state. When the detection result is snow- free state, the snow depth is zero by default, otherwise the snow depth is retrieved with the help of classical algorithms. This article is structured as follows. Section II introduces the snow depth detection method based on SVM. Next, in Section III, experimental validation and discussion are presented. Finally, Section IV concludes this article. Fig. 1. Classical GNSS-R snow depth retrieval model. (a) Diagram of the geometric relationship of GNSS-R altimetry. HU et al.: SVM-BASED SNOW DETECTION ALGORITHM FOR GNSS-R SNOW DEPTH RETRIEVALS (b) Model predictions for GPS S1C multipath from snow-free state (black) and snow-covered state (red). frequency, in hertz. The strength of the SNR depends on the shape and dielectric constant of the reﬂector, which indicates that there are differences in the SNR received by the GNNS antenna for different reﬂector objects [23], [24]. The oscillation frequency f can often be used to track changes in snow depth. The geometric relationship of the established height model is schematically shown in Fig. 1(a), where H is the antenna height, and h is the reﬂector height, i.e., the distance from the antenna to the reﬂecting surface. The relationship between frequency and reﬂector height can be expressed as I. INTRODUCTION This work was supported in part by the National Natural Science Foundation of China under Grant 52071199, in part by the Shanghai Natural Science FoundationunderGrant19ZR1422800,andinpartbytheNationalKeyResearch and Development Plan under Grant 2019YFD0901303. (Yuan Hu and Xintai Yuan are co-ﬁrst authors.) (Corresponding author: Wei Liu.) Yuan Hu, Xintai Yuan, Qingsong Hu, and Zhihao Jiang are with the Shang- hai Ocean University, Shanghai 201306, China (e-mail: y-hu@shou.edu.cn; xt-yuan@foxmail.com; qshu@shou.edu.cn; 1512330397@qq.com). Wei Liu is with the Merchant Marine College, Shanghai Maritime University, Shanghai 201306, China (e-mail: liu@sreil.com). Jens Wickert is with the Department of Geodesy, German Research Centre for Geosciences, 14473 Potsdam, Germany, and also with the Institute of Geodesy and Geoinformation Science, Technische Universität Berlin, 10623 Berlin, Germany (e-mail: wickert@gfz-potsdam.de). Digital Object Identiﬁer 10.1109/JSTARS.2022.3193113 Digital Object Identiﬁer 10.1109/JSTARS.2022.3193113 II. SNOW DETECTION ALGORITHM BASED ON SVM As the GNSS signal is gradually expanding its coverage, a large number of geodetic receivers are being installed. Since 1994, the International GNSS Service (IGS) Center has made publicly available high-quality GNSS data and products [21] that have been used in a variety of ﬁelds, such as earth science research. Besides, the EarthScope Plate Boundary Observation (PBO, http://pbo.unavco.org) program has built hundreds of permanent GPS stations and can provide GPS observations with 15-s temporal resolution. The receivers record observations, including SNR, which can be decomposed into a slowly varying trend term and a high-frequency varying multipath oscillation term. Assuming a planar and leveled reﬂecting surface, the latter affected by multipath can be modeled as [22] f = 2h/λ (2) (2) where λ is the wave length. As shown in Fig. 1(b), it can be seen that the two SNR arcs collected from snow-free state and snow- covered state obviously show different oscillation frequencies. Actually, there are more differences between them, and machine learning was introduced to solve this problem. In view of the mapping relationship between SNR arcs and reﬂecting surfaces, SVM can be used to perform machine learning on the collected SNR arcs and then output the detection results to determine whether the reﬂecting surface is snow-free state or snow-covered state. SNR = A cos (f sin E + ϕ) (1) (1) where E is the satellite elevation angle; A is the amplitude; and ϕ is the phase delay between the direct and reﬂected signals. f is the oscillation frequency, which is not an ordinary temporal IEEE JOURNAL OF SELECTED TOPICS IN APPLIED EARTH OBSERVATIONS AND REMOTE SENSING, VOL. 15, 2022 6048 Fig. 2. Optimal classiﬁcation surface of SVM. Fig. 3. Introduction to the surrounding environment of the station. (a) Envi- ronment map of the northern area of P351 station. (b) Environment map of the northern area of Galena Summit station. Fig. 2. Optimal classiﬁcation surface of SVM. The SVM [25] is a machine learning algorithm for binary classiﬁcation of input samples in a supervised learning manner, and its decision boundary is based on the input samples using the structure minimization principle to establish the globally optimal maximum margin hyperplane, the principle of which is shown in Fig. 2. The blue circles and red squares represent differenttypesofsamples.ThesolidlineYrepresentstheoptimal hyperplane found, that is, the decision boundary, where w and b are the normal vector of the hyperplane and displacement terms, respectively. II. SNOW DETECTION ALGORITHM BASED ON SVM The dashed lines Y1 and Y2 are the interval boundaries of each type and margin is the classiﬁcation interval indicating the sample differentiation. Fig. 3. Introduction to the surrounding environment of the station. (a) Envi- ronment map of the northern area of P351 station. (b) Environment map of the northern area of Galena Summit station. As in practice, in most cases, the data are not linearly sep- arable, i.e., it is difﬁcult to distinguish the samples with a straight line in the two-dimensional plane. In this case, the hyperplane satisfying the condition simply does not exist, and SVM can solve the problem by introducing kernel functions [26]. Speciﬁcally, SVM ﬁrst completes the computation in the low-dimensional space, then maps the input space to the high-dimensional Hilbert space through the kernel function, and ﬁnally constructs the optimal separation hyperplane in the high-dimensional feature space, so as to separate the nonlinear data that are not well separated on the plane itself. The advantage of the kernel function is that it can be computed in low dimen- sions, whereas the substantial classiﬁcation effect is expressed in high dimensions, avoiding the explicit computation of the inner product in high dimensions. Considering the complexity of the data in this article and the generality of the kernel function, the radial basis kernel (RBF) function is selected A. Station Introduction and Data Selection The P351 station in Blaine County, ID, USA (43.87441 °N, 114.71916 °W, Elevation 2692.6 m) is one of the observa- tion stations of the PBO network and includes a standard TRIMBLE NETRS receiver and Trimble TRM 29659.00 an- tenna. The antenna is about 2 m above the ground and records GPS observations at a sampling interval of 15 s. In principle, it is possible to collect GPS signals in an area of nearly 10 000 m2 around the antenna. Different satellites produce different ground tracks, i.e., the sampling area. Usually, the effective multipath signal is considered to be obtained from the ﬁrst Fresnel zone [14]. Furthermore, the GPS signal acquisition also needs to con- sider the inﬂuence of the surrounding environment. As shown in Fig.3(a),theareaofP351stationissparselyvegetatedandfreeof buildings, so the inﬂuence of shading can be ignored. In addition, due to the antenna gain pattern, the oscillation amplitude of SNR data decreases with increasing elevation angle, i.e., the multipath effect is more obvious at low elevation angles. We used SNR data from 5°–25° elevation angle to expect better results. And the azimuth angle range was selected from 0°–360°. k (x, xi) = exp −g∥x −xi∥2 (3) (3) where the parameter g is self-contained by the RBF kernel function, which affects the degree of ﬁt of the model and, thus, the generalization ability of the model. In addition, the penalty coefﬁcient c, which affects the degree of ﬁt of the model and, thus, the generalization ability of the model. The area where P351 station is located experienced snowpack accumulation–ablation–accumulation in 2014, satisfying the re- quirement to perform a complete cycle of detection of snow-free B. GNSS-R Snow Depth Retrieval Experiments Combined With SVM B. GNSS-R Snow Depth Retrieval Experiments Combined With SVM In the classical algorithm, the least squares ﬁtting (LSF) method and the Lomb–Scargle periodogram (LSP) [27], [28] spectral analysis method were used to retrieve the snow depth. The LSF method was used to separate the low-frequency trend terms from the high-frequency oscillation terms, and the LSP spectral analysis method was able to extract the frequencies in the oscillation terms for the subsequent snow depth retrieval. The results of snow depth retrieval for P351 station in 2014 based on the classical algorithm are shown in Fig. 4(a). The horizontal coordinates indicate time and the vertical coordinates indicate snow depth. The snow depth retrieval based on the classical algorithm is highly consistent with the in-situ snow depth provided by the SNOTEL network, but seems to show large variations in some local areas. From the error maps, it can be observed that the main retrieval errors are mainly distributed in the thick snow depth range and snow-free state. Figs. 5 and 6 show the topography around the station and the spatial distribution of snow depth retrieval results, respectively. It can be clearly seen that the retrieval results are difﬁcult to get the retrieval results with snow depth of zero. On the one hand, when the snow depth is close to the height of the antenna, the low-frequency trend term and high-frequency oscillation term will be difﬁcult to separate the spectrum and the size of the Fresnel zone will be decreased. On the other hand, the snow depth retrieval in snow-free state and shallow snow state is also affected by ground cover and electromagnetic penetration bias [29], [30]. However, if the ground truth information can be determined, it is possible to transform the snow depth re- trieval problem into a ground state detection problem. When the ground is snow-free state, the snow depth retrieval result is set to zero by default, which seems to avoid retrieval errors. Additionally, we observed a blank area in Fig. 6, which may be due to the fact that no qualiﬁed SNR data were collected in this area. Fig. 4. Analysis of GNSS-R snow depth retrieval results for GPS S1C SNR at P351 station. (a) Results of GNSS-R snow depth retrieval based on classical algorithm. (b) Results of GNSS-R snow depth retrieval based on SVM+classical algorithm. HU et al.: SVM-BASED SNOW DETECTION ALGORITHM FOR GNSS-R SNOW DEPTH RETRIEVALS 6049 Fig. 4. Analysis of GNSS-R snow depth retrieval results for GPS S1C SNR at P351 station. (a) Results of GNSS-R snow depth retrieval based on classical algorithm. (b) Results of GNSS-R snow depth retrieval based on SVM+classical algorithm. TABLE I C M C R SVM state and snow-covered state. As shown in Fig. 3(b), the Galena Summit station (43.87497 °N, 114.71363 °W, Elevation 2676 m) of the SNOTEL network, located about 0.5 km in a straight line from P351 station, recorded many in-situ snow data, which provided reference data for validating the experiments. B. GNSS-R Snow Depth Retrieval Experiments Combined With SVM TABLE I CONFUSION MATRIX OF CLASSIFICATION RESULTS OF SVM CLASSIFICATION MODEL set to 2 and 0.5, respectively, via the cross-validation and grid search algorithms. TABLE I CONFUSION MATRIX OF CLASSIFICATION RESULTS OF SVM CLASSIFICATION MODEL As mentioned in Section II, different reﬂecting surfaces will leadtovariabilityintheSNR.Essentially,theSVMclassiﬁcation model is used to detect satellite signals collected in different snow conditions. The 20 000 SNR arc samples from different snow depths collected at P351 station in 2014 were used as input predictors for the SVM classiﬁcation model, which contained 10 000 snow-covered state samples set as positive class (DOY 1–148, 295–365) and 10 000 snow-free state samples set as negative class (DOY 149–294). The ratio of training dataset and test dataset is 4:1. It should be noted that the input SNR arcs are constrained to be in the elevation angle range of 5°–25°. In addition, the parameter g and the penalty coefﬁcient c are two key independent parameters of the SVM classiﬁcation model that need to be considered. In this experiment, c and g were set to 2 and 0.5, respectively, via the cross-validation and grid search algorithms. Combined with the in-situ snow depth, the detection accuracy of test dataset can reach about 96% overall, with the true positive rate and true negative rate of about 97% and 95%, respectively, as shown in Table I. The area under curve area is approxi- mately 0.96. Statistically, the SVM classiﬁcation model can well IEEE JOURNAL OF SELECTED TOPICS IN APPLIED EARTH OBSERVATIONS AND REMOTE SENSING, VOL. 15, 2022 6050 Fig. 5. DEM of P351 station. Fig. 6. Spatial distribution of snow depth retrieval on DOY 200. Fig. 7. Analysis of GNSS-R snow depth retrieval results combined with SVM classiﬁcation model. (a) Statistics of detection results based on SVM classiﬁca- tion model. (b) Accuracy of different transitional period division standards. the snow state transition period by comparing with the actual snow depth. In fact, if we set the threshold value to 50%, i.e., adopt the majority principle, then only one day of detection resultsinthisexperimentdoesnotmeettherequirements,andthe improvement for the accuracy of the ﬁnal snow depth retrieval Fig. 7. Analysis of GNSS-R snow depth retrieval results combined with SVM classiﬁcation model. (a) Statistics of detection results based on SVM classiﬁca- tion model. (b) Accuracy of different transitional period division standards. Fig 5 DEM of P351 station Fig. 5. DEM of P351 station. Fig. 7. B. GNSS-R Snow Depth Retrieval Experiments Combined With SVM Analysis of GNSS-R snow depth retrieval results combined with SVM classiﬁcation model. (a) Statistics of detection results based on SVM classiﬁca- tion model. (b) Accuracy of different transitional period division standards. the snow state transition period by comparing with the actual snow depth. In fact, if we set the threshold value to 50%, i.e., adopt the majority principle, then only one day of detection resultsinthisexperimentdoesnotmeettherequirements,andthe improvement for the accuracy of the ﬁnal snow depth retrieval will be more obvious on this basis. Fig. 6. Spatial distribution of snow depth retrieval on DOY 200. In order to perform a more in-depth analysis of the perfor- mance of the proposed detection algorithm in different snow depth ranges, especially the transition period between snow-free state and snow-covered, we set three transition snow depth ranges: [0, 5], [0, 10], and [0, 20]. As shown in Fig. 7(b), the horizontal coordinates represent the range of snow depths for the different transition periods delineated. The ordinates represent the detection accuracy for different snow depth ranges under different delineated intervals. It can be seen that the detection ac- curacies of both snow-free state and snow-covered state after ex- cluding the set transition period are above 95%, but the detection accuracy of the transition period is not ideal. Taking [0, 5] as an example, the detection accuracies of the snow-free state and the snow-covered state are 95% and 99%, respectively. However, the detection accuracy in the transition period was only 76%, which may be related to the spatial heterogeneity of snow distribution and temperature. The detection results are introduced into the snow depth retrieval to optimize the initial snow depth retrieval results, as shown by the blue dots in Fig. 4(b). The statistics identify SNR arcs from different reﬂecting surfaces and detect ground truth information. It should be noted that in this study, the ground state was divided into two states of no snow with zero snow depth and a snow-covered state with snow depth higher than zero. In addition, since the proposed algorithm is applied in GNSS-R snow depth retrieval experiments, the snow depth is recorded at a frequency of 1 d, so for the detection results, we are concerned with the ﬁnal conclusion of each day. During the experiment, the daily accuracy achieved good performance. REFERENCES Differences in snowfall and topography between GNSS sta- tions and recording stations will affect the retrieval accuracy. In the snow depth retrieval experiment, we use the daily average snow depth data provided by the SNOTEL network as the reference data source to evaluate the retrieval results. Although the distance between the two places is very close, the snowpack has temporal and spatial variability. After all, the snow data of the two places are not completely consistent, which will become an error source for snow depth retrieval. [1] T. A. Erickson, M. W. Williams, and A. Winstral, “Persistence of topo- graphic controls on the spatial distribution of snow in rugged mountain terrain, Colorado, United States,” Water Resour. Res., vol. 41, no. 4, Apr. 2005, Art. no. W04014, doi: 10.1029/2003WR002973. [2] M. C. Serreze, M. P. Clark, R. L. Armstrong, D. A. McGinnis, and R. S. Pulwarty, “Characteristics of the western United States snowpack from snowpack telemetry (SNOTEL) data,” Water Resour. Res., vol. 35, no. 7, pp. 2145–2160, Dec. 1999, doi: 10.1175/2008JHM981.1. [3] M. Takala et al., “Estimating northern hemisphere snow water equivalent for climate research through assimilation of space-borne radiometer data and ground-based measurements,” Remote Sens. Environ., vol. 115, no. 12, pp. 3517–3529, Dec. 2011, doi: 10.1016/j.rse.2011.08.014. Snow depth, as a slowly changing meteorological data, has a seasonal snowpack accumulation cycle of accumulation– ablation–accumulation unless a blizzard is encountered. During the alternating periods of accumulation and ablation states, snowpack is subject to external inﬂuences, such as temperature or wind, that may lead to rapid changes in the state of snow-free and snow-covered on the ground. In special cases, SNR arcs collected on the same day may contain ground information of both snow-free state and snow-covered state, which makes retrieval more challenging. [4] J. P. Gong, R. J. Ma, D. A. Li, and J. M. Zhao, “GNSS multipath mitigation method based on K-means classiﬁcation in urban environment,” J. Indian Soc. Remote Sens., vol. 50, no. 5, pp. 805–813, May 2022, doi: 10.1007/s12524-022-01494-y. [5] M. Martín-Neira, “A passive reﬂectometry and interferometry system (PARIS): Application to ocean altimetry,” ESA J., vol. 17, pp. 331–355, 1993. [6] A. Bilich and K. M. Larson, “Mapping the GPS multipath environment using the signal-to-noise ratio (SNR),” Radio Sci., vol. 42, no. 6, Mar. 2007, Art. no. RS6003, doi: 10.1029/2008RS003839. [7] M. D. Jacobson, “Dielectric-covered ground reﬂectors in GPS multipath reception—Theory and measurement,” IEEE Geosci. Remote Sens. Lett., vol. REFERENCES 5, no. 3, pp. 396–399, Jul. 2008, doi: 10.1109/LGRS.2008.917130. [8] K. M. Larson, E. D. Gutmann, V. U. Zavorotny, J. J. Braun, M. W. Williams, and F. G. Nievinski, “Can we measure snow depth with GPS receivers?,” Geophysical Res. Lett., vol. 36, no. 17, Sep. 2009, Art. no. L17502, doi: 10.1029/2009GL039430. ACKNOWLEDGMENT The GNSS data for P351 station were provided by the PBO network.1 The measured snow depth was provided by the SNO- TEL network.2 B. GNSS-R Snow Depth Retrieval Experiments Combined With SVM The daily ground state detection conclusions consist of the detection results of multiple SNR arcs for the day, and we consider the detection results reliable when a particular result exceeds 75% of the sample size for the day. Combined with the measured snow depth, Fig. 7(a) shows the distribution of the error rate of the daily detection results compared to the real ground state during the experimental period. The statistical results show that out of 365 days in the experimental period, only eight days (DOY 147–149, 167, 294, 296, 298, 304) exceeded the set threshold, and almost all of these 8 days were found to be concentrated in identify SNR arcs from different reﬂecting surfaces and detect ground truth information. It should be noted that in this study, the ground state was divided into two states of no snow with zero snow depth and a snow-covered state with snow depth higher than zero. In addition, since the proposed algorithm is applied in GNSS-R snow depth retrieval experiments, the snow depth is recorded at a frequency of 1 d, so for the detection results, we are concerned with the ﬁnal conclusion of each day. During the experiment, the daily accuracy achieved good performance. The daily ground state detection conclusions consist of the detection results of multiple SNR arcs for the day, and we consider the detection results reliable when a particular result exceeds 75% of the sample size for the day. Combined with the measured snow depth, Fig. 7(a) shows the distribution of the error rate of the daily detection results compared to the real ground state during the experimental period. The statistical results show that out of 365 days in the experimental period, only eight days (DOY 147–149, 167, 294, 296, 298, 304) exceeded the set threshold, and almost all of these 8 days were found to be concentrated in HU et al.: SVM-BASED SNOW DETECTION ALGORITHM FOR GNSS-R SNOW DEPTH RETRIEVALS 6051 TABLE II STATISTICS OF THE RETRIEVAL ACCURACY OF DIFFERENT ALGORITHMS 3) Compared with current topography correction algorithms, the algorithm proposed in this article does not rely on any priori ground measurement data. The SVM classi- ﬁcation model can learn the topography environment of the retrieved region from historical SNR data to improve the matching with each other. Therefore, the algorithm is theoretically universal and applicable to different snow scenarios. Moreover, this study broadens the application scope of GNSS signal and provides a reference for the subsequent application of SNR in the detection ﬁeld. 3) Compared with current topography correction algorithms, the algorithm proposed in this article does not rely on any priori ground measurement data. The SVM classi- ﬁcation model can learn the topography environment of the retrieved region from historical SNR data to improve the matching with each other. Therefore, the algorithm is theoretically universal and applicable to different snow scenarios. Moreover, this study broadens the application scope of GNSS signal and provides a reference for the subsequent application of SNR in the detection ﬁeld. of the retrieval accuracy of different algorithms are recorded in Table II. The root-mean-square error (RMSE) of the snow depth retrieval results combined with the SVM classiﬁcation model is about 15 cm, which is about 25% less than the 20 cm of the classical algorithm. It can be seen that the scheme proposed in this article is feasible, which can effectively reduce the retrieval error in snow-free state and improve the accuracy of snow depth retrieval. of the retrieval accuracy of different algorithms are recorded in Table II. The root-mean-square error (RMSE) of the snow depth retrieval results combined with the SVM classiﬁcation model is about 15 cm, which is about 25% less than the 20 cm of the classical algorithm. It can be seen that the scheme proposed in this article is feasible, which can effectively reduce the retrieval error in snow-free state and improve the accuracy of snow depth retrieval. 1[Online]. Available: https://xenon.colorado.edu/portal/ 2[Online]. Available: https://wcc.sc.egov.usda.gov/ IEEE JOURNAL OF SELECTED TOPICS IN APPLIED EARTH OBSERVATIONS AND REMOTE SENSING, VOL. 15, 2022 6052 IEEE JOURNAL OF SELECTED TOPICS IN APPLIED EARTH OBSERVATIONS AND REMOTE SENSING, VOL. 15, 2022 [14] K. M. Larson and F. G. Nievinski, “GPS snow sensing: Results from the earthscope plate boundary observatory,” GPS Solutions, vol. 17, no. 1, pp. 41–52, Jan. 2013, doi: 10.1007/s10291-012-0259-7. Xintai Yuan was born in Guangdong, China, in 1998. He received the B.E. degree in mechanical engi- neering from Shantou University, Shantou, China, in 2020. He is currently working toward the master’s de- gree in mechanical engineering with the Department of Electrical Engineering, Shanghai Ocean Univer- sity, Shanghai, China. [15] J. S. Löfgren, R. Haas, and H.-G. Scherneck, “Sea level time series and ocean tide analysis from multipath signals at ﬁve GPS sites in different parts of the world,” J. Geodyn., vol. 80, pp. 66–80, Oct. 2014, doi: 10.1016/j.jog.2014.02.012. [16] N. Roussel et al., “Sea level monitoring and sea state estimate using a single geodetic receiver,” Remote Sens. Environ., vol. 171, pp. 261–277, Dec. 2015, doi: 10.1016/j.rse.2015.10.011. His research interests are focused on the use of re- mote sensing techniques, such as GNSS reﬂectometry applied to global climate change. j [17] S. Zhang, X. Wang, and Q. Zhang, “Avoiding errors attributable to topog- raphy in GPS-IR snow depth retrievals,” Adv. Space Res., vol. 59, no. 6, pp. 1663–1669, Mar. 2017, doi: 10.1016/j.asr.2016.12.031. [18] S. Tabibi, F. Geremia-Nievinski, and T. van Dam, “Statistical com- parison and combination of GPS, GLONASS, and multi-GNSS mul- tipath reﬂectometry applied to snow depth retrieval,” IEEE Trans. Geosci. Remote Sens., vol. 55, no. 7, pp. 3773–3785, Jul. 2017, doi: 10.1109/TGRS.2017.2679899. Wei Liu received the B.Sc. and M.Sc. degrees in automation and instrument engineering from North- eastern University, Shenyang, China, in 2003 and 2006, respectively, and the Ph.D. degree in guidance, navigation and control from Shanghai Jiao Tong Uni- versity, Shanghai, China, in 2011. Wei Liu received the B.Sc. and M.Sc. degrees in automation and instrument engineering from North- eastern University, Shenyang, China, in 2003 and 2006, respectively, and the Ph.D. degree in guidance, navigation and control from Shanghai Jiao Tong Uni- versity, Shanghai, China, in 2011. [19] Z. Li, P. Chen, N. Zheng, and H. Liu, “Accuracy analysis of GNSS- IR snow depth inversion algorithms,” Adv. Space Res., vol. 67, no. 4, pp. 1317–1332, Feb. 2021, doi: 10.1016/j.asr.2020.11.021. He is currently a Professor of communication and navigation with Shanghai Maritime University, Shanghai. IEEE JOURNAL OF SELECTED TOPICS IN APPLIED EARTH OBSERVATIONS AND REMOTE SENSING, VOL. 15, 2022 From 2015 to 2016, he was with the De- partment for Geodesy, German Research Centre for Geosciences, Potsdam, Germany. His research inter- ests include global navigation satellite systems (GNSS) signal processing, GNSS reﬂectometry, and GNSS-related interference studies. [20] Y. Li, X. Chang, K. Yu, S. Wang, and J. Li, “Estimation of snow depth using pseudorange and carrier phase observations of GNSS single- frequency signal,” GPS Solutions, vol. 23, no. 4, pp. 1–13, Oct. 2019, doi: 10.1007/s10291-019-0912-5. [21] O. Montenbruck et al., “The multi-GNSS experiment (MGEX) of the International GNSS Service (IGS)—Achievements, prospects and chal- lenges,” Adv. Space Res., vol. 59, no. 7, pp. 1671–1697, Apr. 2017, doi: 10.1016/j.asr.2017.01.011. Qingsong Hu received the Ph.D. degree in engi- neering from the Department of Automatic Control, Tongji University, Shanghai, China, in 2007. Qingsong Hu received the Ph.D. degree in engi- neering from the Department of Automatic Control, Tongji University, Shanghai, China, in 2007. He is currently a Professor with Shanghai Ocean University, Shanghai. His main research interests in- clude the mine Internet of Things, marine intelligent detection technology, instrumentation, and automa- tion. [22] K. M. Larson, E. E. Small, E. Gutmann, A. L. Bilich, J. J. Braun, and V. U. Zavorotny, “Use of GPS receivers as a soil moisture network for water cycle studies,” Geophysical Res. Lett., vol. 35, no. 24, Dec. 2008, Art. no. L24405, doi: 10.1029/2008GL036013. He is currently a Professor with Shanghai Ocean University, Shanghai. His main research interests in- clude the mine Internet of Things, marine intelligent detection technology, instrumentation, and automa- tion. [23] K. M. Larson, E. E. Small, E. Gutmann, A. Bilich, P. Axelrad, and J. Braun, “Using GPS multipath to measure soil moisture ﬂuctuations: Initial results,” GPS Solutions, vol. 12, no. 3, pp. 173–177, Jul. 2008, doi: 10.1007/s10291-007-0076-6. [24] C. C. Chew, E. E. Small, K. M. Larson, and V. U. Zavorotny, “Vege- tation sensing using GPS-interferometric reﬂectometry: Theoretical ef- fects of canopy parameters on signal-to-noise ratio data,” IEEE Trans. Geosci. Remote Sens., vol. 53, no. 5, pp. 2755–2764, May 2014, doi: 10.1109/TGRS.2014.2364513. Jens Wickert received the bachelor’s degree in physics from Technical University Dresden, Dresden, Germany, in 1991, and the Ph.D. degree in geo- physics/meteorology from Karl-Franzens-University Graz, Graz, Austria, in 2002. Jens Wickert received the bachelor’s degree in physics from Technical University Dresden, Dresden, Germany, in 1991, and the Ph.D. degree in geo- physics/meteorology from Karl-Franzens-University Graz, Graz, Austria, in 2002. [25] N. IEEE JOURNAL OF SELECTED TOPICS IN APPLIED EARTH OBSERVATIONS AND REMOTE SENSING, VOL. 15, 2022 Guenther and M. Schonlau, “Support vector machines,” Stata J., vol. 16, no. 4, pp. 917–937, Dec. 2016, doi: 10.1177/1536867X1601600407. [26] O. Okwuashi, C. E. Ndehedehe, D. N. Olayinka, A. Eyoh, and H. Attai, “Deep support vector machine for PoISAR image classiﬁca- tion,” Int. J. Remote. Sens., vol. 42, no. 17, pp. 6502–6540, Sep. 2021, doi: 10.1080/01431161.2021.1939910. He was a Principal Investigator of the pioneering GPS radio occultation experiment aboard the German CHAMP, and he was also with several German geo- science research institutes. He is currently a joint Pro- fessor of global navigation satellite systems (GNSS) remote sensing, navigation, and positioning with the German Research Centre for Geosciences GFZ, Potsdam, Germany, and with the Technical University of Berlin, Berlin, Germany, and also a Chair of the Science Advisory Group of the GEROS-ISS mission for GNSS-reﬂectometry. In addition, he is also the Deputy GFZ Section Head Space Geodetic Techniques and the GFZ Speaker of the Atmosphere and Climate Research Program of the German Helmholtz Association. He has authored or coauthored more than 160 ISI listed publications on GNSS earth observation and received several research awards. [27] N. R. Lomb, “Least-squares frequency analysis of unequally spaced data,” Astrophys. Space Sci., vol. 39, no. 2, pp. 447–462, Oct. 1976, doi: 10.1007/BF00648343. [28] J. D. Scargle, “Studies in astronomical time series analysis. II-Statistical aspects of spectral analysis of unevenly spaced data,” Astrophysical J., vol. 263, pp. 835–853, Dec. 1982, doi: 10.1086/160554. pp [29] J. L. McCreight, E. E. Small, and K. M. Larson, “Snow depth, density, and SWE estimates derived from GPS reﬂection data: Validation in the western U.S.,” Water Resour. Res., vol. 50, no. 8, pp. 6892–6909, Aug. 2014, doi: 10.1002/2014WR015561. [30] K. Boniface, J. Braun, J. McCreight, and F. Nievinski, “Comparison of snow data assimilation system with GPS reﬂectometry snow depth in the western United States,” Hydrological Processes, vol. 29, no. 10, pp. 2425–2437, May 2015, doi: 10.1002/hyp.10346. lite system-reﬂectometry. Zhihao Jiang was born in Jiangsu, China, in 1998. He received the Bachelor of Engineering degree in mechanical engineering and automation from the Nanjing Institute of Technology, Nanjing, China, in 2020. He is currently working toward the master’s degree in electrical engineering with Shanghai Ocean University, Shanghai, China. His research interests are focused on the ocean remote sensing by global navigation satellite system- reﬂectometry signal. IV. CONCLUSION In this study, the SNR arcs were used for snow depth retrieval experiments and the feasibility of snow detection on the ground based on SVM and the effect of improving the retrieval accuracy in the snow-free state are analyzed. This study has the following contributions. [9] K. Yu, W. Ban, X. Zhang, and X. Yu, “Snow depth estimation based on multipath phase combination of GPS triple-frequency signals,” IEEE Trans. Geosci. Remote Sens., vol. 53, no. 9, pp. 5100–5109, Sep. 2015, doi: 10.1109/TGRS.2015.2417214. [10] S. Jin, X. Qian, and H. Kutoglu, “Snow depth variations estimated from GPS-Reﬂectometry: A case study in Alaska from L2P SNR data,” Remote Sens., vol. 8, no. 1, Jan. 2016, Art. no. 63, doi: 10.3390/rs8010063. 1) Since the SNR arcs collected by the GNSS antenna in the snow-free state and the snow-covered state are different, SVM can be used to detect the ground state. The detection results are highly consistent with the measured snow depth data, the detection accuracy of the samples can reach 96%, and the detection accuracy of the daily snow state during the experiment can reach 98% within the set threshold range. [11] S. Vey, A. Güntner, J. Wickert, T. Blume, H. Thoss, and M. Ramatschi, “Monitoring snow depth by GNSS reﬂectometry in built-up areas: A case study for Wettzell, Germany,” IEEE J. Sel. Topics Appl. Earth Observ. Remote Sens., vol. 9, no. 10, pp. 4809–4816, Oct. 2016, doi: 10.1109/JS- TARS.2016.2516041. [12] S. Zhang et al., “GiRsnow: An open-source software for snow depth re- trievalsusingGNSSinterferometricreﬂectometry,”GPSSolutions,vol.25, no. 2, pp. 1–8, Feb. 2021, doi: 10.1007/s10291-021-01096-0. [13] Y. Hu, X. Yuan, W. Liu, J. Wickert, Z. Jiang, and R. Haas, “GNSS-IR model of sea level height estimation combining variational mode decom- position,” IEEE J. Sel. Topics Appl. Earth Observ. Remote Sens., vol. 14, pp. 10405–10414, 2021, doi: 10.1109/JSTARS.2021.3118398. 2) With the aid of daily detection of snow states on the ground, it is possible to achieve constraints on the initial snow retrieval results, especially in the snow-free state. The RMSE of the optimized snow depth retrieval results is reduced from 20 to 15 cm, which is 25% less than the initial results. 1[Online]. Available: https://xenon.colorado.edu/portal/ 2[Online]. Available: https://wcc.sc.egov.usda.gov/ Yuan Hu received the Ph.D. degree in pattern recog- nition & intelligent system from Shanghai Jiao Tong University, Shanghai, China, in 2011. IEEE JOURNAL OF SELECTED TOPICS IN APPLIED EARTH OBSERVATIONS AND REMOTE SENSING, VOL. 15, 2022 His research interests include sea ice remote sensing using global navigation satel- Zhihao Jiang was born in Jiangsu, China, in 1998. He received the Bachelor of Engineering degree in mechanical engineering and automation from the Nanjing Institute of Technology, Nanjing, China, in 2020. He is currently working toward the master’s degree in electrical engineering with Shanghai Ocean University, Shanghai, China. Zhihao Jiang was born in Jiangsu, China, in 1998. He received the Bachelor of Engineering degree in mechanical engineering and automation from the Nanjing Institute of Technology, Nanjing, China, in 2020. He is currently working toward the master’s degree in electrical engineering with Shanghai Ocean University, Shanghai, China. Yuan Hu received the Ph.D. degree in pattern recog- nition & intelligent system from Shanghai Jiao Tong University, Shanghai, China, in 2011. She is currently an Associate Professor of electrical engineering with Shanghai Ocean University, Shang- hai. Her research interests include signal process- ing, computer science, and GNSS-related application studies. Her research interestsare focused on the areas of GNSS signal processing, GNSS reﬂectometry, and the earth deformation studies. Yuan Hu received the Ph.D. degree in pattern recog- nition & intelligent system from Shanghai Jiao Tong University, Shanghai, China, in 2011. Yuan Hu received the Ph.D. degree in pattern recog- nition & intelligent system from Shanghai Jiao Tong University, Shanghai, China, in 2011. She is currently an Associate Professor of electrical engineering with Shanghai Ocean University, Shang- hai. Her research interests include signal process- ing, computer science, and GNSS-related application studies. Her research interestsare focused on the areas of GNSS signal processing, GNSS reﬂectometry, and the earth deformation studies. His research interests are focused on the ocean remote sensing by global navigation satellite system- reﬂectometry signal. His research interests include sea ice remote sensing using global navigation satel-
https://openalex.org/W4238276018	https://journal.ahmareduc.or.id/index.php/AMHJ/article/download/7/7	Indonesian	null	Penyuluhan Berpengaruh terhadap Peningkatan Pengetahuan Ibu Hamil Tentang Senam Hamil	Ahmar Metastasis Health Journal	2,021	cc-by-sa	4,305	AHMAR METASTASIS HEALTH JOURNAL Available online at: http://journal.ahmareduc.or.id/index.php/AMHJ Vol. 1. No. 1. Juni 2021, Halaman 36-42 P-ISSN: 2797-6483 E-ISSN: 2797-4952 Available online at: http://journal.ahmareduc.or.id/index.php/AMHJ Vol. 1. No. 1. Juni 2021, Halaman 36-42 P-ISSN: 2797-6483 E-ISSN: 2797-4952 Penyuluhan Berpengaruh terhadap Peningkatan Pengetahuan Ibu Hamil Tentang Senam Hamil Artika Dewie1, Anna Veronica Pont2, Hasnah3 1 Prodi DIII Kebidanan, Poltekkes Kemenkes Palu, Palu, Sulawesi Tengah, Indonesia. 2 Prodi DIV Kebidanan, Poltekkes Kemenkes Palu, Palu, Sulawesi Tengah, Indonesia. 3 UPTD Puskesmas Pagimana, Banggai, Sulawesi Tengah, Indonesia. A R T I C L E I N F O O R I G I N A L A R T I C LE Article Type: Research Article History: Received: 5/26/2021 Accepted: 6/23/2021 ABSTRACT Introduction: One of the methods used to introduce pregnancy exercise in the community, especially pregnant women, is through counseling about pregnancy exercise. The purpose of the study was to determine the effect of pregnancy exercise counseling on increasing knowledge of pregnant women in the working area of the Lobu Health Center. Methods: This type of research is quasi-experimental with one group pretest posttest design, the sample of 30 respondents was determined using purposive sampling technique. The measurement of knowledge uses a questionnaire. Data analysis using wilcoxon test. Results: The results of the test using Wilcoxon ρ-value < 0.001, indicating that there was a significant increase in knowledge of pregnant women respondents after being given counseling about pregnancy exercise. Conclution: It is recommended that pregnant women be more proactive in participating in pregnancy exercise counseling and for health workers it is necessary to promote pregnancy exercise Keywords : Counseling, Knowledge, Pregnancy Exercise. O R I G I N A L A R T I C LE ABSTRACT Introduction: One of the methods used to introduce pregnancy exercise in the community, especially pregnant women, is through counseling about pregnancy exercise. The purpose of the study was to determine the effect of pregnancy exercise counseling on increasing knowledge of pregnant women in the working area of the Lobu Health Center. Methods: This type of research is quasi-experimental with one group pretest posttest design, the sample of 30 respondents was determined using purposive sampling technique. The measurement of knowledge uses a questionnaire. Data analysis using wilcoxon test. Results: The results of the test using Wilcoxon ρ-value < 0.001, indicating that there was a significant increase in knowledge of pregnant women respondents after being given counseling about pregnancy exercise. Conclution: It is recommended that pregnant women be more proactive in participating in pregnancy exercise counseling and for health workers it is necessary to promote pregnancy exercise Article History: Received: 5/26/2021 Accepted: 6/23/2021 Kata Kunci : Penyuluhan, Pengetahuan, Senam Hamil. Artika Dewie1, Anna Veronica Pont2, Hasnah3 1 Prodi DIII Kebidanan, Poltekkes Kemenkes Palu, Palu, Sulawesi Tengah, Indonesia. 2 Prodi DIV Kebidanan, Poltekkes Kemenkes Palu, Palu, Sulawesi Tengah, Indonesia. 3 UPTD Puskesmas Pagimana, Banggai, Sulawesi Tengah, Indonesia. Keywords : Counseling, Knowledge, Pregnancy Exercise. Corresponding author Email: dewieartika@gmail.com Keywords : Counseling, Knowledge, Pregnancy Exercise. PENDAHULUAN Selama kehamilan akan terjadi perubahan fisik, fungsi tubuh dan psikologis, yang disebabkan oleh perubahan sistem hormonal dalam tubuh yang akan mempengaruhi sistem organ lainnya. Petugas kesehatan yang akan memberikan konsultasi tentang aktifitas fisik sehari – hari dan latihan fisik ringan pada ibu hamil perlu memahami perubahan ini. Latihan fisik merupakan suatu bentuk aktifitas fisik yang dilakukan secara terstruktur dan terencana, dengan tujuan untuk meningkatkan kebugaran, salah satu contohnya adalah senam. Dengan melakukan aktifitas fisik secara teratur, terukur dengan gerakan-gerakan yang sesuai, maka akan mengurangi keluhan-keluhan yang terjadi selama kehamilan (Kementerian Kesehatan Republik Indonesia, 2014). p ) Senam hamil bukan merupakan hal baru di Indonesia dan dalam sosialisasinya masih berlangsung sampai saat ini melalui petugas kesehatan, majalah, dan media-media cetak lainnya, namun masih banyak masyarakat belum mengetahui tentang senam hamil. Masih ada ibu hamil yang tidak menyadari manfaat olahraga selama kehamilan, bahkan menganggap bahwa senam selama hamil tidak aman (Lee, et al., 2020). Sebuah penelitian sebelumnya menyatakan bahwa dewasa ini, pengetahuan tentang pentingnya olahraga selama kehamilan dalam hal ini adalah senam hamil, masih kurang diberikan kepada wanita usia subur, hingga menyebabkan kesalahan persepsi (Alvis, et al., 2019). Program pembangunan kesehatan di Indonesia dewasa ini masih diprioritaskan pada upaya peningkatan derajat kesehatan pada kelompok paling rentan yaitu ibu hamil, ibu bersalin, ibu nifas dan bayi pada masa perinatal. Hal ini karena masih tingginya Angka Kematian Ibu dan Angka Kematian Bayi (Kementerian Kesehatan Republik Indonesia, 2014). Tingginya angka kematian tersebut antara lain disebabkan oleh rendahnya pengetahuan ibu dalam perawatan kesehatan ibu serta pengenalan tanda bahaya obstetri dan neonatal. Upaya meningkatkan pengetahuan ibu dapat dilakukan melalui penyuluhan kesehatan. Penyuluhan adalah penyampaian informasi dari sumber informasi kepada seseorang atau sekelompok orang mengenai berbagai hal yang berkaitan dengan suatu program (Kementerian Kesehatan Republik Indonesia, 2011). ) Jumlah ibu hamil di Indonesia tahun 2017 sebanyak 5.320.550 dan Provinsi Sulawesi Tengah sebanyak 69.417 ibu hamil (Kementerian Kesehatan Republik Indonesia, 2018). Sementara Seksi Kesehatan Keluarga dan KB Dinas Kesehatan Banggai melaporkan jumlah ibu hamil di Kabupaten Banggai tahun 2017 sebanyak 7.941 ibu hamil, dan di Kecamatan Lobu sebanyak 81 ibu hamil (Dinas Kesehatan Kabupaten Banggai, 2018). ABSTRAK Corresponding author Email: dewieartika@gmail.com Corresponding author Email: dewieartika@gmail.com Pendahuluan: Salah satu cara yang digunakan untuk memperkenalkan senam hamil di masyarakat, khususnya ibu hamil adalah melalui penyuluhan senam hamil. Tujuan penelitian untuk mengetahui pengaruh penyuluhan senam hamil terhadap peningkatan pengetahuan ibu hamil diwilayah kerja Puskesmas Lobu. Metode: Jenis penelitian quasi experiment dengan rancangan one group pretest posttest, Sampel sejumlah 30 responden yang ditentukan dengan menggunakan tehnik purposive sampling Pengukuran pengetahuan menggunakan kuesioner. Analisa data yaitu wilcoxon. Hasil: Hasil nilai ρ < 0,001, menunjukkan bahwa terdapat peningkatan pengetahuan yang signifikan pada responden ibu hamil setelah diberikan penyuluhan mengenai senam hamil. Kesimpulan: Disarankan pada ibu hamil agar lebih proaktif dalam mengikuti penyuluhan senam hamil dan bagi tenaga kesehatan perlu melakukan promosi senam hamil secara berkesinambungan. 36 PENDAHULUAN y ( p gg ) Puskesmas Lobu merupakan salah satu fasilitas pelayanan kesehatan dasar di Kecamatan Pagimana, membawahi 10 desa dan dilengkapi ruang pertemuan yang biasa digunakan untuk kegiatan penyuluhan dan senam hamil.Hasil studi pendahuluan yang dilakukan di unitKesehatan Ibu dan Anak (KIA) Puskesmas tersebut, tercatat 76 Ibu hamil di wilayah kerja Puskesmas Lobu yang memeriksakan diri tetapi tidak pernah mengikuti senam hamil. Hasil wawancara cepat dengan 16 ibu hamil, mereka berpendapat bahwa senam hamil adalah hal yang biasa saja dan tidak penting dan 10 orang ibu hamil mengatakan bahwa tidak perlu melakukan senam hamil karena setiap harinya sudah melakukan pekerjaan rumah tangga seperti memasak, mencuci, mengasuh anak dan lain-lain. Hasil konfirmasi dengan bidan koordinator di Puskesmas Lobu diketahui bahwa selama tahun 2018 tidak pernah diadakan penyuluhan senam hamil di Puskesmas Lobu. Berdasarkan informasi diatas, maka peneliti tertarik untuk meneliti pengaruh penyuluhan terhadap peningkatan pengetahuan tentang senam hamil pada ibu hamil di wilayah kerja Puskesmas Lobu Kabupaten Banggai. Diharapkan dengan peningkatan pengetahuan mengenai senam hamil, maka ibu-ibu hamil akan tergerak untuk melakukan senam hamil. METODE PENELITIAN Metode yang digunakan pada penelitian ini adalah quasi experiment dengan rancangan One group pretest posttest yaitu penelitian tanpa kelompok pembanding (kontrol), tapi dilakukan observasi awal (pretest) sehingga memungkinkan mengetahui perubahan-perubahan yang terjadi setelah adanya experiment (Notoatmodjo, 2010). Cara kerja penelitian ini adalah terlebih 37 dahulu menilai pengetahuan penyuluhan dengan metode ceramah dengan media diskusi. Penelitian dilaksanakan di wilayah kerja Puskesmas Lobu, pada bulan Januari 2019. Populasi penelitian ini adalah ibu hamil yang berdomisili diwilayah kerja Puskesmas Lobu. dahulu menilai pengetahuan responden mengenai Senam Hamil, kemudian melakukan luhan dengan metode ceramah dengan media leaflet dan slide powerpoint Penelitian dilaksanakan di wilayah kerja Puskesmas Lobu, pada bulan Januari an ini adalah ibu hamil yang berdomisili diwilayah kerja Puskesmas responden mengenai Senam Hamil, kemudian melakukan dan slide powerpoint diikuti dengan Penelitian dilaksanakan di wilayah kerja Puskesmas Lobu, pada bulan Januari - Juni an ini adalah ibu hamil yang berdomisili diwilayah kerja Puskesmas Pengambilan sampel dilakukan dengan menggunakan tehnik didasarkan pada pertimbangan yang dibuat oleh peneliti berdasarkan sifat populasi yang sudah diketahui sebelumnya penelitian ini yaitu ibu dengan Puskesmas Lobu, kehamilan normal tanpa penyulit, tulis.Sampel ditetapkan sejumlah 30 orang. penelitian ini adalah Penyuluhan variabel) adalah pengetahuan ibu kuesioner yang telah diuji validitas dan reliabilitasnya menggunakan distribusi frekuensi dan wilcoxon. Untuk melihat kemaknaan perhitungan uji statistik, digunakan derajat kepercayaan 95%, dengan nlai ρ ˂ 0,005. dan narasi. Pengambilan sampel dilakukan dengan menggunakan tehnik purposive sampling didasarkan pada pertimbangan yang dibuat oleh peneliti berdasarkan sifat sudah diketahui sebelumnya (Notoatmodjo, 2010). Adapun ibu dengan umur kehamilan ≥ 20 minggu, memeriksakan kehamilan normal tanpa penyulit, bersedia menjadi responden dan bisa baca el ditetapkan sejumlah 30 orang.Variabel bebas (independent variabel) enyuluhan mengenai senam hamil dan variabel terikat adalah pengetahuan ibu tentang senam hamil yang diukur dengan menggunakan kuesioner yang telah diuji validitas dan reliabilitasnya. Analisa data distribusi frekuensi dan uji bivariat menggunakan uji statistik ihat kemaknaan perhitungan uji statistik, digunakan derajat kepercayaan ˂ 0,005.Hasil penelitian dilaporkan dengan menggunakan diagram, tabel purposive sampling yaitu didasarkan pada pertimbangan yang dibuat oleh peneliti berdasarkan sifat-sifat atau ciri . Adapun kriteria inklusi pada ≥ 20 minggu, memeriksakan kehamilannya di bersedia menjadi responden dan bisa baca (independent variabel) dalam senam hamil dan variabel terikat (dependent tentang senam hamil yang diukur dengan menggunakan . Analisa data secara univariat statistik non parametrik yaitu ihat kemaknaan perhitungan uji statistik, digunakan derajat kepercayaan Hasil penelitian dilaporkan dengan menggunakan diagram, tabel HASIL PENELITIAN Berdasarkan penelitian yang telah dilakukan maka dapat berikut: penelitian yang telah dilakukan maka dapat dijelaskan hal dijelaskan hal-hal sebagai Berdasarkan penelitian yang telah dilakukan maka dapat berikut: penelitian yang telah dilakukan maka dapat dijelaskan hal dijelaskan hal-hal sebagai Diagram 1. Distribusi Frekuensi K Frekuensi Karakteristik Responden di Wilayah Kerja Wilayah Kerja Puskesmas Lobu. Sumber data : Data primer 2019 Diagram 1 menunjukkan distribusi Desa Lobu tahun 2019. Terlihat tahun sejumlah 80 %, sebagian besar berpendidikan Sekolah Dasar (SD) sejumlah 66,7 %, hampir seluruh responden sedang hamil anak ke dua atau lebih ( dan sebagian besar ibu hamil trimester II yang memiliki umur kehamilan 20 sejumlah 56,7 %. Umur 80.0% 20.0% 20 - 35 > 35 menunjukkan distribusi frekuensi karakteristik responden Desa Lobu tahun 2019. Terlihat dari 30 responden, hampir seluruh responden berumur 20 tahun sejumlah 80 %, sebagian besar berpendidikan Sekolah Dasar (SD) sejumlah 66,7 %, hampir seluruh responden sedang hamil anak ke dua atau lebih (multipara) sejumlah 86,7 % dan sebagian besar ibu hamil trimester II yang memiliki umur kehamilan 20 Pendidikan Paritas Trimester 66.7% 20.0% 10.0% 3.3% 13.3% 86.7% SD SMP SMA Sarjana Primi Multi frekuensi karakteristik responden di Wilayah Kerja hampir seluruh responden berumur 20 – 35 tahun sejumlah 80 %, sebagian besar berpendidikan Sekolah Dasar (SD) sejumlah 66,7 %, multipara) sejumlah 86,7 % dan sebagian besar ibu hamil trimester II yang memiliki umur kehamilan 20 – 28 minggu Trimester 56.7% 43.3% Dua Tiga Sumber data : Data primer 2019 Umur 80.0% 20.0% 20 - 35 > 35 Pendidikan Paritas Trimester 66.7% 20.0% 10.0% 3.3% 13.3% 86.7% SD SMP SMA Sarjana Primi Multi Trimester 56.7% 43.3% Dua Tiga Sumber data : Data primer 2019 Diagram 1 menunjukkan distribusi Desa Lobu tahun 2019. Terlihat tahun sejumlah 80 %, sebagian besar berpendidikan Sekolah Dasar (SD) sejumlah 66,7 %, hampir seluruh responden sedang hamil anak ke dua atau lebih ( dan sebagian besar ibu hamil trimester II yang memiliki umur kehamilan 20 sejumlah 56,7 %. menunjukkan distribusi frekuensi karakteristik responden Desa Lobu tahun 2019. HASIL PENELITIAN Distribusi Frekuensi P senam hamil pada Responden di Frekuensi Pengetahuan sebelum dan sesudah penyuluhan pada Responden di Wilayah Kerja Puskesmas Lobu sebelum dan sesudah penyuluhan tentang Sumber: Data primer 2019 Kurang Baik Sebelum Sesudah 33.3% 0.0% 66.7% 100.0% Sesudah 100.0% Sumber: Data primer 2019 Diagram 2 menunjukkan tentang senam hamil sebelum diberi penyuluhan terdapat hampir setengah dari %. Namun setelah diberikan penyuluhan mengenai senam hamil, terlihat bahwa seluruh ibu hamil (30 responden) memiliki pengetahuan yang baik mengenai senam hamil (100 %). menunjukkan dari 30 responden, distribusi frekuensi pengetahuan ibu hamil tentang senam hamil sebelum diberi penyuluhan mengenai senam hamil menunjukkan masih terdapat hampir setengah dari ibu hamil yang memiliki pengetahuan kurang baik sejumlah %. Namun setelah diberikan penyuluhan mengenai senam hamil, terlihat bahwa seluruh ibu (30 responden) memiliki pengetahuan yang baik mengenai senam hamil (100 %). distribusi frekuensi pengetahuan ibu hamil mengenai senam hamil menunjukkan masih u hamil yang memiliki pengetahuan kurang baik sejumlah 33,3 %. Namun setelah diberikan penyuluhan mengenai senam hamil, terlihat bahwa seluruh ibu (30 responden) memiliki pengetahuan yang baik mengenai senam hamil (100 %). Tabel 1. Perbandingan Pengetahuan sebelum dan sesudah penyuluhan tentang Senam Hamil di Wilayah Kerja Puskesmas Lobu Pengetahuan sebelum dan sesudah penyuluhan tentang Senam Hamil di Wilayah Kerja Puskesmas Lobu. Pengetahuan sebelum dan sesudah penyuluhan tentang Senam Hamil di Wilayah Kerja Puskesmas Lobu Keterangan Pengetahuan setelah Penyuluhan < Pengetahuan sebelum Penyuluhan Pengetahuan setelah Penyuluhan > Pengetahuan sebelum Penyuluhan Pengetahuan setelah Penyuluhan = Pengetahuan sebelum Penyuluhan Sumber : Data primer 2019 di Wilayah Kerja Puskesmas Lobu. Pengetahuan setelah Penyuluhan < Pengetahuan sebelum Pengetahuan setelah Penyuluhan > Pengetahuan sebelum Pengetahuan setelah Penyuluhan = Pengetahuan sebelum N 0 30 0 Tabel 1 menunjukkan perbandingan pengetahuan mengenai senam hamil sebelum dan sesudah penyuluhan tentang senam hamil.Terdapat 30 pengetahuan mengenai senam hamil yang lebih baik daripada sebelum penyuluhan. Tabel 1 menunjukkan perbandingan pengetahuan mengenai senam hamil sebelum dan sesudah penyuluhan tentang senam hamil.Terdapat 30 responden ibu hamil dengan hasil pengetahuan mengenai senam hamil yang lebih baik daripada sebelum penyuluhan. Tabel 1 menunjukkan perbandingan pengetahuan mengenai senam hamil sebelum dan ibu hamil dengan hasil pengetahuan mengenai senam hamil yang lebih baik daripada sebelum penyuluhan. Tabel 2. Hasil Uji Statistik Pengaruh Penyuluhan terhadap Peningkatan Pengetahuan Tentang Senam Hamil di Wilayah Kerja Penyuluhan Sebelum penyuluhan Sesudah penyuluhan Sumber : Data primer 2019 Pengaruh Penyuluhan terhadap Peningkatan Pengetahuan Tentang Wilayah Kerja Puskesmas Lobu. HASIL PENELITIAN Terlihat dari 30 responden, hampir seluruh responden berumur 20 tahun sejumlah 80 %, sebagian besar berpendidikan Sekolah Dasar (SD) sejumlah 66,7 %, hampir seluruh responden sedang hamil anak ke dua atau lebih (multipara) sejumlah 86,7 % dan sebagian besar ibu hamil trimester II yang memiliki umur kehamilan 20 frekuensi karakteristik responden di Wilayah Kerja hampir seluruh responden berumur 20 – 35 tahun sejumlah 80 %, sebagian besar berpendidikan Sekolah Dasar (SD) sejumlah 66,7 %, multipara) sejumlah 86,7 % dan sebagian besar ibu hamil trimester II yang memiliki umur kehamilan 20 – 28 minggu Diagram 1 menunjukkan distribusi Desa Lobu tahun 2019. Terlihat tahun sejumlah 80 %, sebagian besar berpendidikan Sekolah Dasar (SD) sejumlah 66,7 %, hampir seluruh responden sedang hamil anak ke dua atau lebih ( dan sebagian besar ibu hamil trimester II yang memiliki umur kehamilan 20 sejumlah 56,7 %. menunjukkan distribusi frekuensi karakteristik responden Desa Lobu tahun 2019. Terlihat dari 30 responden, hampir seluruh responden berumur 20 tahun sejumlah 80 %, sebagian besar berpendidikan Sekolah Dasar (SD) sejumlah 66,7 %, hampir seluruh responden sedang hamil anak ke dua atau lebih (multipara) sejumlah 86,7 % dan sebagian besar ibu hamil trimester II yang memiliki umur kehamilan 20 frekuensi karakteristik responden di Wilayah Kerja hampir seluruh responden berumur 20 – 35 tahun sejumlah 80 %, sebagian besar berpendidikan Sekolah Dasar (SD) sejumlah 66,7 %, multipara) sejumlah 86,7 % dan sebagian besar ibu hamil trimester II yang memiliki umur kehamilan 20 – 28 minggu Diagram 1 menunjukkan distribusi Desa Lobu tahun 2019. Terlihat tahun sejumlah 80 %, sebagian besar berpendidikan Sekolah Dasar (SD) sejumlah 66,7 %, hampir seluruh responden sedang hamil anak ke dua atau lebih ( dan sebagian besar ibu hamil trimester II yang memiliki umur kehamilan 20 sejumlah 56,7 %. menunjukkan distribusi frekuensi karakteristik responden Desa Lobu tahun 2019. Terlihat dari 30 responden, hampir seluruh responden berumur 20 tahun sejumlah 80 %, sebagian besar berpendidikan Sekolah Dasar (SD) sejumlah 66,7 %, hampir seluruh responden sedang hamil anak ke dua atau lebih (multipara) sejumlah 86,7 % dan sebagian besar ibu hamil trimester II yang memiliki umur kehamilan 20 frekuensi karakteristik responden di Wilayah Kerja hampir seluruh responden berumur 20 – 35 tahun sejumlah 80 %, sebagian besar berpendidikan Sekolah Dasar (SD) sejumlah 66,7 %, multipara) sejumlah 86,7 % dan sebagian besar ibu hamil trimester II yang memiliki umur kehamilan 20 – 28 minggu 38 Diagram 2. PEMBAHASAN Pengetahuan menjadi dasar setiap manusia untuk bertindak atau melakukan sesuatu dalam kehidupan. Pengetahuan dipengaruhi oleh beberapa faktor diantaranya adalah faktor usia, pendidikan, ekonomi, pengalaman dan informasi, sosial budaya serta lingkungan (Dewie, 2021). Hasil analisa univariat pada penelitian ini menunjukkan pengetahuan responden sebelum penyuluhan dengan kategori kurang berjumlah 33,3 % responden. Menurut asumsi peneliti, hal ini disebabkan karena responden belum sepenuhnya mengerti dan mendapatkan informasi yang lengkap tentang senam hamil dilingkungan masyarakat, misalnya tenaga kesehatan baik yang ditingkat Puskesmas maupun dari bidan desa kurang memberikan penyuluhan mengenai senam hamil. Asumsi ini berdasarkan hasil konfirmasi dengan bidan koordinator Puskesmas Lobu pada saat pengambilan data awal bahwa selama setahun terakhir tidak pernah diadakan penyuluhan senam hamil di Puskesmas Lobu. Sedangkan responden dengan kategori pengetahuan baik sebelum penyuluhan senam hamil sebanyak 66,4 responden. Menurut asumsui peneliti, responden yang berpengetahuan baik dikarenakan sudah pernah mengikuti penyuluhan dan pelatihan senam hamil pada kehamilan sebelumnya. Setelah diberikan penyuluhan terjadi peningkatan pengetahuan yang signifikan yaitu 100 % responden berada pada kategori baik. Menurut asumsi peneliti, peningkatan pengetahuan pada semua responden dalam penelitian ini dipengaruhi oleh beberapa faktor antara lain pemilihan metode penyuluhan yaitu metode ceramah, memberikan leaflet disertai diskusi dan tanya jawab sehingga responden pun memahami apa yang disampaikan. Selain itu materi senam hamil ditampilkan melalui slide powerpoint dimana responden lebih antusias untuk mendengarkan dan memberi respon yang baik. Hasil uji bivariat menggunakan wilcoxon diperolah hasil nilai ρ < 0,001, menunjukkan bahwa terdapat peningkatan pengetahuan yang signifikan pada responden ibu hamil setelah diberikan penyuluhan mengenai senam hamil. Kelas ibu hamil merupakan salah satu kegiatan yang dilakukan oleh Puskesmas sebagai usaha untuk memberi pemahaman kepada ibu hamil dan keluarga mengenai hal-hal yag akan dilewati selama hamil hingga melahirkan dan masa nifas, untuk bayi maupun ibu dan keluarga (Departemen Kesehatan Republik Indonesia, 2009). Senam hamil adalah salah satu program dari kelas ibu hamil yang dilakukan untuk memberikan aktivitas fisik ringan pada ibu hamil. Aktifitas fisk dalam hal ini senam hamil dapat meningkatkan kebugaran fisik, menjaga berat badan, mencegah serta mengurangi nyeri panggul dan punggung bawah pada ibu hamil. Senam hamil juga terbukti mengurangi durasi pada fase persalinan, total durasi persalinan serta menurunkan resiko terjadinya induksi persalinan (Lee, et al., 2020). Olahraga tiga kali setiap minggu dengan durasi selama 30 menit dapat memberikan manfaat yang besar pada ibu dan juga berpengaruh positif pada kesehatan bayi (Alvis et al., 2019). HASIL PENELITIAN n Median (min-max) 30 73,00 (40-86) 100 (93-100) Pengaruh Penyuluhan terhadap Peningkatan Pengetahuan Tentang ρ-Value <0,001 Tabel 2. Hasil Uji Statistik Pengaruh Penyuluhan terhadap Peningkatan Pengetahuan Tentang Senam Hamil di Wilayah Kerja Penyuluhan Sebelum penyuluhan Sesudah penyuluhan Pengaruh Penyuluhan terhadap Peningkatan Pengetahuan Tentang Wilayah Kerja Puskesmas Lobu. n Median (min-max) 30 73,00 (40-86) 100 (93-100) Pengaruh Penyuluhan terhadap Peningkatan Pengetahuan Tentang ρ-Value <0,001 Hasil Uji Statistik Pengaruh Penyuluhan terhadap Peningkatan Pengetahuan Tentang amil di Wilayah Kerja Pengaruh Penyuluhan terhadap Peningkatan Pengetahuan Tentang Wilayah Kerja Puskesmas Lobu. Pengaruh Penyuluhan terhadap Peningkatan Pengetahuan Tentang Sumber : Data primer 2019 39 Untuk Uji statistik bivariat, setelah dilakukan uji normalitas, terlihat bahwa uji statistik yang harus digunakan adalah non parametrik, yaitu uji Wilcoxon. Pada tabel 2 Terlihat bahwa sebelum dilakukan penyuluhan, kuesioner responden menunjukkan nilai antara 40 – 86, namun setelah diberikan penyuluhan, nilai kuesioner responden meningkat menjadi 93 – 100. Hasil uji Wilcoxon diperoleh hasil significancy<0,001 (ρ-Value < 0,05) sehingga disimpulkan terdapat perbedaan pengetahuan mengenai senam hamil yang bermakna pada ibu hamil antara sebelum dengan sesudah penyuluhan tentang senam hamil di Wilayah Kerja Puskesmas Lobu. PEMBAHASAN j g g y ( ) Perubahan perilaku kesehatan dapat terjadi melalui pendidikan kesehatan.Baik untuk individu, kelompok maupun masyarakat. Penyuluhan kesehatan adalah salah satu cara pendidikan kesehatan yang dapat meningkatkan pengetahuan (Notoatmodjo, 2012). Penyuluhan diklaim dapat meningkatkan pengetahuan dan juga sikap peserta yang mengikutinya (Yulinda & Fitriyah, 2018). Pengetahuan adalah hasil pengindraan manusia atau hasil tahu seseorang terhadap obyek melalui indra yang dimilikinya (mata, hidung, telinga dan sebagainya). Sebagian besar pengetahuan seseorang diperoleh melalui indra pendengaran (telinga) dan indra penglihatan (mata) (Notoatmodjo, 2012). Dengan dasar pengetahuan, diharapkan perubahan perilaku yang terjadi dapat berlangsung jangka panjang (Dewie, 2021). Pengetahuan ibu hamil mengenai senam hamil membuat ibu menjadi lebih menyadari bahwa senam hamil dapat memberikan manfaat yang optimal pada ibu selama kehamilan, menyiapkan proses persalinan yang lancar dan mempercepat pemulihan ibu dimasa nifas (Kementerian 40 Kesehatan Republik Indonesia, 2014). Semakin baik pengetahuan ibu hamil, maka dirinya akan lebih mengetahui bagaimana harus bersikap ketika menjalani kehamilannya. Penyuluhan kesehatan merupakan suatu proses belajar untuk mengembangkan pengertian yang benar dan sikap yang positif dari individu atau kelompok terhadap kesehatan. Proses penyuluhan tidak dilaksanakan begitu saja tetapi harus dengan perencanaan yang adekuat, menggunakan perangkat – perangkat dan tekhnik yang baik sehingga proses bisa berjalan dengan baik (Syafrudin & Fratidhina, 2009). Hal ini sejalan dengan salah satu penelitian di Sleman yang mengindikasikan bahwa metode penyuluhan dengan metode ceramah dengan alat bantu leaflet dapat meningkatkan tingkat pengetahuan responden (Cahyaningsih, et al., 2013). Adapula hasil penelitian yang menyatakan bahwa penggunaan audio visual dalam memberikan penyuluhan sangat mempermudah seseorang dalam menerima suatu informasi sehingga meningkatkan pengetahuan (Idris & Enggar, 2019). Sebuah penelitian juga mendukung penggunaan leaflet sebagai media penyuluhan dengan menunjukkan bukti peningkatan skor yang signifikan sebelum dan sesudah pemberian penyuluhan kesehatan dengan menggunakan media leaflet (Alvis, et al., 2019). g gg ( ) Pemiihan metode penyuluhan tergantung tujuan yang akan dicapai dalam penyuluhan tersebut. Membicarakan tujuan penyuluhan, berarti akan berkisah masalah perubahan. Pemilihan metode diskusi dan curah pendapat dalam penyuluhan memberikan kesempatan pada sasaran untuk mengemukakan pendapatnya sehingga ikut aktif dalam proses belajar mengajar sehingga terbina komunikasi dua arah (twoway method) (Syafrudin & Fratidhina, 2009). Semakin banyak panca indra yang digunakan dalam memperoleh pengetahuan, maka semakin banyak dan semakin jelas pula pengertian atau pengetahuan yang diperoleh. PEMBAHASAN Menurut penelitian para ahli, panca indra yang paling banyak menyalurkan pengetahuan ke otak adalah mata (kurang lebih 75% - 87%), sedangkan 13% - 25% pengetahuan manusia diperoleh atau disalurkan melalui indra lainnya (Notoatmodjo, 2011). y ( j ) Sejalan dengan penelitian (Andries, et al., 2015) yang menyatakan bahwa dengan pemberian penyuluhan kesehatan yang efektif bahkan langsung dengan gerakan senam hamil dapat mempengaruhi pengetahuan seseorang. Demikian pula penelitian (Kusumawardani, et al., 2012) menyatakan bahwa pada kelompok yang mendapat penyuluhan kesehatan, terjadi peningkatan pengetahuan, sikap, dan praktik yang ditunjukan dengan perubahan skor yang semakin meningkat.Pengetahuan yang sudah baik ini hendaknya dipertahankan dengan menggali lebih mendalam pengetahuan tentang senam hamil dengan cara pemberian informasi melalui promosi kesehatan atau penjelasan dari petugas kesehatan di Puskesmas dilengkapi dengan leaflet, poster atau menggunakan slide powerpoint agar pemberian informasi lebih menarik dan informatifhingga dapat diterima secara maksimal. KESIMPULAN Kesimpulan dari penelitian ini adalah ada pengaruh yang bermakna antara penyuluhan senam hamil terhadap tingkat pengetahuan ibu hamil diwilayah Kerja Puskesmas Lobu. Diharapkan semua ibu hamil meningkatkan motivasi untuk mengikuti penyuluhan senam hamil dan petugas kesehatan lebih giat dalam melakukan promosi mengenai senam hamil dengan menggunakan berbagai media dan menyelenggarakan kegiatan senam hamil secara berkesinambungan. Alvis, M. L., Morris, C. E., Garrard, T. L., Hughes, A. G., Hunt, L., Koester, M. M., Yocum, I. C., & Tinius, R. A. (2019). Educational Brochures Influence Beliefs and Knowledge Regarding Exercise during Pregnancy: A Pilot Study. International Journal of Exercise Science, 12(3), 581–589. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/31156748%0Ahttp://www.pubmedcentral.nih.gov/artic lerender.fcgi?artid=PMC6533107 Andries, S., Adam, S., & Montolalu, A. (2015). Pengaruh Penyuluhan Tentang Senam Hamil Terhadap Peningkatan Pengetahuan Ibu Hamil. Jurnal Ilmiah Bidan, 3(2), 66–71. Cahyaningsih, I., Wiedyaningsih, C., & Kristina, S. A. (2013). Pengaruh Penyuluhan Terhadap REFERENSI Alvis, M. L., Morris, C. E., Garrard, T. L., Hughes, A. G., Hunt, L., Koester, M. M., Yocum, I. C., & Tinius, R. A. (2019). Educational Brochures Influence Beliefs and Knowledge Regarding Exercise during Pregnancy: A Pilot Study. International Journal of Exercise Science, 12(3), 581–589. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/31156748%0Ahttp://www.pubmedcentral.nih.gov/artic lerender.fcgi?artid=PMC6533107 Andries, S., Adam, S., & Montolalu, A. (2015). Pengaruh Penyuluhan Tentang Senam Hamil Terhadap Peningkatan Pengetahuan Ibu Hamil. Jurnal Ilmiah Bidan, 3(2), 66–71. Cahyaningsih, I., Wiedyaningsih, C., & Kristina, S. A. (2013). Pengaruh Penyuluhan Terhadap 41 Tingkat Pengetahuan Masyarakat Tentang ANalgetik di Kecamatan Cangkringan Sleman. Mutiara Medika, 13(2), 98–104. Departemen Kesehatan Republik Indonesia. (2009). Pedoman Pelaksanaan Kelas Ibu Hamil. Jakarta: Departemen Kesehatan Republik Indonesia. Dewie, A. (2021). Pengetahuan dan Sikap Tentang Tanda Bahaya Kehamilan Berhubungan Dengan Pemanfaatan Buku KIA. Jambi Medical Journal, 9(2), 138-146. Retrieved from https://online-journal.unja.ac.id/kedokteran/article/view/12841 j j Dinas Kesehatan Kabupaten Banggai. (2018). Profil Kesehatan Kabupaten Banggai Tahun 2017. Banggai: Dinas Kesehatan Kabupaten Banggai. Idris, I., & Enggar, E. (2019). Pengaruh Penyuluhan Menggunakan Audio Visual Tentang Asi Eksklusif Terhadap Pengetahuan Dan Sikap Ibu Hamil Di Puskesmas Singgani Kota Palu. Jurnal Bidan Cerdas (JBC), 2(1), 1. Retrieved from https://doi.org/10.33860/jbc.v2i1.159 ( ) ( ) p g j Kementerian Kesehatan Republik Indonesia. (2011). Buku Panduan Kader Posyandu Menuju Keluarga Sadar Gizi. Jakarta: Kementerian Kesehatan Republik Indonesia. Kementerian Kesehatan Republik Indonesia. (2014). Pegangan Fasilitator Kelas Ibu Hamil. Jakarta: Kementerian Kesehatan Republik Indonesia. Kementerian Kesehatan Republik Indonesia. (2018). Profile Kesehatan Indonesia Tahun 2017. Jakarta: Kementerian Kesehatan Republik Indonesia. Kusumawardani, E., Arkhaesi, N., & Hardian, H. (2012). Pengaruh Penyuluhan Kesehatan Terhadap Tingkat Pengetahuan, Sikap Dan Praktik Ibu Dalam Pencegahan Demam Berdarah Dengue Pada Anak. In Fakultas Kedokteran Diponegoro. g p g Lee, C. F., Lin, Y. H., Chi, L. K., Lin, H. M., & Huang, J. P. (2020). The Evidence Base in Exercise Knowledge of Pregnant Women: A Latent Class Analysis. Worldviews on Evidence-Based Nursing, 17(6), 437–447. Retrieved from https://doi.org/10.1111/wvn.12466 Notoatmodjo, S. (2010). Metode Penelitian (1st ed.). Jakarta: Rineka Cipta. j ( ) ( ) p Notoatmodjo, S. (2011). Kesehatan Masyarakat Ilmu dan Seni. Jakarta: Rineka Cip Notoatmodjo, S. (2012). Promosi Kesehatan dan Perilaku Kesehatan (Edisi Revisi 2012). Jakarta: Rineka Cipta. Syafrudin, & Fratidhina, Y. (2009). Promosi Kesehatan Untuk Mahasiswa Kebidanan. Jakarta: Trans Info Media. Yulinda, A., & Fitriyah, N. (2018). Efektivitas Penyuluhan Metode Ceramah Dan Audiovisual Dalam Meningkatkan Pengetahuan Dan Sikap Tentang sadari di SMKN 5 Surabaya. Jurnal Promkes, 6(2), 116–128. REFERENSI Retrieved from https://e- journal.unair.ac.id/PROMKES/article/viewFile/6439/5917 Dalam Meningkatkan Pengetahuan Dan Sikap Tentang sadari di SMKN 5 Surabaya. Jurnal Promkes, 6(2), 116–128. Retrieved from https://e- journal.unair.ac.id/PROMKES/article/viewFile/6439/5917 42
https://openalex.org/W3168164847	https://eprints.walisongo.ac.id/id/eprint/19634/1/Challenges%20of%20Sharia%20Banking%20Notaries%20in%20Indonesia%27s.pdf	English	null	Challenges of Sharia Banking Notaries in Indonesia's Economic Development in the Global Era	Procedia of Social Sciences and Humanities	2,021	cc-by	2,451	Procedia of Social Sciences and Humanities Proceding of the International Conference on Intellectuals’ Global Responsibility 2020 (ICIGR): Science for Handling the Effects of Covid-19, Facing the New Normal, and Improving Public Welfare Procedia of Social Sciences and Humanities Proceding of the International Conference on Intellectuals’ Global Responsibility 2020 (ICIGR): Science for Handling the Effects of Covid-19, Facing the New Normal, and Improving Public Welfare Procedia of Social Sciences and Humanities Proceding of the International Conference on Intellectuals’ Global Responsibility 2020 (ICIGR): Science for Handling the Effects of Covid-19, Facing the New Normal, and Improving Public Welfare Challenges of Sharia Banking Notaries in Indonesia's Economic Development in the Global Era Ro’fah Setyowati, Bagas Heradhyaksa rofah@live.undip.ac.id, bagashera@walisongo.ac.id Faculty of Law, Universitas Diponegoro. Faculty of Sharia and Law, UIN Walisongo Semarang Abstract. Notaries have a strategic position in making Islamic banking contracts. The reason is that the notary is responsible for the correctness of the contract construction to fulfill the terms of the agreement and the sharia principles. This study aims to find the philosophy of juridical consequences of the notary profession relationship with Islamic banking, which is associated with challenges in the global era. This research uses a philosophical, juridical, and empirical approach. The analysis results show that a notary who has sharia competence and understands and also carries out the philosophy of juridical consequences of the profession is very much needed. This is due to the growing challenges in developing Islamic banking globally, particularly about competition due to advances in information technology. Keywords: Opportunities, Notaries, Islamic Banking, Indonesia, Global Era. ISSN 2722-0672 (online), https://pssh.umsida.ac.id. Published by Universitas Muhammadiyah Sidoarjo Copyright (c) 2021 Author (s). This is an open-access article distributed under the terms of Creative Commons Attribution License (CC BY). To view a copy of this license, visit https://creativecommons.org/licenses/by/4.0/. 2. Discussion A Notary is obliged to provide services by existing regulations unless there is a reason to refuse it. (7) Besides, as a public official, a Notary Public must uphold the precautionary principle, because the responsibility of a Notary for deeds he makes is for life. In doing authentic deeds, notaries must prioritize the principle of prudence, especially concerning acts regarding agreements. This is because deeds regarding arrangements generally have legal consequences if there is a violation of the contract by the parties. Not a few deeds made by notaries have problems at a later date. If there is a sharia banking dispute arising from a declared invalid and null and void contract, the notary cannot be separated from responsibility for the incident. This is because the position of notarial deeds is significant as evidence in dispute resolution.(8) Therefore, a notary may not neglect the principle of prudence in determining legal actions in a deed. As well as making this precautionary principle the main principle in carrying out their duties as a public official. The notary must genuinely be responsible for the deed's accuracy, including one in the credit agreement or financing to banks.(9) This legal responsibility comes from the Notary Position Law, Criminal Law, and Civil Law.(10) The notary is required to be able to make a balance between the rights and obligations of the parties who agree. This is because later the agreement is a rule that the parties in question will obey.(11) In the banking world, especially Islamic banking, notaries have the authority to make administrative legal actions. Notaries can make various kinds of contracts or agreements regarding financing, lending, and borrowing, buying and selling, leasing, auction minutes, and contracts required by the parties. Notaries are also always needed to obey and obey any regulations that govern their position and other laws. This is intended so that the public can truly interpret the Notary profession as one of the noble and dignified jobs. Therefore, there are professional values that must be obeyed by them, namely as follows: a) Honesty, b) Authentic; c) Responsible; d) Moral independence; e) Moral courage.(12) If it is related to Islamic banking, the notary's responsibility is to control the scope of notary law, criminal law, civil law, and moral ethics and practice Islamic law, especially those related to muamalah. ISSN 2722-0672 (online), https://pssh.umsida.ac.id. Published by Universitas Muhammadiyah Sidoarjo Copyright (c) 2021 Author (s). This is an open-access article distributed under the terms of Creative Commons Attribution License (CC BY). To view a copy of this license, visit https://creativecommons.org/licenses/by/4.0/. Procedia of Social Sciences and Humanities Proceding of the International Conference on Intellectuals’ Global Responsibility 2020 (ICIGR): Science for Handling the Effects of Covid-19, Facing the New Normal, and Improving Public Welfare 1. Introduction Indonesia's Islamic banking industry has excellent potential to become a global player in Islamic finance. The first reason is that the large Muslim population is a potential customer of the Islamic financial industry. The latest census conducted by the government shows that 87.1% of the Indonesian people or around 270 million people adhere to Islam.(1) The increase in Indonesia's sovereign credit rating to investment grade will increase investor interest in investing in the domestic financial sector, including the Islamic finance industry. It has abundant natural resources that can be used as underlying transactions for the Islamic financial.(2) Globalization has many consequences. One of them is the expansion of equity and various risk financing activities. This will pave the way for further growth in the Islamic banking system(3). Globalization has many consequences. One of them is the expansion of equity and various risk financing activities. This will pave the way for further growth in the Islamic banking system.(4) This view is logical, considering that the contract is made, the notary has full responsibility, regarding the correctness of the contract construction, so that the terms of the agreement are fulfilled, both from the substance of Islamic law, as well as the format and systematics, based on the relevant legislation. Berangkat Based on some of these thoughts and references, it is essential to deepen the Islamic banking Notary profession's challenges, especially in the global era. This research is intended to explain the phenomena that occur in human activities. This aims to reinforce the hypothesis to help formulate new theories(5). This study aims to provide a new theory for notaries in Islamic banking is facing challenges in the cyber era. The research method used is the library research method. Library data is obtained through library research sourced from official documents.(6) 57/332 57/332 Procedia of Social Sciences and Humanities Proceding of the International Conference on Intellectuals’ Global Responsibility 2020 (ICIGR): Science for Handling the Effects of Covid-19, Facing the New Normal, and Improving Public Welfare deeds. (15) By observing developments in several countries, both those with Common Law and Civil Law styles, many countries have empowered their notaries' function and role in electronic transactions. Therefore, Indonesia must stimulate notary services in electronic transactions, even up to the implementation of notary services itself electronically. (16) deeds. (15) By observing developments in several countries, both those with Common Law and Civil Law styles, many countries have empowered their notaries' function and role in electronic transactions. Therefore, Indonesia must stimulate notary services in electronic transactions, even up to the implementation of notary services itself electronically. (16) World Bank research results(17) The 2014 Global Findex Database shows that only around 36.1% of the population aged over 15 years have used formal financial institutions, with only 28.7% of the people in rural areas over 15 having an account with a legal financial institution. This data confirms that the market potential for Islamic banking development is still huge, especially in Indonesia. This shows that the opportunity for Islamic banking notaries is still huge. This is because the market potential is still immense, but technological developments never stop. Therefore, Muslim Notaries who have the awareness to participate in the Islamic banking industry's development need to continue to strive to strengthen five attitudes or characters, namely believing, understanding, practicing, disseminating, and being patient in finding solutions. Even though there is no valid data on the number of Notaries who already have sharia eligibility, it can be assumed that there are not many notaries who are sharia eligible. This is based on introducing the concept and operation of Islamic financial institutions and the making of the contract has not been significant enough to be held specifically for Notaries. This is the actual role expected of the Notaries in the development of Islamic banking. Another thing of concern is related to sharia notaries in Islamic banking. Indonesian Ulema Council Fatwa No. 1 of 2004 states that bank interest includes Riba and Haram. Meanwhile, interest, of course, involves conventional banks. Meanwhile, to become a partner of a sharia bank, a notary must be a partner of a conventional bank. This means that a notary must make a deed relating to the Ribawi case. ISSN 2722-0672 (online), https://pssh.umsida.ac.id. Published by Universitas Muhammadiyah Sidoarjo Copyright (c) 2021 Author (s). This is an open-access article distributed under the terms of Creative Commons Attribution License (CC BY). To view a copy of this license, visit https://creativecommons.org/licenses/by/4.0/. Procedia of Social Sciences and Humanities This study proposes that the Indonesian Ulema Council be able to make a fatwa so that it becomes a legal reference stating that those who become partners with sharia banks must be notaries with sharia compliance. The characteristics are as follows. First, do not become a partner of a conventional bank. Second, understanding sharia laws, especially related to Islamic bank contracts. and to make it happen, a competency test in accordance with sharia compliance is required. 2. Discussion Believing and implementing Islamic law principles is an expected role for a sharia banking notary from a philosophical perspective. OJK has understood the need for philosophical-juridical consequences. Therefore, it is suitable for the 2015-2019 Sharia Banking Roadmap(13) and the 2017-2019 Sharia Financial Roadmap(14) has been stated in the work program, in the form of Capacity Building for Human Resources. The sub-program is aimed at developing a Certification Program and Continuing Professional Education. The program's action is to encourage a certification program and Continuing Professional Education for the profession that contains Islamic finance material. The current condition shows that many occupations are still related to the financial industry, such as accountants, appraisers, actuaries, notaries, legal consultants, and other disciplines, which do not understand Islamic finance. Therefore, the Financial Services Authority needs to encourage a certification program and Continuing Professional Education for professions that contain Islamic finance material.(14) Another challenge in the notary profession is related to the cyber or electronic era. This gave rise to a cyber notary. Section 15 sub-section 3 of the Notary Law's amendment stipulates that notaries also have other powers that are regulated in statutory regulations. In the elucidation of section 15 sub-section 3, the other powers referred to include the authority to certify transactions conducted electronically or cyber notary. The concept of a cyber notary can temporarily be interpreted as a notary who carries out his / her duties or authority based on information technology, which is related to the notary's duties and functions, especially in doing 58/332 Procedia of Social Sciences and Humanities Proceding of the International Conference on Intellectuals’ Global Responsibility 2020 (ICIGR): Science for Handling the Effects of Covid-19, Facing the New Normal, and Improving Public Welfare still not ideal. This conclusion is based on a philosophical approach by exploring the relationship between the notary concept, Islamic banking, and the global era. In simple language, the opportunities and challenges for Islamic banking notaries increase linearly. Therefore, Muslim Notaries are expected to understand and do their best to fulfill both the desired role and the essential role to contribute to the development of Islamic banking. 3. Conclusion The financial services industry, particularly banking, has significantly improved people's spiritual welfare, especially the Muslim community in Indonesia. Therefore, as a profession that is inherent in contract making, the notary has both opportunities and significant challenges in the development of the Islamic banking industry. The growing number of Islamic banking service office networks and the relatively small number of asset customers shows the potential for the Islamic banking market is still very large. Meanwhile, the challenges of notaries in Islamic banking are also getting more significant. Some of the demands required by a notary for Islamic banking include: 1) The notary must believe that the concept of the non-ribawi transaction is the design of Allah SWT, which has the highest benefit value compared to other ideas at any time, including in the global era; 2) Notaries must understand and be skilled in expressing their knowledge related to their functions and positions, with instruments developing in the worldwide period; 3) Notaries should also practice this concept in their daily transaction activities by global developments; 4) The notary is also obliged to convey or ensure the superiority of this non-ribawi bank concept to the parties facing him; 5) Notaries must be patient and participate in fighting by the era if the application of the idea is 59/332 ISSN 2722-0672 (online), https://pssh.umsida.ac.id. Published by Universitas Muhammadiyah Sidoarjo Copyright (c) 2021 Author (s). This is an open-access article distributed under the terms of Creative Commons Attribution License (CC BY). To view a copy of this license, visit https://creativecommons.org/licenses/by/4.0/. References 1. Statistik BP. Hasil Sensus Penduduk 2010. Jakarta: Badan Pusat Statistik; 2011. 10 1. Statistik BP. Hasil Sensus Penduduk 2010. Jakarta: Badan Pusat Statistik; 2011. 10 p. 2. Alamsyah H. Perkembangan dan Prospek Perbankan Syariah Indonesia: Tantangan Dalam Menyongsong MEA 2015. p. 1. 2. Alamsyah H. Perkembangan dan Prospek Perbankan Syariah Indonesia: Tantangan Dalam Menyongsong MEA 2015. p. 1. 3. Marzuki SN. Bank Syariah Dindonesia (Peluang dan Tantangan Di Era Globalisasi). J Ekon Syariah. 2018;I(1):79–90. 3. Marzuki SN. Bank Syariah Dindonesia (Peluang dan Tantangan Di Era Globalisasi). J Ekon Syariah. 2018;I(1):79–90. 4. Yusup DK. Peran Notaris Dalam Praktek Perjanjian Bisnis Di Perbankan Syariah (Tinjauan Dari Perspektif Hukum Ekonomi Syariah). J Al-’Adalah. XXI(4):701. 5. Soekanto S. Pengantar Penelitian Hukum. Jakarta: Penerbit Universitas Indonesia; 1986. 10 p. 6. Ali Z. Metode Penelitian Hukum. Jakarta: Sinar Grafika; 2014. 107 p. 7. Setiawan W. Sikap Profesionalisme Notaris dalam Pembuatan Akta Otentik. Media Notariat. 2004 May;25. 8. Masriani YT. The Position of Notariial Deed in the Syaria Economic Dispute. Mimb Huk. 2016;28(1):162–73. ( ) 9. Muhammad AK. Etika Profesi Hukum. Bandung: Citra Aditya Bakti; 2001. 94 p. 10. Latumeten P. Pertanggungjawaban Hukum Profesi Notaris. 2014. 11. Adjie H. Kebatalan dan Pembatalan Akta Notaris. Bandung: Refika Aditama; 2011. 126 p. 12. Fuady M. Profesi Mulia. Bandung: Citra Aditya Bakti; 2005. 4 p. 3. OJK. Roadmap Perbankan Syariah. Jakarta; 2017. 14. OJK. Roadmap Keuangan Syariah. Jakarta; 2019. 15. Nurita E. Cyber Notary. Bandung: Refika Aditama; 2012. xii. 16. Makarim E. Notaris dan Transaksi Elektronik. 2nd ed. Jakarta: Rajawali Press; 2013. 133 p. 17. Demirguc-Kunt, Leora Klapper. The Global Findex Database. 2014. 60/332
https://openalex.org/W1969973578	https://opencommons.uconn.edu/cgi/viewcontent.cgi?article=1077&context=uchcres_articles	English	null	Borrelia burgdorferi Requires the Alternative Sigma Factor RpoS for Dissemination within the Vector during Tick-to-Mammal Transmission	PLOS pathogens	2,012	cc-by	14,868	Borrelia burgdorferi Requires the Alternative Sigma Factor RpoS for Dissemination within the Vector during Tick-to-Mammal Transmission Star M. Dunham-Ems University of Connecticut School of Medicine and Dentistry Star M. Dunham-Ems University of Connecticut School of Medicine and Dentistry Melissa J. Caimano University of Connecticut School of Medicine and Dentistry Melissa J. Caimano University of Connecticut School of Medicine and Dentistry Justin D. Radolf University of Connecticut School of Medicine and Dentistry ollow this and additional works at: https://opencommons.uconn.edu/uchcres_articles Follow this and additional works at: https://opencommons.uconn.edu/uchcres_articles Part of the Life Sciences Commons, and the Medicine and Health Sciences Commons Part of the Life Sciences Commons, and the Medicine and Health Sciences Commons Part of the Life Sciences Commons, and the Medicine and Health Sciences Commons UCHC Articles - Research University of Connecticut Health Center Research UCHC Articles - Research University of Connecticut Health Center Research University of Connecticut OpenCommons@UConn University of Connecticut OpenCommons@UConn Dunham-Ems, Star M.; Caimano, Melissa J.; and Radolf, Justin D., "Borrelia burgdorferi Requires the Alternative Sigma Factor RpoS for Dissemination within the Vector during Tick-to-Mammal Transmission" (2012). UCHC Articles - Research. 78. https://opencommons.uconn.edu/uchcres_articles/78 Recommended Citation Dunham-Ems, Star M.; Caimano, Melissa J.; and Radolf, Justin D., "Borrelia burgdorferi Requires the Alternative Sigma Factor RpoS for Dissemination within the Vector during Tick-to-Mammal Transmission" (2012). UCHC Articles - Research. 78. https://opencommons.uconn.edu/uchcres_articles/78 Borrelia burgdorferi Requires the Alternative Sigma Factor RpoS for Dissemination within the Vector during Tick-to-Mammal Transmission Abstract * E-mail: jradolf@up.uchc.edu (BB0763) and the DNA-binding protein and Fur orthologue, BosR (BB0647) [10–12]. Additional levels of control are provided by the small RNAs hfq (bb0268) and dsrA (bb0577) as well as the carbon storage regulator csrA (bb0184) [12]. Based upon micro- array analysis of mammalian host-adapted spirochetes [13], it was proposed that RpoS functions as a gatekeeper for controlling the upregulation of mammalian host-phase genes (e.g., ospC) and the downregulation of tick-phase genes (e.g., ospA). By quantitative real time PCR (qRT-PCR), we established that rpoS is induced during the nymphal blood meal but is not expressed by spirochetes within replete larvae or unfed nymphs, thereby delineating the RpoS ON (fed nymph) and OFF (fed larvae and unfed nymph) states during the tick phase of the enzootic cycle [13,14]. Expression patterns of RpoS-dependent genes in the tick, however, reflect more than a simple ON/OFF bipartite regulation. For example, OspC is induced within the midgut during nymphal feeding but not by all spirochetes [14–16]. In addition, OspA continues to be expressed at high levels in the midgut after engorgement and is ‘‘OFF’’ only after organisms have completely mammalian host-adapted [14,16– 18]. Within the mammal, RpoS-dependent genes are required to establish infection and presumably continue to be expressed until spirochetes are acquired by naı¨ve larvae [12,14,19,20]. Abstract While the roles of rpoSBb and RpoS-dependent genes have been studied extensively within the mammal, the contribution of the RpoS regulon to the tick-phase of the Borrelia burgdorferi enzootic cycle has not been examined. Herein, we demonstrate that RpoS-dependent gene expression is prerequisite for the transmission of spirochetes by feeding nymphs. RpoS-deficient organisms are confined to the midgut lumen where they transform into an unusual morphotype (round bodies) during the later stages of the blood meal. We show that round body formation is rapidly reversible, and in vitro appears to be attributable, in part, to reduced levels of Coenzyme A disulfide reductase, which among other functions, provides NAD+ for glycolysis. Our data suggest that spirochetes default to an RpoS-independent program for round body formation upon sensing that the energetics for transmission are unfavorable. Citation: Dunham-Ems SM, Caimano MJ, Eggers CH, Radolf JD (2012) Borrelia burgdorferi Requires the Alternative Sigma Factor RpoS for Dissemination within the Vector during Tick-to-Mammal Transmission. PLoS Pathog 8(2): e1002532. doi:10.1371/journal.ppat.1002532 Editor: Jenifer Coburn, Medical College of Wisconsin, United States of America Received October 10, 2011; Accepted December 28, 2011; Published February 16, 2012 pyright: 2012 Dunham-Ems et al. This is an open-access article distributed under the terms of the Creative Commons Attribution restricted use, distribution, and reproduction in any medium, provided the original author and source are credited. ham-Ems et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits tion, and reproduction in any medium, provided the original author and source are credited. Copyright: 2012 Dunham-Ems et al. This is an open-access article distributed under the terms of the Creative Commons Att unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This research was supported by NIH R01AI029735 (JDR and MJC) and 3R01AI029735-20S1 23 (JDR and MJC), NIH R03AI085248 (MJC), NIH U54AI057159 (SDE), the 1 National Research Fund for 2 Tick-Borne Diseases (MJC), and the Quinnipiac University School of Health Sciences Faculty Research Award (CHE). SDE is the recipient of the New England Career Development award. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Competing Interests: The authors have declared that no competing interests exist. M. Dunham-Ems1, Melissa J. Caimano1, Christian H. Eggers2, Justin D. Radolf1,3,4,5* 1 Department of Medicine, University of Connecticut Health Center, Farmington, Connecticut, United States of America, 2 Department of Biomedical Sciences, Quinnipiac University, Hamden, Connecticut, United States of America, 3 Department of Pediatrics, University of Connecticut Health Center, Farmington, Connecticut, United States of America, 4 Department of Genetics and Developmental Biology, University of Connecticut Health Center, Farmington, Connecticut, United States of America, 5 Department of Immunology, University of Connecticut Health Center, Farmington, Connecticut, United States of America PLoS Pathogens \| www.plospathogens.org February 2012 \| Volume 8 \| Issue 2 \| e1002532 Author Summary Lyme disease, caused by the spirochetal pathogen Borrelia burgdorferi, is the most prevalent arthropod-borne infec- tion in the United States. In order to maintain itself in nature, B. burgdorferi must cycle between its arthropod vector, Ixodes ticks, and a mammalian reservoir, usually a small rodent. Previous studies have demonstrated that the alternative sigma factor RpoS is essential for B. burgdorferi to infect a mammalian host, whereas a role within the tick has never been examined. In this study, we determined that one or more RpoS-dependent genes are required for B. burgdorferi to disseminate through the tick. Using a combination of microscopy techniques, we show that RpoS-deficient organisms are confined to the lumen of the tick midgut during nymphal feeding where they form round bodies, while wild-type spirochetes remain elon- gated and traverse the midgut to enter the hemolymph and salivary glands en route to the mammalian host. contours of RpoS-dependent gene expression within larval and nymphal life stages and demonstrate, for the first time, that RpoS is required for the transmission of Lyme disease spirochetes by feeding nymphs. Although RpoS-deficient spirochetes replicate at wild-type levels during the nymphal blood meal, mutant organisms display an aberrant morphology, previously referred to as round bodies or cysts [22–24], as feeding progresses. We show that round body formation is rapidly reversible, and in vitro appears to be attributable, in part, to reduced levels of Coenzyme A disulfide reductase, which among other functions, provides NAD+ for glycolysis. Our findings reveal a previously unsuspected role for RpoSBb in the physiological adaptations required for tick-to- mammal transmission as well as the existence of a proposed default survival mode (round body formation) triggered when spirochetes are unable to produce sufficient energy for dissemina- tion within the nymph. Borrelia Requires RpoS for Tick Transmission required for the dissemination of spirochetes during nymphal feeding. While it has long been known that ospC is induced during nymphal feeding [15], there has been controversy as to whether this lipoprotein functions in the tick or the mammal [19,25,26]. The strategy we devised also enabled us to clarify the site of OspC function. Because spirochetes lacking either RpoS or OspC are avirulent by needle inoculation [26,27], we used the immersion method [28] to introduce wild-type (WT, CE162), DrpoS (CE174), and DospC (CE303) isolates into naı¨ve larvae (Table 1); larvae infected by immersion feeding were allowed to molt into nymphs that then were fed on naı¨ve mice. As described in Materials and Methods and summarized in Table 2, we cultured (i) hemolymph at 72 h post-placement to monitor penetration through the midgut; (ii) skin excised from the bite site at 24, 48 and 72 h post-repletion to assess penetration through the salivary glands and transmission; and (iii) distant tissues at 2 and 4 weeks post-repletion to assess dissemination within the mouse. WT spirochetes were isolated from hemolymph at 72 h post-placement, from the bite site at 48 and 72 h post-repletion and from multiple tissues at 2 and 4 weeks post-repletion. The DrpoS isolate was not recovered from either hemolymph or distal sites, while the DrpoS-complemented isolate (CE467) was recovered from both. In contrast to DrpoS organisms, DospC Bb were recovered from hemolymph at 72 h post-placement and the bite site but only at 48 h post-repletion. Importantly, the time frames during which WT and DospC organisms were recovered from hemolymph cultures were highly similar in each experiment (n = 3; data not shown), suggesting that the cultured samples contained comparable numbers of organisms. Given that RpoS is essential for the stress responses of many bacteria [2,6], it is possible that spirochetes lacking this sigma factor were unable to survive within the midgut during feeding. qPCR and semi-solid plating, however, revealed virtually identical burdens of WT, DrpoS, and DospC isolates in unfed and replete nymphs; furthermore, similar incubation times were required for the recovery of WT and DrpoS organisms by semi-solid plating (Figure S1; p$0.05). Comparable results were obtained using fluorescent DrpoS mutant and complemented strains described in subsequent sections. Thus, B. burgdorferi requires one or more RpoS- dependent genes to traverse the midgut, while OspC functions exclusively within the mammal. Author Summary Introduction Bacterial genomes typically encode multiple alternative sigma factors that reversibly associate with the RNA polymerase apoenzyme to promote the transcription of specific subsets of genes in response to changing environmental conditions [1]. One of the best studied alternative sigma factors is RpoS, the master regulator of the general stress response in E. coli (RpoSEc) [2,3]. Regulation of rpoSEc is multifaceted, occurring at the transcriptional and translational levels, as well by proteolysis [2,3]. Induction of the RpoSEc regulon occurs in response to a variety of stressors, including low nutrient availability, high osmolarity, reactive oxygen intermediates and low pH [4]. Up to 10% of the genome may be regulated either directly or indirectly by RpoSEc with the composition of the regulon determined by the specific stressor [5]. RpoS orthologs exist in diverse Proteobacteria, including numerous pathogenic species, and have been shown to control genes involved in a wide range of adaptive processes [6]. For example, RpoS is essential for biofilm formation in Pseudomonas aeruginosa [7], growth and survival of Legionella pneumophila within phagolysosomes [8], and infectivity of mice by Salmonella enterica [9]. B. burgdorferi (Bb), the Lyme disease spirochete, uses just three sigma factors, s70, RpoN, and RpoS, to modulate its transcrip- tome during its enzootic cycle. Transcription of rpoSBb is directly activated by the alternative sigma factor RpoN (BB0450) with the aid of two enhancer proteins: the response regulator Rrp2 While the roles of rpoSBb and individual RpoS-dependent genes have been studied extensively within the mammal [21], the contribution of the RpoS regulon to the tick-phase of the enzootic cycle has yet to be examined. In this study, we defined the February 2012 \| Volume 8 \| Issue 2 \| e1002532 1 Borrelia Requires RpoS for Tick Transmission Results Lyme disease spirochetes require RpoS-dependent genes other than ospC to traverse the nymphal midgut RpoS-dependent gene expression by spirochetes in fed nymphs differs from that of mammalian host-adapted organisms adapted organisms were expressed at equal or higher levels by DrpoS-gfp spirochetes compared to WT-gfp (Figures 1C and S2B). Confocal microscopy of WT spirochetes harboring fluorescent transcriptional reporters for flaB or ospA was performed as an additional means of confirming that RpoS-repressed tick-phase genes are expressed throughout the nymphal blood meal. As shown in Figures 1D and 1E, the overall density and distribution of the two reporter strains within fed nymphal midguts were indistinguishable. Collectively, our findings indicate that genes whose expression is upregulated by RpoS are likely to be expressed both during and after transmission by nymphs while RpoS-dependent repression appears to occur only after spir- ochetes have fully adapted to the mammalian host. adapted organisms were expressed at equal or higher levels by DrpoS-gfp spirochetes compared to WT-gfp (Figures 1C and S2B). Confocal microscopy of WT spirochetes harboring fluorescent transcriptional reporters for flaB or ospA was performed as an additional means of confirming that RpoS-repressed tick-phase genes are expressed throughout the nymphal blood meal. As shown in Figures 1D and 1E, the overall density and distribution of the two reporter strains within fed nymphal midguts were indistinguishable. Collectively, our findings indicate that genes whose expression is upregulated by RpoS are likely to be expressed both during and after transmission by nymphs while RpoS-dependent repression appears to occur only after spir- ochetes have fully adapted to the mammalian host. g The above results established the importance of defining the RpoS regulon during the tick-phase of the enzootic cycle. At the present time, however, delineation of the spirochete’s transcrip- tome within ticks is not technically possible. Therefore, we used qRT-PCR to measure transcript levels for a panel of 10 RpoS- upregulated and 6 RpoS-repressed tick-phase genes derived from our microarray analysis of WT and DrpoS spirochetes cultivated within dialysis membrane chambers (DMCs) [13]. Representative results are shown in Figure 1, while results for the remainder of the panel are presented in Figure S2. All eight genes previously shown to be absolutely RpoS-dependent in DMC-cultivated spirochetes were induced during nymphal feeding with little to no transcript detectable in either fed larvae or unfed nymphs. Two of the upregulated Borrelia genes in our panel (bb0728/cdr and bb0670/ cheW3) were selected because they were transcribed by both RpoS and s70 [13]. In accord with these findings, transcripts for both genes were significantly enhanced in fed compared to unfed nymphs. Lyme disease spirochetes require RpoS-dependent genes other than ospC to traverse the nymphal midgut We began by devising an experimental strategy to test our hypothesis that one or more genes within the RpoS regulon are We began by devising an experimental strategy to test our hypothesis that one or more genes within the RpoS regulon are Table 1. Borrelia burgdorferi strains used in this study. Table 1. Borrelia burgdorferi strains used in this study. Strain Description Antibiotic Resistance1 Reference CE162 Wild-type 297 clone NA [27] CE174 CE162 DrpoS; non-fluorescent rpoS mutant Erm [27] CE303 CE162 DospC; non-fluorescent ospC mutant Kan This study CE467 CE174+rpoS/pCE320; non-fluorescent RpoS-complement Erm and Kan [36] Bb914 CE162 with PflaB-gfp in cp26; fluorescent wild-type Gent [30] Bb1058 CE174 with PflaB-gfp in cp26; fluorescent rpoS mutant Gent and Erm This study SE186 Bb1058+ rpoS/pCE320; fluorescent RpoS-complement Gent, Erm, and Kan This study CE56 CE162+PflaB-gfp/pCE320; fluorescent flaB transcriptional reporter Kan [59] CE103 CE162+PospA-gfp/pCE320; fluorescent ospA transcriptional reporter Kan [36] CE309 CE162 Dcdr; non-fluorescent Erm [29] CE1655 CE309+cdr/pCE323; non-fluorescent CoADR-complement Erm and Kan [29] 1Antibiotic resistance determined by growing spirochetes in the presence of the following antibiotics: erythromycin (Erm, 0.06 mg/ml); kanamycin (Kan, 400 mg/ml); and/ or gentamycin (Gent, 50 mg/ml). doi:10.1371/journal.ppat.1002532.t001 February 2012 \| Volume 8 \| Issue 2 \| e1002532 2 Borrelia Requires RpoS for Tick Transmission Table 2. One or more RpoS-dependent gene products, independent of ospC, are required for spirochete’s to penetrate into the hemolymph; see Figure S1 for spirochete burden analyses. Strain Description Hemolympha,b,c Bite Siteb,d Distal Tissue Sitesb,e 24 h 48 h 72 h Earf Joint Bladder Heart CE162 WT 21/25 ND ND ND 7/7 6/7 7/7 6/7 Bb914 WT-gfp 35/37 0/24 8/20 13/20 25/25 25/25 25/25 24/25 CE303 DospC 31/34 0/20 7/20 0/20 0/5 0/5 0/5 0/5 CE174 DrpoS 0/39 ND ND ND 0/7 0/7 0/7 0/7 Bb1058 DrpoS-gfp 0/56 0/24 0/24 0/24 0/25 0/25 0/25 0/25 CE467 DrpoS+rpoS 22/25 ND ND ND 7/7 7/7 7/7 7/7 SE186 DrpoS-gfp+rpoS 44/47 0/24 3/24 9/24 24/25 25/25 25/25 23/25 aHemolymph was collected from feeding nymphs 72 h post-placement and cultured in BSK-II. The denominator represents the total number of ticks analyzed. bCultures were monitored for the presence of spirochetes by dark field microscopy for 8 weeks. cThe time frames in which organisms (WT, WT-gfp, DospC, DrpoS+rpoS, and DrpoS-gfp+rpoS) were recovered from the hemolymph was highly similar in each experiment (n = 3). Lyme disease spirochetes require RpoS-dependent genes other than ospC to traverse the nymphal midgut fEar punches were performed at 2 and 4 weeks post-feeding and cultured in BSK-II. doi:10.1371/journal.ppat.1002532.t002 RpoS-dependent gene expression by spirochetes in fed nymphs differs from that of mammalian host-adapted organisms It was interesting to note, however, that in fed larvae, when RpoS is OFF, both cdr and cheW3 were expressed at levels comparable to those in fed nymphs. Particularly noteworthy, all six tick-phase genes repressed by RpoS in the mammal were well expressed during the nymphal blood meal; in fact, three (ospA, bb0365, and bba52) were expressed at their highest levels within fed nymphs. PLoS Pathogens \| www.plospathogens.org Lyme disease spirochetes require RpoS-dependent genes other than ospC to traverse the nymphal midgut dSkin of a C3H/HeJ mouse (4–6 sites per mouse) was excised from the site where a nymph was attached at the indicated time post-repletion. A minimum of 3 mice were tested per isolate. The denominator represents the total number of bite sites analyzed; ND = not determined. eMice were sacrificed 4 weeks post-inoculation and the indicated tissues cultured in BSK-II. The denominator represents the total number of mice analyzed per isolate. fEar punches were performed at 2 and 4 weeks post-feeding and cultured in BSK-II. doi:10.1371/journal.ppat.1002532.t002 Table 2. One or more RpoS-dependent gene products, independent of ospC, are required for spirochete’s to penetrate into the hemolymph; see Figure S1 for spirochete burden analyses. Table 2. One or more RpoS-dependent gene products, independent of ospC, are required for spirochete’s to penetrate into the hemolymph; see Figure S1 for spirochete burden analyses. Table 2. One or more RpoS-dependent gene products, independent of ospC, are required for spirochete’s to penetrate into the hemolymph; see Figure S1 for spirochete burden analyses. Strain Description Hemolympha,b,c Bite Siteb,d Distal Tissue Sitesb,e 24 h 48 h 72 h Earf Joint Bladder Heart CE162 WT 21/25 ND ND ND 7/7 6/7 7/7 6/7 Bb914 WT-gfp 35/37 0/24 8/20 13/20 25/25 25/25 25/25 24/25 CE303 DospC 31/34 0/20 7/20 0/20 0/5 0/5 0/5 0/5 CE174 DrpoS 0/39 ND ND ND 0/7 0/7 0/7 0/7 Bb1058 DrpoS-gfp 0/56 0/24 0/24 0/24 0/25 0/25 0/25 0/25 CE467 DrpoS+rpoS 22/25 ND ND ND 7/7 7/7 7/7 7/7 SE186 DrpoS-gfp+rpoS 44/47 0/24 3/24 9/24 24/25 25/25 25/25 23/25 aHemolymph was collected from feeding nymphs 72 h post-placement and cultured in BSK-II. The denominator represents the total number of ticks analyzed. bCultures were monitored for the presence of spirochetes by dark field microscopy for 8 weeks. cThe time frames in which organisms (WT, WT-gfp, DospC, DrpoS+rpoS, and DrpoS-gfp+rpoS) were recovered from the hemolymph was highly similar in each experiment (n = 3). dSkin of a C3H/HeJ mouse (4–6 sites per mouse) was excised from the site where a nymph was attached at the indicated time post-repletion. A minimum of 3 mice were tested per isolate. The denominator represents the total number of bite sites analyzed; ND = not determined. eMice were sacrificed 4 weeks post-inoculation and the indicated tissues cultured in BSK-II. The denominator represents the total number of mice analyzed per isolate. Spirochetes introduced into larvae by immersion undergo biphasic dissemination during nymphal feeding undergo biphasic dissemination during nymphal feeding Lyme disease spirochetes disseminate through the midguts of naturally-infected nymphs in two stages: an initial adherence- mediated migration phase during which non-motile spirochetes advance as networks toward the basolateral surface of the epithelium, followed by a transition into motile organisms at or near the basement membrane which then access the hemocoel [30]. The data in Table 2 suggested that spirochetes lacking RpoS are defective in one or both of these phases. In order to examine the contribution of RpoS to biphasic dissemination, we first established that transmission of WT-gfp Bb by nymphs infected as larvae by immersion recapitulates the sequence of events previously described for naturally-infected nymphs [30]; a detailed schematic indicating how confocal images of unfed and fed midguts were acquired is presented in Figure S3. Spirochete burdens in unfed and fed nymphs infected by immersion were comparable to those detected in nymphs infected by the natural route (Figure S4A; p = 1.000). By confocal microscopy, the distribution of spirochetes within the midguts of unfed and feeding nymphs infected by either method were highly similar (Figure S4B and [30]). The kinetics of transmission by nymphs infected by either route, based on the recovery of organisms in hemolymph, also were indistinguish- able (Table 2 and [30]). To corroborate the above expression data, we also examined the same panel of Bb genes in fed nymphs that had been infected as larvae by immersion with either WT-gfp (Bb914) or DrpoS-gfp (Bb1058) isolates. Little to no transcript was detected for 8 of the 10 RpoS-upregulated genes in fed nymphs infected with DrpoS-gfp organisms compared to their WT-gfp counterparts (Figures 1A and S2A). Expression of cdr and cheW3 was reduced significantly in nymphs infected with the mutant compared to WT, esta- blishing definitively that these genes are partially-dependent on RpoS for expression during the nymphal blood meal (Figure 1B and [29]). Consistent with results obtained using naturally- infected ticks, the genes repressed by RpoS in mammalian host- February 2012 \| Volume 8 \| Issue 2 \| e1002532 3 Borrelia Requires RpoS for Tick Transmission Figure 1. Contours of the RpoSBb regulon in I. scapularis. qRT-PCR analysis of (A) absolutely and (B) partially RpoS-dependent genes and (C) RpoS-repressed genes selected from microarray data derived from Bb cultivated within DMCs [13]. A representative sampl shown; data for the remaining genes are presented in Figure S2. PLoS Pathogens \| www.plospathogens.org February 2012 \| Volume 8 \| Issue 2 \| e1002532 Spirochetes introduced into larvae by immersion undergo biphasic dissemination during nymphal feeding In addition, many of the mutant organisms located between cells were thin, ragged and blebbing (Figure 2B, ‘midway’ panel). At 72 h, the difference between midguts harboring WT-gfp and DrpoS-gfp isolates was striking (Figure 3). Whereas WT-gfp organisms formed typical confluent networks (Figure 3A; WT-gfp), DrpoS-gfp Bb were rarely observed (Figure 3B, DrpoS-gfp ‘coronal’), a result ostensibly discordant with the comparable spirochete burdens detected in fed nymphs infected with either strain (Figures S1B and S1C). We conjectured that DrpoS-gfp organisms were confined to the lumen at the late feeding time point but that the blood meal was interfering with their visualization. We confirmed this assumption by carefully removing a portion (,30–40%) of the blood meal from isolated 72 h-fed midguts and acquiring transverse optical sections from distal portions of diverticula to reduce light scattering and absorption by the luminal contents (Figures S3B–3D). As shown placement, however, there was a marked reduction in the overall density of DrpoS-gfp Bb on the luminal surfaces and between epithelial cells (Figure 2B). In addition, many of the mutant organisms located between cells were thin, ragged and blebbing (Figure 2B, ‘midway’ panel). At 72 h, the difference between midguts harboring WT-gfp and DrpoS-gfp isolates was striking (Figure 3). Whereas WT-gfp organisms formed typical confluent networks (Figure 3A; WT-gfp), DrpoS-gfp Bb were rarely observed (Figure 3B, DrpoS-gfp ‘coronal’), a result ostensibly discordant with the comparable spirochete burdens detected in fed nymphs infected with either strain (Figures S1B and S1C). We conjectured that DrpoS-gfp organisms were confined to the lumen at the late feeding time point but that the blood meal was interfering with their visualization. We confirmed this assumption by carefully removing a portion (,30–40%) of the blood meal from isolated 72 h-fed midguts and acquiring transverse optical sections from distal portions of diverticula to reduce light scattering and absorption by the luminal contents (Figures S3B–3D). As shown Spirochetes introduced into larvae by immersion undergo biphasic dissemination during nymphal feeding Expression profiling was performed using fed larvae, unfed and fed nym been naturally-infected with WT Bb as well as fed nymphs that had been infected as larvae by immersion with either WT-gfp or DrpoS Borrelia Requires RpoS for Tick T Figure 1. Contours of the RpoSBb regulon in I. scapularis. qRT-PCR analysis of (A) absolutely and (B) partially RpoS-dependent upregulated genes and (C) RpoS-repressed genes selected from microarray data derived from Bb cultivated within DMCs [13]. A representative sample of genes is shown; data for the remaining genes are presented in Figure S2. Expression profiling was performed using fed larvae, unfed and fed nymphs that had been naturally-infected with WT Bb as well as fed nymphs that had been infected as larvae by immersion with either WT-gfp or DrpoS-gfp isolates. Figure 1. Contours of the RpoSBb regulon in I. scapularis. qRT-PCR analysis of (A) absolutely and (B) partially RpoS-dependent upregulated genes and (C) RpoS-repressed genes selected from microarray data derived from Bb cultivated within DMCs [13]. A representative sample of genes is shown; data for the remaining genes are presented in Figure S2. Expression profiling was performed using fed larvae, unfed and fed nymphs that had been naturally-infected with WT Bb as well as fed nymphs that had been infected as larvae by immersion with either WT-gfp or DrpoS-gfp isolates. February 2012 \| Volume 8 \| Issue 2 \| e1002532 February 2012 \| Volume 8 \| Issue 2 \| e1002532 4 Borrelia Requires RpoS for Tick Transmission Borrelia Requires RpoS for Tick Transmission Borrelia Requires RpoS for Tick Transmission Values represent the average flaB-normalized transcript copy number 6 standard error of the mean (SEM) for each gene; values are considered significantly different when p is #0.05 (indicated by asterisks). Composite confocal images through the full thickness of nymphal midguts at 72 h post-placement showing the distribution of spirochetes expressing gfp under the control of the (D) flaB or (E) ospA promoter. A detailed schematic indicating how confocal images of fed midguts were acquired is presented in Figure S3. Here and elsewhere, green represents GFP+ spirochetes while red indicates midgut epithelial cells labeled with FM4-64; scale bars = 25 mm. doi:10.1371/journal.ppat.1002532.g001 placement, however, there was a marked reduction in the overall density of DrpoS-gfp Bb on the luminal surfaces and between epithelial cells (Figure 2B). DrpoS organisms are confined to the midgut lumen and develop into an aberrant morphotype during nymphal feeding Having confirmed the suitability of the immersion method, we proceeded to characterize DrpoS spirochetes within nymphal midguts. For these experiments, we generated fluorescent versions of our previously characterized DrpoS mutant CE174 (DrpoS-gfp; Bb1058) and complemented strain (DrpoS-gfp+rpoS; SE186) (Table 1 and Figure S5). At the outset, we confirmed using qPCR and semi- solid plating that DrpoS-gfp Bb, like its non-fluorescent counterpart, replicated to wild-type levels within feeding nymphs (Figure S1B) but was unable to traverse the midgut or establish infection (Table 2). As expected, the complement was fully virulent by tick- and needle-inoculation (Tables 2 and S2). By confocal microscopy, the distribution of DrpoS-gfp Bb in midguts isolated from unfed nymphs did not differ from that of WT-gfp organisms (Figure 2A; also see Figure S3). By 48 h post- Figure 2. Spirochetes lacking RpoS are distributed normally within unfed nymphal midguts but are destroyed between epithelial cells early during feeding. The leftmost images in each panel depict the full composites (basement membrane to basement membrane) of the midgut, while 3-mm composite images show spirochetes at the luminal surface, midway through the epithelium, and at the basement membrane; scale bars = 25 mm. A detailed schematic indicating how confocal images of unfed and 48 h-fed midguts were acquired is presented in Figure S3. (A) In unfed midguts, WT-gfp and DrpoS-gfp are similarly distributed. (B) In midguts isolated at 48 h post-placement, WT-gfp organisms form aggregates, while DrpoS-gfp located between epithelial cells are destroyed; arrows indicate ragged, blebbing Bb. Numbers in lower right hand corner indicate the optical depth of the image. Inset in the DrpoS-gfp midway panel depicts a 1-mm optical section showing thin, ragged and blebbing organisms between epithelial cells; see also Figure S4 for a comparison of midguts infected with WT-gfp Bb by natural versus immersion methods. doi:10.1371/journal.ppat.1002532.g002 Figure 2. Spirochetes lacking RpoS are distributed normally within unfed nymphal midguts but are destroyed between epithelial cells early during feeding. The leftmost images in each panel depict the full composites (basement membrane to basement membrane) of the midgut, while 3-mm composite images show spirochetes at the luminal surface, midway through the epithelium, and at the basement membrane; scale bars = 25 mm. A detailed schematic indicating how confocal images of unfed and 48 h-fed midguts were acquired is presented in Figure S3. (A) In unfed midguts, WT-gfp and DrpoS-gfp are similarly distributed. DrpoS organisms are confined to the midgut lumen and develop into an aberrant morphotype during nymphal feeding (B) In midguts isolated at 48 h post-placement, WT-gfp organisms form aggregates, while DrpoS-gfp located between epithelial cells are destroyed; arrows indicate ragged, blebbing Bb. Numbers in lower right hand corner indicate the optical depth of the image. Inset in the DrpoS-gfp midway panel depicts a 1-mm optical section showing thin, ragged and blebbing organisms between epithelial cells; see also Figure S4 for a comparison of midguts infected with WT-gfp Bb by natural versus immersion methods. Figure 2. Spirochetes lacking RpoS are distributed normally within unfed nymphal midguts but are destroyed between epithelial cells early during feeding. The leftmost images in each panel depict the full composites (basement membrane to basement membrane) of the midgut, while 3-mm composite images show spirochetes at the luminal surface, midway through the epithelium, and at the basement membrane; scale bars = 25 mm. A detailed schematic indicating how confocal images of unfed and 48 h-fed midguts were acquired is presented in Figure S3. (A) In unfed midguts, WT-gfp and DrpoS-gfp are similarly distributed. (B) In midguts isolated at 48 h post-placement, WT-gfp organisms form aggregates, while DrpoS-gfp located between epithelial cells are destroyed; arrows indicate ragged, blebbing Bb. Numbers in lower right hand corner indicate the optical depth of the image. Inset in the DrpoS-gfp midway panel depicts a 1-mm optical section showing thin, ragged and blebbing organisms between epithelial cells; see also Figure S4 for a comparison of midguts infected with WT-gfp Bb by natural versus immersion methods. doi:10.1371/journal.ppat.1002532.g002 February 2012 \| Volume 8 \| Issue 2 \| e1002532 PLoS Pathogens \| www.plospathogens.org PLoS Pathogens \| www.plospathogens.org 5 Borrelia Requires RpoS for Tick Transmission Figure 3. RpoS is required to form networks during nymphal feeding. (A) At 72 h post-placement, WT-gfp organisms form networks that encase midgut epithelial cells and extend to the basement membrane. (B) DrpoS-gfp Bb are not observed in intact midguts imaged in the ‘coronal’ plane but are visualized within the lumens of midguts that had been punctured to remove a portion of their contents and imaged by acquiring transverse optical sections from distal portions of the diverticula to reduce light scattering and absorption by the blood meal. A detailed schematic illustrating how coronal and transverse confocal images were acquired is presented in Figure S3. DrpoS organisms are confined to the midgut lumen and develop into an aberrant morphotype during nymphal feeding Interestingly, WT round bodies also were observed sporadically in midguts at the 72 h time point, examples of which are shown in Figure 6A. DrpoS organisms are confined to the midgut lumen and develop into an aberrant morphotype during nymphal feeding (C) Complementation restores the ability of DrpoS- gfp Bb to form networks and extend to the basement membrane; see also Figure S5 for complementation verification by protein profile analysis. The leftmost images in each panel depict the full composites (basement membrane to luminal surface) of the midgut, while 3-mm composite images show spirochetes at the luminal surface, midway through the epithelial layer, and at the basement membrane. Numbers in lower right hand corner indicate the optical depth of the image; scale bars = 25 mm. Representative cryosections of 72 h-fed midguts infected with WT-gfp and DrpoS-gfp isolates are presented in Figure S6. doi:10.1371/journal.ppat.1002532.g003 Figure 3. RpoS is required to form networks during nymphal feeding. (A) At 72 h post-placement, WT-gfp organisms form networks that encase midgut epithelial cells and extend to the basement membrane. (B) DrpoS-gfp Bb are not observed in intact midguts imaged in the ‘coronal’ plane but are visualized within the lumens of midguts that had been punctured to remove a portion of their contents and imaged by acquiring transverse optical sections from distal portions of the diverticula to reduce light scattering and absorption by the blood meal. A detailed schematic illustrating how coronal and transverse confocal images were acquired is presented in Figure S3. (C) Complementation restores the ability of DrpoS- gfp Bb to form networks and extend to the basement membrane; see also Figure S5 for complementation verification by protein profile analysis. The leftmost images in each panel depict the full composites (basement membrane to luminal surface) of the midgut, while 3-mm composite images show spirochetes at the luminal surface, midway through the epithelial layer, and at the basement membrane. Numbers in lower right hand corner indicate the optical depth of the image; scale bars = 25 mm. Representative cryosections of 72 h-fed midguts infected with WT-gfp and DrpoS-gfp isolates are presented in Figure S6. doi:10.1371/journal.ppat.1002532.g003 in Figure 3B (DrpoS-gfp, transverse panel), the lumens of DrpoS-gfp- infected midguts were, in fact, crowded with organisms. Epifluorescence microscopy of intact and cryosectioned midguts (Figures 4B and S6B, respectively) and silver-staining of paraffin- embedded specimens (Figures 4E and 4H) also revealed lumens packed with DrpoS-gfp spirochetes at the 72 h time point. Importantly, complementation restored the ability of DrpoS-gfp Bb to form networks (Figures 3C, 4C, 4F and 4I). entities. PLoS Pathogens \| www.plospathogens.org February 2012 \| Volume 8 \| Issue 2 \| e1002532 Loss of RpoS and CoADR exacerbate the formation of round bodies in vitro Spirochetes cultivated under nutrient-limiting conditions form morphotypes [22–24,31,32] resembling the round bodies observed in nymphal midguts. These reports led us to hypothesize that RpoS-deficiency creates a metabolic lesion that promotes the formation of round bodies during the nymphal blood meal. To test this idea, we took advantage of the observation by Alban et al. [32] that spirochetes form round bodies during a relatively short incubation period in RPMI. As shown in Figure 7, the percentage of WT-gfp round bodies increased steadily over 4 days, reaching a plateau of ,50% on day 3; a significantly higher percentage of DrpoS-gfp spirochetes formed round bodies on days 1 through 4 (Figure 7, p#0.002 for each day). Complementation of DrpoS-gfp significantly reduced the percentages of round bodies to levels lower than those observed with WT-gfp on days 3 and 4 (Figures 7 and S7, p#0.01 for both days). Moreover, we confirmed by TEM that round bodies formed by WT-gfp and DrpoS-gfp isolates in RPMI were identical to each other (Figure 6B) and closely resembled those in nymphal midguts (Figure 6A). Previously, Xu Closer inspection of silver-stained paraffin sections at high magnification revealed that the vast majority of DrpoS-gfp organisms no longer displayed typical spirochete morphology but, instead, were round and larger in diameter than a typical spirochete in cross-section (compare insets in Figures 4G and 4H). We next used transmission electron microscopy (TEM) of ultra- thin sections to better characterize this aberrant morphotype. At 48 h post-placement, there was no discernible difference between the populations of WT-gfp and DrpoS-gfp organisms (Figures 5A and 5B). By 72 h post-placement, however, a striking difference was readily apparent (Figures 5C and 5D). In contrast to WT organisms, DrpoS-gfp Bb were predominantly membranous, spherical structures containing irregularly-shaped vesicles and protoplasmic cylinders (Figures 5C, 5D, and 6A). Based on the nomenclature proposed by Brorson et al. [23], we hereafter use the term ‘round bodies’ to denote these structurally heterogeneous February 2012 \| Volume 8 \| Issue 2 \| e1002532 6 Borrelia Requires RpoS for Tick Transmission Figure 4. Spirochetes lacking RpoS are confined to the lumen and develop into round bodies during the later stages of feeding. (A–C) Representative epifluorescence images of midguts isolated at 72 h post-placement containing (A) WT-gfp, (B) DrpoS-gfp or (C) RpoS- complemented isolates; midguts appear white when imaged by dark-field microscopy. PLoS Pathogens \| www.plospathogens.org Loss of RpoS and CoADR exacerbate the formation of round bodies in vitro (D–I) Representative silver-stained paraffin-embedded sections of midguts isolated from 72 h-fed nymphs infected with (D and G) WT-gfp, (E and H) DrpoS-gfp or (F and I) RpoS-complemented isolates. Insets in (G) and (H) are enlargements of the boxed regions showing WT-gfp spirochetes and DrpoS-gfp round bodies, respectively; scale bars: A– C = 50 mm; D–F = 25 mm; and G–I = 10 mm. doi:10.1371/journal.ppat.1002532.g004 Figure 4. Spirochetes lacking RpoS are confined to the lumen and develop into round bodies during the later stages of feeding. (A–C) Representative epifluorescence images of midguts isolated at 72 h post-placement containing (A) WT-gfp, (B) DrpoS-gfp or (C) RpoS- complemented isolates; midguts appear white when imaged by dark-field microscopy. (D–I) Representative silver-stained paraffin-embedded sections of midguts isolated from 72 h-fed nymphs infected with (D and G) WT-gfp, (E and H) DrpoS-gfp or (F and I) RpoS-complemented isolates. Insets in (G) and (H) are enlargements of the boxed regions showing WT-gfp spirochetes and DrpoS-gfp round bodies, respectively; scale bars: A– C = 50 mm; D–F = 25 mm; and G–I = 10 mm. doi:10.1371/journal.ppat.1002532.g004 formed round bodies at levels lower than those of WT-gfp on days 3 and 4 (Figures 7 and S7, p#0.002 on both days). et al. [33] proposed that DospC spirochetes have a defective membrane surface. It was thus surprising to find that the percentage of DospC round bodies was actually lower than WT levels at days 3 and 4 and that the rate of round body formation was comparable to that observed for WT-gfp organisms (data not shown). Round bodies elongate rapidly upon addition of BSK-II Round bodies elongate rapidly upon addition of BSK-II Alban et al. [32] noted that round bodies formed in RPMI elongate within seconds following the addition of normal rabbit serum (NRS). We sought to confirm this observation, which suggests that neither the metabolic lesion that triggers round body formation nor the resulting rearrangement of the spirochete’s protoplasmic cylinder involve alterations in the cell envelope that require extensive biosynthetic repair. In our hands, the addition of NRS did not promote round body recovery. However, the addition of an equal volume of Barbour-Stoenner-Kelly medium (BSK-II), either with or without NRS, induced the rapid (#3 s) recovery of WT-gfp and DrpoS-gfp round bodies (Video S1). Interestingly, one or two highly refractile inclusions often could be observed within round bodies formed in RPMI by both Bb isolates; these structures remained prominent even after bacteria had fully elongated. Based on visualization of 32 round bodies formed by In Bb, the enzyme CoADR, encoded by cdr, contributes to the regeneration of NAD+ for glycolysis and the maintenance of the pool of Coenzyme A for biosynthetic functions [34,35]. Our finding that transcription of cdr, one of the few metabolic genes within the RpoS regulon, is markedly reduced in DrpoS-gfp Bb during nymphal feeding (Figure 1B) suggested that diminished levels of CoADR might be involved in the enhanced formation of round bodies by DrpoS-gfp in RPMI. As shown in Figure 7, the percentages of round bodies formed by Dcdr and DrpoS-gfp Bb were not significantly different on days 1 and 2 (p.0.6 for both days); by days 3 and 4, however, the rate of round body formation by Dcdr organisms significantly outpaced both WT and DrpoS spirochetes (p#0.0006 and p,0.002 for WT and DrpoS, respectively, on both days). As with DrpoS-gfp Bb, complemented Dcdr spirochetes PLoS Pathogens \| www.plospathogens.org February 2012 \| Volume 8 \| Issue 2 \| e1002532 7 Borrelia Requires RpoS for Tick Transmission Figure 5. DrpoS Bb form round bodies within nymphal midguts during the later stages of feeding. Representative TEM images of nymphal midguts containing WT-gfp and DrpoS-gfp spirochetes isolated at (A–B) 48 or (C-D) 72 h post-placement. Color overlays are used to highlight normal spirochetes (red), round bodies (blue), and peritrophic membranes (PM, green); scale bars = 2 mm. doi:10.1371/journal.ppat.1002532.g005 Figure 5. DrpoS Bb form round bodies within nymphal midguts during the later stages of feeding. Round bodies elongate rapidly upon addition of BSK-II Representative TEM images of nymphal midguts containing WT-gfp and DrpoS-gfp spirochetes isolated at (A–B) 48 or (C-D) 72 h post-placement. Color overlays are used to highlight normal spirochetes (red), round bodies (blue), and peritrophic membranes (PM, green); scale bars = 2 mm. doi:10.1371/journal.ppat.1002532.g005 WT-gfp and DrpoS-gfp Bb (16 per isolate) in RPMI, we found that each recovered into a single spirochete (Video S1). spirochetes against ‘‘conventional’’ external stressors (e.g., reactive oxygen species, low pH, high salt) [27] previously led us to conclude that RpoSBb is not a central regulator of a general stress response [13]. The findings presented here, however, argue that RpoS-mediated alterations in the spirochete’s transcriptome include physiologic adaptations required for dissemination within the vector. To further the comparison between round bodies formed within ticks and RPMI, we next investigated whether round bodies within midguts isolated at 72 h post-placement also recover upon the addition of BSK-II. Indeed, as shown in Figure 8A, tick-derived DrpoS round bodies elongated into spirochetes within minutes. Importantly, the addition of RPMI did not promote recovery (Figure 8B). ospC is widely regarded as the prototypical RpoS-dependent gene [12]. While there is universal agreement that OspC is essential for the ability of Bb to establish murine infection, there is lingering controversy as to whether this surface lipoprotein functions within the vector or the mammal [38]. Pal et al. [25] reported that spirochetes lacking OspC were unable to penetrate the salivary glands of feeding nymphs. Rosa and colleagues [19,26], on the other hand, visualized DospC organisms within salivary glands and demonstrated that OspC was required for survival within mice following needle-inoculation. Our data, derived entirely by tick-inoculation, are in accord with those of the Rosa group. We found that spirochetes lacking OspC were recovered from hemolymph and reached the tissue surrounding the bite site with kinetics indistinguishable from those of WT Bb but were cleared from the skin within 48–72 h post-repletion, a time frame which closely mirrors that previously reported using needle-inoculation [26]. The inability of DrpoS Bb to traverse the midgut, therefore, cannot be attributed to the lack of OspC. We recently reported that spirochetes lacking cdr possess a growth defect within feeding nymphs and are avirulent by needle inoculation [29]. Thus, RpoS-dependent genes fall into three PLoS Pathogens \| www.plospathogens.org Discussion Studies involving RpoSBb have focused primarily on the contribution of RpoS-dependent genes to virulence in mammals [21]. The role of RpoSBb during the tick-phase of the enzootic cycle, on the other hand, has been largely unexplored despite multiple lines of evidence suggesting that genes controlled by this alternative sigma factor might function during tick-to-mammal transmission [12,13,36]. In this study, we demonstrate that DrpoS spirochetes remain confined to the midgut throughout the blood meal, and, thus, are unable to progress beyond the initial stage of dissemination. Unexpectedly, RpoS deficient spirochetes replicat- ing within the midguts of feeding nymphs undergo a dramatic morphologic transformation comparable to that induced by nutritional deprivation in vitro. The high degree of phylogenetic divergence between RpoSBb and its orthologs in Proteobacteria [37], the ostensible lack of overlap between the RpoS regulons in Bb and E. coli [13], and the finding that RpoSBb does not protect PLoS Pathogens \| www.plospathogens.org February 2012 \| Volume 8 \| Issue 2 \| e1002532 8 Borrelia Requires RpoS for Tick Transmission Figure 6. Wild-type and DrpoS round bodies are structurally similar both within feeding nymphs and under nutrient-limiting conditions in vitro. Representative TEM images of DrpoS-gfp and WT-gfp spirochetes and round bodies in (A) nymphal midguts isolated at 48 or 72 h post-placement or (B) in vitro-derived organisms following incubation in BSK-II or RPMI for 3 days. In panels A and B, arrows indicate examples of protoplasmic cylinders; scale bars = 500 nm. doi:10.1371/journal.ppat.1002532.g006 Figure 6. Wild-type and DrpoS round bodies are structurally similar both within feeding nymphs and under nutrient-limit conditions in vitro. Representative TEM images of DrpoS-gfp and WT-gfp spirochetes and round bodies in (A) nymphal midguts isolated at 48 72 h post-placement or (B) in vitro-derived organisms following incubation in BSK-II or RPMI for 3 days. In panels A and B, arrows indicate example protoplasmic cylinders; scale bars = 500 nm. d i 10 1371/j l t 1002532 006 Figure 6. Wild-type and DrpoS round bodies are structurally similar both within feeding nymphs and under nutrient-limiting conditions in vitro. Representative TEM images of DrpoS-gfp and WT-gfp spirochetes and round bodies in (A) nymphal midguts isolated at 48 or 72 h post-placement or (B) in vitro-derived organisms following incubation in BSK-II or RPMI for 3 days. In panels A and B, arrows indicate examples of protoplasmic cylinders; scale bars = 500 nm. PLoS Pathogens \| www.plospathogens.org February 2012 \| Volume 8 \| Issue 2 \| e1002532 Discussion Maintaining a large population of organisms that are both tick- and mammalian host-adapted could be advantageous for Bb by enhancing the probability that the small number of spirochetes that reach the basement membrane are ‘‘transmission ready’’ while ensuring that the large number of organisms remaining in the midgut can resume the fully tick-adapted state post-repletion. As a prelude to defining the contribution of RpoS during the tick-phase of the enzootic cycle, we first confirmed that the transmission of spirochetes by ticks infected by immersion faithfully reproduces the spatio-temporal sequence of events previously described for organisms acquired naturally [30,53]. Discernible differences between WT-gfp and DrpoS-gfp Bb, first 0243) to maintain maximal fitness throughout all tick life stages [51] because these gene products enable spirochetes to import and utilize glycerol, an increasingly important carbon source within the arthropod as the availability of glucose diminishes during digestion of the blood meal. In addition, the continued expression of tick- phase borrelial surface adhesins, such as OspA [17,52], along with the inhibition of motility by components elaborated by the midgut during the nymphal blood meal, may facilitate the close association of Bb with midgut epithelium, a prerequisite for network formation and adherence-mediated migration [30]. Maintaining a large population of organisms that are both tick- and mammalian host-adapted could be advantageous for Bb by enhancing the probability that the small number of spirochetes that reach the basement membrane are ‘‘transmission ready’’ while ensuring that the large number of organisms remaining in the midgut can resume the fully tick-adapted state post-repletion. As a prelude to defining the contribution of RpoS during the tick-phase of the enzootic cycle, we first confirmed that the transmission of spirochetes by ticks infected by immersion faithfully reproduces the spatio-temporal sequence of events previously described for organisms acquired naturally [30,53]. Discernible differences between WT-gfp and DrpoS-gfp Bb, first The most striking aspect of the DrpoS phenotype, the transformation from typical spirochetes to round bodies, did not become apparent until 48–72 h post-attachment. During this late stage of feeding, the nutritional requirements of midgut epithelial cells immensely increases as the cells switch from undifferentiated reserve cells into hyperactive digestive cells, while spirochetes replicate extensively around them [30,56]. Presumably, an intense competition for blood meal nutrients ensues at this interface. Discussion doi:10.1371/journal.ppat.1002532.g006 broad categories: (i) ospC-like genes that are required solely within the mammal; (ii) genes, such as bba64 [39,40], that promote dissemination within the tick; and (iii) genes, such as cdr, that function in the arthropod as well as the mammal [29]. possible mechanistic explanation for the limited RpoS-mediated repression observed within fed nymphs is that signals produced during the blood meal activate regulatory pathways that oppose the downregulation of tick-phase genes during transmission. The Hk1/Rrp1 two-component system, which promotes tick-adapta- tion during feeding but is not required for mammalian infection [41–43], is a candidate counter-regulatory pathway. Our findings add to a large body of evidence that the ‘‘classical’’ paradigm for reciprocal regulation of tick- and mammalian host-phase genes (ospA/ospC being the prototypes) is valid only within the mammal [13,14,16,44,45]. A number of recent studies have helped to elucidate the biological significance for the prolonged transition from the tick- to the mammalian host-adapted state [46–51]. As one example, Bb requires the products of the glp operon (bb0240- The qRT-PCR studies described herein brought to light a striking difference in the transcriptional profiles of WT Bb within larval and nymphal ticks as compared to mammalian host-adapted (i.e., DMC-cultivated) organisms: while genes upregulated by RpoS in DMCs also were induced in fed nymphs, genes repressed by RpoS in mammals were expressed at comparable levels by Bb in all tick life stages. A comparison of transcript copy numbers in WT and DrpoS Bb demonstrated that several tick-phase genes (i.e., ospA, bb0240, bb0365, and bba52) are, in fact, subject to some degree of downregulation during the nymphal blood meal. One PLoS Pathogens \| www.plospathogens.org February 2012 \| Volume 8 \| Issue 2 \| e1002532 9 PLoS Pathogens \| www.plospathogens.org Borrelia Requires RpoS for Tick Transmission Figure 7. Loss of RpoS and CoADR exacerbates round body formation in vitro. Bb were incubated in RPMI for 1–4 days. A minimum of 300 organisms were counted per strain for each time point. Experiments were performed in triplicate; error bars represent means 6 SEM. Representative images of fields used to quantify round body formation are shown in Figure S7. The percentages of round bodies formed by DrpoS-gfp and Dcdr isolates were significantly greater than WT on days 1 through 4 (p#0.002). Round body formation by DrpoS-gfp and Dcdr isolates was significantly different on days 3 and 4 (p#0.002). doi:10.1371/journal.ppat.1002532.g007 Figure 7. Discussion Loss of RpoS and CoADR exacerbates round body formation in vitro. Bb were incubated in RPMI for 1–4 days. A minimum of 300 organisms were counted per strain for each time point. Experiments were performed in triplicate; error bars represent means 6 SEM. Representative images of fields used to quantify round body formation are shown in Figure S7. The percentages of round bodies formed by DrpoS-gfp and Dcdr isolates were significantly greater than WT on days 1 through 4 (p#0.002). Round body formation by DrpoS-gfp and Dcdr isolates was significantly different on days 3 and 4 (p#0.002). doi:10.1371/journal.ppat.1002532.g007 apparent at 48 h post-attachment, were dependent upon the positioning of DrpoS-gfp spirochetes relative to the epithelial cells. The degradation of DrpoS-gfp Bb between cells suggests that one or more RpoS-dependent genes are required for protection against noxious elements encountered in the intercellular spaces. That spirochete burdens in fed nymphs infected with DrpoS Bb were not significantly diminished suggests that the destruction of organisms is confined to a small niche within the midgut. Another contrast between WT and mutant Bb at the 48 h time point was the paucity of DrpoS-gfp organisms at the luminal surface. Given that RpoS- deficient Bb accumulate within the lumen as feeding progresses, one might attribute this difference to an attachment defect associated with loss of RpoS-upregulated surface molecules that function cooperatively with tick-phase adhesins. Bioinformatic analysis of the RpoS regulon revealed 38 potential surface adhesins (Table S3); seven are predicted outer membrane- spanning proteins (i.e., b-barrels) based on the computational consensus framework used by Cox et al. [54] while the remainder are lipoproteins [55]. 0243) to maintain maximal fitness throughout all tick life stages [51] because these gene products enable spirochetes to import and utilize glycerol, an increasingly important carbon source within the arthropod as the availability of glucose diminishes during digestion of the blood meal. In addition, the continued expression of tick- phase borrelial surface adhesins, such as OspA [17,52], along with the inhibition of motility by components elaborated by the midgut during the nymphal blood meal, may facilitate the close association of Bb with midgut epithelium, a prerequisite for network formation and adherence-mediated migration [30]. Strain construction Strains CE162, CE174, CE467, CE56, CE103, Bb914, CE309, and CE1655 have been previously described [27,29, 30,36,59] (Table 1). To generate an ospC deletion mutant, we amplified overlapping upstream and downstream regions con- taining ospC (bbb19) and flanking sequences from strain 297 us- ing primers BBB18#1F/BBB19#1R(NgoMIV) and BBB19#1F (NgoMIV)/BBB22#1R, respectively (Table S1). The resulting amplicons were cloned into pCR2.1-TOPO (Invitrogen), re- amplified using the same primers as above, and gel-purified. Purified upstream and downstream amplicons were mixed 1:1 and amplified for 4 cycles with ExTaq in the absence of primer to promote annealing/extension of the overlapping regions followed by 30 cycles in the presence of BBB18#1F and BBB22#1R flanking primers. The joined amplicon was subsequently cloned into pCR2.1-TOPO, digested with EcoRI, and subcloned into similarly digested pUC19 to yield pCE216. A PflgB-kan cassette conferring resistance to kanamycin was amplified from pTA- KanG [60] using PflgB(NgoMIV) and KanR(BamHI) and cloned into pCR2.1-TOPO. The PflgB-kan cassette was liberated by digestion with NgoMIV and BamHI and ligated into NgoMIV- BglII digested pCE216 to yield pCE217. Approximately 10 mg of gel-purified ospC::kanR insert, released from pCE217 by digestion with NgoMIV and BamHI, was electro-transformed into CE162 (Table 1) as previously described [61]. A transformant (CE303) was selected in BSK-II medium containing the appropriate antibiotic and subsequently confirmed by PCR to be an ospC deletion mutant using the BBB18#1F (extd) and BBB22#1R primers (Table S1). Bb1058, the fluorescent rpoS mutant, was generated by electro-transforming CE174 with pMC1916 [30] followed by selection in BSK-II containing erythromycin (0.06 mg/ml) and gentamycin (5 mg/ml). Inactivation of rpoS was confirmed by PCR using the primers PrpoS-59 and rpoS-3 (Table S1). To generate the fluorescent DrpoS complement, competent Bb1058 cells were transformed with a wild-type copy of rpoS contained on pCE320 as previously described [27]. A transformant, SE186, was selected in BSK-II medium containing kanamycin, erythromycin, and gentamycin and subsequently confirmed to contain rpoS by PCR using the primers rpoS- prom(extd) and RpoS-39XbaI (Table S1). Complementation was confirmed by the production of OspC and DbpA following temperature-shift as assessed by silver-stain and immunoblot, respectively, as previously described [27]. A fundamental question posed by this work is whether round body formation is a unique stress response to nutrient limitation or an aberration of the laboratory with no role in the natural ecology of the spirochete. Strain construction Formation of round bodies by WT spirochetes argues for its biological relevance as does the sheer complexity of the process, its elegant recovery mechanism, and the stress-related circumstances in which it is observed. Round body formation by WT spirochetes in RPMI can be conceptualized as a response to inadequate energy generation. In contrast, round body formation by WT spirochetes within the midguts of feeding nymphs is sporadic; perhaps the competition between spirochete and epithelial cells for micro-nutrients in the blood meal is particularly intense in localized regions of the midgut. What is clear is that, in both settings, physiologic adaptations mediated by RpoS inhibit round body formation. This formulation implies that (i) the apparatus for round body formation and recovery is poised to operate within the background during feeding but is suppressed either directly or indirectly by an RpoS-dependent gene product and (ii) spirochetes default to round bodies upon sensing that the energetics for transmission (i.e., transition to motility) are unfavorable. Underlying many studies of round body formation in the literature has been the presumption that its primary importance relates to persistence within the mammal [22,24,31]. We propose that round body formation has evolved to support the tick phase of the cycle and predict that there are circumstances, as yet undefined, when spirochetes within the tick resort to this survival program on a large scale in order to maintain a population of transmissible organisms. Borrelia Requires RpoS for Tick Transmission midguts to recover within minutes after the addition of BSK-II (i.e., without replication) strongly supports this contention. To shed light on this unusual morphotype, we turned to an in vitro system in which WT Bb transform into round bodies during several days of incubation in RPMI [32]. Although the culture conditions obviously differ considerably from the nutrient-replete midgut, the round bodies formed by WT and DrpoS Bb in vitro closely resemble those observed in feeding nymphs. Several other features of this in vitro system are particularly instructive and support its utility for elucidating the in vivo morphotype. First, round bodies elongate rapidly in the presence of BSK-II, indicating that their cell envelopes are intact. Second, spirochete numbers do not increase in this system, demonstrating that round body formation is divorced from replication. Third, and most importantly, compared to WT organisms, DrpoS Bb exhibited a pronounced tendency to form round bodies, suggesting that the metabolic lesion in feeding nymphs is operative in vitro. This supposition led us to examine cdr, whose enzymatic product, CoADR, provides the cofactor NAD+ for glycolysis as a byproduct. While the Dcdr phenotype provides proof of principle that an energy-related gene product is involved in round body formation, additional genes within the RpoS regulon also may contribute to this phenomenon. serum (Pel-Freeze Biologicals). Selection and maintenance of B. burgdorferi strains was performed in media supplemented with the appropriate antibiotics (Table 1). The plasmid content of all isolates was monitored as previously described [58]. Standard temperature-shift experiments and growth curves were performed as previously described [13]. Escherichia coli strains were maintained in Luria-Bertani broth (LB) (1% tryptone, 0.5% yeast extract, 1% NaCl) with the appropriate antibiotic. Selection was performed on LB agar plates (LB with 1.5% agar) supplemented with the appropriate antibiotic. DNA manipulations and routine cloning Routine molecular cloning and plasmid propagation were performed using E. coli Top10 cells (Invitrogen). Routine and high-fidelity PCR amplification reactions were performed using Choice Taq (Denville Scientific) and Takara ExTaq (Fisher Scientific), respectively. Plasmid DNAs were purified using Qiagen Midi and Spin Prep kits. Nucleotide sequencing was performed by Agencourt Bioscience Corp. Ethics statement All animal experimentation was conducted following NIH guidelines for housing and care of laboratory animals and in accordance with University of Connecticut Health Center regulations after review and approval by the UCHC Institutional Animal Care and Use Committee. Discussion Within this same 24 h window, a small percentage of organisms that reach the basement membrane become motile, a transition that enables them to access the hemocoel but likely also substantially adds to their energy requirements. Spirochetes lacking RpoS replicate to WT levels within the midguts of feeding nymphs and exhibit growth kinetics equivalent to those of WT as demonstrated by qPCR and semi- solid plating. These findings argue strongly that the formation of round bodies by DrpoS Bb cannot be attributed to their inability to utilize the blood meal for biosynthetic purposes but rather suggest that loss of RpoS creates a rapidly reversible metabolic lesion that provokes the structural rearrangements that give rise to round bodies. The ability of DrpoS round bodies derived from tick Figure 8. Round bodies within fed nymphal midguts recover into elongated spirochetes. DrpoS-infected nymphs were removed at 72 h post-placement and midguts dissected into RPMI. (A) The addition of BSK-II induced the recovery of round bodies into elongated spirochetes. (B) Round bodies do not recover when midguts were submerged in RPMI. Scale bars = 10 mm. See also the Video S1 of the rapid recovery of in vitro-derived organisms. doi:10.1371/journal.ppat.1002532.g008 Figure 8. Round bodies within fed nymphal midguts recover into elongated spirochetes. DrpoS-infected nymphs were removed at 72 h post-placement and midguts dissected into RPMI. (A) The addition of BSK-II induced the recovery of round bodies into elongated spirochetes. (B) Round bodies do not recover when midguts were submerged in RPMI. Scale bars = 10 mm. See also the Video S1 of the rapid recovery of in vitro-derived organisms. doi:10.1371/journal.ppat.1002532.g008 PLoS Pathogens \| www.plospathogens.org February 2012 \| Volume 8 \| Issue 2 \| e1002532 PLoS Pathogens \| www.plospathogens.org PLoS Pathogens \| www.plospathogens.org 10 Borrelia Requires RpoS for Tick Transmission PLoS Pathogens \| www.plospathogens.org Assessment of B. burgdorferi survival by quantitative culture and qPCR Spirochete burdens were assessed by quantitative PCR (qPCR) using individual pools of 10 unfed and 5 replete nymphs. Total genomic DNA was isolated from surface-sterilized nymphs using the Gentra Puregene Yeast and Bacteria kit (Qiagen) according to the manufacturer’s instructions. DNAs were diluted 1:10 in water and analyzed using a TaqMan-based assay for flaB ([30] and Table S1). In some experiments, the viability of spirochetes within replete nymphs (5 per strain) was assessed by semi-solid plating in pBSK medium as previously described [61]. Generation of Bb infected I. scapularis ticks To generate naturally-infected ticks, 300 to 400 pathogen-free I. scapularis larvae (Oklahoma State University) were placed on infected C3H/HeJ mice 2 to 3 weeks after needle inoculation with CE162 as described above; larvae were allowed to feed to repletion and then held in an environmental incubator until they had molted. For experiments using Bb isolates that were avirulent by syringe, see above, naı¨ve larvae were infected according to the immersion method described by Policastro and Schwan [28], fed on naı¨ve C3H/HeJ mice, and allowed to molt into nymphs. Assessment of Bb infectivity by needle inoculation Assessment of Bb infectivity by needle inoculation Infectivity was assessed using 4–6 week old female C3H/HeJ mice (five per group, per isolate) that were inoculated intradermally with 16104 spirochetes. Infection was assessed at 2 and 4 weeks post- inoculation by serology and cultivation of tissues (ears, skin, joints, hearts, and/or bladders) in BSK-II containing sulfamethoxazole (0.05 mg/ml), phosphomycin (0.02 mg/ml), rifampin (0.05 mg/ml), trimethoprim (0.01 mg/ml) and amphotericin (0.0025 mg/ml). Cultures were monitored weekly by dark-field microscopy. Transmission Electron Microscopy (TEM) py For TEM, fed midguts were isolated at 48 and 72 h post- placement and immersed for 3 h at 4uC in 0.1 M sodium cacodylate buffer (CAC) containing 4% paraformaldehyde and 2.5% glutaraldehyde. The midguts then were embedded in 1% agarose solution in 0.1 M CAC buffer. After solidification of the agarose, the midguts were sliced into .1 mm fragments, transferred to fresh 0.1 M CAC buffer and incubated overnight at 4uC. The specimens then were rinsed with fresh 0.1 M CAC buffer and post-fixed for 2 h in 0.1 M CAC buffer containing 1% OsO4, and 0.8% potassium ferricyanide. Individual midgut slices were washed in water, stained with 1% uranyl acetate, dehydrated in ascending ethanol solutions, and embedded in PolyBed812. Thin sections (70 nm) were cut on an ultramicrotome (Leica), collected on formvar coated slot grids, and stained in 2.5% uranyl acetate and lead citrate. The stained thin sections were viewed on Processing of nymphal midguts for light microscopy For fluorescence microscopy, intact midguts were isolated from unfed and partially-fed (48 and 72 h post-placement) nymphs and labeled with FM4-64 (2 ng/ml in PBS; Invitrogen) as described previously [30]. Confocal microscopy was performed on a Zeiss LSM-510; serial Z-series images were acquired in both red and green channels [30]. Briefly, our standard methodology for acquiring serial Z-series images involves obtaining 1-mm optical sections through the depth of the midgut that can be resolved by a confocal microscope (,50 mm). The basement membrane repre- sents the first image in each Z-series; subsequent optical sections were acquired through the epithelial layer towards the lumen (Figure S3A). For midguts isolated from unfed and 48 h-fed nymphs, optical sections were acquired through the full thickness of each specimen (i.e., from basement membrane to basement membrane, Figure S3A). As feeding progresses, influx of the blood meal and expansion of the luminal space precludes our ability to image through the full thickness (.100 mm) of individual diverticula. Consequently, optical sections of 72 h-fed midguts were acquired through a single epithelial layer (i.e., from the basement membrane to the luminal surface, Figure S3B). Representative composites of three consecutive 1-mm optical sections acquired (i) at the luminal surface, (ii) midway through the epithelium, and (iii) at the basement membrane are used to present the distribution of spirochetes through the midgut; the midway point for each specimen was determined during image processing based on the thickness of the epithelial layer. Routine optical sections of unfed and fed midguts were acquired in the coronal plane (Figure S3C). To visualize DrpoS-gfp organisms within 72 h- fed midguts, however, two additional steps were required to reduce light scattering and absorption by the luminal contents. First, we carefully removed a portion (,30–40%) of the blood meal to minimize interference from the blood meal and reduce the overall thickness of the midgut. Individual diverticula were then imaged in the transverse plane beginning at or near the tip (Figure S3D). A minimum of 20 Bb-infected nymphal midguts were examined at each time point. Epifluorescence microscopy was performed on an Olympus BX41 microscope equipped with a Retiga Exi (QImaging) camera; images were acquired using a 406 (1.4 NA) oil immersion objective with QCapture software v. 2.1 (QImaging). Fed midguts also were processed for paraffin- embedding and silver staining using the Wharthin-Starry method as previously described [30]. Assessment of spirochete dissemination in ticks and transmission to mice Groups of 10 to 12 infected unfed nymphs were confined to capsules affixed to the backs of naı¨ve C3H/HeJ mice [14]. At 72 h post-placement, approximately 5–6 feeding nymphs were forcible removed, surface-sterilized, and hemolymph collected for cultur- ing in BSK-II as previously described [30], while the remaining ticks were allowed to feed to repletion. At 24, 48 and 72 h post- repletion, groups of mice (3–4 per strain, per time point) used as a blood meal source for infected nymphs were sacrificed and their capsules removed. For each mouse, a 3 cm62 cm region of skin encompassing the feeding site was demarcated using a grid. The excised skin then was cut into equal portions, rinsed in 70% ethanol, and cultured in BSK-II. Bb burdens in replete nymphs were determined using qPCR as described above. PLoS Pathogens \| www.plospathogens.org Culture and maintenance of bacterial strains B. burgdorferi isolates used in these studies (Table 1) were cultivated in modified BSK-II [57] supplemented with 6% rabbit February 2012 \| Volume 8 \| Issue 2 \| e1002532 11 Borrelia Requires RpoS for Tick Transmission gene. Expression levels for each gene of interest were normalized against copies of flaB determined using a TaqMan-based assay (Table S1) performed using iQ Supermix. essment of Bb infectivity by needle inoculation Round body formation and recovery Serum starvation experiments were performed as previously described [32]. Briefly, aliquots (,400 ml) of temperature-shifted cultures at late logarithmic phase were transferred to 15 ml of RMPI-1640 (Invitrogen, catalogue #11875) at a final density of 1.56107 spirochetes/ml) and incubated at 37uC. At 24 h intervals, organisms were enumerated by darkfield microscopy and spirochete morphology was assessed by either darkfield (non- fluorescent isolates) or epifluorescence (fluorescent isolates) mi- croscopy. To assess the ultrastructure of round bodies, spirochetes cultivated in RPMI or BSK-II for 3 days were centrifuged at 6800 g, rinsed in PBS, and resuspended in 0.1 M CAC buffer containing 4% paraformaldehyde and 2.5% glutaraldehyde. The organisms were incubated on ice for 15 minutes then centrifuged at 80006g. Cell pellets were embedded in agarose and processed for examination by TEM as described above. In vitro generated round bodies were recovered by the addition of an equal volume of BSK-II after 4 days of cultivation in RPMI. Recovery was monitored in real-time using StreamPix software (Norpix) on an Olympus BX41 microscope equipped with a Retiga Exi camera. Videos were generated using QuickTime Player v.7.7 (Apple, Inc.). The recovery of round bodies in nymphs at 72 h post- placement was performed by first dissecting the midgut in RPMI. The diverticula were then detached from the stomach using a scalpel blade. A cover slip was then applied to the cover glass and real-time imaging was performed for the RPMI control. Round body recovery in nymphal midguts was induced by adding 10 ml BSK-II to the dissected midguts. Recovery was monitored in real- time as described above. Figure S2 Contours of the RpoSBb regulon in I. scapu- laris. qRT-PCR analysis of (A) absolutely and (B) partially RpoS- dependent upregulated genes selected from microarray data derived from Bb cultivated within DMCs [13]. Expression profiling was performed using fed larvae, unfed and fed nymphs that had been naturally-infected with WT Bb as well as fed nymphs that had been infected as larvae by immersion with either WT-gfp or DrpoS-gfp isolates. Values represent the average flaB-normalized transcript copy number 6 SEM for each gene; average values are considered significantly different when p is #0.05 (indicated by asterisks). (TIF) Figure S3 Cartoon illustrating the scheme used to acquire optical sections through unfed and fed nymph- al midguts isolated at 48 and 72 h post-placement. Statistical analysis Statistical analyses were performed on qPCR, spirochete viability, and round body formation using Prism 5 (GraphPad Software, Inc.). Student’s t tests were used to calculate the significance of the qPCR and spirochete viability (p#0.05 was considered significant) and is indicated in the figures and/or legends. Unpaired student’s t test with two-tailed p values were used to compare round body formation between different Bb isolates at each of the time points studied. For qRT-PCR, the normalized copy number values for each gene of interest were compared in Prism using an unpaired t-test with two-tailed p values and a 95% confidence interval. qRT-PCR For expression studies involving naturally-infected ticks, total RNA was isolated from ,150 fed larvae or unfed nymphs and ,75 engorged nymphs that had been infected as larvae by feeding on a mouse infected with CE162. For studies comparing gene expression in WT-gfp (Bb914) and DrpoS-gfp (Bb1058) isolates, total RNA was isolated from ,60 engorged nymphs that had been infected as larvae by immersion and then fed as nymphs on naı¨ve mice as described above. RNA was isolated from infected ticks using TRIzol reagent (Invitrogen) as previously described [14]. cDNAs (+RT) were assayed in quadruplicate using iQ SYBR Green Supermix (BioRad) and gene specific primers (Table S1). Transcript copy numbers were calculated using the iCycler post- run analysis software based on internal standard curves for each February 2012 \| Volume 8 \| Issue 2 \| e1002532 12 Borrelia Requires RpoS for Tick Transmission a transmission electron microscope (Hitachi H-7650) at 80 kV. Images were acquired using an AMT camera and Image Capture Engine v6.01 software (Advanced Microscopy Techniques). All images were processed using ImageJ v.1.42 software [62]. In Figure 5, color overlays were added using Adobe Photoshop (CS4). OspC. (A) Spirochete burdens of WT, DrpoS, RpoS complement- ed RpoS Compl) and DospC before and after feeding on naı¨ve C3H/HeJ mice. The nymphs used in these experiments were used in Table 2. WT-gfp and DrpoS-gfp (B) burdens determined by qPCR and (C) viability, assessed by semi-solid plating, are highly similar in unfed (black) and fed (gray) nymphs. Values represent the means 6 SEMs from three independent experiments. (TIF) Round body formation and recovery Serial Z-series confocal images were generated by obtaining 1- mm optical sections through the depth of unfed and fed midguts as described in Materials and Methods. (A) Cartoon depicting the scheme used to image individual diverticulum from unfed and 48 h-fed nymphal midguts; for these types of specimens, optical sections were acquired in the coronal plane through the full thickness of a midgut. (B) Cartoon depicting the scheme used to acquire images of an individual diverticulum from a 72 h-fed midgut in the coronal and transverse planes. (C) Zoom-in of coronal views for 72 h-fed nymphal midguts infected with either WT or DrpoS organisms illustrating the depths at which fluorescent spirochetes can be visualized by confocal microscopy. (D) Zoom-in of 72 h-fed nymphal midguts infected with DrpoS organisms imaged in the transverse plane after a portion of the blood meal had been removed; note the difference in the luminal space in the coronal and transverse planes. Solid-line arrows are used to indicate the laser beam, while a dashed line and ‘‘eye’’ are used to represent what is detected by the microscope’s photomultiplier tube. Abbreviations: LS, luminal surface; MW, midway through the epithelial layer; and BM, basement membrane. Bioinformatics Candidate surface-exposed lipoproteins within the RpoS regulon [13] were identified using the database created by Setubal et al. [55]. Non-surface exposed lipoproteins then were excluded based on published data [49,63,64]. Candidate RpoS-dependent b-barrel-outer membrane spanning proteins (OMPs) were identi- fied and filtered using the consensus computational framework recently used to identify OMPs in Treponema pallidum [54]. The proteins identified by two or more OMP localization/topology programs were designated OMP candidates (Table S3). Figure S4 Spirochetes introduced by immersion un- dergo biphasic dissemination. (A) Spirochete burdens are identical in unfed and fed nymphs that acquired WT Bb as larvae by immersion or naturally by feeding on infected mice. Burdens were determined by qPCR; values represent the means 6 SDs from three independent experiments. (B) The distribution of WT- gfp spirochetes in nymphs infected as larvae by immersion is highly similar to that of spirochetes within naturally-infected nymphs [30]. Composite images depicting midguts of nymphs infected by immersion are the same as those presented in Figures 2 and 3, while the images of midguts isolated from naturally-infected nymphs were taken from [30] and used with permission. The leftmost images in each panel ‘‘Full Composite’’ depict the full thickness of the midgut, while 3-mm composite images show spirochetes at the luminal surface, midway through the epithelial layer, and at the basement membrane. A detailed schematic indicating how confocal images of unfed and fed midguts were acquired is presented in Figure S3. Green PLoS Pathogens \| www.plospathogens.org Table S3 Known and/or putative RpoS-dependent tick midgut adhesins. (DOC) Video S1 Round bodies generated in vitro rapidly elongate into spirochetes. Spirochetes were transformed into round bodies by culturing either WT-gfp or DrpoS-gfp isolates in RPMI for 4 days. The video is arranged into two segments depicting the rapid transformation of WT-gfp or DrpoS-gfp round bodies into normal spirochetes after the addition of BSK-II (see the upper left hand corner for the time stamp). (MOV) Figure S5 Complementation of DrpoS Bb with a wild- type copy of rpoS restores expression of RpoS-dependent genes following temperature-shift. Whole cell B. burgdorferi lysates were prepared from WT (Bb914), DrpoS (Bb1058) and RpoS-complemented (SE186) isolates, separated by SDS-PAGE and stained with silver as described in Materials and Methods. (TIF) Figure S6 DrpoS organisms have an altered morphology and distribution pattern within 72 h-fed midguts. Rep- resentative composite images of cryosectioned midguts infected with (A) WT-gfp or (B) DrpoS-gfp Bb; green represents spirochetes expressing GFP while red indicates midgut epithelial cells labeled with FM4-64; scale bars = 25 mm. (TIF) Acknowledgments We would like to thank the personnel of the Histology facility at UCHC and Maya Yankova of the EM facility at UCHC. We would like to extend our deep appreciation to Drs. Juan Salazar and Zhu Wang for their helpful assistance with the statistics. We are also indebted to Amit Luthra for his help predicting the surface localization of RpoS-dependent proteins. We would like to thank the personnel of the Histology facility at UCHC and Maya Yankova of the EM facility at UCHC. We would like to extend our deep appreciation to Drs. Juan Salazar and Zhu Wang for their helpful assistance with the statistics. We are also indebted to Amit Luthra for his help predicting the surface localization of RpoS-dependent proteins. Figure S7 Loss of RpoS and CoADR enhances round body formation under nutrient-limiting conditions in vitro. Representative images of (A) WT-gfp, (B) DrpoS-gfp, (C) complemented DrpoS-gfp, (D) Dcdr, and (E) complemented Dcdr isolates after 3 days in RPMI; scale bar = 50 mm. References 1. Gruber TM, Gross CA (2003) Multiple sigma subunits and the partitioning of bacterial transcription space. Annu Rev Microbiol 57: 441–466. 15. Schwan TG, Piesman J, Golde WT, Dolan MC, Rosa PA (1995) Induction of an outer surface protein on Borrelia burgdorferi during tick feeding. Proc Natl Acad Sci U S A 92: 2909–2913. p p 2. Hengge-Aronis R (2002) Recent insights into the general stress response regulatory network in Escherichia coli. J Mol Microbiol Biotechnol 4: 341–346. 16. Ohnishi J, Piesman J, de Silva AM (2001) Antigenic and genetic heterogeneity of Borrelia burgdorferi populations transmitted by ticks. Proc Natl Acad Sci U S A 98: 670–675. 3. Battesti A, Majdalani N, Gottesman S (2011) The RpoS-Mediated General Stress Response in Escherichia coli. Annu Rev Microbiol 65: 189–213. Stress Response in Escherichia coli. Annu Rev Microbiol 65: 189–213 17. Pal U, de Silva AM, Montgomery RR, Fish D, Anguita J, et al. (2000) Attachment of Borrelia burgdorferi within Ixodes scapularis mediated by outer surface protein A. J Clin Invest 106: 561–569. 4. Hengge-Aronis R (2002) Signal transduction and regulatory mechanisms involved in control of the ss (RpoS) subunit of RNA polymerase. Microbiol Mol Biol Rev 66: 373–395. 18. Fikrig E, Barthold SW, Flavell RA (1993) OspA vaccination of mice with established Borrelia burgdorferi infection alters disease but not infection. Infect Immun 61: 2553–2557. 5. Klauck E, Typas A, Hengge R (2007) The sS subunit of RNA polymerase as a signal integrator and network master regulator in the general stress response in Escherichia coli. Sci Prog 90: 103–127. 6. Dong T, Schellhorn HE (2010) Role of RpoS in virulence of pathogens. Infect Immun 78: 887–897. 19. Grimm D, Tilly K, Byram R, Stewart PE, Krum JG, et al. (2004) Outer-surface protein C of the Lyme disease spirochete: A protein induced in ticks for infection of mammals. Proc Natl Acad Sci U S A 101: 3142–3147. 7. Whiteley M, Parsek MR, Greenberg EP (2000) Regulation of quorum sensing by RpoS in Pseudomonas aeruginosa. J Bacteriol 182: 4356–4360. of mammals. Proc Natl Acad Sci U S A 101: 3142–3147. 20. Gilmore RD, Jr., Howison RR, Schmit VL, Nowalk AJ, Clifton DR, et al. (2007) Temporal expression analysis of the Borrelia burgdorferi paralogous gene family 54 genes BBA64, BBA65, and BBA66 during persistent infection in mice. Infect Immun 75: 2753–2764. 8. Author Contributions Conceived and designed the experiments: SMDE MJC CHE JDR. Performed the experiments: SMDE MJC CHE. Analyzed the data: SMDE MJC CHE JDR. Contributed reagents/materials/analysis tools: SMDE MJC CHE JDR. Wrote the paper: SMDE MJC JDR. Conceived and designed the experiments: SMDE MJC CHE JDR. Performed the experiments: SMDE MJC CHE. Analyzed the data: SMDE MJC CHE JDR. Contributed reagents/materials/analysis tools: SMDE MJC CHE JDR. Wrote the paper: SMDE MJC JDR. Supporting Information Figure S1 Spirochete persistence and survival in I. scapularis are not affected by loss of either RpoS or February 2012 \| Volume 8 \| Issue 2 \| e1002532 13 Borrelia Requires RpoS for Tick Transmission represents GFP+ spirochetes and red indicates midgut epithelial cells labeled with FM4-64; scale bars = 25 mm. Images of naturally infected nymphs are reproduced with permission from Live imaging reveals a biphasic mode of dissemination of Borrelia burgdorferi within ticks; published in Volume 119, Issue 12 (December 1,2009), J Clin Invest. 2009; 119(12):3652–3665. doi10.117/JCI39401. Copyright 2009, American Society for Clinical Investigation [30]. (TIF) References Molofsky AB, Swanson MS (2004) Differentiate to thrive: lessons from the Legionella pneumophila life cycle. Mol Microbiol 53: 29–40. 9. Fang FC, Libby SJ, Buchmeier NA, Loewen PC, Switala J, et al. (1992) The alternative sigma factor katF (rpoS) regulates Salmonella virulence. Proc Natl Acad Sci U S A 89: 11978–11982. 21. Norris SJ, Coburn J, Leong JM, Hu LT, Hook M (2010) Pathobiology of Lyme Disease Borrelia. In: Samuels DS, Radolf JD, eds. Borrelia: Molecular Biology, Host Interaction and Pathogenesis. Norfolk, UK: Caister Academic Press. pp 293–325. 10. Ouyang Z, Kumar M, Kariu T, Haq S, Goldberg M, et al. (2009) BosR (BB0647) governs virulence expression in Borrelia burgdorferi. Mol Microbiol 74: 1331–1343. 22. Brorson O, Brorson SH (1998) In vitro conversion of Borrelia burgdorferi to cystic forms in spinal fluid, and transformation to mobile spirochetes by incubation in BSK-H medium. Infection 26: 144–150. 11. Hyde JA, Shaw DK, Smith Iii R, Trzeciakowski JP, Skare JT (2009) The BosR regulatory protein of Borrelia burgdorferi interfaces with the RpoS regulatory pathway and modulates both the oxidative stress response and pathogenic properties of the Lyme disease spirochete. Mol Microbiol 74: 1344–1355. 23. Brorson O, Brorson SH, Scythes J, MacAllister J, Wier A, et al. (2009) Destruction of spirochete Borrelia burgdorferi round-body propagules (RBs) by the antibiotic tigecycline. Proc Natl Acad Sci U S A 106: 18656–18661. 12. Samuels DS (2011) Gene Regulation in Borrelia burgdorferi. Annu Rev Microbiol 65: 479–499. 24. Brorson O, Brorson SH (1997) Transformation of cystic forms of Borrelia burgdorferi to normal, mobile spirochetes. Infection 25: 240–246. 13. Caimano MJ, Iyer R, Eggers CH, Gonzalez C, Morton EA, et al. (2007) Analysis of the RpoS regulon in Borrelia burgdorferi in response to mammalian host signals provides insight into RpoS function during the enzootic cycle. Mol Microbiol 65: 1193–1217. 25. Pal U, Yang X, Chen M, Bockenstedt LK, Anderson JF, et al. (2004) OspC facilitates Borrelia burgdorferi invasion of Ixodes scapularis salivary glands. J Clin Invest 113: 220–230. 14. Mulay VB, Caimano MJ, Iyer R, Dunham-Ems S, Liveris D, et al. (2009) Borrelia burgdorferi bba74 is expressed exclusively during tick feeding and is regulated by both arthropod- and mammalian host-specific signals. J Bacteriol 191: 2783–2794. 26. Tilly K, Krum JG, Bestor A, Jewett MW, Grimm D, et al. (2006) Borrelia burgdorferi OspC protein required exclusively in a crucial early stage of mammalian infection. Infect Immun 74: 3554–3564. Table S1 Oligonucleotides used in this study. (DOC) represents GFP+ spirochetes and red indicates midgut epithelial cells labeled with FM4-64; scale bars = 25 mm. Images of naturally infected nymphs are reproduced with permission from Live imaging reveals a biphasic mode of dissemination of Borrelia burgdorferi within ticks; published in Volume 119, Issue 12 (December 1,2009), J Clin Invest. 2009; 119(12):3652–3665. doi10.117/JCI39401. Copyright 2009, American Society for Clinical Investigation [30]. (TIF) Table S2 Virulence of B. burgdorferi strains following needle-inoculation. (DOC) Borrelia Requires RpoS for Tick Transmission (2011) Borrelia burgdorferi Requires Glycerol for Maximum Fitness During The Tick Phase of the Enzootic Cycle. PLoS Pathog 7: e1002102. 33. Xu Q, McShan K, Liang FT (2008) Essential protective role attributed to the surface lipoproteins of Borrelia burgdorferi against innate defences. Mol Microbiol 69: 15–29. y g 52. Yang XF, Pal U, Alani SM, Fikrig E, Norgard MV (2004) Essential role for OspA/B in the life cycle of the Lyme disease spirochete. J Exp Med 199: 641–648. 34. Boylan JA, Hummel CS, Benoit S, Garcia-Lara J, Treglown-Downey J, et al. (2006) Borrelia burgdorferi bb0728 encodes a coenzyme A disulphide reductase whose function suggests a role in intracellular redox and the oxidative stress response. Mol Microbiol 59: 475–486. 53. Pal U, Fikrig E (2010) Tick Interactions. In: Samuels DS, Radolf JD, eds. Borrelia: Molecular Biology, Host Interaction and Pathogenesis. Norfolk, UK: Caister Academic Press. pp 273–292. 35. Gherardini F, Boylan J, Lawrence K, Skare J (2010) Metabolism and Physiology of Borrelia. In: Samuels DS, Radolf JD, eds. Borrelia Molecular biology, host interaction, and pathogenesis. Norfolk, UK: Caister Academic Press. pp 103–138. 54. Cox DL, Luthra A, Dunham-Ems S, Desrosiers DC, Salazar JC, et al. (2010) Surface immunolabeling and consensus computational framework to identify candidate rare outer membrane proteins of Treponema pallidum. Infect Immun 78: 5178–5194. 36. Caimano MJ, Eggers CH, Gonzalez CA, Radolf JD (2005) Alternate sigma factor RpoS is required for the in vivo -specific repression of Borrelia burgdorferi plasmid lp54-borne ospA and lp6.6 genes. J Bacteriol 187: 7845–7852. 55. Setubal JC, Reis M, Matsunaga J, Haake DA (2006) Lipoprotein computational prediction in spirochaetal genomes. Microbiology 152: 113–121. 37. Chiang SM, Schellhorn HE (2010) Evolution of the RpoS regulon: origin of RpoS and the conservation of RpoS-dependent regulation in bacteria. J Mol Evol 70: 557–571. 56. Balashov YS (1972) Blood-sucking ticks (Ixodidae)-vectors of disease of man and animals. Misc Pub Entomol Soc Am 8: 161–376. 57. Pollack RJ, Telford SR, Spielman A (1993) Standardization of medium for culturing Lyme disease spirochetes. J Clin Microbiol 31: 1251–1255. 38. Radolf JD, Caimano MJ (2008) The long strange trip of Borrelia burgdorferi outer- surface protein C. Mol Microbiol 69: 1–4. 58. Eggers CH, Caimano MJ, Clawson ML, Miller WG, Samuels DS, et al. Borrelia Requires RpoS for Tick Transmission 27. Caimano MJ, Eggers CH, Hazlett KR, Radolf JD (2004) RpoS is not central to the general stress response in Borrelia burgdorferi but does control expression of one or more essential virulence determinants. Infect Immun 72: 6433–6445. 45. Hodzic E, Feng S, Freet KJ, Barthold SW (2003) Borrelia burgdorferi population dynamics and prototype gene expression during infection of immunocompetent and immunodeficient mice. Infect Immun 71: 5042–5055. 28. Policastro PF, Schwan TG (2003) Experimental infection of Ixodes scapularis larvae (Acari: Ixodidae) by immersion in low passage cultures of Borrelia burgdorferi. J Med Entomol 40: 364–370. 46. Xu H, He M, He JJ, Yang XF (2010) Role of the surface lipoprotein BBA07 in the enzootic cycle of Borrelia burgdorferi. Infect Immun 78: 2910–2918. 47. Xu H, He M, Pang X, Xu ZC, Piesman J, et al. (2010) Characterization of the highly regulated antigen BBA05 in the enzootic cycle of Borrelia burgdorferi. Infect Immun 78: 100–107. 29. Eggers CH, Caimano MJ, Malizia RA, Kariu T, Cusack B, et al. (2011) The coenzyme A disulfide reductase of Borrelia burgdorferi is important for rapid growth throughout the enzootic cycle and essential for infection of the mammalian host. Mol Microbiol 82: 679–697. 48. Pal U, Dai J, Li X, Neelakanta G, Luo P, et al. (2008) A differential role for BB0365 in the persistence of Borrelia burgdorferi in mice and ticks. J Infect Dis 197: 148–155. 30. Dunham-Ems SM, Caimano MJ, Pal U, Wolgemuth CW, Eggers CH, et al. (2009) Live imaging reveals a novel, biphasic mode of dissemination of Borrelia burgdorferi within ticks. J Clin Invest 119: 3652–3665. 49. Promnares K, Kumar M, Shroder DY, Zhang X, Anderson JF, et al. (2009) Borrelia burgdorferi small lipoprotein Lp6.6 is a member of multiple protein complexes in the outer membrane and facilitates pathogen transmission from ticks to mice. Mol Microbiol 74: 112–125. 31. Miklossy J, Kasas S, Zurn AD, McCall S, Yu S, et al. (2008) Persisting atypical and cystic forms of Borrelia burgdorferi and local inflammation in Lyme neuroborreliosis. J Neuroinflammation 5: 40–58. 50. Kumar M, Yang X, Coleman AS, Pal U (2010) BBA52 facilitates Borrelia burgdorferi transmission from feeding ticks to murine hosts. J Infect Dis 201: 1084–1095. 32. Alban PS, Johnson PW, Nelson DR (2000) Serum-starvation-induced changes in protein synthesis and morphology of Borrelia burgdorferi. Microbiology 146: 119–127. 51. Pappas CJ, Iyer R, Petzke MM, Caimano MJ, Radolf JD, et al. Borrelia Requires RpoS for Tick Transmission (2002) Identification of loci critical for replication and compatibility of a Borrelia burgdorferi cp32 plasmid and use of a cp32-based shuttle vector for the expression of fluorescent reporters in the Lyme disease spirochaete. Mol Microbiol 43: 281–295. 39. Patton TG, Dietrich G, Dolan MC, Piesman J, Carroll JA, et al. (2011) Functional analysis of the Borrelia burgdorferi bba64 gene product in murine infection via tick infestation. PLoS One 6: e19536. 40. Gilmore RD, Jr., Howison RR, Dietrich G, Patton TG, Clifton DR, et al. (2010) The bba64 gene of Borrelia burgdorferi, the Lyme disease agent, is critical for mammalian infection via tick bite transmission. Proc Natl Acad Sci U S A 107: 7515–7520. 59. Eggers CH, Caimano MJ, Radolf JD (2004) Analysis of promoter elements involved in the transcriptional initiation of RpoS-dependent Borrelia burgdorferi genes. J Bacteriol 186: 7390–7402. 41. Rogers EA, Terekhova D, Zhang H-M, Hovis KM, Schwartz I, et al. (2009) Rrp1, a cyclic-di-GMP-producing response regulator, is an important regulator of Borrelia burgdorferi core cellular functions. Mol Microbiol 71: 1551–1573. 60. Bono JL, Elias AF, Kupko JJ, Stevenson B, Tilly K, et al. (2000) Efficient targeted mutagenesis in Borrelia burgdorferi. J Bacteriol 182: 2445–2452. 61. Samuels DS (1995) Electrotransformation of the spirochete Borrelia burgdorferi. Electrotransformation protocols for microorgansims. Meth Mol Biol 47: 253–259. 42. He M, Ouyang Z, Troxell B, Xu H, Moh A, et al. (2011) Cyclic di-GMP is essential for the survival of the lyme disease spirochete in ticks. PLoS Pathog 7: e1002133. 62. Rasband WS (2009) ImageJ. Available: http://rsb.info.nih.gov/ij/ U.S. National Institues of Health. 43. Caimano MJ, Kenedy MR, Kairu T, Desrosiers D, Harman M, et al. (2011) The hybrid sensory histidine kinase Hk1 (BB0420) of Borrelia burgdorferi is part of a two-component system essential for survival in feeding larval and nymphal Ixodes scapularis ticks. Infect Immun 79: 3117–3130. 63. Mulay V, Caimano MJ, Liveris D, Desrosiers DC, Radolf JD, et al. (2007) Borrelia burgdorferi BBA74, a periplasmic protein associated with the outer membrane, lacks porin-like properties. J Bacteriol 189: 2063–2068. p 44. Montgomery RR, Malawista SE, Feen KJM, Bockenstedt LK (1996) Direct demonstration of antigenic substitution of Borrelia burgdorferi ex vivo: exploration of the paradox of the early immune response to outer surface proteins A and C in Lyme disease. J ExpMed 183: 261–269. 64. Banik S, Terekhova D, Iyer R, Pappas CJ, Caimano MJ, et al. References PLoS Pathogens \| www.plospathogens.org PLoS Pathogens \| www.plospathogens.org February 2012 \| Volume 8 \| Issue 2 \| e1002532 February 2012 \| Volume 8 \| Issue 2 \| e1002532 14 February 2012 \| Volume 8 \| Issue 2 \| e1002532 Borrelia Requires RpoS for Tick Transmission (2011) BB0844, an RpoS-regulated protein, is dispensable for Borrelia burgdorferi infectivity and maintenance in the mouse-tick infectious cycle. Infect Immun 79: 1208–1217. PLoS Pathogens \| www.plospathogens.org February 2012 \| Volume 8 \| Issue 2 \| e1002532 PLoS Pathogens \| www.plospathogens.org 15
https://openalex.org/W2103393505	https://rbej.biomedcentral.com/counter/pdf/10.1186/1477-7827-9-147	English	null	New algorithm for OHSS prevention	Reproductive biology and endocrinology	2,011	cc-by	4,066	* Correspondence: drvagpapanikolaou@yahoo.gr 1Human Reproduction & Genetics Foundation, Adrianoupoleos 6, 55133 Kalamaria, Thessaloniki, Greece Full list of author information is available at the end of the article © 2011 Papanikolaou et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. New algorithm for OHSS prevention Evangelos G Papanikolaou1,7*, Peter Humaidan2, Nikos Polyzos3, Sofia Kalantaridou4, Sahar Kol5, Claudio Benadiva6, Herman Tournaye3 and Basil Tarlatzis7 Abstract Ovarian hyperstimulation syndrome (OHSS) still remains a life-threatening complication of in vitro fertilization treatment (IVF), keeping patients and especially those, who previously experienced OHSS, from attempting infertility treatment and childbearing. The recent implementation of four new modalities: the GnRH antagonist protocol, GnRH agonist (GnRHa) triggering of ovulation, blastocyst transfer and embryo/oocyte vitrification, renders feasible the elimination of OHSS in connection with ovarian hyperstimulation for IVF treatment. The proposed current algorithm is based on the number of follicles developed after ovarian stimulation, setting a cut-off level at the development of 18 or more follicles. Further, fulfilling this criterion, the algorithm is based on four decision-making points: the final day of patient work-up, the day of triggering final oocyte maturation, day-1 post oocyte pick-up (OPU) and day-5 post OPU. y p If the physician decides to administer hCG for final oocyte maturation regardless the type of analogue used, he has the option on day-1 to either freeze all embryos or to proceed to day-5. On this day, based on the clinical condition of the patient, a decision should be made to either transfer a single blastocyst or to vitrify all blastocysts available. However, this strategy will not guarantee an OHSS free luteal phase especially if a pregnancy occurs. If the physician decides to trigger ovulation with GnRHa, feasible only with the antagonist protocol, embryos can be cultured until day-5. On this day a transfer can be performed with no risk of OHSS and spare blastocysts may be vitrified. Alternatively, on day-1 or day-2 post OPU, all embryos could be frozen. Hopefully, in a near future, GnRHa triggering and vitrification of oocytes will become everyday practice. Only the combined use of a GnRH antagonist protocol with GnRHa triggering and subsequent single blastocyst transfer or embryo/oocyte freezing will completely abolish the risk of OHSS after ovarian hyperstimulation. Papanikolaou et al. Reproductive Biology and Endocrinology 2011, 9:147 http://www.rbej.com/content/9/1/147 Papanikolaou et al. Reproductive Biology and Endocrinology 2011, 9:147 http://www.rbej.com/content/9/1/147 Papanikolaou et al. Reproductive Biology and Endocrinology 2011, 9:147 http://www.rbej.com/content/9/1/147 Background the production of endogenous HCG from the implant- ing embryo [3]. Therefore, two types of OHSS have been identified: the early onset OHSS which is self-lim- ited in case no pregnancy occurs, and the late onset OHSS which develops ten days or more after the egg retrieval [4]. In contrast to the early OHSS the late onset OHSS is poorly correlated to the ovarian response after stimulation. The most feared complication of IVF-related ovarian sti- mulation for the patient as well as the doctor is the development of ovarian hyperstimulation syndrome (OHSS) [1]; a syndrome, which in its severe form leads to hospitalization and in the worst case scenario fatal complications. The incidence of clinically significant OHSS is 2-3%, however, milder forms of OHSS might develop in up to 20-30% of all IVF patients [2]. All late onset OHSS cases are related to pregnancy and these cases often require hospitalization. Unfortu- nately these late OHSS cases render the prediction of OHSS a difficult task [5] and the methods used to pre- dict the condition prior to stimulation have been shown having limited success [6]. The basis for OHSS development is the development of multiple follicles. Once this criterion is fulfilled, the second factor needed for the development of the severe form of the disease is either the exogenous administra- tion of HCG for final oocyte maturation - as is the cur- rent practice - or the establishment of a pregnancy and The protocol of choice for potential high-responder patients prone to develop OHSS should be the GnRH antagonist protocol, as it has been shown to decrease the incidence of OHSS significantly [7,8]. Furthermore, it allows the utilization of a GnRHa to induce final Page 2 of 5 Papanikolaou et al. Reproductive Biology and Endocrinology 2011, 9:147 http://www.rbej.com/content/9/1/147 Page 2 of 5 Papanikolaou et al. Reproductive Biology and Endocrinology 2011, 9:147 http://www.rbej.com/content/9/1/147 Papanikolaou et al. Reproductive Biology and Endocrinology 2011, 9:147 http://www.rbej.com/content/9/1/147 four time-points: the final day of patient work-up, the day of ovulation triggering, day-1 post-OPU and day-5 post-OPU (Figure 1). However, the OHSS reducing strategy obviously already starts when the physician evaluates the patient’s ovarian reserve and thus the risk for hyperstimulation prior to stimulation [6]. oocyte maturation, which has recently regained interest. Final day of patient work-up (Decision point 1) Final day of patient work-up (Decision point 1) A mandatory step in the ovarian stimulation for IVF is firstly the gonadotropin dose and secondly the protocol. Regarding the prediction of hyper-response, recent evi- dence suggests the superiority of AMH over antral folli- cle count [11,12]. In a prospective cohort of 262 IVF cycles with 8% moderate and severe OHSS, Lee et al showed that an AMH cut-off value of 3.36 ng/mL gave a sensitivity of 90.5% and a specificity of 81.3% to pre- dict OHSS [11,12]. Regarding the antral follicle count, Kwee et al have shown that a cut off level of > 14 antral follicles gave the highest sensitivity (82%) and specificity (89%) and also the highest accuracy. With a prevalence of 15% for high response (defined as > 20 oocytes in an At the same time, the development of the vitrification procedure has improved the embryo survival rate as compared to the classical method of slow freezing [10]. Thus, considering the different modalities that the phy- sician has available, we below propose an algorithm for OHSS high-risk patients. The algorithm may easily be applied according to the preferences of the doctor and his patient and refers to both GnRH-analogues, GnRH agonist as well as GnRH antagonist. Background The pooled evidence shows that by triggering with GnRHa in patients co-treated with a GnRH antagonist protocol, not only is OHSS minimized, but also this concept allows embryo transfer in the hyper-responding patient with a reproductive outcome comparable to that seen after hCG triggering as long as adequate luteal sup- port can be achieved [9]. Algorithm The algorithm is based on two decision making time periods: the follicular phase and the luteal phase; and D-0 D-HCG OPU+1 OPU+5 Figure 1 New proposed algorithm for OHSS prevention and treatment. The upper pathway (traditional) applies to both the GnRH agonist and GnRH antagonist protocol. The lower pathway (innovative) applies only to the GnRH antagonist protocol. Figure 1 New proposed algorithm for OHSS prevention and treatment. The upper pathway (traditional) applies to both the GnRH agonist and GnRH antagonist protocol. The lower pathway (innovative) applies only to the GnRH antagonist protocol. Page 3 of 5 Papanikolaou et al. Reproductive Biology and Endocrinology 2011, 9:147 http://www.rbej.com/content/9/1/147 IVF treatment), the accuracy was 88% [13]. According to these thresholds, a woman considered to be at high risk of developing OHSS should be stimulated with low doses of gonadotropins and preferably co-treated with a GnRH antagonist. Nevertheless, individualization of doses and protocol utilized depends on the clinical experience of the treating physician. IVF treatment), the accuracy was 88% [13]. According to these thresholds, a woman considered to be at high risk of developing OHSS should be stimulated with low doses of gonadotropins and preferably co-treated with a GnRH antagonist. Nevertheless, individualization of doses and protocol utilized depends on the clinical experience of the treating physician. (ii) Innovative way with GnRH Agonist triggering the newer option, using GnRHa triggering, minimizes the risk of OHSS and secures the appropriate matura- tion of oocytes. The oocyte pick-up after GnRHa trig- gering should be performed within 34-35 hours. However, GnRHa triggering is possible only when using a GnRH antagonist protocol and requires modified luteal support in order to be as efficient as hCG trigger- ing [19-21]. The complete eradication of OHSS has made the GnRHa triggering concept the protocol of choice in oocyte donation cycles [22]. In parallel, in IVF with fresh embryo transfer, a new meta-analysis indi- cates that if luteal support is modified with either a bolus of hCG on the day of OPU, alternate doses of rec- LH, or intense luteal support with intramuscular proges- terone and estradiol patches, the delivery rate is com- parable to that seen after hCG triggering [9]. Although we use certain cut-off values and our clini- cal experience to avoid hyperstimulation, we many times fail and the patients ends-up with an excessive ovarian response. (i) Traditional way with HCG-triggering hCG for triggering of final oocyte maturation has been the gold standard treatment for decades, clinically approved and highly efficient. This triggering concept is the only one to be applied in patients co-treated with a long GnRH agonist protocol. However, OHSS may still occur despite administration of lower doses of hCG instead of the standard 10,000 IU dose [18]. Specifically in high-risk patients the OHSS incidence might reach up to 30% in case of embryo transfer and the achieve- ment of a pregnancy [19]. On the other hand the pre- sence of exogenous HCG for 8 days in circulation during the luteal phase [18] secures the appropriate function of the corpora lutea and hence the implanta- tion of the transferred embryo. Day of Triggering final oocyte maturation (Decision 2) Day of Triggering final oocyte maturation (Decision 2) Once the patient has met the criterion set above, the clinician has two options for triggering ovulation: either to administer a lower dose of HCG (250 mcg rec-HCG or 3,300 to 5000 IU urinary hCG), which is the tradi- tional way, applicable in GnRH agonist as well as GnRH antagonist protocols [16]; or to administer GnRHa for final oocyte maturation (0.2 mg Triptorelin or 0.5 mg Buserelin or 1 mg Leuprolide), the innovative way, applicable only in the Antagonist protocol [17]. y (i) Traditional way after HCG-triggering y gg g If the patient on the planned day of triggering already has developed signs or symptoms of early OHSS it is advisable to freeze all embryos at the 2PN stage or at the cleavage stage (day-2/3) and to cancel the embryo transfer. With appropriate counseling the patient will not regard a total freeze as a failure, but rather as a pre- ventive measure, ensuring her health. Importantly, with a good cryo-program her chances of obtaining a preg- nancy will not be reduced [23]. Alternatively, given that the patient can tolerate a milder hyperstimulation, a blastocyst transfer can be planned, if her condition does not worsen during the observational period. Neverthe- less, the risk of late onset OHSS cannot be excluded with this approach. (i) Traditional way with HCG-triggering Algorithm As mentioned, Papanikolaou and col- leagues have shown that the first prerequisite for OHSS development is the development of multiple fol- licles and specifically more than 18 follicles with a dia- meter above 11 mm and/or above 5000 pg/ml Estradiol [2,14]. Thus, from the late follicular phase and onwards, it becomes clear which patient is poten- tially at high-risk of developing OHSS. Therefore, a modification of the ovarian stimulation should be applied, either decreasing the dose of gonadotropins or administering the triggering bolus of hCG earlier (when three follicles reach the size of 17 mm). In GnRH antagonist protocols, this has been shown to be more efficient compared to later administration at a larger follicular size [15,16]. Both these actions intend to induce the regression of small and medium sized follicles initially recruited, which will decrease the risk of OHSS. Day-1 in luteal phase post-OPU (Decision 3) Day-1 in luteal phase post-OPU (Decision 3) On the first day after the oocyte retrieval, a decision should be made whether to proceed with luteal phase support and subsequently embryo transfer or to freeze all embryos. On this day, the physician will have per- formed an ultrasound measuring the quantity of fluid in the pouch of Douglas and the ovarian volume; he should also assess the physical condition of the patient i.e. the degree of pain, discomfort, use of painkillers, and breathing difficulty [15]. In addition, the number of fer- tilized oocytes will be known. Taking these facts into account and adding the efficacy of the cryopreservation program of the unit, a decision can be made whether to proceed with the embryo transfer or to freeze all embryos. (i) Traditional way after hCG-triggering (i) Traditional way after hCG-triggering 7. Griesinger G, Diedrich K, Tarlatzis BC, Kolibianakis EM: GnRH-antagonists in ovarian stimulation for IVF in patients with poor response to gonadotrophins, polycystic ovary syndrome, and risk of ovarian hyperstimulation: a meta-analysis. Reprod Biomed Online 2006, 13:628-638. 7. Griesinger G, Diedrich K, Tarlatzis BC, Kolibianakis EM: GnRH-antagonists in ovarian stimulation for IVF in patients with poor response to gonadotrophins, polycystic ovary syndrome, and risk of ovarian hyperstimulation: a meta-analysis. Reprod Biomed Online 2006, 13:628-638. on day-5 if the patient confirms her good physical con- dition and blood tests and/or ultrasound examination are reassuring, a single blastocyst transfer should be per- formed. Possible supernumerary blastocysts should be vitrified. Importantly, the patient has to be informed that the risk of late OHSS still exists. In contrast, if the patient has developed signs of early onset OHSS, all blastocysts should be cryopreserved. 8. Al-Inany HG, Abou-Setta AM, Aboulghar M: Gonadotrophin-releasing hormone antagonists for assisted conception: a Cochrane review. Reprod Biomed Online 2007, 14:640-649. 8. Al-Inany HG, Abou-Setta AM, Aboulghar M: Gonadotrophin-releasing hormone antagonists for assisted conception: a Cochrane review. Reprod Biomed Online 2007, 14:640-649. 9. Humaidan P, Kol S, Papanikolaou EG, on behalf of the “The Copenhagen GnRH Agonist Triggering Workshop Group: GnRH agonist for triggering of final oocyte maturation: time for a change of practice? Hum Reprod Update 2011, 17:510-524. 9. Humaidan P, Kol S, Papanikolaou EG, on behalf of the “The Copenhagen GnRH Agonist Triggering Workshop Group: GnRH agonist for triggering of final oocyte maturation: time for a change of practice? Hum Reprod Update 2011, 17:510-524. 10. Loutradi KE, Kolibianakis EM, Venetis CA, Papanikolaou EG, Pados G, Bontis I, Tarlatzis BC: Cryopreservation of human embryos by vitrification or slow freezing: a systematic review and meta-analysis. Fertil Steril 2008, 90:186-193. 10. Loutradi KE, Kolibianakis EM, Venetis CA, Papanikolaou EG, Pados G, Bontis I, Tarlatzis BC: Cryopreservation of human embryos by vitrification or slow freezing: a systematic review and meta-analysis. Fertil Steril 2008, 90:186-193. (ii) Innovative way after GnRH Agonist triggering usually no signs of early OHSS will be present and hence the transfer of a single or even two blastocysts in older patients will not increase the risk of late onset OHSS [9]. Supernumerary blastocysts can be vitrified for future use. 11. Acknowledgements W ld l k h 13. Kwee J, Elting ME, Schats R, McDonnell J, Lambalk CB: Ovarian volume and antral follicle count for the prediction of low and hyper responders with in vitro fertilization. Reprod Biol Endocrinol 2007, 15;5:9. 13. Kwee J, Elting ME, Schats R, McDonnell J, Lambalk CB: Ovarian volume and antral follicle count for the prediction of low and hyper responders with in vitro fertilization. Reprod Biol Endocrinol 2007, 15;5:9. We would like to thank Mrs. Vlachou Christina for analyzing reference papers and final editing of the manuscript. 14. Papanikolaou EG, Tournaye H, Verpoest W, Camus M, Vernaeve V, Van Steirteghem A, Devroey P: Early and late ovarian hyperstimulation syndrome: early pregnancy outcome and profile. Hum Reprod 2005, 20:636-641. 14. Papanikolaou EG, Tournaye H, Verpoest W, Camus M, Vernaeve V, Van Steirteghem A, Devroey P: Early and late ovarian hyperstimulation syndrome: early pregnancy outcome and profile. Hum Reprod 2005, 20:636-641. (ii) Innovative way after GnRH Agonist triggering (ii) Innovative way after GnRH Agonist triggering As mentioned above, in the new studies focusing on intense luteal support [19] or supplementation with LH-activity [20,21,24] pregnancy rates are comparable to the standard HCG triggering protocol and at the same time OHSS is eliminated. Therefore, a fresh Page 4 of 5 Page 4 of 5 Papanikolaou et al. Reproductive Biology and Endocrinology 2011, 9:147 http://www.rbej.com/content/9/1/147 embryo transfer with freezing of surplus embryos might be a more preferred strategy instead of a total freeze. Nevertheless, for those yet not convinced about the reproductive outcome after GnRH agonist trigger- ing followed by modified luteal phase support, a total freeze is still an option. Authors’ contributions EGP conceived that concept and wrote the manuscript, PH wrote the manuscript, NPP revised the manuscript, SK revised the manuscript, SK revised the manuscript, HT revised the manuscript, CB wrote the manuscript, BT revised the manuscript. All authors read and approved the final manuscript. Day-5 in luteal phase post-OPU (Decision 4) Received: 22 February 2011 Accepted: 3 November 2011 Received: 22 February 2011 Accepted: 3 November 2011 Published: 3 November 2011 Received: 22 February 2011 Accepted: 3 November 2011 Published: 3 November 2011 Prior to a day-5 transfer, an ultrasound should be per- formed in combination with blood tests (Hematocrit, WBC, PLT, PT, aPTT, Fibrinogen, D-Dimers, Urea, Creatinine, ALT, AST, gGT, ALP, K, Na, Total Protein, Albumin) to identify patients at high risk of developing severe late onset OHSS. If the evaluation is reassuring a single blastocyst transfer can be performed, followed by vitrification of spare blastocysts [25]. If a patient is con- sidered at high risk of developing OHSS - as might often be the case if hCG triggering has been utilized - the embryo transfer should be cancelled. For units working with slow freezing only or having low blastocyst development rates, an alternative option is to freeze half of the embryos at the cleavage stage and allow the rest to develop into blastocysts. If the condition of the patient permits it, a blastocyst transfer is performed and surplus embryos are cryopreserved. Competing interests The authors declare that they have no competing interests. (i) Traditional way after hCG-triggering Lee TH, Liu CH, Huang CC, Wu YL, Shih YT, Ho HN, Yang YS, Lee MS: Serum anti-Müllerian hormone and estradiol levels as predictors of ovarian hyperstimulation syndrome in assisted reproduction technology cycles. Hum Reprod 2008, 23:160-167. 11. Lee TH, Liu CH, Huang CC, Wu YL, Shih YT, Ho HN, Yang YS, Lee MS: Serum anti-Müllerian hormone and estradiol levels as predictors of ovarian hyperstimulation syndrome in assisted reproduction technology cycles. Hum Reprod 2008, 23:160-167. 12. La Marca A, Giulini S, Tirelli A, Bertucci E, Marsella T, Xella S, Volpe A: Anti- Müllerian hormone measurement on any day of the menstrual cycle strongly predicts ovarian response in assisted reproductive technology. Hum Reprod 2007, 22:766-770. 12. La Marca A, Giulini S, Tirelli A, Bertucci E, Marsella T, Xella S, Volpe A: Anti- Müllerian hormone measurement on any day of the menstrual cycle strongly predicts ovarian response in assisted reproductive technology. Hum Reprod 2007, 22:766-770. 1Human Reproduction & Genetics Foundation, Adrianoupoleos 6, 55133 Kalamaria, Thessaloniki, Greece. 2The Fertility Clinic Odense University Hospital (OUH) Boulevard 29, entrance 55 5000 Odense C, Denmark. 3Centrum voor Reproductieve Geneeskunde, UZ Brussel, Flemish Free university of Brussels, Belgium. 4Department of Obstetrics and Gynecology, University of Ioannina, Greece. 5Department of Obstetrics and Gynecology, IVF Unit, Rambam Medical Center, Haifa, Israel. 6Center for Advanced Reproductive Services, University of Connecticut School of Medicine, Department of Obstetrics and Gynecology, Farmington, Connecticut, USA. 7Assisted Reproduction Unit, 1st Department of Obstetrics and Gynecology, Aristotle University of Thessaloniki, Greece. References 1. Rizk B, Aboulghar MA: Classification, pathophysiology and management of ovarian hyperstimulation syndrome. In In-Vitro Fertilization and Assisted Reproduction. Edited by: Brinsden P. New York, NY: Parthenon Publishing Group; 1999:131-155. 2. Papanikolaou EG, Pozzobon C, Kolibianakis EM, Camus M, Tournaye H, Fatemi HM, Van Steirteghem A, Devroey P: Incidence and prediction of ovarian hyperstimulation syndrome in women undergoing gonadotropin-releasing hormone antagonist in vitro fertilization cycles. Fertil Steril 2006, 85:112-120. 3. Orvieto R: Can we eliminate severe ovarian hyperstimulation syndrome? Hum Reprod 2005, 20:320-322. 3. Orvieto R: Can we eliminate severe ovarian hyperstimulation syndrome? Hum Reprod 2005, 20:320-322. p 4. Mathur RS, Akande VA, Keay SD, Hunt LP, Jenkins JM: Distinction between early and late ovarian hyperstimulation syndrome. Fertil Steril 2000, 73:901-907. 4. Mathur RS, Akande VA, Keay SD, Hunt LP, Jenkins JM: Distinction between early and late ovarian hyperstimulation syndrome. Fertil Steril 2000, 73:901-907. 5. Kol S: Prediction of ovarian hyperstimulation syndrome: why predict if we can prevent! Hum Reprod 2003, 18:1557-1558. 5. Kol S: Prediction of ovarian hyperstimulation syndrome: why predict if we can prevent! Hum Reprod 2003, 18:1557-1558. 6. Papanikolaou EG, Humaidan P, Polyzos NP, Tarlatzis B: Identification of the high-risk patient for ovarian hyperstimulation syndrome: Semin. Reprod Med 2010, 28:458-462. Author details 1H R d 1Human Reproduction & Genetics Foundation, Adrianoupoleos 6, 55133 Kalamaria, Thessaloniki, Greece. 2The Fertility Clinic Odense University Hospital (OUH) Boulevard 29, entrance 55 5000 Odense C, Denmark. 3Centrum voor Reproductieve Geneeskunde, UZ Brussel, Flemish Free university of Brussels, Belgium. 4Department of Obstetrics and Gynecology, University of Ioannina, Greece. 5Department of Obstetrics and Gynecology, IVF Unit, Rambam Medical Center, Haifa, Israel. 6Center for Advanced Reproductive Services, University of Connecticut School of Medicine, Department of Obstetrics and Gynecology, Farmington, Connecticut, USA. 7Assisted Reproduction Unit, 1st Department of Obstetrics and Gynecology, Aristotle University of Thessaloniki, Greece. 15. Humaidan P, Quartarolo J, Papanikolaou EG: Preventing ovarian hyperstimulation syndrome: guidance for the clinician. Fertil Steril 2010, 94:389-400. 15. Humaidan P, Quartarolo J, Papanikolaou EG: Preventing ovarian hyperstimulation syndrome: guidance for the clinician. Fertil Steril 2010, 94:389-400. 16. Schmidt D, Maier D, Nulsen J, Benadiva CA: Reducing the dose of hCG in high responders does not affect the outcomes of in vitro fertilization. Fertil Steril 2004, 82:841-846. 16. Schmidt D, Maier D, Nulsen J, Benadiva CA: Reducing the dose of hCG in high responders does not affect the outcomes of in vitro fertilization. Fertil Steril 2004, 82:841-846. 17. Kol S, Itskovitz-Eldor J: Sever OHSS; Yes there is a strategy to prevent it! Hum Reprod 2000, 15:2226-2227. 17. Kol S, Itskovitz-Eldor J: Sever OHSS; Yes there is a strategy to prevent it! Hum Reprod 2000, 15:2226-2227. Page 5 of 5 Page 5 of 5 Papanikolaou et al. Reproductive Biology and Endocrinology 2011, 9:147 http://www.rbej.com/content/9/1/147 18. Symosium MAboulghar: Update on prediction and management of OHSS. Prevention of OHSS. Reprod Biomed Online 2009, 19:33-42. 19. Engmann L, DiLuigi A, Schmidt D, Nulsen J, Maier D, Benadiva C: The use of gonadotropin-releasing hormone (GnRH) agonist to induce oocyte maturation after cotreatment with GnRH antagonist in high-risk patients undergoing in vitro fertilization prevents the risk of ovarian hyperstimulation syndrome: a prospective randomized controlled study. Fertil Steril 2008, 89:84-91. 20. Humaidan P, Ejdrup Bredkjaer H, Westergaard LG, Yding Andersen C: 1,500 IU human chorionic gonadotropin administered at oocyte retrieval rescues the luteal phase when gonadotropin-releasing hormone agonist is used for ovulation induction: a prospective, randomized, controlled study. Fertil Steril 2010, 93:847-854. 21. Author details 1H R d Papanikolaou EG, Verpoest W, Fatemi H, Tarlatzis B, Devroey P, Tournaye H: A novel method of luteal supplementation with recombinant luteinizing hormone when a gonadotropin-releasing hormone agonist is used instead of human chorionic gonadotropin for ovulation triggering: a randomized prospective proof of concept study. Fertil Steril 2011, 95:1174-1177. 22. Bodri D, Guillen JJ, Galindo A, Mataro D, Pujol A, Coll O: Triggering with human chorionic gonadotropin or a gonadotropin-releasing hormone agonist in gonadotropin-releasing hormone antagonist-treated oocyte donor cycles: findings of a large retrospective cohort study. Fertil Steril 2009, 91:365-371. 23. Saragusty J, Arav A: Current progress in oocyte and embryo cryopreservation by slow freezing and vitrification. Reproduction 2011, 141:1-19. 24. Castillo JC, Dolz M, Bienvenido E, Abad L, Casan EM, Bonilla-Musoles F: Cycles triggered with GnRH agonist: exploring low-dose HCG for luteal support. Reprod Biomed Online 2010, 20:175-181. 25. Papanikolaou EG, Kolibianakis EM, Tournaye H, Venetis CA, Fatemi H, Tarlatzis B, Devroey P: Live birth rates after transfer of equal number of blastocysts or cleavage-stage embryos in IVF. A systematic review and meta-analysis. Hum Reprod 2008, 23:91-99. doi:10.1186/1477-7827-9-147 Cite this article as: Papanikolaou et al.: New algorithm for OHSS prevention. Reproductive Biology and Endocrinology 2011 9:147. Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit
https://openalex.org/W3018118933	https://europepmc.org/articles/pmc7188773?pdf=render	English	null	Clinically Relevant Extended-Spectrum β-Lactamase–Producing Escherichia coli Isolates From Food Animals in South Korea	Frontiers in microbiology	2,020	cc-by	7,848	Clinically Relevant Extended-Spectrum β-Lactamase–Producing Escherichia coli Isolates From Food Animals in South Korea Jihyun Song1†, Sung-Suck Oh2†, Junghee Kim2, Sukyoung Park1 and Jinwook Shin1* 1 Department of Microbiology, College of Medicine, Inha University, Incheon, South Korea, 2 Incheon Research Institute of Public Health and Environment, Incheon, South Korea Jihyun Song1†, Sung-Suck Oh2†, Junghee Kim2, Sukyoung Park1 and Jinwook Shin1* 1 Department of Microbiology, College of Medicine, Inha University, Incheon, South Korea, 2 Incheon Research Institute of Public Health and Environment, Incheon, South Korea Extended-spectrum β-lactam antimicrobials have been broadly used in food animals and humans to control infectious diseases. However, the emergence and rapid spread of extended-spectrum β-lactamase (ESBL)–producing Enterobacteriaceae, mainly Escherichia coli, have seriously threatened global health in recent decades. In this study, we determined the prevalence, antimicrobial susceptibility, and genetic properties of ESBL-producing E. coli (ESBL-EC) strains isolated from food animals in South Korea. A total of 150 fecal samples from healthy chickens (n = 34), pigs (n = 59), and cattle (n = 57) were screened from January to July 2018. Among these, 77 non-duplicate cefotaxime-resistant ESBL-EC strains were isolated from 32 chicken, 41 pig, and 4 cattle samples, with the corresponding occurrence rates of 94.1, 69.5, and 7.0%, respectively. All the isolates showed multidrug resistance (MDR) and produced at least one type of β-lactamase, including CTX-M (98.7%) and TEM (40.3%). CTX-M-14 (53.1%), CTX-M-55 (53.7%), and CTX-M-65 (50.0%) were the predominant genotypes in the chicken, pig, and cattle samples, respectively. Multilocus sequence typing revealed 46 different sequence types (STs), including the human-associated extraintestinal pathogenic E. coli ST131 (n = 2), ST10 (n = 5), ST38 (n = 1), ST410 (n = 4), ST354 (n = 2), ST58 (n = 3), ST117 (n = 1), and ST457 (n = 1). To the best of our knowledge, this is the ﬁrst report of pandemic E. coli ST131 in non-human isolates in South Korea. Our results demonstrate the high prevalence and diversity of MDR-ESBL- EC in food animals and highlight them as potential pathogenic ESBL-EC reservoirs that may pose a high risk to human health. Citation: Song J, Oh S-S, Kim J, Park S and Shin J (2020) Clinically Relevant Extended-Spectrum β-Lactamase–Producing Escherichia coli Isolates From Food Animals in South Korea. Edited by: Kwangcheol Casey Jeong, University of Florida, United States Edited by: Kwangcheol Casey Jeong, University of Florida, United States Reviewed by: Jian-Hua Liu, South China Agricultural University, China Raies Mir, United States Department of Agriculture (USDA), United States Correspondence: Jinwook Shin shin001@inha.ac.kr †These authors have contributed equally to this work Kwangcheol Casey Jeong, University of Florida, United States Reviewed by: Jian-Hua Liu, South China Agricultural University, China Raies Mir, United States Department United States Department of Agriculture (USDA), United States Correspondence: Jinwook Shin shin001@inha.ac.kr †These authors have contributed equally to this work †These authors have contributed equally to this work Specialty section: This article was submitted to Food Microbiology, a section of the journal Frontiers in Microbiology Received: 21 November 2019 Accepted: 18 March 2020 Published: 22 April 2020 Keywords: Escherichia coli, extended-spectrum β-lactamase, multidrug resistance, food animal, South Korea ORIGINAL RESEARCH published: 22 April 2020 doi: 10.3389/fmicb.2020.00604 Keywords: Escherichia coli, extended-spectrum β-lactamase, multidrug resistance, food animal, South Korea 1ftp://ftp.ncbi.nlm.nih.gov/pathogen/Antimicrobial_resistance Antimicrobial Susceptibility Testing Antimicrobial Susceptibility Testing Antimicrobial susceptibilities testing was performed by the disk diﬀusion method in accordance with the guidelines of the Clinical and Laboratory Standards Institute (CLSI, M100-S27) (CLSI, 2017) using commercial disks (Oxoid) supplemented with 21 antimicrobial agents as follows: gentamicin (10 µg), amikacin (30 µg), ertapenem (10 µg), imipenem (10 µg), meropenem (10 µg), cefazolin (30 µg), cefotaxime (30 µg), ceftazidime (30 µg), cefepime (30 µg), cefoxitin (30 µg), ciproﬂoxacin (5 µg), nalidixic acid (30 µg), trimethoprim-sulfamethoxazole (1.25/23.75 µg), tigecycline (15 µg), aztreonam (30 µg), ampicillin (10 µg), piperacillin (100 µg), amoxicillin– clavulanic acid (20/10 µg), ampicillin–sulbactam (10/10 µg), chloramphenicol (30 µg), and tetracycline (30 µg). Mueller– Hinton agar plate (Difco Laboratories, Detroit, MI, United States) was swabbed with a pure ESBL-EC suspension adjusted to a 0.5 McFarland standard. The disks were placed on the agar using a disk dispenser (Oxoid), and the plate was incubated at 37◦C for 24 h. The zone diameters of growth inhibition were measured using an electronic caliper. All of the results were interpreted according to the zone diameter breakpoints of the CLSI guidelines (CLSI, 2017) except that the tigecycline results were interpreted according to the European Committee for Antimicrobial Susceptibility Testing (EUCAST) breakpoint version 7.1 (EUCAST, 2017; Supplementary Table S1). Escherichia coli ATCC 25922 was used as the reference strain. Multidrug resistance was determined as non-susceptibility to at least one agent in ≥3 antimicrobial classes, and extensive drug resistance (XDR) was deﬁned as non-susceptibility to at least one agent in all but ≤2 classes (Magiorakos et al., 2012). In this study, we evaluated the prevalence, antimicrobial susceptibility, and molecular genetic features of ESBL-EC strains isolated from food animals in South Korea. Furthermore, we assessed the epidemiological relatedness of the clonal populations to human-associated E. coli STs according to a national surveillance program. Isolation and Identiﬁcation of ESBL-EC From Food Animals A total of 150 healthy food animals, including 34 chickens, 59 pigs, and 57 cattle, were obtained from 28, 34, and 53 farms (115 in total), respectively, across the country in South Korea. Fecal samples were collected from the intestinal tracts of individual animals slaughtered at the slaughterhouses. For E. coli isolation, 0.1 g of the samples was inoculated to 9 mL of Tryptone Soya Broth (Oxoid, Basingstoke, United Kingdom) containing 0.4 µg/mL vancomycin (Wako Pure Chemical Industries, Hyogo, Japan) and incubated at 37◦C for 4 h. INTRODUCTION Extended-spectrum β-lactam antimicrobials have been widely used to treat bacterial infections both in humans and animals. Since the ﬁrst extended-spectrum β-lactamase (ESBL) was described in Germany in 1983, the global spread of ESBL-producing Escherichia coli (ESBL-EC), including the pandemic E. coli sequence type (ST) 131 clone, has led to a rapid increase in the population of Front. Microbiol. 11:604. doi: 10.3389/fmicb.2020.00604 April 2020 \| Volume 11 \| Article 604 Frontiers in Microbiology \| www.frontiersin.org 1 ESBL-Producing E. coli in Food Animals Song et al. A loopful of each enrichment was streaked on MacConkey screening plate supplemented with 2 µg/mL cefotaxime and incubated at 37◦C for 24 h. Subsequently, one pink or reddish colony suspected of comprising E. coli from each fecal sample was randomly selected using a sterile platinum loop and cultured on CHROMagar ESBL (CHROMagar, Paris, France) at 37◦C for 24 h. One dark pink to reddish single colony selected from each plate was grown on Tryptone Soya Agar (Oxoid) at 37◦C for 4 h, and the pure isolates were used for further characterization. The species of the isolates were identiﬁed using matrix-assisted laser desorption ionization–time of ﬂight mass spectrometry (Bruker Daltonik GmbH, Bremen, Germany) with score values ≥2.0. Extended- spectrum β-lactamase production was conﬁrmed via the double- disk synergy test using disks containing amoxicillin–clavulanic acid (20/10 µg), cefotaxime (30 µg), cefepime (30 µg), and ceftazidime (30 µg). ESBL-EC strains worldwide (Pitout and Laupland, 2008; Rogers et al., 2011). The most widespread ESBLs are CTX-M–type β-lactamases, which can be divided into ﬁve major groups (CTX- M groups 1, 2, 8, 9, and 25) (Bonnet, 2004; Canton et al., 2012; Bevan et al., 2017), and at least 214 CTX-M variants have been detected1 accessed January 24, 2020. Among these, CTX-M-15 in the CTX-M group 1 and CTX-M-14 in the CTX-M group 9 are prevalent in most countries (Bevan et al., 2017). Likewise, both variants have been predominantly detected in clinical ESBL-EC isolates in South Korea (Song et al., 2009; Kim et al., 2019). ESBL-EC strains worldwide (Pitout and Laupland, 2008; Rogers et al., 2011). The most widespread ESBLs are CTX-M–type β-lactamases, which can be divided into ﬁve major groups (CTX- M groups 1, 2, 8, 9, and 25) (Bonnet, 2004; Canton et al., 2012; Bevan et al., 2017), and at least 214 CTX-M variants have been detected1 accessed January 24, 2020. INTRODUCTION Among these, CTX-M-15 in the CTX-M group 1 and CTX-M-14 in the CTX-M group 9 are prevalent in most countries (Bevan et al., 2017). Likewise, both variants have been predominantly detected in clinical ESBL-EC isolates in South Korea (Song et al., 2009; Kim et al., 2019). g As ESBL-EC strains are rising in humans, they have also been increasingly isolated from food animals in diﬀerent geographical regions, including China (Rao et al., 2014), Germany (Laube et al., 2013), Netherlands (Hordijk et al., 2013), Tunisia (Maamar et al., 2016), and United States (Markland et al., 2019). Moreover, multidrug-resistant (MDR) ESBL-EC pathogens, which pose a serious threat to human health due to the limited treatment options, extensively disseminate among food animals (Ho et al., 2011; Vitas et al., 2018), which are considered to be the primary reservoirs of antimicrobial-resistant enteric bacteria, although the routes of transmission to humans are unclear. Such bacteria can presumably pass through the food chain or via close contact and can colonize the intestines of humans (Carattoli, 2008). In fact, the same genetic elements and/or STs have been observed between human and food animal isolates of ESBL-EC (Moodley and Guardabassi, 2009; Leverstein-van Hall et al., 2011; Tamang et al., 2013a; Hammerum et al., 2014; Dahms et al., 2015), suggesting the possibility of clonal and genetic transmissions between these settings. Previous studies conducted in South Korea have mainly focused on the prevalence and characteristics of ESBL genes of E. coli isolates from food animals (Tamang et al., 2013b; Shin et al., 2017), but their relatedness to human-associated clonal lineages has rarely been investigated. Genes Total DNAs of ESBL-EC isolates were extracted and puriﬁed using G-spin Total DNA Extraction Kit (iNtRON Biotechnology, Seongnam, South Korea) according to the manufacturer’s instructions. To identify the ESBL alleles 1ftp://ftp.ncbi.nlm.nih.gov/pathogen/Antimicrobial_resistance April 2020 \| Volume 11 \| Article 604 Frontiers in Microbiology \| www.frontiersin.org 2 Song et al. ESBL-Producing E. coli in Food Animals of the isolates, the extracts were subjected to polymerase chain reaction (PCR) analyses using the speciﬁc primer pairs, including blaTEM, blaSHV, and blaCTX-M groups 1, 2, 9, and 25 (Supplementary Table S2). After the bidirectional Sanger sequencing of the amplicons by BIOFACT (Daejeon, South Korea), the resultant sequences were compared with the published β-lactamase gene sequences from the GenBank database of the NCBI using the online BLAST program2. 2https://blast.ncbi.nlm.nih.gov/Blast.cgi 3https://enterobase.warwick.ac.uk Conjugation and Plasmid Typing Assay Conjugation and Plasmid Typing Assay Conjugation assay was performed with 24 blaCTX-M-55- positive donors isolated from food animals and E. coli J53-AziR recipient strains. Transconjugants were selected on MacConkey agar plates containing 2 µg/mL of cefotaxime and 100 µg/mL of sodium azide. The presence of the blaCTX-M-55 gene in the transconjugants was conﬁrmed by PCR analysis using the CTX-M-1F/R primer pair described in Supplementary Table S2. Replicon typing of the transconjugant plasmids were tested for the major plasmid incompatibility groups among Enterobacteriaceae (HI1, HI2, I1-Iγ, X, L/M, N, FIA, FIB, W, Y, P, FIC, A/C, T, FIIAS, F, K, and B/O) using a PCR-based replicon typing method (Carattoli et al., 2005). The IncF plasmids containing FIB were further analyzed for distinguishing variants by a replicon sequence typing (RST) scheme (Supplementary Table S2) as previously described (Villa et al., 2010). The results of sequences were compared with the plasmid multilocus sequence typing (MLST) database deposited at http://pubmlst.org/plasmid/. The number of plasmids in the transconjugants was solved by pulsed ﬁeld gel electrophoresis (PFGE) with S1 nuclease (Thermo Fisher Scientiﬁc, Waltham, MA, United States) digestion of total DNA using the CHEF MAPPER system (Bio-Rad Laboratories, Hercules, CA, United States). Characterization of ESBL Genes Next, the genotypes responsible for ESBL production were investigated using PCR-based sequencing for blaTEM, blaSHV, and blaCTX-M groups 1, 2, 9, and 25. The most prevalent ESBL genotype was represented by blaCTX-M group 1 (46/77, 59.7%), followed by blaCTX-M group 9 (31/77, 40.3%) (Table 1). The sequence identities with the β-lactamase gene sequences of the NCBI database using BLAST search were as follows: blaTEM-1 (>99.5%), blaCTX-M-1 (>99.8%), blaCTX-M-3 (99.9%), blaCTX-M-15 (>99.4%), blaCTX-M-55 (>99.3%), blaCTX-M-14 (>99.7%), and blaCTX-M-65 (>99.6%). All sequenced blaTEM amplicons belonged to non-ESBL blaTEM-1 (31/77, 40.3%), whereas blaSHV and blaCTX-M groups 2 and 25 were not detected. The predominant CTX-M–type ESBLs were CTX-M-14 in chickens (17/32, 53.1%), CTX-M-55 in pigs (22/41, 53.7%), and CTX-M-65 in cattle (2/4, 50.0%). CTX-M-1, CTX-M-3, and CTX-M-15 were also identiﬁed at various frequencies in each animal species. The ESBL gene proﬁle of an individual isolate is given in Supplementary Table S4. In addition, we carried out the conjugation assay with azide-resistant E. coli J53 recipient strain, focusing on blaCTX-M-55. The transmission of blaCTX-M-55 gene was observed in 14 of 24 blaCTX-M-55-positive ESBL-EC isolates (Supplementary Table S6). The J53 transconjugants were further analyzed using PCR-based replicon typing. F, FIB, I1-Iγ, K, N, and FIA replicons were identiﬁed in 12, 10, 5, 3, 2, and 1 transconjugants (Supplementary Table S6). The RST discriminated F1 (9/10, 90.0%) and F20 (1/10, 10.0%) plasmids among the IncF plasmids containing FIB. S1-PFGE Prevalence and Antimicrobial Susceptibility of ESBL-EC Strains in Food Animals To investigate the prevalence of ESBL-EC strains in food animals, we cultured a total of 150 fecal samples from 34 chickens, 59 pigs, and 57 cattle on MacConkey agar plates containing cefotaxime (2 µg/mL) and then puriﬁed single colonies on CHROMagar ESBL plates. Among them, 77 non- duplicate cefotaxime-resistant E. coli strains were isolated from 32 chickens (94.1%), 41 pigs (69.5%), and 4 cattle (7.0%). Antimicrobial susceptibility testing of 21 antimicrobial substances from 14 classes showed that all of the isolates were not susceptible to cefotaxime (extended-spectrum cephalosporin class), cefazolin (non-extended-spectrum cephalosporin class), and ampicillin and piperacillin (penicillin class), indicating MDR (≥4 classes) phenotypes; however, they were susceptible to ertapenem, meropenem, cefoxitin, and tigecycline (Figure 1 and Supplementary Table S3). High frequencies of amikacin (76/77, 98.7%) and imipenem (76/77, 98.7%) susceptibilities were also observed. Although there was no XDR strain, three pig isolates (EC21, EC37, and EC43) showed intermediate resistance or resistance to at least one agent in all but three antimicrobial classes investigated, as described in Supplementary Tables S4, S5. 3https://enterobase.warwick.ac.uk 2https://blast.ncbi.nlm.nih.gov/Blast.cgi Phylogenetic Characterization The MLST-based phylogenetic tree showed that ESBL-EC isolates were branched into three major clusters, among which Cluster I and Cluster II consisted of 89.2% of subgroup A and 92% of subgroup B1, respectively, and Cluster III included all of the subgroups B2 and D (Figure 2). Phylogenetic Characterization y g The DNA extracts of the isolates were used for multiplex PCR targeting chuA, yjaA, and the DNA fragment TspE4.C2, as described previously (Clermont et al., 2000). Phylogroups A and B1 are typically commensal, whereas groups B2 and D are extraintestinal virulence-associated strains (Johnson et al., 2001). The ST genotypes of the isolates were determined by PCR-based sequencing of the housekeeping genes (adk, fumC, gyrB, icd, mdh, purA, and recA) followed by MLST in accordance with the Enterobase protocol and database3 (Wirth et al., 2006). The sequences of seven housekeeping genes were aligned using Clustal W, and the phylogenetic tree was constructed using MEGA version X based on the maximum- likelihood method using Kimura’s two-parameter model with gamma distribution and invariant sites. The E. coli phylogeny was estimated by a bootstrap analysis with 1,000 replicates (Kumar et al., 2018). April 2020 \| Volume 11 \| Article 604 Frontiers in Microbiology \| www.frontiersin.org 3 ESBL-Producing E. coli in Food Animals Song et al. FIGURE 1 \| Antimicrobial susceptibility and multidrug resistance proﬁles of cefotaxime-resistant Escherichia coli isolates from food animals in South Korea. (A) Antimicrobial susceptibilities were analyzed by the disk agar diffusion method. GEN, gentamicin; AMK, amikacin; ETP, ertapenem; IPM, imipenem; MEM, meropenem; CFZ, cefazolin; CTX, cefotaxime; CAZ, ceftazidime; FEP, cefepime; FOX, cefoxitin; CIP, ciproﬂoxacin; NAL, nalidixic acid; SXT, trimethoprim-sulfamethoxazole; TGC, tigecycline; ATM, aztreonam; AMP, ampicillin; PIP, piperacillin; AMC, amoxicillin–clavulanic acid; SAM, ampicillin–sulbactam; CHL, chloramphenicol; TET, tetracycline; R, resistant; I, intermediate resistant; S, susceptible. (B) Multidrug resistance was determined as non-susceptibility to at least one agent in three or more antimicrobial classes. MDR, multidrug resistance. FIGURE 1 \| Antimicrobial susceptibility and multidrug resistance proﬁles of cefotaxime-resistant Escherichia coli isolates from food animals in South Korea. (A) Antimicrobial susceptibilities were analyzed by the disk agar diffusion method. GEN, gentamicin; AMK, amikacin; ETP, ertapenem; IPM, imipenem; MEM, meropenem; CFZ, cefazolin; CTX, cefotaxime; CAZ, ceftazidime; FEP, cefepime; FOX, cefoxitin; CIP, ciproﬂoxacin; NAL, nalidixic acid; SXT, trimethoprim-sulfamethoxazole; TGC, tigecycline; ATM, aztreonam; AMP, ampicillin; PIP, piperacillin; AMC, amoxicillin–clavulanic acid; SAM, ampicillin–sulbactam; CHL, chloramphenicol; TET, tetracycline; R, resistant; I, intermediate resistant; S, susceptible. (B) Multidrug resistance was determined as non-susceptibility to at least one agent in three or more antimicrobial classes. MDR, multidrug resistance. analysis revealed that 10 transconjugants (EC4, EC7, EC10, EC16, EC18, EC19, EC31, EC33, EC39, and EC62) contained one plasmid, whereas four (EC17, EC21, EC35, and EC40) contained two plasmids. DISCUSSION In contrast, F1 types, which are commonly found to carry ESBL genes (Brolund, 2014), were most predominantly identiﬁed in this study. a A77V substitution from blaCTX-M-15 but also horizontal gene transfer. Plasmids play a critical role in the global dissemination of ESBL genes (Wang et al., 2018). The blaCTX-M-55 gene was frequently found on IncF, IncI1, and IncHI2 plasmids of E. coli in many countries, including China (Yang et al., 2014; Wang et al., 2018), France (Lupo et al., 2018), and United States (McGann et al., 2016). Similarly, the presence of I1-Iγ, F, and P plasmids in blaCTX-M-55–positive ESBL-EC isolates from pigs has been documented in South Korea (Tamang et al., 2013b). This study showed that most of the blaCTX-M-55–positive transconjugants (12/14, 85.7%) carried IncF replicon in combination with other types, including FIB, I1-Iγ, K, N, and/or FIA. The diversity of plasmid types was increased in comparison with those in previous report (Tamang et al., 2013b), which may reﬂect more inﬂux of various antimicrobial-resistant genes. Among IncF plasmids in the blaCTX-M-55-positive E. coli isolates from animals in China, F33 plasmids were the most prevalent replicon STs (Yang et al., 2015). In contrast, F1 types, which are commonly found to carry ESBL genes (Brolund, 2014), were most predominantly identiﬁed in this study. reported to be mostly susceptible to tigecycline and amikacin (Morosini et al., 2006; Denisuik et al., 2019), and similarly, the same phenotype was also found in food animal isolates in this study (Figure 1A and Supplementary Table S3). y g pp y The epidemiology of CTX-M β-lactamases has been globally changed (Bevan et al., 2017). CTX-M-55, which diﬀers from CTX-M-15 by one nucleotide at 239 resulting in A77V substitution, displayed enhanced cephalosporin-hydrolyzing activity and structural stability (He et al., 2015). The population of CTX-M-55–producing ESBL-EC strains in China is showing increasing trends in both human and food animals (Hu et al., 2013; Rao et al., 2014; Zhang et al., 2014). In South Korea, CTX- M-15 and CTX-M-14 had been reported to be the predominant CTX-M β-lactamases in ESBL-EC isolates from food animals (Tamang et al., 2013b; Shin et al., 2017), however, CTX-M-55 was most prevalently detected in companion animals (Hong et al., 2019) and raw retail chickens (Park et al., 2019). In this study, we found that food animal ESBL-EC predominantly produced the CTX-M-55 enzyme (Table 1), suggesting that the CTX-M-55 may be supplanting CTX-M-15. DISCUSSION coli isolates Chicken Pig Cattle Total (n = 32) (n = 41) (n = 4) (n = 77) TEM-1 10 (31.3) 21 (51.2) 0 31 (40.3) CTX-M groups 32 (100) 40 (97.6) 4 (100) 76 (98.7) CTX-M group 1 10 (31.3) 33 (80.5) 2 (50.0) 45 (58.4) CTX-M group 9 21 (65.6) 7 (17.1) 2 (50.0) 30 (39.0) CTX-M group 1 and group 9 1 (3.1) 0 0 1 (1.3) CTX-M genotypes 5 (15.6) CTX-M-1 0 0 5 (6.5) CTX-M-3 0 1 (2.4) 0 1 (1.3) CTX-M-15 4 (12.5) 10 (24.4) 1 (25.0) 15 (19.5) CTX-M-55 1 (3.1) 22 (53.7) 1 (25.0) 24 (31.2) CTX-M-14 16 (50.0) 4 (9.8) 0 20 (26.0) CTX-M-65 5 (15.6) 3 (7.3) 2 (50.0) 10 (13.0) CTX-M-15 and CTX-M-14 1 (3.1) 0 0 1 (1.3) TABLE 2 \| Phylogroups of cefotaxime-resistant Escherichia coli isolates from food animals. Phylogroup Number (%) of cefotaxime-resistant E. coli isolates Chicken (n = 32) Pig (n = 41) Cattle (n = 4) Total (n = 77) A 16 (50.0) 20 (48.8) 1 (25.0) 37 (48.1) B1 6 (18.8) 17 (41.5) 2 (50.0) 25 (32.5) B2 0 2 (4.9) 0 2 (2.6) D 10 (31.3) 2 (4.9) 1 (25.0) 13 (16.9) reported to be mostly susceptible to tigecycline and amikacin (Morosini et al., 2006; Denisuik et al., 2019), and similarly, the same phenotype was also found in food animal isolates in this study (Figure 1A and Supplementary Table S3). The epidemiology of CTX-M β-lactamases has been globally changed (Bevan et al., 2017). CTX-M-55, which diﬀers from CTX-M-15 by one nucleotide at 239 resulting in A77V substitution, displayed enhanced cephalosporin-hydrolyzing acti it and structural stabilit (He et al 2015) The population TABLE 3 \| MLST analysis of cefotaxime-resistant Escherichia coli isolates from food animals. MLST Number (%) of cefotaxime-resistant E. DISCUSSION coli isolates Chicken Pig Cattle Total (n = 32) (n = 41) (n = 4) (n = 77) ST10 3 (9.4) 2 (4.9) 0 5 (6.5) ST48 3 (9.4) 4 (9.8) 0 7 (9.1) ST58 0 3 (7.3) 0 3 (3.9) ST93 2 (6.3) 0 0 2 (2.6) ST101 0 7 (17.1) 0 7 (9.1) ST131 0 2 (4.9) 0 2 (2.6) ST155 2 (6.3) 0 0 2 (2.6) ST354 2 (6.3) 0 0 2 (2.6) ST362 2 (6.3) 0 0 2 (2.6) ST410 0 4 (9.8) 0 4 (5.2) ST542 0 2 (4.9) 0 2 (2.6) ST5728 0 0 2 (50.0) 2 (2.6) ST5853 2 (6.3) 0 0 2 (2.6) Once detected ST 14 (43.8) 17 (41.5) 2 (50.0) 33 (42.9) Not determined ST 2 (6.3) 0 0 2 (2.6) ST, sequence type. TABLE 3 \| MLST analysis of cefotaxime-resistant Escherichia coli isolates from food animals. TABLE 1 \| Extended-spectrum β-lactamase genotypes of cefotaxime-resistant Escherichia coli isolates from food animals. TABLE 2 \| Phylogroups of cefotaxime-resistant Escherichia coli isolates from food animals. Phylogroup Number (%) of cefotaxime-resistant E. coli isolates Chicken (n = 32) Pig (n = 41) Cattle (n = 4) Total (n = 77) A 16 (50.0) 20 (48.8) 1 (25.0) 37 (48.1) B1 6 (18.8) 17 (41.5) 2 (50.0) 25 (32.5) B2 0 2 (4.9) 0 2 (2.6) D 10 (31.3) 2 (4.9) 1 (25.0) 13 (16.9) a A77V substitution from blaCTX-M-15 but also horizontal gene transfer. Plasmids play a critical role in the global dissemination of ESBL genes (Wang et al., 2018). The blaCTX-M-55 gene was frequently found on IncF, IncI1, and IncHI2 plasmids of E. coli in many countries, including China (Yang et al., 2014; Wang et al., 2018), France (Lupo et al., 2018), and United States (McGann et al., 2016). Similarly, the presence of I1-Iγ, F, and P plasmids in blaCTX-M-55–positive ESBL-EC isolates from pigs has been documented in South Korea (Tamang et al., 2013b). This study showed that most of the blaCTX-M-55–positive transconjugants (12/14, 85.7%) carried IncF replicon in combination with other types, including FIB, I1-Iγ, K, N, and/or FIA. The diversity of plasmid types was increased in comparison with those in previous report (Tamang et al., 2013b), which may reﬂect more inﬂux of various antimicrobial-resistant genes. Among IncF plasmids in the blaCTX-M-55-positive E. coli isolates from animals in China, F33 plasmids were the most prevalent replicon STs (Yang et al., 2015). DISCUSSION The distribution of phylogroups showed that subgroup A was predominant (37/77, 48.1%), followed by subgroups B1 (25/77, 32.5%), D (13/77, 16.9%), and B2 (2/77, 2.6%) in ESBL-EC isolates from food animals (Table 2), thus suggesting the higher distribution of commensal groups A and B1 than pathogenic groups B2 and D. However, the subgroups were diﬀerentially distributed among the animal species. The chicken isolates mainly belonged to subgroups A and D, whereas the pig isolates mostly belonged to subgroups A and B1. Through MLST analysis of a total of 77 isolates, we determined 46 distinct E. coli STs (21 chickens, 24 pigs, and 3 cattle), among which ST10 and ST48 were found in both chicken and pig isolates, and 2 unknown STs (Table 3). The proportions of STs detected only once among the isolates were 43.8% (14/32) in chickens, 41.5% (17/41) in pigs, and 50.0% (2/4) in cattle. The highest number of chicken isolates belonged to ST10 or ST48 (3/32, 9.4% each). Among the pig isolates, ST101 was the most prevalent lineage (7/41, 17.1%) and pandemic ST131 was also identiﬁed (2/41, 4.9%). The prevalence of ESBL-EC in food animals (94.1% in chickens, 69.5% in pigs, and 7.0% in cattle) in this study was found to be higher than those reported in previous investigations before and after 2010 (33.3% in chickens, 21.5% in pigs, and 0.2% in cattle) (Tamang et al., 2013b; Lim et al., 2015) in South Korea. In the susceptibility testing for 14 antimicrobial classes, all of the isolates showed the MDR phenotypes with a resistance range of 4–11 classes (Figure 1B). In general, ESBLs can hydrolyze extended-spectrum cephalosporins and monobactams but not carbapenems and cephamycins and are inhibited by β-lactamase inhibitors (Canton et al., 2012). Consistent with these properties, ESBL-EC isolates in this study showed a relatively high susceptibility to ertapenem, imipenem, meropenem, cefoxitin, amoxicillin −clavulanic acid, and ampicillin −sulbactam (Figure 1A and Supplementary Table S3). In addition, ESBL- producing Enterobacteriaceae among clinical isolates have been April 2020 \| Volume 11 \| Article 604 Frontiers in Microbiology \| www.frontiersin.org 4 ESBL-Producing E. coli in Food Animals Song et al. TABLE 1 \| Extended-spectrum β-lactamase genotypes of cefotaxime-resistant Escherichia coli isolates from food animals. bla genotype Number (%) of cefotaxime-resistant E. DISCUSSION Together, these results suggest the two-way spread of resistant bacteria: food animals may be getting them from humans, hospital waste, and the environment or from their feed and fodder. ETHICS STATEMENT Ethical review and approval was not required for the animal study because we used livestock feces from slaughterhouses, not livestock itself. DATA AVAILABILITY STATEMENT All datasets generated for this study are included in the article/Supplementary Material. ACKNOWLEDGMENTS We would like to thank Drs. Junyoung Kim, Young Ah Kim, and Hyukmin Lee for their helpful discussions and the Research Institute of Bacterial Resistance at Yonsei University College of Medicine for providing services. In conclusion, our results demonstrate the increasing occurrence and clonal diversity of MDR-ESBL-EC strains in food animals. These strains include pathogenic human-associated lineages, such as the E. coli ST131 clone. To explore the possible origin of the two ST131 strains found in pigs, it would be interesting to compare their core genome sequences with Bonnet, R. (2004). Growing group of extended-spectrum β-lactamases: the CTX-M enzymes. Antimicrob. Agents Chemother. 48, 1–14. doi: 10.1128/aac.48.1.1-14. 2004 SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb. 2020.00604/full#supplementary-material FUNDING This research was supported by the Korea Centers for Disease Control and Prevention (2017ER540301), the Basic Science Research Program through the National Research Foundation (NRF) of Korea funded by the Ministry of Education (2018R1A6A1A03025523), and Inha University Research Grant (2018). AUTHOR CONTRIBUTIONS S-SO and JSh contributed conception and design of the study. JSo, S-SO, and JK collected the samples and performed the experiments. JSh wrote the manuscript. All authors analyzed the data, contributed to manuscript revision, read and approved the submitted version. DISCUSSION The blaCTX-M-55 gene associated with cefotaxime-resistant phenotype was transferable from 14 of 24 blaCTX-M-55–positive ESBL-EC isolates to other E. coli strain by conjugation as described in Supplementary Table S6, suggesting that food animals may acquire blaCTX-M-55 through In this study, the food animal ESBL-EC isolates mainly belonged to commensal groups A or B1. Most of each phylogroup to which they belonged was allocated to a distinct cluster, whereas there was no diﬀerence between animal species allocations (Figure 2), suggesting that the phylogenetic relationships among the ESBL-EC STs may be closely related to their phylogroups regardless of the host animal species. Notably, the diﬀerent breed composition of cattle has been reported to be associated with gut microbiota structure and β-lactam resistance (Fan et al., 2019), suggesting the impact of animal genetics on the antimicrobial-resistant bacteria proﬁle even within the same species. We further detected various clonal STs in these isolates April 2020 \| Volume 11 \| Article 604 Frontiers in Microbiology \| www.frontiersin.org Frontiers in Microbiology \| www.frontiersin.org 5 Song et al. ESBL-Producing E. coli in Food Animals FIGURE 2 \| Phylogenetic tree of cefotaxime-resistant Escherichia coli isolates from chickens, pigs, and cattle. The maximum likelihood phy constructed using Mega X software based on the seven housekeeping genes (adk, fumC, gyrB, icd, mdh, purA, and recA) and Kimura’s 2- Bootstrap support percentages (1,000 replicates) were indicated in the different branches. Scale bar at the bottom represents the genetic FIGURE 2 \| Phylogenetic tree of cefotaxime-resistant Escherichia coli isolates from chickens, pigs, and cattle. The maximum likelihood phylogenetic tree was constructed using Mega X software based on the seven housekeeping genes (adk, fumC, gyrB, icd, mdh, purA, and recA) and Kimura’s 2-parameter model. Bootstrap support percentages (1,000 replicates) were indicated in the different branches. Scale bar at the bottom represents the genetic distance. The phylogroup and sequence type (ST) of each isolate were displayed. Black, gray, and white squares represent chicken, pig, and cattle, respectively. ND, not determined ST. April 2020 \| Volume 11 \| Article 604 Frontiers in Microbiology \| www.frontiersin.org 6 ESBL-Producing E. coli in Food Animals Song et al. those of other ST131 from humans in South Korea. Given the possibility of direct transmission of antimicrobial resistance to humans through the food chain, this study also demonstrates the importance of understanding the dynamics of MDR E. coli in food animals. by MLST analysis. DISCUSSION The rate of STs detected only once was 42.9%, suggesting that food animals possess a wider variety of MDR-ESBL-EC STs. Interestingly, among them, ST131, ST10, ST38, ST410, ST354, ST58, and ST117 have been reported as major extraintestinal pathogenic E. coli (ExPEC) lineages, which cause an extraintestinal infection in human (Manges et al., 2019). Rarely reported ST457 also belongs to ExPEC (Seni et al., 2018). The globally predominant ExPEC ST131 belongs to the highly virulent phylogroup B2 and causes both community-onset and hospital-onset infections (Nicolas-Chanoine et al., 2014). It commonly produces ESBLs and is highly associated with MDR, including resistance to ﬂuoroquinolone. The epidemiology and characteristics of the ST131 clonal group have mostly been investigated in human clinical isolates, and animal and environmental clones have been identiﬁed only in a few studies worldwide. Similarly in South Korea, E. coli ST131 was found to be the most prevalent clone in patients with urinary tract infections and bacteremia and commonly harbored blaCTX-M-15 and blaCTX-M-14 (Lee et al., 2010; Cha et al., 2016; Kim H. et al., 2017, Kim Y.A. et al., 2017). There have been few reports on E. coli ST131 from food animals (Nicolas-Chanoine et al., 2014). To the best of our knowledge, this is the ﬁrst report of the presence of E. coli ST131 in food animals in South Korea. We identiﬁed two pig E. coli ST131 isolates, which harbored both blaCTX-M-65 and blaTEM-1 genes and had MDR phenotypes. Human E. coli ST131 carrying both blaCTX-M-65 and blaTEM-1 has been detected in Germany (Cullik et al., 2010). Clonal populations of ST410 are present in humans, companion animals, livestock, and the environment (Falgenhauer et al., 2016) and pose a high risk of causing ExPEC outbreaks in hospitals worldwide (Roer et al., 2018). ST10, ST58, and ST117 lineages have also been detected in both humans and food animals (Manges et al., 2015; McKinnon et al., 2018). In addition, ST38 and ST101 were present in chicken and pig samples analyzed in this study, respectively, which are more related to hospital-onset than to community- onset infections in South Korea (Yoo et al., 2013; Kim et al., 2016). Extended-spectrum β-lactamase genes may circulate among food animals, farm workers, and the farm environment (Tamang et al., 2013a), but also cefotaxime-resistant bacteria can be isolated from food animals raised without cephalosporins (Mir et al., 2016, 2018), suggesting the animal acquisition of antimicrobial resistance from the environment. REFERENCES Bonnet, R. (2004). Growing group of extended-spectrum β-lactamases: the CTX-M enzymes. Antimicrob. Agents Chemother. 48, 1–14. doi: 10.1128/aac.48.1.1-14. 2004 Bevan, E. R., Jones, A. M., and Hawkey, P. M. (2017). Global epidemiology of CTX- M β-lactamases: temporal and geographical shifts in genotype. J. Antimicrob. Chemother. 72, 2145–2155. doi: 10.1093/jac/dkx146 Bevan, E. R., Jones, A. M., and Hawkey, P. M. (2017). Global epidemiology of CTX- M β-lactamases: temporal and geographical shifts in genotype. J. Antimicrob. Chemother. 72, 2145–2155. doi: 10.1093/jac/dkx146 Brolund, A. (2014). Overview of ESBL-producing Enterobacteriaceae from a Nordic perspective. Infect. Ecol. Epidemiol. 4:24555. doi: 10.3402/iee.v4.24555 Brolund, A. (2014). Overview of ESBL-producing Enterobacteriaceae from a Nordic perspective. Infect. Ecol. Epidemiol. 4:24555. doi: 10.3402/iee.v4.24555 April 2020 \| Volume 11 \| Article 604 Frontiers in Microbiology \| www.frontiersin.org 7 ESBL-Producing E. coli in Food Animals Song et al. Hu, Y. Y., Cai, J. C., Zhou, H. W., Chi, D., Zhang, X. F., Chen, W. L., et al. (2013). Molecular typing of CTX-M-producing Escherichia coli isolates from environmental water, swine feces, specimens from healthy humans, and human Canton, R., Gonzalez-Alba, J. M., and Galan, J. C. (2012). CTX-M enzymes: origin and diﬀusion. Front. Microbiol. 3:110. doi: 10.3389/fmicb.2012.00110 Carattoli, A. (2008). Animal reservoirs for extended spectrum β-lactamase producers. Clin. Microbiol. Infect. 14, 117–123. doi: 10.1111/j.1469-0691.2007. 01851.x p y patients. Appl. Environ. Microbiol. 79, 5988–5996. doi: 10.1128/AEM.01740-13 Johnson, J. R., Delavari, P., Kuskowski, M., and Stell, A. L. (2001). Phylogenetic distribution of extraintestinal virulence-associated traits in Escherichia coli. J. Infect. Dis. 183, 78–88. doi: 10.1086/317656 Carattoli, A., Bertini, A., Villa, L., Falbo, V., Hopkins, K. L., and Threlfall, E. J. (2005). Identiﬁcation of plasmids by PCR-based replicon typing. J. Microbiol. Methods 63, 219–228. doi: 10.1016/j.mimet.2005.03.018 Kim, H., Kim, Y. A., Park, Y. S., Choi, M. H., Lee, G. I., and Lee, K. (2017). Risk Factors and Molecular features of sequence type (ST) 131 extended-spectrum β- lactamase-producing Escherichia coli in community-onset bacteremia. Sci. Rep. 7:14640. doi: 10.1038/s41598-017-14621-4 Cha, M. K., Kang, C. I., Kim, S. H., Cho, S. Y., Ha, Y. E., Wi, Y. M., et al. (2016). Comparison of the microbiological characteristics and virulence factors of ST131 and non-ST131 clones among extended-spectrum β-lactamase- producing Escherichia coli causing bacteremia. Diagn. Microbiol. Infect. Dis. 84, 102–104. doi: 10.1016/j.diagmicrobio.2015.10.015 Kim, Y. A., Kim, J. J., Kim, H., and Lee, K. (2017). REFERENCES Community-onset extended- spectrum-β-lactamase-producing Escherichia coli sequence type 131 at two Korean community hospitals: the spread of multidrug-resistant E. coli to the community via healthcare facilities. Int. J. Infect. Dis. 54, 39–42. doi: 10.1016/j. ijid.2016.11.010 Clermont, O., Bonacorsi, S., and Bingen, E. (2000). Rapid and simple determination of the Escherichia coli phylogenetic group. Appl. Environ. Microbiol. 66, 4555–4558. doi: 10.1128/aem.66.10.4555-4558.2000 CLSI (2017). Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing. 27th ed. CLSI supplement M100. Wayne, PA: Clinical and Laboratory Standards Institute. Kim, K. G., Jeong, J., Kim, M. J., Park, D. W., Shin, J. H., Park, H. J., et al. (2019). Prevalence and molecular epidemiology of ESBLs, plasmid-determined AmpC- type β-lactamases and carbapenemases among diarrhoeagenic Escherichia coli isolates from children in Gwangju Korea: 2007-16. J. Antimicrob. Chemother. 74, 2181–2187. doi: 10.1093/jac/dkz175 Cullik, A., Pfeifer, Y., Prager, R., von Baum, H., and Witte, W. (2010). A novel IS26 structure surrounds blaCTX-M genes in diﬀerent plasmids from German clinical Escherichia coli isolates. J. Med. Microbiol. 59, 580–587. doi: 10.1099/ jmm.0.016188-0 Kim, S., Sung, J. Y., Cho, H. H., Kwon, K. C., and Koo, S. H. (2016). Characteristics of the molecular epidemiology of CTX-M-Producing Escherichia coli isolated from a tertiary hospital in Daejeon Korea. J. Microbiol. Biotechnol. 26, 1643– 1649. doi: 10.4014/jmb.1603.03063 Dahms, C., Hubner, N. O., Kossow, A., Mellmann, A., Dittmann, K., and Kramer, A. (2015). Occurrence of ESBL-producing Escherichia coli in livestock and farm workers in Mecklenburg-Western Pomerania Germany. PLoS One 10, e0143326. doi: 10.1371/journal.pone.0143326 Kumar, S., Stecher, G., Li, M., Knyaz, C., and Tamura, K. (2018). MEGA X: molecular evolutionary genetics analysis across computing platforms. Mol. Biol. Evol. 35, 1547–1549. doi: 10.1093/molbev/msy096 Denisuik, A. J., Karlowsky, J. A., Adam, H. J., Baxter, M. R., Lagace-Wiens, P. R. S., Mulvey, M. R., et al. (2019). Dramatic rise in the proportion of ESBL-producing Escherichia coli and Klebsiella pneumoniae among clinical isolates identiﬁed in Canadian hospital laboratories from 2007 to 2016. J. Antimicrob. Chemother. 74, 64–71. doi: 10.1093/jac/dkz289 Laube, H., Friese, A., von Salviati, C., Guerra, B., Kasbohrer, A., Kreienbrock, L., et al. (2013). Longitudinal monitoring of extended-spectrum- β-lactamase/AmpC-producing Escherichia coli at German broiler chicken fattening farms. Appl. Environ. Microbiol. 79, 4815–4820. doi: 10.1128/AEM.00856-13 EUCAST (2017). The European Committee on Antimicrobial Susceptibility Testing. Breakpoint tables for interpretation of MICs and zone diameters. Version 7.1. Sweden: EUCAST. Lee, M. Y., Choi, H. J., Choi, J. REFERENCES Y., Song, M., Song, Y., Kim, S. W., et al. (2010). Dissemination of ST131 and ST393 community-onset, ciproﬂoxacin-resistant Escherichia coli clones causing urinary tract infections in Korea. J. Infect. 60, 146–153. doi: 10.1016/j.jinf.2009.11.004 Falgenhauer, L., Imirzalioglu, C., Ghosh, H., Gwozdzinski, K., Schmiedel, J., Gentil, K., et al. (2016). Circulation of clonal populations of ﬂuoroquinolone- resistant CTX-M-15-producing Escherichia coli ST410 in humans and animals in Germany. Int. J. Antimicrob. Agents 47, 457–465. doi: 10.1016/j.ijantimicag. 2016.03.019 Leverstein-van Hall, M. A., Dierikx, C. M., Cohen Stuart, J., Voets, G. M., van den Munckhof, M. P., van Essen-Zandbergen, A., et al. (2011). Dutch patients, retail chicken meat and poultry share the same ESBL genes, plasmids and strains. Clin. Microbiol. Infect. 17, 873–880. doi: 10.1111/j.1469-0691.2011.03497.x Fan, P., Nelson, C. D., Driver, J. D., Elzo, M. A., and Jeong, K. C. (2019). Animal breed composition is associated with the hindgut microbiota structure and β- lactam resistance in the multibreed Angus-Brahman herd. Front. Microbiol. 10:1846. doi: 10.3389/fmicb.2019.01846 Lim, J. S., Choi, D. S., Kim, Y. J., Chon, J. W., Kim, H. S., Park, H. J., et al. (2015). Characterization of Escherichia coli-producing extended-spectrum β-lactamase (ESBL) isolated from chicken slaughterhouses in South Korea. Foodborne Pathog. Dis. 12, 741–748. doi: 10.1089/fpd.2014.1921 Hammerum, A. M., Larsen, J., Andersen, V. D., Lester, C. H., Skovgaard Skytte, T. S., Hansen, F., et al. (2014). Characterization of extended-spectrum β- lactamase (ESBL)-producing Escherichia coli obtained from Danish pigs, pig farmers and their families from farms with high or no consumption of third- or fourth-generation cephalosporins. J. Antimicrob. Chemother. 69, 2650–2657. doi: 10.1093/jac/dku180 Lupo, A., Saras, E., Madec, J. Y., and Haenni, M. (2018). Emergence of blaCTX- M-55 associated with fosA, rmtB and mcr gene variants in Escherichia coli from various animal species in France. J. Antimicrob. Chemother. 73, 867–872. doi: 10.1093/jac/dkx489 Maamar, E., Hammami, S., Alonso, C. A., Dakhli, N., Abbassi, M. S., Ferjani, S., et al. (2016). High prevalence of extended-spectrum and plasmidic AmpC β-lactamase-producing Escherichia coli from poultry in Tunisia. Int. J. Food Microbiol. 231, 69–75. doi: 10.1016/j.ijfoodmicro.2016.05.001 He, D., Chiou, J., Zeng, Z., Liu, L., Chen, X., Zeng, L., et al. (2015). Residues distal to the active site contribute to enhanced catalytic activity of variant and hybrid β-lactamases derived from CTX-M-14 and CTX-M-15. Antimicrob. Agents Chemother. 59, 5976–5983. doi: 10.1128/AAC.04920-14 Magiorakos, A. P., Srinivasan, A., Carey, R. B., Carmeli, Y., Falagas, M. E., Giske, C. G., et al. (2012). REFERENCES 60, 1136–1151. doi: 10.1111/j.1365-2958.2006.05172.x Park, H., Kim, J., Ryu, S., and Jeon, B. (2019). Predominance of blaCTX-M-65 and blaCTX-M-55 in extended-spectrum β-lactamase-producing Escherichia coli from raw retail chicken in South Korea. J. Glob. Antimicrob. Resist. 17, 216–220. doi: 10.1016/j.jgar.2019.01.005 Yang, Q. E., Sun, J., Li, L., Deng, H., Liu, B. T., Fang, L. X., et al. (2015). IncF plasmid diversity in multi-drug resistant Escherichia coli strains from animals in China. Front. Microbiol. 6:964. doi: 10.3389/fmicb.2015.00964 Pitout, J. D., and Laupland, K. B. (2008). Extended-spectrum β-lactamase- producing Enterobacteriaceae: an emerging public-health concern. Lancet Infect. Dis. 8, 159–166. doi: 10.1016/S1473-3099(08)70041-0 Yang, X., Liu, W., Liu, Y., Wang, J., Lv, L., Chen, X., et al. (2014). F33:A-: B-, IncHI2/ST3, and IncI1/ST71 plasmids drive the dissemination of fosA3 and blaCTX-M-55/-14/-65 in Escherichia coli from chickens in China. Front. Microbiol. 5:688. doi: 10.3389/fmicb.2014.00688 Rao, L., Lv, L., Zeng, Z., Chen, S., He, D., Chen, X., et al. (2014). Increasing prevalence of extended-spectrum cephalosporin-resistant Escherichia coli in food animals and the diversity of CTX-M genotypes during 2003-2012. Vet. Microbiol. 172, 534–541. doi: 10.1016/j.vetmic.2014.06.013 Yoo, J. S., Kim, H. M., Koo, H. S., Yang, J. W., Yoo, J. I., Kim, H. S., et al. (2013). Nosocomial transmission of NDM-1-producing Escherichia coli ST101 in a Korean hospital. J. Antimicrob. Chemother. 68, 2170–2172. doi: 10.1093/jac/ dkt126 Roer, L., Overballe-Petersen, S., Hansen, F., Schonning, K., Wang, M., Roder, B. L., et al. (2018). Escherichia coli sequence type 410 is causing new international high-risk clones. mSphere 3:e00337-18. doi: 10.1128/mSphere.00337-18 Zhang, J., Zheng, B., Zhao, L., Wei, Z., Ji, J., Li, L., et al. (2014). Nationwide high prevalence of CTX-M and an increase of CTX-M-55 in Escherichia coli isolated from patients with community-onset infections in Chinese county hospitals. BMC Infect. Dis. 14:659. doi: 10.1186/s12879-014-0659-0 Rogers, B. A., Sidjabat, H. E., and Paterson, D. L. (2011). Escherichia coli O25b- ST131: a pandemic, multiresistant, community-associated strain. J Antimicrob Chemother. 66, 1–14. doi: 10.1093/jac/dkq415 Seni, J., Peirano, G., Okon, K. O., Jibrin, Y. B., Mohammed, A., Mshana, S. E., et al. (2018). The population structure of clinical extra-intestinal Escherichia coli in a teaching hospital from Nigeria. Diagn. Microbiol. Infect. Dis. 92, 46–49. doi: 10.1016/j.diagmicrobio.2018.04.001 Conﬂict of Interest: The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. Shin, S. W., Jung, M., Won, H. G., Belaynehe, K. REFERENCES Multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria: an international expert proposal for interim standard deﬁnitions for acquired resistance. Clin. Microbiol. Infect. 18, 268–281. doi: 10.1111/j.1469-0691.2011.03570.x Ho, P. L., Chow, K. H., Lai, E. L., Lo, W. U., Yeung, M. K., Chan, J., et al. (2011). Extensive dissemination of CTX-M-producing Escherichia coli with multidrug resistance to ’critically important’ antibiotics among food animals in Hong Kong, 2008-10. J. Antimicrob. Chemother. 66, 765–768. doi: 10.1093/jac/ dkq539 Manges, A. R., Geum, H. M., Guo, A., Edens, T. J., Fibke, C. D., and Pitout, J. D. D. (2019). Global extraintestinal pathogenic Escherichia coli (ExPEC) lineages. Clin. Microbiol. Rev. 32:CMR.00135-18. doi: 10.1128/CMR.00135-18 Hong, J. S., Song, W., Park, H. M., Oh, J. Y., Chae, J. C., Shin, S., et al. (2019). Clonal spread of extended-spectrum cephalosporin-resistant Enterobacteriaceae between companion animals and humans in South Korea. Front. Microbiol. 10:1371. doi: 10.3389/fmicb.2019.01371 Manges, A. R., Harel, J., Masson, L., Edens, T. J., Portt, A., Reid-Smith, R. J., et al. (2015). Multilocus sequence typing and virulence gene proﬁles associated with Escherichia coli from human and animal sources. Foodborne Pathog. Dis. 12, 302–310. doi: 10.1089/fpd.2014.1860 Hordijk, J., Wagenaar, J. A., van de Giessen, A., Dierikx, C., van Essen- Zandbergen, A., Veldman, K., et al. (2013). Increasing prevalence and diversity of ESBL/AmpC-type β-lactamase genes in Escherichia coli isolated from veal calves from 1997 to 2010. J. Antimicrob. Chemother. 68, 1970–1973. doi: 10. 1093/jac/dkt132 Markland, S., Weppelmann, T. A., Ma, Z., Lee, S., Mir, R. A., Teng, L., et al. (2019). High prevalence of cefotaxime resistant bacteria in grazing beef cattle: a cross sectional study. Front. Microbiol. 10:176. doi: 10.3389/fmicb.2019.00176 April 2020 \| Volume 11 \| Article 604 Frontiers in Microbiology \| www.frontiersin.org 8 ESBL-Producing E. coli in Food Animals Song et al. β-lactamase in clinical isolates of Escherichia coli from Korea. J. Med. Microbiol. 58, 261–266. doi: 10.1099/jmm.0.004507-0 β-lactamase in clinical isolates of Escherichia coli from Korea. J. Med. Microbiol. 58, 261–266. doi: 10.1099/jmm.0.004507-0 McGann, P., Snesrud, E., Maybank, R., Corey, B., Ong, A. C., Cliﬀord, R., et al. (2016). Escherichia coli harboring mcr-1 and blaCTX-M on a novel IncF plasmid: ﬁrst report of mcr-1 in the United States. Antimicrob. Agents Chemother. 60, 4420–4421. doi: 10.1128/AAC.01103-16 Tamang, M. D., Nam, H. M., Gurung, M., Jang, G. C., Kim, S. R., Jung, S. C., et al. (2013a). REFERENCES Molecular characterization of CTX-M β-lactamase and associated addiction systems in Escherichia coli circulating among cattle, farm workers, and the farm environment. Appl. Environ. Microbiol. 79, 3898–3905. doi: 10. 1128/AEM.00522-13 McKinnon, J., Roy Chowdhury, P., and Djordjevic, S. P. (2018). Genomic analysis of multidrug-resistant Escherichia coli ST58 causing urosepsis. Int. J. Antimicrob. Agents 52, 430–435. doi: 10.1016/j.ijantimicag.2018. 06.017 Tamang, M. D., Nam, H. M., Kim, S. R., Chae, M. H., Jang, G. C., Jung, S. C., et al. (2013b). Prevalence and molecular characterization of CTX-M β-lactamase- producing Escherichia coli isolated from healthy swine and cattle. Foodborne Pathog. Dis. 10, 13–20. doi: 10.1089/fpd.2012.1245 Mir, R. A., Weppelmann, T. A., Johnson, J. A., Archer, D., Morris, J. G. Jr., et al. (2016). identiﬁcation and characterization of cefotaxime resistant bacteria in beef cattle. PLoS One 11:e0163279. doi: 10.1371/journal.pone.0163279 Villa, L., Garcia-Fernandez, A., Fortini, D., and Carattoli, A. (2010). Replicon sequence typing of IncF plasmids carrying virulence and resistance determinants. J. Antimicrob. Chemother. 65, 2518–2529. doi: 10.1093/jac/dkq347 Mir, R. A., Weppelmann, T. A., Teng, L., Kirpich, A., Elzo, M. A., Driver, J. D., et al. (2018). Colonization dynamics of cefotaxime resistant bacteria in beef cattle raised without cephalosporin antibiotics. Front. Microbiol. 9:500. doi: 10.3389/fmicb.2018.00500 Moodley, A., and Guardabassi, L. (2009). Transmission of IncN plasmids carrying blaCTX-M-1 between commensal Escherichia coli in pigs and farm workers. Antimicrob. Agents Chemother. 53, 1709–1711. doi: 10.1128/AAC.01014-08 Vitas, A. I., Naik, D., Perez-Etayo, L., and Gonzalez, D. (2018). Increased exposure to extended-spectrum β-lactamase-producing multidrug-resistant Enterobacteriaceae through the consumption of chicken and sushi products. Int. J. Food Microbiol. 269, 80–86. doi: 10.1016/j.ijfoodmicro.2018.01.026 Morosini, M. I., Garcia-Castillo, M., Coque, T. M., Valverde, A., Novais, A., Loza, E., et al. (2006). Antibiotic coresistance in extended-spectrum-β-lactamase- producing Enterobacteriaceae and in vitro activity of tigecycline. Antimicrob. Agents Chemother. 50, 2695–2699. doi: 10.1128/AAC.00155-06 Wang, J., Zeng, Z. L., Huang, X. Y., Ma, Z. B., Guo, Z. W., Lv, L. C., et al. (2018). Evolution and comparative genomics of F33:A-:B- plasmids carrying blaCTX- M-55 or blaCTX-M-65 in Escherichia coli and Klebsiella pneumoniae isolated from animals, food products, and humans in China. mSphere 3:e00137-18. doi: 10.1128/mSphere.00137-18 Nicolas-Chanoine, M. H., Bertrand, X., and Madec, J. Y. (2014). Escherichia coli ST131, an intriguing clonal group. Clin. Microbiol. Rev. 27, 543–574. doi: 10. 1128/CMR.00125-13 Wirth, T., Falush, D., Lan, R., Colles, F., Mensa, P., Wieler, L. H., et al. (2006). Sex and virulence in Escherichia coli: an evolutionary perspective. Mol. Microbiol. Frontiers in Microbiology \| www.frontiersin.org REFERENCES M., Yoon, I. J., and Yoo, H. S. (2017). Characteristics of transmissible CTX-M- and CMY-Type β-lactamase- producing Escherichia coli isolates collected from pig and chicken farms in South Korea. J. Microbiol. Biotechnol. 27, 1716–1723. doi: 10.4014/jmb.1610. 10006 Copyright © 2020 Song, Oh, Kim, Park and Shin. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Song, W., Lee, H., Lee, K., Jeong, S. H., Bae, I. K., Kim, J. S., et al. (2009). CTX- M-14 and CTX-M-15 enzymes are the dominant type of extended-spectrum April 2020 \| Volume 11 \| Article 604 Frontiers in Microbiology \| www.frontiersin.org Frontiers in Microbiology \| www.frontiersin.org 9
https://openalex.org/W2914728325	https://europepmc.org/articles/pmc6374354?pdf=render	English	null	Ceratophysella species from mushrooms in China (Collembola, Hypogastruridae)	ZooKeys	2,019	cc-by	3,909	ZooKeys 822: 67–77 (2019) doi: 10.3897/zookeys.822.30880 http://zookeys.pensoft.net ZooKeys 822: 67–77 (2019) doi: 10.3897/zookeys.822.30880 http://zookeys.pensoft.net ZooKeys 822: 67–77 (2019) doi: 10.3897/zookeys.822.30880 http://zookeys.pensoft.net la species from mushro RESEARCH ARTICLE Ceratophysella species from mushrooms in China (Collembola, Hypogastruridae) Wanda Maria Weiner1, Zhijing Xie2,3, Yu Li4, Xin Sun2,4,5 1 Institute of Systematics and Evolution of Animals, Polish Academy of Sciences, Sławkowska 17, 31-016 Kraków, Poland 2 Key laboratory of Wetland Ecology and Environment, Northeast Institute of Geography and Agroecology, Chinese Academy of Sciences, Changchun 130102, China 3 University of Chinese Academy of Sciences, Beijing 100049, China 4 Engineering Research Center of Chinese Ministry of Education for Edible and Medicinal Fungi, Jilin Agricultural University, Changchun 130118, China 5 J.F. Blumenbach Institute of Zoology and Anthropology, University of Göttingen, 37073 Göttingen, Germany Corresponding author: Xin Sun (sunxin@iga.ac.cn) Academic editor: L. Deharveng \| Received 27 October 2018 \| Accepted 21 January 2019 \| Published 5 February 2019 http://zoobank.org/DDE9A97D-0715-45E8-879F-8FCE235F8335 Citation: Weiner WM, Xie Z, Li Y, Sun X (2019) Ceratophysella species from mushrooms in China (Collembola, Hypogastruridae). ZooKeys 822: 67–77. https://doi.org/10.3897/zookeys.822.30880 Academic editor: L. Deharveng \| Received 27 October 2018 \| Accepted 21 January 2019 \| Published 5 February 2019 http://zoobank.org/DDE9A97D-0715-45E8-879F-8FCE235F8335 Citation: Weiner WM, Xie Z, Li Y, Sun X (2019) Ceratophysella species from mushrooms in China (Collembola, Hypogastruridae). ZooKeys 822: 67–77. https://doi.org/10.3897/zookeys.822.30880 Citation: Weiner WM, Xie Z, Li Y, Sun X (2019) Ceratophysella species from mushrooms in China (Collembola, Hypogastruridae). ZooKeys 822: 67–77. https://doi.org/10.3897/zookeys.822.30880 Abstract Four species of the genus Ceratophysella living on mushrooms are reported from China, including a new species, Ceratophysella skarzynskii Weiner & Sun, sp. n., which is described from alpine mushrooms. The new species belongs to the Ceratophysella group of species with a dorsal chaetotaxy of type B and differs from the other species in a combination of characters. Ceratophysella skarzynskii sp. n. is distinguished by its small body size (maximum length 1.09 mm), number of peg-like s-chaetae (30–32) in the ventral sensory file, the trilobed apical vesicle of antennal segment IV, five modified chaetae on dens, and serrated dorsal chaetae. A key to the Chinese species of the genus has been provided. Keywords Ceratophysella skarzynskii sp. n., key, new species, taxonomy Copyright Wanda Maria Weiner et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Wanda Maria Weiner et al. / ZooKeys 822: 67–77 (2019) 68 Introduction The genus Ceratophysella Börner, 1932 is distributed worldwide, having more than 130 species (Bellinger et al. 1996–2018). The main diagnostic characters for the genus are the pigmented body, 8 + 8 ocelli, body chaetae mostly differentiated into micro- and macrochaetae, an eversible integumental sac usually present between antennal seg ments III and IV, the ventral side of antennal segment IV with a sensory file often well-developed of short, erect, curved, and flattened at tips s-chaetae, unguiculus with broad basal lamella, furca well developed, mucro usually boat-like with a spoon-like apex and distinct lamella, and anal spines usually long and curved. Until now, fourteen species of the genus Ceratophysella have been reported from China (Zhao 1992, Shen 1993, Liu et al. 1998, Zhao et al. 1997, Jia et al. 2010). As a common group of Collembola living on mushrooms, species have often caused sig nificant economic damage in China (Wei 2002; Zhu 2012). Within a large collection of the mushroom Collembola in China, three known species, C. communis (Folsom, 1898), C. denticulata (Bagnall, 1941), C. liguladorsi (Lee, 1974), and the new species described here, C. skarzynskii sp. n., are reported in the present paper. Materials and methods Specimens were collected by hand using a brush and stored in ethanol; they were then cleared in lactic acid and KOH, and mounted in Marc André II medium. Drawings and measurements were made using a phase contrast microscope LEICA DM2500 equipped with a camera lucida. Mushroom species were determined by the third author, Yu Li. Taxonomy Ceratophysella skarzynskii Weiner & Sun, sp. n. http://zoobank.org/BA3F1CD5-D62C-4AFA-8450-26FB928994CA Figs 1–3, Table 1 Ceratophysella skarzynskii Weiner & Sun, sp. n. http://zoobank.org/BA3F1CD5-D62C-4AFA-8450-26FB928994CA Figs 1–3, Table 1 Type material. Holotype: preadult male, China: Jilin: Changbai Mountains, alt. 2000 m, on Russula sp., leg. Xin Sun, 29 July 2015. Paratypes: 10 females and one juvenile, the same data as holotype. Type material: the holotype and 8 paratypes are housed in IGA-CAS, China, two paratypes in ISEA-PAS, Poland. Diagnosis. Dorsal chaetotaxy of type B with serrated chaetae. Maximal length 1.09 mm. Antennal segment IV with bi- or trilobed apical vesicle and ventral sensory file with 30–32 peg-like s-chaetae. Dens with seven chaetae, five of them modified. iii Description. Body length 0.9–1.09 mm (holotype: 1.07 mm). Body colour violet or blue in alive specimens, grey or grey-black in alcohol, ventrally pale. Granulation rather coarse, 10–14 granules between chaetae p1 on Abd. V (Yosii’s parameter). iii Description. Body length 0.9–1.09 mm (holotype: 1.07 mm). Body colour violet or blue in alive specimens, grey or grey-black in alcohol, ventrally pale. Granulation rather coarse, 10–14 granules between chaetae p1 on Abd. V (Yosii’s parameter). Antennae. Ant. IV with bilobed or trilobed apical vesicle (av), subapical organite (or), dorso-lateral microsensillum (ms), seven cylindrical, subequal sensilla (dorsal S0, S1–4, dorsolateral S7–8), ca. 30 small, peg-like sensilla and one subcylindrical sensil lum in ventral sensory file (sensory rasp) (Fig. 2A, B). Ant. III-organ with two long (external) and two short (internal) curved sensilla (Fig. 2A). Microsensillum on ant. III present. Eversible sac between Ant. III–IV present (Fig. 2B). Ant. I with seven chaetae, Ant. II with 13 chaetae. Head. Ocelli 8 + 8. Postantennal organ 1.5 times as large as single ocellus with four lobes of which the anterior pair is larger than the posterior pair (Fig. 2C). Accessory boss present (Fig. 2C). Head. Ocelli 8 + 8. Postantennal organ 1.5 times as large as single ocellus with four lobes of which the anterior pair is larger than the posterior pair (Fig. 2C). Accessory boss present (Fig. 2C). Labrum with 5, 5, 4 chaetae, four prelabrals present. Head of maxilla of the C. armatatype. Maxillary outer lobe with two sublobal hairs. Labium of the C. armata type, with five papillae (A–E) and six proximal chaetae. Guard chaetae a1, b1–2, d2, e2 and lateral processus (l.p.) as accessory papillae with short terminal sensillum. Guards b3–4, d3–4, and e1–6 with long sensilla. Abbreviations used in the descriptions: Abd. abdominal segments, Ant. antennal segments, av apical vesicle AIIIO sensory organ of Ant. III, l.p. lateral processus on labial palp, ms s-microsetae (ms) (microsen sillum), or subapical organite PAO postantennal organ, S sensillum, s-chaetae sensorial chaetae on Th. and Abd. Th. thoracic segments, VT ventral tube, IGA-CAS Northeast Institute of Geog raphy and Agroecology, Chi nese Academy of Sciences; ISEA-PAS Institute of Systematics and Evolution of Animals, Polish Academy of Sciences. Terminology for the descriptions follows that given in Fjellberg (1984, 1999), Babenko et al. (1994), and Thibaud et al. (2004). Ceratophysella species from mushrooms in China 69 Taxonomy Dorsal guards b3–4, d3–4, and e3 distally expanded and flattened. l Chaetotaxy. Differentiation of dorsal chaetae into micro-/meso- and macrochaetae quite distinct (Figs 1A, 1B, 2D, 3C). Arrangement of chaetae on head typical for the genus, spine-like chaetae absent. Cephalic chaetae d2, 4, v2, p3,4, g1, 5, l01, l11 as macro chaetae. Dorsal chaetotaxy of B type (sensu Gisin 1947, Bourgeois and Cassagnau 1972, and Babenko et al. 1994) (Fig. 1B). Chaetae of medium length, pointed and ser rated. Th. I with macrochaetae p4, without p2. Th. II–III with macrochaetae p2 (shifted forward), p5, 6, m5, chaetae m4 and m5 (Th. II with m4’and microsensillum ms), chaetae p4 m6 as sensorial chaetae s, chaetae a2 as long as a3. Abd. I–III with macrochaetae p2,6, sensorial chaetae s = p5. Abd. IV with macrochaetae p1, 3, 6, s-chaetae as p4. Abd. V with macrochaetae p1, 5, 4 + 4 a-chaetae inside two macrochaetae p5 (a2, 2’ absent, chaeta a5 Wanda Maria Weiner et al. / ZooKeys 822: 67–77 (2019) 70 Figure 1. Ceratophysella skarzynskii sp. n. A Chaetotaxy of head and Th. I–III B Chaetotaxy of Abd. I–VI. Scale bars: 0.1 mm. Figure 1. Ceratophysella skarzynskii sp. n. A Chaetotaxy of head and Th. I–III B Chaetotaxy of Abd. I–VI. Scale bars: 0.1 mm. straight above p5) (Figs 1B, 3C). Body s-chaetae relatively long, but shorter than mac rochaetae, only on Abd. V as long as macrochaetae p1, 5 (Figs 1A, B, 3C). 1, 5 Tibiotarsi I, II, III with 19, 19, 18 chaetae respectively, including one acuminate tenent hair A1 each, femora with 13, 13, 12 chaetae, trochanters with 7, 7, 7 chaetae, coxae with 3, 7, 8 chaetae, subcoxae II with 0, 3, 3 chaetae, subcoxaeI with 1, 2, 3 chaetae. Claws with inner tooth and two pairs of lateral teeth. Empodial append age with broad basal lamella and apical filament reaching slightly below inner tooth (ratio empodial filament: inner edge of claw = 0.47) (Fig. 3A). Ventral tube with 4 + 4 chaetae. Retinaculum with 4 + 4 teeth. Furca well developed. Ratio dens + mucro: inner edge of claw III = 2.11: 1, ratio dens: mucro = 1.79: 1. Cuticular skeleton of furca well visible. Anal spines as long as inner edge of claw III slightly curved, situated on basal papil lae, colourless (Figs 1B, 3C). Taxonomy Dens with uniform granulation and seven dorsal chaetae of which five are modified, two strongly thickened and three moderately so; basal macrochaeta longer than others chaetae, shorter than dens (3/5 of its length). Mucro boat-like with clear outer lamella (Fig. 3B, D). Anal spines as long as inner edge of claw III slightly curved, situated on basal papil lae, colourless (Figs 1B, 3C). Ceratophysella species from mushrooms in China 71 Table 1. Morphological characters for C. skarzynski sp. nov. and similar species: C. denisana Yosii, 1954 (Yosii 1956), C. empodialis Babenko, 1994, C. longispina (Tullberg, 1876) and C. scotica (Carpenter & Evans, 1899) after authors and Babenko et al. (1994). Table 1. Morphological characters for C. skarzynski sp. nov. and similar species: C. denisana Yosii, 1954 (Yosii 1956), C. empodialis Babenko, 1994, C. longispina (Tullberg, 1876) and C. scotica (Carpenter & Evans, 1899) after authors and Babenko et al. (1994). Species/characters C. denisana C. skarzynskii C. empodialis C. longispina C. scotica Maximal body size (mm) 1.20 1.09 1.80 2.00 2.00 Yosii’ parameter 14–16 (20) 10–14 8–13 12–16 13–15 Ant.IV: apical vesicle trilobed trilobed simple/slightly bilobed simple/slightly bilobed simple Ant.IV: number of peg-like chaetae in ventral file >50 30–32 20 max. 15 15–20 Maxillar palp: number of sublobal hairs 2 2 2 2 1 Dorsal chaetae smooth serrated rather smooth serrated rather smooth Th.II–III: length of chaetae s = p4/p3 p4 >p3 p4 >p3 p4 >p3 p4≈p3 p4≈p3 Abd. V: chaetae s = p3/p1 p3≈p1 p3≈p1 p3<p1 p3≈p1 p3<p1 Tibiotarsial tenent hair ? pointed pointed ? pointed Empodial appendage : inner edge of claw ±½ ±½ ±1¼ ±½ ±1 Empodial basal lamella : inner edge of claw 1/4 1/5 1/5 ? 1/5 Lateral teeth of claw basal 3 pairs 2 pairs 2 pairs 2 pairs 2 pairs Chaetae on dens: total number/numer of modified chaetae 7/5 7/5 7/2 7(8)/2 7/2 Etymology. The species is cordially dedicated to our colleague and friend Dr Dari usz Skarżyński, a prominent Polish specialist in Hypogastruridae, Collembola. p p p g Remarks. The new species belongs to the armata-group of species, group B (Abd. tergum IV with p1 as macrochaeta) and subgroup B2 (Abd. tergum IV without chaeta p3) (Bourgeois and Cassagnau 1972). Among the species which could belong to this subgroup,C. skarzynskii is most similar to four species: C. denisana (Yosii, 1956), C. empo dialis Babenko, 1994 (in Babenko et al. 1994), C. Taxonomy longispina (Tullberg, 1876), and C. scot ica (Carpenter & Evans, 1899), due to the absence of transformed into spines, spine-like chaetae or spine-like integumentary protuberance on the head or Abd. V. They differ in the shape of the apical vesicle, the number of modified chaetae on the dens, the number of peg-like chaetae in the ventral sensory file on Ant. IV, length of empodial appendage,in the type of dorsal chaetae (serrated or smooth), and in the number of sublobal hairs on maxillary palp (one or two). A comparison of these species is presented in Table 1. Ceratophysella communis (Folsom, 1898) Acorutes communis Folsom, 1898: 52. Syn: Hypogastrura yuasai Yosii, 1954: Yosii 1960. Acorutes communis Folsom, 1898: 52. Syn: Hypogastrura yuasai Yosii, 1954: Yosii 1960. Studied material. China: Henan: Zhengzhou, on Pleurotus ostreatus, 7 specimens on slides and 20 in alcohol, leg. Xin Sun, 05 Dec 2014; China: Henan: Zhumadian, on 72 72 Wanda Maria Weiner et al. / ZooKeys 822: 67–77 (2019) Figure 2. Ceratophysella skarzynskii sp. n. A ant. III and IV dorsal B ant. III and IV dorsal C PAO and eyes D macro-, microchaetae and s-chaeta. Scale bars: 0.01 mm. Figure 2. Ceratophysella skarzynskii sp. n. A ant. III and IV dorsal B ant. III and IV dorsal C PAO and eyes D macro-, microchaetae and s-chaeta. Scale bars: 0.01 mm. Figure 2. Ceratophysella skarzynskii sp. n. A ant. III and IV dorsal B ant. III and IV dorsal C PAO and eyes D macro-, microchaetae and s-chaeta. Scale bars: 0.01 mm. Pleurotus ostreatus, 9 specimens on slides and 50 in alcohol, leg. ZhijingXie, 10 Nov 2017; China: Zhejiang: Pinghu, on Pleurotus ostreatus, 13 specimens on slides and 100 in alcohol, leg. Xin Sun, 07 Dec 2014; China: Sichuan: Qingchuan, on Morchel la esculenta, 4 specimens on slides and 30 in alcohol, leg. Zhijing Xie, 19 Mar 2017. Achorutes denticulatus Bagnall, 1941: 218. Syn: Achorutes armata var. nov. cuspidate Axelson, 1905: Thibaud et al. 2004. Achorutes distinguendus Bagnall, 1941: Thibaud et al. 2004. Achorutes distinguendus Bagnall, 1941: Thibaud et al. 2004. g gh Hypogastrura (Ceratophysella) exilis Yosii, 1956: Yosii 1965.h Hypogastrura (Ceratophysella) afghanistanensis Stach, 1963: Thibaud et al. 2004. Hypogastrura (Ceratophysella) afghanistanensis Stach, 1963: Thibaud et al. 2004. Hypogastrura (Ceratophysella) afghanistanensis Stach, 1963: Thibaud et al. 2004. Studied material. China: Tibet: Lasa, on Ganoderma sp., 9 specimens on slides, leg. Weiping Xiong, 27 Mar 2015. Ceratophysella liguladorsi (Lee, 1974) Hypogastrura liguladorsi Lee, 1974: 95. Hypogastrura liguladorsi Lee, 1974: 95. Ceratophysella denticulata (Bagnall, 1941) Achorutes denticulatus Bagnall, 1941: 218. Syn: Achorutes armata var. nov. cuspidate Axelson, 1905: Thibaud et al. 2004. Achorutes denticulatus Bagnall, 1941: 218. Ceratophysella species from mushrooms in China 73 Figure 3. Ceratophysella skarzynskii sp. n. A tibiotarsus and claw III B dens and mucro C abd. V and VI D set of dens chaetae. Scale bars: 0.01 mm. Figure 3. Ceratophysella skarzynskii sp. n. A tibiotarsus and claw III B dens and mucro C abd. V and VI D set of dens chaetae. Scale bars: 0.01 mm. D set of dens chaetae. Scale bars: 0.01 m Hypogastrura liguladorsi Lee, 1974: 95. Studied material. China: Zhejiang: Wuyi, on Lentinus sp., 11 specimens on slides and 80 in alcohol, leg. Xin Sun, 09 Dec 2014. 74 Wanda Maria Weiner et al. / ZooKeys 822: 67–77 (2019) * following the description in Tamura and Zhao 2008 Acknowledgements We would like to express our thanks to Dariusz Skarżyński, Anatoly Babenko, and Louis Deharveng for their valuable and helpful advice. We are also grateful to Taizo Nakamori for collecting C. communis specimens in the type locality. The present study was mainly supported by funding from the National Basic Research Program of Chi na (No. 2016YFA0602303-1), the National Natural Science Foundation of China (No.: 41571052, 41430857, 41811530086), the Science and Technology Develop ment Plan Project of Jilin Province (No. 20160520051JH), the Postdoctoral Science Foundation of China (No. 2015M570281), the funding provided by the Alexander von Humboldt Foundation, the Youth Innovation Promotion Association, CAS, and the academic exchange program between Chinese and Polish Academies of Sciences. Key to the Chinese species of Ceratophysella In 2007 Wu and Yin proposed a key to the six species of the genus Ceratophysella known from China. Jia et al. (2010) proposed a list of 14 Chinese species of Cerato physella. The present key includes 15 species. Some of the species are not sufficiently described, but the available characters given in the descriptions are sufficient to include them in the key. The characters for C. adexilis have been verified on type material, and for C. communis on fresh material from the type locality (Tokyo). 1 Abd. IV with p1 chaeta shorter than p2 chaeta (A-type)....................................2 – Abd. IV with p1 chaeta longer than p2 chaeta (B-type)......................................9 2 Dens with 6 chaetae...........................................................................................3 – Dens with 7chaetae............................................................................................5 3 Unguiculus as long as 1/2–1 of internal edge of claw.........................................4 – Unguiculus very short, as long as 1/3, claw without teeth.....C. zhangi (Zhao, 1998)* 4 Labial palp with 4 papillae (papilla C absent), macrochaetae rather short, Th. II with p4 = s longer thanmacrochaeta p5.......................C. succinea (Gisin, 1949) – Labial palp with 5 papillae (papillae A–E present), macrochaetae long, Th. II with p4 =shorter than macrochaeta p5..........C. taiguensis Jia, Skarżyński & Li, 2010 5 Body chaetae smooth.........................................................................................6 – Body chaetae serrated.........................................................................................8 6 Dens with thickened chaetae..............................................................................7 – Dens without thickened chaetae...............................C. yinae (Yue & Fu, 2000) 7 Four internal chaetae on dens thickened, Ant. IV with 8 dorsal sensilla............... ......................................................................C. baichengensis Wu & Yin, 2007 – Two internal chaetae on dens thickened, Ant. IV with 7 dorsal sensilla................ .......................................................................................C. adexilis Stach, 1964 8 Abd. V tergum with chaeta a2’ present, Ant. IV with simple apical vesicle, Abd. IV tergum with p5=s equal to ½ macrochaetae..........C. denticulata (Bagnall, 1941) – Abd. V tergum without chaeta a2, Ant. IV with bi- or trilobed apical vesicle, Abd IV with chaeta p5=s equal to ¾ macrochaetae.......C. communis (Folsom, 1898) 9 Abd. V tergum with cuticular projection or medial spines................................10 – Abd. V tergum without such projection or spines............................................11 10 Abd. V tergum with medial cuticular projection, dens with 7 normal chaetae, ventral sensory file (sensory rasp) with ca. 40 peg-like sensilla.............................. .................................................................................. C. liguladorsi (Lee, 1974) – Abd. V tergum with chaetae p1 modified in spines, dens with 7 chaetae among which two modified, central sensory file (sensory rasp) with ca. 25–35 peg-like sensilla............................................................... C. Key to the Chinese species of Ceratophysella duplicispinosa (Yosii, 1954) 11 Head with chaetae d5 and sd5 modified into spines.........................................12 – Head without chaetae modified into spines......................................................13 Ceratophysella species from mushrooms in China 75 12 Length of unguiculus as 1/3 of inner edge of claw, tibiotarsi with A1 (tenent hair) short (= ½ of inner edge of claw)......................................................................... .......................................C. xiaoi (Tamura, 1998) (in: Tamura and Zhao 1998) – Length of unguiculus as 1/2 of inner edge of claw, tibiotarsi with A1 (tenent hair) long (= length of inner edge of claw).....C. anshanensis (Wu & Xie, 2007) 13 Head without a pair of cornea-like convexity...................................................14 – Head with a pair of cornea-like convexity.......................C. sinensis Stach, 1964 14 Dens with thickened chaetae, tibiotarsi with prolonged chaeta A1 as pointed tenent hair, claws with one internal tooth and two pairs of lateral teeth............... ...........................................................................................C. skarzynskii sp. n. – Dens without thickened chaetae, tibiotarsi without prolonged chaeta A1, claws without internal and lateral teeth..................... C. flectochaeta Lin & Xia, 1983 References Axelson WM (1905) Einigeneue Collembolen aus Finnland. Zoologischer Anzeiger 28: 788–794. Babenko AB, Chernova N, Potapov MB, Stebaeva SK (1994) Collembola of Russia and adja cent countries: Family Hypogastruridae. Moscow, Nauka, 336 pp. Bagnall RS (1941) Notes on British Collembola. VIII. The Entomology Monthly Magazine 77: 217–226. Bellinger P, Christiansen KA, Janssens F (1996–2018) Checklist of the Collembola of the World. http://www.collembola.org [Date of access: 22.08.2018] Börner C (1932) Apterygota. In: Brohmer P (Ed.) Fauna von Deutschland, Auflage 4. Leipzig, 136–143. Bourgeois A, Cassagnau P (1972) La différentiation du type ceratophysellien chez les Collem boles Hypogastruridae. Nouvelle Revue d’Entomologie 2: 271–291. Carpenter GH, Evans W (1899) Collembola and Thysanura of the Edinburgh District. Pro ceedings of the Royal Physical Society, Edinburgh 14: 221–266. 76 Wanda Maria Weiner et al. / ZooKeys 822: 67–77 (2019) Fjellberg A (1984) Maxillary structures in Hypogastruridae (Collembola). Annales de la Société Royale Zoologique de Belgique 114: 89–99. g g jellberg A (1999) The Labial Palp in Collembola. Zoologischer Anzeiger 237: 309–330. Folsom JW (1898) Japanese Collembola, Part I. Bulletin of the Essex Institute 29: 51–58. https://doi.org/10.5962/bhl.part.14789 Gisin H (1947) Notes taxonomiques sur quelques espèces des genres Hypogastrurab et Xenylla (Collembola). Mitteilungen der Schweizerischen Entomologischen Gesellschaft 20: 341–344. Gisin H (1949) Notes sur les Collemboles avec description de quatorze espèces et d’un genre nouveaux. Mitteilungen der Schweizerischen Entomologischen Gesellschaft 22: 385–410. Gisin H (1949) Notes sur les Collemboles avec description de quatorze espèces et d un genre nouveaux. Mitteilungen der Schweizerischen Entomologischen Gesellschaft 22: 385–410. Jia J, Skarżyński D, Li Y (2010) Ceratophysella taiguensis sp. nov. (Collembola, Hypogastru ridae) from China, with an annotated checklist of Chinese Ceratophysella Börner, 1932. Zootaxa 2644: 57–63. https://doi.org/10.11646/zootaxa.2644.1.4 Lee BH (1974) Étude de la faune Coréenne des Insectes Collemboles II. Description de quatre es pèces nouvelles de la famille Hypogastruridae. Nouvelle Revue d’Entomologie 4(2): 89–102. Lin S, Xia F (1983) A new species of the genus Ceratophysella (Collembola: Hypogastruridae). Acta Entomologica Sinica 26: 426–427. Liu Y, Hou D, Li Z (1998) A checklist of Collembola species from China. Journal of Southwest Agricultural University 20: 125–131. Shen XC (1993) A checklist of insects from Henan. Chinese Agriculture Science and Techno logical Press, 353 pp. Stach J (1963) Materials to the knowledge of Chinese Collembolan Fauna. Acta zoologica cracoviensia 10: 345–372. Stach J (1964) Materials to the knowledge of Chinese Collembolan fauna. Acta zoologicalcra coviensia 9: 1–26. References Tamura H, Zhao L (1998) Three species of Collembola from Yunnan, Southwest China (Ap terygota). Zoological Research 19(2): 153–159. Thibaud JM, Schulz HJ, Gama Assalino MM da (2004) Hypogastruridae. In: Dunger W (Ed.) Synopses on Palaearctic Collembola, Vol. 4. Abhandlungen und Berichte des Naturkunde museums, Görlitz 75(2): 1–287. Tullberg T (1876) Collembola borealia (NordiskaCollembola). Öfversigtaf Kongliga Vetenska ps-Akademiens Förhandlingar 33: 23–42. Wei W (2002) The harm and prevention of Collembola on Stropharia. Guangxi Tropical Agri culture 2: 17. [In Chinese] Wu D, Xie R (2007) New record of the genus Mitchellania Wray from China (Collembola, Hy pogastruridae) with description of a new species. Acta Zootaxonomica Sinica 32: 287–289. Wu D, Yin W (2007) A new species of Ceratophysella Börner, 1932 (Collembola: Hypogastru ridae), and description of Chinese specimens of Ceratophysella succinea Gisin, 1949. The Pan-Pacific Entomologist 83: 255–256. https://doi.org/10.3956/0031-0603-83.3.255 Yosii R (1954) Springschwänze des Ozé-Naturschutzgebietes. Scientific Researches of the Ozegahara Moor, Tokyo, 777–830. Yosii R (1954) Springschwänze des Ozé-Naturschutzgebietes. Scientific Researches of the Ozegahara Moor, Tokyo, 777–830. Yosii R (1956) Monografie zur höhlen collembolen Japans. Contributions from the Biological Laboratory Kyoto University 3: 1–109. Yosii R (1956) Monografie zur höhlen collembolen Japans. Contributions from the Biological Laboratory Kyoto University 3: 1–109. Ceratophysella species from mushrooms in China 77 Yosii R (1960) Studies on the Collembolan genus Hypogastrura. American Middland Naturalist 64: 257–281. https://doi.org/10.2307/2422661 p g Yosii R (1965) On some Collembola of Japan and adjacent countries. Contributions from the Biological Laboratory Kyoto University 19: 1–71. Yue Q, Fu R (2000) New records and new species of fresh water springtails from China (Col lembola). Acta Entomologica Sinica 43: 394–402. Zhao L (1992) Collembola. In: Yin W (Ed) Subtropical soil animals of China. Science Press, Beijing, 414–457. Zhao L, Tamura H, Ke X (1997) Tentative checklist of collembolan species from China (In secta). Publications of Itako Hydrobiological Station 9: 15–40. Zhu F (2012) Occurrence and comprehensive control measures of Collembola on edible fungi. Edible and medicinal mushrooms 20(5): 310–311.
https://openalex.org/W2904179379	https://www.frontiersin.org/articles/10.3389/fmicb.2018.03276/pdf	English	null	Ameliorating the Metabolic Burden of the Co-expression of Secreted Fungal Cellulases in a High Lipid-Accumulating Yarrowia lipolytica Strain by Medium C/N Ratio and a Chemical Chaperone	Frontiers in microbiology	2,019	cc-by	19,018	Keywords: fungal cellulolytic enzymes, Yarrowia lipolytica, cellulosic biofuel, cellobiohydrolase I, endoglucanase II, lipid metabolism, endoplasmic reticulum stress, chemical chaperone Ameliorating the Metabolic Burden of the Co-expression of Secreted Fungal Cellulases in a High Lipid-Accumulating Yarrowia lipolytica Strain by Medium C/N Ratio and a Chemical Chaperone Hui Wei1, Wei Wang1, Hal S. Alper2, Qi Xu1, Eric P. Knoshaug3, Stefanie Van Wychen1,3, Chien-Yuan Lin1, Yonghua Luo1, Stephen R. Decker1, Michael E. Himmel1 and Min Zhang1,3 Hui Wei1, Wei Wang1, Hal S. Alper2, Qi Xu1, Eric P. Knoshaug3, Stefanie Van Wychen1,3, Chien-Yuan Lin1, Yonghua Luo1, Stephen R. Decker1, Michael E. Himmel1 and Min Zhang1,3 Hui Wei1, Wei Wang1, Hal S. Alper2, Qi Xu1, Eric P. Knoshaug3, Stefanie Van Wychen1,3, Chien-Yuan Lin1, Yonghua Luo1, Stephen R. Decker1, Michael E. Himmel1 and Min Zhang1,3 ORIGINAL RESEARCH published: 09 January 2019 doi: 10.3389/fmicb.2018.03276 Reviewed by: Rodrigo Ledesma-Amaro, Imperial College London, United Kingdom Aleksandra Maria Miro ´nczuk, Wrocław University of Environmental and Life Sciences, Poland Reviewed by: Rodrigo Ledesma-Amaro, Imperial College London, United Kingdom Aleksandra Maria Miro ´nczuk, Wrocław University of Environmental and Life Sciences, Poland *Correspondence: Hui Wei Hui.Wei@nrel.gov Min Zhang Min.Zhang@nrel.gov Specialty section: This article was submitted to Systems Microbiology, a section of the journal Frontiers in Microbiology Specialty section: This article was submitted to Systems Microbiology, a section of the journal Frontiers in Microbiology Received: 21 August 2018 Accepted: 17 December 2018 Published: 09 January 2019 1 Biosciences Center, National Renewable Energy Laboratory, Golden, CO, United States, 2 Department of Chemical Engineering, The University of Texas at Austin, Austin, TX, United States, 3 National Bioenergy Center, National Renewable Energy Laboratory, Golden, CO, United States Edited by: Marie-Joelle Virolle, The National Center for Scientiﬁc Research (CNRS), France Edited by: Marie-Joelle Virolle, The National Center for Scientiﬁc Research (CNRS), France Yarrowia lipolytica, known to accumulate lipids intracellularly, lacks the cellulolytic enzymes needed to break down solid biomass directly. This study aimed to evaluate the potential metabolic burden of expressing core cellulolytic enzymes in an engineered high lipid-accumulating strain of Y. lipolytica. Three fungal cellulases, Talaromyces emersonii-Trichoderma reesei chimeric cellobiohydrolase I (chimeric-CBH I), T. reesei cellobiohydrolase II (CBH II), and T. reesei endoglucanase II (EG II) were expressed using three constitutive strong promoters as a single integrative expression block in a recently engineered lipid hyper-accumulating strain of Y. lipolytica (HA1). In yeast extract- peptone-dextrose (YPD) medium, the resulting cellulase co-expressing transformant YL165-1 had the chimeric-CBH I, CBH II, and EG II secretion titers being 26, 17, and 132 mg L−1, respectively. Cellulase co-expression in YL165-1 in culture media with a moderate C/N ratio of ∼4.5 unexpectedly resulted in a nearly two-fold reduction in cellular lipid accumulation compared to the parental control strain, a sign of cellular metabolic drain. Such metabolic drain was ameliorated when grown in media with a high C/N ratio of 59 having a higher glucose utilization rate that led to approximately twofold more cell mass and threefold more lipid production per liter culture compared to parental control strain, suggesting cross-talk between cellulase and lipid production, both of which involve the endoplasmic reticulum (ER). Most importantly, we found that the chemical chaperone, trimethylamine N-oxide dihydride increased glucose utilization, cell mass and total lipid titer in the transformants, suggesting further amelioration of the metabolic drain. This is the ﬁrst study examining lipid production in cellulase-expressing Y. lipolytica strains under various C/N ratio media and with a chemical chaperone highlighting the metabolic complexity for developing robust, cellulolytic and lipogenic yeast strains. Abbreviations: BGL, β-glucosidase; CBH, cellobiohydrolase; DCW, dry cell weight; DGA, diacylglycerolacyltransferase; ER, endoplasmic reticulum; FAME, fatty acid methyl esters; hp4d, hybrid promoter derived from pXPR2; Te, Talaromyces emersonii; TMAO, trimethylamine N-oxide dihydride; Tr, Trichoderma reesei; Te-Tr, Talaromyces emersonii-Trichoderma reesei; XPR2, alkaline extracellular protease 2; YP, yeast extract-peptone (medium); YPD, yeast extract-peptone-dextrose (medium). Promoters, Signal Peptide, and Terminators for Cellulase Expression Promoters were selected to mimic as closely as possible the optimal ratio of CBH I: CBH II: EG II of 60: 10: 30, or to the optimal ratio for CBH I: EG II of 90: 10 to achieve maximal cellulose degradation (Kallioinen et al., 2014; Wei et al., 2014). Thus the promotors TEFin, GPD, and EXP1 with expression levels of approximately 17, 0.8, and 1.2-fold that of TEF (Damude et al., 2011; Tai and Stephanopoulos, 2013) were chosen to control the expression of individual cellulases. Another recent accomplishment is the engineering of Y. lipolytica to achieve a high yield of lipids or other hydrocarbons; see major reviews focused on the metabolic engineering of Y. lipolytica in the past 5 years (Abghari and Chen, 2014; Gonçalves et al., 2014; Zhu and Jackson, 2015; Ledesma- Amaro and Nicaud, 2016; Xie, 2017; Abdel-Mawgoud et al., 2018; Carsanba et al., 2018). Notably, the mutant pex10 mfe1 leu− ura+ DGA1 was generated as a lipid hyper-accumulator (strain HA1), which can accumulate total lipids up to ∼90% on a cell dry-weight basis (Blazeck et al., 2014). p Details for the promoters, signal peptide and terminators for expressing individual cellulases are described as below. Chimeric CBH I was expressed by the TEFin promoter and the XPR2 terminator (GenBank, accession no. M23353) (Davidow et al., 1987). CBH II was expressed by the GPD promoter (YALI0C06369p; -931 to -1; GenBank nucleotide 158467892) and the Lip2 terminator (GenBank accession no. AJ012632). EG II was expressed by the EXP1 promoter and EXP1 terminator (Damude and Zhu, 2007; Ye et al., 2012). Each of three cellulase genes contains an XPR2 signal peptide (AAGCTCGCTACCGCCTTTACTATTCTCACGGCCGTTCTG GCC) and was codon optimized according to the codon optimization table for Y. lipolytica. The cellulase expression cassette (i.e., CBH I-CBH II-EG II cassette; named as construct 162) was synthesized by GenScript Inc. (Piscataway, NJ, United States) with SalI-PmlI sites on the 5′ end and a KpnI site on its 3′ end (see sequence in the additional ﬁle). This expression cassette was cloned into the vector pUC57 at the SalI and EcoRV sites. Strains, Plasmids, and Culture Medium , , Yarrowia lipolytica strains and plasmids used in this study are described in Table 1. The pedigree of transformants expressing either a single or multiple cellulases is illustrated together with an overview of the experimental characterization of these transformants (Figure 1). Yeast strains were maintained at 28◦C on YPD agar medium that contains 10 g L−1 yeast extract, 20 g L−1 peptone, 20 g L−1 dextrose and 15 g L−1 agar. Citation: Wei H, Wang W, Alper HS, Xu Q, Knoshaug EP, Van Wychen S, Lin C-Y, Luo Y, Decker SR, Himmel ME and Zhang M (2019) Ameliorating the Metabolic Burden of the Co-expression of Secreted Fungal Cellulases in a High Lipid-Accumulating Yarrowia lipolytica Strain by Medium C/N Ratio and a Chemical Chaperone. Front. Microbiol. 9:3276. doi: 10.3389/fmicb.2018.03276 January 2019 \| Volume 9 \| Article 3276 1 Frontiers in Microbiology \| www.frontiersin.org Ameliorating Cellulases Expression Metabolic Burden Wei et al. INTRODUCTION abovementioned Y. lipolytica HA1 strain. These fungal enzymes include the Te-Tr chimeric CBH I (Ilmen et al., 2011; Wei et al., 2014), T. reesei CBH II, and T. reesei endoglucanase II (EG II). For simplicity, the co-expression of chimeric CBH I-CBH II- EG II (triplet cassette of cellulases) in this study is generally referred to as CBH I-CBH II-EG II in the text. Secondly, to investigate whether the co-expression of the core cellulolytic enzymes is a metabolic burden to the high lipid-accumulating strain of Y. lipolytica and whether adjusting the C/N ratio of the growth medium and supplementing the medium with a chemical chaperone can alleviate this metabolic stress. This work provides a new level of rigor for CBP strain development in yeast by exploring the relationship between cellulase production and lipid accumulation. As one of the most abundant renewable resources today, lignocellulosic biomass is under development worldwide to produce biofuels and chemicals. One key bottleneck hindering biofuels development is the high cost of conversion of feedstocks to sugars due to the general recalcitrance of plant cell walls (Himmel et al., 2007). To overcome this hurdle, a process strategy of cellulase production, cell wall polymer hydrolysis, and sugar fermentation in a single step termed consolidated bioprocessing (CBP) has been proposed (Lynd et al., 2005). So far, Saccharomyces cerevisiae and Kluyveromyces marxianus (Fan et al., 2012; Olson et al., 2012; Sun et al., 2012; Chang et al., 2013; Yamada et al., 2013; Kricka et al., 2014), and recently Yarrowia lipolytica (Wei et al., 2014; Guo et al., 2017) have been explored as potential CBP microorganisms. MATERIALS AND METHODS g Yarrowia lipolytica has long been known to be oleaginous and recently, has been engineered to utilize xylose for lipid production (Ledesma-Amaro et al., 2016; Li and Alper, 2016; Markham et al., 2016; Rodriguez et al., 2016). The accumulated lipids can be extracted and upgraded for biodiesel production (Ratledge and Wynn, 2002; Sitepu et al., 2014). Because Y. lipolytica lacks the cellulolytic enzymes needed to break down cellulosic biomass directly (Ryu et al., 2015), eﬀorts have been made recently to express cellulases in this yeast, speciﬁcally CBH I, CBH II, endoglucanase II (EG II), β-D-glucosidase, and xylanase (Boonvitthya et al., 2013; Wang et al., 2014a; Wei et al., 2014; Guo et al., 2015). We note that a consortium co-culture of these cellulase transformants demonstrated synergy in utilizing cellulose (Wei et al., 2014). Most recently, progress has been made in co-expressing CBHs, EGs, and BGLs in Y. lipolytica to mimic the ratio of the main cellulases in the secretome of T. reesei. The resultant strains were shown to grow eﬃciently on industrial cellulose pulp, which is mostly amorphous, but limited growth on recalcitrant crystalline cellulose was also noted (Guo et al., 2017). Strains, Plasmids, and Culture Medium Promoters, Signal Peptide, and Terminators for Cellulase Expression The sequence of the complete expression cassette of construct 162 is provided in Supplementary Materials With the overall goal of developing a CBP platform to produce lipids as a drop-in fuel precursor from cellulosic biomass feedstocks, the objectives of this study are twofold: First, to co-express and evaluate the secretion eﬃciency and cellulolytic functionality of fungal CBH I, CBH II, and EG II in the January 2019 \| Volume 9 \| Article 3276 Frontiers in Microbiology \| www.frontiersin.org 2 Ameliorating Cellulases Expression Metabolic Burden Wei et al. TABLE 1 \| Yarrowia lipolytica strains and plasmids. Strain, plasmids, or constructs Description: (strain genotype and phenotype; cloned genes and MW of encoded proteins) Source Parent and derivative strains Po1f Genotype: MatA, leu2-270, ura3-302, xpr2-322, axp-2; Phenotype: Leu-, Ura-, 1AEP, 1AXP, Suc+ Madzak et al., 2000 Po1g Genotype: MatA, leu2-270, ura3-302::URA3, xpr2-332, axp-2; Phenotype: Leu-, 1AEP, 1AXP, Suc+, pBR platform Madzak et al., 2000 YL101 Previously named as Yl (EG II); expressing Tr eg2 (P07982; 42 kDa) with a hybrid promoter (hp4d) in parent strain Po1g. Wei et al., 2014 YL102 Previously named as Yl (CBH II); expressing Tr cbh2 (P07987; 47 kDa) with a hybrid promoter (hp4d) in parent strain Po1g. Wei et al., 2014 YL151 Previously named as Yl (chimeric CBH I); expressing chimeric cbh1 (Te CBH1 catalytic domain-Tr Linker-Tr CBM1; AAL89553 and P62694; 53 kDa) with a hybrid promoter (hp4d) in parent strain Po1g. Wei et al., 2014 HA1 Genotype: Po1f 1pex10 1mfe1 leu−ura+ DGA1; Phenotype: prevent peroxisome biogenesis and β-oxidation by 1pex10 and 1mfe1, respectively; enhance lipid synthesis by DGA1 overexpression. Blazeck et al., 2014 YL165-1 Genotype: Po1f 1pex10 1mfe1 leu−ura+ DGA1 chimeric cbh1 cbh2 eg2; Phenotype: prevent peroxisome biogenesis and β-oxidation by 1pex10 and 1mfe1, respectively; enhance lipid synthesis by DGA1 overexpression; co-express CBH I (with TEFin promoter) – CBH II (with GPD promoter) – EG II (with EXP1 promoter). This study Plasmids and the cloned genes pYLEX1 (i.e., pINA1269) hybrid promoter (hp4d); selection marker gene (LEU2). Madzak et al., 2000 pYLSC1 (i.e., pINA1296) hybrid promoter (hp4d); secretion signal (XPR2 pre-region); selection marker gene (LEU2). Madzak et al., 2000 pNREL151 Chimeric cbh1 (Te CBH1 catalytic domain-Tr Linker-Tr CBM1; AAL89553 and P62694; 53 kDa) in SﬁI/Xbal cut pYLSC1. Wei et al., 2014 pNREL162 Chimeric cbh1-cbh2-eg2 cassette; cloned in vector pUC57. This work pNREL165 Chimeric cbh1-cbh2-eg2 cassette; cloned in vector pYLEX1. Promoters, Signal Peptide, and Terminators for Cellulase Expression This work The theoretical molecular weight (MW) was calculated based on amino acid sequence (without a signal peptide). CBH, cellobiohydrolase; EG, endoglucanase; Te, Talaromyces emersonii; Tr, T. reesei. Description: (strain genotype and phenotype; cloned genes and MW of encoded proteins) Source Chimeric cbh1-cbh2-eg2 cassette; cloned in vector pUC57. Chimeric cbh1-cbh2-eg2 cassette; cloned in vector pYLEX1 oretical molecular weight (MW) was calculated based on amino acid sequence (without a signal peptide). CBH, cellobiohydrol yces emersonii; Tr, T. reesei. FIGURE 1 \| Pedigree of transformants expressing single or multiple cellulases and the experimental outline for transformant characterization. The details for these strains are described in Table 1. CBH, cellobiohydrolase; EG, endoglucanase; FAME, fatty acid methyl esters. FIGURE 1 \| Pedigree of transformants expressing single or multiple cellulases and the experimental outline for transfor strains are described in Table 1. CBH, cellobiohydrolase; EG, endoglucanase; FAME, fatty acid methyl esters. FIGURE 1 \| Pedigree of transformants expressing single or multiple cellulases and the experimental outline for transformant characterization. The details for these strains are described in Table 1. CBH, cellobiohydrolase; EG, endoglucanase; FAME, fatty acid methyl esters. kit (Yeastern Biotech Co., Taipei, Taiwan) as described previously (Wang et al., 2014a). The transformation mixture was spread on YNB selection plates lacking leucine for the appearance of transformant (Leu+) colonies. and Methods. Finally, to build the Y. lipolytica cellulase secretion construct (named as construct 165), construct 162 and vector pYLEX1 were digested with SalI and KpnI, and ligated together (Figures 2A,B). Cellulosic Substrates for Enzymatic Activity Assays, Screening, and Culturing of Transformants Primers were designed for real-time RT-PCR targeting the codon-optimized sequences for individual cbh1, cbh2, and eg2 genes expressed in Y. lipolytica transformants (Supplementary Table S1). Primers for the reference gene encoding actin (YALI0D08272g) were previously described (Dulermo et al., 2015). Real-time RT-PCR was performed using ABI 7500 Real- Time PCR System (Thermo Fisher Scientiﬁc, Applied Biosystems, Grand Island, NY, United States) and Power SYBR Green PCR Master Mix (Cat. no. 4367659, Applied Biosystems, Grand Island, NY, United States). PCR reactions were performed in triplicate. The relative transcription level of genes was calculated from the Ct value of reference gene and gene-of-interest (Schmittgen and Livak, 2008). Two types of cellulosic substrates were used in this study: (1) Phosphoric acid swollen cellulose (PASC) was prepared from Avicel PH101 (cat. no. 11365, Sigma) using the procedure described in literature (Schulein, 1997; Harris et al., 2010). (1) Phosphoric acid swollen cellulose (PASC) was prepared from Avicel PH101 (cat. no. 11365, Sigma) using the procedure described in literature (Schulein, 1997; Harris et al., 2010). (2) Wet ball-dispersed Avicel was prepared by suspending 1% Avicel in 50 mM acetate buﬀer, pH 4.8 in capped Fisherbrand glass bottles containing sterile glass beads with an average diameter of 0.7 mm and shaking overnight at 180 rpm and 25◦C; the volume ratio of 1% Avicel: glass beads was 5:1, with the volume of glass beads being measured by sterile cylinder. Note that proper dispersion of Avicel is important for testing the optimal functionality of cellulases secreted by transformants. Wet ball milling has been reported to decrease the particle size and increase the surface area of cellulose (Ishikawa and Ide, 1993; Kobayashi et al., 2011) and is often used for particle dispersion of various materials (Hussain et al., 1996; Munkhbayar et al., 2013). (2) Wet ball-dispersed Avicel was prepared by suspending 1% Avicel in 50 mM acetate buﬀer, pH 4.8 in capped Fisherbrand glass bottles containing sterile glass beads with an average diameter of 0.7 mm and shaking overnight at 180 rpm and 25◦C; the volume ratio of 1% Avicel: glass beads was 5:1, with the volume of glass beads being measured by sterile cylinder. Note that proper dispersion of Avicel is important for testing the optimal functionality of cellulases secreted by transformants. SDS–PAGE and Western Blot The supernatant from each strain was collected from YPD pH 4.0 medium when the culture reached an OD600 value of 10. The loading amount per well was 22.5 µL mixed with 7.5 µL 4X loading buﬀer, following the procedures described previously for the SDS–PAGE and western blot analyses (Wei et al., 2014), for which the custom mouse monoclonal anti-CBH I, mouse monoclonal anti-CBH II, and rabbit polyclonal anti-EG II were used. Densitometric analysis of the detected bands was performed in accordance with literature (Savina et al., 2002; Olszanecki et al., 2007; Rashid et al., 2015) using the Quantity One software (Bio- Rad, CA, United States). The band intensities, relative to the corresponding bands in the protein samples of strains expressing Transformation and Selection Symbols and abbreviations: rep (pMB1), origin of replication derived from plasmid pMB1; t, terminator; ∗, ∗∗, and ∗∗∗ indicate the relatively low, middle and high expression levels of promoters, respectively. FIGURE 2 \| Plasmid construction for cellulase expression in Yarrowia lipolytica. (A) Plasmid construct 162 with CBH I-CBH II-EG II cloned in vector pUC57. (B) Plasmid construct 165 with CBH I-CBH II-EG II cloned in the Y. lipolytica expression vector pYLEX1, which carries Leu2 as a selection marker. More details are described in the Materials and Methods. Symbols and abbreviations: rep (pMB1), origin of replication derived from plasmid pMB1; t, terminator; ∗, ∗∗, and ∗∗∗ indicate the relatively low, middle and high expression levels of promoters, respectively. individual cellulases, are presented as the average values from three separate experiments. individual cellulases, are presented as the average values from three separate experiments. similar to the protocol described by Wei and coworkers (Wei et al., 2012). One microgram of puriﬁed total RNA was reverse- transcribed using High-Capacity cDNA Reverse Transcription Kit (Cat. no. 4368814, Applied Biosystems, Grand Island, NY, United States) with random hexamers according to the manufacturer’s instructions. Cellulosic Substrates for Enzymatic Activity Assays, Screening, and Culturing of Transformants Wet ball milling has been reported to decrease the particle size and increase the surface area of cellulose (Ishikawa and Ide, 1993; Kobayashi et al., 2011) and is often used for particle dispersion of various materials (Hussain et al., 1996; Munkhbayar et al., 2013). Transformation and Selection Total RNA was extracted from 60 to 80 mg (wet weight) cell pellets of transformants using Qiagen RNeasy Mini Kit (Valencia, CA, United States). The procedure for cDNA synthesis was Random integrative transformation of Y. lipolytica strain HA1 with NotI-linearized plasmid 165 DNA was conducted using YLOS One Step Transformation system and the YLEX expression January 2019 \| Volume 9 \| Article 3276 Frontiers in Microbiology \| www.frontiersin.org 3 Ameliorating Cellulases Expression Metabolic Burden Wei et al. FIGURE 2 \| Plasmid construction for cellulase expression in Yarrowia lipolytica. (A) Plasmid construct 162 with CBH I-CBH II-EG II cloned in vector pUC57. (B) Plasmid construct 165 with CBH I-CBH II-EG II cloned in the Y. lipolytica expression vector pYLEX1, which carries Leu2 as a selection marker. More details are described in the Materials and Methods. Symbols and abbreviations: rep (pMB1), origin of replication derived from plasmid pMB1; t, terminator; ∗, ∗∗, and ∗∗∗ indicate the relatively low, middle and high expression levels of promoters, respectively. FIGURE 2 \| Plasmid construction for cellulase expression in Yarrowia lipolytica. (A) Plasmid construct 162 with CBH I-CBH II-EG II cloned in vector pUC57. (B) Plasmid construct 165 with CBH I-CBH II-EG II cloned in the Y. lipolytica expression vector pYLEX1, which carries Leu2 as a selection marker. More details are described in the Materials and Methods. Symbols and abbreviations: rep (pMB1), origin of replication derived from plasmid pMB1; t, terminator; ∗, ∗∗, and ∗∗∗ indicate the relatively low, middle and high expression levels of promoters, respectively. FIGURE 2 \| Plasmid construction for cellulase expression in Yarrowia lipolytica. (A) Plasmid construct 162 with CBH I-CBH II-EG II cloned in vector pUC57. (B) Plasmid construct 165 with CBH I-CBH II-EG II cloned in the Y. lipolytica expression vector pYLEX1, which carries Leu2 as a selection marker. More details are described in the Materials and Methods. Symbols and abbreviations: rep (pMB1), origin of replication derived from plasmid pMB1; t, terminator; ∗, ∗∗, and ∗∗∗ indicate the relatively low, middle and high expression levels of promoters, respectively. FIGURE 2 \| Plasmid construction for cellulase expression in Yarrowia lipolytica. (A) Plasmid construct 162 with CBH I-CBH II-EG II cloned in vector pUC57. (B) Plasmid construct 165 with CBH I-CBH II-EG II cloned in the Y. lipolytica expression vector pYLEX1, which carries Leu2 as a selection marker. More details are described in the Materials and Methods. Screening CBH I-CBH II-EG II Co-expressing Transformants on YPD-PASC Plates The capacity of transformants to utilize cellulose was assessed by clearing zones on 0.5% PASC-YPD agar plates (0.5% PASC, Frontiers in Microbiology \| www.frontiersin.org January 2019 \| Volume 9 \| Article 3276 4 Ameliorating Cellulases Expression Metabolic Burden Wei et al. 2.7 g Avicel (i.e., 2.7% w/v), as well as with BGL (Aspergillus niger BGL: Cat. no. E-BGLUC, Megazyme, International Ireland Ltd., Wicklow, Ireland), at a concentration of 2 mg BGL per gram cellulose substrate based on the literature (Selig et al., 2014; Wei et al., 2014; Xu et al., 2017). The BGL used was chromatographically puriﬁed prior to use. The ﬂasks that contained the cell-medium-Avicel-BGL mixtures were incubated in a rotary shaker at 200 rpm and 28◦C for 5 days. The cells were centrifuged, freeze-dried, and subjected to Avicel residue analysis, as described below. Three biological cultures were run for the cell mixtures. 1.0% yeast extract, 2.0% peptone, 2.0% dextrose, 1.5% agar). The plates were inoculated with strains and then incubated at 28◦C for 6 days before Congo Red staining (Wood and Mahalingeshwara Bhat, 1988). Cellulose Utilization by Cellulase Co-expressing Transformants in Mineral Medium Growth Curves and Validation Growth curves were obtained by using a Bioscreen C analyzer (Growth Curves United States, Piscataway, NJ, United States) and a modiﬁed protocol for cell inoculation, growth conditions, and turbidity measurements (Franden et al., 2013; Wang et al., 2014b). In brief, log phase cultures of Y. lipolytica strains were used to inoculate 20 mL YPD pH 4.0 medium (as a cellulase production medium), which had an initial C/N ratio of approximately 4.5 (Martinez-Force and Benitez, 1995; Josefsen et al., 2012), in 150 mL ﬂask for overnight growth at 30◦C and 210 rpm. After overnight growth, the culture reached an OD600 ∼10. Cells were then diluted into fresh YPD pH 4.0 medium at an initial OD600 of 0.25 and distributed into Bioscreen C microplates (three wells per cell line; 300 µL per well). Incubation for the Bioscreen C microplates was performed at 30◦C for 5 days, with absorbance readings taken every 15 min. The turbidity measurement with a wide band ﬁlter (420–580 nm, which is relatively insensitive to color changes) were computer operated with EZ Experiment software. The collected data were exported to spreadsheets of Microsoft Excel, and the turbidity data was averaged from three replicates of cell samples. Basal mineral media have been used to assess the cellulose utilization by ﬁlamentous fungi and yeast (Wei et al., 2013, 2014; Guo et al., 2017). The mineral medium described by Guo et al. (Guo et al., 2017) contained higher essential nutrients, and was used in this study. In addition to CBH and EG, functionally potent BGL is also needed for the conversion of cellulose to glucose (Dashtban and Qin, 2012). In general, Y. lipolytica is viewed as limited in endogenous ability to digest cellobiose, with some cellobiose-consuming wild-type, or substrate-adapted strains being reported (Kurtzman et al., 2011; Mirbagheri et al., 2012; Lane et al., 2015; Ryu et al., 2016). A strategy of adding exogenous BGL was used in previous studies to boost the low BGL activity in T. reesei’s cellulase preparations (Xin et al., 1993; Berlin et al., 2007; Chen et al., 2008). This study follows these previous examples by adding exogenous BGL to the medium so that BGL would not be a limiting factor for cellulose degradation. Brieﬂy, seed cell culture was grown in 20 mL YPD medium in a 125-mL baﬄed ﬂask overnight at 28◦C, followed by centrifugation for cell collection. Enzyme Activity Assay The combined enzymatic activity of co-expressed CBH I-CBH II-EG II was measured by using unconcentrated or concentrated supernatants of yeast cell cultures. The supernatants were collected from YPD cultures after 5 days shaking at 30◦C, 200 rpm. The concentrated crude enzymes were prepared by concentrating 35-fold using ultraﬁltration with a molecular weight cut-oﬀof 10,000-dalton. For enzyme activity measurements, 0.5 mL of the supernatants or concentrated crude enzyme solutions was mixed with 0.5 mL of 1% Avicel suspended in 50 mM acetate buﬀer at pH 4.8. As controls, 0.5 mL of the concentrated crude enzyme was mixed with 0.5 mL acetate buﬀer without the substrate; in parallel, Avicel without enzyme (using ddH2O instead) was also set as controls. The replicate vials containing enzyme-Avicel mixtures or controls were incubated at 50◦C for 1 h, 24 h, and 5 days. The samples were centrifuged at 12,000 rpm for 3 min and the supernatant was ﬁltered through a 0.45-µm ﬁlter. The released sugars were measured by high-performance liquid chromatography (HPLC). The “% Avicel to glucose” conversion rate was calculated as the measured “Total glucose equivalent released g L−1” divided by 5 g L−1, which was the amount of total glucose equivalent released for theoretical 100% Avicel conversion. Quantiﬁcation of Avicel Residues and Cell Weight in Avicel-Yeast Cell Pellets Ce e g ce eas Ce e e s The determination of Avicel residues and cell dry weight was conducted as previously described (Wei et al., 2014; Guo et al., 2017). Brieﬂy, the Avicel-yeast cells from the transformant culture growing on Avicel were centrifuged, washed with ddH2O, freeze- dried, and weighed. The amount of Avicel contained in the pellet was determined by enzymatic digestions using the Cellic CTec2 cellulase enzyme product (Novozyme, Franklin, NC, United States) and cross-checked by diluted acid (2.5% sulfuric acid) hydrolysis of the residues with at 121◦C for 1 h. The total glucose released was measured by HPLC and taken as the corresponding amount of Avicel contained in the Avicel-yeast cell mix. Cell dry weight was calculated by subtracting the amount of Avicel from the weight of Avicel-cell yeast pellet. Glucose Consumption and Lipid Accumulation in Moderate and High C/N Media Two types of media were used to grow the cellulase co-expressing transformants for investigating their glucose consumption and lipid accumulation. One was a moderate C/N ratio medium prepared from the YPD medium supplemented with 10 g L−1 extra glucose (with the ﬁnal glucose of 30 g L−1, thus referred as YPD-3% Glu), with unadjusted pH and a C/N ratio > 4.5 (Martinez-Force and Benitez, 1995; Josefsen et al., 2012). Another was a high C/N ratio medium adapted from literature (Blazeck et al., 2014), which contained 80 g L−1 glucose, 6.7 g L−1 Yeast Nitrogen Base w/o amino acids (containing 5 g L−1 ammonium sulfate, which corresponds to 1.365 g L−1 ammonium, or approximately 38 mM nitrogen), and 0.79 g L−1 CSM supplement (Cat. no. 114500012; MP Biomedicals). The C/N ratio (g/g) of this high C/N ratio medium was approximately 59, with a pH value of 4.7. Both media were sterilized by 0.2-µm ﬁltration. Approximately 2000 Leu+ transformants were recovered on YNB plates lacking leucine. Since the cells we used for transformation were not synchronized, they likely were in diﬀerent stages of their cell cycle. In addition, random insertion-associated positional eﬀects have the potential to lead to a range of size variation among the resulted positive colonies of transformants on the selection plates. Out of these transformants, 20 visually larger colonies were selected as, in general, the large colonies are more likely to be stable transformants from transformation of Y. lipolytica (Orr-Weaver and Szostak, 1983) and colony size has been used as a proxy for ﬁtness in yeast and other microorganisms (Baryshnikova et al., 2010; Wagih et al., 2013). These 20 transformants were further narrowed down to eight transformants based on their higher OD600 values in YNB liquid medium after overnight growth. The single-colony puriﬁed transformants were tested for their ability to hydrolyze cellulose by growth on PASC-YPD agar for 6 days followed by Congo Red staining. The results showed that ﬁve out of the eight transformants (YL165-1, YL165-5, YL165-6, YL165-7, and YL165-8) produced relatively large clearance zones on the PASC-YPD plates after Congo Red staining (Supplementary Figures S1A,B). These transformants were conﬁrmed to express CBH I-CBH II-EG II by real-time RT-PCR (Supplementary Figure S2). Treatment of Yeast Cells With a Chemical Chaperone It is noteworthy that in this study, two colony sizes appeared on the selection plates: large colonies appeared at day 3 after plating while smaller became visible at day 5 to 6 after plating. In addition to the picking of 20 larger colonies as described above, we also picked 20 of the later smaller colonies. However, further culturing of the smaller colonies indicated that all of them were false positive as they did not grow after re-streaking on selection plates, which was not an unusual phenomenon in fungal and yeast transformation caused by transient expression or unstable insertion of target genes and marker into the host genome (Singh A. et al., 2015; Schwarzhans et al., 2016). The chemical chaperone, trimethylamine N-oxide dihydride (TMAO; Cat. no T0514, Sigma), was used to examine its eﬀect on the cell growth and lipid accumulation of Y. lipolytica control strain and the cellulase-expressing transformants. A stock solution of 3 M TMAO was prepared by dissolving the chemical in the culture medium and sterilized by using 0.22 µm ﬁlter. y g Brieﬂy, an overnight growth of transformants and the parent control strains grown in YPD was used to inoculate 50 mL of high C/N ratio medium in 250-mL baﬄed ﬂasks to reach an initial OD of 0.25. The ﬂasks were incubated in a rotary shaker at 200 rpm and 28◦C for 4 days, followed by adding 2.63 mL of sterile 3 M TMAO stock solution to reach a working concentration of 150 mM. The ﬂasks were incubated in a rotary shaker at 200 rpm and 28◦C for another 2 days, then centrifuged for cell harvest. The dose and duration of the chemical chaperone treatment were based on literature (Thanonkeo et al., 2007; Sootsuwan et al., 2013; Lamont and Sargent, 2017). Untreated parallel cultures (without TMAO) were used as controls. The cell pellets were freeze-dried and subjected to FAME analysis, as described previously (Wei et al., 2013). Co-expression of CBH I-CBH II-EG II in Y. lipolytica HA1 Prior to the present work, the enzymes chimeric CBH I, CBH II, and EG II had been expressed in Y. lipolytica individually (Wei et al., 2014). To co-express these three enzymes, plasmid construct 165 was built in the backbone of pYLEX1 vector, and the resultant plasmid was named as pYLEX1-CBH I-CBH II- EG II with LEU2 as selection marker (Figure 2). The plasmid construct 165 was linearized with NotI and transformed into Y. lipolytica HA1. Glucose Consumption and Lipid Accumulation in Moderate and High C/N Media Brieﬂy, an overnight growth of transformants and the parent control strains grown in YPD medium was used to inoculate 50 mL of either YPD-3% Glu medium or the high C/N ratio medium in 250-mL baﬄed ﬂasks to reach an initial OD of 0.25. The ﬂasks were incubated in a rotary shaker at 200 rpm and 28◦C for 6 days, followed by centrifugation for cell harvest. The cell pellets were freeze-dried and subjected to FAME analysis, as described previously (Wei et al., 2013). Cellulose Utilization by Cellulase Co-expressing Transformants in Mineral Medium The cells were washed with sterile ddH2O, and used to inoculate 100 mL of mineral medium in 500-mL baﬄed ﬂasks to reach an initial OD of 1.6 (equivalent to approximately 1 g DCW L−1; DCW basis), which was comparable to the inoculation rate reported in literature (Guo et al., 2017). The mineral medium was supplemented with Basal mineral media have been used to assess the cellulose utilization by ﬁlamentous fungi and yeast (Wei et al., 2013, 2014; Guo et al., 2017). The mineral medium described by Guo et al. (Guo et al., 2017) contained higher essential nutrients, and was used in this study. In addition to CBH and EG, functionally potent BGL is also needed for the conversion of cellulose to glucose (Dashtban and Qin, 2012). In general, Y. lipolytica is viewed as limited in endogenous ability to digest cellobiose, with some cellobiose-consuming wild-type, or substrate-adapted strains being reported (Kurtzman et al., 2011; Mirbagheri et al., 2012; Lane et al., 2015; Ryu et al., 2016). A strategy of adding exogenous BGL was used in previous studies to boost the low BGL activity in T. reesei’s cellulase preparations (Xin et al., 1993; Berlin et al., 2007; Chen et al., 2008). This study follows these previous examples by adding exogenous BGL to the medium so that BGL would not be a limiting factor for cellulose degradation. Brieﬂy, seed cell culture was grown in 20 mL YPD medium in a 125-mL baﬄed ﬂask overnight at 28◦C, followed by centrifugation for cell collection. The cells were washed with sterile ddH2O, and used to inoculate 100 mL of mineral medium in 500-mL baﬄed ﬂasks to reach an initial OD of 1.6 (equivalent to approximately 1 g DCW L−1; DCW basis), which was comparable to the inoculation rate reported in literature (Guo et al., 2017). The mineral medium was supplemented with Cell mass dry weight measurements in shake ﬂask cultures were used to validate the growth curves generated using the Bioscreen C analyzer. Overnight cultures were used to inoculate 200 mL YPD pH 4.0 medium in 1000-mL baﬄed ﬂask to reach an initial OD600 of 0.25. RESULTS AND DISCUSSION 120 h and centrifuged at 4000 g for 5 min. The collected cell pellets were washed three times with sterile distilled-deionized water (ddH2O) to remove medium residues and collected by centrifugation at 4000 g for 5 min. The cell pellets obtained were freeze dried and weighed. Cell weight data were averaged from three replicate samples. Frontiers in Microbiology \| www.frontiersin.org Cellulose Utilization by Cellulase Co-expressing Transformants in Mineral Medium The ﬂasks were incubated in a rotary shaker at 200 rpm and 30◦C for 5 days, during which ten milliliters of the cell cultures were collected after 6, 12, 24, 48, 72, 96, and January 2019 \| Volume 9 \| Article 3276 Frontiers in Microbiology \| www.frontiersin.org 5 Wei et al. Ameliorating Cellulases Expression Metabolic Burden Comparing Cellulase Levels in YL165-1 vs. Single Cellulase Expressing Strains supernatants of transformants of YL165-1, YL165-5, and YL165- 7, the enzyme activities (expressed as released sugars) were only measurable after 24 h and 5 days of hydrolysis, with up to 11% of Avicel being converted to cellobiose and glucose (Table 2, columns 3–4). For transformants YL165-6 and YL165-8, the enzyme activities of their unconcentrated supernatants were the lowest, only converting 3–4% of Avicel to cellobiose and glucose after 5 days of incubation with the Avicel substrates. Thus, these transformants were eliminated from further testing of their supernatants for enzymatic analysis. vs. Single Cellulase Expressing Strains The best performing transformant from those described above in converting Avicel was YL165-1, which was further subjected to detailed analysis for the co-expressed cellulases. Strains YL151, YL102, and YL101, which were previously generated to express individual chimeric CBH I, CBH II, and EG II cellulases (Wei et al., 2014), were used as reference (see Figure 1 for the strain pedigree). Experimentally, these strains were arranged into three subsets and cultured in parallel in YPD media for 5 days. The supernatants were collected from the cultures when the OD600 value reached 10, followed by PAGE and western blot analyses (Figures 3A–C). With the above low percent conversion in the culture supernatants and to better understand the potential enzymatic activity present in these culture supernatants, the culture supernatants of YL165-1, YL165-5, and YL165-7 were concentrated 35×. In the concentrated supernatants, signiﬁcant conversion of Avicel, 13% to 31%, was observed after 1 h. Conversion increased to 56 and 69% for transformant YL165-1 and 50% and 66% for YL165-5 after 24 h and 5 days, respectively (Table 2, columns 5–6), indicating that 35x concentrated cellulases can signiﬁcantly enhance the degradation of cellulose. A western blot using anti-Tr CBH I antibody, which recognizes the T. reesei CBM and linker in the chimeric CBH I, detected a single band with the expected size for chimeric CBH I (Figure 3A, middle panel, lane 4), conﬁrming a successful expression of chimeric CBH I in transformant YL165-1, with a protein titer being 0.8-fold of that for transformant YL151 expressing the single chimeric CBH I (Figure 3A, bottom panel, lanes 4 vs. 2′). Data presented were the average of three biological replicates and the SEM (standard error of the mean) was less than 10%. Glu equiv., glucose equivalent. HA1 (EV), parent control strain HA1 transformed with empty vector. Enzyme Activity Screening of Transformants Co-expressing CBH I-CBH II-EG II Experimentally, these strains were arranged into three subsets and cultured in parallel in YPD media for 5 days. The supernatants were collected from the cultures when the OD600 value reached 10, followed by PAGE and western blot analyses (Figures 3A–C). A western blot using anti-Tr CBH I antibody, which recognizes the T. reesei CBM and linker in the chimeric CBH I, detected a single band with the expected size for chimeric CBH I (Figure 3A, middle panel, lane 4), conﬁrming a successful expression of chimeric CBH I in transformant YL165-1, with a protein titer being 0.8-fold of that for transformant YL151 expressing the single chimeric CBH I (Figure 3A, bottom panel, lanes 4 vs. 2′). The western blot using anti-CBH II antibody also showed a single band of the size expected for CBH II (Figure 3B, middle panel, lane 4), indicating the expression of CBH II in transformant YL165-1, with a titer being 0.7-fold of that for transformant YL102 expressing single CBH II (Figure 3B, bottom panel, lanes 4 vs. 2′′). The western blot using anti-EG II antibody detected a single band with the expected size for EG II (Figure 3C, middle panel, lane 4), validating the successful expression of EG II in transformant YL165-1, with a titer being 3.3-fold of that in transformant a presented were the average of three biological replicates and the SEM (standard error of the mean) was less than 10%. Glu equ ent control strain HA1 transformed with empty vector. Enzyme Activity Screening of Transformants Co-expressing CBH I-CBH II-EG II Further investigation was conducted to measure the enzyme activity of the culture supernatant or concentrated crude enzymes using wet ball-dispersed Avicel as a substrate. Enzymatic conversion of Avicel to cellobiose and glucose was measured after 1 h, 24 h, and 5 days digestion. For unconcentrated January 2019 \| Volume 9 \| Article 3276 Frontiers in Microbiology \| www.frontiersin.org 6 Ameliorating Cellulases Expression Metabolic Burden Wei et al. supernatants of transformants of YL165-1, YL165-5, and YL165- 7, the enzyme activities (expressed as released sugars) were only measurable after 24 h and 5 days of hydrolysis, with up to 11% of Avicel being converted to cellobiose and glucose (Table 2, columns 3–4). For transformants YL165-6 and YL165-8, the enzyme activities of their unconcentrated supernatants were the lowest, only converting 3–4% of Avicel to cellobiose and glucose after 5 days of incubation with the Avicel substrates. Thus, these transformants were eliminated from further testing of their supernatants for enzymatic analysis. With the above low percent conversion in the culture supernatants and to better understand the potential enzymatic activity present in these culture supernatants, the culture supernatants of YL165-1, YL165-5, and YL165-7 were concentrated 35×. In the concentrated supernatants, signiﬁcant conversion of Avicel, 13% to 31%, was observed after 1 h. Conversion increased to 56 and 69% for transformant YL165-1 and 50% and 66% for YL165-5 after 24 h and 5 days, respectively (Table 2, columns 5–6), indicating that 35x concentrated cellulases can signiﬁcantly enhance the degradation of cellulose. Microscopic imaging analysis of the Avicel-crude enzyme mixture after 5 days incubation with 35x concentrated crude enzymes of transformant YL165-1 conﬁrmed the eﬀects of cellulases on the size of Avicel particles. Compared to Avicel granules incubated with no enzymes, the particles of Avicel incubated with 35x concentrated crude enzymes of transformant YL165-1 were found to be much ﬁner (Supplementary Figure S3), conﬁrming deconstruction of the crystalline cellulose particles. Comparing Cellulase Levels in YL165-1 vs. Single Cellulase Expressing Strains The best performing transformant from those described above in converting Avicel was YL165-1, which was further subjected to detailed analysis for the co-expressed cellulases. Strains YL151, YL102, and YL101, which were previously generated to express individual chimeric CBH I, CBH II, and EG II cellulases (Wei et al., 2014), were used as reference (see Figure 1 for the strain pedigree). Comparing Cellulase Levels in YL165-1 vs. Single Cellulase Expressing Strains The western blot using anti-CBH II antibody also showed a single band of the size expected for CBH II (Figure 3B, middle panel, lane 4), indicating the expression of CBH II in transformant YL165-1, with a titer being 0.7-fold of that for transformant YL102 expressing single CBH II (Figure 3B, bottom panel, lanes 4 vs. 2′′). The western blot using anti-EG II antibody detected a single band with the expected size for EG II (Figure 3C, middle panel, lane 4), validating the successful expression of EG II in transformant YL165-1, with a titer being 3.3-fold of that in transformant Microscopic imaging analysis of the Avicel-crude enzyme mixture after 5 days incubation with 35x concentrated crude enzymes of transformant YL165-1 conﬁrmed the eﬀects of cellulases on the size of Avicel particles. Compared to Avicel granules incubated with no enzymes, the particles of Avicel incubated with 35x concentrated crude enzymes of transformant YL165-1 were found to be much ﬁner (Supplementary Figure S3), conﬁrming deconstruction of the crystalline cellulose particles. TABLE 2 \| Enzyme activity of supernatant or concentrated crude enzymes of transformants co-expressing CBH I-CBH II-EG II. Enzyme-Avicel incubation time (50◦C) Y. lipolytica strain 1× supernatant enzyme activity 35× concentrated crude enzyme activity Total Glu equiv. released g L−1 % Avicel to Glu equiv. Total Glu equiv. released g L−1 % Avicel to Glu equiv. 0 h HA1 (EV) 0 0 0 0 YL165-1 0 0 0 0 YL165-5 0 0 0 0 YL165-7 0 0 0 0 1 h HA1 (EV) 0 0 0 1% YL165-1 0 0 1.56 31% YL165-5 0 0 1.19 24% YL165-7 0 0 0.63 13% 24 h HA1 (EV) 0 0 0.04 1% YL165-1 0.30 6% 2.78 56% YL165-5 0.24 5% 2.48 50% YL165-7 0.14 3% 1.34 27% 5 days HA1 (EV) 0.00 0% 0.11 2% YL165-1 0.53 11% 3.45 69% YL165-5 0.50 10% 3.28 66% YL165-7 0.26 5% 1.55 31% Data presented were the average of three biological replicates and the SEM (standard error of the mean) was less than 10%. Glu equiv., glucose equivalent. HA1 (EV), parent control strain HA1 transformed with empty vector. January 2019 \| Volume 9 \| Article 3276 Frontiers in Microbiology \| www.frontiersin.org 7 Ameliorating Cellulases Expression Metabolic Burden Wei et al. FIGURE 3 \| Comparison of cellulase secretion levels between CBH I-CBH II-EG II co-expressing Y. lipolytica transformant YL165-1 and the individual, single cellulase expressing transformants. Comparing Cellulase Levels in YL165-1 vs. Single Cellulase Expressing Strains (A) Supernatant samples for western blot using anti-Tr CBH I antibody. (B) Supernatant samples for western blot using anti-CBH II antibody. (C) Supernatant samples for western blot using anti-EG II antibody. In (A–C), the upper panels show SDS–PAGE gels after staining, and the middle panels show the identically loaded gels used for the western blot, while the bottom panels show the densitometric analysis of the western blots, for which error bars indicate the SEM for three biological replicates; ∗and ∗∗indicate signiﬁcantly different from the reference strains (that expressing single CBH I, CBH II, or EG II) with p < 0.05 and p < 0.01, respectively. Lane 1, strain Po1g (transformed with empty vector) as the parent strain control for YL151, YL102, and YL101. Lane 2′, YL151 expressing chimeric CBH I; lane 2′′, YL102 expressing CBH II; lane 2′′′, YL101 expressing EG II. Lane 3, strain HA1 (transformed with empty vector). Lane 4, YL165-1. Loading amount was 22.5 µL supernatant per well. Cellulase titers (g L-1) are indicated by numbers in the western blot images. FIGURE 3 \| Comparison of cellulase secretion levels between CBH I-CBH II-EG II co-expressing Y. lipolytica transformant YL165-1 and the individual, single cellulase expressing transformants. (A) Supernatant samples for western blot using anti-Tr CBH I antibody. (B) Supernatant samples for western blot using anti-CBH II antibody. (C) Supernatant samples for western blot using anti-EG II antibody. In (A–C), the upper panels show SDS–PAGE gels after staining, and the middle panels show the identically loaded gels used for the western blot, while the bottom panels show the densitometric analysis of the western blots, for which error bars indicate the SEM for three biological replicates; ∗and ∗∗indicate signiﬁcantly different from the reference strains (that expressing single CBH I, CBH II, or EG II) with p < 0.05 and p < 0.01, respectively. Lane 1, strain Po1g (transformed with empty vector) as the parent strain control for YL151, YL102, and YL101. Lane 2′, YL151 expressing chimeric CBH I; lane 2′′, YL102 expressing CBH II; lane 2′′′, YL101 expressing EG II. Lane 3, strain HA1 (transformed with empty vector). Lane 4, YL165-1. Loading amount was 22.5 µL supernatant per well. Cellulase titers (g L-1) are indicated by numbers in the western blot images. YL101 expressing single EG II (Figure 3C, bottom panel, lanes 4 vs. 2′′′). diﬀerent western blot bands in their respective lanes in Figure 3A. Impacts of Cellulase Expression on the Growth of Transformants in YPD Medium An optimal ratio of CBH I, CBH II, and EG II is crucial for an eﬃcient conversion of cellulose to simple sugars. This study achieved a high titer for EG II but a moderate titer for chimeric CBH I, which indicates that further eﬀorts are needed to boost the expression level of CBH I for an optimal CBH I and EG II ratio. CBH I proteins from diﬀerent fungal species vary in their expression levels and speciﬁc activity when expressed in the yeast S. cerevisiae (Ilmen et al., 2011) and Y. lipolytica (Wei et al., 2014; Guo et al., 2017). While the Te-Tr chimeric CBH I showed a likely intrinsic property for its expression limitation (26 mg L−1 under the control of TEFin promoter, which is a TEF promoter combined with its intron) in this study, a higher expression level of CBH I from Neurospora crassa (24 and 95 mg L−1 under control of TEF and a hybrid promoter HTEF, respectively) (Guo et al., 2017) raises hope for further tuning the ratio of CBH I and EG II by including N. crassa CBH I into this platform in future studies. The eﬀects of simultaneous expression of multiple cellulases on the growth of Y. lipolytica was investigated by plotting the turbidity obtained from a Bioscreen C growth assay. Previously, the Bioscreen C instrument has been used to monitor microbial growth curve in terms of optical density of bacteria (Franden et al., 2009, 2013; Wang et al., 2014b) and yeast (Bom et al., 2001; Jung et al., 2015; Gientka et al., 2016). Y. lipolytica transformants expressing single cellulases were similar to their parent strain Po1g (EV) during the exponential growth phase and only show slight diﬀerences during the late growth phase (Figure 5). The growth curve of YL165-1 was similar to its parent control strain HA1 (EV) during exponential growth and was only 11% lower in optical density during the late growth phase (p < 0.05). Meanwhile, there was no signiﬁcant diﬀerence between YL165-1 and HA1 (EV) in the mean size of cells based on the measurement by the Cellometer Vision (Nexcelom Bioscience, Lawrence, MA, United States). These data indicate that the co-expression of CBH I-CBH II-EG II comes at a small cost for the cells in terms of slightly reduced growth rate in acquiring the capacity to degrade and utilize cellulosic substrates. Comparing Cellulase Levels in YL165-1 vs. Single Cellulase Expressing Strains It is noteworthy that recombinant CBH I enzymes in yeast were reported to exhibit variable levels of glycosylation, for which hyper-glycosylation leads to smeared bands in SDS–PAGE imaging (Godbole et al., 1999; Boer et al., 2000; Den Haan et al., 2007; Ilmen et al., 2011). The smeared band pattern of TeTrCBH I in YL151 (Figure 3A) was consistent with our observation in another recent study, which showed that when TeTrCBH I was expressed in the yeast L. starkeyi, it had a relatively high magnitude of glycosylation that led to apparently smeared band of puriﬁed TeTrCBH I band by SDS–PAGE analysis (Xu et al., 2017). Our observation here further showed that the TeTrCBH I co-expressed in YL165-1 vs. YL151 had diﬀerent extents of glycosylation, as reﬂected by the Low Correlation Between Transcriptional and Protein Levels of Cellulase Genes: Intrinsic Nature of Individual Cellulases Affecting Their Co-expression Ratio The above data permitted a quantitation of the co-expressed cellulases in YL165-1. Previously, the titers of single chimeric CBH I, CBH II, and EG II proteins expressed were estimated to be 32, 24, and 40 mg L−1 in strains YL151, YL102, and YL101, respectively, in ﬂask cultures (Wei et al., 2014). Accordingly, the titers of co-expressed CBH I, CBH II, and EG II in YL165-1 can be calculated by multiplying each single protein’s titer (in YL151, Low Correlation Between Transcriptional and Protein Levels of Cellulase Genes: Intrinsic Nature of Individual Cellulases Affecting Their Co-expression Ratio g p The above data permitted a quantitation of the co-expressed cellulases in YL165-1. Previously, the titers of single chimeric CBH I, CBH II, and EG II proteins expressed were estimated to be 32, 24, and 40 mg L−1 in strains YL151, YL102, and YL101, respectively, in ﬂask cultures (Wei et al., 2014). Accordingly, the titers of co-expressed CBH I, CBH II, and EG II in YL165-1 can be calculated by multiplying each single protein’s titer (in YL151, January 2019 \| Volume 9 \| Article 3276 Frontiers in Microbiology \| www.frontiersin.org 8 Ameliorating Cellulases Expression Metabolic Burden Wei et al. YL102, and YL101) with its respective densitometric fold-changes (Figures 3A–C, bottom panels). This gives a protein titer ratio of: which can be explained by a suboptimal CBH I/EG II titer ratio of the expressed enzymes in this study. Protein CBH I : CBH II : EG II = 26 : 17 : 132 mg L−1 (1) = 1.0 : 0.7 : 5.1 Protein CBH I : CBH II : EG II = 26 : 17 : 132 mg L−1 (1) = 1.0 : 0.7 : 5.1 The total titer of these cellulases is 175 mg L−1. Among them, EG II appears to be highly eﬃcient for synthesis and secretion as it was the dominant cellulase based on its titer (132 mg L−1); in contrast, CBH I appears to be less eﬃcient for synthesis and secretion in the obtained transformant (with a titer of 26 mg L−1). Meanwhile, the real-time RT PCR analysis of cDNA samples revealed that: To obtain a direct, visual illustration for the transcriptional and protein levels of these cellulases, the data in Eqns. 1 and 2 were re-plotted in Figure 4, which reveals a substantial discrepancy (i.e., lack of correlation) between the transcriptional and protein levels for chimeric CBH I vs. EG II. Future studies on the co-secretion of chimeric CBH I and EG II under the same promoters are warranted. Comparing Cellulase Levels in YL165-1 vs. Single Cellulase Expressing Strains Furthermore, FAME analysis showed that the total FAME produced by transformant YL165-1 was 0.19 g L−1, while the FAME yield was 31 mg g−1 Avicel consumed (Table 3). Compared with lipid production by other oleaginous microorganisms on cellulose or glucose medium, this FAME yield by YL165-1 is in a similar range. For example, it was reported that the FAME yield of the oleaginous, ﬁlamentous fungus Mucor circinelloides was 57 mg FAME per g Avicel consumed when supplemented with exogenous CBH I and cultured on Avicel substrates (with 33% Avicel being utilized) (Wei et al., 2013). Another report showed the yield of total lipids (which are usually higher than FAME) of the oleaginous yeast Rhodosporidium toruloides was 80 mg lipids per g glucose consumed when cultured in a medium (C/N ratio of 5) using glucose as substrates (Andrade et al., 2012). Note that the FAME proﬁle of YL165-1 in Avicel medium is presented and discussed a later section. Taken together, the transformant YL165-1 was able to maintain a considerable capability for lipid accumulation using cellulose-containing medium, while the cellulose conversion eﬃciency remains as the limiting factor and as a future direction for strain engineering. January 2019 \| Volume 9 \| Article 3276 Cellulose Utilization and Lipid Production by Transformant YL165-1 in Avicel Medium The capability of Y. lipolytica transformant YL165-1 for utilizing cellulose and producing lipids was evaluated by growing the transformant in mineral medium with Avicel as a carbon source. The cultures were harvested at 120 h and the Avicel residues were analyzed. The results showed that YL165-1 consumed 22.8% of the original Avicel content and produced 0.28 g DCW cell biomass per g Avicel consumed (Table 3), while the control strain HA1 (EV) showed no detectable cell growth. Note that the Avicel utilization rate of 22.8% by YL165-1 is on the low end of that observed for Y. lipolytica strains expressing a diﬀerent set of cellulase enzymes (in the range of 22–30%) (Guo et al., 2017), The Bioscreen C has been used to determine the growth curve of Y. lipolytica in recent years (Lazar et al., 2011; Dobrowolski et al., 2016; Miro´nczuk et al., 2016, 2017; Rzechonek et al., 2018). Under the culture condition we used for Bioscreen C analysis, relatively a small proportion of cells changed from typical yeast form to ﬁlamentous form (as examined using Cellometer), which was not signiﬁcant enough to disrupt the turbidity readings. Nevertheless, a cell dry weight method for shake-ﬂask culture was used to validate the results of growth curve obtained via the Bioscreen C, and the data are presented January 2019 \| Volume 9 \| Article 3276 Frontiers in Microbiology \| www.frontiersin.org 9 Ameliorating Cellulases Expression Metabolic Burden Wei et al. FIGURE 4 \| Comparison between the transcript and protein secretion levels for CBH I-CBH II-EG II co-expressed in Y. lipolytica transformant YL165-1. For the relative transcriptional data (in blue color), the transcriptional level for CBH II-encoding gene had the lowest mRNA level and was set at 1. The presented data were collected from three biological replicates, for which error bars indicate the SEM. FIGURE 4 \| Comparison between the transcript and protein secretion levels for CBH I-CBH II-EG II co-expressed in Y. lipolytica transformant YL165-1. For the relative transcriptional data (in blue color), the transcriptional level for CBH II-encoding gene had the lowest mRNA level and was set at 1. The presented data were collected from three biological replicates, for which error bars indicate the SEM. TABLE 3 \| Cell mass and FAME analyses of Y. lipolytica transformant YL165-1 grown on mineral medium containing Avicel as sole carbon sources. Cellulose Utilization and Lipid Production by Transformant YL165-1 in Avicel Medium Cell mass FAME Strain Avicel consumed %[1] Total DCW[2] DCW of newly grown cells[3] Yield[4] Total newly formed FAME[5] FAME% FAME yield g L−1 g L−1 g g−1 Avicel consumed g L−1 DCW basis mg g−1 Avicel consumed YL165-1 22.8 ± 0.56 2.7 ± 0.12 1.7 ± 0.12 0.28 ± 0.02 0.19 ± 0.02 10.2% ± 0.7% 31 ± 3 Data presented as the average of triplicate biological samples with SEM. DCW, dry cell weight. [1] Avicel consumed % was measured described in Methods section; The initial concentration of cellulose in the medium was 27 g L−1. [2] Total DCW was calculated as Dry weight of cell-Avicel pellet – Amount of Avicel consumed. [3] DCW of newly grown cells = Total DCW – Dry weight of initial inoculated cells; the dry weight of initial inoculated cells was 1 g L−1. [4] Cell mass yield was calculated was as g dry weight of newly grown cells per g Avicel consumed. [5] “Total newly formed FAME” = “FAME of total cells” – “FAME of initial inoculated cells.” FIGURE 5 \| Comparison of growth curves between Y. lipolytica transformants expressing individual or multiple cellulases grown in YPD medium. Turbidity data were obtained from Bioscreen C, by which absorbance readings were taken every 15 min. The relative turbidity percentage values for HA1 (EV) and YL165-1 were calculated from the turbidity of the strain cultures at the end-point of the growth curve, with the turbidity of HA1 (EV) being set as 100%. HA1 (EV), strain HA1 transformed with empty vector; Po1g (EV), strain Po1g transformed with empty vector. The bars for SEM from triplicates at time points of 60, 70, 80, 90, 100, and 110 h were shown; ∗indicates signiﬁcantly different from the HA1 (EV) strain with p < 0.05. ate biological samples with SEM. DCW, dry cell weight. [1] Avicel consumed % was measured described in Methods section; The medium was 27 g L−1. [2] Total DCW was calculated as Dry weight of cell-Avicel pellet – Amount of Avicel consumed. [3] DCW of eight of initial inoculated cells; the dry weight of initial inoculated cells was 1 g L−1. [4] Cell mass yield was calculated was as g dry l consumed. [5] “Total newly formed FAME” = “FAME of total cells” – “FAME of initial inoculated cells.” FIGURE 5 \| Comparison of growth curves between Y. Scenario 2. Improved Lipid Production in Cellulase Co-expressing Cells by High C/N Ratio Medium The purpose of using a moderate C/N ratio medium (YPD- 3% Glu, with a C/N ratio > 4.5) was to investigate whether cellulase co-expression causes a drain on lipid production in carbon-nitrogen balanced medium. After 6 days of culturing, most glucose in the medium was consumed by both the parent control strain HA1 (EV) and the transformant YL165-1 cells (Table 4, rows 1–2). Consistent with our above observation that the turbidity of YL165-1 cells was only slightly lower than its parent control strain HA1 (EV) at the late growth phase, the cell mass yield of YL165-1 was only slightly lower than that of HA1 (EV) (10.5 vs. 11.9 DCW g L−1; Table 4). The lipid production capability of Y. lipolytica YL165-1 was also examined in a high C/N ratio medium, with an initial concentration of 80 g L−1 glucose and an initial C/N ratio of 59. For glucose consumption, after 6 days of culturing, the control strain HA1 (EV) and transformant YL165-1 cells consumed 12.6 and 48.0 g L−1 glucose in the medium, respectively (Table 4, rows 3–4). The control strain HA1 (EV) had a relative low OD600 of 5.1 but a high % FAME of 28% (DCW basis) (Table 4), which is consistent with previous reports showing that while a high C/N ratio medium beneﬁted lipid accumulation, it limited the cell growth of Y. lipolytica (Back et al., 2016; Yang et al., 2016). However, the FAME content in transformant YL165-1 was signiﬁcantly lower than that in parent control strain HA1 (EV) (15 vs. 27% FAME, cell dry weight basis; Table 4, rows 1–2). In contrast, cellulase co-expressing transformant YL165-1 grown in high C/N ratio medium showed a completely diﬀerent TABLE 4 \| Cell mass and FAME content in Y. lipolytica transformant YL165-1 cells cultured in moderate and high C/N media with or without a chemical chaperone. Row no. Media and strains Glucose Cell mass FAME Consumed OD600 DCW Yield FAME% Total Yield g L−1 g L−1 g g−1 sugar DCW basis amount g L−1 mg g−1 sugar Moderate C/N medium for FAME production at basal level 1 HA1 (EV) 29.2 ± 0.3 21.2 ± 0.7 11.9 ± 0.5 0.41 ± 0.02 27% ± 2% 3.2 ± 0.2 113 ± 9 2 YL165-1 29.3 ± 0.2 19.4 ± 0.8 10.5 ± 0.4∗ 0.36 ± 0.02∗ 15% ± 2%∗∗ 1.5 ± 0.1∗∗ 52 ± 6∗∗ (Statistical analysis: Row 2 vs. Cellulose Utilization and Lipid Production by Transformant YL165-1 in Avicel Medium lipolytica transformants expressing individual or multiple cellulases grown in YPD medium. Turbidity data were obtained from Bioscreen C, by which absorbance readings were taken every 15 min. The relative turbidity percentage values for HA1 (EV) and YL165-1 were calculated from the turbidity of the strain cultures at the end-point of the growth curve, with the turbidity of HA1 (EV) being set as 100%. HA1 (EV), strain HA1 transformed with empty vector; Po1g (EV), strain Po1g transformed with empty vector. The bars for SEM from triplicates at time points of 60, 70, 80, 90, 100, and 110 h were shown; ∗indicates signiﬁcantly different from the HA1 (EV) strain with p < 0.05. January 2019 \| Volume 9 \| Article 3276 Frontiers in Microbiology \| www.frontiersin.org 10 Ameliorating Cellulases Expression Metabolic Burden Wei et al. Accordingly, the FAME yield of YL165-1 was half of that in HA1 (EV) (52 vs. 113 mg FAME per g sugar; Table 4), suggesting that co-expression of CBH I-CBH II-EG II can compromise lipid accumulation when cultured in carbon-nitrogen balanced medium. in the Supplementary Figure S4. The results indicate that the timeline proﬁling of cell mass weight of the parent control strains and cellulase-expressing transformants follows a similar trend as their growth curves illustrated in Figure 5, conﬁrming that the co-expression of CBH I-CBH II-EG II in YL165-1 comes at a small cost (i.e., mild metabolic burden) for the cells in terms of slightly reduced growth rate the late growth phase. Fatty acid methyl esters analyses show that the major types of fatty acids in both types of cells were C18:1n9, C18:0, C18:2n6, and C16:0 (in order of prominence) with relatively moderate contributions from C16:1n7, C24:0, and C22:0 (Figure 6A). Such a fatty acid pattern is not only consistent with recent observation for the parent strain HA1 in which C18:1, C18:0, and C16:0 being the predominant types (Blazeck et al., 2014), but also consistent with the reported very long-chain fatty acids (VLCFA) content in the wild-type (Katre et al., 2012; Pomraning et al., 2015) and recombinant Y. lipolytica strains (Faure et al., 2010; Tsigie et al., 2012; Zhang et al., 2013). Overall, there is no dramatic diﬀerence between YL165-1 and HA1 (EV) in their FAME proﬁling despite a slight diﬀerence in the ratio of saturated fatty acids (SFA) vs. unsaturated fatty acids (UFA) (Figures 6A,D). Cellulose Utilization and Lipid Production by Transformant YL165-1 in Avicel Medium This relatively mild metabolic burden of cellulase co- expression on the growth of Y. lipolytica is in contrast to the relatively larger burden of cellulase expression on growth of S. cerevisiae, by which CBH expressing S. cerevisiae Y294[Y118p] strain had a 1.4-fold lower maximum speciﬁc growth rate than the reference strain (van Rensburg et al., 2012). Effects of Cellulase Co-expression on Lipid Production: Metabolic Drain and Its Alleviation Scenario 1. Drain of Cellulase Co-expression on Lipid Production of Cells in Medium With Moderate C/N Ratio Scenario 2. Improved Lipid Production in Cellulase Co-expressing Cells by High C/N Ratio Medium row 1) High C/N ratio medium for FAME production at suboptimal level 3 HA1 (EV) 12.6 ± 0.9 5.1 ± 0.2 3.1 ± 0.1 0.25 ± 0.02 28% ± 1% 0.9 ± 0.1 69 ± 7 4 YL165-1 48.0 ± 1.3∗∗ 16.2 ± 0.6 10.1 ± 0.3 0.21 ± 0.01 34% ± 2%∗ 3.4 ± 0.2∗∗ 71 ± 4∗∗ (Statistical analysis: Row 4 vs. row 2) High C/N ratio medium + TMAO for FAME production at optimal level 5 HA1 (EV) 16.1 ± 1.1 6.2 ± 0. 2 4.1 ± 0.3 0.26 ± 0.03 24% ± 1% 1.0 ± 0.1 63 ± 8 6 YL165-1 79.9 ± 0.1∗∗ 35.3 ± 1.2∗∗ 21.9 ± 0.5∗∗ 0.27 ± 0.01∗ 27% ± 2% 5.9 ± 0.3∗∗ 74 ± 5 (Statistical analysis: Row 6 vs. row 4) Data presented as the average of 3 biological replicate samples with SEM. ∗and ∗∗indicate statistical signiﬁcance of p < 0.05 and p < 0.01, respectively. DCW, dry cell weight; Glu, glucose. HA1 (EV), parent control strain HA1 transformed with empty vector; TMAO, trim ethylamine N-oxide dehydrate. January 2019 \| Volume 9 \| Article 3276 11 Frontiers in Microbiology \| www.frontiersin.org Wei et al. Ameliorating Cellulases Expression Metabolic Burden URE 6 \| Fatty acid proﬁles of Y. lipolytica YL165-1 vs. parent control strain HA1 (EV). (A) Cells cultured in YPD-3% glucose medium; (B) cells medium; (C) cells cultured in high C/N ratio medium supplemented with the chemical chaperone TMAO. Data presented were the mean of surements ± SEM. ∗and ∗∗indicate statistical signiﬁcance of p < 0.05 and p < 0.01, respectively, comparing the same fatty acid type betw ent control strain HA1 (EV) for (A–C), or comparing the SFA/UFA ratio (D) or lipid production (E) of the same cell lines in scenarios 2 or 3 again A1 (EV), parent strain HA1 transformed with empty vector; SFA, saturated fatty acids; SFA/UFA, ratio of saturated fatty acids and unsaturated ethylamine N-oxide dihydride; UFA, unsaturated fatty acids. es of Y. lipolytica YL165-1 vs. parent control strain HA1 (EV). (A) Cells cultured in YPD-3% glucose medium; (B) cells cultured in high C/N red in high C/N ratio medium supplemented with the chemical chaperone TMAO. Scenario 2. Improved Lipid Production in Cellulase Co-expressing Cells by High C/N Ratio Medium In summary, as demonstrated in Table 4, TMAO signiﬁcantly enhanced glucose consumption and cell mass yield of YL165-1 that led to higher production yield of lipids (i.e., g total lipid per liter). TMAO has been found to stimulate the cell growth of Escherichia coli (Ishimoto and Shimokawa, 1978), Salmonella typhimurium (Kim and Chang, 1974) and Proteus sp. strain NTHC153 (Strom et al., 1979), likely by acting as a terminal electron acceptor in the respiratory chain (Strom et al., 1979), thus mediating the cell’s energy metabolism and redox balance. This study observed the stimulation of cell growth of Y. lipolytica cellulase-expressing strains; we propose that in addition to the well-documented role of TMAO acting as a chemical chaperon in facilitating protein folding as well as in alleviating ER stress, TMAO may also act as electron acceptor, enhance the cell’s energy metabolism and thus mediate the redox status in the cellulase-expressing Y. lipolytica cells. Future studies are needed to explore this complex mechanism. Future studies are also needed to investigate the mechanism of this eﬀect. pattern of enhanced glucose utilization (i.e., 48.0 g L−1 vs. 12.6 glucose g L−1 for the control strain). This led to twofold improvement in cell mass and a threefold increase in total FAME amount in the control strain (i.e., 10.1 vs. 3.1 g DCW, and 3.4 vs. 0.9 g FAME L−1 in control strain) (Table 4, rows 3–4). pattern of enhanced glucose utilization (i.e., 48.0 g L−1 vs. 12.6 glucose g L−1 for the control strain). This led to twofold improvement in cell mass and a threefold increase in total FAME amount in the control strain (i.e., 10.1 vs. 3.1 g DCW, and 3.4 vs. 0.9 g FAME L−1 in control strain) (Table 4, rows 3–4). Signiﬁcant shifts between SFA and UFA were found in transformant YL165-1, with signiﬁcantly more SFA (C16:0 and C18:0) but much less UFA (C18:1n9 and C18:2n6; Figure 6B), leading to a higher SFA/UFA ratio of 1.03 compared with the control strain’s SFA/UFA ratio of 0.30 (Figure 6D). These data suggest that cellulase co-expression can alter not only the amount but also the composition of the accumulated lipids. Scenario 2. Improved Lipid Production in Cellulase Co-expressing Cells by High C/N Ratio Medium Data presented were the mean of three replicate d ∗∗indicate statistical signiﬁcance of p < 0.05 and p < 0.01, respectively, comparing the same fatty acid type between YL165-1 and V) for (A–C), or comparing the SFA/UFA ratio (D) or lipid production (E) of the same cell lines in scenarios 2 or 3 against those in scenario A1 transformed with empty vector; SFA, saturated fatty acids; SFA/UFA, ratio of saturated fatty acids and unsaturated fatty acids; TMAO, dride; UFA, unsaturated fatty acids. FIGURE 6 \| Fatty acid proﬁles of Y. lipolytica YL165-1 vs. parent control strain HA1 (EV). (A) Cells cultured in YPD-3% glucose medium; (B) cells cultured in high C/N ratio medium; (C) cells cultured in high C/N ratio medium supplemented with the chemical chaperone TMAO. Data presented were the mean of three replicate measurements ± SEM. ∗and ∗∗indicate statistical signiﬁcance of p < 0.05 and p < 0.01, respectively, comparing the same fatty acid type between YL165-1 and parent control strain HA1 (EV) for (A–C), or comparing the SFA/UFA ratio (D) or lipid production (E) of the same cell lines in scenarios 2 or 3 against those in scenario 1. HA1 (EV), parent strain HA1 transformed with empty vector; SFA, saturated fatty acids; SFA/UFA, ratio of saturated fatty acids and unsaturated fatty acids; TMAO, trimethylamine N-oxide dihydride; UFA, unsaturated fatty acids. FIGURE 6 \| Fatty acid proﬁles of Y. lipolytica YL165-1 vs. parent control strain HA1 (EV). (A) Cells cultured in YPD-3% glucose medium; (B) cells cultured in high C/N ratio medium; (C) cells cultured in high C/N ratio medium supplemented with the chemical chaperone TMAO. Data presented were the mean of three replicate measurements ± SEM. ∗and ∗∗indicate statistical signiﬁcance of p < 0.05 and p < 0.01, respectively, comparing the same fatty acid type between YL165-1 and parent control strain HA1 (EV) for (A–C), or comparing the SFA/UFA ratio (D) or lipid production (E) of the same cell lines in scenarios 2 or 3 against those in scenario 1. HA1 (EV), parent strain HA1 transformed with empty vector; SFA, saturated fatty acids; SFA/UFA, ratio of saturated fatty acids and unsaturated fatty acids; TMAO, trimethylamine N-oxide dihydride; UFA, unsaturated fatty acids. January 2019 \| Volume 9 \| Article 3276 Frontiers in Microbiology \| www.frontiersin.org 12 Ameliorating Cellulases Expression Metabolic Burden Wei et al. 4 vs. row 6). Scenario 3. Lipid Production of Cellulase Co-expressing Cells Was Further Enhanced by the Addition of a Chemical Chaperone It has been reported that chemical chaperones, a group of small-molecular-weight compounds, stabilize the conformation and structure of proteins, enhance the protein folding capacity of the ER, and help the traﬃcking of mutated proteins (Vallejo-Becerra et al., 2008). They are capable of reducing ER and oxidative stresses (Sootsuwan et al., 2013; Lamont and Sargent, 2017). Accordingly, we treated cellulase co-expressing transformants and control cells with the chemical chaperone TMAO, and compared the results with those from untreated cells. It is noteworthy that since high C/N ratio medium has been broadly used as a lipid production medium to maximize fatty acids synthesis and lipid accumulation by severely restricting protein synthesis in oleaginous organisms (Back et al., 2016; Yang et al., 2016), the cellulase production of YL165-1 grown in high C/N plus TMAO medium was not assessed in this study. Future studies are needed to investigate any potential eﬀects of TMAO on the cellulase production in engineered Y. lipolytica strains. The TMAO-facilitated lipid production capability of Y. lipolytica YL165-1 was examined in its day 4 culture grown with a high C/N ratio medium. The day 4 cells were treated with 150 mM TMAO for two additional days. For glucose consumption, while TMAO treatment increased that of control strain HA1 (EV) cells by 28%, from 12.6 to 16.1 g glucose L−1 (Table 4, row 3 vs. row 5), it increased that of transformant YL165-1 cells by much larger magnitude of 66% from 48.0 to 79.9 g glucose L−1 (Table 4, row 4 vs. row 6). Accordingly, for cell mass yield, while TMAO treatment increased that of control strain HA1 (EV) cells by 32%, from 3.1 to 4.1 g DCW L−1 (Table 4, row 3 vs. row 5), it increased that of transformant YL165-1 by a much larger magnitude 117%, from 10.1 to 21.9 g DCW L−1 (Table 4, row 4 vs. row 6). Scenario 2. Improved Lipid Production in Cellulase Co-expressing Cells by High C/N Ratio Medium Our observation of increased SFA in YL165-1 grown in high C/N ratio medium is consistent with report that in the green alga Ankistrodesmus falcatus grown under combined stress conditions of nutrients (N, P, and Fe), both saturated fatty acid and lipid accumulation were signiﬁcantly enhanced (Singh P. et al., 2015). The underlying mechanism were that under the stress conditions, unsaturated fatty acids tend to undergo oxidative cleavage, which led to higher saturated fatty acids providing oxidative stability (Sitepu et al., 2013; Singh et al., 2016). For the SFA and UFA proﬁling, TMAO-treated transformant YL165-1 cells in scenario 3, showed a similar pattern as observed in scenario 2, with signiﬁcantly more SFA (C16:0 and C18:0) and less UFA (C18:1n9 and C18:2n6; Figure 6C) that leading to a much higher SFA/UFA ratio of 0.74, compared with the TMAO- treated control strain’s SFA/UFA ratio of 0.31 (Figure 6D). Nevertheless, it is noteworthy the SFA/UFA ratio value of 0.74 in scenario 3 was signiﬁcantly lower that of 1.03 in scenario 2, supporting the literature-reported role of TMAO in reducing the oxidative stress in cells (Sootsuwan et al., 2013; Lamont and Sargent, 2017), which can lead to less oxidative cleavage and less conversion of UFA to SFA. Frontiers in Microbiology \| www.frontiersin.org Effects of Carbon Source and C/N Ratio on the Fatty Acid Composition Proﬁle of YL165-1 The fatty acid proﬁle of YL165-1 cultured in four types of media demonstrates that MM-Avicel medium-grown YL165-1 cells have a unique fatty acid composition diﬀerent from both the moderate C/N medium, and the high C/N medium (with or without TMAO) (Figure 7). MM-Avicel medium-grown YL165- 1 cells have the highest proportion of UFA (C16:1n7, C16:3 and C18:2n6) and lowest SFA (C18:0 and C24:0) than other three media-grown cells (Figure 7). Accordingly, MM-Avicel medium-grown YL165-1 cells have the lowest SFA/UFA ratio. This observation is consistent with a recent study conducted by Dobrowolski et al., which showed that the fatty acid proﬁles in Y. lipolytica strain A101 cultured on various sources of For lipid production, TMAO treatment increased the total FAME production of control strain HA1 (EV) cells by 11%, from 0.9 to 1.0 g FAME L−1 (Table 4, row 3 vs. row 5) and increased that of transformant YL165-1 cells by a much larger magnitude of 74%, from 3.4 to 5.9 g FAME L−1 (Table 4, row January 2019 \| Volume 9 \| Article 3276 Frontiers in Microbiology \| www.frontiersin.org 13 Ameliorating Cellulases Expression Metabolic Burden Wei et al. FIGURE 7 \| Comparison of fatty acid proﬁles of Y. lipolytica YL165-1 cells grown in four types of media with different carbon sources or C/N ratios. Data shown are the mean from three replicate measurements ± standard error of the mean (SEM). ∗indicates the fatty acids for which MM-Avicel medium-grown cells have different relative levels from other media-grown cells. FIGURE 7 \| Comparison of fatty acid proﬁles of Y. lipolytica YL165-1 cells grown in four types of media with different carbon sources or C/N ratios. Data shown are the mean from three replicate measurements ± standard error of the mean (SEM). ∗indicates the fatty acids for which MM-Avicel medium-grown cells have different relative levels from other media-grown cells. crude glycerol were diﬀerent, depending on the types of applied substrate (Dobrowolski et al., 2016). Effects of Carbon Source and C/N Ratio on the Fatty Acid Composition Proﬁle of YL165-1 the modiﬁed YPD-based medium (YPD-3% Glu that contains yeast extract 10 g L−1, peptone 20 g L−1, glucose of 30 g L−1, which led to a FAME level of 27% ± 2% DCW basis for the strain HA1 cells), this study also used a YNB-based medium that contained 80 g L−1 glucose and 1.365 g L−1 ammonium (which led to a FAME level of 28% ± 1% DCW basis for the strain HA1 cells); both media were signiﬁcantly diﬀerent from the YNB-based medium used in the previous study (Blazeck et al., 2014), which contained up to 160 g L−1 glucose and only 0.055 g L−1 ammonium and led to a total lipid level up to 90% DCW basis for the strain HA1 cells. The signiﬁcantly higher C/N ratio of the YNB-based media used in the previous study will likely lead to a higher lipid accumulation for the same strain. Factors Affecting the Lipid Contents of Y. lipolytica and Their Estimation It should be noted that since the focus of this study was to co-express fungal cellulases in Yarrowia, we deviated from the optimal culture conditions (such as fermentation in a bioreactor with controlled pH and improved aeration, etc.) used in the previous studies with this strain (Blazeck et al., 2014). As a result, the measured FAME content data for parent control strain HA1 (EV), which was close to 30% FAME DCW basis in shake ﬂasks in this study, cannot be directly compared to the previously reported values, which was in the range of 71% (Liu et al., 2015) to 90% (Blazeck et al., 2014) generated during fermentation in pH controlled, more eﬃciently aerated bioreactors. Diﬀerence between the media and shaking speeds used in ﬂask culture in this vs. previous studies. The previous study used three types of containers (rotary drum, shake ﬂasks, and bioreactor) in assessing lipid accumulation in Y. lipolytica strain HA1 (Blazeck et al., 2014). For shake ﬂask culturing of HA1, the diﬀerent shaking speeds used in the previous study (225 rpm) vs. this study (200 rpm) should partially account for the diﬀerent levels of lipid accumulation between these two studies as a better agitation/aeration for microbial growth can signiﬁcantly impact the performance in producing metabolites (Liu et al., 2015). Meanwhile, the diﬀerent media used in ﬂask culture in this vs. the previous study would likely account for a larger portion of the reported diﬀerence in lipid accumulation. In addition to Growth conditions (ﬂask vs. bioreactor). Growth conditions play a signiﬁcant role in lipid accumulation in Y. lipolytica, which is exempliﬁed by the observation of a signiﬁcant range in lipid accumulation that spans a 74-fold improvement over the parent strain of HA1, strain Po1f, as illustrated in Figure 1 (Blazeck et al., 2014). Another study reported the bioreactor fermentation increased the lipid content of Y. lipolytica cells by 50% compared to the shake ﬂask (Tai and Stephanopoulos, 2013). Buoyancy of Y. lipolytica cells. As listed in Table 1, both strains Po1f (the parent strain for HA1 used in the study) and Po1g were derived from the wild-type strain W29 (Madzak et al., 2000). For Po1g, its cell mats were found to ﬂoat in water after being scrapped oﬀthe surface of agar medium, as illustrated in the Supplementary Figure S1 of our previous report (Wei et al., 2014). Frontiers in Microbiology \| www.frontiersin.org Mechanisms for Relieving the Metabolic Burden of Cellulase Expression by a High C/N Ratio Medium and the Addition of Chemical Chaperone Proposed Role of ER for the Efﬁcient Co-expression of Cellulases and Lipid Biosynthesis One goal of this study was to examine the dynamic relationship between the production and secretion of heterologous cellulases and the accumulation of lipids. ER is an important organelle for both protein synthesis, folding and secretion, and lipid metabolism. In fact, the synthesis of secretory proteins starts in the ER for correctly integrating nascent proteins and ensuring correct post-translational modiﬁcation and folding, followed by being captured into ER-derived transport vesicles and delivered to the early Golgi (Barlowe and Miller, 2013; den Haan et al., 2013). The high C/N ratio, limited-nitrogen media have been used to cultivate a range of yeast and algae to achieve high levels of lipid accumulation (Li et al., 2008; Chaisawang et al., 2012; Lamers et al., 2012; Sharma et al., 2012; Braunwald et al., 2013; Sitepu et al., 2013; Blazeck et al., 2014; Hirooka et al., 2014; Zhang et al., 2014; Yu et al., 2015; Yang et al., 2016). As observed in this study, the cellulase co-expression route had an unexpected beneﬁt in enhancing lipid accumulation when grown in medium with high C/N ratio; we propose the likely underlying mechanism would be that the high C/N ratio medium eﬀectively lowers the levels of protein synthesis (because of nitrogen limitation) and ER stress, which leads to boosting nitrogen-limitation-induced lipid production, as shown in Figure 6E (scenario 2). The supplement of TMAO into the high C/N ratio medium likely facilitates the protein folding and lowers the ER and oxidative stresses. TMAO may also help mediate the cell energy metabolism and redox balance via acting as an electron acceptor, as suggested by its enhancement of cell growth and cell mass yield. Future studies are warranted to further explore the mechanisms underlying the delicate balance between the cellulase synthesis/secretion and fatty acid-based biofuels production. Endoplasmic reticulum also plays a critical role for lipid metabolism, reﬂected by literature report that (1) a list of 493 candidate proteins (accounting for approximately 9% of the proteome in the yeast S. cerevisiae) were known or predicted to be involved in lipid metabolism and its regulation (Natter et al., 2005), and (2) green ﬂuorescent protein (GFP) tagging coupled with confocal laser scanning microscopy was used to localize the above proteins, the majority of tagged, lipid metabolism- related enzymes localized to ER (92), followed by vesicles (53), mitochondria (27), lipid droplets (23), peroxisomes (17), plasma membrane (14), and Golgi (7) (Natter et al., 2005). Factors Affecting the Lipid Contents of Y. lipolytica and Their Estimation For HA1, a more recent study January 2019 \| Volume 9 \| Article 3276 Frontiers in Microbiology \| www.frontiersin.org 14 Wei et al. Ameliorating Cellulases Expression Metabolic Burden Possible Mechanisms for Relieving the Metabolic Burden by High C/N Ratio Medium and the Addition of Chemical Chaperone showed that a subpopulation of cells had very high buoyancy. The cells ﬂoating on top of the medium were full of lipids determined by using ﬂuorescence microscopy with Nile Red staining, whereas normal cells that settled to the bottom of the tube did not contain as much lipids (Liu et al., 2015). In our study, as illustrated in Supplementary Figure S5, the cellulase- co-expressing transformant YL165-1 showed more ﬂoating cell mass than the control strain HA1 (EV) in shake ﬂask culture. We speculate that the buoyancy of YL165-1 cells likely also aﬀects the oxygen diﬀusion into the medium, providing another rationale for optimizing culture conditions by using higher rpm in ﬂask shaking or bioreactor condition to achieve increased lipid accumulation. High level expression of heterologous proteins in yeast has been previously found to induce signiﬁcant cellular changes, including a decrease in growth rate and the altering of nitrogen and redox metabolisms, and poses a metabolic burden on the host cells (Ruijter et al., 2017). In the case of cellulase expression, expression of heterologous A. aculeatus and Saccharomycopsis ﬁbuligera BGLs had an increasingly negative eﬀect on cell growth of S. cerevisiae as the expressed gene doses increased until a ﬁnal failure to grow (Ding et al., 2017). Equally relevantly, it is reported that high level expression of endogenous and heterologous secreted cellulases can cause ER stress, which subsequently induces the unfolded protein response (UPR), activating related genes to relieve stress in the secretory pathway to improve protein folding eﬃciency and capacity in ﬁlamentous fungi and yeast (Collen et al., 2005; Ilmen et al., 2011; Fan et al., 2015; Van Zyl et al., 2016; Ruijter et al., 2017). The link between ER stress, UPR, and lipid accumulation have been shown in literature that reported ER stress stimulates and increases the level of lipid droplets and protects the yeast cells against the eﬀects of misfolded proteins (Czabany et al., 2008; Fei et al., 2009; Hapala et al., 2011). Mechanisms for Relieving the Metabolic Burden of Cellulase Expression by a High C/N Ratio Medium and the Addition of Chemical Chaperone Proposed Role of ER for the Efﬁcient Co-expression of Cellulases and Lipid Biosynthesis Furthermore, lipid droplets in yeast are not only functionally connected to the ER (Jacquier et al., 2011), its de novo LD biogenesis occurs in ER, and it eventually buds from ER (Jacquier et al., 2013; Choudhary et al., 2015). All these considerations undoubtedly support the ER as the central organelle for protein synthesis, lipid biosynthesis, and lipid droplet formation. Frontiers in Microbiology \| www.frontiersin.org REFERENCES Zygosaccharomyces bailii based on combination of a membrane-active peptide with an oligosaccharide that leads to an impaired glycosylphosphatidylinositol (GPI)-dependent yeast wall protein layer. FEMS Yeast Res. 1, 187–194. Zygosaccharomyces bailii based on combination of a membrane-active peptide with an oligosaccharide that leads to an impaired glycosylphosphatidylinositol (GPI)-dependent yeast wall protein layer. FEMS Yeast Res. 1, 187–194. Zygosaccharomyces bailii based on combination of a membrane-active peptide with an oligosaccharide that leads to an impaired glycosylphosphatidylinositol (GPI)-dependent yeast wall protein layer. FEMS Yeast Res. 1, 187–194. Abdel-Mawgoud, A. M., Markham, K. A., Palmer, C. M., Liu, N., Stephanopoulos, G., and Alper, H. S. (2018). Metabolic engineering in the host Yarrowia lipolytica. Metab. Eng. 50, 192–208. doi: 10.1016/j.ymben.2018.07.016 Boonvitthya, N., Bozonnet, S., Burapatana, V., O’donohue, M. J., and Chulalaksananukul, W. (2013). Comparison of the heterologous expression of Trichoderma reesei endoglucanase II and cellobiohydrolase II in the yeasts Pichia pastoris and Yarrowia lipolytica. Mol. Biotechnol. 54, 158–169. doi: 10. 1007/s12033-012-9557-0 Abghari, A., and Chen, S. (2014). Yarrowia lipolytica as an oleaginous cell factory platform for production of fatty acid-based biofuel and bioproducts. Front. Energy Res. 2:21. doi: 10.3389/FENRG.2014.00021 Andrade, R., Leal, R., Roseiro, J., Reis, A., and Da Silva, T. L. (2012). Monitoring Rhodosporidium toruloides NCYC 921 batch fermentations growing under carbon and nitrogen limitation by ﬂow cytometry. World J. Microbiol. Biotechnol. 28, 1175–1184. doi: 10.1007/s11274-011-0920-2 Braunwald, T., Schwemmlein, L., Graeﬀ-Honninger, S., French, W. T., Hernandez, R., Holmes, W. E., et al. (2013). Eﬀect of diﬀerent C/N ratios on carotenoid and lipid production by Rhodotorula glutinis. Appl. Microbiol. Biotechnol. 97, 6581–6588. doi: 10.1007/s00253-013-5005-8 Back, A., Rossignol, T., Krier, F., Nicaud, J.-M., and Dhulster, P. (2016). High- throughput fermentation screening for the yeast Yarrowia lipolytica with real- time monitoring of biomass and lipid production. Microb. Cell Fact. 15:147. doi: 10.1186/s12934-016-0546-z Carsanba, E., Papanikolaou, S., and Erten, H. (2018). Production of oils and fats by oleaginous microorganisms with an emphasis given to the potential of the nonconventional yeast Yarrowia lipolytica. Crit. Rev. Biotechnol. 38, 1230–1243. doi: 10.1080/07388551.2018.1472065 Barlowe, C. K., and Miller, E. A. (2013). Secretory protein biogenesis and traﬃc in the early secretory pathway. Genetics 193, 383–410. doi: 10.1534/genetics.112. 142810 Chaisawang, M., Verduyn, C., Chauvatcharin, S., and Suphantharika, M. (2012). Metabolic networks and bioenergetics of Aurantiochytrium sp. B-072 during storage lipid formation. Braz. J. Microbiol. 43, 1192–1205. doi: 10.1590/S1517- 838220120003000047 Baryshnikova, A., Costanzo, M., Kim, Y., Ding, H., Koh, J., Touﬁghi, K., et al. (2010). AUTHOR CONTRIBUTIONS MH, MZ, HA, and HW led the project and coordinated the study. HW conceived and designed the experiments. HA provided the lipid hyperaccumulating yeast strains and the genetic background information. HW executed, and WW and QX assisted with DNA construct building, yeast transformation and screening, SDS–PAGE, western blotting analysis, and enzymatic analyses. SVW conducted the FAME analysis. C-YL, YL, and HW designed the primers and conducted the RNA extraction and real-time RT-PCR. SD provided CBH I, CBH II and EG II antibodies, and provided guidance on enzymatic assays. EK provided guidance on yeast culturing techniques and media. HW prepared the initial draft of the manuscript. SD, EK, MH, HA, and MZ edited the manuscript. HW coordinated the manuscript submission. All authors read and approved the ﬁnal manuscript. ACKNOWLEDGMENTS The authors thank Dr. Andrew B. Hill for technical assistance. FUNDING titer of CBH I. Remarkably, when grown in medium with a high C/N ratio and supplemented with a chemical chaperone, the cellulase co-expressing transformant showed a pattern by which the metabolic drain caused by cellulase co-expression and secretion was relieved, and both cell growth and lipid productivity were signiﬁcantly increased, highlighting the eﬀectiveness of the above approaches in rebalancing the protein synthesis and lipid production in Y. lipolytica. This work was authored by Alliance for Sustainable Energy, LLC, the Manager and Operator of the National Renewable Energy Laboratory for the U.S. Department of Energy (DOE) under contract no. DE-AC36-08GO28308. Funding provided by U.S. Department of Energy Oﬃce of Energy Eﬃciency and Renewable Energy Bioenergy Technologies Oﬃce. The views expressed in the article do not necessarily represent the views of the DOE or the U.S. Government. The U.S. Government retains and the publisher, by accepting the article for publication, acknowledges that the U.S. Government retains a non-exclusive, paid-up, irrevocable, worldwide license to publish or reproduce the published form of this work, or allow others to do so, for U.S. Government purposes. SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb. 2018.03276/full#supplementary-material CONCLUSION Our results demonstrated that transformant YL165-1 with an overall high level of cellulase secretion compromises the lipid accumulation of these cells. Such an observation, plus the above literature analysis, prompts us to propose an intrinsic link between cellulase co-expression/secretion and lipid accumulation. An enforced overexpression of secreted cellulases will cause a drain on the ER of yeast cells, leading to competition among the co-expressed cellulases for synthesis and secretion, and lipid production. We have successfully demonstrated the co-expression and secretion of three core fungal cellulases in a high lipid- accumulating engineered strain of the oleaginous yeast, Y. lipolytica, enabling nearly a 23% conversion of Avicel. To the best of our knowledge, it represents the ﬁrst case of exploring the relationship between secreted cellulase expression, cell growth, and lipid production in Y. lipolytica strains. The transformant YL165-1 expressed a relatively high titer of EG II and moderate We have successfully demonstrated the co-expression and secretion of three core fungal cellulases in a high lipid- accumulating engineered strain of the oleaginous yeast, Y. lipolytica, enabling nearly a 23% conversion of Avicel. To the best of our knowledge, it represents the ﬁrst case of exploring the relationship between secreted cellulase expression, cell growth, and lipid production in Y. lipolytica strains. The transformant YL165-1 expressed a relatively high titer of EG II and moderate January 2019 \| Volume 9 \| Article 3276 Frontiers in Microbiology \| www.frontiersin.org 15 Ameliorating Cellulases Expression Metabolic Burden Wei et al. REFERENCES W., Ragghianti, J. J., et al. (2011). High eicosapentaenoic acid producing strains of Yarrowia lipolytica. U.S. Patent No. 7, 077. Guo, Z.-P., Duquesne, S., Bozonnet, S., Cioci, G., Nicaud, J.-M., Marty, A., et al. (2017). Conferring cellulose-degrading ability to Yarrowia lipolytica to facilitate a consolidated bioprocessing approach. Biotechnol. Biofuels 10:132. doi: 10. 1186/s13068-017-0819-8 Damude, H. G., and Zhu, Q. Q. (2007). Delta-8 desaturase and its use in making polyunsaturated fatty acids. U.S. Patent No. 7,256,033. polyunsaturated fatty acids. U.S. Patent No. 7,256,033. Hapala, I., Marza, E., and Ferreira, T. (2011). Is fat so bad? Modulation of endoplasmic reticulum stress by lipid droplet formation. Biol. Cell 103, 271–285. doi: 10.1042/BC20100144 Dashtban, M., and Qin, W. (2012). Overexpression of an exotic thermotolerant β-glucosidase in Trichoderma reesei and its signiﬁcant increase in cellulolytic activity and sacchariﬁcation of barley straw. Microb. Cell Fact. 11:63. doi: 10. 1186/1475-2859-11-63 Harris, P. V., Welner, D., Mcfarland, K., Re, E., Navarro Poulsen, J.-C., Brown, K., et al. (2010). Stimulation of lignocellulosic biomass hydrolysis by proteins of glycoside hydrolase family 61: structure and function of a large, enigmatic family. Biochemistry 49, 3305–3316. doi: 10.1021/bi100009p Davidow, L. S., O’donnell, M. M., Kaczmarek, F. S., Pereira, D. A., Dezeeuw, J. R., and Franke, A. E. (1987). Cloning and sequencing of the alkaline extracellular protease gene of Yarrowia lipolytica. J. Bacteriol. 169, 4621–4629. doi: 10.1128/ jb.169.10.4621-4629.1987 Himmel, M. E., Ding, S. Y., Johnson, D. K., Adney, W. S., Nimlos, M. R., Brady, J. W., et al. (2007). Biomass recalcitrance: engineering plants and enzymes for biofuels production. Science 315, 804–807. doi: 10.1126/science.1137016 den Haan, R., Kroukamp, H., Van Zyl, J.-H. D., and Van Zyl, W. H. (2013). Cellobiohydrolase secretion by yeast: current state and prospects for improvement. Process Biochem. 48, 1–12. doi: 10.1016/j.procbio.2012.11.015 Hirooka, S., Higuchi, S., Uzuka, A., Nozaki, H., and Miyagishima, S. Y. (2014). Acidophilic green alga Pseudochlorella sp. YKT1 accumulates high amount of lipid droplets under a nitrogen-depleted condition at a low-pH. PLoS One 9:e107702. doi: 10.1371/journal.pone.0107702 Den Haan, R., Rose, S. H., Lynd, L. R., and Van Zyl, W. H. (2007). Hydrolysis and fermentation of amorphous cellulose by recombinant Saccharomyces cerevisiae. Metab. Eng. 9, 87–94. doi: 10.1016/j.ymben.2006.08.005 Hussain, M., Oku, Y., Nakahira, A., and Niihara, K. (1996). Eﬀects of wet ball- milling on particle dispersion and mechanical properties of particulate epoxy composites. Mater. Lett. 26, 177–184. REFERENCES doi: 10.1016/0167-577X(95)00223-5 Ding, J., Liang, G., Zhang, K., Hong, J., Zou, S., Lu, H., et al. (2017). Extra metabolic burden by displaying over secreting: growth, fermentation and enzymatic activity in cellobiose of recombinant yeast expressing β-glucosidase. Bioresour. Technol. 254, 107–114. doi: 10.1016/j.biortech.2017. 12.030 Ilmen, M., Den Haan, R., Brevnova, E., Mcbride, J., Wiswall, E., Froehlich, A., et al. (2011). High level secretion of cellobiohydrolases by Saccharomyces cerevisiae. Biotechnol. Biofuels 4:30. doi: 10.1186/1754-6834-4-30 Dobrowolski, A., Mituła, P., Rymowicz, W., and Miro´nczuk, A. M. (2016). Eﬃcient conversion of crude glycerol from various industrial wastes into single cell oil by yeast Yarrowia lipolytica. Bioresour. Technol. 207, 237–243. doi: 10.1016/j. biortech.2016.02.039 Ishikawa, H., and Ide, S. (1993). Method of producing ﬁnely divided ﬁbrous cellulose particles. U.S. Patent NO 5,269,470. Ishimoto, M., and Shimokawa, O. (1978). Reduction of trimethylamine N-oxide by Escherichia coli as anaerobic respiration. Z. Allg. Mikrobiol. 18, 173–181. doi: 10.1002/jobm.3630180304 Dulermo, R., Gamboa-Melendez, H., Ledesma-Amaro, R., Thevenieau, F., and Nicaud, J.-M. (2015). Unraveling fatty acid transport and activation mechanisms in Yarrowia lipolytica. Biochim. Biophys. Acta Mol. Cell Biol. Lipids 1851, 1202–1217. doi: 10.1016/j.bbalip.2015.04.004 Jacquier, N., Choudhary, V., Mari, M., Toulmay, A., Reggiori, F., and Schneiter, R. (2011). Lipid droplets are functionally connected to the endoplasmic reticulum in Saccharomyces cerevisiae. J. Cell Sci. 124, 2424–2437. doi: 10.1242/jcs.076836 Fan, F., Ma, G., Li, J., Liu, Q., Benz, J. P., Tian, C., et al. (2015). Genome-wide analysis of the endoplasmic reticulum stress response during lignocellulase production in Neurospora crassa. Biotechnol. Biofuels 8:66. doi: 10.1186/s13068- 015-0248-5 Jacquier, N., Mishra, S., Choudhary, V., and Schneiter, R. (2013). Expression of oleosin and perilipins in yeast promotes formation of lipid droplets from the endoplasmic reticulum. J. Cell Sci. 126, 5198–5209. doi: 10.1242/jcs.131896 Fan, L. H., Zhang, Z. J., Yu, X. Y., Xue, Y. X., and Tan, T. W. (2012). Self- surface assembly of cellulosomes with two miniscaﬀoldins on Saccharomyces cerevisiae for cellulosic ethanol production. Proc. Natl. Acad. Sci. U.S.A. 109, 13260–13265. doi: 10.1073/pnas.1209856109 Josefsen, L., Droce, A., Sondergaard, T. E., Sorensen, J. L., Bormann, J., Schafer, W., et al. (2012). Autophagy provides nutrients for nonassimilating fungal structures and is necessary for plant colonization but not for infection in the necrotrophic plant pathogen Fusarium graminearum. Autophagy 8, 326–337. doi: 10.4161/auto.18705 Faure, J.-D., Nicaud, J.-M., Bourdenx, B., and Haddouche, R. (2010). Method for the production of very long chain fatty acids (VLCFA) by fermentation with a recombinant Yarrowia SP. U.S. REFERENCES Quantitative analysis of ﬁtness and genetic interactions in yeast on a genome scale. Nat. Methods 7, 1017–1024. doi: 10.1038/nmeth.1534 Chang, J.-J., Ho, F.-J., Ho, C.-Y., Wu, Y.-C., Hou, Y.-H., Huang, C.-C., et al. (2013). Assembling a cellulase cocktail and a cellodextrin transporter into a yeast host for CBP ethanol production. Biotechnol. Biofuels 6:19. doi: 10.1186/1754-6834- 6-19 Berlin, A., Maximenko, V., Gilkes, N., and Saddler, J. (2007). Optimization of enzyme complexes for lignocellulose hydrolysis. Biotechnol. Bioeng. 97, 287– 296. doi: 10.1002/bit.21238 Chen, M., Zhao, J., and Xia, L. (2008). Enzymatic hydrolysis of maize straw polysaccharides for the production of reducing sugars. Carbohydr. Polym. 71, 411–415. doi: 10.1016/j.carbpol.2007.06.011 Blazeck, J., Hill, A., Liu, L., Knight, R., Miller, J., Pan, A., et al. (2014). Harnessing Yarrowia lipolytica lipogenesis to create a platform for lipid and biofuel production. Nat. Commun. 5, 1–10. doi: 10.1038/ncomms4131 Boer, H., Teeri, T. T., and Koivula, A. (2000). Characterization of Trichoderma reesei cellobiohydrolase Cel7A secreted from Pichia pastoris using two diﬀerent promoters. Biotechnol. Bioeng. 69, 486–494. doi: 10.1002/1097-0290(20000905) 69:5<486::AID-BIT3>3.0.CO;2-N Choudhary, V., Ojha, N., Golden, A., and Prinz, W. A. (2015). A conserved family of proteins facilitates nascent lipid droplet budding from the ER. J. Cell Biol. 211, 261–271. doi: 10.1083/jcb.201505067 Collen, A., Saloheimo, M., Bailey, M., Penttila, M., and Pakula, T. M. (2005). Protein production and induction of the unfolded protein response in Trichoderma reesei strain Rutâ€-C30 and its transformant expressing Bom, I. J., Klis, F. M., De Nobel, H., and Brul, S. (2001). A new strategy for inhibition of the spoilage yeasts Saccharomyces cerevisiae and January 2019 \| Volume 9 \| Article 3276 Frontiers in Microbiology \| www.frontiersin.org 16 Ameliorating Cellulases Expression Metabolic Burden Wei et al. Gonçalves, F., Colen, G., and Takahashi, J. (2014). Yarrowia lipolytica and its multiple applications in the biotechnological industry. Sci. World J. 2014:14. doi: 10.1155/2014/476207 endoglucanase I with a hydrophobic tag. Biotechnol. Bioeng. 89, 335–344. doi: 10.1002/bit.20350 Czabany, T., Wagner, A., Zweytick, D., Lohner, K., Leitner, E., Ingolic, E., et al. (2008). Structural and biochemical properties of lipid particles from the yeast Saccharomyces cerevisiae. J. Biol. Chem. 283, 17065–17074. doi: 10.1074/jbc. M800401200 Guo, Z. P., Duquesne, S., Bozonnet, S., Cioci, G., Nicaud, J. M., Marty, A., et al. (2015). Development of cellobiose-degrading ability in Yarrowia lipolytica strain by overexpression of endogenous genes. Biotechnol. Biofuels 8:109. doi: 10.1186/s13068-015-0289-9 Damude, H. G., Gillies, P. J., Macool, D. J., Picataggio, S. K., Pollak, D. M. REFERENCES D., and Velagapudi, V. (2017). Understanding the metabolic burden of recombinant antibody production in Saccharomyces cerevisiae using a quantitative metabolomics approach. Yeast 35, 331–341. doi: 10.1002/yea.3298 Li, Y., Horsman, M., Wang, B., Wu, N., and Lan, C. Q. (2008). Eﬀects of nitrogen sources on cell growth and lipid accumulation of green alga Neochloris oleoabundans. Appl. Microbiol. Biotechnol. 81, 629–636. doi: 10.1007/s00253- 008-1681-1 Liu, L., Pan, A., Spoﬀord, C., Zhou, N., and Alper, H. S. (2015). An evolutionary metabolic engineering approach for enhancing lipogenesis in Yarrowia lipolytica. Metab. Eng. 29, 36–45. doi: 10.1016/j.ymben.2015.02.003 Ryu, S., Hipp, J., and Trinh, C. T. (2016). Activating and elucidating metabolism of complex sugars in Yarrowia lipolytica. Appl. Environ. Microbiol. 82, 1334–1345. doi: 10.1128/AEM.03582-15 Lynd, L. R., Van Zyl, W. H., Mcbride, J. E., and Laser, M. (2005). Consolidated bioprocessing of cellulosic biomass: an update. Curr. Opin. Biotechnol. 16, 577–583. doi: 10.1016/j.copbio.2005.08.009 Ryu, S., Labbe, N., and Trinh, C. T. (2015). Simultaneous sacchariﬁcation and fermentation of cellulose in ionic liquid for eﬃcient production of α-ketoglutaric acid by Yarrowia lipolytica. Appl. Microbiol. Biotechnol. 99, 4237–4244. doi: 10.1007/s00253-015-6521-5 Madzak, C., Treton, B., and Blanchin-Roland, S. (2000). Strong hybrid promoters and integrative expression/secretion vectors for quasi-constitutive expression of heterologous proteins in the yeast Yarrowia lipolytica. J. Mol. Microbiol. Biotechnol. 2, 207–216. Rzechonek, D. A., Day, A. M., Quinn, J., and Miro´nczuk, A. M. (2018). Inﬂuence of ylHog1 MAPK kinase on Yarrowia lipolytica stress response and erythritol production. Sci. Rep. 8:14735. doi: 10.1038/s41598-018-33168-6 Savina, A., Vidal, M., and Colombo, M. I. (2002). The exosome pathway in K562 cells is regulated by Rab11. J. Cell Sci. 115, 2505–2515. Markham, K. A., Cordova, L., Hill, A., and Alper, H. S. (2016). “Yarrowia lipolytica as a cell factory for oleochemical biotechnology,” in Consequences of Microbial Interactions with Hydrocarbons, Oils, and Lipids: Production of Fuels and Chemicals, ed. S. Y. Lee (Cham: Springer International Publishing), 1–18. Schmittgen, T. D., and Livak, K. J. (2008). Analyzing real-time PCR data by the comparative CT method. Nat. Protoc. 3, 1101–1108. doi: 10.1038/nprot.2008.73 Schulein, M. (1997). Enzymatic properties of cellulases from Humicola insolens. J. Biotechnol. 57, 71–81. doi: 10.1016/S0168-1656(97) 00090-4 Martinez-Force, E., and Benitez, T. (1995). Eﬀects of varying media, temperature, and growth rates on the intracellular concentrations of yeast amino acids. Biotechnol. Prog. 11, 386–392. doi: 10.1021/bp00034a004 Schwarzhans, J.-P., Wibberg, D., Winkler, A., Luttermann, T., Kalinowski, J., and Friehs, K. (2016). REFERENCES Patent No 13 510,577 Jung, P. P., Christian, N., Kay, D. P., Skupin, A., and Linster, C. L. (2015). Protocols and programs for high-throughput growth and aging phenotyping in yeast. PLoS One 10:e0119807. doi: 10.1371/journal.pone.0119807 Fei, W., Wang, H., Fu, X., Bielby, C., and Yang, H. (2009). Conditions of endoplasmic reticulum stress stimulate lipid droplet formation in Saccharomyces cerevisiae. Biochem. J. 424, 61–67. doi: 10.1042/BJ200 90785 Kallioinen, A., Puranen, T., and Siika-Aho, M. (2014). Mixtures of thermostable enzymes show high performance in biomass sacchariﬁcation. Appl. Biochem. Biotechnol. 173, 1038–1056. doi: 10.1007/s12010-014-0893-3 Franden, M. A., Pienkos, P. T., and Zhang, M. (2009). Development of a high- throughput method to evaluate the impact of inhibitory compounds from lignocellulosic hydrolysates on the growth of Zymomonas mobilis. J. Biotechnol. 144, 259–267. doi: 10.1016/j.jbiotec.2009.08.006 Katre, G., Joshi, C., Khot, M., Zinjarde, S., and Ravikumar, A. (2012). Evaluation of single cell oil (SCO) from a tropical marine yeast Yarrowia lipolytica NCIM 3589 as a potential feedstock for biodiesel. AMB Express 2:36. doi: 10.1186/ 2191-0855-2-36 Kim, K. E., and Chang, G. W. (1974). Trimethylamine oxide reduction by Salmonella. Can. J. Microbiol. 20, 1745–1748. doi: 10.1139/m74-269 Franden, M. A., Pilath, H. M., Mohagheghi, A., Pienkos, P. T., and Zhang, M. (2013). Inhibition of growth of Zymomonas mobilis by model compounds found in lignocellulosic hydrolysates. Biotechnol. Biofuels 6:99. doi: 10.1186/1754- 6834-6-99 Kobayashi, H., Matsuhashi, H., Komanoya, T., Hara, K., and Fukuoka, A. (2011). Transfer hydrogenation of cellulose to sugar alcohols over supported ruthenium catalysts. Chem. Commun. 47, 2366–2368. doi: 10.1039/c0cc04311g Gientka, I., Bzducha-Wrobel, A., Stasiak-Rozanska, L., Bednarska, A. A., and Blazejak, S. (2016). The exopolysaccharides biosynthesis by Candida yeast depends on carbon sources. Electron. J. Biotechnol. 22, 31–37. doi: 10.1016/j. ejbt.2016.02.008 Kricka, W., Fitzpatrick, J., and Bond, U. (2014). Metabolic engineering of yeasts by heterologous enzyme production for degradation of cellulose and hemicellulose from biomass: a perspective. Front. Microbiol. 5:174. doi: 10.3389/fmicb.2014. 00174 Godbole, S., Decker, S. R., Nieves, R. A., Adney, W. S., Vinzant, T. B., Baker, J. O., et al. (1999). Cloning and expression of Trichoderma reesei cellobiohydrolase I in Pichia pastoris. Biotechnol. Prog. 15, 828–833. doi: 10.1021/bp9901116 Kurtzman, C., Fell, J. W., and Boekhout, T. (2011). The Yeasts: A Taxonomic Study. New York, NY: Elsevier. January 2019 \| Volume 9 \| Article 3276 Frontiers in Microbiology \| www.frontiersin.org 17 Ameliorating Cellulases Expression Metabolic Burden Wei et al. Lamers, P. P., Janssen, M., De Vos, R. REFERENCES C., Bino, R. J., and Wijﬀels, R. (2012). Carotenoid and fatty acid metabolism in nitrogen-starved Dunaliella salina, a unicellular green microalga. J. Biotechnol. 162, 21–27. doi: 10.1016/j.jbiotec. 2012.04.018 Olszanecki, R., Rezzani, R., Omura, S., Stec, D. E., Rodella, L., Botros, F. T., et al. (2007). Genetic suppression of HO-1 exacerbates renal damage: reversed by an increase in the antiapoptotic signaling pathway. Am. J. Physiol. Renal Physiol. 292, F148–F157. doi: 10.1152/ajprenal.00261.2006 Orr-Weaver, T. L., and Szostak, J. W. (1983). Yeast recombination: the association between double-strand gap repair and crossing-over. Proc. Natl. Acad. Sci. U.S.A. 80, 4417–4421. doi: 10.1073/pnas.80.14.4417 Lamont, C. M., and Sargent, F. (2017). Design and characterisation of synthetic operons for biohydrogen technology. Arch. Microbiol. 199, 495–503. doi: 10. 1007/s00203-016-1322-5 Lane, S., Zhang, S., Wei, N., Rao, C., and Jin, Y. S. (2015). Development and physiological characterization of cellobiose-consuming Yarrowia lipolytica. Biotechnol. Bioeng. 112, 1012–1022. doi: 10.1002/bit. 25499 Pomraning, K. R., Wei, S., Karagiosis, S. A., Kim, Y.-M., Dohnalkova, A. C., Arey, B. W., et al. (2015). Comprehensive metabolomic, lipidomic and microscopic proﬁling of Yarrowia lipolytica during lipid accumulation identiﬁes targets for increased lipogenesis. PLoS One 10:e0123188. doi: 10.1371/journal.pone. 0123188 Lazar, Z., Walczak, E., and Robak, M. (2011). Simultaneous production of citric acid and invertase by Yarrowia lipolytica SUC+ transformants. Bioresour. Technol. 102, 6982–6989. doi: 10.1016/j.biortech.2011.04.032 Rashid, M., Runci, A., Polletta, L., Carnevale, I., Morgante, E., Foglio, E., et al. (2015). Muscle LIM protein/CSRP3: a mechanosensor with a role in autophagy. Cell Death Discov. 1:15014. doi: 10.1038/cddiscovery. 2015.14 Ledesma-Amaro, R., Lazar, Z., Rakicka, M., Guo, Z., Fouchard, F., Crutz-Le Coq, A.-M., et al. (2016). Metabolic engineering of Yarrowia lipolytica to produce chemicals and fuels from xylose. Metab. Eng. 38, 115–124. doi: 10.1016/j.ymben. 2016.07.001 Ratledge, C., and Wynn, J. P. (2002). The biochemistry and molecular biology of lipid accumulation in oleaginous microorganisms. Adv. Appl. Microbiol. 51, 1–51. doi: 10.1016/S0065-2164(02)51000-5 Ledesma-Amaro, R., and Nicaud, J.-M. (2016). Yarrowia lipolytica as a biotechnological chassis to produce usual and unusual fatty acids. Prog. Lipid Res. 61, 40–50. doi: 10.1016/j.plipres.2015.12.001 Rodriguez, G. M., Hussain, M. S., Gambill, L., Gao, D., Yaguchi, A., and Blenner, M. (2016). Engineering xylose utilization in Yarrowia lipolytica by understanding its cryptic xylose pathway. Biotechnol. Biofuels 9:149. doi: 10.1186/s13068-016- 0562-6 Li, H., and Alper, H. S. (2016). Enabling xylose utilization in Yarrowia lipolytica for lipid production. Biotechnol. J. 11, 1230–1240. doi: 10.1002/biot.201600210 Ruijter, J. C., Koskela, E. V., Nandania, J., Frey, A. REFERENCES Integration event induced changes in recombinant protein productivity in Pichia pastoris discovered by whole genome sequencing and derived vector optimization. Microb. Cell Fact. 15:84. doi: 10.1186/s12934-016- 0486-7 Mirbagheri, M., Nahvi, I., Emtiazi, G., Mafakher, L., and Darvishi, F. (2012). Taxonomic characterization and potential biotechnological applications of Yarrowia lipolytica isolated from meat and meat products. Jundishapur J. Microbiol. 5, 346–351. Miro´nczuk, A. M., Biegalska, A., and Dobrowolski, A. (2017). Functional overexpression of genes involved in erythritol synthesis in the yeast Yarrowia lipolytica. Biotechnol. Biofuels 10:77. doi: 10.1186/s13068-017-0772-6 Selig, M. J., Thygesen, L. G., and Felby, C. (2014). Correlating the ability of lignocellulosic polymers to constrain water with the potential to inhibit cellulose sacchariﬁcation. Biotechnol. Biofuels 7:159. doi: 10.1186/s13068-014- 0159-x Miro´nczuk, A. M., Rzechonek, D. A., Biegalska, A., Rakicka, M., and Dobrowolski, A. (2016). A novel strain of Yarrowia lipolytica as a platform for value-added product synthesis from glycerol. Biotechnol. Biofuels 9:180. doi: 10.1186/s13068-016-0593-z Sharma, K. K., Schuhmann, H., and Schenk, P. M. (2012). High lipid induction in microalgae for biodiesel production. Energies 5, 1532–1553. doi: 10.3390/ en5051532 Munkhbayar, B., Nine, M. J., Jeoun, J., Bat-Erdene, M., Chung, H., and Jeong, H. (2013). Inﬂuence of dry and wet ball milling on dispersion characteristics of the multi-walled carbon nanotubes in aqueous solution with and without surfactant. Powder Technol. 234, 132–140. doi: 10.1016/j.powtec.2012. 09.045 Singh, A., Taylor, L. E., Vander Wall, T. A., Linger, J., Himmel, M. E., Podkaminer, K., et al. (2015). Heterologous protein expression in Hypocrea jecorina: a historical perspective and new developments. Biotechnol. Adv. 33, 142–154. doi: 10.1016/j.biotechadv.2014.11.009 Natter, K., Leitner, P., Faschinger, A., Wolinski, H., Mccraith, S., Fields, S., et al. (2005). The spatial organization of lipid synthesis in the yeast Saccharomyces cerevisiae derived from large scale green ﬂuorescent protein tagging and high resolution microscopy. Mol. Cell. Proteom. 4, 662–672. doi: 10.1074/mcp. M400123-MCP200 Singh, P., Guldhe, A., Kumari, S., Rawat, I., and Bux, F. (2015). Investigation of combined eﬀect of nitrogen, phosphorus and iron on lipid productivity of microalgae Ankistrodesmus falcatus KJ671624 using response surface methodology. Biochem. Eng. J. 94, 22–29. doi: 10.1016/j.bej.2014.10.019 Singh, P., Kumari, S., Guldhe, A., Misra, R., Rawat, I., and Bux, F. (2016). Trends and novel strategies for enhancing lipid accumulation and quality in microalgae. Renew. Sustain. Energy Rev. 55, 1–16. doi: 10.1016/j.rser.2015. 11.001 Olson, D. G., Mcbride, J. E., Shaw, A. J., and Lynd, L. R. (2012). Recent progress in consolidated bioprocessing. Curr. Opin. Biotechnol. REFERENCES Microb. Cell Fact. 16:126. doi: 10.1186/s12934-017-0742-5 Tai, M., and Stephanopoulos, G. (2013). Engineering the push and pull of lipid biosynthesis in oleaginous yeast Yarrowia lipolytica for biofuel production. Metab. Eng. 15, 1–9. doi: 10.1016/j.ymben.2012.08.007 Yamada, R., Nakatani, Y., Ogino, C., and Kondo, A. (2013). Eﬃcient direct ethanol production from cellulose by cellulase-and cellodextrin transporter-co- expressing Saccharomyces cerevisiae. AMB Express 3, 1–7. doi: 10.1186/2191- 0855-3-34 Thanonkeo, P., Laopaiboon, P., Sootsuwan, K., and Yamada, M. (2007). Magnesium ions improve growth and ethanol production of Zymomonas mobilis under heat or ethanol stress. Biotechnology 6, 112–119. doi: 10.3923/ biotech.2007.112.119 Tsigie, Y. A., Huynh, L. H., Ahmed, I. N., and Ju, Y.-H. (2012). Maximizing biodiesel production from Yarrowia lipolytica Po1g biomass using subcritical water pretreatment. Bioresour. Technol. 111, 201–207. doi: 10.1016/j.biortech. 2012.02.052 Yang, S., Wang, W., Wei, H., Van Wychen, S., Pienkos, P. T., Zhang, M., et al. (2016). Comparison of nitrogen depletion and repletion on lipid production in yeast and fungal species. Energies 9:685. doi: 10.3390/en9090685 Ye, R. W., Sharpe, P. L., and Zhu, Q. (2012). Bioengineering of oleaginous yeast Yarrowia lipolytica for lycopene production. Methods Mol. Biol. 898, 153–159. doi: 10.1007/978-1-61779-918-1_9 Vallejo-Becerra, V., Marín-Zamora, M. E., Vásquez-Bahena, J. M., Rojas- Melgarejo, F., Hidalgo-Lara, M. E., and García-Ruiz, P. A. (2008). Immobilization of recombinant invertase (re-INVB) from Zymomonas mobilis on D-sorbitol cinnamic ester for production of invert sugar. J. Agric. Food Chem. 56, 1392–1397. doi: 10.1021/jf072646h Yu, X., Chen, L., and Zhang, W. (2015). Chemicals to enhance microalgal growth and accumulation of high-value bioproducts. Front. Microbiol. 6:56. doi: 10. 3389/fmicb.2015.00056 Zhang, B., Chen, H., Li, M., Gu, Z., Song, Y., Ratledge, C., et al. (2013). Genetic engineering of Yarrowia lipolytica for enhanced production of trans-10, cis-12 conjugated linoleic acid. Microb. Cell fact. 12:70. doi: 10.1186/1475-2859-12-70 van Rensburg, E., Den Haan, R., Smith, J., Van Zyl, W. H., and Gorgens, J. F. (2012). The metabolic burden of cellulase expression by recombinant Saccharomyces cerevisiae Y294 in aerobic batch culture. Appl. Microbiol. Biotechnol. 96, 197– 209. doi: 10.1007/s00253-012-4037-9 Zhang, C., Zhang, Y., Zhuang, B., and Zhou, X. (2014). Strategic enhancement of algal biomass, nutrient uptake and lipid through statistical optimization of nutrient supplementation in coupling Scenedesmus obliquus-like microalgae cultivation and municipal wastewater treatment. Bioresour. Technol. 171, 71–79. doi: 10.1016/j.biortech.2014.07.060 Van Zyl, J. H. D., Den Haan, R., and Van Zyl, W. H. (2016). Overexpression of native Saccharomyces cerevisiae ER-to-Golgi SNARE genes increased heterologous cellulase secretion. Appl. Microbiol. REFERENCES 23, 396–405. doi: 10.1016/j. copbio.2011.11.026 January 2019 \| Volume 9 \| Article 3276 Frontiers in Microbiology \| www.frontiersin.org 18 Ameliorating Cellulases Expression Metabolic Burden Wei et al. Sitepu, I. R., Garay, L. A., Sestric, R., Levin, D., Block, D. E., German, J. B., et al. (2014). Oleaginous yeasts for biodiesel: current and future trends in biology and production. Biotechnol. Adv. 32, 1336–1360. doi: 10.1016/j.biotechadv.2014. 08.003 Wei, H., Wang, W., Alahuhta, M., Vander Wall, T., Baker, J. O., Taylor, L. E., et al. (2014). Engineering towards a complete heterologous cellulase secretome in Yarrowia lipolytica reveals its potential for consolidated bioprocessing. Biotechnol. Biofuels 7:148. doi: 10.1186/s13068-014-0148-0 Sitepu, I. R., Sestric, R., Ignatia, L., Levin, D., German, J. B., Gillies, L. A., et al. (2013). Manipulation of culture conditions alters lipid content and fatty acid proﬁles of a wide variety of known and new oleaginous yeast species. Bioresour. Technol. 144, 360–369. doi: 10.1016/j.biortech.2013.06.047 Wei, H., Wang, W., Yarbrough, J. M., Baker, J. O., Laurens, L., Van Wychen, S., et al. (2013). Genomic, proteomic, and biochemical analyses of oleaginous Mucor circinelloides: evaluating its capability in utilizing cellulolytic substrates for lipid production. PLoS One 8:e71068. doi: 10.1371/journal.pone.007 1068 Technol. 144, 360–369. doi: 10.1016/j.biortech.2013.06.047 Sootsuwan, K., Thanonkeo, P., Keeratirakha, N., Thanonkeo, S., Jaisil, P., and Yamada, M. (2013). Sorbitol required for cell growth and ethanol production by Zymomonas mobilis under heat, ethanol, and osmotic stresses. Biotechnol. Biofuels 6:180. doi: 10.1186/1754-6834-6-180 Wood, T. M., and Mahalingeshwara Bhat, K. (1988). Methods for measuring cellulase activities. Methods Enzymol. 160, 87–112. doi: 10.1016/0076-6879(88) 60109-1 Strom, A. R., Olafsen, J. A., and Larsen, H. (1979). Trimethylamine oxide: a terminal electron acceptor in anaerobic respiration of bacteria. Microbiology 112, 315–320. Xie, D. (2017). Integrating cellular and bioprocess engineering in the non- conventional yeast yarrowia lipolytica for biodiesel production: a review. Front. Bioeng. Biotechnol. 5:65. doi: 10.3389/fbioe.2017.00065 Sun, J., Wen, F., Si, T., Xu, J.-H., and Zhao, H. (2012). Direct conversion of xylan to ethanol by recombinant Saccharomyces cerevisiae strains displaying an engineered minihemicellulosome. Appl. Environ. Microbiol. 78, 3837–3845. doi: 10.1128/AEM.07679-11 Xin, Z., Yinbo, Q., and Peiji, G. (1993). Acceleration of ethanol production from paper mill waste ﬁber by supplementation with Î2-glucosidase. Enzyme Microb. Technol. 15, 62–65. doi: 10.1016/0141-0229(93)90117-K Xu, Q., Knoshaug, E. P., Wang, W., Alahuhta, M., Baker, J. O., Yang, S., et al. (2017). Expression and secretion of fungal endoglucanase II and chimeric cellobiohydrolase I in the oleaginous yeast Lipomyces starkeyi. January 2019 \| Volume 9 \| Article 3276 Copyright © 2019 Wei, Wang, Alper, Xu, Knoshaug, Van Wychen, Lin, Luo, Decker, Himmel and Zhang. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. REFERENCES Biotechnol. 100, 505–518. doi: 10.1007/s00253-015-7022-2 Zhu, Q., and Jackson, E. N. (2015). Metabolic engineering of Yarrowia lipolytica for industrial applications. Curr. Opin. Biotechnol. 36, 65–72. doi: 10.1016/j.copbio. 2015.08.010 Wagih, O., Usaj, M., Baryshnikova, A., Vandersluis, B., Kuzmin, E., Costanzo, M., et al. (2013). SGAtools: one-stop analysis and visualization of array-based genetic interaction screens. Nucleic Acids Res. 41, W591–W596. doi: 10.1093/ nar/gkt400 Conﬂict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. Wang, W., Wei, H., Alahuhta, M., Chen, X., Hyman, D., Johnson, D. K., et al. (2014a). Heterologous expression of xylanase enzymes in lipogenic yeast Yarrowia lipolytica. PLoS One 9:e111443. doi: 10.1371/journal.pone.0111443 Wang, W., Yang, S., Hunsinger, G. B., Pienkos, P. T., and Johnson, D. K. (2014b). Connecting lignin-degradation pathway with pre-treatment inhibitor sensitivity of Cupriavidus necator. Front. Microbiol. 5:247. doi: 10.3389/fmicb. 2014.00247 Copyright © 2019 Wei, Wang, Alper, Xu, Knoshaug, Van Wychen, Lin, Luo, Decker, Himmel and Zhang. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Copyright © 2019 Wei, Wang, Alper, Xu, Knoshaug, Van Wychen, Lin, Luo, Decker, Himmel and Zhang. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Wei, H., Tucker, M. P., Baker, J. O., Harris, M., Luo, Y., Xu, Q., et al. (2012). Tracking dynamics of plant biomass composting by changes in substrate structure, microbial community, and enzyme activity. Biotechnol. Biofuels 5:20. doi: 10.1186/1754-6834-5-20 January 2019 \| Volume 9 \| Article 3276 Frontiers in Microbiology \| www.frontiersin.org 19
https://openalex.org/W2898410112	https://estudogeral.sib.uc.pt/bitstream/10316/92623/1/MASK_2017%28CTA19%29.pdf	English	null	MASK 2017: ARIA digitally-enabled, integrated, person-centred care for rhinitis and asthma multimorbidity using real-world-evidence	Clinical and translational allergy	2,018	cc-by	17,854	MASK 2017: ARIA digitally‑enabled, integrated, person‑centred care for rhinitis and asthma multimorbidity using real‑world‑evidence J. Bousquet1,2,3, S. Arnavielhe4, A. Bedbrook1, M. Bewick5, D. Laune4, E. Mathieu‑Dupas4, R. Murray6, G. L. Onorato1, J. L. Pépin7,8, R. Picard9, F. Portejoie1, E. Costa10, J. Fonseca11,12, O. Lourenço13, M. Morais‑Almeida14, A. Todo‑Bom15, A. A. Cruz16,17, J. da Silva18, F. S. Serpa19, M. Illario20, E. Menditto21, L. Cecchi22, R. Monti23, L. Napoli24, M. T. Ventura25, G. De Feo26, D. Larenas‑Linnemann27, M. Fuentes Perez28, Y. R. Huerta Villabolos28, D. Rivero‑Yeverino29, E. Rodriguez‑Zagal30, F. Amat31,32, I. Annesi‑Maesano33, I. Bosse34, P. Demoly35, P. Devillier36, J. F. Fontaine37, J. Just31,32, T. P. Kuna38, B. Samolinski39, A. Valiulis40,41, R. Emuzyte42, V. Kvedariene43, D. Ryan44,45, A. Sheikh46, P. Schmidt‑Grendelmeier47, L. Klimek48,49, O. Pfaar48,49, K. C. Bergmann50,51, R. Mösges52,53, T. Zuberbier50,51, R. E. Roller‑Wirnsberger54, P. Tomazic55, W. J. Fokkens56, N. H. Chavannes57, S. Reitsma56, J. M. Anto58,59,60,61, V. Cardona62, T. Dedeu63,64, J. Mullol65,66, T. Haahtela67, J. Salimäki68, S. Toppila‑Salmi67, E. Valovirta69,70, B. Gemicioğlu71, A. Yorgancioglu72,73, N. Papadopoulos74,75, E. P. Prokopakis76, S. Bosnic‑Anticevich77, R. O’Hehir78,79, J. C. Ivancevich80, H. Neffen81, E. Zernotti82, I. Kull83, E. Melen84,85, M. Wickman86, C. Bachert87, P. Hellings3,88,89, S. Palkonen90, C. Bindslev‑Jensen91, E. Eller91, S. Waserman92, M. Sova93, G. De Vries94, M. van Eerd94, I. Agache95, T. Casale96, M. Dykewickz97, R. N. Naclerio98, Y. Okamoto99, D. V. Wallace100 and MASK study group Bousquet et al. Clin Transl Allergy (2018) 8:45 https://doi.org/10.1186/s13601-018-0227-6 Bousquet et al. Clin Transl Allergy (2018) 8:45 https://doi.org/10.1186/s13601-018-0227-6 Clinical and Translational Allergy Open Access Background evidence based approach [1, 23–25] to care pathways using mobile technology in AR and asthma multimorbid- ity [26]. ARIA appears to be close to the patient’s needs but real-life data suggest that few patients follow guide- line recommendations and that they often self-medicate. Moreover, patients frequently using OTC medications dispensed in pharmacies [27]. Shared decision making (SDM) centered around the patient for self-management should be used more often. g Allergic rhinitis (AR) is the most common chronic disease worldwide. Evidence-based guidelines have improved knowledge on rhinitis and made a significant impact on AR management. However, many patients remain inadequately controlled and the costs for society are enormous, in particular due to the major impact of AR on school and work productivity [1, 2]. Unmet needs have identified clearly many gaps. These include (1) sub- optimal rhinitis and asthma control due to medical, cul- tural and social barriers [3, 4], (2) poor understanding of endotypes [5], better characterization of phenotypes and multimorbidities [6], better understanding of gen- der differences [7], (3) assessment of sentinel networks in care pathways for allergen and pollutants exposures, using symptom variation [8], (4) lack of stratification of patients for optimized care pathways [9] and (5) lack of multidisciplinary teams within integrated care pathways, endorsing innovation in real life clinical trials [8] and encouraging patient empowerment [10, 11]. Mobile Airways Sentinel networK (MASK), the Phase 3 ARIA initiative, has been initiated to reduce the global burden of rhinitis and asthma multimorbidity, giving the patient and the health care professional simple tools to better prevent and manage respiratory allergic diseases. More specifically, MASK is focusing on (1) understand- ing the disease mechanisms and the effects of air pollu- tion in allergic diseases and asthma, (2) better appraising the burden incurred by medical needs and indirect costs, (3) the implementation of multi-sectoral care pathways integrating self-care, air pollution and patient’s literacy, using emerging technologies with real world data using the AIRWAYS ICPs algorithm [28], (4) proposing indi- vidualized and predictive medicine in rhinitis and asthma multimorbidity, (5) proposing the basis for a sentinel network at the global level for pollution and allergy and (6) assessing the societal implications of exposure to air pollution and allergens and its consequences on health inequalities globally. g g p p Mobile health (mHealth) is the use of information and communication technology (ICT) for health services and information transfer [12]. Background mHealth, including apps run- ning on consumer smart devices (i.e., smartphones and tablets), is becoming increasingly popular and has the potential to profoundly impact on healthcare [13]. Novel app-based collaborative systems can have an important role in gathering information quickly and improving coverage and accessibility of prevention and treatment [14]. Implementing mHealth innovations may also have disruptive consequences [15], so it is important to test applicability in each individual situation [16]. A rapid growth of the health apps market has been seen with an estimated 325,000 health apps available in 2017 for most fields of medicine [17]. Benefits and drawbacks have been estimated for a number of disease [18]. The application of mHealth solutions can support the provision of high quality care to patients with AR or asthma, to the satisfac- tion of both patients and health care professionals, with a reduction in both health care utilization and costs [19]. Appropriately identifying and representing stakeholders’ interests and viewpoints in evaluations of mHealth is a critical part of ensuring continued progress and innova- tion [20]. Patient, caregiver and clinician evaluations and recommendations play an important role in the develop- ment of asthma mHealth tools to support the provision of asthma management [21]. Smart devices and inter- net-based applications are already used in rhinitis and asthma and may help to address some unmet needs [22]. However, these new tools need to be tested and evaluated for acceptability, usability and cost-effectiveness. The freely available MASK app (the Allergy Diary, Android and iOS) [26] is combined with an inter-oper- able tablet for physicians and other health care profes- sionals (HCPs [29]), using the same extremely simple colloquial language to manage AR (Visual Analogue Scale: VAS) [30, 31]. It is being combined with data on allergen and pollution exposure (POLLAR). MASK will be scaled up using the EU EIP on AHA strategy [32]. Phase 4 is starting in 2018 and will focus on “change management”. MASK is supported by several EU grants and is a WHO GARD (Global Alliance against Chronic Respiratory Diseases) research demonstration project (Table 1). Abstract mHealth, such as apps running on consumer smart devices is becoming increasingly popular and has the potential to profoundly affect healthcare and health outcomes. However, it may be disruptive and results achieved are not always reaching the goals. Allergic Rhinitis and its Impact on Asthma (ARIA) has evolved from a guideline using the best evidence-based approach to care pathways suited to real-life using mobile technology in allergic rhinitis (AR) and asthma multimorbidity. Patients largely use over-the-counter medications dispensed in pharmacies. Shared decision making centered around the patient and based on self-management should be the norm. Mobile Airways Sentinel networK (MASK), the Phase 3 ARIA initiative, is based on the freely available MASK app (the Allergy Diary, Android and iOS platforms). MASK is available in 16 languages and deployed in 23 countries. The present paper provides an over‑ view of the methods used in MASK and the key results obtained to date. These include a novel phenotypic charac‑ terization of the patients, confirmation of the impact of allergic rhinitis on work productivity and treatment patterns in real life. Most patients appear to self-medicate, are often non-adherent and do not follow guidelines. Moreover, the Allergy Diary is able to distinguish between AR medications. The potential usefulness of MASK will be further explored by POLLAR (Impact of Air Pollution on Asthma and Rhinitis), a new Horizon 2020 project using the Allergy Diary. Keywords: App, ARIA, Asthma, Care pathways, MASK, mHealth, Rhinitis Correspondence: jean.bousquet@orange.fr 1 MACVIA‑France, Fondation Partenariale FMC VIA-LR, CHRU Arnaud de Villeneuve, 371 Avenue du Doyen Gaston Giraud, Montpellier, France Full list of author information is available at the end of the article *Correspondence: jean.bousquet@orange.fr 1 MACVIA‑France, Fondation Partenariale FMC VIA-LR, CHRU Arnaud de Villeneuve, 371 Avenue du Doyen Gaston Giraud, Montpellier, France Full list of author information is available at the end of the article © The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Bousquet et al. Clin Transl Allergy (2018) 8:45 Page 2 of 21 Methods Usersh The Allergy Diary is used by people who searched the internet, Apple App store, Google Play or in any other way. The pages of the App are on the Euforea-ARIA web- site (www.euforea.eu/about-us/aria.html). A few users were clinic patients to whom the app was recommended by their physicians. Users were not requested to com- plete the diary for a minimum number of days. However, due to anonymization of data, no specific information on the route of access to the app could be gathered [33, 34]. f Allergic Rhinitis and its Impact on Asthma (ARIA) has evolved from an evidence-based guideline using the best Bousquet et al. Clin Transl Allergy (2018) 8:45 Page 3 of 21 Bousquet et al. Clin Transl Allergy Table 1 European Union and World Health Organization links of ARIA and MASK Date WHO EU ARIA 1999 Workshop WHO HQ 2003–2013 CC rhinitis and asthma Montpellier 2012– GARD demonstration project WHO HQ 2004–2010 GA2LEN FP6 2011–2015 MeDALL FP7 MASK 2014– MACVIA-LR DG Santé-CNECT 2014– GARD demonstration project WHO HQ 2014– EIP on AHA B3 DG Santé-CNECT 2015–2016 SPAL Structural and develop‑ ment funds 2015–2017 Sunfrail 2017– Twinning DG Santé-CNECT 2018– POLLAR EIT Health Table 1 European Union and World Health Organization links of ARIA and MASK The first question of the App is “I have allergic rhini- tis”: Yes/No. We tested the sensitivity and specificity of this question [33]. 93.4% users with a positive answer had nasal symptoms versus 12.1% of users with a negative answer. In the first two versions of the App, allergy was not considered in the user’s questionnaire and AR cannot be differentiated from chronic rhinosinusitis. It is now included in the third version of the App (June 2018) and we will be able to answer more appropriately to this ques- tion in the next study. The results of the pilot study were confirmed in over 9000 users. data but also to be considered as an identifier itself [38, 39]. Processing personal data by means of an app, like e.g. App Diary, besides Directive 95/46/EC [37] also Directive 2002/58/EC [40] as amended by Directive 2009/136/EC [41] applies. Geolocation was studied for all people who used the Allergy Diary App from December 2015 to November 2017 and who reported medical outcomes. Privacy assessment impact MASK is available in 23 countries and 16 languages. To date (01-09-2018) the app has been used by over 24,000 people. Privacy impact assessments (PIAs), also known as data protection impact assessments (DPIAs) in EU law, is required by GDPR (Article 35 Working Party (WP35). PIA is a systematic process to assess privacy risks to individuals in the collection, use, and disclosure of their personal data. The GDPR introduced PIAs to identify high risks to the privacy rights of individuals when pro- cessing their personal data. The assessment shall con- tain at least: Ethics and privacy of datah The Allergy Diary is CE1 registered. The terms of use were translated into all languages and customized by law- yers according to the legislation of each country, allow- ing the use of the results for research and commercial purposes. The example of the UK terms of use have been provided in a previous paper [33]. 1. a systematic description of the envisaged process- ing operations and the purposes of the processing, including, where applicable, the legitimate interest pursued by the controller; Methods Usersh In contradis- tinction to noise addition (randomization), k-anonymity [42, 43] is an acceptable method for the anonymization of MASK data (generalization) [44] and results can be used for other databases. Geolocation EU data protection rules have changed since the imple- mentation of the General Data Protection Regulation (Art. 4 para. 1 no. 1 GDPR) [35]. Data anonymization is a method of sanitization for privacy. Anonymization ren- ders personal data “in such a manner that the data sub- ject is not or no longer identifiable” [36]. The European Commission’s Article 29 Working Party (WP29) stated already in 2014 with regards to the Directive 95/46/EC [37] that geolocation information is not only personal 2. an assessment of the necessity and proportionality of the processing operations in relation to the purposes; 3. an assessment of the risks to the rights and freedoms of data subjects and 4. the measures envisaged to address the risks, includ- ing safeguards, security measures and mechanisms to ensure the protection of personal data and to dem- Page 4 of 21 Bousquet et al. Clin Transl Allergy (2018) 8:45 Bousquet et al. Clin Transl Allergy Table 2 Questions on symptoms and impact of symptoms (from Bousquet et al. [33]) Table 2 Questions on symptoms and impact of symptoms (from Bousquet et al. [33]) onstrate compliance with this Regulation taking into account the rights and legitimate interests of data subjects and other persons concerned. When these risks are identified, the GDPR expects that an organization formulates measures to address these risks. Those measures may take the form of technical controls such as encryption or anonymization of data.h The PIA analysis is a self-declarative analysis. In France, the local GDPR representative (Commission Informa- tique et Liberté, CNIL) has provided a software to guide the reflexion around security of personal data and the exposure risks in case of security fails. This software has been used to assess all the risks to be considered through the app uses. The conclusion was that is “negligeable”.hi Transfer of personal data from the App to a print Patients cannot give access to their electronic data to a HCP due to privacy policies. However, they can eas- ily print the daily control of their disease and the medi- cations that they filled in the Allergy Diary as follows (Fig. 3). h The field is moving very fast. In France, June, 10 2018, the modified law “LIL” (Loi Informatique et Liberté, 2018-493, https://www.cnil.fr/fr/loi-78-17-du-6-janvi er-1978-modifiee) was enacted with a special focus on health-related personal data. Allergy Diaryh The app collects information on AR and asthma symp- toms experienced (nasal and ocular) and on disease type (intermittent/persistent) [33] (Table 3). Anonymized and geolocalized users assess daily how symptoms impact their control and AR treatment using the touchscreen functionality on their smart phone to click on five con- secutive VAS (i.e. general, nasal and ocular symptoms, asthma and work) (Table 2; Fig. 1). Users input their daily medications using a scroll list that contains all coun- try-specific OTC and prescribed medications available (Fig. 2). The list populated using IMS data and revised by country experts is continuously revised by country experts. Additional questionnaires MASK also includes EQ-5D (EuroQuol) [46–48], Work Productivity and Activity Impairment Allergic Specific (WPAI-AS) [49] and Control of AR and Asthma Test (CARAT) [50–53]. The Epworth Sleepiness Question- naire [54, 55] is included (June 2018). Adherence to treatment Globally, non-adherence to medications is a major obstacle to the effective delivery of health care. Many mobile phone apps are available to support people to take their medications and to improve medication adherence [57, 58]. However, a recent meta-analysis found that the majority did not have many of the desir- able features and were of low quality [57]. However, it is unknown how people use apps, what is consid- ered adherent or non-adherent in terms of app usage, or whether adherence with an app in anyway reflects adherence with medication or control. There is a high degree of correlation between these VAS measurements. The example of VAS global meas- ured and VAS nose is presented in Fig. 2. Medications A scroll list is available for all OTC and prescribed medications of the 23 countries. The International Non- proprietary Names classification was used for drug nomenclature [56]. 85 INNs and 505 medications were identified (Fig. 1). Geolocation Even if the articulation of GDPR and LIL is still unclear, we can anticipate that the app use will remain risk free. Outcomes Five VAS measurements [VAS-global measured, VAS- nose, VAS-eye, VAS-asthma and VAS-work (Table 4)] and a calculated VAS-global score (VAS-nasal + VAS- ocular divided by 2) were assessed [34]. VAS levels range from zero (not at all bothersome) to 100 (very bother- some). Independency of VAS questions was previously confirmed using the Bland and Altman regression analy- sis [34, 45]. In MASK, we did not use adherence questionnaires but first attempted to assess short-term adherence and then to address the long-term issues. [59]. Digitalized ARIA symptom‑medication score Symptom-medication scores are needed to assess the control of allergic diseases. They are currently being Page 5 of 21 Bousquet et al. Clin Transl Allergy (2018) 8:45 Bousquet et al. Clin Transl Allergy developed for MASK and are being compared with existing ones [60]. MASK algorithm and clinical decision support system best evidence and algorithms to aid patients and health care professionals to jointly determine the treatment and its step-up or step-down strategy for an optimal disease control. Fig. 1 Allergy Diary screens relating to Visual Analogue Scale and medications (from Bousquet et al. [26]) Fig. 1 Allergy Diary screens relating to Visual Analogue Scale and medications (from Bousquet et al. [26]) Fig. 1 Allergy Diary screens relating to Visual Analogue Scale and medications (from Bousquet et al. [26]) Fig. 1 Allergy Diary screens relating to Visual Analogue Scale and medications (from Bousquet et al. [26]) developed for MASK and are being compared with existing ones [60]. best evidence and algorithms to aid patients and health care professionals to jointly determine the treatment and its step-up or step-down strategy for an optimal disease control. MASK algorithm and clinical decision support system The selection of pharmacotherapy for AR patients depends on several factors, including age, prominent symptoms, symptom severity, AR control, patient preferences and cost. Allergen exposure, pollution and resulting symptoms vary, needing treatment Clinical decision support systems (CDSS) are software algorithms that advise health care providers on the diagnosis and management of patients based on the interaction of patient data and medical information, such as prescribed drugs. CDSS should be based on the Bousquet et al. Clin Transl Allergy (2018) 8:45 Page 6 of 21 Fig. 2 Correlation between Visual Analog Scale (VAS) global measured and nasal symptoms (VAS nose) (unpublished) Fig. 2 Correlation between Visual Analog Scale (VAS) global measured and nasal symptoms (VAS nose) (unpublished) • Equity. • Practice. • Ethical considerations. • Evaluation. • Empowerment and participation. • Target population. • Sustainability. • Governance. • Scalability adjustment. In AR, The MASK CDSS is incorporated into an interoperable tablet [29] for HCPs (ARIA Allergy Diary Companion) [10, 26]. This is based on an algorithm to aid clinicians to select pharmacotherapy for AR patients and to stratify their disease severity [26] (Fig. 4). It uses a simple step-up/step-down individual- ized approach to AR pharmacotherapy and may hold the potential for optimal control of symptoms, while minimizing side-effects and costs. However, its use var- ies depending on the availability of medications in the different countries and on resources. The algorithm is now digitalized and available in English (Fig. 5). • Empowerment and participation. • Target population. • Sustainability. • Governance. • Scalability As part of SUNFRAIL, MASK tested the 27 item cri- teria of CHRODIS and was found to be an example of Good Practice [62]. As part of SUNFRAIL, MASK tested the 27 item cri- teria of CHRODIS and was found to be an example of Good Practice [62]. MASK follows the CHRODIS criteria of “Good Practice” The European Commission is co-funding a large col- laborative project named JA-CHRODIS in the context of the 2nd EU Health Programme 2008–2013 [61]. JA- CHRODIS has developed a check-list of 27 items for the evaluation of Good Practices (GP) (http://chrodis.eu/ our-work/04-knowledge-platform/). According to the JA-CHRODIS, a Good Practice has been proven to work well and produce good results, and is therefore recom- mended as a model to be scaled up. The JA-CHRODIS criteria are grouped into nine categories: Validation of the MASK Visual Analogue Scale on cell phones VAS included in the Allergy Diary was found to be a vali- dated tool to assess control in AR patients following COS- MIN guidelines [63] in 1225 users and 14,612 days: internal consistency (Cronbach’s α-coefficient > 0.84 and test– retest > 0.7), reliability (intra-class correlation coefficients), sensitivity and acceptability [64]. In addition, e-VAS had a good reproducibility when users (n = 521) answered the e-VAS twice in less than 3 h. Pilot study of mobile phone technology in AR y p gy A pilot study in 3260 users found that Allergy Diary users were able to properly provide baseline simple phenotypic characteristics. Troublesome symptoms were found mainly in the users with the largest number of symptoms. Around 50% of users with troublesome rhinitis and/or ocular symptoms suffered work impairment. Sleep was impaired by troublesome symptoms and nasal obstruction (Fig. 6). results suggest novel concepts and research questions in AR that may not be identified using classical methods [33]. Bousquet et al. Clin Transl Allergy (2018) 8:45 Page 7 of 21 Fig. 3 Transfer of patient information on a computer and printed information (from Bousquet et al. [46] Fig. 3 Transfer of patient information on a computer and printed information (from Bousquet et al. [46] mHealth tools (i.e. the Allergy Diary and CARAT) in 23 Reference Sites or regions across Europe and Argentina, Australia, Brazil and Mexico [46]. This will improve understanding, assessment of burden, diagno- sis and management of rhinitis in the elderly by com- parison with an adult population. The pilot study has been completed in Germany and the project is fully operative using two protocols (Table 3). Transfer of innovation of AR and asthma multimorbidity in the elderly: Reference Site Twinning (EIP on AHA) AR impairs social life, work and school productivity. Indirect costs associated with lost work productivity are the principal contributor to the total AR costs and result mainly from impaired work performance by pres- enteeism [2]. The severity of AR symptoms was the most consistent disease-related factor associated with impact of AR on work productivity, although ocular symptoms and sleep disturbances may independently affect work The EIP on AHA includes 74 Reference Sites. The aim of this TWINNING was to transfer innovation from the MASK App to other reference sites. The phenotypic characteristics of rhinitis and asthma multimorbidity in adults and the elderly are compared using validated Bousquet et al. Clin Transl Allergy (2018) 8:45 Bousquet et al. Clin Transl Allergy Page 8 of 21 productivity. Overall, the pharmacologic treatment of AR showed a beneficial effect on work productivity. A cross-sectional study using Allergy diary in productivity of uncontrolled AR assessed by VAS [34]. In users with uncontrolled rhinitis (VAS global meas- ured≥50) approximately 90% had some work impair- Fig. 4 Clinical decision support systems consensus for allergic rhinitis (from Bousquet et al. [28]) Fig. 5 CDSS digitalization (submitted) Fig. 4 Clinical decision support systems consensus for allergic rhinitis (from Bousquet et al. [28]) Fig. 4 Clinical decision support systems consensus for allergic rhinitis (from Bousquet et al. [28]) Fig. 4 Clinical decision support systems consensus for allergic rhinitis (from Bousquet et al. [28]) productivity. Overall, the pharmacologic treatment of AR showed a beneficial effect on work productivity. A cross-sectional study using Allergy diary in 1136 users (5659 days) assessed the impact on work productivity of uncontrolled AR assessed by VAS [34]. In users with uncontrolled rhinitis (VAS global meas- ured ≥ 50), approximately 90% had some work impair- ment and over 50% had severe work impairment Fig. 5 CDSS digitalization (submitted) productivity. Overall, the pharmacologic treatment of AR showed a beneficial effect on work productivity. A cross-sectional study using Allergy diary in 1136 users (5659 days) assessed the impact on work productivity of uncontrolled AR assessed by VAS [34]. In users with uncontrolled rhinitis (VAS global meas- ured ≥ 50), approximately 90% had some work impair- ment and over 50% had severe work impairment Fig. 5 CDSS digitalization (submitted) productivity. Overall, the pharmacologic treatment of AR showed a beneficial effect on work productivity. productivity. Overall, the pharmacologic treatment of AR showed a beneficial effect on work productivity. Transfer of innovation of AR and asthma multimorbidity in the elderly: Reference Site Twinning (EIP on AHA) A cross-sectional study using Allergy diary in 1136 users (5659 days) assessed the impact on work productivity of uncontrolled AR assessed by VAS [34]. In users with uncontrolled rhinitis (VAS global meas- ured ≥ 50), approximately 90% had some work impair- ment and over 50% had severe work impairment if A cross-sectional study using Allergy diary in 1136 users (5659 days) assessed the impact on work Bousquet et al. Clin Transl Allergy (2018) 8:45 Page 9 of 21 Bousquet et al. Clin Transl Allergy Fig. 6 Impact of allergic rhinitis depending on the number of symptoms (from Bousquet et al. [33]) Novel phenotypes of allergic diseases Novel phenotypes of allergic diseases Multimorbidity in allergic airway diseases is well known [6], but no data exist regarding the daily dynamics of symptoms. The Allergy Diary assessed the presence and control of daily allergic multimorbidity (asthma, conjunctivitis, rhinitis) and its impact on work produc- tivity in 4025 users and 32,585 days monitored in 19 countries from May 25, 2015 to May 26, 2016. VAS lev- els < 20/100 were categorized as “Low” burden and VAS levels ≥ 50/100 as “High” burden. VAS global measured levels assessing the global control of the allergic disease were significantly associated with daily allergic multi- morbidity. Eight hypothesis-driven patterns were defined based on “Low” and “High” VAS levels. There were < 0.2% days of Rhinitis Low and Asthma High or Conjunctivi- tis High patterns. There were 5.9% days with a Rhinitis High—Asthma Low pattern. There were 1.7% days with a Rhinitis High—Asthma High—Conjunctivitis Low pat- tern. A novel Rhinitis High—Asthma High—Conjunc- tivitis High pattern was identified in 2.9% days and had the greatest impact on uncontrolled VAS global meas- ured and impaired work productivity (Fig. 9). The mobile technology enabled investigation in a novel approach of the intra-individual variability of allergic multimorbidity using days. It identified an unrecognized extreme pattern of uncontrolled multimorbidity [59]. Fig. 6 Impact of allergic rhinitis depending on the number of symptoms (from Bousquet et al. [33]) (VAS-work ≥ 50). There was a significant correla- tion between VAS-global calculated and VAS-work (Rho = 0.83, p < 0.00001, Spearman rank test). The study has been extended to almost 17,000 days and similar results were observed (Fig. 7).h The baseline study found that bothersome symptoms, nasal obstruction and ocular symptoms were involved in work productivity impact [33] (Fig. 8). Treatment of allergic rhinitis using mobile technology with real world data The Allergy Diary includes the WPAI:AS in six EU countries. All consecutive users who completed the VAS-work from June 1 to July 31, 2016 were included in the study [66]. A highly significant correlation was found between Questions 4 (impairment of work) and 9 (impairment of activities) in 698 users (Rho = 0.85). Large observational implementation studies are needed to triangulate the findings from randomized control tri- als (RCTs) as they reflect “real world” everyday practice. We attempted to provide additional and complemen- tary insights into the real-life AR treatment using mobile technology. The Allergy Diary was filled in by 2871 users All these studies combine to confirm the impact of uncontrolled AR on work productivity. Table 3 Twinning protocols (from Bousquet et al., [65]) Protocol 1 Protocol 2 Short version Long version Allergy Diary + + Equation 5D Optional + Physician’s questionnaire + Ethics committee Not needed Needed (obtained in some Reference Sites) Inform consent Terms of Reference on App From with patient’s signature Recruitment Any user Persons attending clinic visits can be included Persons attending clinic visits included with a physician’s diagnosis of allergic disease and allergen sensitization (IgE and/or skin tests) Physician’s questionnaire + Table 3 Twinning protocols (from Bousquet et al., [65]) Protocol 1 Protocol 2 Short version Long version Allergy Diary + + Equation 5D Optional + Physician’s questionnaire + Ethics committee Not needed Needed (obtained in some Reference Sites) Inform consent Terms of Reference on App From with patient’s signature Recruitment Any user Persons attending clinic visits can be included Persons attending clinic visits included with a physician’s diagnosis of allergic disease and allergen sensitization (IgE and/or skin tests) Physician’s questionnaire + Table 3 Twinning protocols (from Bousquet et al., [65]) Table 3 Twinning protocols (from Bousquet et al., [65]) Bousquet et al. Clin Transl Allergy (2018) 8:45 Page 10 of 21 Fig. 7 Correlation between VAS work and VAS global measured, nose, eye and asthma (Bousquet unpublished) Fig. 7 Correlation between VAS work and VAS global measured, nose, eye and asthma (Bousquet unpublished) Fig. 7 Correlation between VAS work and VAS global measured, nose, eye and asthma (Bousquet unpublished) Fig. 8 Impact of symptoms on work, school and daily activities (from Bousquet et al. [33]) Fig. 8 Impact of symptoms on work, school and daily activities (from Bousquet et al. [33]) Fig. Treatment of allergic rhinitis using mobile technology with real world data 8 Impact of symptoms on work, school and daily activities (from Bousquet et al. [33]) Bousquet et al. Clin Transl Allergy (2018) 8:45 Bousquet et al. Clin Transl Allergy Page 11 of 21 who reported 17,091 days of VAS in 2015 and 2016. between classes (intranasal corticosteroid use containing Fig. 9 VAS levels in severe rhinitis depending on multimorbidity (from Bousquet et al. [60]) Fig. 10 Treatments received in MAS (from Bousquet et al. [59]) Fig. 9 VAS levels in severe rhinitis depending on multimorbidity (from Bousquet et al. [60]) Fig. 9 VAS levels in severe rhinitis depending on multimorbidity (from Bousquet et al. [60]) Fig. 9 VAS levels in severe rhinitis depending on multimorbidity (from Bousquet et al. [60]) Fig. 10 Treatments received in MAS (from Bousquet et al. [59]) between classes (intranasal corticosteroid use containing medications and oral H1-antihistamines). The control of days differed between no (best control), single or multi- ple treatments (worst control) (Fig. 10). The study con- firms the usefulness of the Allergy Diary in accessing and assessing everyday use and practice in AR [59]. who reported 17,091 days of VAS in 2015 and 2016. Medications were reported for 9634 days. The assess- ment of days appeared to be more informative than the course of the treatment as, in real life, patients rarely use treatment on a daily basis; rather, they appear to increase treatment use with the loss of symptom control and to stop it when symptoms disappear. The Allergy Diary allowed the differentiation between treatments within or Adherence to medications was studied in almost 7000 users reporting medications. 1770 users reported over Bousquet et al. Clin Transl Allergy (2018) 8:45 Page 12 of 21 pollution and allergy and (6) assess the societal implica- tions of the interaction. 7 days of VAS between January 1, 2016 and August 31, 2016 and a major lack of adherence to treatment was observed for all medications (Menditto et al., in preparation). 7 days of VAS between January 1, 2016 and August 31, 2016 and a major lack of adherence to treatment was observed for all medications (Menditto et al., in preparation). POLLAR will use the freely existing application for AR monitoring (Allergy Diary, 14,000 users, TLR8) com- bined with a new tool allowing queries on allergen and pollen (TLR2) and existing pollution data. Global applicability of MASK and POLLAR, and their benefits Although MASK has been devised to optimize care path- ways in rhinitis and asthma multimorbidity, its applica- bility is far more extensive (Table 4). MASK in the pharmacy Multidisciplinary integrated care is necessary to reduce the burden of chronic diseases. A significant proportion of patients with AR self-manage their condition and often the pharmacist is the first HCP that a person with nasal symptoms contacts [66, 67]. Pharmacists are trusted in the community and are easily accessible. As such, phar- macists are an important part of the multidisciplinary healthcare team, acting at different steps of rhinitis care pathways. y Google Trends (GT) searches trends of specific que- ries in Google and reflects the real-life epidemiology of AR. We compared GT terms related to allergy and rhi- nitis in all European Union countries, Norway and Swit- zerland from January 1, 2011 to December, 20 2016. An annual and clear seasonality of queries was found in most countries but the terms ‘hay fever’, ‘allergy’ and ‘pollen’— show cultural differences [70]. Using longitudinal data in different countries and multiple terms, we identified an awareness-related spike of searches (December 2016) [70]. In asthma, GTs can identify spikes of mortality as was found in Australia and Kuwait in 2016. However, the usual peaks of asthma during allergen exposure or virus infections cannot be easily monitored [71]. Pharmacists are important in many areas of interven- tion in AR: • Recognizing (identification).i i • Risk assessment/stratification. • OTC treatment.i • Manage refils. i • Patient education. • Referral to a physician. • Administration of topical treatment technique and adherence to treatment. • Administration of topical treatment technique and adherence to treatment. • Administration of topical treatment technique and adherence to treatment. Simple algorithms and tools are essential in the routine implementation of these steps. A first approach was made by ARIA in the pharmacy [68] and is currently being updated using MASK. POLLAR (Impact of air POLLution on Asthma and Rhinitis) AR and asthma are impacted by allergens and air pollu- tion. However, interactions between air pollution, sleep [55, 69] and allergic diseases are insufficiently under- stood. POLLAR aims at combining emerging technolo- gies [search engine TLR2 (technology readiness level); pollution sampler TLR6, App TLR9] with machine learn- ing to (1) understand effects of air pollution in AR and its impact on sleep, work, asthma, (2) propose novel care pathways integrating pollution and patient’s literacy, (3) study sleep, (4) improve work productivity, (5) pro- pose the basis for a sentinel network at the EU level for For MASK, several steps have been achieved. Treatment of allergic rhinitis using mobile technology with real world data Machine learning will be used to assess the relationship between air pollution and AR comparing polluted and non-pol- luted areas in 6 EU countries. Data generated in 2018 will be confirmed in 2019 and extended by the individ- ual assessment of pollution (Canarin®, portable sensor, TLR6) in AR and sleep apnea patients used as a control group having impaired sleep. The geographic information system GIS will map the results.i of Allergy and Airways Diseases Patients’ Associations; EIP on AHA: European Innovation Partnership on AHA; EIP: European Innovation Partnership; EQ-5D: Euroquol; GARD: WHO Global Alliance against Chronic Respiratory Diseases; GDPR: General Data Protection Regulation; GIS: geographic information sys‑ tem; GP: Good Practice; GT: Google Trends; HCP: health care professional; ICP: integrated care pathway; IMS: Institute of Medical Science; JA-CHRODIS: Joint Action on Chronic Diseases and Promoting Healthy Ageing across the Life Cycle; MACVIA-LR: contre les MAladies Chroniques pour un VIeillissement Actif MASK The Allergy Diary has the potential to improve the control of allergic diseases and to significantly improve work productiv‑ ity at the EU level For public health purposes, a perfect patient characterization in real life is needed to identify the prevalence, burden and costs incurred by patients in order to improve quality of care and optimize health care planning and policies For public health purposes, a perfect patient characterization in real life is needed to identify the prevalence, burden and costs incurred by patients in order to improve quality of care and optimize health care planning and policies Inequities still exist in the EU for allergic diseases prevalence and burden (not only sex/gender inequi‑ ties). POLLAR will attempt to understand them and to propose policies and health promotion strategies Inequities still exist in the EU for allergic diseases prevalence and burden (not only sex/gender inequi‑ ties). POLLAR will attempt to understand them and to propose policies and health promotion strategies Conclusion MASK is a novel approach to obtain real-life data con- cerning rhinitis and asthma multimorbidity and to help patients and physicians for a better SDM. It can be used for multiple purposes in a friendly manner in order to improve the control of allergic diseases in a cost-effective approach. Page 13 of 21 Bousquet et al. Clin Transl Allergy (2018) 8:45 Bousquet et al. Clin Transl Allergy Abbreviations AHA: active and healthy ageing; AIRWAYS ICPs: integrated care pathways for airway diseases; AR: allergic rhinitis; ARIA: Allergic Rhinitis and Its Impact on Asthma; CARAT: Control of Allergic Rhinitis and Asthma Test; CDSS: clinical decision support system; CNIL: Commission Informatique et Liberté; CRD: Chronic Respiratory Disease; DG CONNECT: Directorate General for Com‑ of Allergy and Airways Diseases Patients’ Associations; EIP on AHA: European Innovation Partnership on AHA; EIP: European Innovation Partnership; EQ-5D: Euroquol; GARD: WHO Global Alliance against Chronic Respiratory Diseases; GDPR: General Data Protection Regulation; GIS: geographic information sys‑ tem; GP: Good Practice; GT: Google Trends; HCP: health care professional; ICP: integrated care pathway; IMS: Institute of Medical Science; JA-CHRODIS: Joint Table 4 Global applicability of MASK Applicability MASK Clinical practice Physicians will be able to read the files of the patients in order to Optimize treatment for the patient and, in particular, the current or the next pollen season Assess and increase the adherence to treatment Help for shared decision making Prescribe allergen immunotherapy (AIT) more rapidly when the patient is not controlled despite optimal pharmacologic treatment Determine the efficacy of AIT in patients The Allergy Diary is an essential tool to provide personalized medicine in AR and asthma Change management The first results of MASK indicate that many patients are uncontrolled and non-adherent to treatment Moreover, they appear to use their medications as needed and not as a regular basis as prescribed Change management is needed Patient empowerment Better understanding of the symptoms Sentinel network linking aerobiology data and control Improved adherence Self-management Patient empowerment Messages sent by the App Clinical trials For RCTs, it is essential to have clarity on definitions, and relevant tools. MASK Observational studies are of key importance to confirm RCTs and bring new hypotheses for the treat‑ ment of AR and asthma Observational studies are of key importance to confirm RCTs and bring new hypotheses for the treat‑ ment of AR and asthma Controlled trials designed with a uniform approach will be more easily evaluated by the Health Technology Assessment agencies (such as NICE) for reimbursement. The Allergy Diary uses EQ-5D, a validated measure of utility Controlled trials designed with a uniform approach will be more easily evaluated by the Health Technology Assessment agencies (such as NICE) for reimbursement. The Allergy Diary uses EQ-5D, a validated measure of utility Better understanding of direct and indirect costs Controlled trials designed with a uniform approach will help to synchronize data from real-life world regarding clinical effects and safety/tolerability of new drugs (post-marketing pharmacovigilance A uniform definition and a collaborative approach to epidemiological, genetic and mechanistic research are important and will be enhanced by the stratification of patients using the Allergy Diary Different levels of phenotype characterization (granularity) can be applied to assess phenotypic char‑ acterization in old age subjects In epidemiologic population studies, standardized definitions and tools are fundamental. The Allergy Diary allows novel approaches combining classical cross-sectional and longitudinal studies with real life studies in large populations In epidemiologic population studies, standardized definitions and tools are fundamental. The Allergy Diary allows novel approaches combining classical cross-sectional and longitudinal studies with real life studies in large populations AR and asthma represent a major burden for the employers, and the estimated annual costs in the EU range from 30 to 60 B€. Better control of the disease was shown to reduce costs. The Allergy Diary has the potential to improve the control of allergic diseases and to significantly improve work productiv‑ ity at the EU level AR and asthma represent a major burden for the employers, and the estimated annual costs in the EU range from 30 to 60 B€. Better control of the disease was shown to reduce costs. Conclusion The Allergy Diary allows To better stratify the patients needing AIT To assess the efficacy of AIT during the trial To assess the efficacy when AIT is stopped Observational studies are of key importance to confirm RCTs and bring new hypotheses for the treat‑ ment of AR and asthma Registration and reimbursement of medicines Controlled trials designed with a uniform approach will be more easily evaluated by the Health Technology Assessment agencies (such as NICE) for reimbursement. The Allergy Diary uses EQ-5D, a validated measure of utility Better understanding of direct and indirect costs Controlled trials designed with a uniform approach will help to synchronize data from real-life world regarding clinical effects and safety/tolerability of new drugs (post-marketing pharmacovigilance Research on mechanisms and genetics A uniform definition and a collaborative approach to epidemiological, genetic and mechanistic research are important and will be enhanced by the stratification of patients using the Allergy Diary Different levels of phenotype characterization (granularity) can be applied to assess phenotypic char‑ acterization in old age subjects Epidemiology In epidemiologic population studies, standardized definitions and tools are fundamental. The Allergy Diary allows novel approaches combining classical cross-sectional and longitudinal studies with real life studies in large populations Employers AR and asthma represent a major burden for the employers, and the estimated annual costs in the EU range from 30 to 60 B€. Better control of the disease was shown to reduce costs. The Allergy Diary has the potential to improve the control of allergic diseases and to significantly improve work productiv‑ ity at the EU level Public health planning For public health purposes, a perfect patient characterization in real life is needed to identify the prevalence, burden and costs incurred by patients in order to improve quality of care and optimize health care planning and policies Reduction of inequities Inequities still exist in the EU for allergic diseases prevalence and burden (not only sex/gender inequi‑ ties). POLLAR will attempt to understand them and to propose policies and health promotion strategies Table 4 Global applicability of MASK Table 4 Global applicability of MASK Author details 1 1 MACVIA‑France, Fondation Partenariale FMC VIA-LR, CHRU Arnaud de Villeneuve, 371 Avenue du Doyen Gaston Giraud, Montpellier, France. 2 INSERM U 1168, VIMA: Ageing and Chronic Diseases Epidemiological and Public Health Approaches, Villejuif, Université Versailles St-Quentin-en- Yvelines, UMR-S 1168, Montigny le Bretonneux, France. 3 Euforea, Brussels, Belgium. 4 KYomed-INNOV, Montpellier, France. 5 iQ4U Consultants Ltd, London, UK. 6 MedScript Ltd, Dundalk, Co Louth, Ireland. 7 Laboratoire HP2, Grenoble, INSERM, U1042, Université Grenoble Alpes, Grenoble, France. 8 CHU de Grenoble, Grenoble, France. 9 Conseil Général de l’Economie Ministère de l’Economie, de l’Industrie et du Numérique, Paris, France. 10 UCIBIO, REQUINTE, Faculty of Pharmacy and Competence Center on Active and Healthy Ageing, University of Porto (Porto4Ageing), Porto, Portugal. 11 Center for Health Technology and Services Research‑ CINTESIS, Faculdade de Medicina, Universidade do Porto, Porto, Portugal. 12 Medida, Lda, Porto, Portugal. 13 Faculty of Health Sciences and CICS – UBI, Health Sciences Research Centre, University of Beira Interior, Covilhã, Portugal. 14 Allergy Center, CUF Descober‑ tas Hospital, Lisbon, Portugal. 15 Imunoalergologia, Centro Hospitalar Universitário de Coimbra and Faculty of Medicine, University of Coimbra, Coimbra, Portugal. 16 ProAR – Nucleo de Excelencia em Asma, Federal University of Bahia, Vitória da Conquista, Brazil. 17 WHO GARD Planning Group, Salvador, Brazil. 18 Allergy Service, University Hospital of Federal University of Santa Catarina (HU-UFSC), Florianópolis, Brazil. 19 Asthma Reference Center, Escola Superior de Ciencias da Santa Casa de Misericordia de Vitoria, Vitória, Esperito Santo, Brazil. 20 Division for Health Innovation, Campania Region and Federico II University Hospital Naples (R&D and DISMET), Naples, Italy. 21 CIRFF, Federico II University, Naples, Italy. 22 SOS Allergology and Clinical Immunology, USL Toscana Centro, Prato, Italy. 23 Department of Medical Sciences, Allergy and Clinical Immunology Unit, University of Torino & Mauriziano Hospital, Torino, Italy. 24 Consortium of Pharmacies and Services COSAFER, Salerno, Italy. 25 Unit of Geriatric Immunoallergology, University of Bari Medical School, Bari, Italy. 26 Department of Medicine, Surgery and Dentistry “Scuola Medica Salernitana”, University of Salerno, Salerno, Italy. 27 Center of Excellence in Asthma and Allergy, Hospital Médica Sur, México City, Mexico. 28 Mexico City, Mexico. 29 Puebla, Puebla, Mexico. 30 Ciutad Mexico, Mexico. 31 Allergology Department, Centre de l’Asthme et des Allergies Hôpital d’Enfants Armand-Trousseau (APHP), Paris, France. 32 UPMC Univ Paris 06, UMR_S 1136, Institut Pierre Louis d’Epidémiologie et de Santé Publique, Sorbonne Universités, Equipe EPAR, 75013 Paris, France. Authors’ contributions All authors are MAKS members and have contributed to the design of the pro‑ ject. Many authors also included users and disseminated the project in their own country. All authors read and approved the final manuscript. Abbreviations 98 Johns Hopkins School of Medicine, Baltimore, MD, USA. 99 Department of Otorhinolaryngology, Chiba University Hospital, Chiba, Japan. 100 Nova Southeastern University, Fort Lauderdale, Florida, USA. A k l d t (Fighting chronic diseases for AHA); MASK: Mobile Airways Sentinel networK; MeDALL: Mechanisms of the Development of ALLergy (FP7); mHealth: mobile health; NCD: non-communicable disease; OTC: over the counter; PIA: privacy Impact Assessment; POLLAR: Impact of air POLLution on Asthma and Rhinitis; QOL: quality of life; SCUAD: severe chronic upper airway disease; TRL: technol‑ ogy readiness level; TWINNING: transfer of innovation of mobile technology; VAS: Visual Analogue Scale; WHO: World Health Organization; WPAI-AS: Work Productivity and Activity Questionnaire. Abbreviations of Allergy and Airways Diseases Patients’ Associations; EIP on AHA: European Innovation Partnership on AHA; EIP: European Innovation Partnership; EQ-5D: Euroquol; GARD: WHO Global Alliance against Chronic Respiratory Diseases; GDPR: General Data Protection Regulation; GIS: geographic information sys‑ tem; GP: Good Practice; GT: Google Trends; HCP: health care professional; ICP: integrated care pathway; IMS: Institute of Medical Science; JA-CHRODIS: Joint Action on Chronic Diseases and Promoting Healthy Ageing across the Life Cycle; MACVIA-LR: contre les MAladies Chroniques pour un VIeillissement Actif AHA: active and healthy ageing; AIRWAYS ICPs: integrated care pathways for airway diseases; AR: allergic rhinitis; ARIA: Allergic Rhinitis and Its Impact on Asthma; CARAT: Control of Allergic Rhinitis and Asthma Test; CDSS: clinical decision support system; CNIL: Commission Informatique et Liberté; CRD: Chronic Respiratory Disease; DG CONNECT: Directorate General for Com‑ munications Networks, Content & Technology; DG Santé: Directorate General for Health and Food Safety; DG: Directorate General; EFA: European Federation Page 14 of 21 Page 14 of 21 Bousquet et al. Clin Transl Allergy (2018) 8:45 Bousquet et al. Clin Transl Allergy (2018) 8:45 University of Edinburgh, Medical School, Edinburgh, UK. 46 Centre of Medical Informatics, Usher Institute of Population Health Sciences and Informatics, The University of Edinburgh, Edinburgh, UK. 47 Allergy Unit, Department of Dermatology, University Hospital of Zurich, Zürich, Switzerland. 48 Center for Rhinology and Allergology, Wiesbaden, Germany. 49 Department of Otorhinolaryngology, Head and Neck Surgery, Universitätsmedizin Mannheim, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany. 50 Comprehensive Allergy‑Centre‑Charité, Department of Dermatol‑ ogy and Allergy, Charité - Universitätsmedizin Berlin, Berlin, Germany. 51 Global Allergy and Asthma European Network (GA2LEN), Berlin, Germany. 52 Institute of Medical Statistics, and Computational Biology, Medical Faculty, University of Cologne, Cologne, Germany. 53 CRI-Clinical Research International-Ltd, Hamburg, Germany. 54 Department of Internal Medicine, Medical University of Graz, Graz, Austria. 55 Department of ENT, Medical University of Graz, Graz, Austria. 56 Department of Otorhinolaryngology, Academic Medical Centre, Amsterdam, The Netherlands. 57 Department of Public Health and Primary Care, Leiden University Medical Center, Leiden, The Netherlands. 58 ISGlobAL, Centre for Research in Environmental Epidemiology (CREAL), Barcelona, Spain. 59 IMIM (Hospital del Mar Research Institute), Barcelona, Spain. 60 CIBER Epidemiología y Salud Pública (CIBERESP), Barcelona, Spain. 61 Universitat Pompeu Fabra (UPF), Barcelona, Spain. 62 Allergy Section, Department of Internal Medicine, Hospital Vall ‘dHebron & ARADyAL Research Network, Barcelona, Spain. 63 AQuAS, Barcelona, Spain. 64 EUREGHA, European Regional and Local Health Association, Brussels, Belgium. Abbreviations 65 Rhinology Unit and Smell Clinic, ENT Department, Hospital Clínic, University of Barcelona, Barcelona, Spain. 66 Clinical and Experimental Respiratory Immunoallergy, IDIBAPS, CIBERES, University of Barcelona, Barcelona, Spain. 67 Skin and Allergy Hospital, Helsinki University Hospital, Helsinki, Finland. 68 Association of Finnish Pharmacists, Helsinki, Finland. 69 Department of Lung Diseases and Clinical Immunology, University of Turku, Turku, Finland. 70 Terveystalo Allergy Clinic, Turku, Finland. 71 Department of Pulmonary Diseases, Cerrahpasa Faculty of Medicine, Istanbul University, Istanbul, Turkey. 72 Department of Pulmonary Diseases, Faculty of Medicine, Celal Bayar University, Manisa, Turkey. 73 GARD Executive Committee, Manisa, Turkey. 74 Center for Pediatrics and Child Health, Institute of Human Development, Royal Manchester Children’s Hospital, University of Manchester, Manchester, UK. 75 Allergy Department, 2nd Pediatric Clinic, Athens General Children’s Hospital “P&A Kyriakou”, University of Athens, 11527 Athens, Greece. 76 Department of Otorhinolaryngology, University of Crete School of Medicine, Heraklion, Greece. 77 Woolcock Institute of Medical Research, University of Sydney and Sydney Local Health District, Glebe, NSW, Australia. 78 Department of Allergy, Immunology and Respiratory Medicine, Alfred Hospital and Central Clinical School, Monash University, Melbourne, VIC, Australia. 79 Department of Immunology, Monash University, Melbourne, VIC, Australia. 80 Servicio de Alergia e Immunologia, Clinica Santa Isabel, Buenos Aires, Argentina. 81 Director of Center of Allergy, Immunology and Respiratory Diseases, Santa Fe, Argentina Center for Allergy and Immunology, Santa Fe, Argentina. 82 Universidad Católica de Córdoba, Córdoba, Argentina. 83 Department of Clinical Science and Education, Karolinska Institutet, Södersjukhuset, Stockholm, Sweden. 84 Sachs’ Children and Youth Hospital, Södersjukhuset, Stockholm, Sweden. 85 Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden. 86 Centre for Clinical Research Sörmland, Uppsala University, Eskilstuna, Sweden. 87 Upper Airways Research Laboratory, ENT Department, Ghent University Hospital, Ghent, Belgium. 88 Department of Otorhinolaryngology, Univ Hospitals Leuven, Louvain, Belgium. 89 Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands. 90 EFA European Federation of Allergy and Airways Diseases Patients’ Associations, Brussels, Belgium. 91 Department of Dermatology and Allergy Centre, Odense University Hospital, Odense Research Center for Anaphylaxis (ORCA), Odense, Denmark. 92 Department of Medicine, Clinical Immunology and Allergy, McMaster University, Hamilton, ON, Canada. 93 University Hospital Olomouc, Olomouc, Czech Republic. 94 Peercode BV, Geldermalsen, The Netherlands. 95 Faculty of Medicine, Transylvania University, Brasov, Romania. 96 Division of Allergy/Immunology, University of South Florida, Tampa, USA. 97 Section of Allergy and Immunology, Saint Louis University School of Medicine, Saint Louis, MO, USA. Mask Study Group 1 3 FER Simons325, V Siroux326, JC Sisul327, I Skrindo378, D Solé328, D Somekh329, M Sondermann330, T Sooronbaev331, M Sova332, M Sorensen333, M Sorlini334, O Spranger139, C Stellato118, R Stelmach335, R Stukas336, J Sunyer14–17, J Strozek193, A Szylling193, JN Tebyriçá337, M Thibaudon338, T To339, A Todo-Bom340, PV Tomazic341, S Toppila-Salmi163, U Trama342, M Triggiani118, C Suppli Ulrik343, M Urrutia-Pereira344, R Valenta345, A Valero346, A Valiulis347, E Valovirta348, M van Eerd119, E van Ganse349, M van Hague350, O Vandenplas351, MT Ventura352, G Vezzani353, T Vasankari354, A Vatrella118, MT Verissimo211, F Viart78, G Viegi355, D Vicheva356, T Vontetsianos357, M Wagenmann358, S Walker359, D Wallace360, DY Wang361, S Waserman362, T Werfel363, M Westman364, M Wickman191, DM Williams365, S Williams366, N Wilson, J Wright367, P Wroczynski40, P Yakovliev368, BP Yawn369, PK Yiallouros370, A Yorgancioglu371, OM Yusuf372, HJ Zar373, L Zhang374, N Zhong200, ME Zernotti375, M Zidarn376, T Zuberbier35, C Zubrinich259, A Zurkuhlen377 Mask Study Group J Bousquet1–3, PW Hellings4, W Aberer5, I Agache6, CA Akdis7, M Akdis7, MR Alberti8, R Almeida9, F Amat10, R Angles11, I Annesi-Maesano12, IJ Ansotegui13, JM Anto14–17, S Arnavielle18, E Asayag19, A Asarnoj20, H Arshad21, F Avolio22, E Bacci23, C Bachert24, I Baiardini25, C Barbara26, M Barbagallo27, I Baroni28, BA Barreto29, X Basagana14, ED Bateman30, M Bedolla-Barajas31, A Bedbrook2, M Bewick32, B Beghé33, EH Bel34, KC Bergmann35, KS Bennoor36, M Benson37, L Bertorello23, AZ Białoszewski38, T Bieber39, S Bialek40, C Bindslev-Jensen41, L Bjermer42, H Blain43,44, F Blasi45, A Blua46, M Bochenska Marciniak47, I Bogus- Buczynska47, AL Boner48, M Bonini49, S Bonini50, CS Bosnic-Anticevich51, I Bosse52, J Bouchard53, LP Boulet54, R Bourret55, PJ Bousquet12, F Braido25, V Briedis56, CE Brightling57, J Brozek58, C Bucca59, R Buhl60, R Buonaiuto61, C Panaitescu62, MT Burguete Cabañas63, E Burte3, A Bush64, F Caballero- Fonseca65, D Caillot67, D Caimmi68, MA Calderon69, PAM Camargos70, T Camuzat71, G Canfora72, GW Canonica25, V Cardona73, KH Carlsen74, P Carreiro- Martins75, AM Carriazo76, W Carr77, C Cartier78, T Casale79, G Castellano80, L Cecchi81, AM Cepeda82, NH Chavannes83, Y Chen84, R Chiron68, T Chivato85, E Chkhartishvili86, AG Chuchalin87, KF Chung88, MM Ciaravolo89, A Ciceran90, C Cingi91, G Ciprandi92, AC Carvalho Coehlo93, L Colas94, E Colgan95, J Coll96, D Conforti97, J Correia de Sousa98, RM Cortés-Grimaldo99, F Corti100, E Costa101, MC Costa-Dominguez102, AL Courbis103, L Cox104, M Crescenzo105, AA Cruz106, A Custovic107, W Czarlewski108, SE Dahlen109, C Dario110, J da Silva111, Y Dauvilliers112, U Darsow113, F De Blay114, G De Carlo115, T Dedeu116, M de Fátima Emerson117, G De Feo118, G De Vries119, B De Martino120, N de Paula Motta Rubini121, D Deleanu122, P Demoly12,68, JA Denburg123, P Devillier124, S Di Capua Ercolano125, N Di Carluccio66, A Didier126, D Dokic127, MG Dominguez- Silva128, H Douagui129, G Dray103, R Dubakiene130, SR Durham131, G Du Toit132, MS Dykewicz133, Y El-Gamal134, P Eklund135, E Eller41, R Emuzyte136, J Farrell95, A Farsi81, J Ferreira de Mello Jr137, J Ferrero138, A Fink-Wagner139, A Fiocchi140, WJ Fokkens141, JA Fonseca142, JF Fontaine143, S Forti97, JM Fuentes-Perez144, JL Gálvez-Romero145, A Gamkrelidze146, J Garcia-Aymerich14, CY García- Cobas147, MH Garcia-Cruz148, B Gemicioğlu149, S Genova150, C George151, JE Gereda152, R Gerth van Wijk153, RM Gomez154, J Gómez-Vera155, S González Diaz156, M Gotua157, I Grisle158, M Guidacci159, NA Guldemond160, Z Gutter161, MA Guzmán162, T Haahtela163, J Hajjam164, L Hernández165, JO’B Hourihane166, YR Huerta-Villalobos167, M Humbert168, G Iaccarino169, M Illario170, JC Ivancevich171, EJ Jares172, E Jassem173, SL Johnston174, G Joos175, KS Jung176, M Jutel177, I Kaidashev178, O Kalayci179, AF Kalyoncu180, J Karjalainen181, P Kardas182, T Keil183, PK Keith184, M Khaitov185, N Khaltaev186, J Kleine-Tebbe187, L Klimek188, ML Kowalski189, M Kuitunen190, I Kull191, P Kuna47, M Kupczyk47, V Kvedariene192, E Krzych-Fałta193, P Lacwik47, D Larenas-Linnemann194, D Laune18, D Lauri195, J Lavrut196, LTT Le197, M Lessa198, G Levato199, J Li200, P Lieberman201, A Lipiec193, B Lipworth202, KC Lodrup Carlsen203, R Louis204, O Lourenço205, JA Luna-Pech206, K Maciej47, A Magnan94, B Mahboub207, D Maier208, A Mair209, I Majer210, J Malva211, E Mandajieva212, P Manning213, E De Manuel Keenoy214, GD Marshall215, MR Masjedi216, JF Maspero217, E Mathieu- Dupas18, JJ Matta Campos218, AL Matos219, M Maurer220, S Mavale-Manuel221, O Mayora97, MA Medina-Avalos222, E Melén223, E Melo-Gomes26, EO Meltzer224, E Menditto225, J Mercier226, N Miculinic227, F Mihaltan228, B Milenkovic229, G Moda230, MD Mogica-Martinez231, Y Mohammad232, I Momas233,234, S Montefort235, R Monti236, D Mora Bogado237, M Morais-Almeida238, FF Morato-Castro239, R Mösges240, A Mota-Pinto241, P Moura Santo242, J Mullol243, L Münter244, A Muraro245, R Murray246, R Naclerio247, R Nadif3, M Nalin28, L Napoli248, L Namazova-Baranova249, H Neffen250, V Niedeberger251, K Nekam252, A Neou253, A Nieto254, L Nogueira-Silva255, M Nogues2,256, E Novellino257, TD Nyembue258, RE O’Hehir259, C Odzhakova260, K Ohta261, Y Okamoto262, K Okubo263, GL Onorato2, M Ortega Cisneros264, S Ouedraogo265, I Pali-Schöll266, S Palkonen115, P Panzner267, NG Papadopoulos268, HS Park269, A Papi270, G Passalacqua271, E Paulino272, R Pawankar273, S Pedersen274, JL Pépin275, AM Pereira276, M Persico277, O Pfaar278,279, J Phillips280, R Picard281, B Pigearias282, I Pin283, C Pitsios284, D Plavec285, W Pohl286, TA Popov287, F Portejoie2, P Potter288, AC Pozzi289, D Price290, EP Prokopakis291, R Puy259, B Pugin292, RE Pulido Ross293, M Przemecka47, KF Rabe294, F Raciborski193, R Rajabian-Soderlund295, S Reitsma141, I Ribeirinho296, J Rimmer297, D Rivero-Yeverino298, JA Rizzo299, MC Rizzo300, C Robalo-Cordeiro301, F Rodenas302, X Rodo14, M Rodriguez Gonzalez303, L Rodriguez-Mañas304, C Rolland305, S Rodrigues Valle306, M Roman Rodriguez307, A Romano308, E Rodriguez-Zagal309, G Rolla310, RE Roller- Wirnsberger311, M Romano28, J Rosado-Pinto312, N Rosario313, M Rottem314, D Ryan315, H Sagara316, J Salimäki317, B Samolinski193, M Sanchez-Borges318, J Sastre-Dominguez319, GK Scadding320, HJ Schunemann58, N Scichilone321, P Schmid-Grendelmeier322, FS Serpa323, S Shamai240, A Sheikh324, M Sierra96, 1University Hospital, Montpellier, France. Mask Study Group 1 3 2MACVIA-France, Fondation partenariale FMC VIA-LR, Montpellier, France. 3VIMA. INSERM U 1168, VIMA : Ageing and chronic diseases Epidemiological and public health approaches, Villejuif, Université Versailles St-Quentin-en-Yvelines, UMR-S 1168, Montigny le Bretonneux, France and Euforea, Brussels, Belgium. 4Laboratory of Clinical Immunology, Department of Microbiology and Immunology, KU Leuven, Leuven, Belgium. 5Department of Dermatology, Medical University of Graz, Graz, Austria. 6Transylvania University Brasov, Brasov, Romania. 7Swiss Institute of Allergy and Asthma Research (SIAF), University of Zurich, Davos, Switzer‑ land. 8Project Manager, Chairman of the Council of Municipality of Salerno, Italy. 9Center for Health Technology and Services Research- CINTESIS, Faculdade de Medicina, Universidade do Porto; and Medida, Lda Porto, Portugal. 10Allergology department, Centre de l’Asthme et des Allergies Hôpital d’Enfants Armand-Trousseau (APHP); Sorbonne Université, UPMC Univ Paris 06, UMR_S 1136, Institut Pierre Louis d’Epidémiologie et de Santé Publique, Equipe EPAR, Paris, France. 11Innovación y nuevas tecnologías, Salud Sector sanitario de Barbastro, Barbastro, Spain. 12Epidemiology of Allergic and Respiratory Diseases, Department Institute Pierre Louis of Epidemiology and Public Health, INSERM and Sorbonne Université, Medical School Saint Antoine, Paris, France 13Department of Allergy and Immunology, Hospital Quirón Bizkaia, Erandio, Spain. 14ISGlobAL, Centre for Research in Environmental Epidemiology (CREAL), Barcelona, Spain. 15IMIM (Hospital del Mar Research Institute), Barcelona, Spain. 16CIBER Epidemiología y Salud Pública (CIBERESP), Barcelona, Spain. 17Universitat Pompeu Fabra (UPF),Barcelona, Spain. 18KYomed INNOV, Montpellier, France. 19Argentine Society of Allergy and Immunopathology, Buenos Aires, Argentina. 20Clinical Immunology and Allergy Unit, Department of Medicine Solna, Karolinska Institutet, Stockholm, and Astrid Lindgren Children’s Hospital, Department of Pediatric Pulmonology and Allergy, Karolinska University Hospital, Stockholm, Sweden. 21David Hide Asthma and Allergy Research Centre, Isle of Wight, United Kingdom. 22Regionie Puglia, Bari, Italy. 23Regione Liguria, Genoa, Italy. 24Upper Airways Research Laboratory, ENT Dept, Ghent University Hospital, Ghent, Belgium. 25Allergy and Respiratory Diseases, Ospedale Policlinico San Martino, University of Genoa, Italy. 26PNDR, Portuguese National Programme for Respiratory Diseases, Faculdade de Medicina de Lisboa, Lisbon, Portugal. 27Director of the Geriatric Unit, Department of Internal Medicine (DIBIMIS), University of Palermo, Italy. 28Telbios SRL, Milan, Italy. 29Universidade do Estado do Pará, Belem, Brazil. 30Department of Medicine, University of Cape Town, Cape Town, South Africa. 31Hospital Civil de Guadalajara Dr Juan I Menchaca, Guadalarara, Mexico. 32iQ4U Consultants Ltd, London, UK. 33Section of Respiratory Disease, Department of Oncology, Haematology and Respiratory Diseases, University of Modena and Reggio Emilia, Modena, Italy. 34Depart‑ ment of Respiratory Medicine, Academic Medical Center (AMC), University of Amsterdam, The Netherlands. Author details 1 33 Epidemi‑ ology of Allergic and Respiratory Diseases, Department Institute Pierre Louis of Epidemiology and Public Health, INSERM, UPMC Sorbonne Université, Medical School Saint Antoine, Paris, France. 34 La Rochelle, France. 35 Depart‑ ment of Respiratory Diseases, Montpellier University Hospital, Montpellier, France. 36 UPRES EA220, Pôle des Maladies des Voies Respiratoires, Hôpital Foch, Université Paris-Saclay, Suresnes, France. 37 Reims, France. 38 Division of Internal Medicine, Asthma and Allergy, Barlicki University Hospital, Medical University of Lodz, Lodz, Poland. 39 Department of Prevention of Environmen‑ tal Hazards and Allergology, Medical University of Warsaw, Warsaw, Poland. 40 Clinic of Children’s Diseases, and Institute of Health Sciences Department of Public Health, Vilnius University Institute of Clinical Medicine, Vilnius, Lithuania. 41 European Academy of Paediatrics (EAP/UEMS-SP), Brussels, Belgium. 42 Clinic of Children’s Diseases, Faculty of Medicine, Vilnius University, Vilnius, Lithuania. 43 Faculty of Medicine, Vilnius University, Vilnius, Lithuania. 44 Woodbrook Medical Centre, Loughborough, UK. 45 Allergy and Respiratory Research Group, Usher Institute of Population Health Sciences and Informatics, Acknowledgements None. Acknowledgements None. Page 15 of 21 Bousquet et al. Clin Transl Allergy (2018) 8:45 Bousquet et al. Clin Transl Allergy Mask Study Group 1 3 131Allergy and Clinical Immunology National Heart and Lung Institute, Imperial College London, UK. 132Guy’s and st Thomas’ NHS Trust, Kings College London, UK. 133Section of Allergy and Immunology, Saint Louis University School of Medicine, Saint Louis, Missouri, USA. 134Pediatric Allergy and Immunology Unit, Children’s Hospital, Ain Shams University, Cairo, Egypt. 135Department of Computing Science, Umeå University, Sweden and Four Computing Oy, Finland. 136Clinic of Children’s Diseases, Faculty of Medicine, Vilnius University, Vilnius, Lithuania. 137University of São Paulo Medical School, Sao Paulo, Brazil 138Andalusian Agency for Healthcare Quality, Seville, Spain. 139Global Allergy and Asthma Platform GAAPP, Vienna, Austria. 140Division of Allergy, Department of Pediatric Medicine - The Bambino Gesù Children’s Research Hospital Holy see, Rome, Italy. 141Department of Otorhinolaryngol‑ ogy, Amsterdam, University Medical Centres, AMC, Amsterdam the Netherlands. 142CINTESIS, Center for Research in Health Technologies and Information Systems, Faculdade de Medicina da Universidade do Porto, Porto, Portugal and MEDIDA, Lda, Porto, Portugal 143Allergist, Reims, France. 144Hospital general regional 1 “Dr Carlos Mc Gregor Sanchez Navarro” IMSS, Mexico City, Mexico. 145Regional hospital of ISSSTE, Puebla, Mexico. 146National Center for Disease Control and Public Health of Georgia, Tbilisi, Georgia. 147Guadalarara, Mexico. 148Allergy Clinic, National Institute of Respiratory Diseases, Mexico City, Mexico. 149Department of Pulmonary Diseases, Istanbul University-Cerrahpasa, Cerrahpasa Faculty of Medicine, Istambul,Turkey. 150Allergology unit, UHATEM “NIPirogov”, Sofia, Bulgaria. 151Medical University, Faculty of Public Health, Sofia. 152Allergy and Immunology Division, Clinica Ricardo Palma, Lima, Peru. 153Department of Internal Medicine, section of Allergology, Erasmus MC, Rotterdam, The Netherlands. 154Allergy & Asthma Unit, Hospital San Bernardo Salta, Argentina. 155Allergy Clinic, Hospital Regional del ISSSTE ‘Lic. López Mateos’, Mexico City, Mexico. 156Head and Professor, Centro Regional de Excelencia CONACYT y WAO en Alergia, Asma e Inmunologia Hospital Universitario Universidad Autónoma de Nuevo León Lund, Sweden. 43Department of Geriatrics, Montpellier University Hospital, Montpellier, France. 44EA 2991, Euromov, University Montpellier, France. 45Department of Pathophysiology and Transplantation, University of Milan, IRCCS Fondazione Ca’Granda Ospedale Maggiore Policlinico, Milan, Italy. 46Argentine Association of Respiratory Medicine, Buenos Aires, Argentina. 47Division of Internal Medicine, Asthma and Allergy, Barlicki University Hospital, Medical University of Lodz, Poland. 48Pediatric Department, University of Verona Hospital, Verona, Italy. 49Department of Public Health and Infectious Diseases, Sapienza University of Rome, Italy. 50Second University of Naples and Institute of Translational Medicine, Italian National Research Council. 51Woolcock Institute of Medical Research, University of Sydney and Woolcock Emphysema Centre and and Sydney Local Health District, Glebe, NSW, Australia. 52Allergist, La Rochelle, France. Mask Study Group 1 3 53Associate professor of clinical medecine, Laval’s University, Quebec city, Head of medecine department, Hôpital de la Malbaie, Quebec, Canada. 54Quebec Heart and Lung Institute, Laval University, Québec City, Quebec, Canada. 55Centre Hospitalier Valenciennes, France. 56Head of Department of Clinical Pharmacy of Lithuanian University of Health Sciences, Kaunas, Lithuania. 57Institute of Lung Health, Respiratory Biomedical Unit, University Hospitals of Leicester NHS Trust, Leicestershire, UK; Department of Infection, Immunity and Inflammation, University of Leicester, Leicester, UK. 58Department of Health Research Methods, Evidence and Impact, Division of Immunology and Allergy, Department of Medicine, McMaster University, Hamilton, ON, Canada. 59Chief of the University Pneumology Unit- AOU Molinette, Hospital City of Health and Science of Torino, Italy. 60Universitätsmedizin der Johannes Gutenberg- Universität Mainz, Mainz, Germany. 61Pharmacist, Municipality Pharmacy, Sarno, Italy. 62University of Medicine and Pharmacy Victor Babes, Timisoara, Romania. 63Instituto de Pediatria, Hospital Zambrano Hellion Tec de Monterrey, Monterrey, Mexico. 64Imperial College and Royal Brompton Hospital, London, UK. 65Centro Medico Docente La Trinidad, CaRacas, Venezuela. 66Regional Director Assofarm Campania and Vice President of the Board of Directors of Cofaser, Salerno, Italy 67Service de pneumologie, CHU et université d’Auvergne, Clermont-Ferrand, France. 68Department of Respiratory Diseases, Montpellier University Hospital, France. 69Imperial College London - National Heart and Lung Institute, Royal Brompton Hospital NHS, London, UK. 70Federal University of Minas Gerais, Medical School, Department of Pediatrics, Belo Horizonte, Brazil 71Assitant Director General, Montpellier, Région Occitanie, France. 72Mayor of Sarno and President of Salerno Province, Director, Anesthesiology Service, Sarno “Martiri del Villa Malta” Hospital, Italy. 73Allergy Section, Department of Internal Medicine, Hospital Vall d’Hebron & ARADyAL Spanish Research Network, Barcelona, Spain. 74Department of Paediatrics, Oslo University Hospital and University of Oslo, Oslo, Norway. 75CEDOC, Integrated Pathophysiological Mechanisms Research Group, Nova Medical School, Campo dos Martires da Patria, Lisbon, and Serviço de Imunoalergologia, Centro Hospitalar de Lisboa Central, EPE, Lisbon, Portugal. 76Regional Ministry of Health of Andalusia, Seville, Spain. 77Allergy and Asthma Associates of Southern California, Mission Viejo, CA, USA. 78ASA - Advanced Solutions Accelerator, Clapiers, France. 79Division of Allergy/Immunology, University of South Florida, Tampa, Fla, USA. 80Celentano pharmacy, Massa Lubrense, Italy. 81SOS Allergology and Clinical Immunology, USL Toscana Centro, Prato, Italy. 82Allergy and Immunology Laboratory, Metropolitan University Hospital, Branquilla, Columbia. 83Department of Public Health and Primary Care, Leiden University Medical Center, Leiden, The Netherlands 84Capital Institute of Pediatrics, Chaoyang district, Beijing, China. 85School of Medicine, University CEU San Pablo, Madrid, Spain. Mask Study Group 1 3 156Head and Professor, Centro Regional de Excelencia CONACYT y WAO en Alergia, Asma e Inmunologia, Hospital Universitario , Universidad Autónoma de Nuevo León, Monterrey NL, Mexico. 157Center of Allergy and Immunology, Georgian 100FIMMG (Federazione Italiana Medici di Medicina Generale), Milan, Italy. 101UCIBIO, REQUINTE, Faculty of Pharmacy and Competence Center on Active and Healthy Ageing of University of Porto(Porto4Ageing), Porto, Portugal. 102Mexico City, Mexico. 103IMT Mines Alès, Unversité Montpellier, Alès, France. 104Department of Medicine, Nova Southeastern University, Davie, University of Miami Dept of Medicine, Miami, Florida, USA. 105Regional Director Assofarm Campania and Vice President of the Board of Directors of Cofaser, Salerno, Italy. 106ProAR – Nucleo de Excelencia em Asma, Federal University of Bahia, Brasil and WHO GARD Planning Group, Brazil. 107Centre for Respiratory Medicine and Allergy, Institute of Inflammation and Repair, University of Manchester and University Hospital of South Manchester, Manchester, UK. 108Medical Consulting Czarlewski, Levallois, France. 109The Centre for Allergy Research, The Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden. 110Azienda Provinciale per i Servizi Sanitari di Trento (APSS-Trento), Italy. 111Department of Internal Medicine, Federal University of Santa Catarina, Trindade, Florianópolis, Santa Catarina, Brazil. 112Sleep Unit, Department of Neurology, Hôpital Gui-de-Chauliac Montpellier, Inserm U1061, France. 113Department of Dermatology and Allergy, Technische Universität München, Munich, Germany; ZAUM-Center for Allergy and Environment, Helmholtz Center Munich, Technische Universität München, Munich, Germany. 114Allergy Division, Chest Disease Department, University Hospital of Strasbourg, Strasbourg, France. 115EFA European Federation of Allergy and Airways Diseases Patients’ Associations, Brussels, Belgium 116AQuAS, Barcelna, Spain & EUREGHA, European Regional and Local Health Association, Brussels, Belgium 117Policlínica Geral do Rio de Janeiro, Rio de Janeiro – Brasil 118Department of Medicine, Surgery and Dentistry “Scuola Medica Salernitana”, University of Salerno, Salerno, Italy. 119Peercode BV, Geldermalsen,The Netherlands. 120Social workers oordinator, Sorrento, Italy. 121Federal University of the State of Rio de Janeiro, School of Medicine and Surgery, Rio de Janeiro, Brazil 122Allergology and Immunology Discipline, “Iuliu Hatieganu” University of Medicine and Pharmacy, Cluj-Napoca, Romania. 123Department of Medicine, Division of Clinical Immunology and Allergy, McMaster University, Hamilton, Ontario, Canada. 124Laboratoire de Pharmacologie Respiratoire UPRES EA220, Hôpital Foch, Suresnes, Université Versailles Saint-Quentin, Université Paris Saclay, France. 125Farmacie Dei Golfi Group, Massa Lubrense, Italy. 126Rangueil- Larrey Hospital, Respiratory Diseases Department, Toulouse, France. 127University Clinic of Pulmology and Allergy, Medical Faculty Skopje, R Macedonia. 128Mexico City, Mexico. 129Service de Pneumo-Allergologie, Centre Hospitalo-Universitaire de Béni-Messous, Algiers, Algeria. 130Clinic of infectious, chest diseases, dermatology and allergology, Vilnius University, Vilnius, Lithuania. Mask Study Group 1 3 35Comprehensive Allergy Center Charité, Department of Dermatology and Allergy, Charité - Universitätsmedizin Berlin; Global Allergy and Asthma European Network (GA2LEN), Berlin, Germany. 36Deptt of Respiratory Medicine, National Institute of Diseases of the Chest and Hospital, Dhaka, Bangladesh. 37Centre for Individualized Medicine, Department of Pediatrics, Faculty of Medicine, Linköping, Sweden. 38Depart‑ ment of Prevention of Environmental Hazards and Allergology, Medical University of Warsaw, Poland. 39BIEBER. Department of Dermatology and Allergy, Rheinische Friedrich-Wilhelms-University Bonn, Bonn, Germany 40Dept of Biochemistry and Clinical Chemistry, Faculty of Pharmacy with the Division of Laboratory Medicine, Warsaw Medical University, Warsaw, Poland. 41Department of Dermatology and Allergy Centre, Odense University Hospital, Odense Research Center for Anaphylaxis (ORCA), Odense, Denmark. 42Department of Respiratory Medicine and Allergology, University Hospital, Page 16 of 21 Page 16 of 21 Bousquet et al. Clin Transl Allergy (2018) 8:45 Bousquet et al. Clin Transl Allergy (2018) 8:45 Bousquet et al. Clin Transl Allergy (2018) 8:45 100FIMMG (Federazione Italiana Medici di Medicina Generale), Milan, Italy. 101UCIBIO, REQUINTE, Faculty of Pharmacy and Competence Center on Active and Healthy Ageing of University of Porto(Porto4Ageing), Porto, Portugal. 102Mexico City, Mexico. 103IMT Mines Alès, Unversité Montpellier, Alès, France. 104Department of Medicine, Nova Southeastern University, Davie, University of Miami Dept of Medicine, Miami, Florida, USA. 105Regional Director Assofarm Campania and Vice President of the Board of Directors of Cofaser, Salerno, Italy. 106ProAR – Nucleo de Excelencia em Asma, Federal University of Bahia, Brasil and WHO GARD Planning Group, Brazil. 107Centre for Respiratory Medicine and Allergy, Institute of Inflammation and Repair, University of Manchester and University Hospital of South Manchester, Manchester, UK. 108Medical Consulting Czarlewski, Levallois, France. 109The Centre for Allergy Research, The Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden. 110Azienda Provinciale per i Servizi Sanitari di Trento (APSS-Trento), Italy. 111Department of Internal Medicine, Federal University of Santa Catarina, Trindade, Florianópolis, Santa Catarina, Brazil. 112Sleep Unit, Department of Neurology, Hôpital Gui-de-Chauliac Montpellier, Inserm U1061, France. 113Department of Dermatology and Allergy, Technische Universität München, Munich, Germany; ZAUM-Center for Allergy and Environment, Helmholtz Center Munich, Technische Universität München, Munich, Germany. 114Allergy Division, Chest Disease Department, University Hospital of Strasbourg, Strasbourg, France. 115EFA European Federation of Allergy and Airways Diseases Patients’ Associations, Brussels, Belgium 116AQuAS, Barcelna, Spain & EUREGHA, European Regional and Local Health Association, Brussels, Belgium 117Policlínica Geral do Rio de Janeiro, Rio de Janeiro – Brasil 118Department of Medicine, Surgery and Dentistry “Scuola Medica Salernitana”, University of Salerno, Salerno, Italy. Mask Study Group 1 3 119Peercode BV, Geldermalsen,The Netherlands. 120Social workers oordinator, Sorrento, Italy. 121Federal University of the State of Rio de Janeiro, School of Medicine and Surgery, Rio de Janeiro, Brazil 122Allergology and Immunology Discipline, “Iuliu Hatieganu” University of Medicine and Pharmacy, Cluj-Napoca, Romania. 123Department of Medicine, Division of Clinical Immunology and Allergy, McMaster University, Hamilton, Ontario, Canada. 124Laboratoire de Pharmacologie Respiratoire UPRES EA220, Hôpital Foch, Suresnes, Université Versailles Saint-Quentin, Université Paris Saclay, France. 125Farmacie Dei Golfi Group, Massa Lubrense, Italy. 126Rangueil- Larrey Hospital, Respiratory Diseases Department, Toulouse, France. 127University Clinic of Pulmology and Allergy, Medical Faculty Skopje, R Macedonia. 128Mexico City, Mexico. 129Service de Pneumo-Allergologie, Centre Hospitalo-Universitaire de Béni-Messous, Algiers, Algeria. 130Clinic of infectious, chest diseases, dermatology and allergology, Vilnius University, Vilnius, Lithuania. 131Allergy and Clinical Immunology National Heart and Lung Institute, Imperial College London, UK. 132Guy’s and st Thomas’ NHS Trust, Kings College London, UK. 133Section of Allergy and Immunology, Saint Louis University School of Medicine, Saint Louis, Missouri, USA. 134Pediatric Allergy and Immunology Unit, Children’s Hospital, Ain Shams University, Cairo, Egypt. 135Department of Computing Science, Umeå University, Sweden and Four Computing Oy, Finland. 136Clinic of Children’s Diseases, Faculty of Medicine, Vilnius University, Vilnius, Lithuania. 137University of São Paulo Medical School, Sao Paulo, Brazil 138Andalusian Agency for Healthcare Quality, Seville, Spain. 139Global Allergy and Asthma Platform GAAPP, Vienna, Austria. 140Division of Allergy, Department of Pediatric Medicine - The Bambino Gesù Children’s Research Hospital Holy see, Rome, Italy. 141Department of Otorhinolaryngol‑ ogy, Amsterdam, University Medical Centres, AMC, Amsterdam the Netherlands. 142CINTESIS, Center for Research in Health Technologies and Information Systems, Faculdade de Medicina da Universidade do Porto, Porto, Portugal and MEDIDA, Lda, Porto, Portugal 143Allergist, Reims, France. 144Hospital general regional 1 “Dr Carlos Mc Gregor Sanchez Navarro” IMSS, Mexico City, Mexico. 145Regional hospital of ISSSTE, Puebla, Mexico. 146National Center for Disease Control and Public Health of Georgia, Tbilisi, Georgia. 147Guadalarara, Mexico. 148Allergy Clinic, National Institute of Respiratory Diseases, Mexico City, Mexico. 149Department of Pulmonary Diseases, Istanbul University-Cerrahpasa, Cerrahpasa Faculty of Medicine, Istambul,Turkey. 150Allergology unit, UHATEM “NIPirogov”, Sofia, Bulgaria. 151Medical University, Faculty of Public Health, Sofia. 152Allergy and Immunology Division, Clinica Ricardo Palma, Lima, Peru. 153Department of Internal Medicine, section of Allergology, Erasmus MC, Rotterdam, The Netherlands. 154Allergy & Asthma Unit, Hospital San Bernardo Salta, Argentina. 155Allergy Clinic, Hospital Regional del ISSSTE ‘Lic. López Mateos’, Mexico City, Mexico. Mask Study Group 1 3 207Department of Pulmonary Medicine, Rashid Hospital, Dubai, UAE. 208Biomax Informatics AG, Munich, Germany. 209Director Gerneral for Health and Social Care, Scottish Government, Edinburgh, UK. 210Department of Respiratory Medicine, University of Bratislava, Bratislava, Slovakia. 211Coimbra Institute for Clinical and Portugal; Ageing@Coimbra EIP-AHA Reference Site, Coimbra, Portugal. 212Medical center Iskar Ltd Sofia, Bulgaria. 213Department of Medicine (RCSI), Bon Secours Hospital, Glasnevin, Dublin, Ireland. 214Kronikgune, International Centre of Excellence in Chronicity Research Barakaldo, Bizkaia, Spain 215Division of Clinical Immunology and Allergy, Laboratory of Behavioral Immunology Research, The University of Mississippi Medical Center, Jackson, Mississippi, USA. 216Tobacco Control Research Centre;Iranian Anti Tobacco Association, Tehran, Iran. 217Argentine Association of Allergy and Clinical Immunology, Buenos Aires, Argentina. 218Mexico City, Mexico. 219University of Southeast Bahia, Brazil. 220Allergie-Centrum-Charité at the Department of Dermatology and Allergy, Charité - Universitätsmedizin Berlin, Germany 221Maputo Central Hospital--Department of Paediatrics, Mozambique. 222Veracruz, Mexico. 223Sachs’ Children and Youth Hospital, Södersjukhuset, Stockholm and Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden. 224Allergy and Asthma Medical Group and Research Center, San Diego, California, USA. 225CIRFF, Federico II University, Naples, Italy. 226Department of Physiology, CHRU, University Montpellier, Vice President for Research, PhyMedExp, INSERM U1046, CNRS UMR 9214, France. 227Croatian Pulmonary Society. 228National Institute of Pneumology M Nasta, Bucharest, Romania. 229Clinic for Pulmonary Diseases, Clinical Center of Serbia, Faculty of Medicine, University of Belgrade, Serbian Association for Asthma and COPD, Belgrade, Serbia. 230Regione Piemonte, Torino, Italy. 231Col Jardines de Sta Monica, Tlalnepantla, Mexico. 232National Center for Research in Chronic Respiratory Diseases, Tishreen University School of Medicine, Latakia, Syria. 233Department of Public health and health products, Paris Descartes University-Sorbonne Paris Cité, EA 4064 and Paris Municipal Department of social action, childhood, and health, Paris, France. 234Paris municipal Department of social action, childhood, and health, Paris, France. 235Lead Respiratory Physician Mater Dei Hospital Malta, Academic Head of Dept and Professor of Medicine University of Malta, Deputy Dean Faculty of Medicine and Surgery University of Medicine, La Valette, Malta. 236Department of Medical Sciences, Allergy and Clinical Immunology Unit, University of Torino & Mauriziano Hospital, Torino, Italy. 237Instituto de Prevision Social IPS HC, Socia de la SPAAI, Tesorera de la SLAAI, Asuncion, Paraguay. 238Allergy Center, CUF Descobertas Hospital, Lisbon, Portugal. 239Universidade de São Paulo, São Paulo, Brazil. 240Institute of Medical Statistics, and Computational Biology, Medical Faculty, University of Cologne, Germany and CRI-Clinical Research International-Ltd, Hamburg, Germany. 241General Pathology Institute, Faculty of Medicine, University of Coimbra, Portugal; Ageing@Coimbra EIP-AHA Reference Site, Coimbra, Portugal. Mask Study Group 1 3 86David Tvildiani Medical University - AIETI Highest Medical School, David Tatishvili Medical Center Tbilisi, Georgia. 87Pulmonolory Research Institute FMBA, Moscow, Russia and GARD Executive Committee, Moscow, Russia. 88National Heart & Lung Institute, Imperial College, London, UK. 89Specialist social worker, Sorrento, Italy. 90Argentine Federation of Otorhinolaryngology Societies, Buenos Aires, Argentina. 91Eskisehir Osmangazi University, Medical Faculty, ENT Department, Eskisehir,Turkey. 92Medicine Department, IRCCS-Azienda Ospedaliera Universitaria San Martino, Genoa, Italy. 93Universidade Federal da Bahia, Escola de Enfermagem, Brazil. 94Plateforme Transversale d’Allergologie, Institut du Thorax, CHU de Nantes, Nantes, France. 95LANUA International Healthcare Consultancy, Northern Ireland, UK. 96Innovación y nuevas tecnologías, Salud Sector sanitario de Barbastro, Barbastro, Spain. 97Innovation and Research Office, Department of Health and Social Solidarity, Autonomous Province of Trento, Italy. 98Life and Health Sciences Research Institute (ICVS), School of Medicine University of Minho Braga Portugal; ICVS/3B’s PT Government Page 17 of 21 Page 17 of 21 Page 17 of 21 Bousquet et al. Clin Transl Allergy (2018) 8:45 Bousquet et al. Clin Transl Allergy (2018) 8:45 Bousquet et al. Clin Transl Allergy (2018) 8:45 Association of Allergology and Clinical Immunology, Tbilisi, Georgia. 158Latvian Association of Allergists, Center of Tuberculosis and Lung Diseases, Riga, Latvia. 159Federal District Base Hospital Institute, Brasília, Brazil. 160Institute of Health Policy and Management iBMG, Erasmus University, Rotterdam, The Nether‑ lands 161University Hospital Olomouc – National eHealth Centre, Czech Republic. 162Immunology and Allergy Division, Clinical Hospital, University of Chile, Santiago, Chile. 163Skin and Allergy Hospital, Helsinki University Hospital, University of Helsinki, Helsinki, Finland. 164Centich : centre d’expertise national des technologies de l’information et de la communication pour l’autonomie, Gérontopôle autonomie longévité des Pays de la Loire, Conseil régional des Pays de la Loire, Centre d’expertise Partenariat Européen d’Innovation pour un vieillissement actif et en bonne santé, Nantes, France. 165Autonomous University of Baja California, Ensenada, Baja California, Mexico. 166Department of Paediatrics and Child Health, University College Cork, Cork, Ireland. 167Hospital General Regional 1 “Dr. Carlos MacGregor Sánchez Navarro” IMSS, Mexico City, Mexico. 168Université Paris-Sud; Service de Pneumologie, Hôpital Bicêtre; Inserm UMR_S999, Le Kremlin Bicêtre, France. 169Dipartimento di medicina, chirurgia e odontoiatria, università di Salerno, Italy. 170Division for Health Innovation, Campania Region and Federico II University Hospital Naples (R&D and DISMET) Naples, Italy. 171Servicio de Alergia e Immunologia, Clinica Santa Isabel, Buenos Aires, Argentina. 172President, Libra Foundation, Buenos Aires, Argentina. 173Medical University of Gdańsk, Department of Allergology, Gdansk, Poland. Mask Study Group 1 3 174Airway Disease Infection Section, National Heart and Lung Institute, Imperial College; MRC & Asthma UK Centre in Allergic Mechanisms of Asthma, London, UK. 175Dept of Respiratory Medicine, Ghent University Hospital, Ghent, Belgium. 176Hallym University College of Medicine, Hallym University Sacred Heart Hospital, Gyeonggi-do, South Korea. 177Department of Clinical Immunology, Wrocław Medical University, Poland. 178Ukrainina Medical Stomatological Academy, Poltava, Ukraine. 179Pediatric Allergy and Asthma Unit, Hacettepe University School of Medicine, Ankara, Turkey. 180Hacettepe University, School of Medicine, Department of Chest Diseases, Immunology and Allergy Division, Ankara, Turkey. 181Allergy Centre, Tampere University Hospital, Tampere, Finland. 182First Department of Family Medicine, Medical University of Lodz, Poland. 183Institute of Social Medicine, Epidemiology and Health Economics, Charité - Universitätsmedizin Berlin, Berlin, and Institute for Clinical Epidemiology and Biometry, University of Wuerzburg, Germany. 184Department of Medicine, McMaster University, HealthSciences Centre 3V47, West, Hamilton, Ontario, Canada. 185National Research Center, Institute of Immunology, Federal Medicobiological Agency, Laboratory of Molecular immunology, Moscow, Russian Federation. 186GARD Chairman, Geneva, Switzerland. 187Allergy & Asthma Center Westend, Berlin, Germany. 188Center for Rhinology and Allergology, Wiesbaden, Germany. 189Department of Immunology and Allergy, Healthy Ageing Research Center, Medical University of Lodz, Lodz, Poland. 190Children’s Hospital and University of Helsinki, Finland. 191Centre for Clinical Research Sörmland, Uppsala University, Eskilstuna, Sweden. 192Faculty of Medicine, Vilnius University, Vilnius, Lithuania. 193Department of Prevention of Envinronmental Hazards and Allergology, Medical University of Warsaw, Poland. 194Center of Excellence in Asthma and Allergy, Médica Sur Clinical Foundation and Hospital, México City,, Mexico. 195Presidente CMMC, Milano, Italy. 196Head of the Allergy Department of Pedro de Elizalde Children’s Hospital, Buenos Aires, Argentina. 197University of Medicine and Pharmacy, Hochiminh City, Vietnam. 198Federal University of Bahia, Brazil. 199Sifmed, Milano, Italy. 200State Key Laboratory of Respiratory Diseases, Guangzhou Institute of Respiratory Disease, the First Affiliated Hospital of Guangzhou Medical University, Guangzhou, China. 201Departments of Internal Medicine and Pediatrics (Divisions of Allergy and Immunology), University of Tennessee College of Medicine, Germantown, TN, USA. 202Scottish Centre for Respiratory Research, Cardiovascular & Diabetes Medicine, Medical Research Institute, Ninewells Hospital, University of Dundee, UK. 203Oslo University Hospital, Department of Paediatrics, Oslo, and University of Oslo, Faculty of Medicine, Institute of Clinical Medicine, Oslo, Norway. 204Department of Pulmonary Medicine, CHU Sart-Tilman, and GIGA I3 research group, Liege, Belgium. 205Faculty of Health Sciences and CICS – UBI, Health Sciences Research Centre, University of Beira Interior, Covilhã, Portugal. 206Department of Philosophical, Methodological and Instrumental Disciplines, CUCS, University of Guadalajara, Guadalajara, Mexico. Mask Study Group 1 3 365Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, NC, USA. 366International Primary Care Respiratory Group IPCRG, Aberdeen, Scotland. 367Bradford Institute for Health Research, Bradford Royal Infirmary, Bradford, UK. 368Allergologyst - Medical College of Medical Faculty, Thracian University, Stara Zagora, Bulgaria. 369Department of Research, Olmsted Medical Center, Rochester, Minnesota, USA. 370Cyprus International Institute for Environmental & Public Health in Association with Harvard School of Public Health, Cyprus University of Technology, Limassol, Cyprus; Department of Pediatrics, Hospital “Archbishop Makarios III”, Nicosia, Cyprus. 371C l l B U D f P l l M T k 372Th Medicine; Messerli Research Institute of the University of Veterinary Medicine and Medical University, Vienna, Austria.267Department of Immunology and Allergology, Faculty of Medicine and Faculty Hospital in Pilsen, Charles University in Prague, Pilsen, Czech Republic. 268Division of Infection, Immunity & Respiratory Medicine, Royal Manchester Children’s Hospital, University of Manchester, Manchester, UK, and Allergy Department, 2nd Pediatric Clinic, Athens General Children’s Hospital “P&A Kyriakou,” University of Athens, Athens, Greece. 269Department of Allergy and Clinical Immunology, Ajou University School of Medicine, Suwon, South Korea. 270Respiratory Medicine, Department of Medical Sciences, University of Ferrara, Ferrara, Italy. 271Allergy and Respiratory Diseases, Ospedale Policlino San Martino -University of Genoa, Italy. 272Farmacias Holon, Lisbon, Portugal. 273Department of Pediatrics, Nippon Medical School, Tokyo, Japan. 274University of Southern Denmark, Kolding, Denmark. 275Université Grenoble Alpes, Laboratoire HP2, Grenoble, INSERM, U1042 and CHU de Grenoble, France. 276Allergy Unit, CUF-Porto Hospital and Institute; Center for Research in Health Technologies and information systems CINTESIS, Universidade do Porto, Portugal. 277Sociologist, municipality area n33, Sorrento, Italy. 278Center for Rhinology and Allergology, Wiesbaden, Germany. 279Department of Otorhinolaryngology, Head and Neck Surgery, Universitätsmedizin Mannheim, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany. 280Centre for empowering people and communites, Dublin, UK. 281Conseil Général de l’Economie Ministère de l’Economie, de l’Industrie et du Numérique, Paris, France. 282Société de Pneumologie de Langue Française, Espace francophone de Pneumologie, Paris, France. 283Département de pédiatrie, CHU de Grenoble, Grenoble France. 284Medical School, University of Cyprus, Nicosia, Cyprus. 285Children’s Hospital Srebrnjak, Zagreb, School of Medicine, University J.J. Strossmayer, Osijek, Croatia. 286Karl Landsteiner Institute for Clinical and Experimental Pneumology, Hietzing Hospital, Vienna, Austria. 287University Hospital ‘Sv. Ivan Rilski’”, Sofia, Bulgaria. 288Allergy Diagnostic and Clinical Research Unit, University of Cape Town Lung Institute, Cape Town, South Africa. 289Vice-Presidente of IML, Milano, Italy. Mask Study Group 1 3 242Federal University of Bahia, Brazil. 243Rhinology Unit & Smell Clinic, ENT Department, Hospital Clínic; Clinical & Experimental Respiratory Immunoallergy, IDIBAPS, CIBERES, University of Barcelona, Spain. 244Danish Commitee for Health Education, Copenhagen East, Denmark. 245Food Allergy Referral Centre Veneto Region, Department of Women and Child Health, Padua General University Hospital, Padua, Italy. 246Director, Medical Communications Consultant, MedScript Ltd, Dundalk, Co Louth, Ireland and Honorary Research Fellow, OPC, Cambridge, UK Ireland. 247Johns Hopkins School of Medicine, Baltimore, Maryland, USA. 248General Manager of COFASER - Pharmacy Services Consortium, Salerno, Italy. 249Scientific Centre of Children’s Health under the MoH, Moscow, Russian National Research Medical University named Pirogov, Moscow, Russia. 250Director of Center of Allergy, Immunology and Respiratory Diseases, Santa Fe, Argentina Center for Allergy and Immunology, Santa Fe, Argentina. 251Dept of Otorhinolaryngology, Medical University of Vienna, AKH, Vienna, Austria. 252Hospital of the Hospitaller Brothers in Buda, Budapest, Hungary. 253Die Hautambulanz and Rothhaar study center, Berlin, Germany. 254Neumología y Alergología Infantil, Hospital La Fe, Valencia, Spain. 255Center for Health Technology and Services Research - CINTESIS and Department of Internal Medicine, Centro Hospitalar Sao Joao, Porto, Portugal. 256Caisse d’assurance retraite et de la santé au travail du Languedoc-Roussillon (CARSAT-LR), Montpellier, France. 257Director of Department of Pharmacy of University of Naples Federico II, Naples, Italy. 258ENT Department, University Hospital of Kinshasa, Kinshasa, Congo. 259Department of Allergy, Immunology and Respiratory Medicine, Alfred Hospital and Central Clinical School, Monash University, Melbourne, Victoria, Australia; Department of Immunology, Monash University, Melbourne, Victoria, Australia. 260Medical center “Research expert”, 261 Page 18 of 21 Page 18 of 21 Bousquet et al. Clin Transl Allergy (2018) 8:45 Bousquet et al. Clin Transl Allergy (2018) 8:45 Bousquet et al. Clin Transl Allergy (2018) 8:45 Department of Immunology, Faculty of Medicine, University of Manitoba, Winnipeg, Manitoba, Canada. 326INSERM, Université Grenoble Alpes, IAB, U 1209, Team of Environmental Epidemiology applied to Reproduction and Respiratory Health, Université Joseph Fourier, Grenoble, France. 327Sociedad Paraguaya de Alergia Asma e Inmunologı´a, Paraguay. 328Division of Allergy, Clinical Immunology and Rheumatology, Department of Pediatrics, Federal University of São Paulo, São Paulo, Brazil. 329European Health Futures Forum (EHFF), Dromahair, Ireland. 330ENT, Aachen, Germany. 331Kyrgyzstan National Centre of Cardiology and Internal medicine, Euro-Asian respiratory Society, Bishkek, Kyrgyzstan. 332University Hospital Olomouc, Czech Republic. 333Department of Paediatric and Adolescent medicine, University Hospital of North Norway, Tromsø, Paediatric Research Group, Deptarment of Clinical Medicine, Faculty of Health Sciences, UiT The Arctic University of Norway, Tromsø, Norway. Mask Study Group 1 3 334Presidente, IML (Lombardy Medical Initiative), Bergamo, Italy. 335Pulmonary Division, Heart Institute (InCor), Hospital da Clinicas da Faculdade de Medicina da Universidade de Sao Paulo, Sao Paulo, Brazil. 336Public Health Institute of Vilnius University, Vilnius, Lithuania. 337Universi‑ dade Federal do Estado do Rio de Janeiro, Rio de Janeiro - Brazil 338RNSA (Réseau National de Surveillance Aérobiologique), Brussieu, France. 339The Hospital for Sick Children, Dalla Lana School of Public Health, University of Toronto, Canada. 340Imunoalergologia, Centro Hospitalar Universitário de Coimbra and Faculty of Medicine, University of Coimbra, Portugal. 341Depart‑ ment of ENT, Medical University of Graz, Austria. 342Campania Region, Division on Pharmacy and devices policy, Naples, Italy. 343Department of Respiratory Medicine, Hvidovre Hospital & University of Copenhagen, Denmark. 344Universidade Federal dos Pampas, Uruguaiana, Brazil. 345Division of Immunopathology, Department of Pathophysiology and Allergy Research, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria. 346Pneumology and Allergy Department CIBERES and Clinical & Experimental Respiratory Immunoallergy, IDIBAPS, University of Barcelona, Spain. 347Vilnius University Institute of Clinical Medicine, Clinic of Children’s Diseases, and Institute of Health Sciences, Department of Public Health, Vilnius, Lithuania; European Academy of Paediatrics (EAP/UEMS-SP), Brussels, Belgium. 348Department of Lung Diseases and Clinical Immunology Allergology, University of Turku and Terveystalo allergy clinic, Turku, Finland. 349PELyon; HESPER 7425, Health Services and Performance Resarch - Université Claude Bernard Lyon, France.350Immunology and Allergy Unit, Department of Medicine Solna, Karolinska Institutet and University Hospital, Stockholm. 351Department of Chest Medicine, Centre Hospitalier Universitaire UCL Namur, Université Catholique de Louvain, Yvoir, Belgium. 352University of Bari Medical School, Unit of Geriatric Immunoallergology, Bari, Italy. 353Pulmonary Unit, Department of Medical Specialties, Arcispedale SMaria Nuova/IRCCS, AUSL di Reggio Emilia, Italy. 354FILHA, Finnish Lung Association, Helsinki, Finland. 355Pulmonary Environmental Epidemiology Unit, CNR Institute of Clinical Physiology, Pisa, Italy ; and CNR Institute of Biomedicine and Molecular Immunology “A Monroy”, Palermo, Italy. 356Medical University, Plovdiv, Bulgaria, Department of Otorhinolaryngology, Plovdiv, Bulgaria. 357Sotiria Hospital, Athens, Greece. 358Dept of Otorhinolaryngology, Universitätsklinikum Düsseldorf, Germany. 359Asthma UK, Mansell street, London, UK. 360Nova Southeastern University, Fort Lauderdale, Florida, USA. 361Department of Otolaryngology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore. 362Department of Medicine, Clinical Immunology and Allergy, McMaster University, Hamilton, Ontario, Canada. 363Division of Immunodermatology and Allergy Research, Department of Dermatology and Allergy, Hannover Medical School, Hannover, Germany. 364Department of Medicine Solna, Immunology and Allergy Unit, Karolinska Institutet and Department of ENT diseases, Karolinska University Hospital, Stockholm, Sweden. Competing interests OP reports grants and personal fees from ALK-Abelló, Allergopharma, Stallergenes Greer, HAL Allergy Holding B.V./HAL Allergie GmbH, Bencard Allergie GmbH/Allergy Therapeutics, Lofarma, Biotech Tools S.A., Laboratorios LETI/LETI Pharma, Anergis S.A., grants from Biomay, Nuvo, Circassia, Glaxo Smith Kline, personal fees from Novartis Pharma, MEDA Pharma, Mobile Chamber Experts (a GA2LEN Partner), Pohl-Boskamp, Indoor Biotechnologies, grants from, outside the submitted work. AMTB reports grants and personal fees from Novartis, Boehringer Ingelheim, Mundipharma, GSK (GlaxoSmithKline), personal fees from Teva Pharma, AstraZeneca, grants from Leti, outside the submitted work. SW reports personnal fees from Merck, GSK, Novartis, Behring, Shire, Sanofi, Barid Aralez, Mylan Meda, Pediapharm outside the submitted work. 3. Bousquet J, Bachert C, Canonica GW, Casale TB, Cruz AA, Lockey RJ, et al. Unmet needs in severe chronic upper airway disease (SCUAD). J Allergy Clin Immunol. 2009;124(3):428–33. 4. Bousquet J, Mantzouranis E, Cruz AA, Ait-Khaled N, Baena-Cagnani CE, Bleecker ER, et al. Uniform definition of asthma severity, con‑ trol, and exacerbations: document presented for the World Health Organization Consultation on Severe Asthma. J Allergy Clin Immunol. 2010;126(5):926–38. 5. De Greve G, Hellings PW, Fokkens WJ, Pugin B, Steelant B, Seys SF. Endotype-driven treatment in chronic upper airway diseases. Clin Transl Allergy. 2017;7:22. 6. Cingi C, Gevaert P, Mosges R, Rondon C, Hox V, Rudenko M, et al. Multi- morbidities of allergic rhinitis in adults: European Academy of Allergy and Clinical Immunology task force report. Clin Transl Allergy. 2017;7:17. 7. Frohlich M, Pinart M, Keller T, Reich A, Cabieses B, Hohmann C, et al. Is there a sex-shift in prevalence of allergic rhinitis and comorbid asthma from childhood to adulthood? A meta-analysis. Clin Transl Allergy. 2017;7:44. 8. Bousquet J, Addis A, Adcock I, Agache I, Agusti A, Alonso A, et al. Inte‑ grated care pathways for airway diseases (AIRWAYS-ICPs). Eur Respir J. 2014;44(2):304–23. 9. Hellings PW, Fokkens WJ, Bachert C, Akdis CA, Bieber T, Agache I, et al. Positioning the principles of precision medicine in care pathways for allergic rhinitis and chronic rhinosinusitis—an EUFOREA-ARIA-EPOS- AIRWAYS ICP statement. Allergy. 2017;72(9):1297–305. 10. Bousquet J, Schunemann HJ, Fonseca J, Samolinski B, Bachert C, Canonica GW, et al. MACVIA-ARIA Sentinel NetworK for allergic rhinitis (MASK-rhinitis): the new generation guideline implementation. Allergy 2015;70(11):1372–92. 11. Hellings PW, Fokkens WJ, Akdis C, Bachert C, Cingi C, Dietz de Loos D, et al. Uncontrolled allergic rhinitis and chronic rhinosinusitis: where do we stand today? Allergy. 2013;68(1):1–7. 12. mHealth. Competing interests SBA reports personal fees from Boehringer Ingelheim, GSK, AstraZeneca, TEVA, grants from TEVA, MEDA outside the submitted work. JB reports personal fees and other from Chiesi, Cipla, Hikma, Menarini, Mundipharma, Mylan, Novartis, Sanofi-Aventis, Takeda, Teva, Uriach, other from Kyomed, outside the submitted work. AAC reports grants and personal fees from GlaxoSmithKline, personal fees from Boehrinher Ingelheim, personal fees from AstraZeneca, personal fees from Novartis, personal fees from Merk, Sharp & Dohma, personal fees from MEDA Pharma, personal fees from EUROFARMA, personal fees from Sanofi Aventis, outside the submitted work. MD reports other from Allergan, outside the submitted work. WF reports grants from Meda, outside the submitted work. TH reports personal fees from Mundipharma, Novartis, and Orion Pharma, outside the submitted work. JJ reports grants and personal fees from novartis, ALK abello, personal fees from thermofischer, astra zeneca outside the submitted work. PK reports personal fees from Adamed, Boehringer Ingelheim, AstraZeneca, Chiesi, FAES, Berlin Chemie, Novartis, Polp‑ harma, Allergopharma, outside the submitted work. VK has received payment for consultancy from GSK and for lectures from Stallergens, Berlin-CHemie outside the submitted work. DLL reports personal fees from GSK, Astrazeneca, MEDA, Boehringer Ingelheim, Novartis, Grunenthal, UCB, Amstrong, Siegfried, DBV Technologies, MSD, Pfizer, grants from Sanofi, Astrazeneca, Novartis, UCB, GSK, TEVA, Chiesi, Boehringer Ingelheim, outside the submitted work. RM reports personal fees from ALK, grants from ASIT biotech, Leti, BitopAG, Hulka, Ursapharm, Optima; personal fees from allergopharma, Nuvo, Meda, Friulchem, Hexal, Servier, Bayer, Johnson&Johnson, Klosterfrau, GSK, MSD, FAES, Stada, UCB, Allergy Therapeutics; grants and personal fees from Bencard, Stallergenes; grants, personal fees and non-financial support from Lofarma; non-financial support from Roxall, Atmos, Bionorica, Otonomy, Ferrero; per‑ sonal fees and non-financial support from Novartis. NP reports personal fees from Novartis, Faes Farma, BIOMAY, HAL, Nutricia Research, Menarini, Novartis, MEDA, Abbvie, MSD, Omega Pharma, Danone, grants from Menarini, outside the submitted work. JLP reports grants from Air Liquide Foundation, AGIR à dom, Astrazeneca, Fisher & Paykel, Mutualia, Philips, Resmed, Vitalaire, other from AGIR à dom, Astrazeneca, Boehringer Ingelheim, Jazz Pharmaceutical, Night Balance, Philips, Resmed, Sefam, outside the submitted work. References 1. Bousquet J, Khaltaev N, Cruz AA, Denburg J, Fokkens WJ, Togias A, et al. Allergic rhinitis and its impact on asthma (ARIA) 2008 update (in col‑ laboration with the World Health Organization, GA(2)LEN and AllerGen). Allergy. 2008;63(Suppl 86):8–160. 2. Vandenplas O, Vinnikov D, Blanc PD, Agache I, Bachert C, Bewick M, et al. Impact of rhinitis on work productivity: a systematic review. J Allergy Clin Immunol Pract. 2018;6(4):1274–86. Mask Study Group 1 3 Clin Transl Allergy (2018) 8:45 Institute of Otolaryngology, Beijing, China. 375Universidad Católica de Córdoba, Córdoba, Argentina. 376University Clinic of Respiratory and Allergic Diseases, Golnik, Slovenia. 377Gesundheitsregion KölnBonn - HRCB Projekt GmbH, Kohln, Germany. 378Akershus University Hospital, Department of Otorhinolaryngol‑ ogy, Akershus, Norway. Institute of Otolaryngology, Beijing, China. 375Universidad Católica de Córdoba, Córdoba, Argentina. 376University Clinic of Respiratory and Allergic Diseases, Golnik, Slovenia. 377Gesundheitsregion KölnBonn - HRCB Projekt GmbH, Kohln, Germany. 378Akershus University Hospital, Department of Otorhinolaryngol‑ ogy, Akershus, Norway. Funding 18. Lee L, Sheikh A. Understanding stakeholder interests and perspectives in evaluations of health IT. Stud Health Technol Inf. 2016;222:53–62. 19. Geryk LL, Roberts CA, Sage AJ, Coyne-Beasley T, Sleath BL, Carpenter DM. Parent and clinician preferences for an asthma app to promote adolescent self-management: a formative study. JMIR Res Protoc. 2016;5(4):e229. Competing interests New horizons for health through mobile technologies. Global Observatory for eHealth series—Vol. 3 WHO Library Cataloguing-in-Pub‑ lication Data. 2011; http://www.who.int/goe/publications/goe_mheal th_web.pdf. Accessed 30 Sept 2018. 13. Ozdalga E, Ozdalga A, Ahuja N. The smartphone in medicine: a review of current and potential use among physicians and students. J Med Internet Res. 2012;14(5):e128. 14. Freifeld CC, Chunara R, Mekaru SR, Chan EH, Kass-Hout T, Ayala Iacucci A, et al. Participatory epidemiology: use of mobile phones for community- based health reporting. PLoS Med. 2010;7(12):e1000376. 15. Keijser W, de-Manuel-Keenoy E, d’Angelantonio M, Stafylas P, Hobson P, Apuzzo G, et al. DG Connect funded projects on information and com‑ munication technologies (ICT) for old age people: Beyond Silos, CareWell and SmartCare. J Nutr Health Aging. 2016;20(10):1024–33. Mask Study Group 1 3 290Centre of Academic Primary Care, Division of Applied Health Sciences, University of Aberdeen, Aberdeen, United Kingdom ; Observational and Pragmatic Research Institute, Singapore, Singapore. 291Department of Otorhinolaryngology University of Crete School of Medicine, Heraklion, Greece. 292European Forum for Research and Education in Allergy and Airway Diseases (EUFOREA), Brussels, Belgium. 293Cancun, Quintana Roo, Mexico. 294LungenClinic Grosshansdorf, Airway Research Center North, Member of the German Center for Lung Research (DZL), Grosshansdorf, Germany Department of Medicine, Christian Albrechts University, Airway Research Center North, Member of the German Center for Lung Research (DZL), Kiel, Germany. 295Department of Nephrology and Endocrinology, Karolinska University Hospital, Stockholm, Sweden. 296Farmácia São Paio, Vila Nova de Gaia, Porto, Portugal. 297St Vincent’s Hospital and University of Sydney, Sydney, New South Wales, Australia. 298Puebla, Mexico. 299Serviço de Pneumologia-Hosp das Clinicas UFPE-EBSERH, Recife, Brazil. 300Universidade Federal de São Paulo, São Paulo, Brazil. 301Centre of Pneumology, Coimbra University Hospital, Portugal. 302Polibienestar Research Institute, University of Valencia, Valencia, Spain. 303Pediatric Allergy and Clinical Immunology, Hospital Angeles Pedregal, Mexico City, Mexico. 304Getafe University Hospital Department of Geriatrics, Madrid, Spain. 305Association Asthme et Allergie, Paris, France. 306Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil. 307Primary Care Respiratory Research Unit Institutode Investigación Sanitaria de Palma IdisPa, Palma de Mallorca, Spain. 308Allergy Unit, Presidio Columbus, Rome, Catholic University of Sacred Heart, Rome and IRCCS Oasi Maria SS, Troina, Italy. 309Mexico City, Mexico. 310Regione Piemonte, Torino, Italy. 311Medical University of Graz, Department of Internal Medicine, Graz, Austria. 312Serviço de Imunoalergolo‑ gia Hospital da Luz Lisboa Portugal. 313Hospital de Clinicas, University of Parana, Brazil. 314Division of Allergy Asthma and Clinical Immunology, Emek Medical Center, Afula, Israel. 315Honorary Clinical Research Fellow, Allergy and Respiratory Research Group, The University of Edinburgh, Edinburgh, UK. 316Showa University School of Medicine, Tokyo, Japan. 317Association of Finnish Pharmacies. 318Allergy and Clinical Immunology Department, Centro Médico-Docente la, Trinidad and Clínica El Avila, Caracas, Venezuela. 319Faculty of Medicine, Autnonous University of Madrid, Spain. 320The Royal National TNE Hospital, University College London, UK. 321DIBIMIS, University of Palermo, Italy. 322Allergy Unit, Department of Dermatology, University Hospital of Zurich, Zürich, Switzerland. 323Asthma Reference Center, Escola Superior de Ciencias da Santa Casa de Misericordia de Vitoria - Esperito Santo, Brazil. 324THe Usher Institute of Population Health Sciences and Informatics The University of Page 19 of 21 Page 19 of 21 Page 19 of 21 Bousquet et al. Clin Transl Allergy (2018) 8:45 Bousquet et al. Availability of data and materials 16. Mozaffar H, Cresswell KM, Williams R, Bates DW, Sheikh A. Exploring the roots of unintended safety threats associated with the introduction of hospital ePrescribing systems and candidate avoidance and/or mitiga‑ tion strategies: a qualitative study. BMJ Qual Saf. 2017;26(9):722–733. y Not applicable. Ethics approval and consent to participate Not applicable. 17. Talboom-Kamp EP, Verdijk NA, Harmans LM, Numans ME, Chavannes NH. An eHealth platform to manage chronic disease in primary care: an innovative approach. Interact J Med Res. 2016;5(1):e5. Publisher’s Note Allergic Rhinitis and its Impact on Asthma (ARIA) guide‑ lines—2016 revision. J Allergy Clin Immunol. 2017;140(4):950–8. 43. El Emam K, Dankar FK, Issa R, et al. A globally optimal k-anonymity method for the de-identification of health data. J Am Med Inform Assoc. 2009;16:670–82. 26. Bousquet J, Hellings PW, Agache I, Bedbrook A, Bachert C, Bergmann KC, et al. ARIA 2016: care pathways implementing emerging technologies for predictive medicine in rhinitis and asthma across the life cycle. Clin Transl Allergy. 2016;6:47. 44. Samreth D, Arnavielhe S, Ingenrieth F, Bedbrook A, Onorato GL, Murray R, et al. Geolocation with respect to personal privacy for the Allergy Diary app—a MASK study. World Allergy Organ J. 2018;11(1):15. https://doi. org/10.1186/s40413-018-0194-3. y 27. Lombardi C, Musicco E, Rastrelli F, Bettoncelli G, Passalacqua G, Canonica GW. The patient with rhinitis in the pharmacy. A cross-sectional study in real life. Asthma Res Pract. 2015;1:4. 45. Bland JM, Altman DG. Statistical methods for assessing agree‑ ment between two methods of clinical measurement. Lancet. 1986;1(8476):307–10. 28. Bousquet J, Schunemann HJ, Hellings PW, Arnavielhe S, Bachert C, Bedbrook A, et al. MACVIA clinical decision algorithm in adolescents and adults with allergic rhinitis. J Allergy Clin Immunol. 2016;138(2):367–74 (e2). 46. Bousquet J, Agache I, Aliberti MR, Angles R, Annesi-Maesano I, Anto JM, et al. Transfer of innovation on allergic rhinitis and asthma multimorbid‑ ity in the elderly (MACVIA-ARIA)—EIP on AHA Twinning Reference Site (GARD research demonstration project). Allergy. 2018;73(1):77–92. 29. Bourret R, Bousquet J, Mercier J, Camuzat T, Bedbrook A, Demoly P, et al. MASK rhinitis, a single tool for integrated care pathways in allergic rhinitis. World Hosp Health Serv. 2015;51(3):36–9. 47. Konig HH, Bernert S, Angermeyer MC, Matschinger H, Martinez M, Vilagut G, et al. Comparison of population health status in six european coun‑ tries: results of a representative survey using the EQ-5D questionnaire. Med Care. 2009;47(2):255–61. 30. Hellings PW, Muraro A, Fokkens W, Mullol J, Bachert C, Canonica GW, et al. A common language to assess allergic rhinitis control: results from a survey conducted during EAACI 2013 Congress. Clin Transl Allergy. 2015;5:36. 48. Smith AF, Pitt AD, Rodruiguez AE, Alio JL, Marti N, Teus M, et al. The economic and quality of life impact of seasonal allergic conjunctivitis in a Spanish setting. Ophthalmic Epidemiol. 2005;12(4):233–42. 31. Klimek L, Bergmann KC, Biedermann T, Bousquet J, Hellings P, Jung K, et al. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in pub‑ lished maps and institutional affiliations. 20. Bousquet J, Chavannes NH, Guldemond N, Haahtela T, Hellings PW, Sheikh A. Realising the potential of mHealth to improve asthma and allergy care: how to shape the future. Eur Respir J. 2017;49(5):1700447. 20. Bousquet J, Chavannes NH, Guldemond N, Haahtela T, Hellings PW, Sheikh A. Realising the potential of mHealth to improve asthma and allergy care: how to shape the future. Eur Respir J. 2017;49(5):1700447. Received: 31 July 2018 Accepted: 7 September 2018 Bousquet et al. Clin Transl Allergy (2018) 8:45 Page 20 of 21 Page 20 of 21 Bousquet et al. Clin Transl Allergy (2018) 8:45 40. Directive 2002/58/EC of the European Parliament and of the Council of 12 July 2002 concerning the processing of personal data and the protec‑ tion of privacy in the electronic communications sector (Directive on privacy and electronic communications). Off J Eur Commun L 201, 37; 31 July 2002. 21. Lau AY, Arguel A, Dennis S, Liaw ST, Coiera E. “Why Didn’t it Work?” Lessons from a randomized controlled trial of a web-based personally controlled health management system for adults with asthma. J Med Internet Res. 2015;17(12):e283. 22. Simpson AJ, Honkoop PJ, Kennington E, Snoeck-Stroband JB, Smith I, East J, et al. Perspectives of patients and healthcare professionals on mHealth for asthma self-management. Eur Respir J. 2017. https://doi. org/10.1183/13993003.01966-2016. 41. Directive 2009/136/EC of The European Parliament and of the Council of 25 November 2009 amending Directive 2002/22/EC on universal service and users’ rights relating to electronic communications networks and services, Directive 2002/58/EC concerning the processing of personal data and the protection of privacy in the electronic communications sec‑ tor and Regulation (EC) No 2006/2004 on cooperation between national authorities responsible for the enforcement of consumer protection laws. Off J Eur Union, L 337, 11; 18 December 2009. 23. Bousquet J, Van Cauwenberge P, Khaltaev N. Allergic rhinitis and its impact on asthma. J Allergy Clin Immunol. 2001;108(5 Suppl):S147–334. 24. Brozek JL, Bousquet J, Baena-Cagnani CE, Bonini S, Canonica GW, Casale TB, et al. Allergic Rhinitis and its Impact on Asthma (ARIA) guidelines: 2010 revision. J Allergy Clin Immunol. 2010;126(3):466–76. f 42. Sweeney L. k-anonymity: a model for protecting privacy. Int J Uncertain Fuz Knowl Syst. 2002;10:557–70. 25. Brozek JL, Bousquet J, Agache I, Agarwal A, Bachert C, Bosnic-Anticevich S, et al. Publisher’s Note Visual analogue scales (VAS): measuring instruments for the documentation of symptoms and therapy monitoring in cases of allergic rhinitis in everyday health care: position Paper of the German Society of Allergology (AeDA) and the German Society of Allergy and Clinical Immunology (DGAKI), ENT Section, in collaboration with the working group on Clinical Immunology, Allergology and Environmental Medicine of the German Society of Otorhinolaryngology, Head and Neck Surgery (DGHNOKHC). Allergo J Int. 2017;26(1):16–24. 49. Bousquet J, VandenPlas O, Bewick M, Arnavielhe S, Bedbrook A, Murray R, et al. The Work Productivity and Activity Impairment Allergic Specific (WPAI-AS) Questionnaire using mobile technology: the MASK study. J Investig Allergol Clin Immunol. 2018;28(1):42–4. 50. Azevedo P, Correia de Sousa J, Bousquet J, Bugalho-Almeida A, Del Giacco SR, Demoly P, et al. Control of Allergic Rhinitis and Asthma Test (CARAT): dissemination and applications in primary care. Prim Care Respir J. 2013;22(1):112–6. 32. Bousquet J, Farrell J, Crooks G, Hellings P, Bel EH, Bewick M, et al. Scaling up strategies of the chronic respiratory disease programme of the Euro‑ pean Innovation Partnership on Active and Healthy Ageing (Action Plan B3: Area 5). Clin Transl Allergy. 2016;6:29. 51. Fonseca JA, Nogueira-Silva L, Morais-Almeida M, Azevedo L, Sa-Sousa A, Branco-Ferreira M, et al. Validation of a questionnaire (CARAT10) to assess rhinitis and asthma in patients with asthma. Allergy. 2010;65(8):1042–8. 33. Bousquet J, Caimmi DP, Bedbrook A, Bewick M, Hellings PW, Devillier P, et al. Pilot study of mobile phone technology in allergic rhinitis in Euro‑ pean countries: the MASK-rhinitis study. Allergy. 2017;72(6):857–65. 52. Nogueira-Silva L, Martins SV, Cruz-Correia R, Azevedo LF, Morais-Almeida M, Bugalho-Almeida A, et al. Control of allergic rhinitis and asthma test— a formal approach to the development of a measuring tool. Respir Res. 2009;10:52. 34. Bousquet J, Bewick M, Arnavielhe S, Mathieu-Dupas E, Murray R, Bed‑ brook A, et al. Work productivity in rhinitis using cell phones: the MASK pilot study. Allergy. 2017;72(10):1475–84. 53. van der Leeuw S, van der Molen T, Dekhuijzen PN, Fonseca JA, van Gemert FA, Gerth van Wijk R, et al. The minimal clinically important dif‑ ference of the control of allergic rhinitis and asthma test (CARAT): cross- cultural validation and relation with pollen counts. NPJ Prim Care Respir Med. 2015;25:14107. 35. Aristodimou A, Antoniades A, Pattichis CS. Privacy preserving data pub‑ lishing of categorical data through k-anonymity and feature selection. Healthc Technol Lett. 2016;3(1):16–21. 36. Publisher’s Note Aldeen YA, Salleh M, Razzaque MA. A comprehensive review on privacy preserving data mining. Springerplus. 2015;4:694. 54. Johns MW. Reliability and factor analysis of the Epworth Sleepiness Scale. Sleep. 1992;15(4):376–81. 37. Protection of personal data. Article 29 data protection working party. Opinion 05/2014 on anonymisation techniques. European Commission Justice Data Protection. 2014;0829/14/EN WP216. http://ec.europa.eu/ justice/data-protection/index_en.htm. Accessed 30 Sept 2018. 55. Leger D, Annesi-Maesano I, Carat F, Rugina M, Chanal I, Pribil C, et al. Allergic rhinitis and its consequences on quality of sleep: an unexplored area. Arch Intern Med. 2006;166(16):1744–8. 56. Kopp-Kubel S. International Nonproprietary Names (INN) for pharmaceu‑ tical substances. Bull World Health Organ. 1995;73(3):275–9. 38. Regulation (EU) 2016/679 of the European Parliamant and of the Council of 27 April 2016 on the protection of natural persons with regard to the processing of personal data and on the free movement of such data, and repealing Directive 95/46/EC (General Data Protection Regulation). Offi‑ cial Organ of the European Union. 2016. http://eur-lex.europa.eu/legal -content/EN/TXT/PDF/?uri=CELEX:32016R0679&from=EN. Accessed 30 Sept 2018. 57. Santo K, Richtering SS, Chalmers J, Thiagalingam A, Chow CK, Redfern J. Mobile phone apps to improve medication adherence: a systematic stepwise process to identify high-quality apps. JMIR Mhealth Uhealth. 2016;4(4):e132. 58. Thakkar J, Kurup R, Laba TL, Santo K, Thiagalingam A, Rodgers A, et al. Mobile telephone text messaging for medication adherence in chronic disease: a meta-analysis. JAMA Intern Med. 2016;176(3):340–9. 39. Article 28 EU General Data Protection Regulation (EU-GDPR). 2018. https ://www.eugdpr.org/. Accessed 30 Sept 2018. Page 21 of 21 Page 21 of 21 Bousquet et al. Clin Transl Allergy (2018) 8:45 Bousquet et al. Clin Transl Allergy (2018) 8:45 59. Bousquet J, Arnavielhe S, Bedbrook A, Alexis-Alexandre G, van Eerd M, Murray R, et al. Treatment of allergic rhinitis using mobile technol‑ ogy with real world data: the MASK observational pilot study. Allergy. 2018;73(9):1763–74. 65. Bousquet J, Devillier P, Anto JM, Bewick M, Haahtela T, Arnavielhe S, et al. Daily allergic multimorbidity in rhinitis using mobile technology: a novel concept of the MASK study. Allergy. 2018;73(8):1622–31. 66. Tan R, Cvetkovski B, Kritikos V, Price D, Yan K, Smith P, et al. Identifying the hidden burden of allergic rhinitis (AR) in community pharmacy: a global phenomenon. Asthma Res Pract. 2017;3:8. 60. Devillier P, Chassany O, Vicaut E, de Beaumont O, Robin B, Dreyfus JF, et al. The minimally important difference in the Rhinoconjunctivitis Total Symptom Score in grass-pollen-induced allergic rhinoconjunctivitis. Allergy. Publisher’s Note 2014;69(12):1689–95. 67. Carr WW, Yawn BP. Management of allergic rhinitis in the era of effective over-the-counter treatments. Postgrad Med. 2017;129(6):572–80. 68. Members of the Workshop. ARIA in the pharmacy: management of allergic rhinitis symptoms in the pharmacy. Allergic rhinitis and its impact on asthma. Allergy. 2004;59(4):373–87 61. Onder G, Palmer K, Navickas R, Jureviciene E, Mammarella F, Strandz‑ heva M, et al. Time to face the challenge of multimorbidity. A European perspective from the joint action on chronic diseases and promoting healthy ageing across the life cycle (JA-CHRODIS). Eur J Intern Med. 2015;26(3):157–9. 69. Bousquet J, Cruz A, Robalo-Cordeiro C. Obstructive sleep apnoea syn‑ drome is an under-recognized cause of uncontrolled asthma across the life cycle. Rev Port Pneumol. 2016;22(1):1–3 62. Bousquet J, Onorato GL, Bachert C, Barbolini M, Bedbrook A, Bjermer L, et al. CHRODIS criteria applied to the MASK (MACVIA-ARIA Sentinel NetworK) Good Practice in allergic rhinitis: a SUNFRAIL report. Clin Transl Allergy. 2017;7:37. y 70. Bousquet J, Agache I, Anto JM, Bergmann KC, Bachert C, Annesi-Maesano I, et al. Google Trends terms reporting rhinitis and related topics differ in European countries. Allergy 2017;72(8):1261–6. 71. Bousquet J, O’Hehir RE, Anto JM, D’Amato G, Mösges R, Hellings PW, Van Eerd M, Sheikh A. Assessment of thunderstorm-induced asthma using Google Trends. J Allergy Clin Immunol. 2017;140(3):891–3. 63. Mokkink LB, Terwee CB, Patrick DL, Alonso J, Stratford PW, Knol DL, et al. The COSMIN study reached international consensus on taxonomy, ter‑ minology, and definitions of measurement properties for health-related patient-reported outcomes. J Clin Epidemiol. 2010;63(7):737–45. 64. Caimmi D, Baiz N, Tanno LK, Demoly P, Arnavielhe S, Murray R, et al. Valida‑ tion of the MASK-rhinitis visual analogue scale on smartphone screens to assess allergic rhinitis control. Clin Exp Allergy. 2017;47(12):1526–33. • fast, convenient online submission • thorough peer review by experienced researchers in your ﬁeld • rapid publication on acceptance • support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year • At BMC, research is always in progress. Learn more biomedcentral.com/submissions Ready to submit your research ? 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https://openalex.org/W2099281321	https://ars.copernicus.org/articles/7/123/2009/ars-7-123-2009.pdf	English	null	Efficient modelling of IC conducted emission for power integrity analysis	Advances in radio science	2,009	cc-by	5,825	2 IC conducted emission models The three IC conducted emission models employed in this section describe the conducted emission behaviour of the supply system of an IC. The model with the highest level of complexity is generated with the so-called EXPO tool (Hes- idenz and Steinecke, 2005). Efﬁcient modelling of IC conducted emission for power integrity analysis R. Kazemzadeh1,2, S. Ludwig1,2, Lj. Radi´c-Weissenfeld1,2, and W. Mathis1 1Leibniz University of Hannover Institute of Electromagnetic Theory, Hannover, Germany 2Fraunhofer Research Institution for Electronic Nano Systems, Advanced System Engineering, Paderborn, Germany R. Kazemzadeh1,2, S. Ludwig1,2, Lj. Radi´c-Weissenfeld1,2, and W. Mathis1 1Leibniz University of Hannover Institute of Electromagnetic Theory, Hannover, Germany 2Fraunhofer Research Institution for Electronic Nano Systems, Advanced System Engineering, Paderborn, Germany et al., 1994). For the estimation of the power integrity be- haviour of integrated circuits (IC), IC Conducted Emission Models are created. Depending on the precision and simu- lation speed demands of the designer, different complexity levels for these models exist. Section 2 shows the necessity of reducing the complexity of certain IC conducted emission models and describes the structure of such models. The mod- elling methodologies of the two models with lower complex- ity, gained by reducing the size of the complex model, will be described in Sects. 3 and 4. In Sect. 5 simulation results of the three models are presented and compared. Section 6 gives a summary of the paper. Abstract. In this paper two methodologies to reduce the complexity of IC conducted emission models for Power In- tegrity analysis in ICs are presented. The methodologies dif- fer concerning the applicability in simulation tools, complex- ity and accuracy of the generated models. The ﬁrst methodol- ogy uses a complex model and reduces its order to generate a model with a fewer number of elements. This methodol- ogy therefore involves a model order reduction approach. A second minimum complexity, module based modelling ap- proach is introduced for rough estimations, as the order re- duced model is still too complex for some applications. The two methodologies are applied to an IC conducted emis- sion model of two digital modules of a 32 Bit microcon- troller. The results of the three models are compared and discussed. Fields of application for the introduced modelling approaches are the estimation of the magnitude and time be- haviour of the supply current as well as the determination of the number and position of the IC’s supply pins. Adv. Radio Sci., 7, 123–126, 2009 www.adv-radio-sci.net/7/123/2009/ © Author(s) 2009. This work is distributed under the Creative Commons Attribution 3.0 License. Adv. Radio Sci., 7, 123–126, 2009 www.adv-radio-sci.net/7/123/2009/ © Author(s) 2009. This work is distributed under the Creative Commons Attribution 3.0 License. Adv. Radio Sci., 7, 123–126, 2009 www.adv-radio-sci.net/7/123/2009/ © Author(s) 2009. This work is distributed under the Creative Commons Attribution 3.0 License. Advances in Radio Science 1 Introduction g p y g on an existing chip. of up to hundreds of line models (SLM) power integrity behaviour is preserved with these models, the accuracy i components and controlled current sources (Fig 3) 124[H mo ide R. Kazemzadeh et al.: Efﬁcient modelling of IC conducted emission for power integrity analysis necke, 2005]. on an existing chip. of up to hundreds of line models (SLM) p g y p with these models, the accuracy i components and controlled current sources (Fig 3) Due to the relatively high complexity o the model gained with this approach applicability in power integrity analysi tools is limited. IC conducted emission models with a smaller number of element are required to lower the computationa diminished to a certain degree compared t the complex model. 3 Modelling of an order reduced IC Conducted Emission Model Fig.3 MOR reduced IC conducted emission model Fig. 3. MOR reduced IC conducted emission model. Fig.1 Section of a functional module of the EXPO model consisting of a large number VDD PIN VSS PIN VSS PIN VDD PIN Fig. 1. Section of a functional module of the EXPO model consist- ing of a large number of SLMs. forming a mesh (Fig.1). The SLM therefore represents the basic element of the EXPO model. Each SLM contains a power distribution network (PDN) consisting of passive R, L, C elements, and an independent current source (Fig. 2) [Dhia et al., 2006]. models with a smaller number o are required to lower the com Conducted Emission Mode Fig.3 MOR reduced IC co i i d l Fig. 3. MOR reduced IC conducted emission model. Fig.1 Section of a functional module of the EXPO model consisting of a large number Fig. 1. Section of a functional module of the EXPO model consist- ing of a large number of SLMs. p ( g ) [Dhia et al., 2006]. adequate for the implementation in EDA tools by reducing the complexity of th EXPO model are presented here. While th computational effort is reduced and th ability to obtain information about th power integrity behaviour is preserve with these models, the accuracy i diminished to a certain degree compared t the complex model. 1 Introduction 3 Modelling of an order reduced IC Conducted Emission Model Th fi d lli h d l i l a model order reduction (MOR) approach The objective of the MOR modellin methodology is to reduce the size of th considered model and thereby it complexity while approximating th properties at the pins of the origina network [Ludwig, 2008b]. For MOR th electrical network of the IC conducte emission model has to be described wit the help of the modified nodal analysis as system of differential algebraic equations Here the number of equations specifies th order of the system. To use MOR in IC d t d i i d l ffi i tl 4 Module-related IC Conducted Emission Model For a rough estimation of the power integrity behaviour where low simulation time takes priority over preciseness, the MOR modelling approach still leads to models that are too complex. For these applications a simplified IC conducted emission model with a very low number of elements is required. Such a model can be generated either by measurements or in a simulative approach and contains only one has to preserve their main properties. This is for example achieved by using moment matching, as we investigated in Radi´c-Weissenfeld (2009). Here we use the block Arnoldi algorithm, where every system matrix is directly reduced, re- sulting in a drastically reduced order of the system. It can be shown that the obtained transfer function of the considered network matches the original transfer function within the in- vestigated frequency band. In a last step, the order reduced circuit equations have to be synthesised as an electrical net- work for simulations with power analysis tools. The network synthesis method we proposed in Ludwig (2008b) generates a network with a low number of nodes and is exported as a SPICE netlist. By reducing the complexity of the EXPO model by means of MOR, the EXPO model’s SLM structure is not maintained in the ﬁnal synthesis step, resulting in a model consisting of G, C components and controlled current sources (Fig. 3). of SLMs It consists of several modules, each describing a specific functionality on the chip (e.g. digital, analogue, memory). Each module consists of up to hundreds of identical supply line models (SLM) forming a mesh (Fig.1). The SLM therefore represents the basic element of the EXPO model. a model order reduction (MOR) app The objective of the MOR mo certain preparations have to be made network's description as we have sh SLM for each functional module, i.e elements of the PDN and one cur 4 Module-related IC conducted emission model The objective of the MOR modellin methodology is to reduce the size of th considered model and thereby it complexity while approximating th properties at the pins of the origina network [Ludwig, 2008b]. For MOR th electrical network of the IC conducte emission model has to be described wit the help of the modified nodal analysis as system of differential algebraic equations p , [Ludwig, 2008a]. The network descriptio of the IC conducted emission model ca now be reduced by the use of MOR algorithms. Order reduction of system descriptions of networks has to preserv their main properties. This is for exampl achieved by using moment matching, a we investigated in [Radić-Weissenfeld 2008]. Here we use the block Arnold source [UTE 47A EMC Task Force, 2002] (Fig. 4). To meet the low complexity demands of the model, a curve fitting approach is used to obtain the R, L, C parameters of the PDN. The fitting equation for an impedance Zmn For a rough estimation of the power integrity behaviour where low simulation time takes priority over preciseness, the MOR modelling approach still leads to models that are too complex. For these applications a simpliﬁed IC con- ducted emission model with a very low number of elements is required. Such a model can be generated either by mea- surements or in a simulative approach and contains only one SLM for each functional module, i.e. the elements of the PDN and one current source (UTE 47A EMC Task Force, 2002) (Fig. 4). To meet the low complexity demands of the model, a curve ﬁtting approach is used to obtain the R, L, C parameters of the PDN. 1 Introduction Each SLM contains a power distribution network (PDN) Fig.2 Supply line model (SLM) consisting Passive Distribution Network (PDN) IIA Internal Activity (IA) LVDD RVDD LVSS RVSS VDD PIN VSS PIN Fig. 2. Supply line model (SLM) consisting of PDN and IA. on the Internal modu VSS PIN erefore represents the basic EXPO d l E h SLM Passive Distribution Network (PDN) power distribution network (PD Fig 2 Supply line model (SLM) consist Fig. 2. Supply line model (SLM) consisting of PDN and IA. an independent current source (Fig. 2) [Dhia et al., 2006]. IIA Internal Activity (IA) LVDD RVDD L R VDD PIN VSS PIN While the PDN describes the parasitic electromagnetic effects of the on-chip power supply, the current source characterises the internal activity (IA), i.e. the simultaneous switching noise (SSN) of the transistors. The parameters of the passive elements of the PDN as well as the prescribed waveforms of the current Due to the relatively high complexity of the model gained with this approach, applicability in power integrity analy- sis tools is limited. IC conducted emission models with a smaller number of elements are required to lower the compu- tational effort. Two methodologies to gain models adequate for the implementation in EDA tools by reducing the com- plexity of the EXPO model are presented here. While the computational effort is reduced and the ability to obtain in- formation about the power integrity behaviour is preserved with these models, the accuracy is diminished to a certain degree compared to the complex model. 1 Introduction It consists of several modules, each describing a speciﬁc functionality on the chip (e.g. digital, analogue, memory). Each module consists of up to hundreds of identical supply line models (SLM) forming a mesh (Fig. 1). The SLM there- fore represents the basic element of the EXPO model. Each SLM contains a power distribution network (PDN) consist- ing of passive R, L, C elements, and an independent current source (Fig. 2) (Dhia et al., 2006). The ongoing development in the area of electronic circuit in- tegration is accompanied by new, outstanding challenges in the ﬁeld of low power design. This leads to more severe con- straints in the ﬁeld of power integrity, especially in regard to the ever decreasing supply voltages in modern electronic devices, circuits and systems. For a reduction of time-to- market by ﬁrst-time right designs these low power and power integrity constraints need to be considered in an early stage of the design ﬂow. To achieve this, the designer is reliant on modern, power-conscious EDA tools and accurate mod- els that give insight to the power consumption and integrity behaviour at a high abstraction level of the design (Landman While the PDN describes the parasitic electromagnetic ef- fects of the on-chip power supply, the current source charac- terises the internal activity (IA), i.e. the simultaneous switch- ing noise (SSN) of the transistors. The parameters of the passive elements of the PDN as well as the prescribed wave- forms of the current sources vary from module to module, but are the same for all SLMs within one functional module. The parameters of the model are determined either during the IC design phase or by taking measurements on an existing chip. Correspondence to: R. Kazemzadeh (reza.kazemzadeh@pb.izm.fraunhofer.de) Published by Copernicus Publications on behalf of the URSI Landesausschuss in der Bundesrepublik Deutschland e.V. Published by Copernicus Publications on behalf of the URSI Landesausschuss in der Bundesrepub y Copernicus Publications on behalf of the URSI Landesausschuss in der Bundesrepublik Deutschl R. Kazemzadeh et al.: Efﬁcient modelling of IC conducted emission for power integrity analysis necke, 2005]. g p y g on an existing chip. f up to hundreds of line models (SLM) power integrity behaviour is preserve with these models, the accuracy i components and controlled current sources (Fig 3) R. Kazemzadeh et al.: Efﬁcient modelling of IC conducted emission for power integrity analysis necke, 2005]. Passive Distribution Network (PDN) are the same for all SLMs within one functional module. The parameters of the 3 Modelling of an order reduced IC conducted emission model Fig.2 Supply line model (SLM) consisting of PDN and IA While the PDN describes the parasitic electromagnetic effects of the on-chip power supply, the current source characterises the internal activity (IA), i.e. the simultaneous switching noise (SSN) of the transistors. The parameters of the passive elements of the PDN as well as the prescribed waveforms of the current sources vary from module to module, but model are determined either during the IC The ﬁrst modelling methodology involves a model order reduction (MOR) approach. The objective of the MOR modelling methodology is to reduce the size of the consid- ered model and thereby its complexity while approximat- ing the properties at the pins of the original network (Lud- wig, 2008b). For MOR the electrical network of the IC con- ducted emission model has to be described with the help of the modiﬁed nodal analysis as a system of differential alge- braic equations. Here the number of equations speciﬁes the order of the system. To use MOR in IC conducted emis- sion models efﬁciently, certain preparations have to be made in the network’s description, as we have shown in Ludwig (2008a). The network description of the IC conducted emis- sion model can now be reduced by the use of MOR algo- rithms. Order reduction of system descriptions of networks Here the number of equations specifies th order of the system. To use MOR in IC conducted emission models efficiently The ﬁtting equation for an impedance Zmn deﬁned as the voltage Vm between the VDD and VSS Pins at module m di- vided by the input current In at the VDD pin at module n is y certain preparations have to be made in th network's description, as we have shown in Zmn(s) = Vm In = amn + s · bmn + cmn s (1) n th wn i (1) [Ludwig, 2008a]. The network description of the IC conducted emission model can now be reduced by the use of MOR where the coefﬁcients amn, bmn and cmn are obtained from the simulation results of the of the EXPO model. The relation between the coefﬁcients and the Rmn, Lmn, Cmn parameters are as follows algorithms. Order reduction of system descriptions of networks has to preserve their main properties. parameters are as R 5 Simulation results y complexity is e modules can not be considered for this type of model. g p supply voltage VDD1, ref=1.5V for the three models at the VDD1-Pin of the first digital module. Again the MOR model as well as the For the Z12 parameter, describing the coupling between the two modules, Fig. 6 depicts good correlation between the EXPO model and the MOR model. A Z12 curve is not pro- vided for the module-related model, as coupling effects be- tween modules can not be considered for this type of model. 1- mn mn mn mn mn mn c C b L a R = = = (2) For every module, Zmn, m=n, is determined The coefficients for m≠n are For the analysis, the three proposed methodologies were ap- plied to an IC conducted emission model describing two dig- ital modules of a 32-Bit IC. The model consists of 12 SLMs with 120 elements and 51 nodes and is generated with the EXPO tool (Hesidenz and Steinecke, 2005). This model was reduced with the MOR approach to a model containing 64 elements and 14 nodes. this approach compared to the MOR approach. 10 5 10 6 10 7 Z11 EXPO model Z11 MOR model Z11 Module-related model Again, the MOR model as well as the module-related model show a very good matching with the EXPO model. Figure 7 shows the transient response of the supply voltage VDD1,ref=1.5 V for the three models at the VDD1-Pin of the ﬁrst digital module. determined. The coefficients for m≠n are not considered, as coupling effects between modules are neglected for this low complexity approach Since the netlist of the module-related model for rough power integrity estimation contains only 8 elements and 6 nodes, complexity is even further reduced with this approach compared to the MOR approach. 10 2 10 3 10 4 Z11/Ohm 1.8 Again, the MOR model as well as the module-related model show a very good matching with the EXPO model. 1.4 1.6 The main properties of the three models are summarised in Table 1. The simulation time is reduced signiﬁcantly for the MOR and the module-related approach. complexity approach. For the determination of the internal activity of the SLMs, the external currents at the pins of the model are obtained thro gh sim lation Follo ing this the All three models were simulated in HSpice. Kazemzadeh et al.: Efﬁcient modelling of IC conducted emission for power integrity analysis this approach compar approach The relation between the coefficients and the Rmn, Lmn, Cmn parameters are as follows Iint (jω) = Iext (jω) · 1 −ω2LC + jωRC (3) 5 Simulation results Since the netlist of the module-related model for rough power integrity estimation contains only 8 elements and 6 nodes, complexity is even further reduced with obtained from the simulation results of the of the EXPO model. The relation between the coefficients and the R L C Iint (jω) = Iext (jω) · 1 −ω2LC + jωRC (3) Since the netlist of the module-related model for rough power integrity estimation een (3) ed curve is not provided for the module- related model as coupling effects between Fig. 6 Impedance curves Z12 of the EXPO model and MOR model Fig. 6. Impedance curves Z12 of the EXPO model and MOR model. Kazemzadeh et al.: Efﬁcient modelling of IC conducted emission for power integrity analysis this approach compar approach For the Z12 parameter, describing the coupling between the two modules, Fig. 6 depicts good correlation between the EXPO model and the MOR model. A Z12 curve is not pro- vided for the module-related model, as coupling effects be- tween modules can not be considered for this type of model. Figure 7 shows the transient response of the supply voltage VDD1,ref=1.5 V for the three models at the VDD1-Pin of the Passive Distribution Network Passive Distribution Network Passive Distribution Network Internal Activity Internal Activity VDD VSS VDD VSS VDD VSS Internal Activity Digital Model Block Analog Model Block I/O Model Block Iint Iint Iint Passive Distribution Network Passive Distribution Network Passive Distribution Network Internal Activity Internal Activity VDD VSS VDD VSS VDD VSS Internal Activity Digital Model Block Analog Model Block I/O Model Block Iint Iint Iint Fig.4 Module-related model with one SLM (encircled) for each functional module Fig. 4. Module-related model with one SLM (encircled) for each functional module. 5 Simulation Results F th l i th th d Fig. 5 Impedance curves Z11 of the EXPO model, MOR model, and module-related model 10 3 10 4 10 5 10 6 10 7 10 8 10 9 10 10 10 -1 10 0 10 1 10 2 10 3 10 4 10 5 10 6 10 7 Z11 EXPO model Z11 MOR model Z11 Module-related model Z11/Ohm Fig. 5. Impedance curves Z11 of the EXPO model, MOR model, and module-related model. Fig. 5 Impedance curves Z11 of the EXPO model, MOR model, and module-related model 10 3 10 4 10 5 10 6 10 7 10 8 10 9 10 10 10 -1 10 0 10 1 10 2 10 3 10 4 10 5 10 6 10 7 Z11 EXPO model Z11 MOR model Z11 Module-related model Z11/Ohm Fig. 5. Impedance curves Z11 of the EXPO model, MOR model, and module-related model. Analog Model Block Analog Model Block Z11 EXPO model Z11 MOR model Z11 Module-related model Passive Distribution Network Passive Distribution Network Passive Distribution Network Passive Distribution Network Fig. 5 Impedance curves Z11 of the EXPO model, MOR model, and module-related model Fig. 5. Impedance curves Z11 of the EXPO model, MOR model, and module-related model. Fig.4 Module related model with one SLM (encircled) for each functional module Fig. 4. Module-related model with one SLM (encircled) for each functional module. external currents are con frequency domain and mu model, MOR model, an model www.adv-radio-sci.net/7/123/2009/ Passive Distribution Network (PDN) are the same for all SLMs within one functional module. The parameters of the 3 Modelling of an order reduced IC conducted emission model This is for example hi d b i hi Rmn = amn Lmn = bmn Cmn = c−1 mn (2) algorit descrip their m Rmn = amn Lmn = bmn Cmn = c−1 mn serv ampl (2) serv l (2) g g, ated in [Radić-Weissenfeld www.adv-radio-sci.net/7/123/2009/ functional module. The d l d i d i Adv. Radio Sci., 7, 123–126, 2009 g g, ated in [Radić-Weissenfeld www.adv-radio-sci.net/7/123/2009/ Kazemzadeh et al.: Efﬁcient modelling of IC conducted emission for power integrity analysis this approach compar approach Kazemzadeh et al.: Efﬁcient modelling of IC conducted emission for power integrity analysis this approach compar approach R. Kazemzadeh et al.: Efﬁcient modelling of IC conducted emission for power integrity analysis this approach compa approach. 125 OR Passive Distribution Network Passive Distribution Network Passive Distribution Network Internal Activity Internal Activity VDD VSS VDD VSS VDD VSS Internal Activity Digital Model Block Analog Model Block I/O Model Block Iint Iint Iint Passive Distribution Network Passive Distribution Network Passive Distribution Network Internal Activity Internal Activity VDD VSS VDD VSS VDD VSS Internal Activity Digital Model Block Analog Model Block I/O Model Block Iint Iint Iint Fig.4 Module-related model with one SLM (encircled) for each functional module defined as the voltage Vm between the VDD and VSS Pins at module m divided by the input current In at the VDD pin at module n is s c b s a I V s Z mn mn mn n m mn + ⋅ + = = ) ( (1) where the coefficients amn, bmn and cmn are obtained from the simulation results of the of the EXPO model. The relation between the coefficients and the Rmn, Lmn, Cmn parameters are as follows 1- mn mn mn mn mn mn c C b L a R = = = (2) For every module, Zmn, m=n, is d t i d Th ffi i t f ≠ Fig. 4. Module-related model with one SLM (encircled) for each functional module. For every module, Zmn, m=n, is determined. The coefﬁcients for m̸=n are not considered, as coupling effects between modules are neglected for this low complexity approach. For the determination of the internal activity of the SLMs, the external currents at the pins of the model are obtained through simulation. Following this, the external currents are converted into the frequency domain and multiplied with the transfer function of the previously determined PDN of the SLM, resulting in the internal current of the SLM of the model. Iint (jω) = Iext (jω) · 1 −ω2LC + jωRC (3) 5 Simulation results For the analysis, the three proposed methodologies were ap- plied to an IC conducted emission model describing two dig- ital modules of a 32-Bit IC. The model consists of 12 SLMs with 120 elements and 51 nodes and is generated with the EXPO tool (Hesidenz and Steinecke, 2005). This model was reduced with the MOR approach to a model containing 64 elements and 14 nodes. pp Fig. Kazemzadeh et al.: Efﬁcient modelling of IC conducted emission for power integrity analysis this approach compar approach 5 Impedance curves Z11 of the EXPO model, MOR model, and module-related model All three models were simulated in HSpice. Fig. 5 shows the impedance curves Z11 of one digital module for all three models. The Z11 curve of the MOR model as well as the curve of the module- related model show very good matching with the curve of the model generated with the EXPO Tool. For the Z12 parameter, describing the coupling between the two modules, Fig. 6 depicts good correlation between the EXPO model and the MOR model. A Z12 curve is not provided for the module- related model, as coupling effects between modules can not be considered for this type of model. 10 3 10 4 10 5 10 6 10 7 10 8 10 9 10 10 10 -1 10 0 10 1 10 2 10 3 10 4 10 5 10 6 10 7 Z11 EXPO model Z11 MOR model Z11 Module-related model Z11/Ohm Fig. 5. Impedance curves Z11 of the EXPO model, MOR model, and module-related model. 10 3 10 4 10 5 10 6 10 7 10 8 10 9 10 10 10 -1 10 0 10 1 10 2 10 3 10 4 10 5 Simulation Results 5 10 6 10 7 For the analysis, the three proposed methodologies were applied to an IC conducted emission model describing two digital modules of a 32-Bit IC. The model consists of 12 SLMs with 120 elements and 51 nodes and is generated with the EXPO tool [Hesidenz and Steinecke, 2005]. This model was reduced with the MOR approach to a model containing 64 elements and 14 nodes. Z12 EXPO model Z12 MOR model F/Hz Since the netlist of the module-related model for rough power integrity estimation contains only 8 elements and 6 nodes, complexity is even further reduced with this approach compared to the MOR approach. Fig. 6 Impedance curves Z12 of the EXPO model and MOR model Fig. 7 shows the transient response of the supply voltage VDD1, ref=1.5V for the three models at the VDD1-Pin of the first digital module. 10 5 10 6 10 7 Z11 EXPO model Z11 MOR model Z11 Module-related model Again, the MOR model as well as the module-related model show a very good t hi ith th EXPO d l Fig. 6. Impedance curves Z12 of the EXPO model and MOR model. Kazemzadeh et al.: Efﬁcient modelling of IC conducted emission for power integrity analysis this approach compar approach 5 Simulation Results F h l i h h d All three models were simulated in HSpice. Fig. 5 shows the impedance curves Z11 of one digital module for all three models. The Z11 curve of the MOR model as well as the curve of the module- related model show very good matching with the curve of the model generated with the EXPO Tool. For the Z12 parameter, describing the coupling between the two modules, Fig. 6 depicts good correlation between the EXPO model and the MOR model. A Z12 curve is not provided for the module- related model, as coupling effects between 10 3 10 4 10 5 10 6 10 7 10 8 10 9 10 10 10 -1 10 0 10 1 10 2 10 3 10 4 10 5 10 6 10 7 Z12 EXPO model Z12 MOR model F/Hz Fig. 6 Impedance curves Z12 of the EXPO model and MOR model Fig. 6. Impedance curves Z12 of the EXPO model and MOR model. re simulated i the impedanc al module for a Z12 EXPO model Z12 MOR model and VSS Pins at module m divided by the input current In at the VDD pin at module n i For every module, Zmn, m=n, is determined. The coefﬁcients for m̸=n are not considered, as coupling effects between modules are neglected for this low complexity approach. methodologies were applied to an IC conducted emission model describing two digital modules of a 32-Bit IC. The model is s c b s a I V s Z mn mn mn n m mn + ⋅ + = = ) ( (1) where the coefficients amn, bmn and cmn are obtained from the simulation results of the For the determination of the internal activity of the SLMs, the external currents at the pins of the model are obtained through simulation. Following this, the external currents are converted into the frequency domain and multiplied with the transfer function of the previously determined PDN of the SLM, resulting in the internal current of the SLM of the model. consists of 12 SLMs with 120 elements and 51 nodes and is generated with the EXPO tool [Hesidenz and Steinecke, 2005]. This model was reduced with the MOR approach to a model containing 64 elements and 14 nodes. obtained from the simulation results of the of the EXPO model. References Dhia, S. B., Ramdani, M., and Sicard, E.: Electromagnetic Compat- ibility in Intergrated Circuits, Springer-Verlag, New York, 2006. French committee UTE 47A EMC Task Force: Cookbook for In- tegrated Circuit model ICEM, project number 62014-3, 2002, available at: http://www.ute-fr.com/FR/, last accessed 2009. Hesidenz, D. and Steinecke, T.: Chip-Package EMI Modeling and Simulation Tool “EXPO”, presented at the 2005 EMC Compo, Munich, Germany, 2005. Landman, P. E.: Low Power Architectural Design, PhD disserta- tion, Electrical Engineering and Computer Sciences, University of California, Berkeley, CA, 1994. Ludwig, S., Radi´c-Weissenfeld, Lj., Mathis, W., and John, W.: Ef- ﬁcient Model Reduction of Passive Electrical Networks with a Large Number of Independent Sources, presented at the Inter- national Symposium on Circuits and Systems (ISCAS), Seattle, Washington, USA, 2008. Ludwig, S., Radi´c-Weissenfeld, Lj., Mathis, W., and John, W.: Efﬁ- cient passive network description of IC conducted emission mod- els for model reduction, Adv. Radio Sci., 6, 133–137, 2008, http://www.adv-radio-sci.net/6/133/2008/. estimation of conducted emission, whereas for the EXPO and the MOR model, having all IC pins modelled, pin-related es- timation of emission is possible. estimation of conducted emission, whereas for the EXPO and the MOR model, having all IC pins modelled, pin-related es- timation of emission is possible. Radi´c-Weissenfeld, Lj., Ludwig, S., Mathis, W., and John, W.: Two-step Order Reduction of IC Conducted Emission Models, presented at the 2008 Asia-Paciﬁc EMC Week, Singapore, to be published, 2009. parameters are as R 5 Simulation results y complexity is e Figure 5 shows the impedance curves Z11 of one digital module for all three models. The Z11 curve of the MOR model as well as the curve of the module-related model show very good matching with the curve of the model generated with the EXPO Tool. Fig 5 Impedance curves Z11 of the EXPO 10 3 10 4 10 5 10 6 10 7 10 8 10 9 10 10 10 -1 10 0 10 1 0.8 1 1.2 Vdd/V For the module-related model in this work, two digi- tal modules of the complex EXPO model were reduced to one SLM for each module. Since the SLM possesses only two pins, the model is merely suitable for module-related VDD EXPO model Adv. Radio Sci., 7, 123–126, 2009 external currents are con frequency domain and mu model, MOR model, an model www.adv-radio-sci.net/7/123/2009/ R. Kazemzadeh et al.: Efﬁcient modelling of IC conducted emission for power integrity analysis y g EXPO model. R. Kazemzadeh et al.: Efﬁcient modelling of IC conducted emission for power integrity analysis y g EXPO model. 126m 0 0.5 1 1.5 2 2.5 3 3.5 4 x 10 -8 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 VDD EXPO model VDD MOR model VDD Module-related model Vdd/V Fig. 7 Transient response of the EXPO model, MOR model, and module-related model Fig. 7. Transient response of the EXPO model, MOR model, and module-related model. While it is possible to make a point about the distur- bances at the IC pins with both modelling approaches, only the MOR model provides conclusive information about pin- coupling effects. This information is lost for the module- related model. The MOR and module-related model were compared with the EXPO model and close alignments were observed in the time and frequency domain. A signiﬁcant speed-up in simu- lation time was achieved for the MOR model in comparison to the EXPO model, and even more so for the module-related model. Vdd/V Acknowledgements. Represented research and development work is carried out in the frame of the MEDEA+ project A701 PARACHUTE project (Parasitic Extraction and Optimization for Efﬁcient Microelectronic System Design and Application). This particular research was supported by the BMBF (Bun- desministerium fuer Bildung und Forschung) Federal Republic of Germany under 01M 3169 A, 01M 3169 D and 01M 3169 E. The responsibility for this publication is held by the authors only. Fig. parameters are as R 5 Simulation results y complexity is e 7 Transient response of the EXPO model, MOR model, and module-related d l Fig. 7. Transient response of the EXPO model, MOR model, and module-related model. The main properties of the three models are summarised in Table 1 The simulation Table 1. Complexity of the EXPO model, MOR model and module- related model. 4 The main properties of the three models are summarised in Table 1. The simulation time is reduced significantly for the MOR and the module-related approach. For the module-related model in this work, two digital modules of the complex EXPO model were reduced to one SLM for each module. Since the SLM possesses only two Table 1. Complexity of the EXPO model, MOR model and module- related model. EXPO MOR Module-related Model complexity El. compon. nodes 110 51 64 14 8 6 HSpice simulation time AC-Analysis Transient 100% 100% 42% 74% 19% 13% Accuracy – Pin-related – Pin-related – Module- related – Crosstalk considered – Crosstalk considered – Crosstalk not considered The main properties of the three models are summarised in Table 1 The simulation Table 1. Complexity of the EXPO model, MOR model and module- related model. 6 Conclusion This paper proposes two methodologies to reduce the com- plexity of large IC conducted emission models. The ﬁrst methodology uses MOR algorithms, while the second method uses a ﬁtting algorithm to obtain the parameters of a minimum complexity module-related model from simulation results of the complex model. Adv. Radio Sci., 7, 123–126, 2009 www.adv-radio-sci.net/7/123/2009/
https://openalex.org/W2285502193	https://pmr.lf1.cuni.cz/media/pdf/pmr_2012113030189.pdf	English	null	Does Prenatal Methamphetamine Exposure Induce Cross-sensitization to Cocaine and Morphine in Adult Male Rats?	Prague Medical Report	2,012	cc-by	7,722	189) 189) Prague Medical Report / Vol. 113 (2012) No. 3, p. 189–205 © Charles University in Prague – Karolinum Press, Prague 2012 Does Prenatal Methamphetamine Exposure Induce Cross-sensitization to Cocaine and Morphine in Adult Male Rats? Šlamberová R., Yamamotová A., Pometlová M., Schutová B., Hrubá L., Nohejlová-Deykun K., Nová E., Macúchová E. Department of Normal, Pathological and Clinical Physiology, Third Faculty of Medicine, Charles University in Prague, Prague, Czech Republic Šlamberová R., Yamamotová A., Pometlová M., Schutová B., Hrubá L., Nohejlová-Deykun K., Nová E., Macúchová E. Department of Normal, Pathological and Clinical Physiology, Third Faculty of Medicine, Charles University in Prague, Prague, Czech Republic Received January 23, 2012; Accepted June 25, 2012. Received January 23, 2012; Accepted June 25, 2012. Key words: Prenatal drug exposure – Methamphetamine – Cocaine – Morphine – Open field – Plantar test – Conditioned place preference – Locomotion – Nociception – Drug-seeking behavior Abstract: The aim of the present study was to examine the cross-sensitization induced by prenatal methamphetamine (MA) exposure to challenge dose of cocaine or morphine. Rat mothers received a daily injection of MA (5 mg/kg) or saline throughout the gestation period. Adult male offspring (prenatally MA- or saline-exposed) were divided to groups with challenge doses of saline (1 ml/kg), cocaine (5 mg/kg) or morphine (5 mg/kg). Behavior in unknown environment was examined in Laboras, nociception in Plantar test, and active drug-seeking behavior in conditioned place preference (CPP). Our data demonstrate that cocaine increased the exploratory activity in Laboras test in prenatally saline-exposed, but decreased it in prenatally MA-exposed rats. An analgesic effect of cocaine was demonstrated only by the tail withdrawal and it was independent of the prenatal drug exposure. CPP test showed that prenatal MA exposure induced rather tolerance than sensitization to cocaine. In contrast to cocaine effects, morphine decreased rearing activity in both, prenatally MA-exposed and saline-exposed rats, and locomotion only in prenatally MA-exposed rats in the Laboras. In the Plantar This study was supported by grant # NS10509-3/2009 from Internal Agency of the Ministry of Health of the Czech Republic, grant # 305/09/0126 from Grant Agency of the Czech Republic, project CSM 110 from Ministry of Education, Youth and Sports and project 264706/SVV/2012 from Charles University in Prague. Mailing Address: Assoc. Prof. Romana Šlamberová, MD., PhD., Department of Normal, Pathological and Clinical Physiology, Third Faculty of Medicine, Charles University in Prague, Ke Karlovu 4, 120 00 Prague 2, Czech Republic; Phone: + 420 224 902 713; Fax: + 420 224 902 750; e-mail: rslamber@lf3.cuni.cz © Charles University in Prague – Karolinum Press, Prague 2012 Prague Medical Report / Vol. 113 (2012) No. 3, p. Does Prenatal Methamphetamine Exposure Induce Cross-sensitization to Cocaine and Morphine in Adult Male Rats? 189–205 190) test, the results demonstrated that morphine had an analgesic effect in prenatally saline-exposed rats but this effect was suppressed in prenatally MA-exposed rats. In the CPP test morphine induced drug-seeking behavior, which however was not affected by prenatal drug exposure. Thus, our data demonstrate that there is a cross-effect between prenatal MA exposure and the challenge dose of other drug in adulthood, however drug-seeking behavior is not increased by prenatal MA exposure as we expected. Šlamberová R. et al. Introduction Methamphetamine (MA) is one of the most common “hard” drugs abused by pregnant women (Marwick, 2000), which is also one of the most frequently used illicit drugs in the Czech Republic (Vavřínková et al., 2001). Since psychoactive drugs are able to cross blood-brain barrier (one of the most impervious barriers of the body), the placental barrier is even easier to go through. MA is a powerfully addictive psychostimulant that metabolizes slowly and its effect is long-lasting (8 to 24 h) (Marwick, 2000). This might be the reason that makes it so popular. The research of the long-term effects of prenatal MA exposure is not fully understood yet. Our laboratory specializes in investigation of the effects of drugs (especially MA) on rat mothers and their progeny. Our previous studies demonstrated that administration of MA during pregnancy attenuates maternal behavior of rat mothers (Šlamberová et al., 2005) and impairs postnatal development of their pups (Šlamberová et al., 2006). Further, we found that prenatal MA exposure with respect of adult MA challenge impairs the learning abilities tested in Morris water maze (Schutová et al., 2008), affects anxiety behavior (Schutová et al., 2009), alters pain sensitivity in Plantar tests in sex-specific manner (Yamamotová et al., 2011) and seizure susceptibility in adult male and female rats (Šlamberová et al., 2009; Bernášková et al., 2011). In our most recent studies we demonstrated that there is a sensitizing effect of prenatal MA exposure to the same drug in adulthood (Bubeníková-Valešová et al., 2009; Šlamberová et al., 2011a, b). There are studies showing that abuse of one drug may increase sensitivity to another drug. This effect is called cross-sensitization (Bartoletti et al., 1985; He and Grasing, 2004; Valvassori et al., 2007). Cross-sensitization between amphetamine and cocaine was first demonstrated on changes of locomotor activity (Shuster et al., 1977; Bonate et al., 1997). Repeated pretreatment with amphetamine was shown to enhance the acquisition (Horger et al., 1992) and escalation of cocaine self-administration (Ferrario and Robinson, 2007). Microinjections of amphetamine into the ventral tegmental area were shown to increase cocaine self-administration under a progressive ratio procedure and to enhance reinstatement of cocaine seeking (Suto et al., 2002). Valvassori et al. (2007) found that rats chronically treated with methylphenidate showed augmented locomotor sensitization to D-amphetamine in the adolescent period. Introduction Other studies demonstrated that cross-sensitization may be induced not only between related drugs, such as cocaine and amphetamines 191) Prague Medical Report / Vol. 113 (2012) No. 3, p. 189–205 (psychostimulants), but also between unrelated drugs, such as between opioids and cocaine (Leri et al., 2003; He and Grasing, 2004) or between endocannabinoids and cocaine (Arnold, 2005) or opioids (Fattore et al., 2005), respectively. Furthermore, it was shown (Malanga and Kosofsky, 2003) that rodents exposed to various abused drugs in utero, became sensitized to the rewarding effects of drugs in adulthood. For example they responded to lower doses of drug than control animals. Increased predisposition of drug abuse in adulthood has been shown in prenatally cocaine-exposed (Heyser et al., 1992; Rocha et al., 2002; Estelles et al., 2006), cannabinoid-exposed (Vela et al., 1998) and morphine- exposed offspring (Gagin et al., 1997) relatively to controls. They showed increased drug-seeking behavior in both “self-administration test” and “conditioned place preference test” (CPP). CPP is one of the most widespread drug reward tests (for review see Tzschentke, 1998). Based on Pavlovian conditioning principles, CPP reflects a preference for a context due to the contiguous association between the context and a drug-associated stimulus. It also presents important advantages, among which the possibility to reveal both reward and aversion, to test animals in a drug-free state and to allow simultaneous determination of locomotor activity (Fattore et al., 2005). Thus, the above mentioned studies suggest that prenatal drug exposure may induce “cross-sensitization” in adulthood regardless to the type of a drug. This might suggest that prenatal drug exposure induces general predisposition to drug addiction in adulthood. To validate our hypothesis that prenatal drug exposure induces predisposition to drug abuse in general and may therefore induce cross-sensitization, two drugs were tested in adult prenatally MA-exposed male rats: (1) drug with similar mechanism of action (cocaine) that affects dopamine, serotonin and noradrenalin systems similar to MA, and (2) drug with different mechanism of action (morphine) that affects mostly opioid receptors. Tests examining drug reward, nociception and behavior changes induced by prenatal MA exposure were used for demonstration of cross-sensitization in adulthood. Effect of Prenatal Methamphetamine Exposure on Cross-sensitization Methods All experimental procedures implemented in this report were reviewed and approved by the Institutional Animal Care and Use Committee and were in agreement with the Czech Government Requirements under the Policy of Humans Care of Laboratory Animals (No. 246/1992) and with the regulations of the Ministry of Agriculture of the Czech Republic (No. 311/1997). Prenatal and postnatal animal care Adult female Wistar rats (250–300 g) were delivered by Anlab (Prague, the Czech Republic) from Charles River Laboratories International, Inc. Animals were housed 4–5 per cage and left undisturbed for a week in a temperature-controlled (22–24 °C) colony room with free access to food and water on a 12 h (light): Prague Medical Report / Vol. 113 (2012) No. 3, p. 189–205 192) 12 h (dark) cycle with lights on at 06:00 a.m. The phase of females’ estrous cycle was determined by vaginal lavage smears after one week of adaptation period. The smears were examined by light microscopy. Thereafter, female rats that were at the onset of estrous phase of the estrous cycle (Turner and Bagnara, 1976) were housed with sexually mature males overnight. There were always, one female and one male in a cage. The next morning females were smeared for the presence of sperm and returned to their previous home cages. This was counted as gestational day (GD) 1. Dams were randomly assigned to MA-treated and saline-treated (controls). On GD 1 the daily injections started and continued till the day of delivery, which usually occurred on GD 22 (for details see Šlamberová et al., 2005). MA (Sigma-Aldrich®) was injected subcutaneously (s.c.) in a dose of 5 mg/kg, saline was injected s.c. at the same time in the same volume as MA. The day of delivery was counted as postnatal day (PD) 0. On PD 21, pups were weaned and group-housed by sex. Animals were left undisturbed until adulthood. Always one male rat (PD 70–90) per group was used in each experiment from each litter to avoid litter effects. Females were used in other experiments that will be presented as a part of another study. Open field test – Laboras In total 64 adult male rats (n=8) were tested in Laboras apparatus (Metris B.V., Netherlands) for natural behavior. Laboras is a fully automatic system for continuous behavior recognition and tracking in small rodents. Šlamberová R. et al. Methods Single animal was placed to the plexiglass cage (45×30×30 cm) filled with bedding material, that was covered and equipped as normal home cage with food and water available ad libitum. The cage was placed on a triangular sensor platform (95×75×75 cm). The platform was connecting the basis of the system with a computer for continuous recording of different types of activities during the time of Open field testing. The movements’ records afterwards were analyzed by Laboras software. Challenge dose of the tested drugs [cocaine (5 mg/kg); morphine (5 mg/kg)] or saline (1 ml/kg) was administered immediately prior to placing the animal to the Laboras cage to both of the prenatal groups (prenatally MA-exposed and prenatally saline-exposed). Each animal was tested separately. The behavior was monitored for 1 hour. The duration of each behavior was analyzed within six 10-minute intervals. Following parameters were analyzed in all animals during the 1-h period of testing: the duration of locomotion, rearing (exploratory behavior) and total distance travelled. Effect of Prenatal Methamphetamine Exposure on Cross-sensitization Plantar test Other 32 animals (n=8) were used to test nociception. Plantar test (Plantar test; Ugo Basile, Comerio, Italy) was used to measure pain threshold. A beam generator, which is controlled by the experimenter under the floor of the plexiglass box 193) Prague Medical Report / Vol. 113 (2012) No. 3, p. 189–205 (size 27×17×14 cm) allows to stimulate the sole (planta) of the paw in a freely moving rat. The latency of paw withdrawal from painful heat stimulus was measured for each of the four paws. Latency to withdrawal of the tail was measured as a modified method of the Tail-flick test. The maximal intensity was set to 90 and cut-off time was 22 s to prevent tissue damage. Four measures were done in 15-minute intervals. First measure (time 0) was used as a control without drug challenge. When the first measure was finished, challenge dose of the tested drug [cocaine (5 mg/kg); morphine (5 mg/kg)] was injected. Next measures were performed 15, 30, and 45 minutes after the drug administration. Thus, the effect of challenge dose of cocaine or morphine was compared during time period of 45 minutes in prenatally MA- and saline-exposed male rats. Conditioned place preference (CPP) Another 32 adult male rats (n=8) were used to test drug [cocaine (5 mg/kg); morphine (5 mg/kg)] reward conditioning and how it is affected by prenatal drug exposure. The CPP apparatus dimensions and general procedures were modified from the work of Sanchez et al. (2003). The apparatus and the methodological procedure were in detail described in our previous study (Šlamberová et al., 2011b). ) The place conditioning procedure consisted of three phases: pre-exposure, conditioning, and the CPP test as in work of Mueller and Stewart (2000). Pre-exposure: On the Day 1, animals received a single pre-exposure test in which they were placed in the center choice chamber with the doors open to allow access to the entire apparatus for 15 min. The amount of time spent in each chamber was monitored and used to assess unconditioned preferences. Conditioning: During the following conditioning phase (8 days), rats were assigned to receive drug [cocaine (5 mg/kg); morphine (5 mg/kg)] in one of the two chambers and saline in the second one. Half of each group started the experiment on the drug-paired side and half on the saline-paired side. After administration of drug (cocaine or morphine) or saline, animal was allowed to explore the specific chamber for 1 hour. The center chamber was never used during conditioning and was blocked by doors. CPP test: Two days after the last conditioning trial (Day 12), a test for CPP was given. Animals were placed in the center choice chamber with the doors opened and allowed free access to the entire apparatus for 15 min. The time spent in each chamber and number of entries was recorded to assess individual preferences. No injections were given during the CPP test, maintaining the same procedure as that used during the pre-exposure test. Active drug- seeking behavior was developed if the animal preferred the chamber that was associated with the drug injection. 194) Prague Medical Report / Vol. 113 (2012) No. 3, p. 189–205 Statistical analyses Data from each adult challenge (cocaine and morphine, respectively) were analyzed separately (n=8). Laboras. All behavioral activities were evaluated separately. Three-way ANOVA (between factors: prenatal drug, challenge drug; within factors: 10-minute intervals) was used. Plantar test. Average of measurements of both forelimbs (left and right), both hind limbs and all tail measurements, respectively, were used for statistical analyses. Two-way ANOVA (between factor: prenatal drug exposure; within factor: 15-minute intervals) was used. CPP test. Conditioned place preference (CPP) Three-way ANOVA (between factors: prenatal exposure, chamber with or without challenge drug; within factor: time – before vs. after conditioning) was used to analyze differences in number of entries to chambers and total time spent in the specific chamber. Bonferroni post-hoc test was used when appropriate. Differences were considered significant if p<0.05. All statistical data in this report are presented as [F (N-1,n-N)=xx.xx; p<0.0x], where F = test criterion of ANOVA, N-1 = degrees of freedom of groups, n-N = degrees of freedom of individual subjects, p = probability level. Cocainei Open field test – Laboras Four groups were tested in Laboras: prenatally saline- and MA-exposed rats treated with cocaine or saline in adulthood. Locomotion (Figure 1A). Cocaine challenge dose (5 mg/kg) increased the time of locomotion in prenatally saline-exposed rats, while it did not change it in prenatally MA-exposed rats [F(1,28)=6.59; p<0.05]. In addition, prenatally MA-exposed rats without cocaine challenge displayed increased locomotion in the first 10-minute interval relative to prenatally saline-exposed rats without cocaine [F(5,140)=4.12; p<0.01] or prenatally MA-exposed rats with cocaine challenge dose [F(5,140)=5.05; p<0.001]. Rearing (Figure 1B). Cocaine challenge dose (5 mg/kg) increased the time spent with rearing in prenatally saline-exposed rats, while did not change it in prenatally MA-exposed rats [F(1,28)=6.59; p<0.05]. In addition, prenatally MA-exposed rats with cocaine displayed decreased rearing activity relative to prenatally MA-exposed rats without cocaine in the first two 10-minute intervals [F(5,140)=3.67; p<0.01]. Distance (Figure 1C). Cocaine challenge dose (5 mg/kg) increased the total distance moved in prenatally saline-exposed rats, while did not change it in prenatally Šlamberová R. et al. 195) Prague Medical Report / Vol. 113 (2012) No. 3, p. 189–205 MA-exposed rats [F(1,28)=5.90; p<0.05]. In addition, prenatally MA-exposed rats without cocaine challenge displayed increased distance in the first 10-minute interval relative to prenatally saline-exposed rats without cocaine [F(5,140)=3.83; p<0.01] or prenatally MA-exposed rats with cocaine challenge dose [F(5,140)=2.80; p<0.05]. Effect of Prenatal Methamphetamine Exposure on Cross-sensitization p<0.05]. Figure 1 – The effect of prenatal MA exposure (5 mg/kg) and challenge dose of cocaine in adulthood (5 mg/kg) on locomotion (A), rearing (B) and distance (C) in Laboras test. Values are means ± SEM (n=8). p<0.05 vs. animals regardless of prenatal exposure without cocaine treatment (effect of cocaine challenge dose) +p<0.05 vs. prenatally saline-exposed rats regardless of cocaine treatment (effect of prenatal MA exposure) Figure 1 – The effect of prenatal MA exposure (5 mg/kg) and challenge dose of cocaine in adulthood (5 mg/kg) on locomotion (A), rearing (B) and distance (C) in Laboras test. Values are means ± SEM (n=8). p<0.05 vs. animals regardless of prenatal exposure without cocaine treatment (effect of cocaine challenge dose) +p<0.05 vs. prenatally saline-exposed rats regardless of cocaine treatment (effect of prenatal MA exposure) Figure 1 – The effect of prenatal MA exposure (5 mg/kg) and challenge dose of cocaine in adulthood (5 mg/kg) on locomotion (A), rearing (B) and distance (C) in Laboras test. Values are means ± SEM (n=8). p<0.05 vs. Cocainei animals regardless of prenatal exposure without cocaine treatment (effect of cocaine challenge dose) +p<0.05 vs. prenatally saline-exposed rats regardless of cocaine treatment (effect of prenatal MA exposure) Effect of Prenatal Methamphetamine Exposure on Cross-sensitization Prague Medical Report / Vol. 113 (2012) No. 3, p. 189–205 196) Šlamberová R. et al. Plantar test All animals received one cocaine dose after first measurement in the time 0 to see possible analgesic effect of cocaine and how it differs based on the prenatal exposure. Time 0 was used as control measurement in the Plantar test. There was no difference induced by prenatal MA exposure and no analgesic effect induced by cocaine challenge dose (5 mg/kg) in forelimbs (Figure 2A) or hind limbs (Figure 2B). In the tail (Figure 2C) the cocaine challenge dose showed the analgesic effect in the 30th and 45th minute after cocaine administration [F(3,42)=7.11; p<0.0001]. This analgesic effect was independent of the prenatal drug exposure. Figure 2 – The effect of prenatal MA exposure (5 mg/kg) and challenge dose of cocaine in adulthood (5 mg/kg) on nociception in the forelimbs (A), the hind limbs (B) and the tail (C) examined with Plantar test. Values are means ± SEM (n=8). p<0.05 vs. first (control) measure (analgesic effect of cocaine) Figure 2 – The effect of prenatal MA exposure (5 mg/kg) and challenge dose of cocaine in adulthood (5 mg/kg) on nociception in the forelimbs (A), the hind limbs (B) and the tail (C) examined with Plantar test. Values are means ± SEM (n=8). p<0.05 vs. first (control) measure (analgesic effect of cocaine) ( ) p<0.05 vs. first (control) measure (analgesic effect of cocaine) Šlamberová R. et al. 197) Prague Medical Report / Vol. 113 (2012) No. 3, p. 189–205 Conditioned place preference (CPP) After cocaine conditioning, prenatally saline-exposed rats displayed higher number of entries [F(1,42)=5.15; p<0.01] and increased total time spent in the chamber [F(1,42)=3.80; p<0.05] associated with cocaine (5 mg/kg) in the CPP test than in the chamber associated with saline administration. This increase was not apparent in rats prenatally exposed to MA (Table 1). Table 1 – Effect of the drug challenge dose on time spent in a chamber associated with the drug and number of entries to the chamber associated with the drug Table 1 – Effect of the drug challenge dose on time spent in a chamber associated with the drug and number of entries to the chamber associated with the drug Time Entries control MA control MA Cocaine 5 mg/kg  –  – Morphine 5 mg/kg     Arrows show increased visits (entries) or time spent in the chamber associated with the drug. Plantar test Dash means no difference in the number of visits (entries) or time spent in the chamber associated with the drug or chamber associated with saline Arrows show increased visits (entries) or time spent in the chamber associated with the drug. Dash means no difference in the number of visits (entries) or time spent in the chamber associated with the drug or chamber associated with saline Effect of Prenatal Methamphetamine Exposure on Cross-sensitization Morphinei Open field test – Laboras Four groups were tested in Laboras: prenatally saline- and MA-exposed rats treated with morphine or saline in adulthood. Locomotion (Figure 3A). Morphine challenge dose (5 mg/kg) decreased the time of locomotion in prenatally MA-exposed rats, while did not change it in prenatally saline-exposed rats during the first 30 minutes of the Laboras test [F(5,140)=4.80; p<0.01]. In addition, prenatally MA-exposed rats without morphine challenge spent more time by locomotion than prenatally saline-exposed rats without morphine in adulthood during the first 30 minutes of the Laboras test [F(5,140)=4.37; p<0.001]. This prenatal MA-induced difference was not apparent in the groups of animals, who received morphine challenge in adulthood. Rearing (Figure 3B). Morphine challenge dose (5 mg/kg) decreased the time spent by rearing in both, prenatally saline-exposed and prenatally MA-exposed rats during the first 30 minutes of the Laboras test [F(5,140)=8.38; p<0.0001]. Prenatal MA exposure did not induce any difference between groups. Distance (Figure 3C). Morphine challenge dose (5 mg/kg) decreased the total distance moved in prenatally MA-exposed rats, while did not change it in prenatally saline-exposed rats during the first 30 minutes of the Laboras test [F(5,140)=3.72; 198) Prague Medical Report / Vol. 113 (2012) No. 3, p. 189–205 Figure 3 – The effect of prenatal MA exposure (5 mg/kg) and challenge dose (5 mg/kg) on locomotion (A), rearing (B) and distance (C) in Laboras test. Valu p<0.05 vs. animals regardless of prenatal exposure without morphine treatm dose) +p<0.05 vs. prenatally saline-exposed rats regardless of morphine treatment Figure 3 – The effect of prenatal MA exposure (5 mg/kg) and challenge dose of morphine in adulthood (5 mg/kg) on locomotion (A), rearing (B) and distance (C) in Laboras test. Values are means ± SEM (n=8). p<0.05 vs. animals regardless of prenatal exposure without morphine treatment (effect of morphine challenge dose) +p<0.05 vs. prenatally saline-exposed rats regardless of morphine treatment (effect of prenatal MA exposure)) Figure 3 – The effect of prenatal MA exposure (5 mg/kg) and challenge dose of morphine in adulthood (5 mg/kg) on locomotion (A), rearing (B) and distance (C) in Laboras test. Values are means ± SEM (n=8). p<0.05 vs. animals regardless of prenatal exposure without morphine treatment (effect of morphine challenge dose) +p<0.05 vs. Effect of Prenatal Methamphetamine Exposure on Cross-sensitization Morphinei prenatally saline-exposed rats regardless of morphine treatment (effect of prenatal MA exposure)) Figure 3 – The effect of prenatal MA exposure (5 mg/kg) and challenge dose of morphine in adulthood (5 mg/kg) on locomotion (A), rearing (B) and distance (C) in Laboras test. Values are means ± SEM (n=8). p<0.05 vs. animals regardless of prenatal exposure without morphine treatment (effect of morphine challenge dose) +p<0.05 vs. prenatally saline-exposed rats regardless of morphine treatment (effect of prenatal MA exposure)) ) +p<0.05 vs. prenatally saline-exposed rats regardless of morphine treatment (effect of prenatal MA expo p<0.05]. In addition, prenatally MA-exposed rats without morphine challenge walked longer distance than prenatally saline-exposed rats without morphine in adulthood during the first 10 minutes of the Laboras test [F(5,140)=5.61; p<0.0001]. This prenatal MA-induced difference was not apparent in the groups of animals, who received morphine challenge in adulthood. p<0.05]. In addition, prenatally MA-exposed rats without morphine challenge walked longer distance than prenatally saline-exposed rats without morphine in adulthood during the first 10 minutes of the Laboras test [F(5,140)=5.61; p<0.0001]. This prenatal MA-induced difference was not apparent in the groups of animals, who received morphine challenge in adulthood. Šlamberová R. et al. 199) Prague Medical Report / Vol. 113 (2012) No. 3, p. 189–205 Conditioned place preference (CPP) After morphine conditioning, both, prenatally saline-exposed and prenatally MA-exposed rats displayed higher number of entries [F(1,42)=33.42; p<0.001] and increased total time spent in the chamber [F(1,42)=50.39; p<0.0001] associated with morphine (5 mg/kg) in the CPP test than in the chamber associated with saline administration. This effect showing increased drug-seeking behavior did not differ in respect of the prenatal drug exposure (Table 1). Plantar test All animals received one morphine dose after first measurement in the time 0 to see possible analgesic effect of morphine and how it differs based on the prenatal exposure. Time 0 was used as control measurement in the Plantar test. Forelimbs (Figure 4A). Morphine induced analgesic effect in forelimbs of prenatally saline-exposed rats that increases with the time [F(3,42)=6.69; p<0.001]. In prenatally MA-exposed rats was the analgesic effect of morphine postponed and started after 30 minutes from morphine administration. In addition, prenatal MA exposure induced decrease in the pain threshold [F(1,14)=3.32; p<0.05]. Hind limbs (Figure 4B). Morphine induced analgesic effect in hind limbs of prenatally saline-exposed rats that increased with the time that however started 30 minutes after morphine administration [F(3,42)=3.72; p<0.05]. Morphine did not have an analgesic effect in prenatally MA-exposed rats. In addition, prenatal MA exposure induced decrease in the pain threshold in hind limbs [F(1,14)=3.34; p<0.05]. Tail (Figure 4C). In the tail morphine induced fast growing analgesia in both, prenatally saline-exposed and prenatally MA-exposed rats [F(3,42)=43.54; p<0.0001]. This analgesic effect of morphine did not differ based on the prenatal drug exposure. Conditioned place preference (CPP) Šlamberová R. et al. Discussion Our data demonstrate differences in the effect of cocaine and morphine in all conducted tests. Cocaine increased the activity in the Laboras test in prenatally saline-exposed, but decreased it in prenatally MA-exposed rats. Cocaine had an analgesic effect only on the tail and this effect was independent of the prenatal drug exposure. Also, prenatal MA exposure induced rather tolerance to cocaine then sensitization in the CPP test. On the other hand, morphine decreased locomotion and distance traveled in prenatally MA-exposed rats and the rearing activity in both, the prenatally MA-exposed and prenatally saline-exposed rats in the Laboras. In the Plantar test the results demonstrate that morphine had an analgesic effect in prenatally saline-exposed rats, but this effect was lowered or suppressed in prenatally MA-exposed rats on forelimbs and hind limbs. In the CPP 200) Prague Medical Report / Vol. 113 (2012) No. 3, p. 189–205 test morphine induced drug-seeking behavior, which however did not d h l d Figure 4 – The effect of prenatal MA exposure (5 mg/kg) and challenge dose of morphine in ad (5 mg/kg) on nociception in the forelimbs (A), the hind limbs (B) and the tail (C) examined with Values are means ± SEM (n=8) p<0.05 vs. first (control) measure (analgesic effect of morphine) +p<0.05 vs. prenatally saline-exposed rats (effect of prenatal MA exposure) Figure 4 – The effect of prenatal MA exposure (5 mg/kg) and challenge dose of morphine in adulthood (5 mg/kg) on nociception in the forelimbs (A), the hind limbs (B) and the tail (C) examined with Plantar test. Values are means ± SEM (n=8) p<0.05 vs. first (control) measure (analgesic effect of morphine) +p<0.05 vs. prenatally saline-exposed rats (effect of prenatal MA exposure) test morphine induced drug-seeking behavior, which however did not differ in respect to the prenatal drug exposure. The behavioral data showing increased locomotion, rearing, as well as distance travelled by prenatally saline-exposed rats in the Laboras test are in agreement Šlamberová R. et al. 201) Prague Medical Report / Vol. 113 (2012) No. 3, p. 189–205 with many studies demonstrating increased locomotor activity in animals after administration of psychostimulants (Carey and Gui, 1997; Tzschentke and Schmidt, 1998; Carey and Damianopoulos, 2006; Ago et al., 2008). However, this increase was not apparent in animals prenatally exposed to MA in the present study. Discussion This is an interesting finding showing effect of prenatal MA exposure on possible development of tolerance to cocaine administration. Similar effect was shown by study of Peltier et al. (1996) as discussing below. In contrast to cocaine, our results show morphine-induced decrease in locomotion which did not differ based on the prenatal exposure and is in agreement with many studies of others (Vezina and Stewart, 1987; Nazarian et al., 1999; Patti et al., 2005). The data showing changes in nociception are in agreement with many studies showing analgesic effect of both, morphine and cocaine. Morphine is well known analgesic drug often used in oncology (Vadalouca et al., 2008). Psychostimulants, including cocaine, were used as local as well as general anaesthetics for decades, even though they are not so often used for systemic application recently (Fleming et al., 1990; Young and MacKenzie, 1992). Our findings showing decreased analgesic effect on forelimbs and hind limbs but not the tail in prenatally MA-exposed relative to prenatally saline-exposed rats suggest long-term effect of prenatal MA exposure affecting especially supraspinal analgesic mechanisms (Zemlan et al., 1984; Roerig and Fujimoto, 1988). The anti-analgesic effect of prenatal MA exposure is supported by our previous studies (Šlamberová et al., 2011b; Yamamotová et al., 2011) as well as studies of others (Chen et al., 2010). Chen et al. suggested that prenatal MA exposure could predispose an alteration in the development of nociceptive neuronal network, which leads to a long-lasting status of hypersensitivity to pain stimulations in the offspring. In contrast to our expectation, our data from the CPP test demonstrate no increase induced by prenatal MA exposure in the active drug-seeking behavior. In contrast to our findings, studies of drug abuse in prenatally cocaine-exposed adult rats (Heyser et al., 1992; Rocha et al., 2002; Estelles et al., 2006) showed increased predisposition to drug abuse in self-administration and CPP tests. These inconsistencies of findings with our model of prenatal MA exposure suggest that cocaine is more potent drug for causing long-term addiction than MA. Even more, we demonstrate rather decreased preference of the cocaine-associated chamber in prenatally MA-exposed rats which would in other words suggest tolerance to cocaine conditioning in prenatally MA-exposed rats. To the best of our knowledge, there is only one study supporting our finding demonstrating tolerance to cocaine induced by MA. Peltier et al. Effect of Prenatal Methamphetamine Exposure on Cross-sensitization Discussion (1996) demonstrated that chronic treatment with psychostimulants, such as amphetamine and MA, produces cross-tolerance to both the discriminative and reinforcing effects of cocaine. Study of Gygi et al. (1996) suggesting that MA causes tolerance to serotonergic effects of psychostimulants might also support our results. It was repeatedly shown that MA and amphetamine affect the noradrenergic system of 202) Prague Medical Report / Vol. 113 (2012) No. 3, p. 189–205 the CNS the most, the dopaminergic system less, and at minimum the serotonergic system. On the other hand, cocaine seems to affect the serotonergic system the most (Fleckenstein et al., 2000; Rothman et al., 2001; Shoblock et al., 2003). This might be an explanation for our results. g p With respect to the effect of morphine in the CPP, our present data demonstrated increased drug seeking behavior that however did not differ based on the prenatal drug exposure. As mentioned above, there are studies available (Heyser et al., 1992; Rocha et al., 2002; Estelles et al., 2006) showing increased drug-seeking behavior of cocaine or morphine in animals prenatally exposed to cocaine, which is in disagreement to our results. On the other hand, prenatal morphine exposure was shown not to affect self-administration of morphine or cocaine in adulthood (Riley and Vathy, 2006; Vathy et al., 2007). In addition, study of Vela et al. (1998) demonstrated sex differences of cross-effect between prenatal cannabinoids exposure and morphine self-administration in adulthood. Specifically, adult female rats born from mothers that were daily treated with delta9- tetrahydrocannabinol (THC) during gestation and lactation periods, exhibited a statistically significant increase in the rate of acquisition of intravenous morphine self-administration behavior when compared with females born from vehicle- exposed mothers, an effect that did not exist in THC-exposed male offspring. Thus, it seems that the possibility of increased drug-seeking of drugs in adulthood depends on the drug that was the animal exposed prenatally and also on the sex. Therefore, our future studies are planned to examine the cross-effects between prenatal MA exposure and sensitivity to the drugs in adult females. With respect to the effect of morphine in the CPP, our present data demonstrated increased drug seeking behavior that however did not differ based on the prenatal drug exposure. Discussion As mentioned above, there are studies available (Heyser et al., 1992; Rocha et al., 2002; Estelles et al., 2006) showing increased drug-seeking behavior of cocaine or morphine in animals prenatally exposed to cocaine, which is in disagreement to our results. On the other hand, prenatal morphine exposure was shown not to affect self-administration of morphine or cocaine in adulthood (Riley and Vathy, 2006; Vathy et al., 2007). In addition, study of Vela et al. (1998) demonstrated sex differences of cross-effect between prenatal cannabinoids exposure and morphine self-administration in adulthood. Specifically, adult female rats born from mothers that were daily treated with delta9- tetrahydrocannabinol (THC) during gestation and lactation periods, exhibited a statistically significant increase in the rate of acquisition of intravenous morphine self-administration behavior when compared with females born from vehicle- exposed mothers, an effect that did not exist in THC-exposed male offspring. Thus, it seems that the possibility of increased drug-seeking of drugs in adulthood depends on the drug that was the animal exposed prenatally and also on the sex. Therefore, our future studies are planned to examine the cross-effects between prenatal MA exposure and sensitivity to the drugs in adult females. In conclusion, our data demonstrate that there is a cross-effect between prenatal MA exposure and the challenge dose of other drug in adulthood, however drug-seeking behavior is not increased by prenatal MA exposure as we expected. Ago, Y., Arikawa, S., Yata, M., Yano, K., Abe, M., Takuma, K., Matsuda, T. (2008) Antidepressant-like effects of the glucocorticoid receptor antagonist RU-43044 are associated with changes in prefrontal dopamine in mouse models of depression. Neuropharmacology 55, 1355–1363. Bernášková, K., Matějovská, I., Šlamberová, R. (2011) Postnatal challenge dose of methamphetamine amplifies anticonvulsant effects of prenatal methamphetamine exposure on epileptiform activity induced by electrical stimulation in adult male rats. Exp. Neurol. 229, 282–287. References Ago, Y., Arikawa, S., Yata, M., Yano, K., Abe, M., Takuma, K., Matsuda, T. (2008) Antidepressant-like effects of the glucocorticoid receptor antagonist RU-43044 are associated with changes in prefrontal dopamine in mouse models of depression. Neuropharmacology 55, 1355–1363. Arnold, J. C. (2005) The role of endocannabinoid transmission in cocaine addiction. Pharmacol. Biochem. Behav. 81, 396–406. Bartoletti, M., Gaiardi, M., Gubellini, C., Bacchi, A., Babbini, M. (1985) Cross-sensitization to the excitatory effect of morphine in post-dependent rats. Neuropharmacology 24, 889–893. Bernášková, K., Matějovská, I., Šlamberová, R. (2011) Postnatal challenge dose of methamphetamine amplifies anticonvulsant effects of prenatal methamphetamine exposure on epileptiform activity induced by electrical stimulation in adult male rats. Exp. Neurol. 229, 282–287. Bonate, P. L., Swann, A., Silverman, P. B. (1997) Behavioral sensitization to cocaine in the absence of altered brain cocaine levels. Pharmacol. Biochem. Behav. 57, 665–669. Bubeníková-Valešová, V., Kačer, P., Syslová, K., Rambousek, L., Janovský, M., Schutová, B., Hrubá, L., Šlamberová, R. (2009) Prenatal methamphetamine exposure affects the mesolimbic dopaminergic system and behavior in adult offspring. Int. J. Dev. Neurosci. 27, 525–530. Arnold, J. C. (2005) The role of endocannabinoid transmission in cocaine addiction. Pharmacol. Biochem. Beh 81, 396–406. Bubeníková-Valešová, V., Kačer, P., Syslová, K., Rambousek, L., Janovský, M., Schutová, B., Hrubá, L., Šlamberová, R. (2009) Prenatal methamphetamine exposure affects the mesolimbic dopaminergic system and behavior in adult offspring. Int. J. Dev. Neurosci. 27, 525–530. Bartoletti, M., Gaiardi, M., Gubellini, C., Bacchi, A., Babbini, M. (1985) Cross-sensitization to the excitatory effect of morphine in post-dependent rats. Neuropharmacology 24, 889–893. B ášk á K M ěj ká I Šl b á R (2011) P l h ll d f h h i lifi References 406, 1–13 transporters: pharmacological consequences and implications for neurotoxicity. Eur. J. Pharmacol. 406, 1–13. Fleming, J. A., Byck, R., Barash, P. G. (1990) Pharmacology and therapeutic applications of cocaine. Anesthesiology 73, 518–531. Gagin, R., Kook, N., Cohen, E., Shavit, Y. (1997) Prenatal morphine enhances morphine-conditioned place preference in adult rats. Pharmacol. Biochem. Behav. 58, 525–528. Gygi, M. P., Gygi, S. P., Johnson, M., Wilkins, D. G., Gibb, J. W., Hanson, G. R. (1996) Mechanisms for tolerance to methamphetamine effects. Neuropharmacology 35, 751–757. He, S., Grasing, K. (2004) Chronic opiate treatment enhances both cocaine-reinforced and cocaine-seeking behaviors following opiate withdrawal Drug Alcohol Depend 75 215–221 He, S., Grasing, K. (2004) Chronic opiate treatment enhances both cocaine-reinforced and cocaine-seeking behaviors following opiate withdrawal. Drug Alcohol Depend. 75, 215–221. behaviors following opiate withdrawal. Drug Alcohol Depend. 75, 215–221. Heyser, C. J., Goodwin, G. A., Moody, C. A., Spear, L. P. (1992) Prenatal cocaine exposure attenuates cocaine- induced odor preference in infant rats. Pharmacol. Biochem. Behav. 42, 169–173. Horger, B. A., Giles, M. K., Schenk, S. (1992) Preexposure to amphetamine and nicotine predisposes rats to self-administer a low dose of cocaine. Psychopharmacology (Berl.) 107, 271–276. Leri, F., Flores, J., Rajabi, H., Stewart, J. (2003) Effects of cocaine in rats exposed to heroin. Neuropsychopharmacology 28, 2102–2116. Malanga, C. J., Kosofsky, B. E. (2003) Does drug abuse beget drug abuse? Behavioral analysis of addiction liability in animal models of prenatal drug exposure. Brain Res. Dev. Brain Res. 147, 47–57. Marwick, C. (2000) NIDA seeking data on effect of fetal exposure to methamphetamine. JAMA 283, 2225–2226. Mueller, D., Stewart, J. (2000) Cocaine-induced conditioned place preference: reinstatement by priming injections of cocaine after extinction. Behav. Brain Res. 115, 39–47. Nazarian, A., Rodarte-Freeman, A. L., McDougall, S. A. (1999) Dopaminergic modulation of kappa opioid- mediated ultrasonic vocalization, antinociception, and locomotor activity in the preweanling rat. Behav. Neurosci. 113, 816–825. Patti, C. L., Frussa-Filho, R., Silva, R. H., Carvalho, R. C., Kameda, S. R., Takatsu-Coleman, A. L., Cunha, J. L., Abilio, V. C. (2005) Behavioral characterization of morphine effects on motor activity in mice. Pharmacol. Biochem. Behav. 81, 923–927. Peltier, R. L., Li, D. H., Lytle, D., Taylor, C. M., Emmett-Oglesby, M. W. (1996) Chronic d-amphetamine or methamphetamine produces cross-tolerance to the discriminative and reinforcing stimulus effects of cocaine. J. Pharmacol. Exp. Ther. 277, 212–218. Riley, M. A., Vathy, I. References Ago, Y., Arikawa, S., Yata, M., Yano, K., Abe, M., Takuma, K., Matsuda, T. (2008) Antidepressant-like effects of the glucocorticoid receptor antagonist RU-43044 are associated with changes in prefrontal dopamine in mouse models of depression. Neuropharmacology 55, 1355–1363. Arnold, J. C. (2005) The role of endocannabinoid transmission in cocaine addiction. Pharmacol. Biochem. Behav. 81, 396–406. Bartoletti, M., Gaiardi, M., Gubellini, C., Bacchi, A., Babbini, M. (1985) Cross-sensitization to the excitatory effect of morphine in post-dependent rats. Neuropharmacology 24, 889–893. Bernášková, K., Matějovská, I., Šlamberová, R. (2011) Postnatal challenge dose of methamphetamine amplifies anticonvulsant effects of prenatal methamphetamine exposure on epileptiform activity induced by electrical stimulation in adult male rats. Exp. Neurol. 229, 282–287. Bonate, P. L., Swann, A., Silverman, P. B. (1997) Behavioral sensitization to cocaine in the absence of altered brain cocaine levels. Pharmacol. Biochem. Behav. 57, 665–669. nate, P. L., Swann, A., Silverman, P. B. (1997) Behavioral sensitization to cocaine in the absence of altered brain cocaine levels. Pharmacol. Biochem. Behav. 57, 665–669. Bubeníková-Valešová, V., Kačer, P., Syslová, K., Rambousek, L., Janovský, M., Schutová, B., Hrubá, L., Šlamberová, R. (2009) Prenatal methamphetamine exposure affects the mesolimbic dopaminergic system and behavior in adult offspring. Int. J. Dev. Neurosci. 27, 525–530. Šlamberová R. et al. 203) Prague Medical Report / Vol. 113 (2012) No. 3, p. 189–205 Carey, R., Gui, J. (1997) A simple and reliable method for the positive identification of pavlovian conditioned cocaine effects in open-field behavior. J. Neurosci. Methods 73, 1–8. Carey, R. J., Damianopoulos, E. N. (2006) Cocaine conditioning and sensitization: the habituation factor. Pharmacol. Biochem. Behav. 84, 128–133. Chen, J. Y., Yeh, G. C., Tao, P. L., Kuo, C. T., Chen, K. B., Wen, Y. R. (2010) Prenatal exposure to methamphetamine alters the mechanical withdrawal threshold and tonic hyperalgesia in the offspring. Neurotoxicology 31, 432–438. Estelles, J., Rodriguez-Arias, M., Maldonado, C., Aguilar, M. A., Minarro, J. (2006) Gestational exposure to cocaine alters cocaine reward. Behav. Pharmacol. 17, 509–515. Fattore, L., Deiana, S., Spano, S. M., Cossu, G., Fadda, P., Scherma, M., Fratta, W. (2005) Endocannabinoid system and opioid addiction: behavioural aspects. Pharmacol. Biochem. Behav. 81, 343–359. Ferrario, C. R., Robinson, T. E. (2007) Amphetamine pretreatment accelerates the subsequent escalation of cocaine self-administration behavior. Eur. Neuropsychopharmacol. 17, 352–357. Fleckenstein, A. E., Gibb, J. W., Hanson, G. R. (2000) Differential effects of stimulants on monoaminergic transporters: pharmacological consequences and implications for neurotoxicity. Eur. J. Pharmacol. References (2006) Mid- to late gestational morphine exposure does not alter the rewarding properties of morphine in adult male rats. Neuropharmacology 51, 295–304. Effect of Prenatal Methamphetamine Exposure on Cross-sensitization 204) Prague Medical Report / Vol. 113 (2012) No. 3, p. 189–205 Rocha, B. A., Mead, A. N., Kosofsky, B. E. (2002) Increased vulnerability to self-administer cocaine in mice prenatally exposed to cocaine. Psychopharmacology (Berl.) 163, 221–229. Roerig, S. C., Fujimoto, J. M. (1988) Morphine antinociception in different strains of mice: relationship of supraspinal-spinal multiplicative interaction to tolerance. J. Pharmacol. Exp. Ther. 247, 603–608. Rothman, R. B., Baumann, M. H., Dersch, C. M., Romero, D. V., Rice, K. C., Carroll, F. I., Partilla, J. S. (2001) Amphetamine-type central nervous system stimulants release norepinephrine more potently than they release dopamine and serotonin. Synapse 39, 32–41. Sanchez, C. J., Bailie, T. M., Wu, W. R., Li, N., Sorg, B. A. (2003) Manipulation of dopamine d1-like receptor activation in the rat medial prefrontal cortex alters stress- and cocaine-induced reinstatement of conditioned place preference behavior. Neuroscience 119, 497–505. Schutová, B., Hrubá, L., Pometlová, M., Deykun, K., Šlamberová, R. (2008) Impact of methamphetamine administered prenatally and in adulthood on cognitive functions of male rats tested in Morris water maze. Prague Med. Rep. 109, 62–70. Schutová, B., Hrubá, L., Pometlová, M., Šlamberová, R. (2009) Impact of prenatal and acute methamphetamine exposure on behaviour of adult male rats. Prague Med. Rep. 110, 67–78. Shoblock, J. R., Sullivan, E. B., Maisonneuve, I. M., Glick, S. D. (2003) Neurochemical and behavioral differences between d-methamphetamine and d-amphetamine in rats. Psychopharmacology (Berl.) 165, 359–369. Shuster, L., Yu, G., Bates, A. (1977) Sensitization to cocaine stimulation in mice. Psychopharmacology (Berl.) 52, 185–190. Šlamberová, R., Charousová, P., Pometlová, M. (2005) Methamphetamine administration during gestation impairs maternal behavior. Dev. Psychobiol. 46, 57–65. Šlamberová, R., Pometlová, M., Charousová, P. (2006) Postnatal development of rat pups is altered by prenatal methamphetamine exposure. Prog. Neuropsychopharmacol. Biol. Psychiatry 30, 82–88. Šlamberová, R., Schutová, B., Matějovská, I., Bernášková, K., Rokyta, R. (2009) Effects of a single postnatal methamphetamine administration on NMDA-induced seizures are sex- and prenatal exposure-specific. Naunyn Schmiedebergs Arch. Pharmacol. 380, 109–114. Šlamberová, R., Schutová, B., Hrubá, L., Pometlová, M. (2011a) Does prenatal methamphetamine exposure affect the drug-seeking behavior of adult male rats? Behav. Brain Res. 224, 50–86. Šlamberová, R., Yamamotová, A., Schutová, B., Hrubá, L., Pometlová, M. References (2011b) Impact of prenatal methamphetamine exposure on the sensitivity to the same drug in adult male rats. Prague Med. Rep. 112, 102–114. Suto, N., Austin, J. D., Tanabe, L. M., Kramer, M. K., Wright, D. A., Vezina, P. (2002) Previous exposure to VTA amphetamine enhances cocaine self-administration under a progressive ratio schedule in a D1 dopamine receptor dependent manner. Neuropsychopharmacology 27, 970–979. Turner, C. D., Bagnara, J. T. (1976) Endocrinology of the ovary. In: General Endocrinology, eds. Turner, C. D., Bagnara, J. T., pp. 450–495, W. B. Saunders Company, Philadelphia. Tzschentke, T. M. (1998) Measuring reward with the conditioned place preference paradigm: a comprehens review of drug effects, recent progress and new issues. Prog. Neurobiol. 56, 613–672. Tzschentke, T. M., Schmidt, W. J. (1998) The development of cocaine-induced behavioral sensitization is affected by discrete quinolinic acid lesions of the prelimbic medial prefrontal cortex. Brain Res. 795, 71–76. Vadalouca, A., Moka, E., Argyra, E., Sikioti, P., Siafaka, I. (2008) Opioid rotation in patients with cancer: a review of the current literature. J. Opioid Manag. 4, 213–250. Valvassori, S. S., Frey, B. N., Martins, M. R., Reus, G. Z., Schimidtz, F., Inacio, C. G., Kapczinski, F., Quevedo, J. (2007) Sensitization and cross-sensitization after chronic treatment with methylphenidate in adolescent Wistar rats. Behav. Pharmacol. 18, 205–212. Šlamberová R. et al. 205) Prague Medical Report / Vol. 113 (2012) No. 3, p. 189–205 Vathy, I., Šlamberová, R., Liu, X. (2007) Foster mother care but not prenatal morphine exposure enhances cocaine self-administration in young adult male and female rats. Dev. Psychobiol. 49, 463–473. Vavřínková, B., Binder, T., Živný, J. (2001) Characteristics of a population of drug dependent pregnant women in the Czech Republic. Ceska Gynekol. 66, 285–291. (in Czech) Vela, G., Martin, S., Garcia-Gil, L., Crespo, J. A., Ruiz-Gayo, M., Javier Fernandez-Ruiz, J., Garcia-Lecumberri, C Pelaprat, D., Fuentes, J. A., Ramos, J. A., Ambrosio, E. (1998) Maternal exposure to delta9- Vela, G., Martin, S., Garcia-Gil, L., Crespo, J. A., Ruiz-Gayo, M., Javier Fernandez-Ruiz, J., Garcia-Lecumberri, C., Pelaprat, D., Fuentes, J. A., Ramos, J. A., Ambrosio, E. (1998) Maternal exposure to delta9- tetrahydrocannabinol facilitates morphine self-administration behavior and changes regional binding to central mu opioid receptors in adult offspring female rats. Brain Res. 807, 101–109. tetrahydrocannabinol facilitates morphine self-administration behavior and changes regional binding to central mu opioid receptors in adult offspring female rats. Brain Res. 807, 101–109. Vezina, P., Stewart, J. Effect of Prenatal Methamphetamine Exposure on Cross-sensitization Vathy, I., Šlamberová, R., Liu, X. (2007) Foster mother care but not prenatal morphine exposure enhance cocaine self-administration in young adult male and female rats. Dev. Psychobiol. 49, 463–473. References (1987) Morphine conditioned place preference and locomotion: the effect of confinement during training. Psychopharmacology (Berl.) 93, 257–260. Yamamotová, A., Hrubá, L., Schutová, B., Rokyta, R., Šlamberová, R. (2011) Perinatal effect of methamphetamine on nociception in adult Wistar rats. Int. J. Dev. Neurosci. 29, 85–92. Young, E. R., MacKenzie, T. A. (1992) The pharmacology of local anesthetics – a review of the literature. J. Can. Dent. Assoc. 58, 34–42. Zemlan, F. P., Kow, L. M., Pfaff, D. W. (1984) Analgesia after lesions of nucleus reticularis magnocellularis: differential effect on supraspinal versus spinal pain reflexes. Pain 18, 221–237.
https://openalex.org/W4389350206	http://jurnal.unprimdn.ac.id/index.php/BIP/article/download/3972/2622	Indonesian	null	SEMIOTIKA RIFFATERRE DALAM PUISI “LAGU SEORANG GERILYA” KARYA W.S. RENDRA	Bahasa Indonesia Prima/Bahasa Indonesia Prima (Online)	2,023	cc-by-sa	3,327	SEMIOTIKA RIFFATERRE DALAM PUISI “LAGU SEORANG GERILYA” KARYA W.S. RENDRA SEMIOTIKA RIFFATERRE DALAM PUISI “LAGU SEORANG GERILYA” KARYA W.S. RENDRA Fairuz Annisa Zahrania, Rini Febrianti Susilob, Rina Ratihc Fakultas Keguruan Ilmu Pendidikan, Universitas Ahmad Dahlan azahraniannisa82@gmail.com, brinifebriantisusilo79453@gmail.com, crina.sudaryani@pbsi.uad.ac.id Artikel History: Submitted: 10 Juli 2023; Revised: 18 Agustus 2023; Accepted: 29 September 2023 10.34012/bip.v4i1.2708 BIP: Jurnal Bahasa Indonesia Prima Creative Commons Attribution-Share Alike 4.0 International License. ISSN: 2684-6780 (online), ISSN: 2088-365X (Print) http://jurnal.unprimdn.ac.id/index.php/BIP ABSTRAK - Puisi yang berjudul “Lagu Seorang Gerilya” merupakan karya sastrawan hebat yang mampu mengungkapkan isi pikirannyamenjadi sebuah bait-bait puisi yang indah yang menjadikan penulis tertarik untuk meneliti puisi dari W.S Rendra. Tujuan penelitian ini mendeskripsikan puisi lagu seoerang gerilya menggunakan kajian semiotika riffatere. Penelitian ini menggunakan pendekatan deskriptif kualitatif dengan mendeskripsikan fakta-fakta yang kemudian dianalisis. Data penelitian berupa kata, frasa, klausa, kalimat yang terdapat pada teks puisi. langkah-langkah pembacaan semiotik riffatere: (1) Pembacaan heuristik dan pembacaan hermeneutik, (2) Ketidaklangsungan ekspresi, (3) Menentukan matriks, model, dan varian, (4) Hipogram puisi Lagu seorang Gerilya karya W.S. Rendra, dan (5) Menyimpulkan data dan menulis laporan. Hasil penelitian ini mendeskripsikan makna yang ingin disampaikan penyair melalui puisi adalah tentang kekasihnya hingga akhir hayatnya di medan perang. Matriks dari puisi lagu seorang gerilya adalah perjuangan, ketahanan, penindasan. Model pada puisi lagu seorang gerilya menggunakan makna simbolis dan metaforis. K t k i P i i L S G il S i tik Riff t K t F Kata kunci: Puisi Lagu Seorang Gerilya, Semiotika Riffatere, Kata, Frasa ABSTRACT - The poem entitled "Song of a Guerrilla" is a great literary work that is able to express the contents of his thoughts into beautiful poetic verses that make the author interested in researching poetry from W.S Rendra. The purpose of this study is to describe a guerrilla song poem using the study of riffatere semiotics. This research uses a qualitative descriptive approach by describing the facts that are then analyzed. Research data in the form of words, phrases, clauses, sentences contained in poetry texts. Riffatere semiotic reading steps: (1) Heuristic reading and hermeneutic reading, (2) Indirectness of expression, (3) Determining matrices, models, and variants, (4) Hypogram of W.S. Rendra's song of a guerrilla poem, and (5) Summing up data and writing reports. The results of this study describe the meaning that poets want to convey through poetry is about their lovers until the end of their lives on the battlefield. The matrix of a guerrilla's song poem is struggle, resilience, oppression. SEMIOTIKA RIFFATERRE DALAM PUISI “LAGU SEORANG GERILYA” KARYA W.S. RENDRA The model on a guerrilla's song poem uses symbolic and metaphorical meanings. Keywords: Poem Song A Guerrilla, Semiotics Riffatere, Words, Phrases 183 makna pada puisi “Lagu Seorang Gerilya” Karya WS. Rendra. Metode pembacaan Semiotik Riffaterre ini melibatkan tahap pembacaan puisi secara hermeneutik dan heuristik, ketidaklangsungan ekspresi ditemukan. Setelah tahap ini, puisi dipahami lebih mudah dengan mengidentifikasi matriks, model, varian, dan hipogramnya. Ratih Sebaliknya, Ratih (2016:5) menjelaskan bahwa semiotika Riffaterre menawarkan metode pemaknaan khusus, yaitu dengan menganggap karya sastra sebagai sistem tanda-tanda, istilahnya menghasilkan makna tanda-tanda. Auden (dalam Pradopo, 2017:6) menyatakan bahwa puisi merupakan pernyataan perasaan yang bercampur baur. Sejalan dengan pernyataan Auden, Septiani (2021:97) menyatakan bahwa puisi merupakan karya yang benar-benar dihasilkan oleh seseorang berdasar pada pengalamannya dan belum pernah diciptakan sebelumnya. Menurut Ade dkk (2017) mengkaji puisi merupakan hasil yang diperoleh setelah proses analisis puisi. Proses ini menggunakan langkah- langkah tertentuk untuk menghasilkan kajian yang obyektif terhadap puisi yang dianalisis. A. Pendahuluan Puisi sebagai salah satu genre dari karya sastra memiliki makna yang tersembunyi di dalam tanda-tanda (Diana, 2018). Perwujudan makna suatu karya sastra dikatakan berhasil apabila makna yang ingin disampaikan penulis dapat dipahami dengan baik oleh pembacanya (Latifah, 2020). Oleh karena itu, perlu teori Semiotik untuk memecahkan tanda dan memahami makna dalam sebuah puisi. Penelitian relevan terkait kajian Semiotik Riffaterre telah dilakukan oleh beberapa peneliti, diantaranya penelitian Noviana dkk (2020: 143-160) yang membahas “Pemaknaan Lirik Lagu “Shabondama” Karya Ujo Noguchi”; Mandala dkk. (2021) yang membahas Lagu Sakura Karya Naotaro Moriyama; Huri dkk. (2017) yang membahas “Puisi Dongeng Marsinah” karya Sapardi Djoko Damono. Persamaan penelitian terdahulu dengan penelitian ini terletak pada objeknya yaitu Puisi “Lagu Seorang Gerilya” Karya WS Rendra. Adapun tujuan penelitian ini adalah mendeskripsikan makna puisi “Lagu Seorang Gerilya” menggunakan kajian semiotika riffattere. Kata "semiotika" berasal dari kata Yunani seme, yang berarti "tanda", dan semeion, yang berarti "tanda". Nasution (2014: 5) memberikan penjelasan tentang bidang yang menyelidiki tanda dan lambang, sistem lambang, dan proses perlambangannya. B. Metode Penelitian Metode yang digunakan adalah metode deskriptif kualitatif. Deskriptif kualitatif adalah suatu proses penelitian untuk memahami fenomena-fenomena manusia atau sosial dengan menciptakan gambar yang menyeluruh dan kompleks yang disajikan dengan kata-kata (Walidin, Saifullah & Tabrani, 2015:77). Pendekatan ini biasanya digunakan dalam penelitian yang berfokus pada pemahaman mendalam tentang persepsi, pengalaman, sikap, keyakinan, atau interaksi individu atau kelompok dalam konteks tertentu. Dengan menggunakan metode penelitian kualitatif Menganalisis puisi dapat menggunakan beberapa pendekatan, namun Semiotik Riffaterre merupakan pendekatan yang tepat untuk menemukan 184 sementara dari jauh resimen tank penindas terdengar menderu. memungkinkan peneliti untuk menggali kekayaan dan kompleksitas puisi, mengungkapkan makna subjektif yang mungkin tidak dapat diukur secara kuantitatif. Malam bermandi cahaya matahari, kehijauan menyelimuti medan perang yang membara. Di dalam hujan tembakan mortir, kekasihku, engkau menjadi pelangi yang agung dan syahdu Malam bermandi cahaya matahari, kehijauan menyelimuti medan perang yang membara. Di dalam hujan tembakan mortir, kekasihku, engkau menjadi pelangi yang agung dan syahdu Data penelitian ini berupa kata, frasa, klausa atau kalimat yang terdapat dalam baris atau bait puisi, sedangkan sumber data penelitian adalah teks puisi “Lagu Seorang Gerilya” yang terdapat pada laman buku berjudul Potret Pembangunan Dalam Puisi Karya W.S. Data diidentifikasi kemudian dianalisis dengan cara menginterpretasikan puisi “Lagu Seorang Gerilya” karya WS. Rendra dengan cara menghubungkan temuan penelitian dengan teori Semiotik Riffaterre dan hipogramnya. Data dianalisis dengan langkah-langkah berikut: Peluruku habis Peluruku habis dan darah muncrat dari dadaku. Maka di saat seperti itu kamu menyanyikan lagu-lagu perjuangan bersama kakek-kakekku yang telah gugur di dalam berjuang membela rakyat jelata (1) Pembacaan heuristik dan pembacaan hermeneutik. 1). Pembacaan heuristik dan pembacaan hermeneutic Dalam pembacaan heuristik, puisi dibaca berdasarkan konvensi bahasa atau sistem bahasa sesuai dengan kedudukan bahasa sebagai sistem semiotik tingkat pertama. 2). Ketidaklangsungan ekspresi 3). Menentukan matriks, model, dan varian 4). Hipogram puisi lagu seorang gerilya karya w.s. rendra, dan Pada puisi “Lagu Seorang Gerilya” karya W.S. Rendra Puisi menceritakan tentang kekasih seorang gerilya yang menjalani perjuangan dengan semangat dan keindahan di tengah kekerasan perang. Kata pohon pisang, sinar matahari, dan pelangi melambangkan keindahan dan ketenangan dalam konteks yang penuh dengan konflik. Puisi ini juga menyoroti pengorbanan para pejuang dan menggambarkan perjuangan mereka melalui lagu-lagu perjuangan. 5). Menyimpulkan data dan menulis laporan. C. Hasil Dan Pembahasan Lagu Seorang Gerilla karya W.S. Rendra C. Hasil Dan Pembahasan Lagu Seorang Gerilla karya W.S. Rendra C. Hasil Dan Pembahasan Engkau melayang jauh, kekasihku. Engkau mandi cahaya matahari. Aku di sini memandangmu, menyandang senapan, berbendera pusaka. Engkau melayang jauh, kekasihku. Engkau mandi cahaya matahari. Aku di sini memandangmu, menyandang senapan, berbendera pusaka. Pendekatan heuristik dalam analisis sastra melibatkan pemahaman dan penafsiran teks berdasarkan intuisi, perasaan, dan pengalaman pribadi pembaca. Pada puisi “Lagu Seorang Gerilya” karya W.S. Rendra puisi tersebut Di antara pohon-pohon pisang di kampung kita yang berdebu, engkau berkudung selendang katun di kepalamu. Di antara pohon-pohon pisang di kampung kita yang berdebu, engkau berkudung selendang katun di kepalamu. Engka menjadi s at keindahan engkau berkudung selendang katun di kepalamu. Engkau menjadi suatu keindahan, 185 secara menyeluruh. Dalam pembacaan hermeneutik, harus mempertimbangkan konteks sejarah, budaya, dan sosial di mana teks tersebut dihasilkan. Dalam konteks puisi "Lagu Seorang Gerilla" karya W.S. Rendra. Pada puisi “Lagu seorang Gerilya” memiliki konteks sejarah dan politik yang terdapat W.S. Rendra merupakan seorang penyair Indonesia yang aktif pada era Orde Baru di Indonesia. Konteks sejarah ini penting untuk memahami puisi ini, karena dapat dihubungkan dengan perlawanan dan perjuangan rakyat terhadap penindasan dan kekerasan pada masa itu. Puisi “Lagu seorang Gerilya” mengacu pada kehidupan seorang gerilyawan atau pejuang dalam perang. Konteks sejarah tentang perang dan konflik, baik di tingkat lokal maupun internasional, dapat memberikan pemahaman yang lebih dalam tentang tema dan pesan yang ingin disampaikan oleh puisi ini. mengidentifikasi perjuangan dan semangat. Pada saat membaca puisi “Lagu Seorang Gerilya” mungkin merasakan rasa identifikasi dan keterhubungan dengan tema perjuangan dan semangat dalam puisi ini. Puisi ini dapat memicu emosi pembaca yang terkait dengan keberanian, ketahanan, dan semangat perlawanan terhadap penindasan. Respons terhadap imajinasi dan gambaran visual pada puisi “Lagu Seorang Gerilya” puisi ini mengandung gambaran visual yang kuat, seperti "mandi cahaya matahari" dan "malam bermandi cahaya matahari". Pembaca mungkin merespons secara intuitif dan estetis terhadap imajinasi yang dihasilkan oleh kata-kata tersebut, dan merasakan keindahan dan kekuatan imajinatif puisi. Relevansi dengan konteks sejarah dan politik pada puisi “Lagu Seorang Gerilya” pembaca yang memiliki pengetahuan tentang konteks sejarah dan politik pada masa Orde Baru di Indonesia mungkin melihat hubungan antara puisi ini dengan perjuangan dan penindasan yang dialami pada masa itu. Pembaca dapat merasakan ketegangan dan perasaan urgensi dalam puisi ini berdasarkan pengalaman atau pengetahuan mereka tentang konteks tersebut. a. Penggantian arti Dalam puisi "Lagu Seorang Gerilya" karya W.S. Rendra, terdapat beberapa penggantian arti yang dapat ditemukan yaitu: /Engkau menjadi suatu keindahan/, /sementara dari jauh/ /sementara dari jauh/ /resimen tank penindas terdengar menderu/. C. Hasil Dan Pembahasan Pengalaman pribadi dalam perjuangan atau konflik pada puisi “Lagu seorang Gerilya” pembaca yang memiliki pengalaman pribadi terkait perjuangan, konflik, atau penindasan mungkin membawa pemahaman dan emosi mereka sendiri ke dalam pembacaan puisi ini. Puisi ini dapat membangkitkan kenangan, refleksi, atau pemikiran tentang pengalaman yang serupa dalam kehidupan pembaca. Analisis hermeneutik adalah pendekatan kritik sastra yang berfokus Pada puisi “Lagu Seorang Gerilya” terdapat simbolisme dan metafora. Puisi ini menggunakan simbolisme dan metafora untuk menggambarkan perjuangan, keindahan, dan kekerasan. Simbol-simbol seperti senapan, bendera pusaka, tank penindas, pohon pisang, dan pelangi dapat ditafsirkan secara simbolis dalam konteks puisi ini. Metafora seperti "mandi cahaya matahari" dan "malam bermandi cahaya matahari" juga dapat dianalisis untuk memahami makna dan hubungannya dengan tema puisi. Pada puisi “Lagu Seorang Gerilya” Terdapat gaya dan teknik puisi. Dalam analisis hermeneutik, kita dapat memperhatikan gaya dan teknik puisi yang digunakan oleh penyair. Misalnya, apakah terdapat penggunaan repetisi, rima, ritme, atau perangkat sastra lainnya yang dapat Analisis hermeneutik adalah pendekatan kritik sastra yang berfokus pada pemahaman dan penafsiran teks 186 /Aku di sini memandangmu/, /Aku di sini memandangmu/, memberikan pengaruh pada pemahaman puisi ini. Struktur puisi, seperti penggunaan bait-bait atau bagian-bagian tertentu, juga dapat dianalisis dalam konteks pembacaan hermeneutik. /menyandang senapan, berbendera pusaka/. Pada larik “Berbendera pusaka” kata pusaka adalah kata yang memiliki arti literal sebagai warisan budaya atau benda bersejarah yang diwariskan. Namun, dalam puisi ini, bendera pusaka mengambil arti simbolik yang melambangkan nilai-nilai luhur, semangat patriotik, dan semangat juang yang dijunjung tinggi oleh gerilyawan. Pada puisi “Lagu Seorang Gerilya” terdapat konteks budaya dan sosial. Puisi ini mencerminkan budaya dan situasi sosial pada masa itu. Pertimbangkan nilai-nilai, norma, dan konteks budaya yang mungkin memengaruhi pemahaman dan penafsiran puisi ini. 4. /Di antara pohon-pohon pisang di kampung// kita yang berdebu/, g g p a. Penggantian arti /engkau berkudung selendang katun di kepalamu/. /engkau menjadi pelangi yang agung dan syahdu/ /engkau menjadi pelangi yang agung dan syahdu/ Larik “Malam bermandi cahaya matahari": frasa ini melibatkan penyimpangan arti karena malam secara harfiah tidak bisa bermandikan cahaya matahari. Penyimpangan ini digunakan untuk menciptakan kontras dan penekanan visual atas pengalaman yang tidak biasa dan luar biasa dalam konteks perang. 2. /Di antara pohon-pohon pisang di kampung kita yang berdebu/, /engkau berkudung selendang katun di kepalamu/. 2. /Di antara pohon-pohon pisang di kampung kita yang berdebu/, /engkau berkudung selendang katun di kepalamu/. /Engkau menjadi suatu keindahan/, /sementara dari jauh/ /sementara dari jauh/ c. Penciptaan arti 1. /Lagu Seorang Gerilya/ Pada larik “Resimen tank penindas" kata "Tank penindas" mengacu pada alat perang yang digunakan untuk menindas dan mengontrol rakyat. Kata "resimen" digunakan untuk menyebutkan jumlah tank yang banyak dan menggambarkan penindasan yang sistematis dan terorganisir. Kata "lagu" dalam puisi ini tidak hanya merujuk pada komposisi musik, tetapi juga melambangkan semangat perjuangan dan penegasan identitas gerilyawan. Dalam konteks puisi ini, "lagu" menjadi simbol perlawanan dan keberanian dalam menghadapi penindasan. 2. /Engkau melayang jauh, kekasihku/. /Engkau mandi cahaya matahari/. /Aku di sini memandangmu/, /menyandang senapan, berbendera pusaka/. 5. /Malam bermandi cahaya matahari/, /kehijauan menyelimuti medan perang yang membara/. /Di dalam hujan tembakan mortir, kekasihku/, 5. /Malam bermandi cahaya matahari/, /kehijauan menyelimuti medan perang yang membara/. /Aku di sini memandangmu/, /menyandang senapan, berbendera pusaka/. /Di dalam hujan tembakan mortir, kekasihku/, Pada larik “Engkau mandi cahaya matahari” frasa ini menggantikan arti harfiah mandi, mengarah pada makna metaforis yang melambangkan pemurnian, pembaruan, atau transformasi spiritual. Mandi cahaya matahari mencerminkan kekuatan alam dan spiritualitas yang memperkuat keberanian dan semangat dalam perjuangan. /engkau menjadi pelangi yang agung dan syahdu/ Pada larik “pelangi yang agung dan syahdu" Pelangi biasanya diasosiasikan dengan keindahan alam yang mencolok dan ceria. Namun, dalam puisi ini, pelangi digunakan sebagai metafora untuk menggambarkan keindahan yang agung dan syahdu yang muncul di tengah situasi perang dan konflik. 3. /Engkau melayang jauh, kekasihku/. /Engkau mandi cahaya matahari/. 3. /Engkau melayang jauh, kekasihku/. /Engkau mandi cahaya matahari/. 187 Larik “Peluruku habis dan darah muncrat dari dadaku" Penyimpangan arti terjadi di sini karena peluru dan darah tidak bisa secara harfiah "habis" atau "muncrat" dari tubuh seseorang. Namun, penyimpangan ini digunakan untuk menggambarkan kondisi fisik dan keadaan yang parah akibat konflik dan perjuangan. b. Penyimpangan arti Dalam puisi "Lagu Seorang Gerilya" karya W.S. Rendra, terdapat beberapa penyimpangan arti yang dapat diamati yaitu: 1. /Engkau melayang jauh, kekasihku/. /Engkau mandi cahaya matahari/. /Aku di sini memandangmu/, 4. /Malam bermandi cahaya matahari/, /kehijauan menyelimuti medan perang yang membara/. /Di dalam hujan tembakan mortir, kekasihku/, /engkau menjadi pelangi yang agung dan syahdu/ /menyandang senapan, berbendera pusaka/. y /kehijauan menyelimuti medan perang yang membara/. Larik “Mandi cahaya matahari" frasa ini mengandung penyimpangan arti karena manusia tidak bisa mandi dalam cahaya matahari secara harfiah. Namun, dalam puisi ini, frasa tersebut digunakan untuk menggambarkan pengalaman spiritual atau pembaruan dalam suasana perang yang membara. /Di dalam hujan tembakan mortir, kekasihku/, c. Penciptaan arti /resimen tank penindas terdengar menderu/. Dalam puisi "Lagu Seorang Gerilya" karya W.S. Rendra, terdapat penciptaan arti yang kuat dan mendalam yaitu: Larik “Tank penindas" pada kata "penindas" digunakan sebagai penyimpangan arti karena tank sebagai objek tidak memiliki sifat penindas secara harfiah. Penyimpangan ini digunakan untuk menekankan peran tank dalam penindasan rakyat dalam konteks perang dan konflik. 1. Pada puisi “Lagu Seorang Gerilya” memiliki imaji visual yang kuat. Puisi ini menghadirkan imaji-imaji visual yang kuat, seperti "mandi cahaya matahari" dan "malam bermandi cahaya matahari". Melalui penggunaan imaji- imaji ini, puisi menciptakan pengalaman sensorik dan membangkitkan perasaan keajaiban dan transendensi dalam konteks perang dan konflik. 3. /Peluruku habis/ /dan darah muncrat dari dadaku/. /Maka di saat seperti itu/ /kamu menyanyikan lagu-lagu perjuangan/ /bersama kakek-kakekku yang telah gugur/ /di dalam berjuang membela rakyat jelata/ /dan darah muncrat dari dadaku/. /Maka di saat seperti itu/ /kamu menyanyikan lagu-lagu perjuangan/ 2. Pada puisi “Lagu Seorang Gerilya” memiliki simbolisme yang kaya. Puisi ini menggunakan simbolisme untuk mengungkapkan makna yang lebih dalam. Contohnya, senapan dan bendera /bersama kakek-kakekku yang telah gugur/ /di dalam berjuang membela rakyat jelata/ 188 pusaka melambangkan semangat perjuangan, pohon pisang melambangkan kehidupan sehari-hari, dan pelangi melambangkan keindahan dan harapan di tengah kegelapan. pusaka melambangkan semangat perjuangan, pohon pisang melambangkan kehidupan sehari-hari, dan pelangi melambangkan keindahan dan harapan di tengah kegelapan. pusaka melambangkan semangat perjuangan, pohon pisang melambangkan kehidupan sehari-hari, dan pelangi melambangkan keindahan dan harapan di tengah kegelapan. memiliki makna khusus seperti “senapan,” “bendera pusaka,” “tank penindas,” dan “pelangi.” Puisi ini menggabungkan kalimat pendek dan panjang untuk menciptakan ritme dan aliran yang bervariasi. 3. Pada puisi “Lagu Seorang Gerilya” memiliki perbandingan dan kontras. Puisi ini menggunakan perbandingan dan kontras untuk menciptakan makna yang kaya. Misalnya, perbandingan antara keindahan kepala yang dibungkus selendang katun dengan suara gemuruh tank penindas menciptakan kontras yang menegaskan ketegangan antara keindahan dan kekerasan. (4) Hipogram Untuk memberikan makna yang lebih penuh dalam pemaknaan sastra, sebuah karya sastra perlu dijajarkan dengan karya sastra lain yang menjadi hipogram atau latar belakang penciptannya. Dalam puisi "Lagu Seorang Gerilya" karya W.S. Rendra terdapat hipogram yang dapat diamati yaitu, terdapat kisah perjuangan. Perjuangan para pahlawan Indonesia dalam meraih kemerdekaan negerinya menjadi hipogram utama dalam puisi ini. Puisi menggambarkan pengalaman perjuangan gerilyawan dan menghidupkan semangat perlawanan terhadap penindasan dan kekerasan. Puisi ini berhipogram pada perjuangan para pahlawan negeri ini dalam meraih kemerdekaan, melawan penjajah, dan mempertahankan kesatuan dan persatuan. Para pahlawan negeri ini bertahan mempertaruhkan nayaw siang dan malam sebagaimana tampak pada baris- baris puisi berikut /Malam bermandi cahaya matahari/, /kehijauan menyelimuti medan perang yang membara/. Di dalam hujan tembakan mortar/. Suasana yang digambrakan dalam baris pusi di atas adalah gambaran nyata para pejuang menghadapi peperangan melawan penjajah. Dengan demikian, hipogram puisi “Lagu Seorang Gerilya” adalah perjuangan para pahlwan Indonesia mempertahankan tanah airnya. 4. Pada puisi “Lagu Seorang Gerilya” memiliki pemilihan kata yang kuat. Puisi ini menggunakan kata-kata yang kuat dan bermakna untuk menciptakan pengalaman yang mendalam. Misalnya, penggunaan kata "penindas" untuk menggambarkan tank, atau "agung dan syahdu" untuk menggambarkan pelangi, memberikan dimensi emosional dan estetika yang lebih dalam pada puisi. 4. Pada puisi “Lagu Seorang Gerilya” memiliki pemilihan kata yang kuat. Puisi ini menggunakan kata-kata yang kuat dan bermakna untuk menciptakan pengalaman yang mendalam. Misalnya, penggunaan kata "penindas" untuk menggambarkan tank, atau "agung dan syahdu" untuk menggambarkan pelangi, memberikan dimensi emosional dan estetika yang lebih dalam pada puisi. (3) Menentukan matriks, model, dan varian Matriks yang digunakan pada puisi “Lagu Seorang Gerilya” karya W.S. Rendra mengangkat tema perjuangan, ketahanan, dan keindahan di tengah konflik dan penindasan. Model pada puisi “Lagu Seorang Gerilya” menggunakan makna simbolis dan metaforis untuk menggambarkan pengalaman dan emosi dalam konteks perjuangan gerilya. Puisi ini menampilkan keindahan bahasa dan penggunaan imaji yang kuat untuk menciptakan gambaran yang visual dan memikat. Varian pada puisi “Lagu seorang Gerilya” puisi tersebut menggunakan kata - kata dengan makna yang kuat dan 189 Diana, Jumianti. (2018). Makna Puisi “Pohon Peradaban” Karya Dinullah Rayes Kajian Semiotika Riffaterre. Jurnal Pena Indonesia. Volume 4, Nomor2, Oktober 2018. Terima Kasih Puji syukur kepada Tuhan Yang Maha Esa, sehingga kami dapat menyelesaikan jurnal ilmiah ini. Tidak lupa juga kami ucapkan terima kasih kepada ibu Rina yang telah membimbing kami, sehingga kami dapat menyelesaikan jurnal ilmiah ini dengan lebih teliti. Dengan demikian, dalam pembuatan penelitian selanjutnya bisa disempurnakan lagi. Linguistics, Literature, and Culture 2.2 (2020): 143-160. Diakses pada : 25 April 2021 Walidin, W., Saifullah, & Tabrani. (2015). Metodologi Penelitian Kualitatif & Grounded Theory. Aceh:FTK Ar- Raniry Press. D. Simpulan Berdasarkan hasil analisis diatas dapat disimpulkan bahwa puisi lagu seorang gerilya menggambarkan peristiwa perang gerilya dan menghadirkan suasana yang penuh emosi keberanian, serta perlawanan. Makna yang ingin disampaikan penyair melalui puisi lagu seorang gerilya adalah tentang kekasihnya hingga akhir hayatnya di medan perang. Huri, Ranti Maretna dkk. (2017). Analisis Semiotika Riffaterre dalam Puisi “Dongeng Marsinah” Karya Sapardi Djoko Damono. Jurnal JBS Jurnal Bahasa dan Sastra. Volume 5, Nomor 1, 2017. Matriks dari puisi lagu seorang gerilya adalah perjuangan, ketahanan, penindasan. Model pada puisi lagu seorang gerilya menggunakan makna simbolis dan metaforis. Noviana, F., & Saifudin, A. Pemaknaan Lirik Lagu Shabondama Karya Ujo Noguchi Berdasarkan Analisis Semiotika Michael Riffaterre. Japanese Research on Linguistics, Literature, and Culture 2.2 (2020): 143-160. Diakses pada : 25 April 2021 Daftar Pustaka Kementerian Pendidikan dan Kebudayaan. (2017). Kamus Besar Bahasa Indonesia (KBBI) Edisi Kelima. Jakarta : Kementerian Pendidikan dan Kebudayaan. W.S. Rendra 2013. POTRET PEMBANGUNAN DALAM PUISI. Bandung: PT Pustaka Jaya. W.S. Latifah, Hani. (2020). Analisis Semiotik dalam Cerpen “Tak Ada yang Gila di Kota ini”. Jurnal Penelitian Humaniora. Volume 25, Nomor 2, Oktober 2020. Ratih, Rina. (2016). Teori dan Aplikasi Semiotik Michael Riffaterre. Yogyakarta: Pustaka Pelajar Ratih, Rina. (2016). Teori dan Aplikasi Semiotik Michael Riffaterre. Yogyakarta: Pustaka Pelajar Pradopo, R. D. (2017). Pengkajian Puisi. Yogyakarta: Gajah Mada University Press. Mandala, dkk. (2021). “Analisis Semiotika Raffaterre dalam Lagu Sakura Karya Naotaro Moriyana”. Jurnal 190 JPBJ, Volume 7, Nomor 2, Juli 2021. Septiani, Eka. (2021). “Analisis Unsur Intrinsik dalam Kumpulan Puisi Goresan Pena Anak Matematika”. Jurnal Pujangga Volume 7, Nomor 1, Juni 2021. Ade, dkk. (2017). “Kajian Puisi”. Universitas Muhammadiyah Prof. DR. Hamka. Ade, 191
https://openalex.org/W2044107439	https://bmcproc.biomedcentral.com/track/pdf/10.1186/1753-6561-5-S7-A1	English	null	IUFRO Tree Biotechnology 2011: "From genomes to integration and delivery"	BMC proceedings	2,011	cc-by	946	IUFRO Tree Biotechnology 2011: “From genomes to integration and delivery” and genetically modified (GM) trees were highlighted. With 340 registered participants, the Conference brought to Brazil most of the world’s brain power in forest tree genomics and biotechnology. An outstanding team of international scientists shared their results and visions on the present and future of this fast moving area of forest science, while a brilliant group of young scientist and students delivered a very energetic and diverse collection of high-quality scientific presentations. Forty two countries were represented at the Conference with almost 100 different laboratories from tens of Uni- versities, research institutions and private companies. Forest trees have unquestionably entered the genomic era. The updated version of the Populus genome, the recently released Eucalyptus grandis genome and the concerted efforts towards the generation of genome sequences for spruces (Picea sp.) and pines (Pinus sp.) by several groups worldwide, are fueling a multitude of inter-disciplinary studies and applications in sustainable forest production and conservation. Time now calls for the integration of scientific fields with an increased sense of urgency for delivery of effective biotechnologies. The IUFRO (International Union of Forestry Research Organizations) Tree Biotechnology biannual conference has established a solid tradition for over 20 years as the official meeting of the IUFRO working group 2.04.06 – Molecular biology of forest trees. This conference has convened scientists and foresters interested in the genet- ics, genomics, molecular biology and physiology of forest trees, and the application of this knowledge to tree improvement and conservation. The Tree Biotechnology Conference has undoubtedly been the premiere interna- tional forum where the most cutting edge research in tree biotechnology developed both in academia and industry is presented. “From genomes to integration and delivery”, this was the theme chosen for the 2011 edi- tion of the IUFRO Tree Biotechnology Conference, first time to be held in South America. Our intention was to promote a more integrated and applied dialogue on tree biotechnology and genomics, beyond the mainstream discussion of the fundamental advances on the genetic mechanisms that underlie tree phenotypes. During the seven days of the Conference 26 invited lectures, 63 oral and 185 poster presentations were delivered, totaling 274 papers made available as extended abstracts into this BMC Proceedings supple- ment. © 2011 Grattapaglia; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Grattapaglia BMC Proceedings 2011, 5(Suppl 7):A1 http://www.biomedcentral.com/1753-6561/5/S7/A1 Grattapaglia BMC Proceedings 2011, 5(Suppl 7):A1 http://www.biomedcentral.com/1753-6561/5/S7/A1 1EMBRAPA Genetic Resources and Biotechnology – Estação Parque Biológico, 70770-910, Brasilia, DF, Brazil Full list of author information is available at the end of the article INTRODUCTION Open Access Open Access IUFRO Tree Biotechnology 2011: “From genomes to integration and delivery” The special workshop on the hot topic of “Geno- mic Selection in tree breeding” and the several reports on whole-genome studies, made this conference edition inaugurate a deliberate effort towards a better integra- tion between the quantitative genomics, the “single- gene” and the system biology approaches to more efficiently unravel the complex relationships between genotypes and phenotypes in forest trees. A field trip to the forest plantations, nurseries and mill of VERACEL Cellulose was a definite highlight and a welcome break from the scientific sessions, providing an overview of some of the advances and challenges facing the transla- tion of research into plantation forestry. In closing this introductory statement, acknowledge- ments are due to the outstanding financial support pro- vided by the competitive grants of the Brazilian Ministry of Science and Technology through the National Research Council (CNPq) and the Ministry of Education through its agency for graduate studies (CAPES). Major support was also provided by EMBRAPA (Brazilian Cor- poration of Agricultural Research), and VERACEL In nine scientific sessions some of the current advances of genomics applied to forest conservation, tree physiology, stress response, molecular breeding, in vitro and propagation technologies, wood development Page 2 of 2 Page 2 of 2 Grattapaglia BMC Proceedings 2011, 5(Suppl 7):A1 http://www.biomedcentral.com/1753-6561/5/S7/A1 Cellulose, the host organizations, together with an exceptional suite of private sponsors. Besides the organi- zations that backed this conference and an active Scien- tific Committee involved in abstract review a number of people were involved in the organization and logistics. The conference would not have been possible without the valuable contributions of all these players. Given the rewarding feedback received after the Con- ference, the original goal of providing an exceptional mix of science, social activities and field exploration in a relaxed atmosphere was truly accomplished. The IUFRO Tree Biotechnology Conference 2011 made a significant contribution to advance the forest biotechnology research community one step ahead on the challenging task of moving from gene and genome discoveries to the delivery of valuable technologies into sustainable forestry. Author details 1EMBRAPA G 1EMBRAPA Genetic Resources and Biotechnology – Estação Parque Biológico, 70770-910, Brasilia, DF, Brazil. 2Graduate Program in Genomics and Biotechnology - Universidade Catolica de Brasilia - SGAN 916 Modulo B, 70790-160 Brasilia, Brazil. Published: 13 September 2011 Published: 13 September 2011 doi:10.1186/1753-6561-5-S7-A1 Cite this article as: Grattapaglia: IUFRO Tree Biotechnology 2011: “From genomes to integration and delivery”. BMC Proceedings 2011 5(Suppl 7):A1. doi:10.1186/1753-6561-5-S7-A1 Cite this article as: Grattapaglia: IUFRO Tree Biotechnology 2011: “From genomes to integration and delivery”. BMC Proceedings 2011 5(Suppl 7):A1. Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit
https://openalex.org/W2619897684	https://dialnet.unirioja.es/descarga/articulo/6334827.pdf	English	null	Cobweb, a serious pathology in mushroom crops: A review	Spanish journal of agricultural research	2,017	cc-by	7,744	Abstract p Funding: MINECO, Spain (Project RTA2010-00011-C02) and FEDER (Project E-RTA2014-00004-C02). JC was the recipient of a fellowship from the FPI-INIA program of Ministerio de Economia y Competitividad. C ti i t t Th th h d l d th t ti i t t i t Funding: MINECO, Spain (Project RTA2010-00011-C02) and FEDER (Project E-RTA2014-00004-C02). JC was the recipient of a fellowship from the FPI-INIA program of Ministerio de Economia y Competitividad. Competing interests: The authors have declared that no competing interests exist. p g p g Correspondence should be addressed to Jaime Carrasco: jaime.carrasco@plants.ox.ac.uk; carraco.jaime@gmail.com p g p g Correspondence should be addressed to Jaime Carrasco: jaime.carrasco@plants.ox.ac.uk; carraco.jaime@gmail.com Cobweb, a serious pathology in mushroom crops: A review Jaime Carrasco1,2, María-Jesús Navarro1 and Francisco J. Gea1 Jaime Carrasco1,2, María-Jesús Navarro1 and Francisco J. Gea1 1Centro de Investigación, Experimentación y Servicios del Champiñón (CIES). 16220 Quintanar del Rey, Cuenca, Spain. 2Department of Plant Scien- ces. University of Oxford. South Parks Road. Oxford, OX1 3RB, UK. OPEN ACCESS REVIEW ARTICLE Abstract Cobweb is a fungal disease of commercially cultivated mushrooms. Several members of the ascomycete genus Cladobotryum sp. have been reported as causal agents. White button mushroom is the most frequently cited host, but a wide range of cultivated edible mushrooms suffer cobweb. The pathology causes production losses and reduces the crop surface available. The parasite produces a great number of harmful conidia that can be released easily and distributed throughout the mushroom farm to generate secondary points of infection. To prevent initial outbreaks, hygiene is of primary importance within the facilities dedicated to mushroom cultivation, while additional measures must be implemented to control and reduce cobweb if there is an outbreak, including chemical and biological methods. This review summarizes and discusses the knowledge available on the historic occurrence of cobweb and its impact on commercial mushroom crops worldwide. Causal agents, disease ecology, including the primary source of infection and the dispersal of harmful conidia are also reviewed. Finally, control treatments to prevent the disease from breaking out are discussed. Additional keywords: Cladobotryum; fungal disease; dispersal; production losses; edible mushroom; control. Authors’ contributions: Conception or design; acquisition, analysis, and interpretation of data; drafting of the manuscript; and coordinating the research project: JC. Critical revision of the manuscript for important intellectual content; administrative, technical, or material support; and supervising the work: JC, MJN and FJG. Obtaining funding: FJG. pp ; p g , g g Citation: Carrasco, J.; Navarro, M. J.; Gea, F. J. (2017). Cobweb, a serious pathology in mushroom crops: A review. Spanish Journal of Agricultural Research, Volume 15, Issue 2, e10R01. https://doi.org/10.5424/sjar/2017152-10143 pp p g g g Citation: Carrasco, J.; Navarro, M. J.; Gea, F. J. (2017). Cobweb, a serious pathology in mushroom crops: A review. Spanish Journal of Agricultural Research, Volume 15, Issue 2, e10R01. https://doi.org/10.5424/sjar/2017152-10143 Received: 22 Jun 2016. Accepted: 29 May 2017 Citation: Carrasco, J.; Navarro, M. J.; Gea, F. J. (2017). Cobweb, a serious pathology in mushroom crops: A review. Spanish Journal of Agricultural Research, Volume 15, Issue 2, e10R01. https://doi.org/10.5424/sjar/2017152-10143 Received: 22 Jun 2016. Accepted: 29 May 2017 Received: 22 Jun 2016. Accepted: 29 May 2017 p y Copyright © 2017 INIA. This is an open access article distributed under the terms of the Creative Commons Attribution (CC-by) Spain 3.0 License. Copyright © 2017 INIA. This is an open access article distributed under the terms of the Creative Commons Attribution (CC-by) Spain 3.0 License. Spanish Journal of Agricultural Research 15(2), e10R01, 11 pages (2017) eISSN: 2171-9292 Spanish Journal of Agricultural Research 15(2), e10R01, 11 pages (2017) eISSN: 2171-9292 Spanish Journal of Agricultural Research 15(2), e10R01, 11 pages (2017) eISSN: 2171-9292 SS https://doi.org/10.5424/sjar/2017152-10143 Jaime Carrasco, María-Jesús Navarro and Francisco J. Gea 2 & Savoie, 2010), Korea (Back et al., 2010), India (Bhatt & Singh, 2002), Ireland (McKay et al., 1999), Japan (Sawada et al., 2005), New Zealand (De Hoog, 1978), Poland (Ślusarski et al., 2012), Serbia (Potočnik et al., 2008), South Africa (Eicker, 1984), Spain (Gea et al., 2012), Taiwan (Kirschner et al., 2007), Turkey (Bora & Özaktan, 2000); UK (Adie et al., 2006) and the USA (Beyer & Kremser, 2004). & Savoie, 2010), Korea (Back et al., 2010), India (Bhatt & Singh, 2002), Ireland (McKay et al., 1999), Japan (Sawada et al., 2005), New Zealand (De Hoog, 1978), Poland (Ślusarski et al., 2012), Serbia (Potočnik et al., 2008), South Africa (Eicker, 1984), Spain (Gea et al., 2012), Taiwan (Kirschner et al., 2007), Turkey (Bora & Özaktan, 2000); UK (Adie et al., 2006) and the USA (Beyer & Kremser, 2004). Various species of filamentous fungi inhabiting soil, decaying wood and wild-mushrooms may cause cobweb: Cladobotryum dendroides (Bull.: Fr.) W. Gams & Hoozem (conidial state of Hypomyces rosellus) is the species historically associated with cobweb in A. bisporus crops, in recent years Cladobotryum mycophilum (Oudem.) W. Gams & Hoozem (conidial state of Hypomyces odoratus) has become the most commonly reported causal agent (Back et al., 2012; Kim et al., 2014; Chakwiya et al., 2015; Carrasco et al., 2016a; Zuo et al., 2016). However, several other species have been reported as causing this pathology in commercial mushroom crops. Historically treated as a minor disease, cobweb is currently considered one of the four most serious diseases of mushroom crops caused by parasitic fungi, together with dry bubble (Lecanicillium fungicola), green mould (Trichoderma aggressivum) and wet bubble (Mycogone perniciosa) (Fletcher & Gaze, 2008). In the mid-1990s, cobweb was reported to be the most serious disease affecting mushroom cultivation in UK and Ireland, where it reached epidemic proportions that involved production losses of up to 40% (Adie et al., 2006). Control methods must be implemented through hygiene measures and by preventing the dissemination of spores, which are dry and easy to dislodge. When not properly treated, conidia will spread within crops, magnifying infection and increasing losses (Adie et al., 2006; Pyck & Grogan, 2015). In this respect, public policies aimed at reducing the use of chemical pesticides through the use of sustainable agriculture practices (e.g. the French “Ecophyto 2018” plan) have led to the intensification of biological control efforts in agriculture. Causal agent Several species belonging to the genus Cladobotryum Nees emend. (syn. Dactylium Nees) can cause cobweb disease in edible mushroom crops (Table 1). They correspond to the conidial or asexual stage of species from the genus Hypomyces (Fries) L.R.Tulasne (Ascomycota, Hypocreales, Hypocreaceae).i Spanish Journal of Agricultural Research Jaime Carrasco, María-Jesús Navarro and Francisco J. Gea Although there have been attempts to identify biological control agents and environmentally-friendly biomolecules that are effective against fungal diseases in mushroom (Potočnik et al., 2010; Kosanović et al., 2013; Gea et al., 2014; Geösel et al., 2014), no efficient bio-treatment to control cobweb disease has been described. In view of this, control of the pathology still relies on the use of chemical fungicides. However, since the sensitivity of mycoparasites to approved pesticides is gradually diminishing and signals of resistance have been detected (McKay et al., 1998; Gea et al., 2005; Grogan, 2006), their use demands judicious management. In short, to optimize integrated disease control, the use of chemicals must be combined with good farming practices and with measures directed towards enhancing hygiene within growing facilities. The prevalence of cobweb disease in commercial mushroom crops has been reported to vary between 6.8 and 28% in Indian A. bisporus facilities (Seth & Dar, 1989; Bhatt & Singh, 2002), 33% in Turkey (Bora & Özaktan, 2000) and, up to 32% in Spanish commercial button mushroom crops (Carrasco et al., 2016a). Its occurrence is associated with final flushes and is conditioned by the season (McKay et al., 1999; Adie, 2000; Desrumeaux, 2005). The occurrence and severity of cobweb gradually increases from the first to the third flush. Although it may be established at any time during the year, it is of particular concern in autumn and winter (Carrasco et al., 2016a). Introduction cycles. First, small, white circular patches appear on the casing soil or basidiomes. These quickly spread by means of a fine grey-white mycelium that resembles a spider web (Carrasco et al., 2016a). Eventually patches of mycelium start to sporulate, producing masses of dry spores that are easy to release when they are physically disturbed, mainly through watering or picking operations − even air currents from air-conditioning systems are sufficiently strong to mobilize the harmful spores (Adie et al., 2006). Once released, conidia are spread throughout the mushrooms facilities by air currents to form secondary colonies on the casing layer or to simultaneously spot the basidiomes (Adie, 2000). As soon as a primary cobweb outbreak is located over the casing or carpophores, it must be treated before sporulation, covering the infected area with thick damp paper to avoid the release of conidia and disease dispersion (Pyck & Grogan, 2015). Many fungal diseases can affect commercial mushroom crops (Fletcher & Gaze, 2008). Among them cobweb is considered one of the most serious diseases for white button mushroom [Agaricus bisporus (Lange) Imbach] cultures, the most widely cultivated species (Royse, 2014). Other edible cultivated mushrooms may also develop the harmful pathology (Gea et al., 2011, 2017; Back et al., 2012; Kim et al., 2012). Its occurrence in commercial crops results in reductions in yield and quality, mainly due to cap spotting, a lesser surface area that can be used for cultivation and to the need for early crop termination when the disease becomes epidemic (Adie, 2000; Adie et al., 2006). Cobweb appears more often at the end of the crop cycle (although the earlier it appears, the more devastating it can be) during the autumn and winter Jaime Carrasco, María-Jesús Navarro and Francisco J. Gea Cobweb disease: a recurrent visitor Cultivated edible mushrooms are susceptible to diseases caused by bacteria, fungi and viruses. Among biotic agents, mycoparasites are responsible for the greatest mushroom crop losses, which have a significant economic impact on industry (Fletcher & Gaze, 2008). Cobweb disease has been known as an edible mushroom crop pathology since the early days of mushroom cultivation (Carrasco, 2016). The pathology has been detected and reported in most edible mushroom- growing countries, including: Australia (Fletcher et al., 2004), Belgium (Desrumeaux, 2005), China (Zuo et al., 2016), Canada (Howard et al., 1994), France (Largeteau Cultivated edible mushrooms are susceptible to diseases caused by bacteria, fungi and viruses. Among biotic agents, mycoparasites are responsible for the greatest mushroom crop losses, which have a significant economic impact on industry (Fletcher & Gaze, 2008). ( y yp yp ) As a rule of thumb, for its correct identification, Cladobotryum spp. must be evaluated by two independent methods: (1) morphology: screening for aurofusarin and camphor odour producers, registering conidia and phialide size as well as taxonomic characters (Carrasco et al., 2016a); and (2) molecular and phylogenetic analysis. The best approach for complete molecular identification involves implementation of multigene phylogenetic analyses, including sequencing of the internal transcribed spacer (ITS) region of ribosomal DNA (rDNA), RNA polymerase subunit I Cobweb disease has been known as an edible mushroom crop pathology since the early days of mushroom cultivation (Carrasco, 2016). The pathology has been detected and reported in most edible mushroom- growing countries, including: Australia (Fletcher et al., 2004), Belgium (Desrumeaux, 2005), China (Zuo et al., 2016), Canada (Howard et al., 1994), France (Largeteau June 2017 • Volume 15 • Issue 2 • e10R01 Mushroom cobweb disease: A review 3 Table 1. Cladobotryum strains associated with cobweb disease in cultivated edible mushrooms. Isolate a Host GenBank Accession b Origin Species Reference Africa TRS9, 11-14, 19, 29, 35-37 A. bisporus KF981169-78 South Africa C. mycophilum Chakwiya et al. (2015) Asia - G. tsugae DQ376084 Taiwan C. semicirculare Kirschner et al. (2007) - - EU340835 India C. asterophorum Unpublished NA P. eryngii JF693809 South Korea C. mycophilum Kim et al. (2012) NA P. eryngii, A. bisporus AB527074 South Korea C. mycophilum Back et al. (2010, 2012) NA H. marmoreus, F. velutipes AB591044 South Korea C. varium Back et al. (2012) NA F. velutipes AB298708 Japan C. varium Unpublished NA F. velutipes AB374290 Japan C. varium Unpublished BAC01-07 A. Spanish Journal of Agricultural Research Cobweb disease: a recurrent visitor bisporus KJ808711-17 South Korea C. mycophilum Unpublished NA A. bisporus EU340384 India C. mycophilum Unpublished C-1 C. comatus KU237239 China C. protrusum Wang et al. (2015) LZ10 G. lucidum KJ942816/KP137364/ KP137365/KP137366 China C. mycophilum Zuo et al. (2016) Europe CM1-6 A. bisporus JQ004732-36 Spain C. mycophilum Gea et al. (2012); Carrasco et al. (2016a) CM7-23 A. bisporus KP698960-73 Spain C. mycophilum Carrasco et al. (2016a) PE32, 40, 53, 57, 68, 70, 71 P. eryngii KP267824-30 Spain C. mycophilum Gea et al. (2017) PE72 P. eryngii JF505112 Spain C. mycophilum Gea et al. (2011, 2017) MGB0003-05 A. bisporus KC964103-104 Spain C. mycophilum Unpublished IMI 267134 A. bisporus Y17095/Y17101/HF911958/ HF911547/HF911744/ HF911637/ HF911854 UK C. dendroides McKay et al. (1999) IMI 359310, 372795-96 A. bisporus HF911959-61/HF911548-49/ HF911638-40/HF911855-57 UK C. dendroides Grogan &Gaze (2000); Tamm & Põldmaa (2013) I.P. 15 A. bisporus HF911962/HF911550/ HF911747/HF911641/ HF911858 Hungary C. mycophilum Tamm & Põldmaa (2013) I.P. 21 A. bisporus HF911963/HF911551/ HF911748/ HF911642/HF911859 Ireland C. mycophilum Tamm & Põldmaa (2013) I.P. 17, 7 A. bisporus HF911964-65/HF911552-53/ HF911749-50/HF911643-44/ HF911860-61 Serbia C. mycophilum Tamm & Põldmaa (2013) I.P. 14-20 A. bisporus HF911986-87/HF911570-71/ HF911774-75/HF911666-67/ HF911883-84 UK C. dendroides Grogan & Gaze (2000); Tamm & Põldmaa (2013) MUCL-28202 A. bisporus Y17092 Luxembourg C. dendroides McKay et al. (1999) Table 1. Cladobotryum strains associated with cobweb disease in cultivated edible mushrooms. Spanish Journal of Agricultural Research June 2017 • Volume 15 • Issue 2 • e10R01 Spanish Journal of Agricultural Research June 2017 • Volume 15 • Issue 2 • e10R01 Jaime Carrasco, María-Jesús Navarro and Francisco J. Gea 4 4 Table 1. Continued Isolate a Host GenBank Accession b Origin Species Reference Z063015,Z15001, Z079004 A. bisporus Y17094, 97, 103 Ireland C. mycophilum McKay et al. (1999) CBS-111.92 A. bisporus Y17098 Germany C. mycophilum McKay et al. (1999) America PSU DC177 A. bisporus HF911770/HF911662/ HF911879 USA C. dendroides Tamm & Põldmaa (2013) CBS-148.46 A. bisporus Y17100/HF911943/ HF911534/ HF911728/HF868622/ HF911838 Canada C. mycophilum McKay et al. (1999); Tamm & Põldmaa (2013) PSU DC26-27 A. bisporus Y17102/HF911947-48/ HF911537-38/HF911733-34/ FN868626/HF911843 USA C. dendroides McKay et al. (1999); Tamm & Põldmaa (2013) PSU DC 294, 0,302,305,306,309,310,315 A. bisporus HF911949-57/HF911539-46/ HF911950/HF911735-43/ HF911736/ USA C. dendroides Tamm & Põldmaa (2013) Oceania Z385044 A. bisporus Y17089 Australia C. astereophorum McKay et al. (1999) Z385037 A. bisporus Y17099 Australia C. mycophilum McKay et al. (1999) CBS 472.71 A. bisporus NR_121423/FN868786/ FN868659/ HF911724/FN868723/ HF911834 New Zealand C. Cobweb disease: a recurrent visitor multiseptatum De Hoog (1978); Põldmaa (2011) Table 1. Continued aCollections listed (IMI: International Mycological Institute, Royal Botanic Garden, Kew, UK; MUCL: BCCM/MUCL - Belgian Co-ordinated Collections of Microorganisms, Belgium; CBS: CBS-KNAW - Westerdijk Fungal Biodiversity Institute, The Nether- lands) and not listed by WFCC (World Federation for Culture Collections). NA: data not available. bGenBank Accession Numbers amplified respectively by ITS, RPB1, RPB2, TEF-1 or protein component of the 60S ribosomal subunit (FG1093) sequences. (RPB1), DNA-dependent RNA polymerase subunit II (RPB2) and translation elongation factor (TEF) genes (Tamm & Põldmaa, 2013; Zuo et al., 2016). bacilliform, cylindrical and often lightly tapered, slightly curved in some cases, with a conspicuous basal hilum in the base. The apex shape and dimensions of the subulate phialides (narrowing towards the apex) vary among species (Fig. 1a-f). When plated on potato dextrose agar (PDA) these fungi develop a greyish-white mycelium with the reverse side of the plate turning yellow in few days. Usually 2-4 weeks later, the plates acquire a deep red colour (Fig. 1m,n,o). This pigment, most probably aurofusarin, is mainly secreted by the hyphae immersed in the growth media (Põldmaa, 2011). However, not every Cladobotryum species provoking cobweb generates the pigment (Potočnik et al., 2008). In vitro, the fungi produce dark, thin walled microsclerotia. Multicellular, globose structures (chlamydospores) have been also reported associated to these microsclerotia (Fig. 1g-l) (Carrasco et al., 2016a). Both are generally associated with the life cycle stage of the fungus that survives under unfavourable conditions (Rogerson & Samuels, 1993). Cladobotryum spp. present verticillated hyphae at the end of which three or four conidiogenous cells, called phialides, are located. Most of the species show a conidial holoblastic ontogeny (in which the apex of the conidiogenous cells is incorporated as part of the generated conidium) through basipetal succession (Grogan & Gaze, 2000; Tamm & Põldmaa, 2013). Conidia, unicellular in origin, usually show from 1 to 3 septa (2 to 4 cells) (Desrumeaux, 2005; Adie et al., 2006). They are hyaline, globose to subglobose, Cladobotryum dendroides (Bull.: Fr.) W. Gams & Hoozem. (syn. Dactylium dendroides) has been the species historically associated with cobweb disease (teleomorph: Hypomyces rosellus (Alb. & Schwein.:Fr.) Tul.). In vitro, it secretes the above described pigment when the strain ages (Põldmaa, 2011). Spanish Journal of Agricultural Research Cobweb disease: a recurrent visitor It is the only species in the genus characterised by a thin-walled sympodial conidiogenous rachis at the phialide tip that apparently is formed after Spanish Journal of Agricultural Research June 2017 • Volume 15 • Issue 2 • e10R01 Mushroom cobweb disease: A review 5 Figure 1. Morphology of C. mycophilum, most cited causal agent of cobweb disease. (a) Conidia clouds engulfing the infected carpophore. (b) Mycelium. (c, d) Conidia (Prot: scar of union to the phialide, hilum basal). (e) Conidia in the my cosphere of A bisporus (1: conidia). (f) Germinating conidia in the mycosphere of A. bisporus. (1: conidia; 2: germinative tubes). (g, h, i) Microsclerotia. (j, k, l) Chla- mydospores. (m, n, o) Front and back side of PDA medium plated with the mycoparasite. Evolution of the colony (2, 4 and 21 days of incubation at 22ºC in darkness). Scale: a=1 cm; b,c,k,l=20 µm; d,e,f,j=10 µm; g=500 µm; h=127 µm; i=30 µm. Figure 1. Morphology of C. mycophilum, most cited causal agent of cobweb disease. (a) Conidia clouds engulfing the infected carpophore. (b) Mycelium. (c, d) Conidia (Prot: scar of union to the phialide, hilum basal). (e) Conidia in the my cosphere of A bisporus (1: conidia). (f) Germinating conidia in the mycosphere of A. bisporus. (1: conidia; 2: germinative tubes). (g, h, i) Microsclerotia. (j, k, l) Chla- mydospores. (m, n, o) Front and back side of PDA medium plated with the mycoparasite. Evolution of the colony (2, 4 and 21 days of incubation at 22ºC in darkness). Scale: a=1 cm; b,c,k,l=20 µm; d,e,f,j=10 µm; g=500 µm; h=127 µm; i=30 µm. Cladobotyum mycophilum Type II was described in the mid-1990s as a highly resistant variety to bencimidazole fungicides (McKay et al., 1999; Grogan & Gaze, 2000). The colonies, which showed higher and earlier sporulation, lost the characteristic camphor odour, and conidia usually presented 1, 2 or 3 septa (Adie, 2000; Grogan & Gaze, 2000). successive conidia are released (Tamm & Põldmaa, 2013). The conidia mostly present 2-3 septa. Cladobotryum mycophilum (Oudem.) W. Gams & Hoozem. (syn. Dactylium mycophilum Oudem.), the anamorph of Hypomyces odoratus G.R.W. Arnold, is currently the most cited causal agent of cobweb. Spanish Journal of Agricultural Research In commercial edible mushroom crops C. protrusum was isolated from infected tissue of Coprinus comatus in commercial mushroom farms of China. Colonies of the pathogen on PDA plates turned ocherous or pinkish. Conidiophores were cylindrical, long clavate, or fusiform and conidia presented 1 to 2 septa (Wang et al., 2015). Cladobotryum spp. has been reported as infecting different species of cultivated edible mushroom, among them: Agaricus bisporus (Gea et al., 2012; Carrasco et al., 2016a), Agaricus bitorquis (Potočnik et al., 2008), Agaricus blazei (Geösel, 2011), Auricularia mesenterica (Eicker et al., 1990), Coprinus comatus (Wang et al., 2015), Flammulina velutipes (Kim et al., 1999), Ganoderma tsugae (Kirschner et al., 2007), Ganoderma lucidum (Zuo et al., 2016), Hypsizygus marmoreus (Back et al., 2012), Pleurotus eryngii (Gea et al., 2011, 2017), Pleurotus ostreatus and Pleurotus pulmonarius (Mignucci et al., 2000). C. semicirculare, an aurofusarin producer characterized by the presence of curved conidia with 0-3 septa, was isolated from commercial Ganoderma tsugae crops in Taiwan (Kirschner et al., 2007). Cobweb disease: a recurrent visitor It has recently been described as parasitizing different edible crops in Africa, Asia and Europe, including Agaricus bisporus, Pleurotus eryngii and Ganoderma lucidum (Back et al., 2010; Chakwiya et al., 2015; Carrasco et al., 2016a; Zuo et al., 2016; Gea et al., 2017). C. mycophilum is also a red pigment producer in vitro (Carrasco et al., 2016a). Colonies generate a camphor odour, whose intensity varies with the age of the strain and the growth medium, and which is perceptible when lifting the lid of the Petri plate (Põldmaa, 2011). Phialide tips are simple and regular, without any evident rachis, and conidia are mostly uniseptate (Carrasco et al., 2016a). C. mycophilum spores start to germinate 2 h after isolation on PDA at room temperature. Spores first undergo constriction of the septum (“septa constricta”), and then grow, acquiring a globose shape from which several germinative tubes are generated. The estimated growth rate of germinative tubes is 11.6 µm/h (Carrasco, 2016). C. varium Nees ex Steud. does not secrete aurofusarin. The species has been described as a causal agent of cobweb disease in Korean edible mushroom crops (F. velutipes, H. marmoreus, P. eryngii) (Back et al., 2012; Kim et al., 2012). C. varium was also reported as pathogenic to A. bisporus in cross pathogenicity tests (Back et al., 2012). Two GenBank sequences of the aurofusarin- producing C. asterophorum have been related to cobweb disease in mushroom crops (McKay et al., 1999; Tamm & Põldmaa, 2013). C. asterophorum has also recently been identified on beech mushrooms in Korea. This species was pathogenic against H. marmoreus, F. velutipes and P. eryngii (Back et al., 2012). C. multiseptatum (de Hoog), also a producer of aurofusarin, was isolated by A. W. Smith as causal agent of cobweb disease in Agaricus brunnescens Peck (an A. Spanish Journal of Agricultural Research June 2017 • Volume 15 • Issue 2 • e10R01 Jaime Carrasco, María-Jesús Navarro and Francisco J. Gea 6 6 bisporus variant), in New Zealand. The strain presented 2-4 cell conidia (1-3 septa) (De Hoog, 1978; Tamm & Põldmaa, 2013). Samuels, 1993, 1994; Kirschner et al., 2007; Põldmaa, 2011). Disease ecology In the wild Certain species of Cladobotryum parasite members of the basidiomycetes group, mostly belonging to the orders Aphyllophorales and Agaricales (Gams & Hoozeman, 1970). In addition, some species are found on different substrates, such as bark, decaying wood or leaf litter. Symptoms Cobweb disease induces both qualitative and quantitative losses in commercial mushroom crops, where it compromises mushroom quality and provokes a significant drop in profitability within the crop cycle (Carrasco et al., 2016a). Pathogenic spores are numerous, dry and easily dislodged by physical contact. Once released, these conidia quickly spread through the air-conditioning systems. Primary outbreaks are characterised by the occurrence of a white, fluffy mycelium over the mushroom beds and infected carpophores. Infected mushrooms usually present discoloration and eventually rot. If not properly controlled, localized outbreaks tend to grow radially outwards over the casing layer, colonizing a larger crop surface and therefore reducing it (Fig. 2). The light, invasive mycelium quickly evolves towards a dense white mass with a mealy texture due to massive sporulation (Adie, 2000). When they age, colonies usually acquire pink-red hues (Tamm & Poldmaa, 2013). Previous reports suggest that the major causes of conidia release are splashing and runoff while watering, and the application of salt through incorrect procedures (Adie & Grogan, 2000). The main measures to prevent conidia dispersal are: avoiding irrigation over or near cobweb patches; covering the patches with thick, damp paper instead of salting; switching off the fans while removing the spent mushroom compost; hermetically closing the doors of growing rooms and, finally using 5 µm ø pore size air filters (HEPA) (Fletcher & Gaze, carra; Pyck & Grogan, 2015; Carrasco et al., 2016a). One of the main causes of harvest depreciation is mushroom discoloration through the action of fungal and bacterial diseases. The secretion of hydrolytic enzymes (combined with mechanical pressure and the formation of penetration structures) and toxic compounds have been related to the interaction between mycoparasites and hosts (Calonje et al., 2000; Abubaker et al., 2013). Certain secondary metabolites produced by fungal parasites are known for being antagonistic towards A. bisporus (Krupke et al., 2003). Mycoparasitic Cladobotryum species produce a wide variety of secondary metabolites with marked activity, including antibacterial, antifungal and repressive effects on cancer cells (Sakemi et al., 2002; Feng et al., 2003; Mitova et al., 2006). Dispersal The key factor that conditions the incidence and severity of the disease in commercial mushroom crops is the spread of harmful conidia within culture facilities (Adie et al., 2006; Fletcher & Gaze, 2008). Mushroom cobweb disease: A review therefore the sale of the product must be conditioned (Adie, 2000; Fletcher & Gaze, 2008). Conversely, compost is not usually considered to be a primary source of infection. During phase II, compost undergoes a high-temperature pasteurisation process to eliminate pathogens (Fletcher & Gaze, 2008). Likewise, spawn or host mycelium is not a source of infection due to high-standard hygiene conditions while the spawn grain is prepared (Desrumeaux, 2005; Fletcher & Gaze, 2008). Brown spots are generated when a single spore lands on the mushroom surface and germinates. These spots usually provoke depression of the cap tissue. From the localized spots a parasitic mycelium emerges that eventually engulfs the whole basidiome. The grey- yellowish spotting is due to the interaction between parasitic mycelium and the host basidiomes; spots progressively discolour the mushroom tissue, which succumbs to wet rot (Adie, 2000) (Fig. 2c). Finally, alternative sources of primary infection are contaminated packages and containers, external visits, vehicles, etc. Contaminated water could be also a source of infection since some mycopathogens, such as Lecanicillium and Mycogone spores, are known to survive in water for many months. Spanish Journal of Agricultural Research Primary source of infection According to literature, casing contamination is frequently considered a source of primary infection (Fletcher & Gaze, 2008). Casing materials artificially inoculated with the pathogen reproduced cobweb disease in A. bisporus and P. eryngii (Carrasco et al., 2016a; Gea et al., 2017). The presence of the host in the casing layer seems necessary to desensitize the dormant spores and to stimulate their germination and the development of Cladobotryum mycelium. In pilot trials, we noted that raw casing material remains healthy after inoculation with a suspension of C. mycophilum conidia, while the same casing colonized by A. bisporus expressed the disease in every replicate (our unpublished data). Likewise, L. fungicola is inhibited by the microflora of the casing layer due to a phenomenon called fungistasis; however, the presence of the host removes the fungistasis to facilitate disease development (Berendsen et al., 2010). Some of the Cladobotryum spp. known to cause cobweb disease in edible mushroom crops have also been reported as parasitizing polyporus and agaricales in the wild (Gams & Hoozemans, 1970; Rogerson & Figure 2. a) Fluffy mycelium growing over the casing layer and A. bisporus basidiomes. b) Mass of conidia engulfing car- pophores. c) Regular grey-yellow spots or decoloration. d) Brown spots with an ill-defined edge. e, f) Mycelium and mass of conidia colonizing P. eryngii basidiomes and casing layer. Tamm & Poldmaa (2013) concluded that the endemic species determines the causal agent of cobweb in commercial crops. This seems to indicate that a primary source of infection may be wild specimens infected near the farm. Under unfavorable conditions, particularly when the relative humidity (RH) is low, most C. dendroides spores do not survive for long periods. However, the fungus produces microsclerotia, resistant structures that can germinate even when they are stored at 0% RH (Lane et al., 1991). High humidity conditions outside the cropping rooms will facilitate survival of the pathogenic conidia and their dispersal through the production area (Carrasco, 2016; Carrasco et al., 2016a). Figure 2. a) Fluffy mycelium growing over the casing layer and A. bisporus basidiomes. b) Mass of conidia engulfing car- pophores. c) Regular grey-yellow spots or decoloration. d) Brown spots with an ill-defined edge. e, f) Mycelium and mass of conidia colonizing P. eryngii basidiomes and casing layer. Primary source of infection Spanish Journal of Agricultural Research June 2017 • Volume 15 • Issue 2 • e10R01 7 Mushroom cobweb disease: A review 7 Mushroom cobweb disease: A review Alternative control methods at the end of the crop cycle through “cooking-out” (65-70 ºC for 9-12 hours) is the best way to ensure correct disinfection (Fletcher & Gaze, 2008). When not possible, it is advisable to clean the empty facility with water and suitable disinfectants. Due to consumer demand and environmental concerns, there is a strong pressure to reduce the use of chemical pesticides (French plan EcoPhyto 2018), which has led to the intensification of biological control in agriculture. It is also possible to prevent disease outbreaks by controlling the humidity and the temperature within mushroom facilities, since few spores of C. dendroides can germinate with a RH lower than 85% , while at low temperatures (17 ºC) development and spread of the disease are unlikely (Desrumeaux, 2005). The antagonistic effect of Pseudomonas for the biocontrol of Cladobotryum dendroides was evaluated in Turkey, where application of P. fluorescens and P. putida (Bora & Özaktan, 2000) increased yields. However, Bacillus subtilis QST 713 (Serenade®) failed to control cobweb disease (C. dendroides) in artificially inoculated experiments (Ślusarski et al., 2012). Chemical control Compost tea from spent mushroom compost and essential oils from aromatic plants have been tested as alternative, environmentally-friendly biomolecules with different degrees of success to cope with fungal diseases (Potocnic et al., 2010; Kosanović et al., 2013; Gea et al., 2014; Geösel et al., 2014). Timorex 66 EC (66% “tea tree oil) showed higher activity than Sonata® (Bacillus pumilus) against C. dendroides, although the efficacy of Timorex was far lower than that of prochloraz-Mn (Potočnik et al., 2010). The application of aerated compost tea from spent mushroom compost was efficient to control dry bubble (Gea et al., 2014), although the results were disappointing when used for cobweb control (unpublished data). Control of cobweb disease is still highly dependent on routine application of fungicides from several chemical groups: prochloraz-Mn (DMI-fungicide, FRAC code: 3) is the fungicide currently recommended by the European Union to treat cobweb (Carrasco, 2016; FRAC, 2016). Chlorothalonil (chloronitrile, FRAC code: M5) is also approved for use in France, Poland and Spain. Two DMI fungicides, imazalil (to control green mould disease) and prochloraz-Mn (for cobweb), are licensed for use in Australian mushroom crops. In South Africa, prochloraz-Mn and thiabendazole are the fungicides approved for use in mushroom (Chakwiya et al., 2015). Thiabendazole (MBC-fungicide, FRAC code: 1) can be used in USA mushroom crops, as well as chlorothalonil formulates. Recently, metrafenone (benzophenone, FRAC code: U8) has been authorized for use in France to fight cobweb disease (FRAC, 2016). Recently, too, metrafenone obtained a temporary approval for use on mushroom crops in Spain. When compared with prochloraz-Mn and chlorothalonil, metrafenone showed higher selectivity towards C. mycophilum in vitro and was the most effective treatment to control cobweb in crop, which suggests that it could be an efficient alternative to prochloraz-Mn (Carrasco et al., 2016b, 2017). Finally, Savić et al. (2012) tested the antifungal activity of organic selenium against C. dendroides. The addition of 70-100 µg/g selenium to the substrate inhibited growth of the mycopathogen and resulted in the enrichment of basidiomes with this trace element. Hygiene Prevention is crucial to precluding the emergence of cobweb and to limiting its impact once installed. Control methods should prevent dispersion of conidia (as previously described), which is the main way of infection (Adie, 2000; Adie & Grogan, 2000; Fletcher & Gaze, 2008). The end of the crop cycle is a crucial time for removal of any residual disease. Wet conidia of Cladobotryum spp. are killed by treatment at 45 ºC for 30 min, but they resist higher temperatures when dry, even up to 100 ºC (Desrumeaux, 2005). Similarly, the pathogenic mycelium, which is susceptible to a 15 min, 40 ºC treatment when wet, requires temperatures of 70 ºC for 15 min when dry (Fletcher & Gaze, 2008). Consequently, in situ thermal disinfection In particular, Cladobotryum spp. causes two types of cap spotting on infected mushrooms, dark-brownish spotting with an ill-defined edge and grey-yellowish spotting (Grogan & Gaze, 2000; Grogan, 2006) (Fig. 2c,d). The spotting can even appear post-harvest, and June 2017 • Volume 15 • Issue 2 • e10R01 Jaime Carrasco, María-Jesús Navarro and Francisco J. Gea 8 Spanish Journal of Agricultural Research j.1364-3703.2010.00627.x Beyer DM, Kremser JJ, 2004. Evaluation of fungicide tolerance and control for three fungal diseases of mushrooms. Mushroom science XVI: Science and cultivation of edible and medicinal fungi, pp. 421-429. Ed by Romaine CP, Keil CB, Rinker DL and Royse DJ. The Pennsylvania State University, USA. Bhatt N, Singh RP, 2002. Cobweb disease of Agaricus bisporus: incidence, losses and effective management. Indian J Mycol Plant Pathol 22: 178-181. Bora T, Ozaktan H, 2000. Biological control of some important mushroom diseases in Turkey by fluorescent Pseudomonads. Proc 15th Int Cong on the Science and Cultivation of Edible Fungi, pp: 689-693. Maastricht, Netherlands, 15-19 May. Acknowledgements Calonje M, Mendoza CG, Cabo AP, Bernardo D, Novaes- Ledieu M, 2000. Interaction between the mycoparasite Verticillium fungicola and the vegetative mycelial phase of Agaricus bisporus. Mycol Res104: 988-992. https://doi. Some images have been taken by Antonio Martinez (CIES) and Jan Dijksterhuis (CBS-KNAW). g p y org/10.1017/S0953756299002154 Carrasco J, 2016. Estudio de la telaraña del champiñón causada por Cladobotryum mycophilum en cultivos españoles. / Study of mushroom cobweb caused by Cladobotryum mycophilum in Spanish crops. 168 pp. PhD thesis, University of Castilla-La Mancha. http://hdl.handle. net/10578/9752 y net/10578/9752 Carrasco J, Navarro MJ, Santos M, Diánez F, Gea FJ, 2016a. Incidence, identification and pathogenicity of Cladobotryum mycophilum, causal agent of cobweb disease on Agaricus bisporus mushroom crops in Spain. An Appl Biol 168: 214-224. https://doi.org/10.1111/aab.12257 Adie BAT, 2000. The biology and epidemiology of the cobweb disease pathogen (Cladobotryum spp.) infecting the cultivated mushroom (Agaricus bisporus). PhD thesis, Imperial College, University of London. Carrasco J, Navarro MJ, Santos M, Gea FJ, 2016b. Chemical control of mushroom cobweb disease caused by Cladobotryum mycophilum. Mushroom science XIX: Science and cultivation of edible and medicinal fungi, pp 448. Amsterdam (The Netherlands). Adie BAT, Grogan H, 2000. The liberation of cobweb (Cladobotryum mycophilum) conidia within a mushroom crop. Proc 15th Int Cong on the Science and Cultivation of Edible Fungi, pp: 595-600. Maastricht, Netherlands, 15-19 May. Carrasco, J, Navarro, MJ, Santos, M, Gea, FJ, 2017. Effect of five fungicides with different modes of action on cobweb disease (Cladobotryum mycophilum) and mushroom yield. An Appl Biol: 10.1111/aab.12352. Adie B, Grogan H, Archer S, Mills P, 2006. Temporal and spatial dispersal of Cladobotryum conidia in the controlled environment of a mushroom growing room. Appl Environ Microbiol 72: 7212-7217. https://doi.org/10.1128/ AEM.01369-06 Chakwiya A, Van der Linde EJ, Korsten, L, 2015. In vitro sensitivity testing of Cladobotryum mycophilum to carbendazim and prochloraz manganese. S Afr J Sci: 111, 1-7. https://doi.org/10.17159/ sajs.2015/20140408 Back CG, Kim YH, Jo WS, Chung H, Jung HY, 2010. Cobweb disease on Agaricus bisporus caused by Cladobotryum mycophilum in Korea. J Gen Plant Pathol 76: 232-235. https://doi.org/10.1007/s10327-010-0236-3 Conclusion and perspectives Certain species from the genus Cladobotryum may generate cobweb disease in a wide range of edible mushroom crops worldwide. Control of this pathology currently relies on prevention and hygiene measures in mushroom farms, together with chemical fungicide treatments. However, the range of available substances approved for mushroom crops is limited by the fungal nature of the host as well as by restrictive legislation. Understanding the mechanisms involved in the interaction between parasite and host is a powerful tool in the design of novel control strategies, including the production of resistant host varieties. However, many questions involving mycoparasites remain unanswered, including the pathway for infection followed by harmful species The sensitivity of mycoparasites to approved pesticides is gradually diminishing (Gea et al., 2005), and symptoms of cobweb resistance have been detected (McKay et al., 1998; Grogan, 2006; Carrasco, 2016). The continuous usage of a given fungicide frequently contributes to pathogen resistance and, consequently, to undermining the value of the active substances available for cobweb control (Chakwiya et al., 2015). In this context, improved hygiene in growing facilities before the disease develops, as well as a better understanding of the pathogen’s behaviour, will lengthen the half-life of available fungicides by streamlining doses to prevent the occurrence of resistant outbreaks (Schwinn & Morton, 1990). June 2017 • Volume 15 • Issue 2 • e10R01 Mushroom cobweb disease: A review 9 Mol Plant Pathol 11: 585-595. https://doi.org/10.1111/ j.1364-3703.2010.00627.x to detect and colonize the host, the molecular basis for the observed symptoms, the molecules implied in the attack on the host tissues, or the mechanisms used to overcome host defences. On the other hand, fungicide alternatives to fight cobweb disease in the form of environmentally-friendly biomolecules are being actively investigated, accompanied by a search for efficient biocontrol agents to cope with Cladobotryum infection. Successful biological control of the fungal diseases would satisfy the mushroom industry’s continuous efforts to minimize the use of chemicals. However, to date, no biocontrol agent has been found to be as effective as approved fungicides. References Abubaker KS, Sjaarda C, Castle AJ, 2013. Regulation of three genes encoding cell-wall-degrading enzymes of Trichoderma aggressivum during interaction with Agaricus bisporus. Can J Microbiol 59: 417-424. https:// doi.org/10.1139/cjm-2013-0173 http://www.frac.info/publications/downloads Gams W, Hoozemans ACM, 1970. Cladobotryum- konidienformen von Hypomyces-arten. Persoonia 6: 95- 110. Kim MK, Lee YH, Cho KM, 2014. Fungicide sensitivity and characterization of cobweb disease on a Pleurotus eryngii mushroom crop caused by Cladobotryum mycophilum. Plant Pathol J 30: 82-89. https://doi.org/10.5423/PPJ. OA 09 2013 0098 Gea FJ, Navarro MJ, Tello JC, 2005. Reduced sensitivity of the mushroom pathogen Verticillium fungicola to prochloraz-manganese in vitro. Mycol Res 109: 741-745. p g y https://doi.org/10.1017/S095375620500242X Gea FJ, Navarro MJ, Suz LM, 2011. First Report of Cladobotryum mycophilum causing cobweb on cultivated king oyster mushroom in Spain. Plant Dis 95: 1030. https://doi.org/10.1094/PDIS-03-11-0255 Kirschner R, Arnold GR, Cheejen C, 2007. Cladobotryum semicirculare sp. nov. (Hyphomycetes) from commercially grown Ganoderma tsugae in Taiwan and other basidiomycota in Cuba. Sydowia 59: 114-124. Kosanović D, Potočnik I, Duduk B, Vukojević J, Stajić M, Rekanović E, Milijašević‐Marčić S, 2013. Trichoderma species on Agaricus bisporus farms in Serbia and their biocontrol. Ann Appl Biol 163: 218-230. https://doi. org/10.1111/aab.12048 Gea FJ, Navarro MJ, Carrasco J, González AJ, Suz LM 2012. First report of cobweb on white button mushroom (Agaricus bisporus) in Spain caused by Cladobotryum mycophilum. Plant Dis 96: 1067. https://doi.org/10.1094/ PDIS 02 12 0120 PDN sajs.2015/20140408 De Hoog GS, 1978. Notes on some fungicolus hyphomycetes and their relatives. Persoonia 10: 33-81. Back CG, Lee CY, Seo GS, Jung HY, 2012. Characterization of species of Cladobotryum which cause cobweb disease in edible mushrooms grown in Korea. Mycobiology 40: 189-194. https://doi.org/10.5941/MYCO.2012.40.3.189 Desrumeaux B, 2005. Cobweb disease: an overview. Pest and diseases. Mushroom business 15: 16-17. Eicker A, 1984. A report on the use of thiabendazole for the control of fungal pathogens of cultivated mushroom. S. Afr. J. Bot 3: 179-183. https://doi.org/10.1016/S0022- Berendsen RL, Baars JJ, Kalkhove SI, Lugones LG, Wösten HA, Bakker PA, 2010. Lecanicillium fungicola: causal agent of dry bubble disease in white-button mushroom. Afr. J. Bot 3: 17 4618(16)30049-3 June 2017 • Volume 15 • Issue 2 • e10R01 Spanish Journal of Agricultural Research Jaime Carrasco, María-Jesús Navarro and Francisco J. Gea 10 benzimidazole resistance profiles. Pest Manag Sci 62: 153-161. https://doi.org/10.1002/ps.1133 Eicker, A, Peng JT, Chen ZC, 1990. A Pseudohansfordia- disease of sawdust-cultivated Auricularia mesenterica in Taiwan. Bot Bull Acad Sinica 31: 205-210. Grogan HM, Gaze RH, 2000. Fungicide resistance among Cladobotryum spp. – causal agents of cobweb disease of the edible mushroom Agaricus bisporus. Mycol Res 104: 357-364. https://doi.org/10.1017/S0953756299001197 Feng Y, Blunt JW, Cole AL, Cannon JF, Robinson WT, Munro MH, 2003. Two novel cytotoxic cyclodepsipeptides from a mycoparasitic Cladobotryum sp. J Org Chem 68: 2002- 2005. https://doi.org/10.1021/jo0263059 g p y 357-364. https://doi.org/10.1017/S0953756299001197 org/10.1111/aab.12048 Krupke OA, Castle AJ, Rinker DL, 2003. The North American mushroom competitor, Trichoderma aggressivum f. aggressivum, produces antifungal compounds in mushroom compost that inhibits mycelial growth of the commercial mushroom Agaricus bisporus. Mycol. Res. 107: 1467–1475. https://doi.org/10.1017/ S0953756203008621 Gea FJ, Carrasco J, Diánez F, Santos M, Navarro, MJ, 2014. Control of dry bubble disease (Lecanicillium fungicola) in button mushroom (Agaricus bisporus) by spent mushroom substrate tea. Eur J Plant Pathol 138: 711-720. https://doi.org/10.1007/s10658-013-0344-y Gea FJ, Carrasco J, Suz LM. Navarro MJ, 2017. Characterization and pathogenicity of Cladobotryum mycophilum in Spanish Pleurotus eryngii mushroom crops and their sensitivity to fungicides. Eur J Plant Pathol 147:129-139. https://doi.org/10.1007/s10658-016- 0986 7 Lane CR, Cooke RC, Burden LJ, 1991. Ecophysiology of Dactylium dendroides – the causal agent of cobweb mould. Proc 14th Int Cong on the Science and Cultivation of Edible Fungi, pp. 365-372. Ed. T.J. Elliott. Rotterdam, The Netherlands. 2005. https://doi.org/10.1021/jo0263059 Howard RJ, Garland JA, Seaman WL, 1994. Chapter 26. Mushrooms. Diseases and pests of vegetable crops in Canada: an illustrated compendium, pp. 363- 379. Entomological Society of Canada & Canadian Phytopathological Society. Ottawa, Ontario. Fletcher JT, Allan J, Seymour GK, 2004, Managing cobweb disease in Australia. Proc 16th Int Cong on the Science and Cultivation of Edible Fungi, pp: 711-715. Miami, FL. U.S.A. Kim HK, Seok SJ, Kim JP, Moon BJ, Terashita T, 1999. Occurrence of disease caused by Cladobotryum varium on Flammulina velutipes in Korea. Korean J Mycol. 27: 415-419. Fletcher JT, Gaze RH, 2008. Mushroom pest and disease control: a color handbook. Ed. Manson Publishing Ltd. Academic Press, San Diego. 192 pp. FRAC (2016) FRAC Code List ©*2016: Fungicides sorted by mode of action (including FRAC Code numbering). http://www.frac.info/publications/downloads Kim MK, Lee, YH, Cho KM, Lee JY, 2012. First report of cobweb disease caused by Cladobotryum mycophilum on the edible mushroom Pleurotus eryngii in Korea. Plant Dis 96: 1374. https://doi.org/10.1094/PDIS-01-12-0015-PDN pp doi.org/10.2298/ABS1204455S Sawada D, Ohmasa M, Fukuda M, Masuno K, Koide H, Tsunoda S, Nakamura K, 2005. Disinfection of some pathogens of mushroom cultivation by photocatalytic treatment. Mycoscience 46: 54-60. https://doi. org/10.1007/S10267-004-0211-Y org/10.2298/PIF0803175P Seth PK, Dar GM, 1989. Studies on Cladobotryum dendroides (Bull: Merat) W. Gams et Hoozem, causing cobweb disease of Agaricus bisporus and its control. Mushroom Sci 12: 711-723. Potočnik I, Vukojević J, Stajić M, Rekanović, E, Stepanović M, Milijašević S, Todorović B, 2010. Toxicity of biofungicide Timorex 66 EC to Cladobotryum dendroides and Agaricus bisporus. Crop Prot 29: 290-294. https://doi. Ślusarski C, Uliński Z, Szumigaj-Tarnowska J, Miszczak A, 2012. Preliminary appraisal of the new preparations for protection of the white button mushroom against fungal diseases. Prog Plant Prot 52: 4. org/10.1016/j.cropro.2009.07.016 Pyck N, Grogan H, 2015. Fungal diseases of mushrooms and their control, 6 pp. Factsheet 04/15. Mush TV Publications. Tamm H, Põldmaa K, 2013. Diversity, host associations and phylogeography of temperate aurofusarin- producing Hypomyces/Cladobotryum including causal agents of cobweb disease of cultivated mushrooms. Fungal Biol 117: 348-367. https://doi.org/10.1016/j. funbio.2013.03.005 Rogerson CT, Samuels GJ, 1993. Polyporicolous species of Hypomyces. Mycologia. 85: 271-272. https://doi. org/10.2307/3760461 Rogerson CT, Samuels GJ, 1994. Agaricicolous species of Hypomyces. Mycologia. 86: 839-866. https://doi. org/10.2307/3760597 org/10.1021/jo051883l Põldmaa K, 2011. Tropical species of Cladobotryum and Hypomyces producing red pigments. Stud Mycol 68: 1–34. https://doi.org/10.3114/sim.2011.68.01 Schwinn FJ, Morton HV., 1990 Antiresistance strategies: design and implementation in practice. Managing Resistance to Agrochemicals, pp. 170-183. Ed. Green M.B., LeBaron H.M. and Moberg W.K. American Chemical Society. Washington DC. https://doi. org/10.1021/bk-1990-0421.ch011 Potočnik I, Rekanović E, Milijašević S, Todorović B, 2008. Morphological and pathogenic characteristics of the fungus Cladobotryum dendroides, the causal agent of cobweb disease of the cultivated mushroom Agaricus bisporus in Serbia. Pestic Fitomed 23: 175-181. https://doi. 0986-7 Geösel A, 2011. The cultivation opportunities and complex comparison survey of Agaricus blazei (Murrill), 21pp. Doctoral dissertation, Budapesti Corvinus Egyetem. Largeteau ML, Savoie JM, 2010. Microbially induced diseases of Agaricus bisporus: biochemical mechanisms and impact on commercial mushroom production. Appl Microbiol Biotechnol 86: 63-73. https://doi.org/10.1007/ s00253-010-2445-2 Geösel A, Szabó A, Akan O, Szarvas J, 2014. Effect of essential oils on mycopathogens of Agaricus bisporus. Proc 8th Conf of Mushroom Biology and Mushroom Products, pp. 530-535. Mushroom society of India (Solan) (Eds.). New Delhi, India. Mckay GJ, Egan D, Morris E, Brown AE, 1998. Identification of benzimidazole resistance in Cladobotryum dendroides using a PCR-based method. Mycol Res 102: 671–676. https://doi.org/10.1017/S095375629700542X Grogan HM, 2006. Fungicide control of mushroom cobweb disease caused by Cladobotryum strains with different Grogan HM, 2006. Fungicide control of mushroom cobweb disease caused by Cladobotryum strains with different June 2017 • Volume 15 • Issue 2 • e10R01 Spanish Journal of Agricultural Research Mushroom cobweb disease: A review 11 from the fungus Cladobotryum varium: fermentation, isolation, structural elucidation, biotransformation and antibacterial activities. J Antibiot 55: 6-18. https://doi. Mckay GJ, Egan D, Morris E, Scott C, Brown AE, 1999. Genetic and morphological characterization of Cladobotryum species causing cobweb disease of mushrooms. Appl Environ Microbiol 65: 606-610. p org/10.7164/antibiotics.55.6 org/10.7164/antibiotics.55.6 Savić M, Aneđelković I, Duvnjak D, Matijasević D, Avramović A, Nikšić M, 2012. The fungistatic activity of organic selenium and its application to the production of cultivated mushrooms Agaricus bisporus and Pleurotus spp. Arch Biol Sci 64: 1455-1463. https:// doi.org/10.2298/ABS1204455S Mignucci JS, Hernández-Bacó C, Rivera-Vargas L, Betancourt C, Alameda M, 2000. Diseases and pests research on oyster mushrooms (Pleurotus spp.) in Puerto Rico. IJMS 3: 21-26. Savić M, Aneđelković I, Duvnjak D, Matijasević D, Avramović A, Nikšić M, 2012. The fungistatic activity of organic selenium and its application to the production of cultivated mushrooms Agaricus bisporus d Pl A h Bi l S i 64 1455 1463 h // Mitova MI, Lang G, Blunt JW, Cummings NJ, Cole AL, Robinson WT, Munro, MH, 2006. Cladobotric acids AF: new cytotoxic polyketides from a New Zealand Cladobotryum sp. J Org Chem 71: 492-497. https://doi. org/10 1021/jo051883l g funbio.2013.03.005 Wang GZ, Guo MP, Bian YB, 2015. First report of Cladobotryum protrusum causing cobweb disease on the edible mushroom Coprinus comatus. Plant Dis 99: 287- 287 https://doi org/10 1094/PDIS 07 14 0757 PDN Royse DJ, 2014. A global perspective on the high five: Agaricus, Pleurotus, Lentinula, Auricularia & Flammulina. Proc 8th Conf on Mushroom Biology and Mushroom Products, pp. 1-6. Mushroom society of India (Solan) (Eds.). New Delhi, India. p 287. https://doi.org/10.1094/PDIS-07-14-0757-PDN Zuo B, Lu BH, Liu XL, Wang Y, Ma GL, Gao J, 2016. First report of Cladobotryum mycophilum causing cobweb on Ganoderma lucidum cultivated in Jilin province, China. Plant Dis 100: 1239. https://doi.org/10.1094/PDIS-12- Sakemi S, Bordner J, Decosta DL, Dekker KA, Hirai H, Inagaki T, Kim Y, Sugiura A, Sutcliffe, JA, Tachikawa K, Truesdell S, Wong JW, Yoshikawa N, Kojima Y, 2002. CJ-15,696 and its analogs, new furopyridine antibiotics Spanish Journal of Agricultural Research June 2017 • Volume 15 • Issue 2 • e10R01
https://openalex.org/W2564455712	http://cds.cern.ch/record/2270931/files/1-s2.0-S0168900216313298-main.pdf	English	null	Measurements of 55 Fe activity in activated steel samples with GEMPix	Nuclear instruments and methods in physics research. Section A, Accelerators, spectrometers, detectors and associated equipment/Nuclear instruments & methods in physics research. Section A, Accelerators, spectrometers, detectors and associated equipment	2,017	cc-by	11,108	1. Introduction DTM or ITM and the activity of some ETM key nuclides (KN). The activities of DTM nuclides in waste packages are estimated by measuring the gamma emitting KNs through gamma-spectrometry measurements from outside the package and applying the SFs to calculate the DTM activities. The SFs can be obtained via experimental measurements (sampling, experimental scaling factors) and through analytical or Monte Carlo calculations (theoretical correlation coeﬃ- cients). The operation of particle accelerators and experimental facilities generates activated equipment and material which, when no longer in use, become radioactive waste. This mostly weakly radioactive waste is usually stored ad interim at the facility premises (to allow for radio- active decay, sorting, conditioning and characterization), before it is sent to a national repository. The waste must be radiologically characterized as the repositories usually require the full radionuclide inventory before it is accepted. Radioactive waste from particle accelerators is mostly made of metallic components coming from accelerators, experimental apparatus, ancillary equipment and sur- rounding infrastructures. Both nuclear power plants and research laboratories, among which CERN, characterize low-level and very- low-level activity waste by exploiting the relationship between easy-to- measure (ETM) nuclides (gamma emitting nuclides whose radioactivity can be readily measured directly by non-destructive assay means), and diﬃcult-to-measure (DTM) nuclides (radionuclides whose radioactivity is diﬃcult to measure directly from outside of the waste package by non-destructive assay means) [1,2]. Some of them are impossible-to- measure (ITM) even in a laboratory. The scaling factor (SF) method is an approach used to evaluate these DTM and ITM nuclides [3]. A scaling factor is the mathematical relationship between the activity of a At CERN, the DTM and ITM radionuclides are discriminated depending on their contribution to the Indice Radiologique d′Acceptation en Stockage (IRAS), which is a hazard factor deﬁned by the French National Agency for Radioactive Waste Management (ANDRA) to establish criteria for waste acceptability in ﬁnal reposi- tories [4]. The radionuclides that contribute for more than 1% to the IRAS are directly measured (direct ETM measurement and Scaling Factors for DTMs) otherwise they are estimated using the Correlation Method (ITMs) [5]. The 55Fe radionuclide is a DTM radionuclide present in iron and steel radioactive waste, and therefore it must be measured according to the speciﬁc characterization procedure [5]. A R T I C L E I N F O In this paper we present a novel method, based on the recently developed GEMPix detector, to measure the 55Fe content in samples of metallic material activated during operation of CERN accelerators and experimental facilities. The GEMPix, a gas detector with highly pixelated read-out, has been obtained by coupling a triple Gas Electron Multiplier (GEM) to a quad Timepix ASIC. Sample preparation, measurements performed on 45 samples and data analysis are described. The calibration factor (counts per second per unit speciﬁc activity) has been obtained via measurements of the 55Fe activity determined by radiochemical analysis of the same samples. Detection limit and sensitivity to the current Swiss exemption limit are calculated. Comparison with radiochemical analysis shows inconsistency for the sensitivity for only two samples, most likely due to underestimated uncertainties of the GEMPix analysis. An operative test phase of this technique is already planned at CERN. Keywords: Radioactive waste X-Ray detectors Micropattern gaseous detectors Radiochemical analysis Timepix GEM Keywords: Radioactive waste X-Ray detectors Micropattern gaseous detectors Radiochemical analysis Timepix GEM Measurements of 55Fe activity in activated steel samples with GEMPix A. Curionia,b, N. Dinara,c, F.P. La Torrea, J. Leidnera,d,⁎, F. Murtasa,e, S. Puddua, M. Silaria a CERN, 1211 Geneva 23, Switzerland b Politecnico di Milano, Piazza Leonardo da Vinci 32, 20133 Milano, Italy Measurements of 55Fe activity in activated steel samples with GEMPix A. Curionia,b, N. Dinara,c, F.P. La Torrea, J. Leidnera,d,⁎, F. Murtasa,e, S. Puddua, M. Silaria a CERN, 1211 Geneva 23, Switzerland b Politecnico di Milano, Piazza Leonardo da Vinci 32, 20133 Milano, Italy c Université de Paris VII, 5 rue Thomas-Mann, 75013 Paris, France d RWTH Aachen, Templergraben 55, 52056 Aachen, Germany e INFN LNF Vi E F i 40 00044 F ti It l a CERN, 1211 Geneva 23, Switzerland b Politecnico di Milano, Piazza Leonardo da Vinci 32, 20133 Milano, Italy c Université de Paris VII, 5 rue Thomas-Mann, 75013 Paris, France d RWTH Aachen, Templergraben 55, 52056 Aachen, Germany e INFN-LNF, Via E. Fermi 40, 00044 Frascati, Italy Nuclear Instruments and Methods in Physics Research A 849 (2017) 60–71 Nuclear Instruments and Methods in Physics Research A 849 (2017) 60–71 Contents lists available at ScienceDirect ⁎ Corresponding author at: CERN, 1211 Geneva 23, Switzerland. E-mail address: johannes.leidner@cern.ch (J. Leidner). http://dx.doi.org/10.1016/j.nima.2016.12.059 Received 21 October 2016; Received in revised form 20 December 2016; Accepted 29 December 2016 Available online 30 December 2016 0168-9002/ © 2017 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/BY/4.0/). http://dx.doi.org/10.1016/j.nima.2016.12.059 Received 21 October 2016; Received in revised form 20 December 2016; Accepted 29 December 2016 ⁎ Corresponding author at: CERN, 1211 Geneva 23, Switzerland. E-mail address: johannes.leidner@cern.ch (J. Leidner). Available online 30 December 2016 0168-9002/ © 2017 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/BY/4.0/). 1. Introduction The activity limit of 55Fe for the disposal of waste as very-low-level radioactive waste (“très faiblement actif” – TFA) towards the French repository is 10 kBq/g (if 55Fe is the only radionuclide present in the waste). The declaration limit, i.e. the activity limit above which 55Fe Nuclear Instruments and Methods in Physics Research A 849 (2017) 60–71 A. Curioni et al. must always be declared in France is 10 Bq/g [4]. The current exemption limit of 55Fe for clearance of materials according to the Swiss legislation is 30 Bq/g [6]. This exemption limit will be increased to 1 kBq/g with the introduction of the new legislation.1 suﬃciently gas tight with a thin layer of epoxy resin. An HVGEM unit [11] controls seven electrical ﬁelds (one per GEM foil, two charge transfer ﬁelds, an induction ﬁeld and a drift ﬁeld), shown schematically in Fig. 2. Except where otherwise noted the chamber is operated at a gain G=3×103 corresponding to a total applied voltage to the GEM foils of 1240 V (~450 V per foil) [12] and a drift ﬁeld of 0.66 kV cm−1. The quad Timepix is mounted on a bespoke quad PCB, and read out using the FITPix system [13] and accompanying Pixelman [14] software. The 55Fe radionuclide is currently assessed at CERN in the TFA waste by means of radiochemical analyses on waste samples. After collection and tracking, the samples are shipped to external companies to perform the analysis. A typical delay time is about 2 months for the work performed by the company, including sample preparation (cut- ting and acid digestion) and liquid scintillation counting. Thanks to the separation of the chemical elements, the scintillation technique reaches a detection limit for 55Fe in metals of about 0.3–0.5 Bq/g [7]. The Timepix [16] is a pixelated silicon detector developed by the Medipix Collaboration [17,18]. It is based on a read-out chip consisting of a 256×256 pixel CMOS ASIC to which diﬀerent pixelated semi- conductor sensors are normally bump-bonded. It has seen wide applications in particle tracking [19,20], as an educational tool [21,22] and in dosimetry [23,24]; it is currently commercially available from various companies. In this application, however, we use a 2×2 array of chips (for a total of 512×512 pixels) without silicon sensor as readout for a triple GEM detector. Each pixel measures 55×55 µm2. 1. Introduction The salient feature of the Timepix is that the processing electronics for each pixel, including a preampliﬁer, discriminator threshold (set at a minimum value of about 1000 electrons for noise free operation) and 13.5 bit pseudo-random counter (counts up to 11,818) ﬁt inside the footprint of the overlying semiconductor pixel. The Timepix contains a global clock which is operated at 48 MHz. Apart from radiochemical analysis, there are a number of options to detect the characteristic 5.9 keV X-rays from 55Fe.2 For example: 1. Si detectors (Si-PIN or SDD), which provide an excellent energy resolution, between 125 and 200 eV FWHM (2–3%), typically for an active area of few tens of square millimeters.3 2. Scintillation detectors, e.g. NaI(Tl) with a thin entry window for the soft X-rays [8] 3. Gaseous detectors, as proportional chambers or Micropattern Gaseous Detectors (MPGD), which give a large eﬀective area (tens of cm2) and an energy resolution of about 20% FWHM at 5.9 keV. One of three modes can be used for each pixel: Counting (Medipix), Time Of Arrival (TOA) and Time Over Threshold (TOT). The mode that has been used for this measurement is the TOT mode. In this mode whenever the pulse is above threshold the pixel counts until the pulse is low again. This allows each pixel to act as a Wilkinson type ADC measuring the discharge time of the preampliﬁer (i.e. the time spent over the threshold). Fig. 3 shows schematically how the TOA and TOT modes of operation work. The Timepix operates with a frame based readout. This means that the chip possesses a digital shutter, and the pixels only count when the shutter is open. After the shutter closes the Timepix is then read out before acquiring a new frame. In this work we have developed a measurement procedure using a novel MPGD detector called GEMPix [9]. 2. The GEMPix detector The GEMPix is a novel detector obtained by coupling two CERN technologies, a small triple Gas Electron Multiplier (GEM) detector (3×3×1.2 cm3 active volume) to a quad Timepix ASIC with 262,144 pixels of 55×55 µm2 area for readout (Fig. 1). GEM detectors are a relatively recent innovation in detector technology invented at CERN by F. Sauli in 1996 [10]. The basic element is a GEM foil, which consists of a 50 µm thick insulating Kapton layer electroplated with a conductive metal on both sides. Small holes are then etched in this foil and a voltage is applied across it. This produces electrical ﬁelds as high as 100 kV cm−1 inside the holes. When an electron traverses the hole, avalanche multiplication takes place giving approximately 20 secondary electrons4 for each primary electron (the exact value depends on gas density, gas mixture and applied electric ﬁeld). The triple GEM conﬁguration used in the GEMPix has gains in the range of 102–104. In the GEMPix the GEM foils are held rigid by gluing them to a frame, and the electrodes supplying the high voltage are arranged to avoid discharges onto the wire bonds of the Timepix readout. On top of the GEM/Timepix region is a 12 mm thick drift volume, topped with a Mylar cathode metallized with a thin aluminum layer (approximately 18 µm in total of which 1 µm is the aluminum layer). The FITPix system and the accompanying Pixelman software are used to readout the GEMPix. The Pixelman can be run using Python scripts that deﬁne the main parameters (thresholds, polarity, trigger type, etc.), initialize the detector and perform the frame readout, typically with a time gate of 1 s. There is also the possibility to use an algorithm inside the Python script to perform an online cluster analysis for a better analysis of the particle interacting in the drift volume. Fig. 4 shows a frame picture taken with 1 s time gate, in which the clusters produced by 5.9 keV X-rays coming from 55Fe and the tracks produced by Compton electrons or cosmic rays can be easily distinguished. 1 New limits to be introduced in the process of the general revision of the Swiss regulations concerning radiation protection: http://www.bag.admin.ch/themen/ strahlung/02883/03200/index.html?lang=fr. 2 A concise review of detectors for X-rays can be found in the X-Ray Data Booklet, Sec. 4.5, available on-line http://xdb.lbl.gov/Section4/Sec_4-5.pdf 3 As an example of oﬀ-the-shelf X-ray detectors: http://amptek.com/x-ray-detector- selection-guide/. 4 This number is the eﬀective multiplication per GEM foil, taking into account that some electrons are captured by the lower side of the GEM foil. The triple GEM conﬁguration with a rather low gain per foil was chosen in order to achieve a reliable system without discharges. 5 Other, cheaper gas mixtures such as Ar:CO2 could be used instead. However, the fast drift velocity of Ar:CO2:CF4 is needed in other applications of the GEMPix to reduce the lateral electron diﬀusion and therefore to increase the cluster analysis performance [15]. 2 A concise review of detectors for X-rays can be found in the X-Ray Data Booklet, Sec. 4.5, available on-line http://xdb.lbl.gov/Section4/Sec_4-5.pdf 1 New limits to be introduced in the process of the general revision of the Swiss regulations concerning radiation protection: http://www.bag.admin.ch/themen/ strahlung/02883/03200/index.html?lang=fr. 4 This number is the eﬀective multiplication per GEM foil, taking into account that some electrons are captured by the lower side of the GEM foil. The triple GEM conﬁguration with a rather low gain per foil was chosen in order to achieve a reliable system without discharges. 3 As an example of oﬀ-the-shelf X-ray detectors: http://amptek.com/x-ray-detector- selection-guide/. 5 Other, cheaper gas mixtures such as Ar:CO2 could be used instead. However, the fast drift velocity of Ar:CO2:CF4 is needed in other applications of the GEMPix to reduce the lateral electron diﬀusion and therefore to increase the cluster analysis performance [15]. 6 Attenuation lengths in iron are 15 µm for 6 keV photons and 2.34 cm for 1.25 MeV photons. These values are calculated using mass attenuation coeﬃcients and the density of iron in NIST data [25]. Tabulated values are available only for certain energies, therefore values for 6 keV and 1.25 MeV are used for 55Fe (5.9 keV X-Rays) and 60Co (1.17 and 1.33 MeV photons) respectively. 3. Experimental technique 55Fe is a radioactive isotope of iron decaying by electron capture to 55Mn with a half-life of 2.7 years. The electron capture is followed by emission of the characteristic 5.9 keV X-rays. Due to their low-energy, the photons are strongly absorbed within a few µm of the material. On the other hand, radionuclides such as 60Co, 54Mn, 44Ti emitting photons of higher energies and therefore longer attenuation lengths, are usually present together with 55Fe in activated metallic waste. A direct measurement of the X-rays of 55Fe thus requires a drastic reduction of the sample thickness in order to decrease the gamma background. For this purpose, the samples need to be ground and a thin layer of metallic powder shall be used for the measurements with the GEMPix. A continuous ﬂow of an Ar:CO2:CF4 (45:15:40 ratio) gas mixture is supplied externally at a rate of 3 l/h.5 The whole system is made This can be shown by calculating the self-attenuation of a sample when measuring the 55Fe X-rays with an underlying background of 60Co photons. Two simplifying assumptions are made: ﬁrst, the problem is one-dimen- sional such that geometrical eﬀects are neglected. Second, 55Fe is equally distributed in the sample such that the emission probability is position- independent in the sample. The same assumption holds for 60Co. The ratio of number of photons escaping from the sample over number of photons 61 A. Curioni et al. Nuclear Instruments and Methods in Physics Research A 849 (2017) 60–71 Fig. 1. The GEMPix detector: (1) external gas supply, (2) external HV connector, (3) Mylar window, (4) frame to hold the GEM foils, (5) FITPix readout. Fig. 1. The GEMPix detector: (1) external gas supply, (2) external HV connector, (3) Mylar window, (4) frame to hold the GE EMPix detector: (1) external gas supply, (2) external HV connector, (3) Mylar window, (4) frame to hold the GEM foils, (5) FITPix readout Fig. 2. The schematic of the detector including the principle dimensions and transport ﬁelds (ED=drift ﬁeld, ET=transfer ﬁelds, EI=induction ﬁeld) [15]. Fig. 3. The TOT mode in the Timepix ASIC measures the time elapsed while the preamp output is high against the threshold discriminator. The TOA mode measures the time from when the preamp goes high against the threshold until the end of the acquisition frame [15]. Fig. 4. 3. Experimental technique The online event display of a frame acquired with a time gate of 1 s. Fig. 2. The schematic of the detector including the principle dimensions and transport ﬁelds (ED=drift ﬁeld, ET=transfer ﬁelds, EI=induction ﬁeld) [15]. Fig. 4. The online event display of a frame acquired with a time gate of 1 s. if the sample thickness is reduced to 100 µm, 15% of the x-rays and almost 100% of the 60Co photons escape from the sample. Therefore, the signal-to-background ratio increases approximately by a factor of 80 (from 0.0019 to 0.15, assuming equal activity for 55Fe and 60Co and modeling the detector as a fully eﬃcient counter without any energy resolution). Fig. 5 shows the fraction calculated by Eq. (1) for 6 keV photons for diﬀerent sample thicknesses. Fig. 3. The TOT mode in the Timepix ASIC measures the time elapsed while the preamp output is high against the threshold discriminator. The TOA mode measures the time from when the preamp goes high against the threshold until the end of the acquisition frame [15]. The operational procedure is the following. A piece of metal from the waste to be examined is selected and cut. This sample is put in a milling machine to produce about 1 g of powder and for this purpose two diﬀerent tools have been used, a DEKEL FP4M and a modiﬁed Astoba. The typical parameters used for the powder production are: rotation velocity of the pin: 400 rot/min, advance velocity: 400 mm/ min, cutter: MTC TiAIN 6 mm Garant (ref SFS 205712 6°). Another method is presently under investigation using a drill with a conical ﬁle to produce the powder. emitted in the sample is then described by: emitted in the sample is then described by: ⎛ ⎝⎜ ⎞ ⎠⎟ ⎡ ⎣ ⎢ ⎛ ⎝⎜ ⎞ ⎠⎟ ⎤ ⎦ ⎥ ∫ N N t x λ dx λ t t λ = 1 exp − = 1 −exp − photons escaped photons emitted cm t , , 0 (1) (1) The sample powder is ﬁltered with a 0.5 mm mesh and placed in a plastic container with dimension 4×4×1 cm3, attached to its bottom with a double-sided tape, and then gently beaten with a tool (parallel punch) to improve the adhesion of the powder to the bottom of the container. The extra powder is ﬁnally removed. 7 This is an approximate average value. The average value has to be smaller than the mesh size but it is diﬃcult to determine due to ﬂuctuations in the grain size and grain shape. 4.1. Online analysis A window in the acquisition software displays the main statistics of the X-ray spectra and the time evolution of the measurements. The output of the cluster analysis is saved in a ﬁle event by event. Nineteen parameters are saved including event time, number of clusters per event, cluster type, cluster size, cluster position and total charge. Two cuts are applied in the analysis (online and oﬄine) using some of these parameters: only clusters ‘heavy blobs’ are used and clusters at the edges of the chips are discarded. Heavy blobs are the typical clusters produced in the GEMPix by low energy X-rays. They fulﬁll the following criteria10 [26]: Fig. 6. The plastic container with the sample powder attached on the bottom. • Minimum inner pixel count: 4 • Minimum ratio of inner and border pixels: 0.5 detection gas (Ar:CO2:CF4, 45:15:40) is supplied by a 50 l bottle equipped with a gas adaptor to reduce the pressure from about 150 bar to about 1 bar. A ﬂowmeter controls the gas ﬂow which is set at 3 l/h. High Voltage (1240 V) is supplied by a NIM Module HVGEM and controlled by a Labview programme. A low voltage supply for the GEMPix (3.3 V) is also necessary. The GEMPix data are acquired by the FITPix module [13] and read out by the Pixelman software (Fig. 8), which includes a Python plugin for code development [14]. The GEMPix detector is placed in a lead shielding box to reduce the background produced by ambient gamma rays. • Maximum deviation from perfect circle11: 1.2 Clusters at the edges of the readout chips are discarded since they might not be fully contained in the chip and therefore their measured parameters can be incorrect. Also, edge eﬀects of the quad Timepix ASIC exist which require an increased discard region at the edges. 8 Attenuation length in aluminum is 32 µm for 6 keV photons. This value is calculated using the mass attenuation coeﬃcient and the density of aluminum in NIST data [25]. 9 This is equal to an energy threshold on the order of 100 eV. 3. Experimental technique The ﬁnal thickness of the sample is around 100 µm.7 Fig. 6 shows the ﬁlled sample container. where t is the thickness of the sample, x is the depth inside the sample and λ is the attenuation length of the x-rays or γ-rays photons in the sample material. For an iron sample with a thickness of 1 cm, only 0.15% of the 55Fe X-rays but 81% of the 60Co photons escape from the sample.6 However The experimental setup is schematically shown in Fig. 7. The 62 m) μ thickness ( 0 50 100 150 200 250 300 350 400 450 500 N N 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 Fig. 5. Fraction of escaped over emitted 6 keV photons versus sample thickness. The corresponding fraction for 1.25 MeV photons (60Co background) is larger than 98% for the shown thickness range. The plotted value is thus approximately equal to the expected signal-to-background ratio. A. Curioni et al. Nuclear Instruments and Methods in Physics Research A 849 (2017) 60–71 Nuclear Instruments and Methods in Physics Research A 849 (2017) 60–71 A. Curioni et al. m) μ thickness ( 0 50 100 150 200 250 300 350 400 450 500 N N 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 Fig. 5. Fraction of escaped over emitted 6 keV photons versus sample thickness. The corresponding fraction for 1.25 MeV photons (60Co background) is larger than 98% for the shown thickness range The plotted value is thus approximately equal to the expected signal to background ratio Fig. 5. Fraction of escaped over emitted 6 keV photons versus sample thickness. The corresponding fraction for 1.25 MeV photons (60Co background) is larger than 98% for the shown thickness range. The plotted value is thus approximately equal to the expected signal-to-background ratio. Fig. 6. The plastic container with the sample powder attached on the bottom. 4.1. Online analysis Therefore clusters must fulﬁll the following position conditions: • 25 < centx < 246 or 266 < centx < 487 • 25 < centy < 246 or 266 < centy < 487 • 25 < centx < 246 or 266 < centx < 487 • 25 < centx < 246 or 266 < centx < 487 • 25 < centy < 246 or 266 < centy < 487 The actual search of the 55Fe content in a metallic sample consists of four separate measurements: 1) calibration of the system using a source of 55Fe to deﬁne the energy window for counting (next section); 2) X-ray counting of the sample; 3) calibration measurement to deﬁne the energy window for counting for the background measurement; 4) measurement of the gamma background from the sample. This is obtained by placing a thin layer of aluminum (approximately 100 µm thick corresponding to 3 attenuation lengths for 6 keV photons8) between sample and detector to absorb the 5.9 keV photons. Standard acquisition parameters set in Pixelman are: one second per frame, threshold of 36 counts,9 software trigger (500 events), ToT mode, internal clock set at 48 MHz and 7200 frames (2 h) for the sample and background measurements. The calibration measurements for sample and background both last 10 min. where centx (centy) is the x (y) coordinate of the centre of the cluster position in pixel count. 10 Using default settings in Pixelman. 11 Deﬁned as ratio of diameter calculated from cluster size and maximum distance within the cluster. g g 11 Deﬁned as ratio of diameter calculated from cluster size and maximum distance within the cluster. 10 Using default settings in Pixelman. 4.2. Oﬄine analysis Fig. 9 shows the energy spectrum of a measurement with a 55Fe source. An energy resolution of about 20% FWHM is typically achieved. A Gaussian function is ﬁt to the data obtained from the calibration measurement with a source. Mean and sigma of this ﬁt deﬁne the counting interval for the analysis of the sample and background measurements: only events within the range “mean ± 2 sigma” are taken into account. Fig. 10 shows the energy spectrum of a sample measurement. 63 A. Curioni et al. lear Instruments and Methods in Physics Research A 849 (2017) 60–7 iochemical analysis and GEMPix results ctivated steel samples were selected to characterize h sample was divided in two pieces: one was sent to f di h i l l i (AMEC [27]) th th samples with high expected background. For the other samples, an estimated background of 50 counts in 7200 s is used instead. This is based on the fact that the lowest measured background in 7200 s is 42 counts, which implies that the true mean value of the lowest back- d i h t l F th th ti ti f 50 t Fig. 8. The Pixelman console [14] showing the 55Fe X-rays observed online during the acquisition. Fig. 7. Measurement setup. Nuclear Instruments and Methods in Physics Research A 849 (2017) 60–71 Fig. 7. Measurement setup. Fig. 7. Measurement setup. Fig. 7. Measurement setup. Fig. 8. The Pixelman console [14] showing the 55Fe X-rays observed online during the acquisition. Fig. 8. The Pixelman console [14] showing the 55Fe X-rays observed online during the acquisition. Fig. 8. The Pixelman console [14] showing the 55Fe X-rays observed online during the acquisition. 5. Samples, radiochemical analysis and GEMPix results h0 Entries 74682 Mean 5316 RMS 1585 / ndf 2 χ 85.17 / 37 Constant 12.3 ± 2154 Mean 2.7 ± 5910 Sigma 2.8 ± 538.4 energy (eV) 0 1000 2000 3000 4000 5000 6000 7000 8000 9000 10000 frequency 0 200 400 600 800 1000 1200 1400 1600 1800 2000 2200 h0 Entries 74682 Mean 5316 RMS 1585 / ndf 2 χ 85.17 / 37 Constant 12.3 ± 2154 Mean 2.7 ± 5910 Sigma 2.8 ± 538.4 Fig. 9. Energy spectrum recorded with a 55Fe calibration source. Peaks are due to 55Fe x-rays (5.9 keV) and Argon escape/ﬂuorescence (2.9 keV/3.0 keV). Here, events within (5910 ± 1077) eV would be counted in the sample and background measurements Fig. 9. Energy spectrum recorded with a 55Fe calibration source. Peaks are due to 55Fe x-rays (5.9 keV) and Argon escape/ﬂuorescence (2.9 keV/3.0 keV). Here, events within (5910 ± 1077) eV would be counted in the sample and background measurements. Fig. 9. Energy spectrum recorded with a 55Fe calibration source. Peaks are due to 55Fe x-rays (5.9 keV) and Argon escape/ﬂuorescence (2.9 k 1077) eV would be counted in the sample and background measurements. 5Fe calibration source. Peaks are due to 55Fe x-rays (5.9 keV) and Argon escape/ﬂuorescence (2.9 keV/3.0 keV). Here, events within (5910 ± and background measurements. Fig. 9. Energy spectrum recorded with a 55Fe calibration source. Peaks are due to 55Fe x-rays (5.9 keV) and Argon escape/ﬂuorescence (2.9 keV/3.0 keV). Here, events within (5910 ± 1077) eV would be counted in the sample and background measurements. uld be counted in the sample and background measurements. h0 Entries 11341 Mean 2144 RMS 2623 energy (eV) 0 1000 2000 3000 4000 5000 6000 7000 8000 9000 10000 frequency 0 200 400 600 800 1000 1200 h0 Entries 11341 Mean 2144 RMS 2623 Fig. 10. Energy spectrum of a sample measurement. Only events within the energy window deﬁned by the source measurement are counted. Radiochemical (Bq/g) 0 200 400 600 800 1000 1200 GEMPix (cts/s) 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 / ndf 2 χ 272.4 / 43 offset 0.0002598 ± 0.0002376 − slope 05 − 1.402e ± 0.0006078 / ndf 2 χ 272.4 / 43 offset 0.0002598 ± 0.0002376 − slope 05 − 1.402e ± 0.0006078 MPix counts per second versus speciﬁc activity as determined by the radiochemical analysis. 5. Samples, radiochemical analysis and GEMPix results samples with high expected background. For the other samples, an estimated background of 50 counts in 7200 s is used instead. This is based on the fact that the lowest measured background in 7200 s is 42 counts, which implies that the true mean value of the lowest back- ground is somewhat larger. Furthermore, the estimation of 50 counts leads to an oﬀset in the ﬁt of the calibration curve (Fig. 11) that is compatible with zero. For the GEMPix analysis, only statistical uncertainties on the number of counts are taken into account. A total of 45 activated steel samples were selected to characterize the technique. Each sample was divided in two pieces: one was sent to an external company for radiochemical analysis (AMEC [27]), the other was reduced to powder as described above and measured with the GEMPix. A correction was applied to the radiochemical results in order to take into account the radioactive decay of 55Fe due to the time diﬀerence between the GEMPix measurements and the radiochemical analysis. Most of the GEMPix measurements lasted 7200 s (2 h), the others were normalized to this duration. The shortest measurement lasted 5700 s. The background was measured as described above for 16 The GEMPix net counts per second (cps) are plotted against the corresponding radiochemical results in Fig. 11. An error-weighted linear ﬁt is used to provide the conversion factor between GEMPix cps and the declared speciﬁc activity from the radiochemical analysis (Bq/g). The slope of the ﬁt provides the calibration factor for the GEMPix: 64 Nuclear Instruments and Methods in Physics Research A 849 (2017) 60–71 A. Curioni et al. h0 Entries 74682 Mean 5316 RMS 1585 / ndf 2 χ 85.17 / 37 Constant 12.3 ± 2154 Mean 2.7 ± 5910 Sigma 2.8 ± 538.4 energy (eV) 0 1000 2000 3000 4000 5000 6000 7000 8000 9000 10000 frequency 0 200 400 600 800 1000 1200 1400 1600 1800 2000 2200 h0 Entries 74682 Mean 5316 RMS 1585 / ndf 2 χ 85.17 / 37 Constant 12.3 ± 2154 Mean 2.7 ± 5910 Sigma 2.8 ± 538.4 Fig. 9. Energy spectrum recorded with a 55Fe calibration source. Peaks are due to 55Fe x-rays (5.9 keV) and Argon escape/ﬂuorescence (2.9 keV/3.0 keV). Here, events within (5910 ± 1077) eV would be counted in the sample and background measurements. 5. Samples, radiochemical analysis and GEMPix results 65 3 3 (3) Thus, if a data point is: ⎛ ⎝⎜ ⎞ ⎠⎟ l l n n n . . = 1−1 9 −1. 65 3 3 (3) ⎛ ⎝⎜ ⎞ ⎠⎟ l l n n n . . = 1−1 9 −1. 65 3 3 (3) 12 Eq. (9) in Ref. [28]. 13 Eq. (12) in the reference. 5. Samples, radiochemical analysis and GEMPix results The slope of the linear ﬁt provides the conversion factor: 1645 ± 38 (Bq/g)/ h0 Entries 11341 Mean 2144 RMS 2623 energy (eV) 0 1000 2000 3000 4000 5000 6000 7000 8000 9000 10000 frequency 0 200 400 600 800 1000 1200 h0 Entries 11341 Mean 2144 RMS 2623 Fig. 10. Energy spectrum of a sample measurement. Only events within the energy window deﬁned by the source measurement are counted. h0 Entries 11341 Mean 2144 RMS 2623 energy (eV) 0 1000 2000 3000 4000 5000 6000 7000 8000 9000 10000 frequency 0 200 400 600 800 1000 1200 h0 Entries 11341 Mean 2144 RMS 2623 Fig. 10. Energy spectrum of a sample measurement. Only events within the energy window deﬁned by the source measurement are counted. Fig. 10. Energy spectrum of a sample measurement. Only events within the energy window deﬁned by the source measurement are counted. Radiochemical (Bq/g) 0 200 400 600 800 1000 1200 GEMPix (cts/s) 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 / ndf 2 χ 272.4 / 43 offset 0.0002598 ± 0.0002376 − slope 05 − 1.402e ± 0.0006078 / ndf 2 χ 272.4 / 43 offset 0.0002598 ± 0.0002376 − slope 05 − 1.402e ± 0.0006078 Fig. 11. GEMPix counts per second versus speciﬁc activity as determined by the radiochemical analysis. The slope of the linear ﬁt provides the conversion factor: 1645 ± 38 (Bq/g)/ (cps). Fig. 11. GEMPix counts per second versus speciﬁc activity as determined by the radiochemical analysis. The slope of the linear ﬁt provides the conversion factor: 1645 ± 38 (Bq/g)/ (cps). 65 Nuclear Instruments and Methods in Physics Research A 849 (2017) 60–71 A. Curioni et al. Difference Radiochem. GEMPix Entries 45 Mean 0.1056 − RMS 1.974 / ndf 2 χ 8.219 / 12 Constant 0.948 ± 4.192 Mean 0.3878 ± 0.1322 − Sigma 0.430 ± 1.911 (Radiochem. - GEMPix) / Sum of Uncertainties 10 − 8 − 6 − 4 − 2 − 0 2 4 6 8 10 Frequency 0 1 2 3 4 5 6 Difference Radiochem. GEMPix Entries 45 Mean 0.1056 − RMS 1.974 / ndf 2 χ 8.219 / 12 Constant 0.948 ± 4.192 Mean 0.3878 ± 0.1322 − Sigma 0.430 ± 1.911 Fig. 12. Comparison between results of radiochemical analysis and GEMPix analysis in units of their uncertainties and a Gaussian ﬁt to the distribution. 5. Samples, radiochemical analysis and GEMPix results Frequency on between results of radiochemical analysis and GEMPix analysis in units of their uncertainties and a Gaussian ﬁt to the distribution. Fig. 12. Comparison between results of radiochemical analysis and GEMPix analysis in units of their uncertainties and a Gaussian ﬁt ⎡ ⎣⎢ ⎤ ⎦⎥ cps Bq g Bq g cps (6. 078 ± 0. 140) × 10 / = 1645 ± 38 / . −4 −1 is (is not) compatible with the background-only hypothesis at 95% C.L. Fig. 15 shows the detection limit as an activity for each number of background counts. The detection limit is well below 10 Bq/g for all background counts of the 45 samples. Note that this is only the statistical detection limit. This detection limit is not reached in practice due to larger systematic uncertainties. The bad quality of the ﬁt (χ2/ndf=272/43) is explained by an underestimation of the uncertainties on the GEMPix counts, since only statistical uncertainties are considered. Fig. 12 shows the distribution of the diﬀerence in units of the sum of the uncertainties of the two methods. The uncertainties of the two methods are added linearly to obtain the uncertainty on the diﬀerence. A Gaussian distribution is expected. The obtained distribution is well ﬁtted by a Gaussian, but the sigma of the ﬁt is larger than 1 and there are some outliers up to 7 sigmas. This indicates most probably an underestimation of the uncertainty, due to the fact that uncertainties on the GEMPix results are small compared to those of the radio- chemical analysis since only statistical uncertainties are taken into account. Fig. 16 compares the GEMPix results with the calculated detection limit. Also, the total number of counts for a sample with an activity of 30 Bq/g is calculated for each number of background counts. For this, the activity of 30 Bq/g is converted to number of counts using the calibration factor and the number of background counts is added. The 95% C.L. single-sided lower limit for the exemption limit plus back- ground is calculated using the tables provided in [28], Eq. (2) for the upper limit for more than 50 counts, and the following Eq. (3)13 for the lower limit, l.l., in case of more than 50 counts, n. ⎛ ⎝⎜ ⎞ ⎠⎟ l l n n n . . = 1−1 9 −1. 6. Sensitivity of the technique, detection limit, conﬁdence levels and uncertainties Thus, if a data point is: In this section, a Conﬁdence Level (C.L.) of 95% is chosen for all calculations. As an example, the sensitivity to a 30 Bq/g limit is calculated as this value is the current Swiss exemption limit for 55Fe. The measured background is not independent of the sample itself since there is a non-negligible contribution from other radionuclides present in the sample. Fig. 13 shows the distribution of the background measured for 16 out of 45 samples. • Below detection limit, the total number of counts is compatible with the background only hypothesis. • Above detection limit, the background only hypothesis is ruled out and we infer that there is some 55Fe content in the sample. • Above detection limit, the background only hypothesis is ruled out and we infer that there is some 55Fe content in the sample. • Below the lower limit of the exemption limit, the activity of the sample is less than 30 Bq/g. • Above the lower limit of the exemption limit, we cannot exclude an activity larger than 30 Bq/g. • Above the lower limit of the exemption limit, we cannot exclude an activity larger than 30 Bq/g. Detection limits of the technique are calculated depending on the background using Poisson statistical methods. The 95%-C.L. single- sided upper limits for each number of background counts is calculated according to [28]: for count numbers, n, smaller than 51 the upper limit is taken directly from a table provided in [28], for larger count numbers the upper limit, u.l., is approximated by a formula given in [28] (12): All statements are at 95%-C.L. All statements are at 95%-C.L. Furthermore, Fig. 16 shows a comparison with radiochemical analysis with diﬀerent marker styles and colors. If a data point is a: • Red upwards pointing triangle: the activity of the sample measured by radiochemical analysis is larger than 30 Bq/g minus its uncer- tainty at 95% C.L. ⎛ ⎝⎜ ⎞ ⎠⎟ u l n n n . . = ( + 1) 1− 1 9( +1) + 1. 65 3 +1 3 (2) (2) • Green downwards pointing triangle, the activity of the sample measured by radiochemical analysis is smaller than 30 Bq/g minus its uncertainty at 95% C.L. Fig. 14 shows the resulting upper limits for each number of background counts. These upper limits are interpreted as the detection limit above which 55Fe content is detected. For example, if 100 background counts and 110 (150) total counts are measured, the result Fig. 14 shows the resulting upper limits for each number of background counts. These upper limits are interpreted as the detection limit above which 55Fe content is detected. For example, if 100 background counts and 110 (150) total counts are measured, the result Results of the radiochemical analysis and of GEMPix are inconsistent 13 Eq. (12) in the reference. 66 A. Curioni et al. Nuclear Instruments and Methods in Physics Research A 849 (2017) 60–71 Background Entries 16 Mean 101.6 RMS 72.41 Background cts (normalized to 7200s) 0 50 100 150 200 250 300 350 400 Frequency 0 0.5 1 1.5 2 2.5 3 3.5 4 Background Entries 16 Mean 101.6 RMS 72.41 Fig. 13. Distribution of the background measured for 16 samples. Background cts 0 50 100 150 200 250 300 Upper Limit (95% CL) of background cts 0 50 100 150 200 250 300 350 50 % 66 % 95 % Fig. 14. The upper limits are calculated for each number of background counts (red points). Above this limit, the detector is sensitive to 55Fe. Below this limit, in the shaded area, background only cannot be excluded. The vertical lines show the maximum background counts for a stated fraction of the measured samples (numbers above the lines). All statements are at 95%-C.L. (For interpretation of the references to color in this ﬁgure legend, the reader is referred to the web version of this article.) g) Background Entries 16 Mean 101.6 RMS 72.41 Background cts (normalized to 7200s) 0 50 100 150 200 250 300 350 400 Frequency 0 0.5 1 1.5 2 2.5 3 3.5 4 Background Entries 16 Mean 101.6 RMS 72.41 Fig. 13. Distribution of the background measured for 16 samples. Fig. 13. Distribution of the background measured for 16 samples. Fig. 13. Distribution of the background measured for 16 samples. Background cts 0 50 100 150 200 250 300 Upper Limit (95% CL) of background cts 0 50 100 150 200 250 300 350 50 % 66 % 95 % Background cts 0 50 100 150 200 250 300 Upper Limit (95% CL) of background cts 0 50 100 150 200 250 300 350 50 % 66 % 95 % Fig. 14. The upper limits are calculated for each number of background counts (red points). Above this limit, the detector is sensitive to 55Fe. Below this limit, in the shaded area, background only cannot be excluded. The vertical lines show the maximum background counts for a stated fraction of the measured samples (numbers above the lines). (For interpretation of the references to color in this ﬁgure legend, the reader is referred to the web version of this article.) Background cts 0 50 100 150 200 250 300 Detection Limit at 95% CL (Bq/g) 1 2 3 4 5 6 7 50 % 66 % 95 % Fig. 15. The upper limits shown in Fig. 14 are background subtracted and then converted to activity using the calibration factor. Above the limit, the detector is sensitive to 55Fe. Below this, in the shaded area, background only cannot be excluded. The vertical lines show the maximum background counts for a stated fraction of the measured samples (numbers above the lines). (For interpretation of the references to color in this ﬁgure legend, the reader is referred to the web version of this article.) Fig. 14. The upper limits are calculated for each number of background counts (red points). Above this limit, the detector is sensitive to 55Fe. Below this limit, in the shaded area, background only cannot be excluded. The vertical lines show the maximum background counts for a stated fraction of the measured samples (numbers above the lines). 7. Improvement on gain stability and HV correction of the GEMPix A procedure for a more stable operation of the detector has been implemented: ambient conditions – pressure, temperature and humid- ity – are monitored and the detector HV is corrected to obtain a stable gain. This procedure increases the gain stability of the detector. A sensor to measure pressure, temperature and humidity has been installed inside the detector. Fig. 17 shows this sensor mounted on the GEMPix readout board. Data recorded by the sensor are used to correct the High Voltage of the GEMPix and thus ensure a more stable GEM gain. First, data are acquired without any HV correction using a 55Fe source. An exponential dependence of the peak position on tempera- ture/pressure (T/P) is expected, as described for example in [29]. Fig. 18 shows measured peak positions for diﬀerent T/P values and an exponential ﬁt to the data. Fig. 17. Sensor for pressure, temperature and humidity (white circle) installed on the GEMPix readout board. The sensor is read out via USB. for the lowest gain for the 55Fe-source (at 1135 V) is diﬃcult since the 55Fe peak is almost in the noise. This results in a very poor ﬁt quality. However, the model ﬁts better in the region of interest for this technique (at 1240 V) and only the slope is used, as explained below. A correction formula for the applied voltage, V, is derived by using the following two equations: for the lowest gain for the 55Fe-source (at 1135 V) is diﬃcult since the 55Fe peak is almost in the noise. This results in a very poor ﬁt quality. However, the model ﬁts better in the region of interest for this technique (at 1240 V) and only the slope is used, as explained below. A correction formula for the applied voltage, V, is derived by using the following two equations: for the lowest gain for the 55Fe-source (at 1135 V) is diﬃcult since the 55Fe peak is almost in the noise. This results in a very poor ﬁt quality. However, the model ﬁts better in the region of interest for this technique (at 1240 V) and only the slope is used, as explained below. for the lowest gain for the 55Fe-source (at 1135 V) is diﬃcult since the 55Fe peak is almost in the noise. This results in a very poor ﬁt quality. All statements are at 95%-C.L. GEMPix raw versus background counts (data points) of the samples are compared with the detection limit (solid red line, compare Fig. 14) and the Swiss exemption limit of 30 Bq/g plus background (black dashed line). Solid black line shows 95%-C.L. lower limit of the exemption limit plus background. Radiochemical analysis results are encoded for sample data points: red upwards (green downwards) pointing triangles denote activity plus uncertainty at 95%-C.L. above (below) exemption limit. Some samples are out of range on the y-axis of this plot but they are well above the detection limit and all are correctly assigned to have an activity larger than 30 Bq/g. (For interpretation of the references to color in this ﬁgure legend, the reader is referred to the web version of this article.) for two of the 45 samples: for sample B the GEMPix states an activity above 30 Bq/g while the radiochemical analysis states an activity below 30 Bq/g. The results for sample A show the inverted situation. Thus, sample A would be falsely declared to have an activity below the exemption limit. However, it is expected that a complete uncertainty investigation of the GEMPix would result in larger uncertainties. The probability to falsely state that an activity is below 30 Bq/g would be decreased. At the same time, a larger uncertainty on the GEMPix results will correspondingly reduce the sensitivity. This is less problematic for larger exemption limits since the majority of 55Fe samples typically has a rather low activity. The future Swiss exemption limit will be 1000 Bq/g. Fig. 17. Sensor for pressure, temperature and humidity (white circle) installed on the GEMPix readout board. The sensor is read out via USB. The complete analysis presented in this section is limited by the fact that only statistical uncertainties on the counting are considered. An investigation of other uncertainties is necessary before deﬁning opera- tional limits with respect to the Swiss exemption limit. Sources of other uncertainties include sample preparation, stability of the GEMPix and positioning of the samples below the GEMPix. All statements are at 95%-C.L. (For interpretation of the references to color in this ﬁgure legend, the reader is referred to the web version of this article.) Background cts 0 50 100 150 200 250 300 Detection Limit at 95% CL (Bq/g) 1 2 3 4 5 6 7 50 % 66 % 95 % Fig. 15. The upper limits shown in Fig. 14 are background subtracted and then converted to activity using the calibration factor. Above the limit, the detector is sensitive to 55Fe. Below this, in the shaded area, background only cannot be excluded. The vertical lines show the maximum background counts for a stated fraction of the measured samples (numbers above the lines). (For interpretation of the references to color in this ﬁgure legend, the reader is referred to the web version of this article.) Background cts 0 50 100 150 200 250 300 Detection Limit at 95% CL (Bq/g) 1 2 3 4 5 6 7 50 % 66 % 95 % Fig. 15. The upper limits shown in Fig. 14 are background subtracted and then converted to activity using the calibration factor. Above the limit, the detector is sensitive to 55Fe. Below this, in the shaded area, background only cannot be excluded. The vertical lines show the maximum background counts for a stated fraction of the measured samples (numbers above the lines). (For interpretation of the references to color in this ﬁgure legend, the reader is referred to the web version of this article.) 67 Nuclear Instruments and Methods in Physics Research A 849 (2017) 60–71 A. Curioni et al. Fig. 16. GEMPix raw versus background counts (data points) of the samples are compared with the detection limit (solid red line, compare Fig. 14) and the Swiss exemption limit of 30 Bq/g plus background (black dashed line). Solid black line shows 95%-C.L. lower limit of the exemption limit plus background. Radiochemical analysis results are encoded for sample data points: red upwards (green downwards) pointing triangles denote activity plus uncertainty at 95%-C.L. above (below) exemption limit. Some samples are out of range on the y-axis of this plot but they are well above the detection limit and all are correctly assigned to have an activity larger than 30 Bq/g. (For interpretation of the references to color in this ﬁgure legend, the reader is referred to the web version of this article.) Fig. 16. 7. Improvement on gain stability and HV correction of the GEMPix H h d l ﬁ b i h i f i f hi To calculate a correction formula for the detector HV, it is necessary to know the dependence of the 55Fe peak position on the HV. This is measured by performing a gain scan. Fig. 19 shows the results and an exponential ﬁt that describes this dependence. In an attempt to cover a large range of the possible gain values also for other applications, not only a 55Fe source but also a 241Am source was used. The determina- tion of the energy deposition and thus TOT counts is more diﬃcult for 241Am (α-source) than for 55Fe (X-ray source). Also, the measurement A correction formula for the applied voltage, V, is derived by using the following two equations: TOT T P TOT p p T P ′( / ) = 5. 9keV* 0* exp ( 1* / ) (4) TOT V A B V ′( ) = * exp( * ) (5) TOT T P TOT p p T P ′( / ) = 5. 9keV* 0* exp ( 1* / ) (4) (5) TOT V A B V ′( ) = * exp( * ) 68 A. Curioni et al. Nuclear Instruments and Methods in Physics Research A 849 (2017) 60–71 Entries 29 Mean 0.3212 RMS 0.003492 / ndf 2 χ 337.9 / 27 p0 0.009 ± 0.231 p1 0.13 ± 32.54 T/P (K/hPa) 0.31 0.312 0.314 0.316 0.318 0.32 0.322 0.324 0.326 0.328 0.33 TOT counts 5000 6000 7000 8000 9000 Entries 29 Mean 0.3212 RMS 0.003492 / ndf 2 χ 337.9 / 27 p0 0.009 ± 0.231 p1 0.13 ± 32.54 Fig. 18. The TOT counts show an exponential dependence on T/P. sum of gem voltages 800 850 900 950 1000 1050 1100 1150 1200 1250 TOT/keV 10 2 10 3 10 / ndf 2 χ 4730 / 16 A 09 − 5.906e ± 07 − 3.515e B 05 − 1.418e ± 0.01904 / ndf 2 χ 4730 / 16 A 09 − 5.906e ± 07 − 3.515e B 05 − 1.418e ± 0.01904 scan with 55Fe (data taken with sum of gem voltages larger than 1100 V) and 241Am sources (data taken with sum of gem voltages smaller than 1100 V). 14 This is a rough energy calibration such that 6000 TOT counts equal 6000 eV (approximate energy of 55Fe X-rays). In principle this should not be necessary since Eq. (4) already contains a calibration to 5.9 keV. However, it is a way to reduce the impact of the poor ﬁt results from Fig. 19 since only the parameter B is used. The impact of this parameter in the T/P range of interest is suﬃciently small to allow for good HV correction (compare Fig. 20). 7. Improvement on gain stability and HV correction of the GEMPix The calibrated activity depends linearly on the number of counts. Therefore, relative uncertainties on the number of counts and the activity are the same. As shown in Fig. 21, a mean TOT value of 5800 (6200) instead of 6000 transforms into a deviation of the number of events and thus on the activity of −1.1%(−0.4%). Thus, activity measurements are stable within approximately 1% with the applied HV correction. It is however not recommended to perform measurements over a longer period without control measurements with a source. This is a rather quick, independent check of the whole measurement system. Large eﬀects will be noticed immediately, and small changes can be corrected by adjusting constant C in Eq. (6). correction (Fig. 20). If the detector is ﬂushed with gas and the HV is on continuously, a gain stability within (6000 ± 200) TOT counts is achieved. Fig. 21 demonstrates the conversion of this deviation of the mean TOT counts into an uncertainty on the number of event counts. The mean value of a Gaussian TOT count distribution with a constant resolution (sigma/mean=constant) and a constant total integral is varied. The integral for a ﬁxed range from 4800 to 7200 TOT counts is presented in dependence of the mean TOT value. The calibrated activity depends linearly on the number of counts. Therefore, relative uncertainties on the number of counts and the activity are the same. As shown in Fig. 21, a mean TOT value of 5800 (6200) instead of 6000 transforms into a deviation of the number of events and thus on the activity of −1.1%(−0.4%). Thus, activity measurements are stable within approximately 1% with the applied HV correction. It is however not recommended to perform measurements over a longer period without control measurements with a source. This is a rather quick, independent check of the whole measurement system. Large eﬀects will be noticed immediately, and small changes can be corrected by adjusting constant C in Eq. (6). ⎛ ⎝⎜ ⎞ ⎠⎟ V B keV TOT A p p B T P V C p B T P = 1 * ln 5. 9 * * 0 − 1 * = + − 1 * set (6) (6) Constants p0, p1, A, B can be found as ﬁt results in Figs. 18 and 19, respectively. The ﬁrst, constant part of Eq. 7. Improvement on gain stability and HV correction of the GEMPix (6) can be split up into the set voltage, Vset, and a new constant C. The calculated result for constant C is not used. Instead, the constant is determined experi- mentally such that the 55Fe peak yields on average 6000 TOT counts.14 Since systematic deviations in the gain stability are observed with this correction, a second-order correction is introduced using the humidity measurement, H. Data are acquired with the T/P-correction of Eq. (6). The analysis is done similarly to the calculation of the T/P-correction. The ﬁnal correction formula to calculate the voltage is: V T P H V V T P V H ( / , ) = + 538 −1709. 03 V hPa K * + 2. 59 m g * set 3 (7 (7) Measurements have been performed over nine days using this Measurements have been performed over nine days using this 7. Improvement on gain stability and HV correction of the GEMPix Entries 29 Mean 0.3212 RMS 0.003492 / ndf 2 χ 337.9 / 27 p0 0.009 ± 0.231 p1 0.13 ± 32.54 T/P (K/hPa) 0.31 0.312 0.314 0.316 0.318 0.32 0.322 0.324 0.326 0.328 0.33 TOT counts 5000 6000 7000 8000 9000 Entries 29 Mean 0.3212 RMS 0.003492 / ndf 2 χ 337.9 / 27 p0 0.009 ± 0.231 p1 0.13 ± 32.54 Fig. 18. The TOT counts show an exponential dependence on T/P. Fig. 18. The TOT counts show an exponential dependence on T/P. Fig. 18. The TOT counts show an exponential dependence on T/P. g p p / sum of gem voltages 800 850 900 950 1000 1050 1100 1150 1200 1250 TOT/keV 10 2 10 3 10 / ndf 2 χ 4730 / 16 A 09 − 5.906e ± 07 − 3.515e B 05 − 1.418e ± 0.01904 / ndf 2 χ 4730 / 16 A 09 − 5.906e ± 07 − 3.515e B 05 − 1.418e ± 0.01904 e gain scan with 55Fe (data taken with sum of gem voltages larger than 1100 V) and 241Am sources (data taken with sum of gem voltages smaller than 1100 V). Fig. 19. Results of the gain scan with 55Fe (data taken with sum of gem voltages larger than 1100 V) and 241Am sources (data taken with sum of gem voltages smaller than 1100 V). e (data taken with sum of gem voltages larger than 1100 V) and 241Am sources (data taken with sum of gem voltages smaller than 1100 V) Fig. 19. Results of the gain scan with 55Fe (data taken with sum of gem voltages larger than 1100 V) and 241Am sources (data taken w Eqs. (4) and (5) are set equal, since TOT T P TOT V ′( / ) = ′( ). The comparison of the two equations thus yields: correction (Fig. 20). If the detector is ﬂushed with gas and the HV is on continuously, a gain stability within (6000 ± 200) TOT counts is achieved. Fig. 21 demonstrates the conversion of this deviation of the mean TOT counts into an uncertainty on the number of event counts. The mean value of a Gaussian TOT count distribution with a constant resolution (sigma/mean=constant) and a constant total integral is varied. The integral for a ﬁxed range from 4800 to 7200 TOT counts is presented in dependence of the mean TOT value. 8. Conclusions The number of event counts is normalized to the number of event t t TOT l f 6000 V ti l li d t i d i ti f th TOT l i th t l t t d i Fi 20 Mean TOT value 5600 5800 6000 6200 6400 Normalized integrated cts from 4800 to 7200 0.93 0.94 0.95 0.96 0.97 0.98 0.99 1 Fig. 21. Conversion of the ﬂuctuation of the mean TOT count value to an uncertainty on the number of event counts. The number of event counts is normalized to the number of event counts at a TOT mean value of 6000. Vertical lines denote maximum deviation of the mean TOT value in the control measurement presented in Fig. 20. Fig. 21. Conversion of the ﬂuctuation of the mean TOT count value to an uncertainty on the number of event counts. The number of event counts is normalized to the number of event counts at a TOT mean value of 6000. Vertical lines denote maximum deviation of the mean TOT value in the control measurement presented in Fig. 20. reduce systematic uncertainties. An operational test phase for this method is planned at CERN. sample takes approximately 4.5 h. Forty-ﬁve samples from CERN's radioactive waste were used to calibrate the detector. Measured counts are converted into speciﬁc activity using a calibration curve based on reference data from radiochemical analyses of the same samples. Acknowledgements Since the background is sample-dependent, the detection limit is also background-dependent and has been accounted for in the calcula- tion of the detection limits. From the forty-ﬁve samples analyzed, good agreement has been found between the radiochemical analysis and the present procedure. As an example, the sensitivity to 30 Bq/g, which is the current Swiss exemption limit for 55Fe, was investigated but only statistical uncertainties of the method were included. The method is potentially sensitive to the exemption limit. A complete uncertainty study is in progress. This will most likely reduce the sensitivity of the method though this will be less important in the future since the activity of most of the samples is small compared to the future Swiss exemption limit of 1 kBq/g. We wish to thank Stuart George (formerly CERN, now with the University of Houston) and Erik Frojd (formerly CERN, now with PSI, Switzerland) for useful discussions. The authors warmly acknowledge Jerome Alozy and Michael Campbell from the Medipix collaboration for the support on the readout electronics. This work was partly supported by the Wolfgang Gentner Programme of the German Ministry of Education and Research and by the Marie Curie Initial Training Network Fellowship of the European Community's Seventh Framework Program under Grant Agreement PITN-GA-4 2011-289198-ARDENT. 8. Conclusions A new method to measure the 55Fe content in radioactive samples using GEMPix, a gas detector with pixelated readout, has been developed. A procedure to measure the background and the total counts of a sample has been designed. In total, measurement of one 69 A. Curioni et al. Nuclear Instruments and Methods in Physics Research A 849 (2017) 60–71 Fig. 20. Measurement with a 55Fe source over nine days. The plots show the time evolution of the TOT counts (top plot), the ratio temperature/pressure (T/P), and the High Voltage (HV). The TOT counts are within 6000 ± 200 if measurements in the heat-up phase of the detector (‘fast rising T’) and after the HV was oﬀare discarded. A. Curioni et al. Nuclear Instruments and Methods in Physics Research A 849 (2017) 60–71 Fig. 20. Measurement with a 55Fe source over nine days. The plots show the time evolution of the TOT counts (top plot), the ratio temperature/pressure (T/P), and the High Voltage (HV). The TOT counts are within 6000 ± 200 if measurements in the heat-up phase of the detector (‘fast rising T’) and after the HV was oﬀare discarded. Mean TOT value 5600 5800 6000 6200 6400 Normalized integrated cts from 4800 to 7200 0.93 0.94 0.95 0.96 0.97 0.98 0.99 1 Fig. 21. Conversion of the ﬂuctuation of the mean TOT count value to an uncertainty on the number of event counts. The number of event counts is normalized to the number of event counts at a TOT mean value of 6000. Vertical lines denote maximum deviation of the mean TOT value in the control measurement presented in Fig. 20. Fig. 20. Measurement with a 55Fe source over nine days. The plots show the time evolution of the TOT counts (top plot), the ratio temperature/pressure (T/P), and the High Voltage (HV). The TOT counts are within 6000 ± 200 if measurements in the heat-up phase of the detector (‘fast rising T’) and after the HV was oﬀare discarded. Mean TOT value 5600 5800 6000 6200 6400 Normalized integrated cts from 4800 to 7200 0.93 0.94 0.95 0.96 0.97 0.98 0.99 1 Fig. 21. Conversion of the ﬂuctuation of the mean TOT count value to an uncertainty on the number of event counts. [1] IAEA, Strategy and Methodology for Radioactive Waste Characterization, IAEA References A correction of the high voltage (and therefore of the gain) as function of temperature, pressure and humidity in the detection volume was developed in order to obtain a more stable system and [1] IAEA, Strategy and Methodology for Radioactive Waste Characterization, IAEA [1] IAEA, Strategy and Methodology for Radioactive Waste Characterization, IAEA 70 A. Curioni et al. uclear Instruments and Methods in Physics Research A 849 (2017) 60 Nuclear Energy Series, IAEA-TECDOC-1537, 2007. [16] X. Llopart, et al., Timepix, a 65k programmable pixel readout chip for arrival time, energy and/or photon counting measurements, Nucl. Instrum. Methods A 581 (2007) 485 (Erratum ibid. A 585 (2008), p. 106). gy [2] IAEA, Determination and use of scaling factors for waste characte gy [2] IAEA, Determination and use of scaling factors for waste characterization in l l l i [2] IAEA, Determination and use of scaling factors for waste characterization in nuclear power plants, IAEA Nuclear Energy Series, NW-T-1.18, 2009. nuclear power plants, IAEA Nuclear Energy Series, NW-T-1.18, 2009. [17] Medipix website: 〈http://medipix.web.cern.ch/medipix/〉, (accessed 01.12.16). [3] INternational Organization For Standardization, Nuclear Energy – Nuclear Fuel Technology – Scaling Factor Method to Determine the Radioactivity of Low- and Intermediate-level Radioactive Waste Packages Generated at Nuclear Power Plants, ISO 21238:2007, ISO, Geneva, 2007. [18] M. Campbell, 10 years of the Medipix2 Collaboration, Nucl. Instrum. Methods A 633 (2011) S1. [19] S.P. George, et al., Measurement of an accelerator based mixed ﬁeld with a Timepix detector, JINST 10 (2015) P03005. [4] ANDRA, Spéciﬁcation, Critères radiologiques d′acceptation des déchets TFA, SUR. SP.AMES.02.0007 (CERN EDMS 1332211), ANDRA (2013). [20] LHCb collaboration, D. Hynds, The Timepix telescope for charged particle tracking, Nucl. Instrum. Methods A 730 (2013) 50. [5] M. Magistris et al., Radionuclide inventory of metallic, TFA radioactive waste, Tech. Note CERN-RP-2015-27-REPORTS-TN EDMS 1501107, 2015. [21] ATLAS MPX, MEDIPIX collaborations, E.H.M. Heijne, et al., Measuring radiation environment in LHC or anywhere else, on your computer screen with Medipix, Nucl. Instrum. Methods A 699 (2013) 198. [6] Ordonnance sur la radioprotection (ORaP), 814.501, Le Conseil Fédéral suisse, 22 juin 1994 (Etat le 1er janvier 2014). [22] T. Whyntie, et al., CERN@school: demonstrating physics with the Timepix detector, Contemp. Phys. (2015) 1. [7] Amec Foster Wheeler’s Analytical Services radiochemical laboratory, Private Communication, 2015. [8] M. Nikl, Scintillation detectors for x-rays, Meas. Sci. Technol. 17 (2006) R37–R54. [23] N. References Stoﬄe, et al., Timepix-based radiation environment monitor measurements aboard the International Space Station, Nucl. Instrum. Methods A 782 (2015) 143. [9] F. Murtas, Applications of triple GEM detectors beyond particle and nuclear physics, JINST 9 (2014) C01058. [24] L. Pinsky, et al., Application of the Medipix2 technology to space radiation dosimetry and hadron therapy beam monitoring, Nucl. Instrum. Methods A 628 (2011) 226. [10] F. Sauli, GEM: a new concept for electron ampliﬁcation in gas detectors, Nucl. Instrum. Methods A 386 (1997) 531. [25] J.H. Hubbell, S.M. Seltzer, Tables of X-ray mass attenuation coeﬃcients and mass energy-absorption coeﬃcients 1 keV to 20 MeV for elements Z=1 to 92 and 48 additional substances of dosimetric interest, NISTIR 5632 (1995). [11] G. Corradi, F. Murtas, D. Tagnani, A novel high-voltage system for a triple GEM detector, Nucl. Instrum. Methods A 572 (2007) 96. [12] M. Alfonsi, et al., High-rate particle triggering with triple-GEM detector, Nucl. Instrum. Methods A 518 (2004) 106. [26] T. Holy, et al., Pattern recognition of tracks induced by individual quanta of ionizing radiation in Medipix2 silicon detector, Nucl. Instrum. Methods A 591 (2008) 287–290. [13] V. Kraus et al., FITPix: Fast Interface for Timepix Pixel detectors, 2011, JINST 6 C01079. [27] Website of AMEC company: 〈http://www.amecfw.com/〉, (accessed 01.12.16). h l ﬁd l f ll b f h l d [14] D. Turecek et al., Pixelman: a multi-platform data acquisition and processing software package for Medipix2, Timepix and Medipix3 detectors, 2011, JINST 6 C01046. [28] N. Gehrels, Conﬁdence limits for small numbers of events in astrophysical data, Astrophys. J. Part 1 303 (1986) 336–346. [29] F. Sauli, et al., Construction, test and commissioning of the triple-gem tracking detector for compass, Nucl. Instrum. Methods A 490 (2002) 177–203. [15] S.P. George, et al., Particle tracking with a Timepix based triple GEM detector, JINST 10 (2015) P11003. 71 71
https://openalex.org/W2889470213	https://www.e3s-conferences.org/articles/e3sconf/pdf/2018/27/e3sconf_nrm2018_00048.pdf	English	null	Legal protection for women environmental activists in urban areas	E3S web of conferences	2,018	cc-by	3,835	Legal protection for women environmental activists in urban areas Tien Handayani Nafi1, Ratih Lestarini1,, Inayati2, Tirtawening1, Succi Wulandhary3, Intan Nurul Aini3, and Dyah Utari4 1Faculty of Law, Universitas Indonesia, Depok, Indonesia 2Faculty of Administrative Science, Universitas Indonesia, Depok, Indonesia 3School of Environmental Science, Universitas Indonesia, Salemba, Indonesia 4Faculty of Health Science, Universitas Pembangunan Nasional Veteran, Jakarta, Indone 1Faculty of Law, Universitas Indonesia, Depok, Indonesia 2Faculty of Administrative Science, Universitas Indonesia, Depok, Indonesia 3School of Environmental Science, Universitas Indonesia, Salemba, Indonesia 4F lt f H lth S i U i it P b N i l V t J k t 1Faculty of Law, Universitas Indonesia, Depok, Indonesia 2Faculty of Administrative Science, Universitas Indonesia, Depok, Indonesia 3School of Environmental Science, Universitas Indonesia, Salemba, Indonesia 4Faculty of Health Science, Universitas Pembangunan Nasional Veteran, Jakarta, Indon , , , 4Faculty of Health Science, Universitas Pembangunan Nasional Veteran, Jakarta, Indonesia Abstract Men and women have equal rights, duties, and degrees in protecting and fighting for the rights of environment. However, in fact, it is men who dominate most conservation and environmental management activities. Even though, women’s roles in environmental conservation activities are also no less great than men’s given their high sense of empathy and care for the environment. This makes women taking more actions to protect the environment itself, but they often get so many threats, physical harass, even murder threats from those who feel threatened by their actions; and it already happened for a long time, and it still does until now. This should be overcome as soon as possible to prevent more women be the next victim. This research used SWOT analysis to determine the strategies for protecting women environmental activists in urban areas. The SWOT calculation results indicate that there is a need for progressive rules in guaranteeing women's rights so as to be able to engage in the process of environmental conservation in their respective regions. Corresponding author: ratihlestarini@yahoo.com © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). E3S Web of Conferences 52, 00048 (2018) CSSPO 2018 E3S Web of Conferences 52, 00048 (2018) CSSPO 2018 https://doi.org/10.1051/e3sconf/20185200048 1 Introduction Issues about women, the last few decades to the present, many fill the discourse in the middle of the life of nation and state in addition to political and economic discourse. This issue is becoming increasingly attractive as awareness of injustice among men and women is increasingly high among our society. In addition, one of the seventeen objectives of the SDGs is gender equality, making it more interesting to continue discussions on this issue of gender equality. Especially at this time the number of women greater than the number of men. On the other hand, women have not yet filled out and occupy influential public sectors in determining the decisions and key state policies [1]. Gender equality is one of the most important aspects of sustainable development. Globally, the phenomenon of gender equality has been long-standing and this can be seen in the achievement of gender equality in some countries of the world from different continents. Until 2015, countries with the highest gender equality are dominated by countries from Continental Europe. However, there are two Asian countries that are able to achieve a very high gender equality, one of which is the ASEAN country, namely E3S Web of Conferences 52, 00048 (2018) CSSPO 2018 https://doi.org/10.1051/e3sconf/20185200048 Singapore [2]. In the pursuit of gender equality, gender justice is the main foundation. Gender equity is a fair process for women and men, to ensure that the process is fair for women and men to take action to stop things that socially and historically discourage women and men from participating in and enjoying the results and the role it plays [3]. Given the value of the UNDP Gender Inequality Index (GII) with the scope of ASEAN, by 2015, gender inequality can be said to have begun to decline. For example in Singapore, gender inequality is ranked 11th in the global scope and the lowest in ASEAN. There is also a Malaysian country whose value of gender inequality lies at the bottom 2 of Singapore. Unfortunately, the value of gender inequality index in Indonesia is ranked second after Cambodia. This means that there are still gaps and gender imbalances in the country of Indonesia in various sectors of life, one of them in the environmental sector. 1 Introduction Gender inequalities in the environment sector in Indonesia can be seen in many activities in the name of 'environmental conservation' and the management and sustainable use of the male-dominated environment [4] [5]. The strong encouragement and motivation of women to fight for environmental sustainability considering their high empathy towards the environment has to be paid with life. According to Global Witness records, by the end of 2016, there were about 200 environmental activists, including female activist, from various countries who died of assassination. Even according to Global Witness, the murder of environmental activists is more 'widespread' than 'developing', which means they must be more vigilant because for the next few years, such killing events will occur in more regions in other countries. Even the number of killings in the year 2016 alone has expanded to 24 countries, more than in 2015 which occurred in only 16 countries [8]. Surely things like this should be addressed as soon as possible so that the number of victims stops growing. Many ways can be done to protect the right of life of environmental activists, one of them by establishing a regulation of protection against them. These regulations may apply in regional and international environments. The hope, with the enactment of this regulation, is to minimize the number of victims as well as the possibility of similar events happening in the years to come so that environmental activists can still fight for rights and obligations to the environment without the need to feel threatened. Based on what had been explained before, the research questions are how is the existing condition of urban women as the environmental activists so far and how do women feel about the enforcement of legal protection which it protects women environmental activists from torture and assasination threats nowadays. The goals of this research are to explore the existing condition of urban women as the environmental activists and also to find out how women feel about the enforcement of legal protection which it protects women environmental activists from torture and assasination threats nowadays. The term gender is used to describe the differences in roles, functions, status, and responsibilities between men and women formed from socio-cultural constructions. The concept of gender equality is a time when women and men have the same status and conditions to fully realize human rights and potential in development in all areas of life. 1 Introduction While gender equality is a fair condition that removes barriers to play a role for women and men through cultural and policy processes [6]. Women's organizations in the field of environment and development have been active in the last fifteen years and have grown considerably over the last decade. The lesson to be learned is the importance of working in an established organization with committed and experienced women with different backgrounds [6]. Results of research by Asteria et. al (2013) indicates the important role of women in overcoming the environmental conflict. Female activists prioritize communication to create harmonious relationships with communication styles that tend to be cooperative considering women have an orientation to maintain relationships in the future and for the 2 E3S Web of Conferences 52, 00048 (2018) CSSPO 2018 https://doi.org/10.1051/e3sconf/20185200048 welfare of society as a whole. Female activists tend to choose mediation and negotiation efforts in environmental conflicts. Female activists in Indonesia will prioritize deliberations to reach agreement on resolving environmental conflicts. It relates to the concept of a self- oriented gender femininity to sustain society sustainably in their community [9]. welfare of society as a whole. Female activists tend to choose mediation and negotiation efforts in environmental conflicts. Female activists in Indonesia will prioritize deliberations to reach agreement on resolving environmental conflicts. It relates to the concept of a self- oriented gender femininity to sustain society sustainably in their community [9]. Not only making some negotiations and mediations to solve the environmental conflicts, women also promoting healthy lifestyle in such many ways and forms as their efforts of the environmental activists. For example, women in Europe has launched a web that promotes healthy lifestyle by giving some tips on maintaining healthy environment for children and baby. The tips are build a baby room with no toxin materials and buy some toxin-free baby and children stuffs to protect children and baby from toxin exposures. Women in developing countries, such as Burkina Faso, also maintaining and purchasing sustainable and healthy lifestyle by making environmentally friendly products with no plastics and no metals [10]. The movement of feminism is a movement of social conflict aimed at breaking down the old values protected by functional structural tradition. Ecofeminism is a movement that serves to restore human consciousness to care about environmental damage due to the disappearance of quality nurturing and maintenance (feminine quality) [9]. 2 Material and method SWOT analysis is a method of determining strategic factors in four areas: Strength (S), Weakness (W), Opportunity (O), and Threat (T) [10]. The SWOT Matrix is a tool to help develop four types of strategies: SO (Strength-Opportunity) strategies, WO (Weakness- Opportunity) strategies, ST strategies (Strength-Threats), and WT (Weakness-Threats) strategies [11]. In the SWOT analysis, the SO (Strength-Opportunity) strategy is the best and for achieving that strategy, and hence relevant factors [12] are required. SO strategies use internal strength to take advantage of external opportunities. The WO strategy aims to improve internal weakness by taking external advantage. The ST strategy uses the company's power to avoid or mitigate the impact of external threats. WT strategy serves to reduce internal weakness and avoid external threats. By using SWOT analysis, four types of strategy will be obtained through internal and external factors, namely aggressive strategy (SO), competitive strategy (ST), conservative strategy (WO), and defense strategy (WT) . The deep interview from many experts to get the judgment to the reality was conducted to support SWOT analysis. 3 Results and discussion T bl 1 SWOT M i 1. Cartesian diagram of SWOT analysis Supporting Rational Strategies Supporting Offensive Stategies Supporting Diversification Strategies Supporting Defensive Strategies Supporting Offensive Stategies Supporting Rational Strategies Supporting Offensive Stategies Supporting Defensive Strategies Supporting Diversification Strategies Supporting Defensive Strategies Supporting Diversification Strategies Fig. 1. Cartesian diagram of SWOT analysis 3 Results and discussion In the case of legal protection for women's environmental activists, there are stronger strengths, weaknesses, opportunities, and threats to be addressed as follows. 1. Strength : The magnitude of the role, caring attitude, and empathy attitude of women to conservation and environmental management activities. 1. Strength : The magnitude of the role, caring attitude, and empathy attitude of women to conservation and environmental management activities. 2. Weakness : Limited opportunities for women to be actively involved in conservation and environmental management activities. 3. Opportunity : Women become more active in voicing their rights as citizens who are also entitled to participate in maintaining and managing the environment even seem to threaten the existence of private projects that tend to damage the environment. 4. Threat : The right to life of women environmental activists is threatened, ranging from just a threat to actually happening cases of torture even to murder. 3 https://doi.org/10.1051/e3sconf/20185200048 E3S Web of Conferences 52, 00048 (2018) CSSPO 2018 The following table presents the SWOT on legal protection cases for women environmental activists. Table 1. SWOT Matrix S (Strength) Internal power factors W (Weakness) Internal weakness factors O (Opportunity) External opportunity factors SO strategy: creating a strategy for women's involvement in conservation activities WO Strategy: creating strategies to increase women's active involvement and role in conservation activities T (Threat) External threat factors Strategy ST: create a strategy using the power of life to deal with threats WT Strategy: Creating a strategy of flexibility of involvement and active participation of women in conservation activities through the granting of a right to life The following table presents the SWOT on legal protection cases for women environmental activists. Table 1. SWOT Matrix S (Strength) Internal power factors W (Weakness) Internal weakness factors O (Opportunity) External opportunity factors SO strategy: creating a strategy for women's involvement in conservation activities WO Strategy: creating strategies to increase women's active involvement and role in conservation activities T (Threat) External threat factors Strategy ST: create a strategy using the power of life to deal with threats WT Strategy: Creating a strategy of flexibility of involvement and active participation of women in conservation activities through the granting of a right to life Fig. 1. Cartesian diagram of SWOT analysis Supporting Rational Strategies Supporting Offensive Stategies Supporting Diversification Strategies Supporting Defensive Strategies The following table presents the SWOT on legal protection cases for women environmental activists. Fig. 1. Cartesian diagram of SWOT analysis The results showed that there are still limitnesses of women’s rules in environmental protections and conservations, whereas their roles and perceptions about environment are also as big as men’s. Those limitnesses make women to more utter their rights as the environmental activists equally as men are. Unfortunately, many of women activitists still feel unsafe about their whereabouts because of their actions. They often get some threats from someone who feel threatened of their actions against environmental destructions. It’s getting worse considering the regulation to protect women from those threats are still strongly unbonding and unprotecting. From here, it is very clear that there has to be some strategies to be made to decrease the limitness towards women’s rules in environmental conservations and protections. In accordance with the results of SWOT analysis based on each strength, weakness, opportunity, and threat, then for this case SO strategy is used. In dealing with and resolving this case, it is necessary to create a strategy for women's involvement in conservation activities. The involvement of women in conservation activities has great potential to realize the main objectives in this activity. The role of women in conservation activities is essentially the same as the conservation actions undertaken by men. However, women have the nature of caring, thorough and meticulous. [13] This is what will eventually complement each other with the male roles in environmental conservation activities and 4 E3S Web of Conferences 52, 00048 (2018) CSSPO 2018 https://doi.org/10.1051/e3sconf/20185200048 actions. Certainly it should also be supported by rights and legal protection for women to participate in conservation activities. actions. Certainly it should also be supported by rights and legal protection for women to participate in conservation activities. The need for a legal protection strategy for women's environmental activists is also important. Through this, there will be no act of women who feel their lives are threatened their due to their involvement in voicing rights and aspirations to the conservation and damage of the environment. Men's gender dominance in the environmental management sector does not necessarily indicate that men have a great empathy and concern for environmental management because, in terms of empathy, it is women who have greater empathy in the environment and the biotic components. Because of its great empathy, women tend to preserve the environment by not exploiting the existing natural resources. Fig. 1. Cartesian diagram of SWOT analysis This is somewhat at odds with the behavior of men who tend to have a sense of 'power' or 'superiority' to their environment so that the possibility of exploitation of nature and its biotic components by men is greater [14]. However, many women still feel that their opportunities to participate actively in environmental activities are still limited or that they can participate as members of environmental groups only [15], whereas in Law Number 32 Year 2009 on Environmental Protection and Management Article 2 has stated that gender differences do not affect management activities or environmental protection because both the rights, obligations, and degrees of men and women are the same in the eyes of the law [16]. Considering the existence of deviations between the policy and its implementation in the field, the emergence of female figures who act as environmental activists aims to achieve gender equality in terms of environmental conservation as stated in the relevant legislation. In the future, if women are given full rights to be able to contribute to environmental conservation, certainly greater and wider potential of environmental sustainability can be realized. The more elements of society that contribute to environmental conservation activities, both oversight and enforcement will certainly ensure the sustainability of the environment. There should be no more parties that restrict the rights of women to get involved in the life of nation and state. Citizens all have inherent rights and it is not justified for those to be restricted. Therefore, gender equality in the above case is very important to be realized. Legal protection for women environmental activists is also the case. Legal safeguards will provide wider space for conservation activities (both coercion and supervision) and contribute to reducing the burden on women themselves. In this context the burden in question is an impact of injustice on the law imposed on women's space. The freedom of space owned by women's environmental conservation activists has become a cornerstone of human rights and the potential for massive environmental conservation. Anticipative action is also required, for example in the aspect of community empowerment so that all agencies together to keep environmental activists. So that the future of women can help maintain and improve the welfare of society and increasing environmental awareness in all aspects of life. E3S Web of Conferences 52, 00048 (2018) CSSPO 2018 Indonesia actually has its own legal protection already to protect women activist’s right and it is well said on Paragraph 66 of Law No. 32 of 2009 on the Protection and Management of the Environment. It says that no environmental activists can be punished by any legal laws. Unfortunately, the implementation of the law is terrible considering there are terrors, physical harass, and even murder threats that still happen to environmental activists, including women, until now; and also the law itself has no explanation on how to protect those activists technically, so most citizens don’t know how to protect people they know, who happens to be women environmental activists. That’s why, to overcome this problem, there’s no need to making any new protection regulations anymore because the problem solving can be done starting with strengthen the laws and its implementation that already exist. First things first, the state should revised the law they already made with putting some technical moves on how to protect activists from threats and physical harass. The state then could increase the public awareness of how important the women presence in any ‘environmental caring, protecting, and keeping’ activities can be by making any public discussions or public demonstrations and citizen, publicly, can participate; hoping that from these strategies, public will aware on how dangerous the risk of being environmental activists could happen and together, they will help to protect the activists from those dangerous risks and the activists themselves are free from those harms that addressed for them and they still can fight for environment without feeling any worries about their safety anymore. Acknowledgements This research is funded by Ministries of Research, Technology and Higher Education/ Kemristekdikti 2018 with Program Penelitian Dasar Ungguan Perguruan Tinggi (PDUPT) 2018 with contract number /UN2.R3.1/HKP05.00/2018 g /UN2.R3.1/HKP05.00/2018 4 Conclusion Legal protection for women engaged in the effort to fight for their rights as citizens entitled to legally manage and protect the environment by channeling their aspirations as environmental activists requires clear instruments. As long as they become environmental activists, the state must be able to protect the existence and rights of women. SWOT calculation results show the importance of women's involvement in conservation of the limb. To achieve this, there needs to be a progressive regulation in ensuring women's rights so as to be able to engage in the process of environmental conservation in their respective regions. This research hopes that woman activist are treated equally and more protected by law in conducting conservation and environmental management movements. 5 https://doi.org/10.1051/e3sconf/20185200048 E3S Web of Conferences 52, 00048 (2018) CSSPO 2018 References 1. Marzuki. Studi tentang kesetaraan gender dalam berbagai aspek. (UNY, Jogjakarta, 2008) 1. Marzuki. Studi tentang kesetaraan gender dalam berbagai aspek. (UNY, Jogjakarta, 2008) 2. United Nations Development Programme. 2015. Human Development Reports: Table 5. Gender Inequalty Index. Retrieved April 3, 2018 from http://hdr.undp.org/en/composite/GII 2. United Nations Development Programme. 2015. Human Development Reports: Table 5. Gender Inequalty Index. Retrieved April 3, 2018 from http://hdr.undp.org/en/composite/GII 3. N.M.D. Widayani, S. Hartati. Kesetaraan dan keadilan gender dalam pandangan perempuan Bali: studi fenomenologis terhadap penulis perempuan Bali. Jurnal Psikologi Undip 13, 2 (2014) 4. I.D.A. Nurhaeni, R. Sugiarti, S. Marwanti, R.D. Pratiwi. Disparitas gender dalam pembangunan pariwisata ramah lingkungan (Gender Disparities in Ecologically Friendly-Tourism Development). PALASTREN Jurnal Studi Gender 10, 1 (2017) 4. I.D.A. Nurhaeni, R. Sugiarti, S. Marwanti, R.D. Pratiwi. Disparitas gender dalam pembangunan pariwisata ramah lingkungan (Gender Disparities in Ecologically Friendly-Tourism Development). PALASTREN Jurnal Studi Gender 10, 1 (2017) 5. Global Witness. 2016. Defenders of the Earth Report: Global Killings of Land and Environmental Defenders in 2016. Retrieved April 3, 2018 from https://www.globalwitness.org/documents/19122/Defenders_of_the_earth_report.pdf. 6. H. Puspitawati. Konsep, teori dan analisis gender. Departemen Ilmu Keluarga dan Konsumen-Institut Pertanian Bogor. (2013) 7. I. Dankelman. Climate Change: Learning from gender analysis and womens’s experiences of organising for sustainable development. Gender and Development 10, (2002) 6 E3S Web of Conferences 52, 00048 (2018) CSSPO 2018 https://doi.org/10.1051/e3sconf/20185200048 8. D. Asteria, E. Suyanti, D. Utari, D. Wisnu. Model of environmental communication with gender perspective in resolving environmental conflict in urban area (study on the role of women’s activist in sustainable environmental conflict management). Procedia Environmental Sciences 20, (2014) 9. M. Samejima, Y. Shimizu, M. Akiyoshi, N. Komoda. SWOT Analysis support tool for verification of business strategy (Graduate School of Information Science and Technology-Osaka Univesity, Japan, 2006) 10. F.R. David. Strategic Management Concepts and Cases (Prentice-Hall Publishing, Francis Marion University, Florance, South Carolina, 2011) 11. S.M. Hatefi. Strategic planning of urban transportation system based on sustainable development dimensions using an integrated SWOT and fuzzy COPRAS approach. Global Journal of Environmental Science and Management 4, 1 (2018) 12. T. Sofiani. Eksistensi Perempuan Pekerja Rumahan dalam Konstelasi Relasi Gender. Muwazah 2, 1 (2010) 13. J. Graca, M.M. Calheiros, A. Oliveira, T.L. Milfont. Why Are Women Less Likely to Support Animal Exploitation than Men? The Mediating Roles of Social Dominance Orientation and Empathy. Personality and Individual Differences 129, (2018) 14. H. Ankesa, S. Amanah, P.S. Asngari. References Partisipasi kelompok perempuan peduli lingkungan dalam penanganan sampah di Sub DAS Cikapundung, Jawa Barat. Jurnal Penyuluhan 12, 2 (2016) 15. Undang-Undang Republik Indonesia Nomor 32 Tahun 2009 Tentang Perlindungan dan Pengelolaan Lingkungan Hidup. Retrieved April 3, 2018 from http://jdih.menlh.go.id/pdf/ind/IND-PUU-1-2009- UU%20No.%2032%20Th%202009_Combine.pdf 16. M. Saleh. Partisipasi perempuan dalam pengelolaan lingkungan hidup. Musawa 6, 2 (2014) 7 7
https://openalex.org/W2002930186	https://www.scielo.br/j/reben/a/vBNhSX4hHSwptNJQDjcJxWd/?lang=pt&format=pdf	Portuguese	null	Enfermeiras-religiosas na luta por espaço no campo da enfermagem	Revista Brasileira de Enfermagem	2,005	cc-by	6,208	RESUMEN Estudio histórico-social, tiene como objetivos: describir las circunstancias de creación de la Unión de Religiosas Enfermeras de Brasil (UREB); analizar las estrategias de las religiosas-enfermeras en busca de espacio en el campo de la enfermería; comentar la importancia de la UREB para los campos de la educación y de la práctica de enfermería. Fuentes primarias: documentos escritos y testimonio oral. Fuentes secundarias: libros, artículos, tesis y disertaciones que tratan sobre el tema. La UREB hay contribuydo mucho para que las enfermeras- religiosas aseguren su participación en los forums de discusión y decisión sobre los diferentes problemas envolviendo la enfermería brasilera. Contribuyó también en el incremento del número de escuelas de enfermería de nivele mediano, así como enviando a las religiosas para las escuelas de enfermería de nivele superior, con vista a inculcar hábitos religiosos a estas enfermeras, instituyendo un nuevo modelo de enfermería católica. Descriptores: Enfermería; Historia de la Enfermería; Escuelas de Enfermería. Rev Bras Enferm 2005 maio-jun; 58(3):361-6. Gomes TO, Almeida Filho AJ, Baptista SS. Enfermeiras-religiosas na luta por espaço no campo da enfermagem. Rev Bras Enferm 2005 maio-jun; 58(3):361-6. Revista Brasileira de Enfermagem REBEn Revista Brasileira de Enfermagem REBEn HISTÓRIA DA ENFERMAGEM Revista Brasileira de Enfermagem Suely de Souza Baptista Doutora em História da Enfermagem. Professora Visitante do DEF, da EEAN/UFRJ. Membro do Nuphebras. Pesquisadora 1B do Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). Antônio José de Almeida Filho Doutor em Enfermagem. Professor assistente do Departamento de Enfermagem Fundamental (DEF) da EEAN/ UFRJ. Membro do Nuphebras. Tatiana de Oliveira Gomes Estudo histórico-social que tem como objetivos: descrever as circunstâncias de criação da União de Religiosas Enfermeiras do Brasil (UREB); analisar as estratégias das enfermeiras religiosas em busca de espaço no campo da enfermagem; comentar a importância da UREB para os campos da educação e da prática da enfermagem. Fontes primárias: documentos escritos e depoimento oral. Fontes secundárias: livros, artigos, teses e dissertações que abordam a temática. A UREB muito contribuiu para que as enfermeiras-religiosas assegurassem sua participação nos fóruns de discussão e decisão de diferentes questões envolvendo a enfermagem brasileira. Ainda contribuiu no incremento no número de escolas de enfermagem de nível médio, bem como no encaminhamento das religiosas para as escolas de enfermagem de nível superior, visando inculcar o habitus religioso nestas enfermeiras, instituindo um novo modelo de enfermagem católica. Descritores: Enfermagem; História da Enfermagem; Escolas de Enfermagem. Enfermeira graduada pela Escola de Enfermagem Anna Nery, da Universidade Federal do Rio de Janeiro (EEAN/UFRJ). Enfermeira residente em Enfermagem Neonatal do Instituto Fernandes Figueira. Membro do Núcleo de Pesquisa de Historia da Enfermagem Brasileira (Nuphebras). ABSTRACT Historic social study that aims to describe the circumstances of creation of the Union of Brazilian Religious Nurses (UBRN); to analyze the strategies of the religious nurses in their seek for space in nursing area; to comment the importance UBRN for education area and nursing practice. Primary sources: written documents and oral report; secondary sources: books, articles, thesis and dissertations related to the thematic. The UBRN contributed for religious nurses have assured their participation in forum and decision of different questions involving the Brazilian nursing. Still it contributed for the increment in the number of nursing schools both in technic and superior level, as well as in directing religious nurses to faculty, aiming to inculcate to those nurses the religious habits, instituting a new model of catholic nursing. Descriptors: Nursing; History of Nursing; Schools, nursing. Rev Bras Enferm 2005 maio-jun; 58(3):361-6. 1. INTRODUÇÃO O objeto deste estudo é o movimento das enfermeiras religiosas em prol da criação e consolidação de uma associação profissional própria, qual seja, a União de Religiosas Enfermeiras do Brasil (UREB), nos anos 40. Assim, o contexto sócio-político e econômico no qual se insere a presente pesquisa é o da década de 40, do século XX. Do período colonial até a Proclamação da República, a Igreja católica constituía-se numa instituição incorporada ao Estado e dentre outras coisas, mantinha o monopólio do cuidado aos doentes e da administração dos hospitais, principalmente nas Santas Casas de Misericórdia, as quais enunciavam um discurso de cunho religioso-caritativo. Porém, a classe médica insatisfeita com esta situação, procurou apoio no movimento positivista, no que foi bem sucedida. Tanto que, 361 Gomes TO, Almeida Filho AJ, Baptista SS. a Igreja perdeu espaço no campo da saúde em virtude do médico assumir, sobretudo, cargos de direção e a prerrogativa de ser o responsável pela admissão e alta dos pacientes(1,2). e freiras exercendo as atividades eclesiásticas, os seminários deficientes em quantidade e qualidade, além da presença do imperador D. Pedro II, que apesar de titular da Igreja, era um católico pouco fervoroso que mantinha fracos vínculos com o Vaticano(1). Desde então, a Igreja passou por reformas internas visando reverter a decadência institucional das décadas anteriores; para isso recrutou e importou novos membros, criou novas dioceses, bem como aumentou o controle episcopal relacionado às atividades clericais. Este panorama também ficou evidenciado, através de uma Carta Pastoral publicada em 1916, por D. Sebastião Leme, apresentando um diagnóstico da situação da Igreja à época, bem como as estratégias necessárias para alterar aquela realidade. Este movimento foi denominado de Igreja da neocristandade(1). A ruptura da Igreja com o Estado foi oficializada na Constituição de 1891 e desde então, ela esteve voltada para reformas internas, as quais a ajudariam a melhorar sua posição no espaço social, bem como a libertaria da relação de dominação do Estado(1,5). Com relação às congregações católicas existentes em serviços hospitalares, ao final do século XIX, houve um movimento organizado pela classe médica, objetivando instituir o médico em cargos de direção e, como conseqüência, as irmãs de caridade não mais puderam assumir tais funções, e a Igreja, então perdeu espaço tanto na prestação de cuidados de enfermagem como na administração dos hospitais a ela não vinculados(6). 1. INTRODUÇÃO O referido modelo atingiu seu apogeu no período de 1930 a 1945, na presidência de Getúlio Vargas, época em que a Igreja católica firmou uma forte aliança com o Estado, embasada, sobretudo, em troca mútua(1). Um dos desdobramentos dessa situação foi o aumento no número de escolas de enfermagem de cunho religioso(2). Dentre elas está a Escola de Enfermeiras do Hospital São Paulo (EEHSP), criada em 1938, por iniciativa de Madre Domeneuca. Cabe destacar que em 1944, ex-alunas desta Escola se reuniram para planejar a fundação da UREB(3,4). Após quatro anos da existência desta Associação, foi então criada a União Católica de Enfermeiras do Brasil (UCEB), a fim de obter maior aproximação com as enfermeiras católicas leigas(3). De outra forma, a criação de escolas de enfermeiras laicas, abalou ainda mais a situação das enfermeiras-religiosas(6). Neste sentido, podemos constatar que até a década de 20, existiam cinco escolas de enfermagem, todas localizadas na região sudeste, sendo uma de cunho evangélico (20%), duas filantrópicas (40%) e duas de caráter federal (40%). Ressalta-se o fato de no ano de 1923 ser criada a Escola de Enfermeiras do DNSP (atual Escola de Enfermagem Anna Nery/UFRJ), a qual em 1931 passou a ser denominada “escola-padrão”, através do Decreto nº 20.109 de 15 de julho de 1931, o qual reconheceu-a como escola oficial padrão para efeito da criação e equiparação de outras escolas de enfermagem. Com isso, somente poderiam exercer a profissão as pessoas que tivessem capital institucionalizado, fornecido pelo diploma de enfermeira adquirido na EAN ou em outra escola a ela equiparada(7,8). Vale lembrar que a criação da UREB deu-se no bojo do movimento católico e também que esta instituição influenciou o campo da educação em enfermagem no Brasil, tanto no âmbito do ensino superior quanto do ensino médio. Desta forma, tal estudo tem como objetivos: descrever as circunstâncias de criação da UREB; analisar as estratégias das religiosas-enfermeiras em busca de espaço no campo da enfermagem e, comentar a importância da UREB para os campos da educação e da prática da enfermagem. O ano de 1916, data do surgimento do modelo da neocristandade, marcou um novo período para a Igreja, com a publicação da carta pastoral, pelo eminente líder da Igreja católica brasileira à época, D. Sebastião Leme, na condição de arcebispo de Recife e Olinda. b 12 de agosto de 1926: Associação Nacional de Enfermeiras Diplomadas (ANED); 1 de junho de 1929: Associação Nacional de Enfermeiras Diplomadas Brasileiras (ANEDB); 7 de agosto de 1944: Associação Brasileira de Enfermeiras Diplomadas (ABED); 21 de agosto de 1954: Associação Brasileira de Enfermagem (ABEn). 1. INTRODUÇÃO Esta carta atentava, entre outros pontos, para a fragilidade da Igreja institucional, sua limitada influência política e para o Estado precário da educação religiosa(1). Além disso, argumentava que a Igreja deveria se organizar, se unificar e pressionar o Governo brasileiro(5). Este estudo, de cunho histórico-social, tem como recorte temporal o período compreendido entre 1942 e 1948. O marco inicial corresponde ao ano de formatura da primeira turma da Escola de Enfermeiras do Hospital São Paulo (EEHSP) e o terminal ao ano de criação da UCEB. Fontes primárias: documentos escritos como atas das diretoras da Escola Anna Nery, estatuto da UREB, ofícios expedidos e o depoimento oral da Madre Marie Domeneuc, localizados no Acervo do Centro de Documentação da Escola de Enfermagem Anna Nery (EEAN); e ofícios localizados no Departamento de Arquivo e Documentação da Casa de Oswaldo Cruz-Fiocruz. Fontes secundárias: artigos, livros, teses e dissertações que abordam o contexto sócio-político do Brasil com destaque para a trajetória da Igreja Católica e da enfermagem, no período estudado. A literatura consultada encontra-se na Biblioteca Setorial de Pós-graduação da EEAN, no Banco de Textos e no acervo “Quem é Quem na História da Enfermagem Brasileira”, ambos do Núcleo de Pesquisa de História da Enfermagem Brasileira (Nuphebras). Vale ressaltar que a análise crítica dos documentos foi realizada face ao contexto no qual os mesmos foram produzidos. Para atingir tais objetivos as estratégias seriam “cristianizar as principais instituições sociais, desenvolver um quadro de intelectuais católicos e alinhar as práticas religiosas populares aos procedimentos ortodoxos”(1). Porém, somente na década de 20 é que o modelo da neocristandade ganhou força e a Igreja passou a ser vista pelos políticos como “um meio de aumentar sua legitimidade aos olhos do povo”(5). Assim é que, surgiram movimentos associativos, visando o fortalecimento da classe religiosa. No caso da enfermagem tal fato pode ser exemplificado pelo resultado do encontro de enfermeiras católicas de diversos países, em Basiléia, Suíça, no ano de 1928, o qual reforçou a necessidade de ser criada uma associação mundial de enfermeiras católicas, qual seja: Comitê Internacional Católico de Enfermeiras e Assistentes Médico-Sociais (CICIAMS). Tal Comitê, organizado por presidentes das Associações já existentes em vários países, tinha como um de seus objetivos: “estimular, em todos os países, a criação e o desenvolvimento de associações profissionais católicas a fim de assegurar apoio moral e espiritual às enfermeiras e assistentes médico- sociais católicas, bem como seu aperfeiçoamento técnico”(3,9). Após muitas lutas e vitórias, veio a falecer em 10 de março de 1998, aos oitenta e seis anos. a Nascida na Bretanha, França, em 11 de novembro de 1911. Titulou-se pela Escola de Enfermagem de Paris em Enfermeira e Visitadora de Higiene. Durante sua vida profissional, entre outras realizações, fundou e assumiu a direção de uma escola de enfermagem para religiosas em São Paulo, fundou a UREB, criou o 1º Curso de Especialização em Enfermagem Obstétrica na EEHSP, fundou uma escola de auxiliares de enfermagem. Em reconhecimento a tais feitos, foi condecorada pelo governo francês, com a “Palmes Academiques. Rev Bras Enferm 2005 maio-jun; 58(3):361-6. 4. A UREB E SUA ATUAÇÃO Na década de 40, dois movimentos associativos se organizaram para assegurar os interesses específicos das enfermeiras-religiosas, quais sejam: a União das Religiosas Enfermeiras do Brasil (UREB) e a União Católica de Enfermeiras do Brasil (UCEB). A UREB foi criada em 1944, por ex-alunas da Escola de Enfermeiras do Hospital São Paulo, e por iniciativa de Madre Marie Domeneuc, fundadora da escola e membro atuante da ABED, para funcionar como órgão técnico indispensável ao apostolado católico(15). Com a promulgação da Constituição de 1934, a Igreja teve uma grande vitória, visto que as principais exigências da LEC foram incorporadas à mesma, inclusive a educação religiosa durante o período escolar e subsídios para as escolas católicas(1,5). Associado a tal fato, o decreto nº 22.257, de 26 de dezembro de 1932, assinado por Vargas, possibilitou às religiosas direitos iguais aos das enfermeiras diplomadas, desde que comprovassem seis ou mais anos de prática em enfermagem. Vale ressaltar que tal direito era restrito ao âmbito hospitalar(6). Em 18 de maio deste mesmo ano, foi fundado o primeiro núcleo da UREB, no Rio de janeiro, durante um encontro de religiosas enfermeiras de várias congregações católicas, contando na ocasião com a orientação do cardeal arcebispo do Rio de Janeiro, D. Jaime de Barros Câmara(3). Em novembro de 1937, após um golpe de Estado, liderado por Vargas, inicia-se o Estado Novo (1937 – 1945), período do governo sob o regime ditatorial, no qual o presidente Getúlio Vargas aboliu a Constituição de 1934, mas sua relação com a Igreja não foi desfeita(5,12). Vale ressaltar que aos quatro dias do mês de outubro de 1938 e anexa à Escola Paulista de Medicina, foi criada por Madre Domeneuc, a primeira escola de enfermagem católica do país, a Escola de Enfermeiras do Hospital São Paulo (EEHSP), a qual iniciou suas atividades em março de 1939, nos moldes da EAN. Destaca-se o fato de que para que as enfermeiras-religiosas que integravam o corpo docente da EEHSP pudessem exercer suas atividades era necessário Em novembro de 1937, após um golpe de Estado, liderado por Vargas, inicia-se o Estado Novo (1937 – 1945), período do governo sob o regime ditatorial, no qual o presidente Getúlio Vargas aboliu a Constituição de 1934, mas sua relação com a Igreja não foi desfeita(5,12). 3. A ERA VARGAS E A (RE)INSERÇÃO DA IGREJA CATÓLICA NO CENÁRIO DE DECISÕES: O APOIO À ENFERMAGEM CATÓLICA Na década de 30 foram criadas cinco escolas de enfermagem: 1 estadual (20%), duas evangélicas (40%) e duas católicas (40%). Podemos afirmar também que durante o Estado Novo, houve um grande crescimento no número de escolas de enfermagem ligadas às congregações católicas. Constata-se ainda que neste período foram criadas dez Escolas de Enfermagem, sendo seis católicas (60%), três estaduais (30%) e 1 municipal (10%). A distribuição geográfica destas escolas demonstra a predominância da região Sudeste sobre as demais: Sudeste - 6 (60%), Nordeste - 2 (20%), Centro Oeste - 1 (10%) e Norte - 1 (10%). É fato também que até 1945 a região Sul não contava com escolas de enfermagem(2). Durante a Era Vargas (1930-1945), a proximidade Igreja-Estado, se tornou efetiva e marcante, e a neocristandade atingiu seu apogeu. Contudo, o apoio da Igreja ao presidente Getúlio Vargas ocorreu não só pelo recebimento de privilégios, mas também por afinidade política, caracterizada pelo fato da Igreja dar ênfase à ordem, ao nacionalismo, ao patriotismo e ao anticomunismo, lemas do Governo Vargas(1). Com o decreto 20.109/31 que reconheceu a EAN como “escola oficial padrão”, o qual afirmava que só seriam reconhecidos como enfermeiros os portadores de diploma fornecido ou revalidado por esta Escola, a Igreja passou a procurar amparo legal para as religiosas que trabalhavam nos hospitais, bem como se organizou para encaminhar as religiosas objetivando a obtenção do diploma de enfermeiras, em virtude das mesmas não possuírem tal título. Para isso, foi fundamental a aproximação de Laís Neto dos Reys, diplomada da turma pioneira da EAN em 1925, com a Igreja católica. Laís dirigiu e organizou a primeira escola a formar religiosas no Brasil, qual seja a Escola de Enfermagem Carlos Chagas. Tal fato tornou-se extremamente importante para defender o espaço da Igreja católica no campo da enfermagem, além de possibilitar a criação de outras escolas de enfermagem sob orientação católica(2,8,10). Cabe destacar que o ano de 1944, foi muito relevante no que tange a situação da ABED (atual ABEn), que naquele momento apresentava o reduzido número de 60 associadas quites(10). Este fato “demonstra que tal associação já não vinha despertando o interesse das enfermeiras”(3). Tal quantitativo representava 4,2% do número total de 1405 enfermeiras diplomadas à época na cidade do Rio de Janeiro(13). 2. A IGREJA CATÓLICA E A ENFERMAGEM BRASILEIRA Em meados do século XIX a Igreja encontrava-se em uma situação de fragilidade, caracterizada, sobretudo, pelo reduzido número de padres Anteriormente ao CICIAMS, cabe ressaltar, a existência a partir de 1926, da atual Associação Brasileira de Enfermagem (ABEn)b, a qual Rev Bras Enferm 2005 maio-jun; 58(3):361-6. 362 Enfermeiras-religiosas na luta por espaço no campo da enfermagem que tivessem seus diplomas revalidados. Após um ano de criação da escola ocorreu o exame de revalidação do diploma de três religiosas: Maria de Fontenelle, Maria Hermana e Madre Domeneuc(6). surgiu a partir da iniciativa da associação de ex-alunas da EAN visando associarem a seus diplomas uma credencial de membro da referida associação profissional(10). Em 1933, o CICIAMS realizou seu 1º Congresso, em Lourdes, na França, e a partir desta data iniciaram-se as atividades do Comitê. Até o ano de 1937, os congressos foram realizados a cada dois anos, quando temporariamente foram interrompidos, devido à 2ª Guerra Mundial, recomeçando em 1946. A partir de então, passaram a ser realizados a cada quatro anos(3,9). Durante o Governo Vargas, principalmente durante o Estado Novo, houve um grande investimento na área da educação, visto que este presidente percebia o ensino como um instrumento necessário para a formação de mão de obra qualificada, bem como um meio eficaz de difundir sua ideologia governamental(2). Tal estratégia teve repercussões no campo da educação em enfermagem, tanto que entre 1930 e 1945 foram criadas treze novas escolas. 3. A ERA VARGAS E A (RE)INSERÇÃO DA IGREJA CATÓLICA NO CENÁRIO DE DECISÕES: O APOIO À ENFERMAGEM CATÓLICA Ressaltando a situação da atual ABEn, Madre Domeneuc reforçou em seu depoimento ao ser questionada acerca do nível da entidade desde sua fundação e a mesma respondeu: “A ABEn nunca tomou a força de uma associação realmente de caráter nacional”(14). Cabe inferir que divergências ideológicas ocorriam entre as lideranças da ABED e as enfermeiras-religiosas, e foi neste contexto, também em 1944, que diplomadas da primeira turma da EEHSP, sob a liderança de Madre Domeneuc mobilizaram-se para a criação da União de Religiosas Enfermeiras do Brasil (UREB), com a finalidade de reunir as enfermeiras-religiosas em um grupo distinto(3,10). Ainda em 1931, foi formalizado o “pacto” entre a Igreja e o Estado, através do decreto nº 19.941, que dispunha acerca da obrigatoriedade da instrução religiosa nos cursos: primário, secundário e normal. Por este decreto, os alunos só poderiam ser dispensados de freqüentar as aulas de religião, se os pais ou responsáveis fizessem tal solicitação(11). Para a Igreja, o Estado deveria seguir sua doutrina social, bem como proteger seus interesses, entre eles a reformulação do sistema educacional. Desta forma, em 1932 foi criada por D. Sebastião Leme, a Liga Eleitoral Católica (LEC)(1), com o objetivo de orientar e organizar o eleitorado católico de como votar, de modo a assegurar poder e prestígio à Igreja Católica(1,5). Rev Bras Enferm 2005 maio-jun; 58(3):361-6. 4. A UREB E SUA ATUAÇÃO Este evento, que contou com a participação de várias religiosas, dentre essas, Madre Marie Domeneuc, recomendou em uma de suas resoluções, a criação de dois tipos de escolas, destinadas ao ensino de enfermagem às religiosas: uma para o ensino superior de enfermagem, conforme previa o estatuto da UREB, que deveria funcionar na Pontifícia Universidade Católica de São Paulo, cuja finalidade seria “o preparo de enfermeiras para a docência a fim de melhorar o nível e a eficiência das escolas católicas”, e outra, de ensino médioc(15). Em julho de 1946, a UREB promoveu o segundo Congresso Nacional de Enfermeiras Religiosas. Nesse conclave, as participantes recomendaram que a UREB aderisse à ABED e, desta forma, colaborasse com as enfermeiras seculares. A UREB foi representada na ABED através do seu Conselho Técnico Administrativo(3). A aproximação com a ABED teve o apoio de Madre Domeneuc, que manifestou preocupação com a “separação em grupos que se prenunciava” na enfermagem. Além do mais, Madre Domeneuc defendia que apenas um congresso de âmbito nacional seria capaz de unir as enfermeiras. Nesse sentido, sugeriu que fosse criado tal congresso, o que aconteceu um ano após, em 1947, com a sua participação efetiva e de outros membros da UREBd, dentre as quais mereceu destaque a presença de Irmã Matilde Nina, a primeira religiosa a obter o diploma de enfermeira no Brasil e fundadora da Escola de Enfermeiras Luíza de Marillac(3,10). A proposta de criação de um estabelecimento de ensino superior de enfermagem não chegou a ser implementada(15). No entanto, no que se refere ao nível médio, na década de 40, foram criados 11 cursos de auxiliar de enfermagem (5 no Distrito Federal, 1 em Santos, 1 em São Paulo, 1 em Petrópolis, 1 em Belo Horizonte, 1 em Porto Alegre e 1 em Juiz de Fora), sendo 4 vinculados a instituições religiosas católicas, correspondendo a 36% do total(16). O investimento das Congregações católicas nesse nível de formação também foi bastante expressivo ao longo da década de 50, pois, dos 45 cursos de auxiliares de enfermagem criados nesse período, 18 (40%) estavam vinculados a instituições católicas. Certamente, este fenômeno manteve nexos com a legalização dessa categoria, através da promulgação da Lei 775/ 49(16). 4. A UREB E SUA ATUAÇÃO O estatuto da UREB previa a criação de um centro de estudos das questões médico-sociais de interesse para o apostolado; de um organismo de assistência médica para assistir as religiosas doentes e de uma Escola Superior, para aperfeiçoar as enfermeiras titulares, encarregadas do ensino da enfermagem ou da direção de serviços de saúde(15). Vale ressaltar que aos quatro dias do mês de outubro de 1938 e anexa à Escola Paulista de Medicina, foi criada por Madre Domeneuc, a primeira escola de enfermagem católica do país, a Escola de Enfermeiras do Hospital São Paulo (EEHSP), a qual iniciou suas atividades em março de 1939, nos moldes da EAN. Destaca-se o fato de que para que as enfermeiras-religiosas que integravam o corpo docente da EEHSP pudessem exercer suas atividades era necessário A administração da UREB era constituída pela Assembléia Geral, composta por todas as sócias fundadoras e efetivas; pelo Conselho Diretor, composto por uma presidente, uma vice-presidente e seis conselheiras eleitas pela Assembléia Geral, com mandato de três anos. O mesmo estatuto definiu que haveria uma Assembléia Ordinária da 363 Gomes TO, Almeida Filho AJ, Baptista SS. Gomes TO, Almeida Filho AJ, Baptista SS. UREB, independente de convocação, na sede social, no dia 18 de julho de cada ano, por se tratar de data comemorativa de São Camilo de Lellis(15). internacional estimulava o discurso nacionalista, por outro, a ameaça de mobilização das massas sustentava o receio de que a conjuntura nacional favorecesse a “comunização” do país e a instauração de uma ‘república sindicalista’(19). Com a finalidade de discutir e estabelecer estratégias para melhor inserção das religiosas no campo da educação em enfermagem, além de ampliar a visibilidade do movimento, a UREB organizou, em novembro de 1944, o Primeiro Congresso Nacional de Enfermeiras Religiosas, em São Paulo, sob a presidência do cardeal arcebispo dessa cidade, D. Carlos Carmelo de Vasconcelos Mota(3). No bojo dessa tensão política que predominava na Capital do Brasil, em junho de 1946, a sede da UREB foi transferida para São Paulo, afastando-se, portanto, do cenário em que se intensificaram os embates políticos no plano nacional. Dessa forma, a UREB foi acolhida pela Escola de Enfermeiras do Hospital São Paulo e, sob a liderança de Madre Domeneuc, definiu importantes diretrizes políticas para assegurar o poder e o prestígio às religiosas enfermeiras. 4. A UREB E SUA ATUAÇÃO Para melhor elucidação, ressalta-se que estes números foram obtidos ao se considerar o vínculo do referido curso com instituições católicas, ou procedida uma associação entre o nome do curso e uma personagem católica ou a figuras santificadas pela Igreja Católica. Ao mesmo tempo, a UREB incentivou as congregações religiosas à criação de escolas de enfermagem de ensino médio e superior e encaminhamento de suas religiosas para as escolas de enfermagem(3). Vale recordar que a diretora da EAN, Laís Netto dos Reys, em outros momentos, também estimulou a presença dessas religiosas nas escolas de enfermagem, sobretudo naquelas sob sua gerência: Escola de Enfermagem Carlos Chagas e Escola Anna Nery. A UREB também criou, em 4 de abril de 1948, na Vila Betânia, em São Paulo, a União Católica de Enfermeiras do Brasil (UCEB), sob a benção apostólica de sua Eminência o Cardeal D. Carlos Carmelo de Vasconcelos Mota, arcebispo de São Paulo, e na presença de sua Excelência D. Antônio Maria Alves Siqueira, Bispo Auxiliar de São Paulo. Criada sob o lema “UBI CARITAS, IBI CHRISTUS, que significa: “onde está a caridade, aí está Cristo”, a UCEB pretendia funcionar como elo de aproximação do entendimento entre as enfermeiras católicas leigas e as religiosas. Assim, cabia à essa organização “desenvolver, proteger e encorajar a vida espiritual, profissional, cultural e social das enfermeiras católicas”(15). Desta forma, podemos constatar que foi bem sucedida a estratégia definida pela UREB, quanto à formação de pessoal de nível médio, incorporando a doutrina católica. Assim, criaram-se as condições necessárias para preservar a luta pela ocupação do espaço de atuação no campo da enfermagem. Um exemplo emblemático do investimento das religiosas enfermeiras na formação de pessoal de nível médio foi a participação de Madre Domeneuc, ainda em 1944, no planejamento da Escola de Enfermeiras Auxiliares São José, a qual foi fundada em março de 1945(3,17). Atendendo as exigências da Lei 775/49, esta passou a ser denominada Escola de Auxiliares de Enfermagem São José, dirigida por religiosas-enfermeiras da Congregação de São José. Mais tarde passou a denominar-se Escola de Enfermagem São José, devido a criação do curso de graduação(3). d Não foi possível a identificação dessas religiosas-enfermeiras no I Congresso Nacional de Enfermagem, nem obter informações relativas ao quantitativo de religiosas nele presentes. cO ensino médio aqui tratado refere-se ao curso de auxiliar de enfermagem. Rev Bras Enferm 2005 maio-jun; 58(3):361-6. 5. CONSIDERAÇÕES FINAIS Dentre as escolas de enfermagem católicas criadas podemos citar a EEHSP, cujas ex-alunas religiosas, e por iniciativa de Madre Domeneuc, criaram a UREB, em 1944, com a finalidade de reunir as religiosas-enfermeiras. Quatro anos mais tarde, a UREB, com o propósito de se aproximar das enfermeiras católicas laicas, fundou a UCEB. Cabe destacar que à época, as divergências ideológicas existentes entre as líderes da atual ABEn e as enfermeiras-religiosas levou estas a criarem uma Associação própria para poderem divulgar a ideologia católica. Adicionalmente, desde 1935, por ocasião do II Congresso Interna- cional de Enfermagem, organizado pelo CICIAMS, realizado em Roma, o Cardeal Pizzardo solicitou às congressistas que votassem algumas resoluções, as quais foram aprovadas por unanimidade e grande entusiasmo. Neste sentido merece relevo a que apresentava como exigência a concentração de esforços das enfermeiras católicas e religiosas no sentido de “reconduzir as almas desgarradas à concepção verdadeiramente cristã da vida, do dever, e do sofrimento”(9,20). Desta forma, podemos identificar que a UREB apesar de ter incentivado a criação de escolas dos dois níveis referidos no Primeiro Congresso Nacional de Enfermeiras Religiosas, só obteve sucesso no nível médio de formação. Por outro lado, sua contribuição foi decisiva no sentido de incentivar as congregações a encaminharem suas religiosas para as escolas de nível superior visando a formação de enfermeiras. Esta providência se tornou de extrema valia, visto que as religiosas trabalhavam na maioria dos hospitais do País e, no entanto, a primeira religiosa-enfermeira havia se diplomado somente no ano de 1936, ou seja, mais de dez anos após a formatura do primeiro grupo de enfermeiras formadas pela EAN o que demonstrava o reduzido quantitativo de enfermeiras-religiosas à época. Nessa linha de pensamento, em atenção às determinações do apostolado católico, tanto a UREB quanto a UCEB, tiveram papel destacado, no intuito de defender através da enfermagem, o “cristianismo autêntico, respeitador de todos os princípios da moral cristã”, considerando a pessoa humana, tanto na sua vida física e psíquica, quanto na sua vida familiar e social(20). É fato que a UREB muito contribuiu para que as enfermeiras-religiosas assegurassem sua participação nos fóruns de discussão e de decisão de diferentes questões envolvendo a enfermagem brasileira. 5. CONSIDERAÇÕES FINAIS Após a Proclamação da República, a Igreja Católica perdeu espaço em todos os campos nos quais, até então, mantinha quase que uma hegemonia, com destaque para os da educação e da saúde. Investindo, de modo a preservar os interesses católicos no campo da enfermagem, a UCEB, através das seções diocesanas, ofereceu aos seus membros várias alternativas de atividades, objetivando a “recristianização da sociedade, no setor de enfermagem”. Para tanto, atribuiu aos membros dessa organização católica algumas responsabilidades, tais como: manter a unidade das enfermeiras católicas na sua profissão; preservar a prática dos ideais cristãos; habilidade para responder algumas questões de princípios cristãos e, retidão na vida pública. Unindo esses atributos, a enfermeira católica estaria apta a “exercer uma influência profunda na difusão, na defesa e na aplicação dos princípios católicos”(15). Alguns anos após, em 1916, a carta pastoral propôs mudanças internas na Igreja e neste sentido, surgiu o modelo da neocristandade, o qual ganhou força a partir do final da década de 20. Neste meio tempo, foi inaugurada, em 1923, a Escola de Enfermeiras do DNSP, a qual ameaçava a atuação das religiosas nos espaços hospitalares. Neste contexto, ressalta-se que o Governo Vargas sustentou uma importante aliança com a Igreja Católica. Neste sentido, observamos um aumento no número de Escolas de Enfermagem Católicas, o que mudou a configuração do campo da educação em enfermagem à época. Vale ressaltar a participação efetiva de Laís Netto dos Reys, ex-aluna da turma pioneira da EAN, católica fervorosa, que muito contribuiu para a criação destas escolas, conseqüentemente elevando o quantitativo de enfermeiras religiosas diplomadas. O investimento da Igreja Católica, através da UREB e, mais tarde, com a participação da UCEB, possibilitou ampliar o período de atuação de religiosas-enfermeiras nas instituições hospitalares, pois estas associações mobilizavam-se para oferecer melhor qualificação às religiosas que atuavam no cuidado de enfermagem, ao mesmo tempo que incentivavam a criação de outras escolas de enfermagem católicas e também de cursos de auxiliares para religiosas. Além disso, o materialismo e o paganismo contemporâneos eram objeto de grande preocupação por parte da Igreja Católica. Provavelmente esta preocupação apoiava-se no crescimento de nações sob a égide comunista, após a segunda guerra mundial, onde prevalecia a divisão geográfica mundial em dois grandes grupos(20). 4. A UREB E SUA ATUAÇÃO O Boletim nº 4, instrumento de divulgação oficial da UCEB, explicita a dimensão do investimento de seus membros para difundir os valores católicos na enfermagem brasileira, ao registrar textualmente: “o pensamento e os esforços individuais, nela [ na UCEB ] se unificam, tornando-se uma força poderosa de ação católica na profissão da enfermagem”(15). Vale ressaltar que uma vez organizada e com o estatuto pronto, a UREB objetivando seu fortalecimento, tratou de filiar-se ao Comitê Internacional Católico de Enfermeiras e Assistentes Médico-Sociais (CICIAMS)(3,10). A UCEB teve sua primeira diretoria eleita durante o II Congresso Nacional de Enfermagem, com Celina Viegas escolhida para a presidência; Cecília Pêcego Coelho, como vice-presidente; Aurea Marques da Silva, 1ª secretária; Flora Mesentier, 2ª secretária e Cecília Sete Torres, como tesoureira. Na oportunidade, Laís Netto dos Reys também foi eleita, por aclamação, presidente de honra da entidade(15). Com o fim da Segunda Guerra Mundial e a derrota do nazi-facismo, verificaram-se mudanças significativas nas relações internacionais, caracterizadas pelo fim da hegemonia européia. À época, teve início uma nova fase histórica, sob o signo da bipolaridade, em que os Estados Unidos e a União Soviética demarcaram vigorosamente o conflito ideológico, na dinâmica das relações internacionais(18). A Guerra Fria colaborava para acentuar o radicalismo dos discursos e das alternativas formuladas na arena política brasileira: por um lado, a conjuntura política A distinção dispensada à diretora da EAN demonstrava reconhecimento pelo empenho de Laís Netto dos Reys, enquanto Rev Bras Enferm 2005 maio-jun; 58(3):361-6. 364 Enfermeiras-religiosas na luta por espaço no campo da enfermagem Enfermeiras-religiosas na luta por espaço no campo da enfermagem importante líder católica da enfermagem brasileira, em função do desenvolvimento da enfermagem religiosa. 10. Barreira IA, Sauthier J, Baptista SS. O movimento associativo das enfermeiras diplomadas na primeira metade do século 20. Rev Bras Enferm 2001; 53(4): 157-73. 5. CONSIDERAÇÕES FINAIS Podemos concluir ainda que a UREB, mediante sua significativa contribuição, no que tange ao incremento do número de escolas de enfermagem, tanto de nível médio como superior, tinha o compromisso de inculcar o habitus religioso nas futuras enfermeiras e auxiliares de enfermagem, instituindo um novo modelo de enfermeira/enfermagem católica. Até meados de 1954, a UREB seguiu com suas atividades vinculadas aos seus objetivos. Porém, anos mais tarde fundiu-se ao Departamento de Saúde, da Conferência dos Religiosos do Brasil (CRB). Em 1956, a UCEB se dissolveu em virtude de seus associados concluírem que não havia no País à época uma associação contra a religião católica, mas sim de caráter neutro. Como seria muito difícil para as religiosas continuarem investindo, de forma eficiente, tanto na UREB como na ABEn, suas afiliadas optaram pela participação efetiva junto à ABEn(3). 7. Sauthier J, Barreira IA. As enfermeiras norte-americanas e o ensino da enfermagem na capital do Brasil: 1921-1931. Rio de Janeiro (RJ): Editora Anna Nery; 1999. Rev Bras Enferm 2005 maio-jun; 58(3):361-6. 8. Bezerra MRM. A enfermagem e a aliança da igreja com o estado: escola de enfermeiras Luiza de Marillac [dissertação]. Rio de Janeiro (RJ): Escola de Enfermagem Anna Nery, Universidade Federal do Rio de Janeiro; 2002. 1. Mainwaring S. A igreja católica e a política no Brasil (1916-1985). São Paulo (SP): Brasiliense; 1989. 2. Baptista SS, Barreira IA. Condições de surgimento das escolas de enfermagem brasileiras (1890-1960). Rev Alternativa Enferm 1997; 1(2): 4-17. 9. Pirovano CA. Sobre o CICIAMS. Rev Bras Enferm 1961; 14(4): 381-84. 1. Mainwaring S. A igreja católica e a política no Brasil (1916-1985). São Paulo (SP): Brasiliense; 1989. 2. Baptista SS, Barreira IA. Condições de surgimento das escolas de enfermagem brasileiras (1890-1960). Rev Alternativa Enferm 1997; 1(2): 4-17. 3. Carvalho AC. Associação Brasileira de Enfermagem 1926-1976 Documentário. Brasília (DF): ABEn; 1976. 4. Souza AR, Baptista SS. A adoção do modelo anglo-americano de ensino de enfermagem na capital paulista. Rev Escola Enferm Anna Nery 2002; 6(2): 211-27. 5. Bruneau TCB. A revolução de 1930: reinteração da igreja no estado. In: Bruneau TCB. Catolicismo em época de transição. São Paulo (SP): Edições Loyola; 1974; p. 77-97. 6. Souza AR, Baptista SS. A criação e o legado da Escola de Enfermeiras do Hospital São Paulo. Rev Enferm Atual 2002; 2(11): 36-41. 4. Souza AR, Baptista SS. A adoção do modelo anglo-americano de ensino de enfermagem na capital paulista. Rev Escola Enferm Anna Nery 2002; 6(2): 211-27. 6. Souza AR, Baptista SS. A criação e o legado da Escola de Enfermeiras do Hospital São Paulo. Rev Enferm Atual 2002; 2(11): 36-41. 3. Carvalho AC. Associação Brasileira de Enfermagem 1926-1976 Documentário. Brasília (DF): ABEn; 1976. Rev Bras Enferm 2005 maio-jun; 58(3):361-6. REFERÊNCIAS 1. Mainwaring S. A igreja católica e a política no Brasil (1916-1985). São Paulo (SP): Brasiliense; 1989. 2. Baptista SS, Barreira IA. Condições de surgimento das escolas de enfermagem brasileiras (1890-1960). Rev Alternativa Enferm 1997; 1(2): 4-17. 4. Souza AR, Baptista SS. A adoção do modelo anglo-americano de ensino de enfermagem na capital paulista. Rev Escola Enferm Anna Nery 2002; 6(2): 211-27. 5. Bruneau TCB. A revolução de 1930: reinteração da igreja no estado. In: Bruneau TCB. Catolicismo em época de transição. São Paulo (SP): Edições Loyola; 1974; p. 77-97. 365 Gomes TO, Almeida Filho AJ, Baptista SS. 16. Almeida Filho AJ. A Escola Anna Nery (EAN) no “front” do campo da educação em enfermagem e o (re) alinhamento de posições de poder (1931-1949) [tese]. Rio de Janeiro (RJ): Escola de Enfermagem Anna Nery, Universidade Federal do Rio de Janeiro; 2004. 11. Cunha LA. A Universidade temporã: da era colonial à era Vargas. 2ª ed. Rio de Janeiro (RJ): Francisco Alves; 1986. 12. Fausto B. História do Brasil. 2ª ed. São Paulo (SP): EDUSP; 1995. 13. Serviço Especial de Saúde Pública. Número de enfermeiras no Brasil, por ano de formatura e por escola, de 1917 a 1950. Departamento de Arquivo e Documentação da COC/FIOCRUZ. Caixa 19; doc 8(4). 17. Revista Brasileira de Enfermagem. Madre Domineuc. Notícias e Comentários. Rev Bras Enferm 1961: 26(3): 261-65. 18. Santos AM, Neves GP, Machado HF, Gonçalves WS. História do Brasil: de terra ignota ao Brasil atual. Rio de Janeiro (RJ): Multimídia; 2002. 14. Rodrigues C, Cozzupde CA, entrevistadores. Madre Marie Domeneuc [entrevistada]. Rio de Janeiro: EEAN; 1988 jul 15. 2 fitas cassetes (120 min). Entrevista concedida ao Acervo de Depoimentos do Centro de Documentação da Escola de Enfermagem Anna Nery, Universidade Federal do Rio de Janeiro. 19. Ferreira MM, Sarmento CE. A república do Brasil: pactos e rupturas. In: Gomes AC, Pandolf DC, Alberti V, organizadores. A República do Brasil. Rio de Janeiro (RJ): Nova Fronteira/CPDOC; 2002. 15. Universidade Federal do Rio de Janeiro. Escola de Enfermagem Anna Nery. Centro de Documentação. Série: Diretoras e outras personalidades. Subsérie: Cecília Pêcego Coelho, 1975-1980. Documento interno. 15. Universidade Federal do Rio de Janeiro. Escola de Enfermagem Anna Nery. Centro de Documentação. Série: Diretoras e outras personalidades. Subsérie: Cecília Pêcego Coelho, 1975-1980. Documento interno. 20. Cruz MA. O estudo da formação profissional da religiosa – enfermeira e os problemas referentes às escolas de enfermeiras em nosso meio. Anais Enferm 1954; 7(2): 85-119. Data do recebimento: 10/11/2004 Data da aprovação: 11/10/2005 366 366
https://openalex.org/W4282828292	https://hal.science/hal-03694895/document	English	null	Platoon-actuated variable area mainstream traffic control for bottleneck decongestion	European journal of control	2,022	cc-by	7,883	Mladen Čičić, Cecilia Pasquale, Silvia Siri, Simona Sacone, Karl Henrik Johansson To cite this version: Mladen Čičić, Cecilia Pasquale, Silvia Siri, Simona Sacone, Karl Henrik Johansson. Platoon-actuated variable area mainstream traﬀic control for bottleneck decongestion. European Journal of Control, 2022, 68 (November), pp.100687. 10.1016/j.ejcon.2022.100687. hal-03694895 Distributed under a Creative Commons Attribution 4.0 International License 1. Introduction up new scenarios in which vehicle-based, or Lagrangian con- trol schemes, can be implemented, as done for instance in [3]. This means that the presence of CAVs can be exploited to regu- late the traffic at the system level, pursuing some global objec- tives. The aim of this work goes in that direction by proposing a platoon-based mainstream traffic control in which the main- stream traffic flow is regulated through the use of truck pla- toons that act by restricting the traffic flow upstream of bottle- necks due to both recurring and non-recurring factors, allow- ing the congestion at bottlenecks to dissipate and improving the throughput of the road. The introduction of Connected and Automated Vehicles (CAVs) into the automotive sector represents one of the tech- nological advances that will most revolutionize the future of road transportation. Although there are still technological chal- lenges to be faced before CAVs can become commonplace in the vehicle market, several studies in the literature recognized that numerous advantages in terms of safety and efficiency will be achieved when these vehicles preponderate over traditional vehicles (see report [1] and the references therein). However, in the near future, traditional vehicles and CAVs will need to coex- ist in mixed traffic, for which new traffic management strategies need to be identified. Truck platooning is a methodology that makes use of vehi- cle automation to create a string of virtually connected trucks that automatically brake, steer, and accelerate based on the ac- tions of the leading vehicle [4]. Specifically, truck platoon- ing originated with the idea of implementing fuel saving poli- cies [5, 6, 7], but recently it has also seen its application for traffic flow control purposes such as the one proposed in this paper. For instance, in [8, 9], truck platoons are modelled as moving bottlenecks and their speed is defined according to proportional-integral feedback regulators in order to mitigate congestion in the mainstream. In [10] the control law proposed in [8] has been embedded in two control schemes, one central- The conventional ways to control vehicular traffic are given by road-based, or Eulerian traffic control schemes. In these control schemes, the control actions to be implemented are de- fined according to traffic conditions, detected at specific loca- tions, and actuated by means of dedicated equipment installed along the infrastructure. Platoon-actuated variable area mainstream traffic control for bottleneck decongestion Mladen ˇCiˇci´ca, Cecilia Pasqualeb, Silvia Sirib, Simona Saconeb, Karl Henrik Johanssonc aUniv. Grenoble Alpes, CNRS, Inria, Grenoble INP, GIPSA-lab, France, (mladen.cicic@gipsa-lab.fr). bDepartment of Informatics, Bioengineering, Robotics and Systems Engineering, University of Genova, Italy. cDivision of Decision and Control System, KTH Royal Institute of Technology, Sweden. ˇCiˇci´ca, Cecilia Pasqualeb, Silvia Sirib, Simona Saconeb, Karl Henrik Johanssonc Abstract In this paper a platoon-actuated mainstream traffic control is proposed to decongest bottlenecks due to recurrent and nonrecurrent events. Indeed, differently from traditional mainstream control strategies, i.e., control strategies applied with fixed actuators, platoon-actuated control can be applied at any location on the freeway. In this work, the control actions to be communicated to the platoons, i.e., speed and configuration, are defined by means of a predictive control law based on traffic and platoon state detected in an area identified immediately upstream of the bottleneck. The main peculiarity of this scheme is that the size of the controlled area is dynamically adjusted based on the predicted congestion at the bottleneck. This approach keeps the control law computation burden low, while not sacrificing much control performance. Specifically, the number of platoons to be controlled and the time at which the platoons begin to be controlled depend from the size of the controlled area. Simulation results reported in the paper show the effectiveness of the proposed scheme, eliminating from 60% to 80% of the delay incurred from congestion compared with the uncontrolled case, depending on the level of traffic. Keywords: mainstream traffic control, bottleneck decongestion, tandem queueing model, platooning coordination HAL Id: hal-03694895 https://hal.science/hal-03694895v1 Submitted on 14 Jun 2022 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License 1. Introduction In the context of freeway traffic, the most popular road-based control strategies are ramp metering, mainstream control (usually via variable speed limits), or route guidance (the interested reader may refer to the survey in [2] for more details). However, the presence of CAVs in traffic opens 1 In this paper the concept of mainstream control is imple- mented using truck platoons as actuators. Therefore, the ba- sic concept of this work is to exploit the presence of platoons in traffic that, properly controlled, i.e. by adjusting their speed and defining the number of lanes they should occupy, are able to regulate the inflow into the bottleneck area in order not to exceed its capacity (i.e. the maximum flow that the bottleneck can discharge), and thus prevent the formation of congestion. Compared with mainstream control with fixed actuators, main- stream control performed with platoons is more flexible allow- ing the management of non-recurring bottlenecks (such as those caused by accidents or roadworks) that may arise at any loca- tion within the freeway stretch. At the same time, it is worth noting that the effectiveness of platoon-based mainstream con- trol depends on several aspects such as the number of platoons present in the freeway stretch, and their distance from the bot- tleneck. ized and one decentralized, in which the control parameters are optimally defined according to the detected traffic conditions. In [11], instead, CAVs that are initially scattered on the road are first collected into platoons, and then used to dissipate stop- and-go waves, improving throughput and homogenizing traffic, while in [12], controlled platoons are exploited to avoid conges- tion and maximize throughput at stationary bottlenecks. Other works investigate the effects of the presence of platoons in the vehicular flow, as in [14] and [15]. The present work takes inspiration from the approach intro- duced in [12] in which the platoon speed and configuration (how many lanes the platoon takes) for each controlled platoon are defined based on a prediction of traffic and platoon state per- formed using the tandem queueing model with moving bottle- necks introduced in [12]. Differently from the work conduced therein, in this paper the control actions (i.e., platoon speed and configuration) are not computed for each truck platoon on the considered road but only for those traveling within a controlled area, which is identified upstream of a bottleneck. 1. Introduction Indeed, the main peculiarity of this work is that the length of the controlled area is time-varying and defined according to the degree of total predicted congestion at the end of the prediction horizon. To this end, a platoon-actuated mainstream traffic control scheme considering these two latter aspects is presented. In particular, the proposed control scheme is of the centralized type and exploits the complete knowledge of both traffic and platoons state in the whole freeway stretch. As shown in the sketch of the proposed control framework depicted in Fig. 1, in this scheme only the platoons that are within the controlled area identified immediately upstream of the bottleneck are con- trolled. Yet, the peculiarity of this approach is that the size of the controlled road segment, based on which the control actions are computed, is dynamically defined according to the severity of the predicted congestion. This paper is organized as follows. Section 2 introduces the motivation and the main features of platoon-based mainstream control, while the control scheme based on the variable-length segment controller is presented in Section 3. Some simula- tion results are introduced in Section 4, in order to show the effectiveness of the proposed control scheme, whereas conclud- ing remarks and future perspectives of platoon-actuated main- stream traffic control are drawn in Section 5. It is worth noting that the size of the control area is a crucial issue for problems of this type, since its length determines some fundamental aspects of the control: 2. Platoon-actuated mainstream traffic control • the number of platoons that can be used as actuators of the mainstream control; • the number of platoons that can be used as actuators of the mainstream control; Freeway traffic control research has produced several strate- gies whose primary goal is to mitigate traffic congestion. These strategies differ in the control variables adopted (e.g., inflow from the on-ramps in ramp metering strategies, splitting rates in routing strategies, etc.) and in the actuators used to imple- ment them, which, as mentioned in the Introduction, are typi- cally placed at fixed locations along the infrastructure. • the distance from which platoons start their control action. • the distance from which platoons start their control action. • the distance from which platoons start their control action. Broadly, the longer the controlled road segment, the greater the likelihood that there will be a sufficient number of platoons to deal with the congestion. Additionally, since the platoons ref- erence speed are lower-bounded by some value, the time each platoon can spend restricting the traffic flow is generally lim- ited. Therefore, a long controlled road segment enables the pla- toons to delay the traffic flow for a longer time, allowing for a more effective action in preventing bottleneck activation. These observations suggest that severe congestion necessitates a long Among these methodologies, the one adopted to directly reg- ulate the vehicular flows traveling on the road is denoted as mainstream control, and is typically designed to prevent the ac- tivation of bottlenecks, generally due to freeway layout (e.g., lane drops, merging zones with on/off-ramps, etc.), see for in- stance the works [16, 17]. As mentioned in [18], this strategy can be implemented in various ways, such as by defining vari- able speed limits, displayed to users through variable message signs placed at significant locations on the freeway, by regu- lating the traffic stream through traffic lights at the roadway or through exploitation of intelligent vehicles. Regardless of the methodology adopted, the objective is to create controlled congestion (of significantly lower intensity and duration than the congestion that would be created in the absence of control) capable of sufficiently reducing the inflow into the bottleneck, thus avoiding its activation. Figure 1: Sketch of the proposed control scheme. Figure 1: Sketch of the proposed control scheme. 2 of the segment on which the control actions are defined, which is dynamically adjusted according to the expected level of con- gestion at the bottleneck. Both the tandem queuing model used to perform the prediction, and the predictive control law in- cluded in the control scheme, are presented below. These two blocks form the inner control loop, whereas the controlled seg- ment length adaptation forms the outer loop, enabling the inner loop to successfully achieve the control goals. controlled road segment in order to be successfully dissipated. However, it should be noted that if the segment size increases, the problem size also increases and thus the computation time required to solve it. 3.1. Description of the control scheme As mentioned above, the aim of this work is to define a main- stream control strategy in which truck platoons are the actuators that operate by slowing down traffic in the mainstream so that the flow reaching the bottleneck does not exceed its capacity. Specifically, in this scheme we assume that the platoons ar- rive randomly, and that their speeds and the number of lanes they occupy are the control inputs, which are defined through a prediction-based control law. p As shown in the control scheme in Fig. 2, the controller re- ceives the current traffic and platoons state measurements and uses them to predict the evolution of the numbers of vehicles accumulated in the queues at the moving and stationary bot- tlenecks, as a function of future control inputs. Then, using a control law derived from the prediction model, the controller computes and communicates to each controlled platoon the ref- erence speed and the number of lanes that it should occupy to decongest the bottleneck downstream of it. Owing to the spe- cific structure of the prediction model, the control action at each prediction time step is calculated directly from the predicted queue lengths at the previous prediction time step. At each con- trol time step, only the first calculated control action is sent to the platoons for execution, acting in a receding horizon manner, and the prediction is repeated at the next control time step. Assuming all the vehicles on the segment (at least approx- imately) share a common constant maximum free-flow speed V, as is the case when a triangular fundamental diagram is used, at time t0 we may predict the outflow from the segment qout s (t\|t0) for t ≤t0 + ∥Xs(t0)∥ V , where ∥Xs(t0)∥= Xout s (t0) −Xin s (t0) is the segment length, using only the traffic state within it, i.e., without the need to know the future inflow to the segment. The segment traffic state consists of the traffic density ρ(x, t0), x ∈Xs(t0), and positions xi(t0) of all bottlenecks i ∈Is(t0) that are within the segment at time t0, xi(t0) ∈Xs(t0). Finally, we also assume that the segment length is constant for the whole duration of the prediction. For conciseness, hereinafter we omit writing the time t0, when the predictions are calculated, wher- ever it is obvious. 3.2. Prediction model In this section the traffic model used to predict the traffic state and to compute the control actions is presented. This predic- tion model is based on the tandem queuing model with mov- ing bottlenecks introduced in [12], and properly extended to represent the traffic behaviour in a road segment. Let s de- note a generic freeway segment coinciding with the interval Xs(t0) = [Xin s (t0), Xout s (t0)], where Xin s (t0) and Xout s (t0) are re- spectively the upstream and the downstream ends of the seg- ment, and t0 is the current time at which we are predicting the queue length evolution. The prediction is based on the current traffic state, i.e., the traffic density ρ, that can be either gath- ered from measurements of the real system or reproduced with a simulation model. • the distance from which platoons start their control action. Moreover, large controlled segments re- quire prediction horizons long enough to track the entire path of platoons, compromising the reliability of the prediction it- self. Therefore, the idea of defining control actions over a zone of varying size allows us to adapt the control problem on the ba- sis of current traffic conditions and platoon availability, thereby reducing the overall computational load of the problem and al- lowing its application for online control purposes. 3.1. Description of the control scheme This is due to the fact that in this frame of reference, there are no delays between the queues and the inflow to each queue is l This third type of queues encapsulates a portion of freeway that is controlled, allowing us to approximate a road segment as a single queueing server. Therefore, we introduce a general for- mulation of the queuing model in which the general element i ∈Is(t0) (stationary bottleneck, moving bottleneck or closed- loop controlled road segment) can be modeled as a queuing server, with the number of queuing vehicles ni(t) evolving in time according to ˙ni(t) = qin i (t) −qout i (t), t ≥ti(t0), i ∈Is(t0) (1) (1) where qin i (t) is the traffic flow arriving at the queue, and qout i (t) the flow discharging from it, qout i (t) =  min n qin i (t), qcap i (t) o , ni(t) = 0, qctr i (t), 0 < ni(t) ≤nctr i (t0), qdis i (t), ni(t) > nctr i (t0). (2) qin i (t) = qout #» i (t), i ∈As(t). (6) (6) Here, qcap i (t) is the maximum capacity of the queue, qctr i (t) and qdis i (t) are the discharging flows from the partially and fully con- gested queue, respectively, nctr i (t0) is defined as the queue length boundary between the partially and fully congested queue, and we have qcap i (t) ≥qctr i (t) ≥qdis i (t). We denote by ti(t0) the first time when the queueing server begins affecting the rest of the road network. If #» i (t) = 0, the inflow to queue i at relative time t is given by (6), and for this upstream-most queue i, we write i = #»0 (t). Otherwise, the input is given as an output of the queue #» i (t) defined by (2). If there are on- and off-ramps between queues i and #» i (t), their net inflow to the road would also be added to qin i (t). Firstly, we model a stationary bottleneck β ∈Is(t0), at po- sition xβ(t) = Xβ ∈Xs(t0), by setting all queue parameters to constant values, nctr β = 0, and qctr β = qdis β < qcap β . 3.1. Description of the control scheme Specifically, as soon as the stationary bottleneck becomes congested, nβ(t) > 0, the maximum outflow is reduced from qcap β to qdis β due to the capacity drop phenomenon. Note that the structure of the chain of queueing servers As(t) representing the bottlenecks varies in time, as demonstrated in Fig. 3. We assume that there is a stationary queue at the down- stream end of the segment, representing either a physical sta- tionary bottleneck, or an encapsulation of the road downstream of the segment. This bottleneck is the downstream-most in the chain for all t, and we formally denote it by #» 0 . Therefore, at time t = t0, we have As(t0) = { #» 0 }. As the prediction time is ad- vanced, more bottlenecks will start affecting the outflow from the road segment, and will therefore be added to As(t) and to the queueing servers chain. Queues i ∈Is(t0) are connected to the chain at its upstream end at time t = ti(t0), Secondly, we model the platoons acting as mov- ing bottlenecks ξ ∈ Is(t), with trajectories xξ(t), xξ(t0) ∈Xs(t0), by assuming constant nctr ξ = 0, and time-varying qdis ξ (t) = qctr ξ (t) = qcap ξ (t). These limits on the overtaking flow, enforced by controlling the formation of the platoons, are used as control inputs, qcap ξ (t) ∈ n qlo, qhio , and set by the control law. ξ n o Finally, we can encapsulate the average behaviour of the closed-loop controlled road segment s as a queuing server with nctr s (t0) = ∥Xs(t0)∥ηctr ≥0, and constant qcap s ≥qctr s ≥qdis s . The choice of parameters and model structure will be elaborated in Section 3.4. ti(t0) = t0 + Xout s (t0) −xi(t0) V , (7) (7) by changing #»i (ti(t0)+) = #»0 (ti(t0)−) and #» i (ti(t0)+) = 0. We as- sume that the moving bottlenecks do not overtake each other until they reach the downstream end of the segment at time tout ξ (t0) for which xξ(tout ξ (t0)) = Xout s (t0). At this time, moving bottleneck ξ is removed from the chain by setting #» #» ξ (tout ξ (t0)+) = 0, and its queue is added to the queue at the downstream end of the segment, by changing #»i (ti(t0)+) = #»0 (ti(t0)−) and #» i (ti(t0)+) = 0. 3.1. Description of the control scheme In this work we use the queuing model to represent three types of queues: stationary bottlenecks, that are the “physical” bottlenecks due to lane drops, traffic accidents, etc., moving The number of platoons to be controlled and the instant at which the platoons begin to be controlled depend on the length Figure 2: The proposed control scheme. 3 Figure 2: The proposed control scheme. 3 bottlenecks, that are the controlled platoons which we use as traffic flow actuators, and closed-loop controlled road segment. bottlenecks, that are the controlled platoons which we use as traffic flow actuators, and closed-loop controlled road segment. bottlenecks, that are the controlled platoons which we use as traffic flow actuators, and closed-loop controlled road segment. This third type of queues encapsulates a portion of freeway that is controlled, allowing us to approximate a road segment as a single queueing server. Therefore, we introduce a general for- mulation of the queuing model in which the general element i ∈Is(t0) (stationary bottleneck, moving bottleneck or closed- loop controlled road segment) can be modeled as a queuing server, with the number of queuing vehicles ni(t) evolving in time according to Essentially, bottlenecks i ∈As(t) ⊂Is(t0) have trajectories that intersect the trajectory of a vehicle that would reach Xout s (t0) at time t travelling at free-flow speed V. A graphical ex- planation of the discussed concepts is given in Fig 3, with τi(t) shown by horizontal dashed coloured lines. At every prediction time t, we order these bottlenecks by increasing τi(t), and denote the bottleneck immediately upstream of bot- tleneck i along the line Xout s (t0) + V · (τ −t) (shown in dotted or dashed black for different t in Fig. 3) as #» i (t), τ #» i (t) < τi(t), and (∄j ∈As(t)), τ #» i (t) < τj(t) < τi(t). If there are no bottlenecks upstream of bottleneck i at relative time t, we write #» i (t) = 0. Therefore, it is much simpler to represent the dynamics of all queues i ∈Is(t0) in “relative” time, i.e., time when a vehicle departing from the position of the bottleneck and travelling at free-flow speed would reach the downstream end of the seg- ment. 3.3. Segment controller the line Xout s (t0) + V · (τ −t), and allows the highest overtak- ing flow that does not exceed any of the downstream queueing servers’ capacity, qhi. In the considered case, qlo corresponds to the traffic flow overtaking a platoon that is taking two lanes out of three, and qhi to that overtaking a platoon that is taking a single lane. The speed of moving bottleneck ξ is controlled so that, if feasible, it reaches Xout s (t0) at time tout ξ (t0) = td ξ(t0), where td ξ(t0) is defined as the minimum time for which µξ(td ξ(t0)) = 0 and nξ(td ξ(t0)) = 0, or otherwise, so that it moves at minimum speed Umin ξ , in which case tout ξ (t0) = tmax ξ (t0). In order to predict the future outflow from the segment, we need to know the future control inputs, i.e., qcap ξ (t), as well as the times when the moving bottlenecks leave the road segment tout ξ (t0). Both of these information sets can be calculated during the prediction process, with qcap ξ (t) depending on the predicted evolution of the queues downstream of platoon ξ up to time t, according to the control law described here. Firstly, the traffic flow overtaking the moving bottleneck ξ only affects the outflow from the segment qout s (t) for t ∈[tξ(t0), tout ξ (t0)], i.e., while moving bottleneck ξ is in the queue chain, so it is enough to define qcap ξ (t) for that time in- terval. The reference speed of all platoons uξ(t0) is constrained to be higher than some minimum speed uξ(t0) ≤Umin ξ , so we know that the platoon should leave the segment at the latest at time tmax ξ (t0), ξ ξ ξ Finally, once the predicted queue lengths and control inputs are calculated until time t = t0 + ∥Xs(t0)∥ V , we can use them to de- termine the control inputs for the real process. The overtaking flow limit applied by platoon ξ at time t0 is thus qcap ξ (t0) = qcap ξ (tξ(t0)\|t0), (12) (12) which will be enforced by having platoon ξ occupy an appro- priate number of lanes. The platoon reference speed is given by which will be enforced by having platoon ξ occupy an appro- priate number of lanes. 3.1. Description of the control scheme We as- sume that the moving bottlenecks do not overtake each other until they reach the downstream end of the segment at time tout ξ (t0) for which xξ(tout ξ (t0)) = Xout s (t0). At this time, moving » Due to the assumption that the free-flow speed V is constant everywhere on the considered road segment, the outflow from the segment qout s (t) at times t ∈[t0, t0 + ∥Xs(t0)∥ V ] can be predicted at time t0 based on the flow of the traffic originating from posi- tion Xout s (t0) −V · (t −t0) at time t0, bottleneck ξ is removed from the chain by setting » #» ξ (tout ξ (t0)+) = 0, and its queue is added to the queue at the downstream end of the segment, qout s (t) = Vρ Xout s (t0) −V · (t −t0), t0 , (3) (3) n #» 0 (tout ξ (t0)+) = n #» 0 (tout ξ (t0)−) + nξ(tout ξ (t0)−) + nπ ξ, (8) (8) and the states and dynamics of those bottlenecks i ∈As(t), for which there exists τi(t) ∈[t0, t] such that where nπ ξ is the passenger-car-equivalent number of vehicles in the platoon itself. This yields a hybrid structure of the pre- diction model, with the continuous dynamics described by a tandem queueing system, and discontinuous dynamics corre- sponding to changing the network structure. xi(τi(t)) = Xout s (t0) + V · (τi(t) −t), (4) As(t) = i ∈Is(t0) (∃τi(t) ∈[t0, t]) . (5) (4) (4) (5) 4 Figure 3: Explanation of the model structure with one stationary bottleneck β at the downstream end of the segment, and two platoons, ξ1 and ξ2. Bottleneck trajectories are shown in coloured lines. Black dotted and dashed lines have slope 1/V and correspond to a single prediction time t. Contents of set As(t) for prediction times t ∈{t1, t2, t3, t4}, indicated by dashed black lines, are displayed. Figure 3: Explanation of the model structure with one stationary bottleneck β at the downstream end of the segment, and two platoons, ξ1 and ξ2. Bottleneck trajectories are shown in coloured lines. Black dotted and dashed lines have slope 1/V and correspond to a single prediction time t. Contents of set As(t) for prediction times t ∈{t1, t2, t3, t4}, indicated by dashed black lines, are displayed. 3.3. Segment controller The platoon reference speed is given by tmax ξ (t0) = t0 + Xout s (t0) −xξ(t0) Umin ξ ≥tout ξ (t0) (9) (9) uξ(t0) =  Xout s (t0)−xξ(t0) td ξ(t0) , ∃td ξ(t0) ≤tmax ξ (t0), Umin ξ , ∄td ξ(t0) ≤tmax ξ (t0). (13) We denote the sum of the lengths of all queues downstream of bottleneck i as µi(t), and define it recursively, (13) µi(t) =  n #»i (t) + µ #»i (t), i ∈Is(t0) \ { #» 0 }, 0, i = #» 0 . (10) (10) 3.4. Closed-loop segment model and length adaptation 3.4. Closed-loop segment model and length adaptation The control law given in the previous section was analysed in [12], concluding that the platoons can be used to improve the throughput of the road segment with a single stationary bottle- neck at its downstream end. This improvement is contingent on having enough platoons available for control, the initial level of congestion not being too high, and the length of the con- trol segment being long enough. The control is able to return the stationary bottleneck to free-flow and improve the outflow The control law governing qcap ξ (t) is given by qcap ξ (t) =  qlo, µξ(t) > 0, qhi, µξ(t) = 0, (11) (11) i.e. the platoon allows the lowest possible overtaking flow qlo in case there is predicted congestion downstream of it along 5 qout s (t) > qdis s , by restricting the traffic flow, as long as the total level of congestion ns does not exceed some value nctr s . We may determine the total level of congestion of a road segment s as the sum of all the queue lengths at the end of the prediction, The road is assumed to have a free-flow speed of V = 100 km/h, and the controllers used Umin ξ = 40 km/h and UX = 20 km/h. The capacity of the stationary bottleneck is qcap β = 4000 veh/h, whereas the capacity of the rest of the road is qmax = 6000 veh/h. Due to capacity drop, once the bottleneck gets congested, its discharging flow drops to qdis β = 3000 veh/h. A platoon taking one and two lanes allows an overtaking flow of qhi = 3600 veh/h and qlo = 2000 veh/h respectively. The ar- rival of platoons at the start of the road is modelled as a Pois- son process with an average gap of 0.0152 h between them. Under these conditions, a preliminary simulation study showed that the controlled road segment can be modelled by adopting a length-dependent nctr s (t0) with ηctr = 15 veh/km, achieving an outflow of qctr s = 3200 veh/h in partially congested conditions. Based on these results, the controlled segment length adaptation was parametrized with η+ = 10 veh/km and η−= 4 veh/km, en- suring that the controlled segment does not become fully con- gested unless the limit on segment length is reached. 3.4. Closed-loop segment model and length adaptation The pla- toons are assumed to be 80 m long, consisting of 1.6 passenger- car-equivalents, due to shorter inter-vehicular distances within them. The inflow of the rest of the traffic takes uniformly distributed values qin(t) ∈[qmin in , qmax in ], changing every 0.012 h, with qmin in = 2000 veh/h and qmax in ∈{4000, 4200, 4400} varying over sets of simulations, yielding average non-platooned traf- fic inflow of ¯qin ∈{3000, 3100, 3200}. In order to ensure a fair comparison, the inflow is halved during the first 0.05 h and fi- nal 0.8 h of the simulation, providing warm-up and cool-down times. ns(t) = µ #» s t + ∥Xs(t)∥ V t ! . (14) (14) Once ns(t) exceeds nctr s , the control is unable to dissipate the congestion without extending the controlled segment. Based on the conclusions of the analysis, and performed sim- ulation experiments, it is apparent that the congestion level limit nctr s depends on the length of the controlled segment ∥Xs(t)∥ap- proximately linearly for a reasonable range of ∥Xs(t)∥, nctr s ≈ηctr∥Xs(t)∥. (15) (15) Parameter ηctr can be identified from simulation experiments, together with identifying the average outflow from the segment qctr s (t) for ns(t) < nctr s (t). While it is beneficial to have the length of the controlled seg- ment always be as large as possible, this leads to increased com- putational burden, due to the need for a longer prediction hori- zon. Therefore, we propose a scheme that dynamically adjusts the controlled segment length in order to keep ns(t) close to nctr s (t). To prevent random perturbations pushing the segment into the fully congested regime, we adopt separate thresholds for extending and shrinking the control segment, respectively η+ and η−, η−< η+ < ηctr. After every control iteration, the segment length is adjusted by updating Xin s as A simulation run example with ¯qin = 3000 veh/h is shown in Fig. 4, depicting colour-coded traffic density profiles of one detail of the simulation. The platoon trajectories are traced by black dots, and the stationary bottleneck and and the upstream limit of the controlled segment are denoted by dashed red lines. As can be seen in Fig. 4a, in case we have no control, the ar- rival of a platoon at the stationary bottleneck causes capacity drop and congestion starts accumulating at the stationary bot- tleneck. 3.4. Closed-loop segment model and length adaptation Both FS and VS control successfully decongest the bottleneck, by creating controlled congestion at an upstream position, starting from the upstream end of the controlled area. The largest difference between the two control schemes is that, in case of FS control, most of the congestion is accumulated very far from the stationary bottleneck, at the start of the road, whereas in case of VS control, the controlled segment is much shorter, and the congestion is accumulated close to the station- ary bottleneck. Furthermore, once the congestion is dissipated, the controlled segment length of VS control is decreased. The shorter controlled segment length reflects in shorter computa- tion time for VS control compared to that of FS control. Xin s (t+T) =  max n Xin,min s , ns(t) η+ o , ns(t) ∥Xs(t)∥> η+, min n Xin,max s , Xin s (t) −UXT o , ns(t) ∥Xs(t)∥< η−, Xin s (t), otherwise. (16) This way, the controlled segment will grow quickly in case there is excess predicted congestion, and slowly shrink in case the platoons in it are predicted to be able to successfully dis- sipate the congestion. The speed of control segment shrinking UX is a control parameter, and is selected to be lower than the minimum platoon reference speed, UX < Umin ξ , in order to en- sure that there is no congestion left outside of the controlled segment after it shrinks. 4. Simulation results We also measured the com- putation time for the two control laws, under the same circum- stances, and compared it between them, with the computation time ratio signifying the ratio between the computation time of VS control divided by the computation time of FS control. The main peculiarity of this approach lies in the fact that the controlled platoons are those travelling in an area identified up- stream of the stationary bottleneck. Specifically, the length of this area is time-varying and defined based on the prediction of congestion at the stationary bottleneck. This aspect allow us to adapt the control law on the basis of the expected congestion and hence to reduce the computational time required. Further- more, in this paper the results obtained by applying the vari- able length controller are compared with those obtained con- sidering a segment with fixed length. The results show that both approaches are effective in decongesting the bottleneck, with a slightly better performance experienced when the con- trol scheme with fixed segment length is applied. However, a substantial improvement of computational time is observed by using the variable length segment controller, while achieving very similar performance. The comparison results are shown in Fig. 5 as box plots, and given in Table 1 as mean and median performance indices. We can see that both control laws achieve a significant reduction of the TTS compared to the NC case, negating from close to 78% of the delay in the lighter traffic case ¯qin = 3000 veh/h, to close to 60% of the delay in the heavier traffic case ¯qin = 3200 veh/h, with FS control performing slightly better than VS control. However, as shown in Fig. 5c, the computation time FS con- trol is much higher than that of VS control, from around 5 times higher in case of lighter traffic, to around 1.5 times higher in case of heavier traffic. This outcome was expected, since the VS control only has to calculate the prediction for a shorter time horizon proportional to the controlled segment length, whereas FS control always calculates the prediction for the full time horizon. In the heavier traffic case, this difference is less no- table, since VS control will also tend to control the full road segment as congestion builds up. 4. Simulation results We tested the effectiveness of the proposed control law in 3 h long macroscopic simulations, on a 20 km long stretch of three- lane highway with a a stationary bottleneck at Xβ = 19.95 km. As in [12], the simulation model used is multi-class CTM with platoons. Two cases of control law (12), (13) were compared against the uncontrolled case (NC): We conducted three sets of 10 simulation runs each, varying the average non-platooned traffic inflow, comparing the perfor- mance of the two control laws with Total Time Spent (TTS) used as the performance metric. We compared the achieved rel- ative delay incurred due to the stationary bottleneck, FS: With fixed controlled segment length, from the beginning of the road, Xin s = 0, to the stationary bottleneck location Xout s = Xβ, and FS: With fixed controlled segment length, from the beginning of the road, Xin s = 0, to the stationary bottleneck location Xout s = Xβ, and TTSXS −TTS0 TTSNC −TTS0 , (17) (17) VS: With varying, dynamically adjusted controlled segment length, and Xin s (t) given by (16). where TTSXS is the TTS achieved using control scheme where TTSXS is the TTS achieved using control scheme 6 (a) No control (NC) (b) Fixed controlled segment length (FS) (a) No control (NC) (a) No control (NC) (b) Fixed controlled segment length (FS) (c) Variable controlled segment length (VS) ure 4: An example simulation run, with a zoomed-in display of the period when congestion starts accumulating at the stationary bottleneck. In the zoome w, platoon trajectories are shown by dotted black lines. 7 (a) No control (NC) (b) Fixed controlled segment length (FS) (b) Fixed controlled segment length (FS) ( ) g g ( ) (c) Variable controlled segment length (VS) (c) Variable controlled segment length (VS) Figure 4: An example simulation run, with a zoomed-in display of the period when congestion starts accumulating at the stationary bottleneck. In the zoomed-in view, platoon trajectories are shown by dotted black lines. Figure 4: An example simulation run, with a zoomed-in display of the period when congestion starts view, platoon trajectories are shown by dotted black lines. 7 7 (a) Absolute delay (b) Relative delay (c) Comp. 4. Simulation results time ratio Figure 5: Simulation results comparing (a) Absolute delay (difference in Total Time Spent compared to TTS0), (b) Relative delay given by (17), and (c) Computation time ratio, for the uncontrolled case (NC) and the two controlled cases, with fixed controlled segment length (FS) and variable segment length (VS), under three different levels of nonplatooned traffic inflow. Filled circles indicate the medians, and boxes stretch from the 25th to the 75th percentile. Data points more than 1.5 times the box length away from the box edges are considered to be outliers, and indicated by empty circles. Whiskers stretch to extreme non-outlier data points. (b) Relative delay (c) Comp. time ratio (a) Absolute delay (c) Comp. time ratio Figure 5: Simulation results comparing (a) Absolute delay (difference in Total Time Spent compared to TTS0), (b) Relative delay given by (17), and (c) Computation time ratio, for the uncontrolled case (NC) and the two controlled cases, with fixed controlled segment length (FS) and variable segment length (VS), under three different levels of nonplatooned traffic inflow. Filled circles indicate the medians, and boxes stretch from the 25th to the 75th percentile. Data points more than 1.5 times the box length away from the box edges are considered to be outliers, and indicated by empty circles. Whiskers stretch to extreme non-outlier data points. ¯qin [veh/h] Total Time Spent [veh h] (TTS0 = 1656 veh h) Relative delay [%] Computation NC FS VS TTSFS−TTS0 TTSNC−TTS0 TTSVS−TTS0 TTSNC−TTS0 time ratio [%] mean median mean median mean median mean median mean median mean median 3000 1918.8 1909.6 1678.8 1668.0 1685.2 1664.5 22.47 22.84 23.50 21.02 20.31 18.58 3100 2306.2 2295.0 1827.1 1849.0 1884.6 1887.4 24.66 28.57 32.82 34.40 39.60 4159 3200 2618.7 2663.4 2039.3 2075.7 2109.8 2159.2 34.66 36.96 42.85 46.50 62.01 68.89 Table 1: Average and median performance indices of the simulation results Table 1: Average and median performance indices of the simulation results lanes. Thus, controlled platoons act as moving bottlenecks, re- ducing the inflow to the stationary bottleneck, returning it to free-flow conditions and keeping it from becoming congested again. XS ∈{FS, VS}, TTSNC is the TTS of the uncontrolled case, and TTS0 is the theoretical minimum TTS in case the stationary bot- tleneck was absent from the road. 4. Simulation results The findings provided by the simulations are rather encour- aging, as they show that even a low presence of connected and automated vehicles, if properly controlled, can positively influ- ence traffic behavior. These results, combined with the fact that the CAVs are likely to continue being a sparse minority com- pared to human-driven vehicles in the near future, further moti- vate the need to pursue research in the direction of the strategy proposed in this paper. The overall goal of future work is to de- fine more sophisticated control schemes, that bring even greater benefits than the proposed approach, or conversely, are easier to implement. Furthermore, validation of the proposed control scheme using microscopic simulations should be performed. References [1] M. Alonso Raposo, et al. The future of road transport - Implications of automated, connected, low-carbon and shared mobility. EUR 29748 EN, Luxembourg: Publications Office of the European Union, 2019. y Luxembourg: Publications Office of the European Union, 2019. [2] S. Siri, C. Pasquale, S. Sacone, A. Ferrara. Freeway traffic control: A survey. In Automatica, 130, 109655, 2021. [3] E. Vinitsky, K. Parvate, A. Kreidieh, C. Wu, A. Bayen. Lagrangian con- trol through deep-rl: Applications to bottleneck decongestion. In Proc. of the 21st IEEE International Conference on Intelligent Transportation Systems, pp. 759–765, 2018. [4] A.K. Bhoopalam, N. Agatz, R. Zuidwijk. Planning of truck platoons: A literature review and directions for future research. In Transportation Research B, 107, pp. 212–228, 2018. [5] K.-Y. Liang, J. Mårtensson, K. H. Johansson. When is it fuel efficient for a heavy duty vehicle to catch up with a platoon? In IFAC Proceedings Volumes, 46, pp. 738–743, 2013. [6] A. Alam, B. Besselink, V. Turri, J. Mårtensson, K.H. Johansson. Heavy- duty vehicle platooning for sustainable freight transportation: a coopera- tive method to enhance safety and efficiency. In IEEE Control Systems, 35, pp. 34–56, 2015. [7] S. van de Hoef, K. H. Johansson, D. V. Dimarogonas. Fuel-efficient en route formation of truck platoons. In IEEE Transactions on Intelligent Transportation Systems 19, pp. 102–112, 2018. [8] C. Pasquale, S. Sacone, S. Siri, A. Ferrara. A new Micro-Macro METANET model for platoon control in freeway traffic networks. In Proc. of the 21th IEEE Intelligent Transportation Systems Conference, pp. 1481–1486, 2018. It is worth noting that these control schemes have a com- putational burden that may increase in scenarios where a large number of vehicles are present. To overcome this issue, devel- opment of further decentralized control schemes can be con- sidered, in which the controller acts on a limited group of pla- toons or even at the individual platoon level. In these schemes, the control actions would be defined on the state measurements observed around the controlled platoons, with minimum com- munication among them. Implementation of this case would be simpler, albeit at the expense of control performance, since pre- diction would be based on estimates using incomplete knowl- edge of the system state. [9] G. Piacentini, C. Pasquale, S. Sacone, S. Siri, A. Ferrara. Multiple moving bottlenecks for traffic control in freeway systems. In Proc. of European Control Conference, pp. 3662–3667, 2019. [10] S. Sacone, C. References Pasquale, S. Siri, A. Ferrara. Centralized and Decentralized Schemes for Platoon Control in Freeway Traffic Systems. In 60th IEEE Conference on Decision and Control, Austin, USA, December 13-15,pp. 2665–2670, 2021. [11] M. ˇCiˇci´c, K. H. Johansson. Stop-and-go wave dissipation using accumu- lated controlled moving bottlenecks in multi-class CTM framework In IEEE 58th Conference on Decision and Control, pp. 3146-3151, 2019. ˇ [12] M. ˇCiˇci´c, X. Xiong, L. Jin, K. H. Johansson. Coordinating vehicle pla- toons for highway bottleneck decongestion and throughput improvement In IEEE Transactions on Intelligent Transportation Systems, 2021. ˇ [13] L. Jin, M. ˇCiˇci´c, S. Amin, K. H. Johansson. Modeling the impact of vehicle platooning on highway congestion: A fluid queuing approach In Proc. 21st International Conference on Hybrid Systems: Computation and Control, pp. 237–246, 2018. 5. Concluding remarks and future perspectives In this paper a platoon-actuated mainstream traffic control scheme is proposed for dissipating the congestion created at a stationary bottleneck. The control is executed by appropriately controlling the platoons present on the road, by controlling their speed and commanding them to occupy a specified number of 8 One direction is to extend the control law to road networks consisting of multiple individually considered segments. In such case, a decentralized control law can be applied, with each segment considered according to the control law presented herein. Another potential extension would be to consider the possibility of creating and controlling platoons of CAVs only where and when useful for control purposes, and then dissolv- ing them when they are no longer needed. Additionally, “clas- sical” traffic control methods, e.g., ramp metering should be considered together with the presented platoon-actuated con- trol. The used modelling framework also lends itself to this purpose, since the ramp flows could easily be included as ad- ditional inflows to the segment. In this configuration, platoon- actuated control and ramp metering would act together to avoid bottleneck activation, as well as to eliminate excess congestion. Indeed, similarly to mainstream control strategies with fixed actuators, platoon-actuated mainstream control regulates traffic flow by creating a moderate controlled congestion but does not completely eliminate congestion in the mainstream, therefore better performances can be obtained by the joint application of ramp metering and the mainstream control via platoons. Acknowledgements [14] M.D. Simoni, C.G. Claudel. A fast simulation algorithm for multiple moving bottlenecks and applications in urban freight traffic management In Transportation Research Part B, 104, pp. 238–255, 2017. The research leading to these results has received funding from VINNOVA within the FFI program under contract 2014- 06200, the Swedish Research Council, the Swedish Founda- tion for Strategic Research, Knut and Alice Wallenberg Foun- dation, and the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation pro- gramme (grant agreement 694209), http://scalefreeback.eu. [15] H. Yu, S. Amin, M Krstic. Stability analysis of mixed-autonomy traffic with CAV platoons using two-class Aw-Rascle model. In Proc. 59th IEEE Conference on Decision and Control, pp. 5659–5664, 2020. [16] X.Y. Lu,S.E. Shladover. Review of variable speed limits and advisories: Theory, algorithms, and practice. In Transportation research record, 2423:1, pp. 15–23, 2014. [17] M. Wang, W. Daamen, S.P. Hoogendoorn, B. van Arem. Connected variable speed limits control and car-following control with vehicle- infrastructure communication to resolve stop-and-go waves. In Journal of Intelligent Transportation Systems, 20:6, pp. 559–572, 2016. [18] R.C. Carlson, I. Papamichail, M. Papageorgiou, Local feedback-based mainstream traffic flow control on motorways using variable speed limits In IEEE Transactions on intelligent transportation systems, 12(4), pp. 1261-1276, 2011. 9
https://openalex.org/W2991279836	https://europepmc.org/articles/pmc6879622?pdf=render	English	null	A Simulated Microgravity Environment Causes a Sustained Defect in Epithelial Barrier Function	Scientific reports	2,019	cc-by	15,362	A Simulated Microgravity Environment Causes a Sustained Defect in Epithelial Barrier Function Rocio Alvarez1,4, Cheryl A. Stork1,2, Anica Sayoc-Becerra1, Ronald R. Marchelletta2,5, G Ki P i k2 3 & D l F M C l 1* OPEN Rocio Alvarez1,4, Cheryl A. Stork1,2, Anica Sayoc-Becerra1, Ronald R. Marchelletta2,5, G Kim Prisk2,3 & Declan F McCole 1* Intestinal epithelial cell (IEC) junctions constitute a robust barrier to invasion by viruses, bacteria and exposure to ingested agents. Previous studies showed that microgravity compromises the human immune system and increases enteropathogen virulence. However, the effects of microgravity on epithelial barrier function are poorly understood. The aims of this study were to identify if simulated microgravity alters intestinal epithelial barrier function (permeability), and susceptibility to barrier- disrupting agents. IECs (HT-29.cl19a) were cultured on microcarrier beads in simulated microgravity using a rotating wall vessel (RWV) for 18 days prior to seeding on semipermeable supports to measure ion flux (transepithelial electrical resistance (TER)) and FITC-dextran (FD4) permeability over 14 days. RWV cells showed delayed apical junction localization of the tight junction proteins, occludin and ZO-1. The alcohol metabolite, acetaldehyde, significantly decreased TER and reduced junctional ZO-1 localization, while increasing FD4 permeability in RWV cells compared with static, motion and flask control cells. In conclusion, simulated microgravity induced an underlying and sustained susceptibility to epithelial barrier disruption upon removal from the microgravity environment. This has implications for gastrointestinal homeostasis of astronauts in space, as well as their capability to withstand the effects of agents that compromise intestinal epithelial barrier function following return to Earth. A microgravity environment, such as that encountered in spaceflight, has been demonstrated to affect basic cel- lular function in complex biological organisms and in cells. Genetic, biochemical and morphological parameters have all been shown to be modulated by a microgravity environment in multiple cell types1–6. A major technical resource in the study of the biological effects of reduced microgravity has been the development of the rotating wall vessel (RWV). The RWV is a rotating bioreactor that maintains cells in a controlled rotation environment where randomization of gravitational vectors creates a low shear simulated microgravity fluid environment. This system serves to model the impact(s) of microgravity or weightlessness on in vitro cultured cells. In addition, since the RWV environment is mixed by gentle rotation, lacks an air-fluid interface despite efficient oxygenation, and maintains laminar fluid flow, it avoids the large shear stress caused by turbulent flow7–12. Since the RWV minimizes the impact of gravitational force, it permits study of the effects of weight, or lack of weight in this case, on biological systems. www.nature.com/scientificreports www.nature.com/scientificreports 1Division of Biomedical Sciences, University of California, Riverside, Riverside, CA, 92521, USA. 2Department of Medicine, University of California, San Diego, La Jolla, CA, 92093, USA. 3Department of Radiology, University of California, San Diego, La Jolla, CA, 92093, USA. 4Present address: Cedars-Sinai Medical Center, 8700 Beverly Blvd, Los Angeles, CA, 90048, USA. 5Present address: Johnson & Johnson Research Laboratories, Janssen Pharmaceuticals Inc., La Jolla, CA, 92093, USA. email: declan.mccole@ucr.edu Scientific Reports \| (2019) 9:17531 \| https://doi.org/10.1038/s41598-019-53862-3 www.nature.com/scientificreports/ www.nature.com/scientificreports/ al.17, and Drummond et al.18, confirmed that HT-29.cl19a and Caco-2 IECs form monolayers when cultured on microcarrier beads, form distinct tight junction and adherens junction complexes and exhibit different patterns of gene expression when cultured in 3-D structures vs. conventional 2-D culture in culture plates (non-polarized). Of the genes upregulated in 3-D cultures, many were associated with epithelial differentiation17,18. This aligns with earlier findings from studies in renal epithelial cells cultured in RWV or on the space shuttle, that showed dramatic changes in morphology that more resembled epithelial cells in vivo compared with 2-D culture, and in the expression of many gene groups including those associated with transcription factors, signaling proteins and cytoskeletal proteins6,19,20. Moreover, a clear morphological consequence of altered gravity was shown as cells cul- tured in the RWV, and to a greater extent on the space shuttle, exhibited increased number and size of microvilli compared with control cells cultured on Earth at normal G. This is an intriguing observation given that epithelial cell culture on Earth is associated with reduced numbers of microvilli compared to epithelial cells in vivo15,19.l p p Another striking observation that was made in astronauts post-flight was that they exhibit immunosuppres- sion of multiple immune cell subtypes21–27. In addition, the suppressive effects of space flight on immune function are compounded as a risk factor for long term habitation in space by evidence from Wilson et al.28, showing increased virulence of a foodborne bacterial pathogen, Salmonella typhimurium after time spent in space. This was complemented by ground-based studies demonstrating altered bacterial virulence or adherence by either bacterial or epithelial cell culture respectively in the simulated microgravity environment of the RWV17,18,29,30.h p p y g y This prompted our interest in understanding how the barrier properties of the single layer of epithelial cells that line the gastrointestinal tract are affected by microgravity. The barrier function of the intestinal epithelium is critical for the maintenance of intestinal homeostasis and when disrupted, can lead to increased permeability to bacterial products, antigens and precipitate inappropriate inflammatory responses. This can greatly increase the risk of infections, and chronic inflammatory conditions including inflammatory bowel disease, celiac disease, Type 1 diabetes and liver disease31–34. Of significance is that these chronic conditions exhibit an increase in intesti- nal permeability prior to the onset of inflammation as shown in animal models and patient studies35–37. www.nature.com/scientificreports/ Therefore, we set out to assess if exposure of intestinal epithelial cells (IEC) to a simulated microgravity environment resulted in a decrease in barrier function and/or increased susceptibility of the barrier following challenge with an agent capable of compromising the barrier. The permeability-inducing agent we chose to investigate was the alcohol metabolite, acetaldehyde. Alcohol increases gastrointestinal macromolecule permeability in normal subjects, in patients with alcoholic liver disease (ALD) and also in animal models38–41. Ingested alcohol undergoes a series of metabolic steps to facilitate its elimination with the generation of acetaldehyde representing the primary, and most toxic, ethanol metabolite. In the gastrointestinal tract, luminal acetaldehyde production can occur via the activity of cytochrome P450 2E1 or alcohol dehydrogenase from the mucosal epithelium or bacterial sources respectively42,43. Studies in rats demonstrated that chronic ethanol administration resulted in high concentrations of acetaldehyde in the colonic lumen. Of note, antibiotic treatment partially attenuated this increase, thus indi- cating a detectable role for gut bacteria in acetaldehyde generation43. With respect to pathologic consequences of ethanol ingestion and metabolism, evidence from in vivo and in vitro studies indicated that acetaldehyde elevates intestinal and cultured IEC permeability to endotoxin or other permeability markers44–46. Epithelial barrier func- tion is principally regulated by apical tight junctions47–49. Mechanistic studies have demonstrated that increases in paracellular permeability caused by ethanol or acetaldehyde exposure are associated with redistribution of tight junction proteins such as occludin and zona occludens-1 (ZO-1), from the intercellular junctions into intracellu- lar compartments50–52. Colocalization of ZO-1, the first identified tight junction plaque protein, and occludin, the first identified tight junction transmembrane protein, at the tight junction is a strong indicator of barrier integrity, and reduction of this membrane colocalization is indicative of a compromised barrier53–55. ZO-1 is a scaffold protein that localizes to the apical tight junction where it maintains cell polarity and is essential to the formation of the tight junctional complex56–58. While ZO-1 knockout mice are embryonic lethal, the importance of ZO-1 to barrier function was demonstrated by decreased transepithelial electrical resistance in siRNA-mediated ZO-1 knockdown studies in epithelial cell lines59. Occludin is a member of the MARVEL (MAL and related proteins for vesicle trafficking and membrane link) domain–containing family of proteins involved in membrane apposition60. Occludin is a tetraspan transmembrane protein that primarily localizes to bicellular tight junctions in association with binding to ZO-161,62. www.nature.com/scientificreports/ While occludin’s importance to barrier function is somewhat controversial, it has been confirmed to contribute to tight junction stabilization and optimal barrier function by restricting macromolecule permeability and modification of claudin-2-mediated cation permeability61. It therefore appears to act primarily as a regulator of tight junctions rather than as an essential structural component58. However, conditions of cellular stress, such as inflammatory cytokine challenge, can induce internalization of ZO-1 and occludin and this action is associated with a compromised epithelial barrier63,64. Here, we investigated if simulated microgravity, generated by culturing IECs in the RWV, altered junction formation and functional barrier properties of polarized epithelial monolayers, and if subsequent exposure to acetaldehyde induced a differential effect on barrier integrity in cells exposed to simulated microgravity conditions. Moreover, our study is the first to investigate if functional changes to epithelial cell barrier properties were sustained over time following removal from the simulated microgravity environment. A Simulated Microgravity Environment Causes a Sustained Defect in Epithelial Barrier Function Rocio Alvarez1,4, Cheryl A. Stork1,2, Anica Sayoc-Becerra1, Ronald R. Marchelletta2,5, G Ki P i k2 3 & D l F M C l 1 OPEN Consequently, the environment could most accurately be referred to as “near weightless- ness”13. One important clarification is that because microgravity simulation experiments can only change the influence of the Earth gravity vector and not its magnitude, true microgravity cannot be fully accomplished with a mechanical simulator14. Therefore, a ground-based simulator such as a RWV has the capacity to model the perception of low-shear microgravity by creating a functional weightlessness as perceived by the organism or cell being investigated11,13.i g g Previous studies investigating the response of epithelial cells to reduced or simulated microgravity identified that HT-29.cl19a colonic epithelial carcinoma cell line clones were capable of forming attachments to extracel- lular matrices following culture in a RWV as efficiently as cultures at normal G15,16. This is important in that it suggests that reduced gravity does not affect the ability of intestinal epithelial cells (IEC) to attach to basement membrane proteins, a fundamental step in the formation of a monolayer. Elegant studies by Honer zu Bentrup et 1Division of Biomedical Sciences, University of California, Riverside, Riverside, CA, 92521, USA. 2Department of Medicine, University of California, San Diego, La Jolla, CA, 92093, USA. 3Department of Radiology, University of California, San Diego, La Jolla, CA, 92093, USA. 4Present address: Cedars-Sinai Medical Center, 8700 Beverly Blvd, Los Angeles, CA, 90048, USA. 5Present address: Johnson & Johnson Research Laboratories, Janssen Pharmaceuticals Inc., La Jolla, CA, 92093, USA. email: declan.mccole@ucr.edu Scientific Reports \| (2019) 9:17531 \| https://doi.org/10.1038/s41598-019-53862-3 Results G h Growth of IECs on microcarrier beads. HT-29.cl19a cells were grown on beads for 18 days in a rotating wall vessel (RWV) as previously shown11,17,18. Under these conditions, HT-29.cl19a cells form a single layer of cells covering each bead as demonstrated successfully in prior studies17. In Fig. 1 we show (A) an uncovered microcarrier bead and (B) a bead around which HT-29.cl19a cells have formed a continuous layer. Figure 1C,D show an uncovered bead and a covered bead respectively that have been embedded in LR White, sectioned and stained with the vital dye, Toluidene Blue65. Figure 1 D shows uptake of Toluidene Blue by cells surrounding the bead indicating that the cells are viable. We also showed that these cells grow as a monolayer around the surface Scientific Reports \| (2019) 9:17531 \| https://doi.org/10.1038/s41598-019-53862-3 www.nature.com/scientificreports/ Figure 1. HT-29.cl19a cells attach and form 3-D monolayers on individual microcarrier beads. HT-29.cl19a intestinal epithelial cells were cultured with microcarrier beads for 18 days under the conditions of simulated microgravity in a rotating wall vessel (RWV) at 16–17.8 rpm. (A,B) Samples were removed from the RWV for observation under an inverted light microscope (Panel A,: day 1, panel B, day 11) and embedded in LR White and stained with Toluidine Blue for EM imaging (Panels C,D, day 1, day 18). Panels A and C show a bead lacking cell attachment. By comparison, panels B and D show that cells attached and grew around individual beads. (D) Cells in panel D were able to take up the vital dye, Toluidene Blue, thus demonstrating viability of cells in situ on the microcarrier bead. Panels C,D shown in cross-section with the center of the hollow bead indicated by an asterisk. (E,F) whole cell-bead aggregates were stained for nuclei (DAPI) and the tight junction protein, occludin, showing an intact layer around the surface of the bead and tight junction formation. (G,H) Cross-section shows that cells grew as a unicellular layer around the outer surface of the bead. (I) Staining of cellular junction formation on cell-bead aggregates shows apico-lateral staining of (J) occludin and (K) actin with (I) nuclei stained by DAPI and (L) showing an overlay image of all three stains. Arrow indicates occludin staining at a cell-cell junction. Figure 1. HT-29.cl19a cells attach and form 3-D monolayers on individual microcarrier beads. Results G h HT-29.cl19a intestinal epithelial cells were cultured with microcarrier beads for 18 days under the conditions of simulated microgravity in a rotating wall vessel (RWV) at 16–17.8 rpm. (A,B) Samples were removed from the RWV for observation under an inverted light microscope (Panel A,: day 1, panel B, day 11) and embedded in LR White and stained with Toluidine Blue for EM imaging (Panels C,D, day 1, day 18). Panels A and C show a bead lacking cell attachment. By comparison, panels B and D show that cells attached and grew around individual beads. (D) Cells in panel D were able to take up the vital dye, Toluidene Blue, thus demonstrating viability of cells in situ on the microcarrier bead. Panels C,D shown in cross-section with the center of the hollow bead indicated by an asterisk. (E,F) whole cell-bead aggregates were stained for nuclei (DAPI) and the tight junction protein, occludin, showing an intact layer around the surface of the bead and tight junction formation. (G,H) Cross-section shows that cells grew as a unicellular layer around the outer surface of the bead. (I) Staining of cellular junction formation on cell-bead aggregates shows apico-lateral staining of (J) occludin and (K) actin with (I) nuclei stained by DAPI and (L) showing an overlay image of all three stains. Arrow indicates occludin staining at a cell-cell junction. of the bead. This was demonstrated by immunofluorescence staining of nuclei and the transmembrane tight junction protein occludin in whole mount imaging of beads and in cross-section (Fig. 1E–H). Furthermore, these monolayers are capable of forming tight junctions as shown by the apico-lateral membrane staining of occludin (Fig. 1I,J)17,51,66,67. The cytoskeletal protein, actin, that plays a critical role in maintaining the architecture of cell junctions, also localized intensely at the apicolateral membrane thus suggesting that polarization is not disrupted (Fig. 1K,L). These data show that HT-29.cl19a IECs are capable of attaching to and growing on microcarrier beads in a manner that facilitates formation of tight junctions. of the bead. This was demonstrated by immunofluorescence staining of nuclei and the transmembrane tight junction protein occludin in whole mount imaging of beads and in cross-section (Fig. 1E–H). Furthermore, these monolayers are capable of forming tight junctions as shown by the apico-lateral membrane staining of occludin (Fig. 1I,J)17,51,66,67. Scientific Reports \| (2019) 9:17531 \| https://doi.org/10.1038/s41598-019-53862-3 Results G h These data suggest that exposure to a Scientific Reports \| (2019) 9:17531 \| https://doi.org/10.1038/s41598-019-53862-3 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 2. Low shear simulated microgravity exposure affects epithelial barrier formation. A) Model of polarized intestinal epithelial monolayers cultured on a semi-permeable support. (B) TER values (days 2–14) of HT-29.cl19a cells grown on semi-permeable inserts following culture under normal cell culture conditions (flask control), cultured on microcarrier beads alone (static control), or cultured on microcarrier beads and exposed to simulated microgravity (RWV). No statistically significant underlying differences in overall TER raw values were observed between conditions. (C) Data are shown as the % change in TER of cells exposed to microgravity vs. static (black) or flask controls (white). No statistically significant difference was observed in % change between the different culture conditions, although the % change in TER of cells from RWV relative to the static control was consistently lower (n = 4). Figure 2. Low shear simulated microgravity exposure affects epithelial barrier formation. A) Model of polarized intestinal epithelial monolayers cultured on a semi-permeable support. (B) TER values (days 2–14) of HT-29.cl19a cells grown on semi-permeable inserts following culture under normal cell culture conditions (flask control), cultured on microcarrier beads alone (static control), or cultured on microcarrier beads and exposed to simulated microgravity (RWV). No statistically significant underlying differences in overall TER raw values were observed between conditions. (C) Data are shown as the % change in TER of cells exposed to microgravity vs. static (black) or flask controls (white). No statistically significant difference was observed in % change between the different culture conditions, although the % change in TER of cells from RWV relative to the static control was consistently lower (n = 4). simulated microgravity environment induces a subtle change in IEC barrier formation that is retained up to 14 days following removal from the simulated microgravity environment. Simulated microgravity alters tight junction protein localization not expression. To understand the molecular changes that might contribute to altered barrier function in cells subjected to simulated micro- gravity, we investigated whether cells cultured in the RWV exhibited changes in expression or localization of the key tight junction proteins, occludin and ZO-1. Localization of occludin and ZO-1 was monitored from day 9–14 post seeding on inserts after removal of cells from beads (RWV, static control) or flasks (flask control). Cells were stained in situ on inserts. Results G h The cytoskeletal protein, actin, that plays a critical role in maintaining the architecture of cell junctions, also localized intensely at the apicolateral membrane thus suggesting that polarization is not disrupted (Fig. 1K,L). These data show that HT-29.cl19a IECs are capable of attaching to and growing on microcarrier beads in a manner that facilitates formation of tight junctions. RWV culture affects barrier formation. Having shown that we could culture IECs on microcarrier beads in the RWV and observe expression of tight junction proteins, we next wanted to perform functional measures of barrier integrity. In order to accomplish this however, we had to resort to growing cells on conventional inserts i.e. a 2–3D format having first exposed them to a simulated microgravity environment by culturing them on microcarrier beads (Fig. 2A). After culture in the RWV for 18 days, cells were removed from microcarrier beads by trypsinization, and seeded onto semi-permeable filter supports at a density of 5 × 105 cells per support. Two separate control cultures were also prepared. A ‘flask’ control was prepared by culturing cells in a T75 flask prior to seeding on filter supports while a ‘static’ control involved culturing cells on microcarrier beads in a culture dish as opposed to the RWV. This control was particularly important as it served as the substrate control for cells in the RWV cultured on the same type of bead. Cells were cultured on inserts up to 14 days and TER was monitored over time. No significant difference was observed in raw TER values over time between the different conditions (Fig. 2B). When the relative change in TER between cells cultured in the RWV and the respective controls was calculated, there was no consistent difference detected vs. cells from a flask not subjected to the simulated micro- gravity environment (Fig. 2C). However, when cells from the RWV were compared with the appropriate substrate control – the static control – the simulated microgravity environment caused a slight but consistent drop in TER over the duration of the experiment (Fig. 2C). The most prominent change in %TER in RWV cells vs. static controls was observed on day 11(−6 ± 3% decrease vs. static; n = 4). This decrease did not reach statistical signif- icance and was not due to differences in seeding density (data not shown). Results G h Levels appeared to recover by day 14 although substantial levels of ZO-1 appeared to be present in the cytoplasm and not at the tight junction. (B,C) Confocal imaging and quantification of total B) ZO-1 and (C) occludin levels was performed using a Zeiss Axioscope 2 upright microscope and Zeiss LSM 5 Image Examiner software respectively. No consistent differences in protein levels were evident between the different conditions over the culture period on semi-permeable supports, although ZO-1 levels were lower in RWV and static controls vs. flask control at day 9, while occludin levels were reduced in the RWV condition at day 11 (p < 0.05; n = 4). igure 3. Delayed tight junction formation in IECs exposed to simulated microgravity. HT-29.cl19a IECs i Figure 3. Delayed tight junction formation in IECs exposed to simulated microgravity. HT-29.cl19a IECs were fixed with 4% paraformaldehyde, immunostained for ZO-1 (green) and occludin (red) and imaged by confocal microscopy. Actin was stained with Phalloidin. Arrows indicate the presence of apical tight junctions. Reduced levels of ZO-1 and occludin were observed in apical tight junctions on day 9 and 11 in IECs exposed to simulated microgravity. Levels appeared to recover by day 14 although substantial levels of ZO-1 appeared to be present in the cytoplasm and not at the tight junction. (B,C) Confocal imaging and quantification of total B) ZO-1 and (C) occludin levels was performed using a Zeiss Axioscope 2 upright microscope and Zeiss LSM 5 Image Examiner software respectively. No consistent differences in protein levels were evident between the different conditions over the culture period on semi-permeable supports, although ZO-1 levels were lower in RWV and static controls vs. flask control at day 9, while occludin levels were reduced in the RWV condition at day 11 (p < 0.05; n = 4). Simulated microgravity increases epithelial barrier susceptibility to challenge. Having shown that culturing IECs in the RWV led to a small drop in TER compared with static controls (c.f. Figure 2), we next inves- tigated if RWV culture pre-disposed IECs to a greater drop in barrier function in response to an agent that com- promises barrier integrity. Acetaldehyde is the major metabolite of alcohol and has been shown to directly reduce barrier function following in vitro administration to IEC cultures, as well as mediate the effects of ethanol adminis- tered in vivo43,44,51. Results G h On day 9, both ZO-1 and occludin localized to the cell membranes in flask control cells, and to a lesser extent in static control cells, as demonstrated by the sharp chicken-wire pattern of staining (Fig. 3A). However, in RWV-cultured cells, both tight junction proteins had a more diffuse sub-membranous localization. This did not appear to be due to a defect in actin localization or expression as actin showed a very clear pattern of staining in RWV-cultured cells similar to flask controls and superior to static controls. The same pattern of diffuse sub-membrane localization of ZO-1 and occludin was observed in RWV cells on day 11. However, by day 14 both ZO-1 and occludin had localized at cell membranes similar to flask and static control cultures. We quantified the staining intensity of each TJ protein over 6 intervals from day 2 to day 14 post-seeding (Fig. 3B,C). A significant decrease in ZO-1 was observed in RWV-treated cells only at day 9 and this subsequently reached equivalent expression with the flask control cells by day 11 (p < 0.05; n = 4; Fig. 3C). A decrease in ZO-1 staining was also observed in the static condition at day 9 but this did not reach significance. While occludin levels were higher in RWV cells at day 2 compared with flask and static controls, they were decreased at day 11 compared with flask controls but reached equivalent levels to both flask and static controls on day 14 (p < 0.05; n = 4; Fig. 3C). Overall, these data suggest that the altered TER in RWV cells is likely due to delayed membrane localization of ZO-1 and occludin rather than reduced expression. This may be due to possible effects on the trafficking of tight junction proteins to the apical membrane and delayed junction formation in IECs subjected to simulated microgravity. Scientific Reports \| (2019) 9:17531 \| https://doi.org/10.1038/s41598-019-53862-3 www.nature.com/scientificreports/ Figure 3. Delayed tight junction formation in IECs exposed to simulated microgravity. HT-29.cl19a IECs were fixed with 4% paraformaldehyde, immunostained for ZO-1 (green) and occludin (red) and imaged by confocal microscopy. Actin was stained with Phalloidin. Arrows indicate the presence of apical tight junctions. Reduced levels of ZO-1 and occludin were observed in apical tight junctions on day 9 and 11 in IECs exposed to simulated microgravity. Results G h static and flask controls (n = 6). (D) IECs at 14-day post-RWV have increased permeability to FD4 in response to acetaldehyde treatment (###p < 0.001 vs. RWV untreated) vs. static and flask controls (p < 0.001, n = 7). Figure 4. Simulated microgravity increases susceptibility of IECs to acetaldehyde-induced barrier dysfunction. (A) IECs grown on inserts for 11 days post-RWV culture exhibit a significant decrease in TER after 5hrs of acetaldehyde treatment (###p < 0.001 vs. RWV untreated) vs. static (p < 0.001) and flask controls (*p < 0.001; n = 7). (B) IECs at 11-day post-RWV also showed an increase in FD4 permeability after acetaldehyde treatment vs. static and flask controls (n = 7). Similarly, panel C shows IECs at 14-day post-RWV exhibited a significant decrease in TER (p < 0.05, n = 6) vs. untreated controls, and a non-significant decrease in TER vs. static and flask controls (n = 6). (D) IECs at 14-day post-RWV have increased permeability to FD4 in response to acetaldehyde treatment (###p < 0.001 vs. RWV untreated) vs. static and flask controls (*p < 0.001, n = 7). Figure 4. Simulated microgravity increases susceptibility of IECs to acetaldehyde-induced barrier dysfunction Random motion does not reproduce the effects of simulated microgravity on alcohol-induced barrier defects. While the static condition served as a valuable substrate control for cells bound to micro- carrier beads, we next determined if physical motion could recapitulate the effects of simulated microgravity in promoting a sustained susceptibility to barrier defects. As a model of physical motion, we cultured cell-bead aggregates in a “Biowiggler” device that can physically thrust structures in solution in 3-dimensions, with reduced unidirectional shear, at adjustable speeds to accommodate cell growth and therefore increased mass of the cell-bead aggregate over time. The Biowiggler has been utilized with a number of different cell culture systems to model the impact of motion on biological properties. We initially performed careful optimization protocols to determine the appropriate duration, speed and vigor of movement in which cell-bead aggregates could be cultured to ensure complete cell coverage of beads and subsequent successful seeding onto transwells (data not shown). HT-29.cl19a IECs were cultured on microcarrier beads for 12 days in the Biowiggler with matching cell-bead aggregates cultured in the RWV until confluent cell-bead aggregates formed (Fig. 5A). Staining for actin indicated appropriate cell cytoskeletal architecture in cell-bead aggregates cultured in the Biowiggler and RWV environments (Fig. Results G h Acetaldehyde vapor was the preferred mode of administration as it delivers a consistent and titrat- able dose of acetaldehyde to cells cultured in neighboring wells68. We treated cells at day 11 and day 14 post-RWV culture in order to assess whether barrier defects in response to challenge were apparent before and after appropriate localization of ZO-1 and occludin to cell membranes in RWV cultured cells (c.f. Figure 3). Exposure of cells on day 11 post-seeding to 0.5% acetaldehyde vapor for 5 hrs led to a significant decrease in TER in RWV cultures compared with flask and control cells (p < 0.001; n = 7; Fig. 4A). No change in TER occurred in untreated cultures over the 5 hr incubation period. In parallel, a significant increase in permeability to 4kD FITC-dextran occurred in RWV cells exposed to acetaldehyde (p < 0.001; n = 7; Fig. 4B). When cells at day 14 post-seeding were exposed to acetaldehyde, similar defects in barrier function were observed as TER was significantly reduced (p < 0.001; n = 6; Fig. 4C) while permeability to FITC-dextran was significantly increased (p < 0.001; n = 7; Fig. 4D). These data indicate that cells cultured in the RWV exhibit a significantly greater susceptibility to an agent that compromises barrier function than control cultures even when tight junction proteins appear to have localized appropriately at the cell membrane (day 14, c.f. Figure 3). Interestingly, FITC-dextran permeability in untreated RWV cells was elevated above the respective controls at day 11 but not day 14 and is thus consistent with data in Fig. 3 indicating maturation of tight junctions in cells cultured in the RWV and subsequently grown on inserts for 14 days. Scientific Reports \| (2019) 9:17531 \| https://doi.org/10.1038/s41598-019-53862-3 www.nature.com/scientificreports/ Figure 4. Simulated microgravity increases susceptibility of IECs to acetaldehyde-induced barrier dysfunction. (A) IECs grown on inserts for 11 days post-RWV culture exhibit a significant decrease in TER after 5hrs of acetaldehyde treatment (###p < 0.001 vs. RWV untreated) vs. static (p < 0.001) and flask controls (*p < 0.001; n = 7). (B) IECs at 11-day post-RWV also showed an increase in FD4 permeability after acetaldehyde treatment vs. static and flask controls (n = 7). Similarly, panel C shows IECs at 14-day post-RWV exhibited a significant decrease in TER (p < 0.05, n = 6) vs. untreated controls, and a non-significant decrease in TER vs. Results G h 5B). Cells were subsequently disassociated from the beads by gentle detachment using Accutase cell detachment solution and seeded at a density of 0.5 × 106 on 12 mm transwells. Cells were cultured for 11 days before exposure to acetaldehyde vapor as previously. Paired cultures were then treated with vehicle control or acetaldehyde vapor (0.5% for 5 hrs) and barrier properties were determined. TER was significantly reduced by acetaldehyde in cultures originating in the RWV compared with the control flask condition as previ- ously (p < 0.05; n = 3; Fig. 5C). However, cells cultured in the Biowiggler showed no significant increase in sus- ceptibility to acetaldehyde compared with control (Fig. 5C). Similarly, the acetaldehyde induction of permeability to FD4 was potentiated in cells previously cultured in the RWV compared with control (p < 0.05; n = 3), but cells cultured in the Biowiggler showed no significant increase in permeability relative to the flask control (Fig. 5D). These data indicate that the sustained increase in susceptibility to acetaldehyde challenge that occurs in cells cul- tured in a simulated microgravity cannot be recapitulated by randomized motion. Expression of epithelial tight junction proteins is unaltered by simulated microgravity with or without acetaldehyde challenge. Increased paracellular permeability to macromolecules such as FITC-dextran (FD4), following challenge of epithelial cells with barrier disrupting agents is often associated with reduced expression of tight junction proteins such as occludin and ZO-1. After being cultured in tissue culture flasks, the Biowiggler or the RWV, HT29 cells were subsequently seeded and grown on transwells as monolayers Scientific Reports \| (2019) 9:17531 \| https://doi.org/10.1038/s41598-019-53862-3 www.nature.com/scientificreports/ Figure 5. Susceptibility to acetaldehyde induction of barrier defects is specific to the simulated microgravity condition. (A) HT-29.cl19a IECs were cultured as microcarrier bead-cell aggregates under established RWV conditions or in a ‘Biowiggler’. Cell-bead aggregate formation was sampled at day 12 and visualized under phase-contrast microscopy. (B) Immunofluorescence imaging shows bead coverage by IECs and formation of cell junctions as shown by actin staining (red). Nuclei were stained with DAPI (blue). (C) Cells were removed from beads or a control flask and cultured on semi-permeable supports (transwells) for 11 days. TER was recorded immediately prior to exposure of cells from each condition to acetaldehyde vapor (5hrs), and the change in TER caused by acetaldehyde was calculated and expressed as the % change in TER from t0 to t5hr. Results G h Cells originally cultured in the RWV, but not the Biowiggler, showed a significantly greater decrease in TER from control flask conditions (p < 0.05 vs. Flask + acetaldehyde condition; n = 3). (D) After 5 hrs of acetaldehyde vapor exposure, permeability to FD4 (over 1 hr) was measured and found to be significantly elevated in cells from the RWV, but not the Biowiggler compared with control cells. Data are expressed as fold change in FD4 permeability (p < 0.05 vs. Flask + acetaldehyde condition; n = 3). Figure 5. Susceptibility to acetaldehyde induction of barrier defects is specific to the simulated microgravity condition. (A) HT-29.cl19a IECs were cultured as microcarrier bead-cell aggregates under established RWV conditions or in a ‘Biowiggler’. Cell-bead aggregate formation was sampled at day 12 and visualized under phase-contrast microscopy. (B) Immunofluorescence imaging shows bead coverage by IECs and formation of cell junctions as shown by actin staining (red). Nuclei were stained with DAPI (blue). (C) Cells were removed from beads or a control flask and cultured on semi-permeable supports (transwells) for 11 days. TER was recorded immediately prior to exposure of cells from each condition to acetaldehyde vapor (5hrs), and the change in TER caused by acetaldehyde was calculated and expressed as the % change in TER from t0 to t5hr. Cells originally cultured in the RWV, but not the Biowiggler, showed a significantly greater decrease in TER from control flask conditions (p < 0.05 vs. Flask + acetaldehyde condition; n = 3). (D) After 5 hrs of acetaldehyde vapor exposure, permeability to FD4 (over 1 hr) was measured and found to be significantly elevated in cells from the RWV, but not the Biowiggler compared with control cells. Data are expressed as fold change in FD4 permeability (p < 0.05 vs. Flask + acetaldehyde condition; n = 3). and subsequently subjected to 0.5% acetaldehyde vapor, as indicated. HT-29.cl19a monolayers were then lysed and processed for Western blotting. Compared to untreated HT-29.cl19a IECs previously cultured in flasks, cells grown in the Biowiggler appeared to have slightly less ZO-1 protein expression, however this was not signifi- cant (Fig. 6A; n = 3–4). Exposure to acetaldehyde of cells cultured in the Biowiggler or RWV had no effect on ZO-1 expression (Fig. 6A). Moreover, RWV culture conditions did not affect occludin expression in either the untreated or acetaldehyde vapor challenged condition (Fig. 6B). Results G h In addition, occludin expression was not signif- icantly altered in cells from flasks or the Biowiggler exposed to acetaldehyde vapor compared to their respective untreated controls. These data suggest that cells subjected to growth conditions in the Biowiggler or the RWV, with or without subsequent exposure to acetaldehyde vapor, do not display substantial changes in the overall protein expression of ZO-1 or occludin tight junction proteins. Acetaldehyde alters TJ protein localization in epithelial cells exposed to simulated microgravity. Immunofluorescence staining for ZO-1 and occludin at day 11 showed similar findings to data in Fig. 3 where untreated RWV cells exhibited delayed localization of these proteins to the tight junctions compared with flask and static controls (Fig. 7A). All three conditions showed disrupted membrane localization of ZO-1 and occludin following 5 hr exposure to acetaldehyde vapor (Fig. 7B). Actin filaments were disrupted in untreated RWV cells on day 11, while acetaldehyde treatment disrupted the actin cytoskeletal in all three culture conditions. At day 14, when RWV cells showed normal tight junction localization of occludin, ZO-1 and an intact actin cytoskeleton in untreated conditions (Fig. 7C), RWV cells showed a greater mislocalization of ZO-1 away from the cell mem- brane following exposure to acetaldehyde vapor than flask or static controls (Fig. 7D). RWV and flask control cells also exhibited greater disruption of actin staining than static controls following acetaldehyde exposure. Overall, these data indicate that exposure to a simulated microgravity environment increases susceptibility of epithelial cells to acetaldehyde-induced functional barrier defects and tight junction reorganization. Scientific Reports \| (2019) 9:17531 \| https://doi.org/10.1038/s41598-019-53862-3 www.nature.com/scientificreports/ Figure 6. Tight junction protein expression levels are not reduced by simulated microgravity conditions. HT-29.cl19a IECs cultured in tissue culture flasks (‘Flask’), the Biowiggler or the rotating wall vessel (RWV), were grown on transwells as monolayers and subsequently subjected to 0.5% acetaldehyde vapor for 5 hours. IEC monolayers were then lysed, processed for Western blotting and probed for the proteins indicated. (A) Representative blots probed for ZO-1, occludin and β-actin. (B) Quantification of densitometric analysis from Western blot experiments performed in duplicate (n = 3–4 independent experiments). Non-contiguous sections of the same blot are indicated by a black line. Figure 6. Tight junction protein expression levels are not reduced by simulated microgravity conditions. l Figure 6. Tight junction protein expression levels are not reduced by simulated microgravity conditions. Results G h HT-29.cl19a IECs cultured in tissue culture flasks (‘Flask’), the Biowiggler or the rotating wall vessel (RWV), were grown on transwells as monolayers and subsequently subjected to 0.5% acetaldehyde vapor for 5 hours. IEC monolayers were then lysed, processed for Western blotting and probed for the proteins indicated. (A) Representative blots probed for ZO-1, occludin and β-actin. (B) Quantification of densitometric analysis from Western blot experiments performed in duplicate (n = 3–4 independent experiments). Non-contiguous sections of the same blot are indicated by a black line. Figure 7. Simulated microgravity potentiates acetaldehyde-induced decrease in epithelial ZO-1. HT-29.cl19a IECs were fixed with 4% paraformaldehyde, immunostained for ZO-1 (green) and occludin (red) and imaged by Zeiss LSM 510. Actin was stained with phalloidin. Nuclear staining was performed using Hoechst 33258. Arrows indicate the position of apical tight junctions. (A) Reduced levels of ZO-1 were observed in apical tight junctions of IECs at 11 days post-RWV vs. static and flask control cells. (B) A greater reduction in junctional ZO-1 was observed in IECs at day 11 post-RWV following 5 hr acetaldehyde treatment versus the reduction seen in both static and flask controls. (C) Unchallenged IECs at day 14 post-RWV show ZO-I and occludin localization to membrane junctions similar to flask and static control conditions. (D) Acetaldehyde challenge (5 hrs) disrupted junctions in all conditions but caused greater disruption of ZO-1 and occludin staining in the day 14 post-RWV condition (n = 3). Figure 7. Simulated microgravity potentiates acetaldehyde-induced decrease in epithelial ZO-1. HT-29.cl19a IECs were fixed with 4% paraformaldehyde, immunostained for ZO-1 (green) and occludin (red) and imaged by Zeiss LSM 510. Actin was stained with phalloidin. Nuclear staining was performed using Hoechst 33258. Arrows indicate the position of apical tight junctions. (A) Reduced levels of ZO-1 were observed in apical tight junctions of IECs at 11 days post-RWV vs. static and flask control cells. (B) A greater reduction in junctional ZO-1 was observed in IECs at day 11 post-RWV following 5 hr acetaldehyde treatment versus the reduction seen in both static and flask controls. (C) Unchallenged IECs at day 14 post-RWV show ZO-I and occludin localization to membrane junctions similar to flask and static control conditions. (D) Acetaldehyde challenge (5 hrs) disrupted junctions in all conditions but caused greater disruption of ZO-1 and occludin staining in the day 14 post-RWV condition (n = 3). www.nature.com/scientificreports/ Not until day 14 did the RWV cells display evidence of ZO-1 staining at lateral cell borders, indicative of differentiated cells. Occludin staining also exhibited delayed lateral membrane localization. This was in contrast to flask and static controls where occludin and ZO-1 had localized to the membranes as early as day 9. The delay in TJ protein localization may be due to a specific defect in trafficking of occludin and ZO-1 or possibly to an overall delay in differentiation of IEC that has been identified previously in a modeled microgravity environment15. In addition, the observed alterations in actin localization may contribute to a defect or delay in appropriate adherens junction protein localization, an event that precedes tight junction formation48,75. Indeed, the cytoskeleton has been pro- posed as a gravity sensor in mammalian cells that lack a specialized organelle, such as a statolith in plants, while actin is capable of acting as a mechanosensor and disruption of actin filaments has been observed in a number of cell types subjected to spaceflight microgravity or simulated microgravity76–79.hii yp j pl g g y g y The significance of our data lies in the novel finding that the low shear simulated microgravity environment of the RWV imprints an altered pattern of tight junction assembly that is retained up to 14 days after removal from the RWV. Although the magnitude of the decrease in TER of RWV cells compared with static controls is modest, it is consistent with observations that although various aspects of gastrointestinal function such as transit time, mucus secretion and nutrient uptake are compromised in astronauts and rodents after time spent in spaceflight, in general they do not suffer from severe GI distress80–84. However, the presence of a modest underlying barrier defect may predispose to a more deleterious response to agents that can enhance barrier defects. This principle of an underlying barrier defect that is not fully manifest until exposed to challenge, underpins much research into the eti- opathogenesis of a number of chronic inflammatory conditions that feature increased intestinal permeability prior to onset of inflammation. These conditions include Crohn’s disease, ulcerative colitis, celiac disease and Type 1 diabetes32. With this in mind, we investigated whether cells previously cultured in the RWV exhibited an increased response to an agent known to compromise barrier function, the alcohol metabolite, acetaldehyde. www.nature.com/scientificreports/ in rats. However, this was not attributed to defects in epithelial proliferation or migration but rather to changes in the cytoskeleton core of the intestinal villi presumably causing villus contraction70. To better understand the effects of microgravity on the functional behavior of the epithelial cells that line the intestinal tract, we exam- ined the impact of simulated microgravity on intestinal epithelial cells cultured in a rotating wall vessel. The RWV generates a low shear simulated microgravity environment that enables epithelial cells to form 3-D cel- lular aggregates with greater in vivo-like characteristics, including tight junctions and extracellular processes such as microvilli, than conventional 2-D culture where cells are grown as flat, two-dimensional monolayers on impermeable surfaces10,12,17,71. This device has also been utilized for the purposes of creating cell cultures for tissue engineering, the study of host-pathogen interactions and drug discovery10,12,72. It is important to note that whereas most studies analyzing 3-D epithelial cultures have used cells grown on a flat impermeable surface as a 2-D control (flask condition in our studies), we removed cells from beads (RWV, static, Biowiggler conditions) and subsequently cultured them in a polarized manner on semi-permeable filters (transwells) in order to perform functional measurements of tight junction integrity. Therefore, these cells were grown in a more advanced system than 2-D as they are capable of correct spatial organization of apical vs. basolateral proteins and formation of tight junctions as determined functionally by measurement of TER, and visually by immunofluorescence staining. This necessary technical approach to facilitate functional and quantifiable measurement of barrier properties, also enabled us to determine if effects of the simulated microgravity environment in the RWV were retained by cells following removal from that environment. We demonstrated that following culture for 18 days in the RWV and subsequent seeding onto semi-permeable supports, that intestinal epithelial cells show delayed formation of tight junctions, as determined by membrane localization of occludin and ZO-1, as well as a minor reduction in barrier function. ZO-1, is a primarily apicolateral localizing protein that is critical for the formation of cell-cell junctions, although in undifferentiated cells, ZO-1 is found in other subcellular sites including the nucleus73,74. In our studies, ZO-1 staining in HT-29.cl19a monolayers generated from RWV-cultured cells showed a primarily cytoplasmic and perinuclear localization thus resembling ZO-1 distribution patterns in undifferentiated cells. Scientific Reports \| (2019) 9:17531 \| https://doi.org/10.1038/s41598-019-53862-3 Discussion Existing data from space flight studies indicate that a microgravity environment can have profound effects on human physiology and lead to clinical symptoms and illnesses including gastroenteritis69. Moreover, animal stud- ies in spaceflight identified morphological changes in the intestine that indicated reduced jejunal villus length Scientific Reports \| (2019) 9:17531 \| https://doi.org/10.1038/s41598-019-53862-3 www.nature.com/scientificreports/ www.nature.com/scientificreports/ structure of the intestinal microbial community observed after 4 weeks of hindlimb unloading by Shi et al.85 were by themselves insufficient to provoke an increase in intestinal permeability. While the mice subjected to hindlimb unloading were subsequently more susceptible to challenge, the nature of the experimental challenge in this study did not selectively modify tight junction function as DSS acts by essentially destroying the epithelial monolayer thus obliterating tight junctions, whereas in our studies acetaldehyde vapor challenge of IECs does not cause chemical stripping of the epithelial lining but rather induces modulation of tight junction protein local- ization while keeping the monolayer intact51,52,88. This was confirmed in our studies by a reduction in, but not a catastrophic loss of, TER in IEC monolayers exposed to acetaldehyde. p y p y With respect to the mechanism(s) of increased susceptibility to acetaldehyde-induced barrier disruption, Sheth et al.52 showed that acetaldehyde destabilizes the interaction of junctional proteins with the actin cytoskel- eton, and this contributes to mislocalization of the tight junction proteins occludin and ZO-1. The molecular changes observed in our study are consistent with our understanding of the effects of acetaldehyde on tight junc- tions. The mechanisms involved in mediating this effect of simulated microgravity on barrier function and the increased susceptibility to alcohol metabolite challenge remain to be determined. They may involve epigenetic modulation of genes coding for proteins involved in junction assembly and protein trafficking, as suggested by the delayed junction localization of ZO-1 and occludin observed in cells cultured in microgravity conditions (c.f. Figure 3). These are attractive avenues for follow-up studies.lf gh p Of relevance to human health on Earth, adaptation to spaceflight shares many similarities with the effects of aging with respect to their impact on human physiology. Both of these factors exert effects at a cellular and molecular level that impact and promote a decline of multiple physiological systems in the body89,90. Pre- and post-flight testing has identified many physiological alterations that also occur in aging. These include bone density loss, muscle atrophy, negative calcium balance, cardiovascular changes, metabolic & endocrine effects, immune suppression and decreased gastrointestinal function84,91,92. One important difference between the effects of spaceflight and aging is that the effects of spaceflight are largely recoverable upon return to Earth, although flight durations have been quite short with the longest sustained period that any human has spent in space being 14 months90. www.nature.com/scientificreports/ Of note, studies of digestive dysfunction in rats during spaceflight identified that altered hepatic and xenobiotic enzyme expression persisted upon return to Earth even after other digestive parameters had alleviated84. Nevertheless, spaceflight and approaches that try to recreate the conditions of spaceflight including head-down bed rest in the case of humans, or devices that simulate microgravity such as the rotating wall vessel in the case of cell cultures, have been used as model systems to study the effects of aging on various physiolog- ical parameters and cell types8,16,93,94. A number of studies have identified increased intestinal permeability to macromolecules such as mannitol, and in particular, significantly elevated permeability in aged rodent intestine following challenge with agents that increase permeability such as phorbol ester50,95,96. Studies of human intestinal permeability in younger vs. elderly adult humans have centered on double sugar and lactulose:mannitol ratios, but while these studies did not show a clear difference between younger vs. older adults, interpretation of these data is complicated by an expected decrease in renal saccharide clearance with age, as well as a small sample size97,98. A study investigating the effects of aging on intestinal permeability in non-human primates identified an increase in macromolecule permeability to horseradish peroxidase and a decrease in the electrical integrity of aging intestine as assessed by potential difference (PD) measurements. Furthermore, these changes were also associated with decreased occludin and ZO-1 levels in colonic mucosal tissue99. Overall, our data share consist- encies with animal studies identifying an increase in intestinal permeability associated with aging, albeit with a requirement for an experimental challenge to fully identify the nature of the barrier defect. Thus, the low-shear simulated microgravity environment generated by the RWV may also serve as a valuable model system to investi- gate the effects of aging on epithelial barrier function as well as other properties of epithelial cells.h gf g g p p p p The data generated using the RWV system are intriguing and although further studies into the mechanism(s) responsible for the observed barrier defect in cells exposed to simulated microgravity are needed, we can spec- ulate on the implications of these findings. www.nature.com/scientificreports/ We tested cells grown on permeable supports at day 11 and day 14 after removal from the RWV. The significance of these time points is that evidence of tight junction assembly, as determined by tight junction protein localization, was not pres- ent at day 11 but was detectable by day 14. At day 11, we observed a clear increase in susceptibility to the effects of acetaldehyde on both TER and permeability to 4kD FITC-dextran. This was consistent with the lack of tight junc- tion protein localization at day 11. The most striking observation was that even at day 14 when ZO-1 and occludin were localized at lateral membranes, that a significant decrease in TER and increase in FITC-dextran permeability was clearly apparent. These data indicate that in spite of appropriate TJ protein localization, that a functional barrier defect was evident following challenge with an agent that compromises epithelial permeability. We were also able to generate data indicating that the sustained susceptibility to a barrier defect (induced by acetaldehyde challenge) was not due to randomized motion but was a specific attribute imparted by the simulated microgravity environment of the RWV. Western blot studies did not show any significant change in expression of total cellular occludin or ZO-1 in unchallenged or acetaldehyde challenged 11-day old epithelial monolayers from control or RWV-cultured cells. When coupled with the immunofluorescence imaging data, this suggests that the functional barrier defects arising from RWV culture are likely mediated by changes in tight junction protein localization, and possibly signaling regulation, rather than alterations in total expression levels of individual junction proteins. g p j p Our observations are further supported by a recent in vivo study using the ground-based hindlimb unloading model of microgravity simulation in mice. This identified changes in gut microbiome composition and increased susceptibility to the dextran sulfate sodium model of chemical colitis in mice subjected to hindlimb unloading85. The same study showed no effect of hindlimb unloading by itself on intestinal permeability as measured by in vivo FITC-dextran permeability. This is consistent with our findings that IECs cultured in the RWV simulated microgravity environment had no significant change in permeability until faced with experimental challenge. www.nature.com/scientificreports/ Moreover, while numerous studies have identified both positive and negative roles for the gut microbiome in modulating intestinal epithelial barrier function86,87, it is worth noting that the changes in composition and Scientific Reports \| (2019) 9:17531 \| https://doi.org/10.1038/s41598-019-53862-3 9 www.nature.com/scientificreports/ www.nature.com/scientificreports/ The capacity of the RWV system to induce an underlying barrier defect in cells that have appropriate tight junction localization may serve as a very useful experimental model to study the pathogenesis of disease conditions associated with underlying barrier defects prior to disease activation, such as inflammatory bowel disease, or patients with increased sensitivity to barrier-disrupting agents such as alcohol. With respect to the potential impact of a microgravity environment on the gastrointestinal tract, these data suggest that intestinal epithelial cells develop a minor barrier defect possibly without significant pathologic manifestations until such time as they are exposed to an agent, either chemical or biological, that can compromise barrier function. This could result in a greatly exaggerated barrier defect that may have dramatic consequences for epithelial integrity and intestinal homeostasis. Specifically, given previous evidence of immunosuppression and increased virulence of pathogenic bacteria in conditions of microgravity, an underlying defect in the intes- tinal epithelial barrier could greatly expedite bacterial colonization of the host, or effects of bacterial products, and give rise to a more rapid and severe infection. This may have particular relevance to long-term space travel and colonization where exposure to a food-borne pathogen may result in a more severe pathology than on Earth. Materials and Methods i i i HT-29.cl19a cells were initially grown as monolayers with fresh McCoy’s 5A media with L-glutamine (Mediatech, Manassas, VA) supplemented with 10% fetal calf serum (Hyclone, Waltham, MA; catalog # SH3007303), Penicillin (500 I.U.) and Streptomycin (10,000 ug/ml) (Mediatech) culture medium in T-75 cell culture flasks (Corning, Inc. Corning, NY) at 37 °C in a 5% CO2 environment in preparation for seeding into the rotating wall vessel (STLV, Synthecon, Inc, Houston, TX). Cells were cultured until approx- imately 70–90% confluent. HT-29.cl19a monolayers were washed once with 0.25% Trypsin-ethylenediamine tetraacetic acid (Gibco, Carlsbad, CA), removed from flasks by treatment with 0.25% Trypsin-EDTA for 15 min- utes at 37 °C and resuspended in fresh culture medium, supplemented with FBS (5%) and L-glutamine (2.5 mM), at a density of 2–2.2 × 105 cells/ml. Cells were assayed for viability by trypan blue exclusion. Cells were introduced to the RWV containing 5 ml/mg of Cytodex-3 microcarrier beads, or 250 mg of Corning microcarrier beads, resulting in a ratio of approximately 10 cells/bead. Cells were cultured in the RWV at a rotation speed adjusted to maintain beads and cells in suspension (16.8–17.8 rpm). HT-29.cl19a cells cultured with beads in a petri-dish in the absence of rotation exposure served as a substrate (Static) control. Fresh medium was replenished by 80–90% of the total volume after 3 days initially and every 24–48 h thereafter. Cell culture in the random motion incubator (Biowiggler). The BiowigglerTM is a PC programmable eight-position stirrer that simplifies cell culture applications (Global Cell Solutions, Charlottesville, VA). Each of eight magnetic drive positions can be independently programmed to stir intermittently, continuously or “wiggle” in a reversing two-way motion. Proprietary LeviTubes (50 mL volume) were preconditioned for 24 hrs prior to cell seeding by adding warm PBS (1×) and placed in the Biowiggler to rotate bidirectionally at 40 rpm inside the CO2 incubator. Phosphate buffer was replaced by 25 mL of pre-warmed McCoys 5A media enriched with 10% FBS. To the LeviTubes and in sequence, 100 mg of reconstituted Corning enhanced attachment microcarrier beads (Corning, cat#3779; Corning Inc., Corning, NY) were added, followed by 5 × 106 HT-29.cl19a cells to a final volume of 40 mL. LeviTubes were placed in the CO2 incubator without shaking to allow the cells to attach to the carrier beads for 5 minutes. For the first 24 hours, LeviTubes were rotated bidirectionally at 40 rpm followed by a 5-minute static rest period. Materials and Methods i i i Microcarrier preparation. Cytodex-3 microcarrier beads (collagen and dextran coated, average 175 μm in size) were purchased from GE Healthcare (Piscataway, NJ) and prepared based on the manufacturer’s protocol. Beads were rehydrated in Ca2+ and Mg2+ free phosphate buffered saline (PBS)(50–100 ml/g) for 3 h at room temperature. Beads were sterilized by removal of the supernatant and 70% ethanol (70–100 ml/g) was added. Beads were washed twice in this manner and incubated in 70% ethanol solution overnight. Prior to use, beads were washed three times in PBS (50 ml/g) and once with culture medium (50 ml/g) before use. Corning enhanced attachment microcarrier beads, (cat#3779, Corning Inc., Corning, NY) were reconstituted in sterile, molecular grade water at a concentration of 200 mg/ml. Scientific Reports \| (2019) 9:17531 \| https://doi.org/10.1038/s41598-019-53862-3 10 www.nature.com/scientificreports/ Cell culture in the rotating wall vessel system. The development of 3-D cell culture model was per- formed as previously described17,71,100. HT-29.cl19a cells were initially grown as monolayers with fresh McCoy’s 5A media with L-glutamine (Mediatech, Manassas, VA) supplemented with 10% fetal calf serum (Hyclone, Waltham, MA; catalog # SH3007303), Penicillin (500 I.U.) and Streptomycin (10,000 ug/ml) (Mediatech) culture medium in T-75 cell culture flasks (Corning, Inc. Corning, NY) at 37 °C in a 5% CO2 environment in preparation for seeding into the rotating wall vessel (STLV, Synthecon, Inc, Houston, TX). Cells were cultured until approx- imately 70–90% confluent. HT-29.cl19a monolayers were washed once with 0.25% Trypsin-ethylenediamine tetraacetic acid (Gibco, Carlsbad, CA), removed from flasks by treatment with 0.25% Trypsin-EDTA for 15 min- utes at 37 °C and resuspended in fresh culture medium, supplemented with FBS (5%) and L-glutamine (2.5 mM), at a density of 2–2.2 × 105 cells/ml. Cells were assayed for viability by trypan blue exclusion. Cells were introduced to the RWV containing 5 ml/mg of Cytodex-3 microcarrier beads, or 250 mg of Corning microcarrier beads, resulting in a ratio of approximately 10 cells/bead. Cells were cultured in the RWV at a rotation speed adjusted to maintain beads and cells in suspension (16.8–17.8 rpm). HT-29.cl19a cells cultured with beads in a petri-dish in the absence of rotation exposure served as a substrate (Static) control. Fresh medium was replenished by 80–90% of the total volume after 3 days initially and every 24–48 h thereafter. Cell culture in the rotating wall vessel system. The development of 3-D cell culture model was per- formed as previously described17,71,100. Materials and Methods i i i This combined period allowed rounded cells make contact with carrier beads (adsorption), followed by flattening of cells on the side of contact (attachment) to promote cell attachment to the microcarriers. Thereafter, cell aggregates continued to grow and proliferate by continued bidirectional rotation at 40 rpm. Media and cell cultures (50 μl aliquots) were monitored every 2 days. Media was changed every day. Harvesting cells from microcarrier beads. Cells were harvested 18 days after initial seeding in the RWV or in respective control environments (Biowiggler; culture flask). Cells cultured with beads were removed from the RWV or petri-dish, allowed to settle, and incubated with Ca2+ and Mg2+ PBS containing 0.02% EDTA (w/v) (50–100 ml/g beads, pH7.6) for 5 minutes at 37 °C. The PBS-EDTA was removed and the beads were incubated with Accutase cell detachment solution (CorningTM cat 25058CI, Corning, NY) at 37 °C with occasional agitation for 15 minutes. After 15 minutes, 30 ml culture medium was added and detached cells separated from beads by filtration through a 100μm nylon filter (Millipore, Bellerica, MA). Detached HT-29.cl19a cells were re-suspended at a density of 2.5 × 105 cells/ml and seeded onto 12 mm semi-permeable polycarbonate inserts (Millipore) in 24-well plates for 9,11 or 14 days for functional measurements of barrier integrity and to assess junction assembly. Cell culture conditions for barrier function assays. Four cell culture growth conditions were used for seeding and growth of epithelial monolayers on transwells for the measurement of barrier properties: Briefly, the multicolor confocal image was separated into its composite colors using the Zeiss LSM 5 image examiner software. The resulting single-color field was exported into ImageJ and mean fluorescence intensity (MFI) was calculated using the Analyze-Measure function. MFI was measured and averaged from 5 fields/replicate from 3–4 independent experiments.if gi p p p Microcarrier bead-cell aggregates were fixed in 4% buffered PFA, washed in PBS (Mg2+/Ca2+) and embed- ded in O.C.T. (VWR). 5–8 μm sections were mounted on microscope slides, washed with PBS (Mg2+/Ca2+), permeabilized with 0.3% Triton in PBS for 30 minutes and consecutively blocked using 10% normal donkey serum in PBS-T (0.02%). Cells were stained using occludin primary antibody (Life Technologies) and after PBS wash, incubated with Alexa-488 conjugated anti-rabbit secondary antibody and Alexa-647 Phalloidin (Jackson ImmunoResearch Labs, West Grove, PA.) Cells were mounted using ProLong® Gold Antifade Reagent with DAPI (Life Technologies, Carlsbad, CA). Bead-cell aggregates were visualized under phase-contrast microscopy (EVOS microscope; AMG/Thermofisher, Waltham, MA). Immunofluorescence staining of bead-cell aggregates were imaged using a Leica DM5500 fluorescent microscope (Leica Microsystems, Inc., Buffalo Grove, IL). Image panels were assembled into complete figures in Adobe Photoshop (San Jose, CA). Western blotting. After acetaldehyde treatment, cells from inserts containing HT-29.cl19a monolayers were suspended in ice-cold lysis buffer (50 mM Tris, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 20 µM NaF, 1 mM EDTA, 1 µg/ml antipain, 1 µg/ml pepstatin, 1 µg/ml leupeptin, 1 mM NaVO3, 100 µg/ml phenylmethylsul- fonyl fluoride). Cells were mechanically detached followed by centrifugation at 10,000 rpm for 10 minutes. Cell lysate supernatants were assayed for protein content using a Bio-Rad protein assay kit (Bio-Rad, Hercules, CA). Equal concentrations of proteins lysates were resuspended in loading buffer (60 mM Tris-Cl pH 6.8, 2% SDS, 5% β-mercaptoethanol, 0.01% bromophenol blue, 10% glycerol) and incubated at 95 oC for 10 minutes. Proteins were separated by SDS-PAGE and transferred onto polyvinylidene fluoride membranes (Millipore). Membranes were blocked with nonfat milk in TBS-T (Tris-buffered saline with 0.1% Tween-20) for 1 h at room temperature prior to addition of primary antibody; ZO-1 (1:1000 dilution, #61-7300, ThermoFisher Scientific, Waltham, MA), or occludin (1:1000 dilution, #71-1500, Invitrogen, Carlsbad, CA), in 1% nonfat milk in TBST. Membranes were incubated overnight at 4 oC101. Membranes were washed 5x for 5 min per wash with TBS-T, then incubated with peroxidase-conjugated secondary antibodies (goat anti-mouse (#115-036-062) or goat anti-rabbit (#111-036- 045); Jackson Immunoresearch Laboratories, Inc. Cell culture conditions for barrier function assays. Four cell culture growth conditions were used for seeding and growth of epithelial monolayers on transwells for the measurement of barrier properties: For permeability studies, HT-29.cl19a cells grown on semi-permeable membranes for 11 and 14 days were incubated with 1 mg/ml FITC-dextran in the apical well. At the end of acetaldehyde treatment, 100ul of media from the basolateral well was removed and fluorescence was measured with a microplate reader (SpectraMax M2, Molecular Devices). Transepithelial permeability was meas- ured as 4-kilodalton fluorescein isothiocyanate-dextran (FITC-dextran; FD4) (Sigma Chemical Co, St. Louis, MO) flux across IEC monolayers. Transepithelial electrical resistance. After 18 days of culture in the presence or absence of simulated microgravity, HT29.cl19a cells were seeded onto 12 mm semi-permeable inserts and cultured for 11 or 14 days. The transepithelial electrical resistance (TER) across HT-29.cl19a monolayers was assessed before and after treat- ment using a voltohmeter (WPI, Sarasota, FL) and electrodes (WPI). To normalize the variation in TER, data were expressed as percentage of pre-treatment TER values. Immunofluorescence microscopy. 2.5 × 105 HT-29.cl19a cells were seeded onto 12 mm Millicell-HA filters for 11 and 14 days prior to acetaldehyde treatment. After treatment, monolayers were fixed with 4% par- aformaldehyde in PBS for 10 minutes at room temperature, permeabilized with 0.1% Triton-X 100 in PBS for 10 minutes, and quenched with 50 mM ammonium chloride for 5 minutes. Between steps, cells were washed (x3) with PBS. Blocking was performed with 1% bovine serum albumin in PBS at 4 °C overnight. Cells were incubated with primary antibodies overnight at 4 °C in a humidified chamber. After PBS washing (x3), second- ary antibodies: Alexa-488 conjugated goat anti-mouse (excitation/emission maxima at 495/519 nm; Molecular Probes, Eugene, OR), Alexa-568 conjugated goat anti-rabbit (excitation/emission maxima at 578/603;Molecular Probes, Eugene, OR) were added at a 1:100 dilution, and Alexa-647 Phalloidin (excitation/emission maxima at 650/668 nm; Molecular Probes, Eugene, OR) was added at 1:25 dilution, in 1% BSA in PBS for 1 hour at 37 °C. After washing (three times with PBS), cells were incubated with Hoechst 33258 (excitation/emission maxima at 352/461 nm; Molecular Probes) in PBS (1:100) for 5 min at 37 °C. The final washing stage consisted of 3 washes with PBS. Then the cells on the filter membrane were transferred onto glass slides and mounted in ProLong Gold Antifade Reagent (Molecular Probes). Confocal microscopy was performed using a Zeiss LSM 510 laser-scanning confocal system on a Zeiss Axioscope 2 upright microscope (Zeiss, Jena, Germany). Quantification of confocal images was performed using ImageJ. Cell culture conditions for barrier function assays. Four cell culture growth conditions were used for seeding and growth of epithelial monolayers on transwells for the measurement of barrier properties: Cell culture conditions for barrier function assays. Four cell culture growth conditions were used for seeding and growth of epithelial monolayers on transwells for the measurement of barrier properties: Flask control. Monolayer controls for HT-29.cl19a were grown in T-75 flasks for 10–18 days prior to seeding on transwell inserts. RWV. As described in detail above, HT-29.cl19a cells were cultured in the RWV at a density of 2.0–2.2 × 105 cells/ml with microcarrier beads in a 10:1 ratio (cells:beads) at a rotation speed adjusted to maintain beads and cells in suspension (16.8–17.8 rpm) for 18 days. RWV. As described in detail above, HT-29.cl19a cells were cultured in the RWV at a density of 2.0–2.2 × 105 cells/ml with microcarrier beads in a 10:1 ratio (cells:beads) at a rotation speed adjusted to maintain beads and cells in suspension (16.8–17.8 rpm) for 18 days. Static control. HT-29.cl19a cells cultured at a density of 2.0–2.2 × 105 cells/ml with microcarrier beads in a 10:1 ratio (cells:beads) in a petri-dish in the absence of rotation exposure. Media changes were performed to coincide with those of the RWV. This condition served as a microcarrier bead substrate control without the effects of the RWV (Static) control. Biowiggler. As described in detail above, 5 × 106 HT-29.cl19a cells in cell culture media were added to custom-specific Levi tubes for pre-attachment prior to defined adsorption and attachment to microcarrier bead phases. Cell aggregates continued to grow and proliferate by continued bidirectional rotation at 40 rpm. Media and cell cultures were monitored every two days up to 18 days. Acetaldehyde treatment studies. Acetaldehyde (200–400 μM)(Sigma Chemical Co, St. Louis, MO), treatment was performed as previously described51,68. After 11 or 14 days of culture post- seeding on transmem- brane inserts, HT29Cl.19a cells were preincubated with PBS containing 1.33 mM CaCl2 and 1 mM MgCl2 for 1 h. Cells were then exposed to 0.5% vapor-phase acetaldehyde in adjacent wells in a sealed 24-well culture plate for 5 h51. Cyanamide (10–50 μM) was added apically to inserts at the start of treatment to inhibit aldehyde dehy- drogenase51. Addition of 0.5% vapor-phase acetaldehyde in the adjacent wells results in an approximate 400 μM acetaldehyde concentration in the buffer incubating the cell monolayers51. Scientific Reports \| (2019) 9:17531 \| https://doi.org/10.1038/s41598-019-53862-3 www.nature.com/scientificreports/ Transepithelial permeability studies. Received: 4 September 2018; Accepted: 31 October 2019; Published: xx xx xxxx Cell culture conditions for barrier function assays. Four cell culture growth conditions were used for seeding and growth of epithelial monolayers on transwells for the measurement of barrier properties: West Grove, PA) diluted at 1:5000 in 1% nonfat milk in TBST for 1 h at room temperature. Immunoreactive proteins were detected using SuperSignal West Pico chemilumi- nescence detection kit (Thermo Scientific, Rockford, IL). Densitometric analysis of Western blots was performed using ImageJ software (National Institutes of Health). Statistical analysis. Data are presented as ± SEM for a series of n experiments. Data are presented as per- centage of respective controls, arbitrary units, or fluorescence units. Statistical analysis was performed by analysis of variance (ANOVA) and Bonferroni post-test using Graphpad Prism Software (GraphPad Software, La Jolla, CA). P values ≤ 0.05 were considered significant. Received: 4 September 2018; Accepted: 31 October 2019; Published: xx xx xxxx Received: 4 September 2018; Accepted: 31 October 2019; Published: xx xx xxxx Scientific Reports \| (2019) 9:17531 \| https://doi.org/10.1038/s41598-019-53862-3 www.nature.com/scientificreports/ References Gene expression in space. Nat Med 5, 359 21. Kaur, I., Simons, E. R., Castro, V. A., Mark Ott, C. & Pierson, D. L. Changes in neutrophil functions in astronauts. Brain Behav Immun 18, 443–450, https://doi.org/10.1016/j.bbi.2003.10.005 (2004).fl p g j 22. Kaur, I., Simons, E. R., Kapadia, A. S., Ott, C. M. & Pierson, D. L. Effect of spaceflight on ability of monocytes to respond to endotoxins of gram-negative bacteria. Clin Vaccine Immunol 15, 1523–1528, https://doi.org/10.1128/CVI.00065-08 (2008).tl 23. Mehta, S. K. et al. Decreased non-MHC-restricted (CD56+) killer cell cytotoxicity after spaceflight. J Appl Physiol (1985) 91 1814–1818, https://doi.org/10.1152/jappl.2001.91.4.1814 (2001). 1814–1818, https://doi.org/10.1152/jappl.2001.91.4.1814 (200 p g j pp 24. Konstantinova, I. V., Rykova, M. P., Lesnyak, A. T. & Antropova, E. A. Immune changes during long-duration missions. J Leukoc Biol 54, 189–201 (1993).li Biol 54, 189–201 (1993). 25. Sonnenfeld, G. Editorial: Space flight modifies T cellactivation-role of microgravity. J Leukoc Biol 92, 1125–1126, https://doi /10 1189/jlb 0612314 (2012) 25. Sonnenfeld, G. Editorial: Space flight modifies T cellactivation-role of microgravity. J Leukoc Biol 92, 1125–1126, https://doi org/10.1189/jlb.0612314 (2012).h g j ( ) 26. Chang, T. T. et al. The Rel/NF-kappaB pathway and transcription of immediate early genes in T cell activation are inhibited by microgravity. J Leukoc Biol 92, 1133–1145, https://doi.org/10.1189/jlb.0312157 (2012). g y p g j 27. Cogoli, A. Signal transduction in T lymphocytes in microgravity. Gravit Space Biol Bull 10, 5–16 (1997). l l fl h l b l d l d l l f l b l 28. Wilson, J. W. et al. Space flight alters bacterial gene expression and virulence and reveals a role for global regulator Hfq. Proc Nat Acad Sci U S A 104, 16299–16304, https://doi.org/10.1073/pnas.0707155104 (2007).f 28. Wilson, J. W. et al. Space flight alters bacterial gene expression and virulence and reveals a role for global regu Acad Sci U S A 104, 16299–16304, https://doi.org/10.1073/pnas.0707155104 (2007).f 29. Nickerson, C. A. et al. Microgravity as a novel environmental signal affecting Salmonella enterica serovar Typhimurium virulence Infect Immun 68, 3147–3152 (2000).l f 30. Rosenzweig, J. A., Ahmed, S., Eunson, J. Jr. & Chopra, A. K. Low-shear force associated with modeled microgravity and spaceflight does not similarly impact the virulence of notable bacterial pathogens. Appl Microbiol Biotechnol 98, 8797–8807, https://doi. org/10.1007/s00253-014-6025-8 (2014). g 31. Turner, J. R. Intestinal mucosal barrier function in health and disease. Nat Rev Immunol 9, 799–809, https://doi.org/10.1038 nri2653 (2009). 32. Arrieta, M. C., Bistritz, L. References 1. Lutwak, L., Whedon, G. D., Lachance, P. A., Reid, J. M. & Lipscomb, H. S. Mineral, electrolyte and nitrogen balance studies of the Gemini-VII fourteen-day orbital space flight J Clin Endocrinol Metab 29 1140–1156 https://doi org/10 1210/jcem-29-9-1140 (1969) 1. Lutwak, L., Whedon, G. D., Lachance, P. A., Reid, J. M. & Lipscomb, H. S. Mineral, electrolyte and nitrogen balance studies of the Gemini-VII fourteen-day orbital space flight. J Clin Endocrinol Metab 29, 1140–1156, https://doi.org/10.1210/jcem-29-9-1140 (1969). 2. Hughes-Fulford, M. Altered cell function in microgravity. Exp Gerontol 26, 247–256 (1991). 1. Lutwak, L., Whedon, G. D., Lachance, P. A., Reid, J. M. & Lipscomb, H. S. Mineral, electrolyte and nitrogen balance studies of the Gemini-VII fourteen-day orbital space flight. J Clin Endocrinol Metab 29, 1140–1156, https://doi.org/10.1210/jcem-29-9-1140 (1969). g g y p 3. Ellis, S., Giometti, C. S. & Riley, D. A. Changes in muscle protein composition induced by disuse atrophy: analysis by two dimensional electrophoresis. Physiologist 28, S159–160 (1985). g g y p 3. Ellis, S., Giometti, C. S. & Riley, D. A. Changes in muscle protein composition induced by disuse atrophy: analysis by two- dimensional electrophoresis. Physiologist 28, S159–160 (1985). p y g 4. Philpott, D. E. et al. Microgravity changes in heart structure and cyclic-AMP metabolism. Physiologist 28, S209–210 (1985). 5. Cogoli, A. & Tschopp, A. Lymphocyte reactivity during spaceflight. Immunol Today 6, 1–4, https://doi.org/10.1016/0167 5699(85)90151-3 (1985).f 4. Philpott, D. E. et al. Microgravity changes in heart structure a p g y g y y g ( ) 5. Cogoli, A. & Tschopp, A. Lymphocyte reactivity during spaceflight. Immunol Today 6, 1–4, https://doi.org/10.1016/0167 5699(85)90151-3 (1985).f 5. Cogoli, A. & Tschopp, A. Lymphocyte reactivity during spaceflight. Immunol Today 6, 1–4, https://doi.org/10.1016/0167- 5699(85)90151-3 (1985).f 6. Hammond, T. G. et al. Mechanical culture conditions effect gene expression: gravity-induced changes on the space shuttle. Physio Genomics 3, 163–173, https://doi.org/10.1152/physiolgenomics.2000.3.3.163 (2000). g y g 7. Schwarz, R. P., Goodwin, T. J. & Wolf, D. A. Cell culture for three-dimensional modeling in rotating-wall vessels: an application o simulated microgravity. J Tissue Cult Methods 14, 51–57 (1992). s u ated c og av ty. J issue Cult ethods , 5 57 ( 99 ). 8. Hammond, T. G. & Hammond, J. M. Optimized suspension culture: the rotating-wall vessel. Am J Physiol Renal Physiol 281, F12–25, https://doi.org/10.1152/ajprenal.2001.281.1.F12 (2001). g y ( ) 8. Hammond, T. G. & Hammond, J. M. Optimized suspension culture: the rotating-wall vessel. Scientific Reports \| (2019) 9:17531 \| https://doi.org/10.1038/s41598-019-53862-3 References Am J Physiol Renal Physiol 281 F12–25, https://doi.org/10.1152/ajprenal.2001.281.1.F12 (2001). p g jp nsworth, B. R. & Lelkes, P. I. Growing tissues in microgravity. Nat M g g y 10. Barrila, J. et al. Organotypic 3D cell culture models: using the rotating wall vessel to study host-pathogen interactions. Nat Rev Microbiol 8, 791–801, https://doi.org/10.1038/nrmicro2423 (2010). p g 11. Nickerson, C. A. et al. Low-shear modeled microgravity: a global environmental regulatory signal affecting bacterial gene expression, physiology, and pathogenesis. J Microbiol Methods 54, 1–11 (2003).h p p y gy p g 12. Navran, S. The application of low shear modeled microgravity to 3-D cell biology and tissue engineering. Biotechnol Annu Rev 14 275–296, https://doi.org/10.1016/S1387-2656(08)00011-2 (2008).i p g 13. Herranz, R. et al. Ground-based facilities for simulation of microgravity: organism-specific recommendations for their use, and recommended terminology. Astrobiology 13, 1–17, https://doi.org/10.1089/ast.2012.0876 (2013). gy gy p g 14. Briegleb, W. Some qualitative and quantitative aspects of the fast-rotating clinostat as a research tool. ASGSB Bull 5, 23–30 (1 14. Briegleb, W. Some qualitative and quantitative aspects of the fast-rotating clinostat as a research tool. ASGSB Bull 5, 23–30 (1992). 15. Goodwin, T. J., Jessup, J. M. & Wolf, D. A. Morphologic differentiation of colon carcinoma cell lines HT-29 and HT-29KM in rotating-wall vessels. In Vitro Cell Dev Biol 28A, 47–60 (1992). g , q q p g , ( ) 15. Goodwin, T. J., Jessup, J. M. & Wolf, D. A. Morphologic differentiation of colon carcinoma cell lines HT-29 and HT-29KM in rotating-wall vessels. In Vitro Cell Dev Biol 28A, 47–60 (1992). g 16. Jessup, J. M. et al. Simulated microgravity does not alter epithelial cell adhesion to matrix and other molecules. Adv Space Res 14 71–76 (1994).h ( ) 17. Honer zu Bentrup, K. et al. Three-dimensional organotypic models of human colonic epithelium to study the early stages of enteric salmonellosis. Microbes Infect 8, 1813–1825, https://doi.org/10.1016/j.micinf.2006.02.020 (2006).h 18. Drummond, C. G., Nickerson, C. A. & Coyne, C. B. A Three-Dimensional Cell Culture Model To Study Enterovirus Infecti Polarized Intestinal Epithelial Cells. mSphere 1, https://doi.org/10.1128/mSphere.00030-15 (2016). 19. Kaysen, J. H. et al. Select de novo gene and protein expression during renal epithelial cell culture in rotating wall vessels is shea stress dependent. J Membr Biol 168, 77–89 (1999). p 20. Hammond, T. G. et al. Gene expression in space. Nat Med 5, 359, https://doi.org/10.1038/7331 (1999). 0. Hammond, T. G. et al. References Role of intestinal permeability and endotoxemia in alcoholic liver disease. Am J Physiol Gastrointest Liver Physiol 286, G881–884, https://doi.org/10.1152/ajpgi.00006.2004 (2004). 46 S k G t l C l i Ch l d O id ti St M di t S i ti Di ti f Ti ht J ti b Eth l d 46. Samak, G. et al. Calcium Channels and Oxidative Stress Mediate a Synergistic Disruption of Tight Junctio Acetaldehyde in Caco-2 Cell Monolayers. Sci Rep 6, 38899, https://doi.org/10.1038/srep38899 (2016). 46. Samak, G. et al. Calcium Channels and Oxidative Stress Mediate a Synergistic Disruption of Tight Junctions by Ethano Acetaldehyde in Caco-2 Cell Monolayers. Sci Rep 6, 38899, https://doi.org/10.1038/srep38899 (2016). y y p g 47. Choi, W., Yeruva, S. & Turner, J. R. Contributions of intestinal epithelial barriers to health and disease. Exp Cell Res 358, 71–77 https://doi.org/10.1016/j.yexcr.2017.03.036 (2017). p g j y ( ) 48. Shen, L., Weber, C. R., Raleigh, D. R., Yu, D. & Turner, J. R. Tight junction pore and leak pathways: a dynamic duo. Annu Rev Physiol 73, 283–309, https://doi.org/10.1146/annurev-physiol-012110-142150 (2011).i 49. Ivanov, A. I., McCall, I. C., Parkos, C. A. & Nusrat, A. Role for actin filament turnover and a myosin II motor in cytoskeleton-driven disassembly of the epithelial apical junctional complex. Mol Biol Cell 15, 2639–2651, https://doi.org/10.1091/mbc.e04-02-0163 (2004). ( ) 50. Ma, T. Y., Nguyen, D., Bui, V., Nguyen, H. & Hoa, N. Ethanol modulation of intestinal epithelial tight junction barrier. Am J Physio 276, G965–974 (1999). , ( ) 51. Atkinson, K. J. & Rao, R. K. Role of protein tyrosine phosphorylation in acetaldehyde-induced disruption of epithelial tight junctions. Am J Physiol Gastrointest Liver Physiol 280, G1280–1288, https://doi.org/10.1152/ajpgi.2001.280.6.G1280 (2001). h h h Th l d l h f ld h d d d ll l 52. Sheth, P., Seth, A., Thangavel, M., Basuroy, S. & Rao, R. K. Epidermal growth factor prevents acetaldehyde-induced paracellular permeability in Caco-2 cell monolayer. Alcohol Clin Exp Res 28, 797–804 (2004). p y y p 53. Furuse, M. et al. Occludin: a novel integral membrane protein localizing at tight junctions. J Cell Biol 123, 1777–1788, https://doi org/10.1083/jcb.123.6.1777 (1993). 54. Furuse, M. et al. Direct association of occludin with ZO-1 and its possible involvement in the localization of occludin at tight junctions. J Cell Biol 127, 1617–1626 (1994).i 55. Stevenson, B. R., Siliciano, J. D., Mooseker, M. S. & Goodenough, D. A. References & Meddings, J. B. Alterations in intestinal permeability. Gut 55, 1512–1520, https://doi.org/10.1136 gut.2005.085373 (2006). g 33. Camilleri, M., Madsen, K., Spiller, R., Greenwood-Van Meerveld, B. & Verne, G. N. Intestinal barrier function in health and gastrointestinal disease. Neurogastroenterol Motil 24, 503–512, https://doi.org/10.1111/j.1365-2982.2012.01921.x (2012).l 34. Luissint, A. C., Parkos, C. A. & Nusrat, A. Inflammation and the Intestinal Barrier: Leukocyte-Epithelial Cell Interactions Junction Remodeling, and Mucosal Repair. Gastroenterology 151, 616–632, https://doi.org/10.1053/j.gastro.2016.07.008 (201 ll d l d l b l h h ’ d d h l bl l f 35. Hollander, D. et al. Increased intestinal permeability in patients with Crohn’s disease and their relatives. A possible etiologic fa Ann Intern Med 105, 883–885 (1986).h 36. Olson, T. S. et al. The primary defect in experimental ileitis originates from a nonhematopoietic source. J Exp Med 203, 541–552, https://doi.org/10.1084/jem.20050407 (2006).i g j 37. Madsen, K. L. et al. Interleukin-10 gene-deficient mice develop a primary intestinal permeability defect in response to enteric microflora. Inflamm Bowel Dis 5, 262–270 (1999).hff l fl ( ) 38. Keshavarzian, A., Fields, J. Z., Vaeth, J. & Holmes, E. W. The differing effects of acute and chronic alcohol on gastric and intestina permeability. Am J Gastroenterol 89, 2205–2211 (1994). p y 39. Purohit, V. et al. Alcohol, intestinal bacterial growth, intestinal permeability to endotoxin, and medical consequences: summary of a symposium. Alcohol 42, 349–361, https://doi.org/10.1016/j.alcohol.2008.03.131 (2008). 40. Starkel, P., Leclercq, S., de Timary, P. & Schnabl, B. Intestinal dysbiosis and permeability: the yin and yang in alcohol dependence and alcoholic liver disease. Clin Sci (Lond) 132, 199–212, https://doi.org/10.1042/CS20171055 (2018). 41. Tamai, H., Horie, Y., Kato, S., Yokoyama, H. & Ishii, H. Long-term ethanol feeding enhances susceptibility of the liver to orally administered lipopolysaccharides in rats. Alcohol Clin Exp Res 26, 75S–80S, https://doi.org/10.1097/01.ALC.0000026981.32386.FD (2002).f 42. Seitz, H. K., Simanowski, U. A., Homann, N. & Waldherr, R. Cell proliferation and its evaluation in the colorectal mucosa: effe ethanol. Z Gastroenterol 36, 645–655 (1998). Scientific Reports \| (2019) 9:17531 \| https://doi.org/10.1038/s41598-019-53862-3 13 www.nature.com/scientificreports/ 43. Ferrier, L. et al. Impairment of the intestinal barrier by ethanol involves enteric microflora and mast cell activation in rodents. Am J Pathol 168, 1148–1154, https://doi.org/10.2353/ajpath.2006.050617 (2006). g j 44. Rao, R. K. Acetaldehyde-induced increase in paracellular permeability in Caco-2 cell monolayer. Alcohol Clin Exp Res 22 1724–1730 (1998). ( ) 45. Rao, R. K., Seth, A. & Sheth, P. Recent Advances in Alcoholic Liver Disease I. References J., Arpin, M., Fanning, A. S. & Louvard, D. The junction-associated protein, zonula occludens-1, localizes t 73. Gottardi, C. J., Arpin, M., Fanning, A. S. & Louvard, D. The junction-associated protein, zonula occludens-1, localizes to the nucleus before the maturation and during the remodeling of cell-cell contacts. Proc Natl Acad Sci U S A 93, 10779–10784 (1996). 73. Gottardi, C. J., Arpin, M., Fanning, A. S. & Louvard, D. The junction-associated protein, zonula occludens-1, localizes to the nucleus before the maturation and during the remodeling of cell-cell contacts. Proc Natl Acad Sci U S A 93, 10779–10784 (1996). 74 M t lt M t l I hi t h i l l i f ZO 1 i th d d l f ti t ith t t d d t t d li 73. Gotta d , C. J., p , ., a g, . S. & ouva d, .h e ju ct o assoc ated p ote , o u a occ ude s , oca es t nucleus before the maturation and during the remodeling of cell-cell contacts. Proc Natl Acad Sci U S A 93, 10779–10784 (19 74. Montalto, M. et al. Immunohistochemical analysis of ZO-1 in the duodenal mucosa of patients with untreated and treated c disease. Digestion 65, 227–233, https://doi.org/10.1159/000063817 (2002). g g , ( ) 74. Montalto, M. et al. Immunohistochemical analysis of ZO-1 in the duodenal mucosa of patients with untreated and treated celiac disease. Digestion 65, 227–233, https://doi.org/10.1159/000063817 (2002). Digestion 65, 227–233, https://doi.org/10.1159/000063817 (2002). g p g 75. Ivanov, A. I., Nusrat, A. & Parkos, C. A. Endocytosis of the apical junctional complex: mechanisms and possible roles in regulation of epithelial barriers. Bioessays 27, 356–365, https://doi.org/10.1002/bies.20203 (2005).h y g 76. Vorselen, D., Roos, W. H., MacKintosh, F. C., Wuite, G. J. & van Loon, J. J. The role of the cytoskeleton in sensing changes in gravity by nonspecialized cells. FASEB J 28, 536–547, https://doi.org/10.1096/fj.13-236356 (2014).i j 77. Galkin, V. E., Orlova, A. & Egelman, E. H. Actin filaments as tension sensors. Curr Biol 22, R96–101, https://doi.org/10.1016/j cub.2011.12.010 (2012).f 78. Hughes-Fulford, M. & Lewis, M. L. Effects of microgravity on osteoblast growth activation. Exp Cell Res 224, 103–109, https://doi org/10.1006/excr.1996.0116 (1996).lf g 79. Meloni, M. A. et al. Space flight affects motility and cytoskeletal structures in human monocyte cell line J-111. Cytoskeleton (Hoboken) 68, 125–137, https://doi.org/10.1002/cm.20499 (2011).l p g 80. Altman, P. L. References Am J Pathol 166, 409–419, https://doi.org/10.1016/s0002-9440(10)62264-x (2005). 64. Bruewer, M. et al. Interferon-gamma induces internalization of epithelial tight junction proteins via a macropinocytosis-like (2005). 64. Bruewer, M. et al. Interferon-gamma induces internalization of epithelial tight junction proteins via a macropinocytosis-like process FASEB J 19 923 933 https //doi org/10 1096/fj 04 3260com (2005) (2005). 64. Bruewer, M. et al. Interferon-gamma induces internalization of epithelial tight junction proteins via a macropinocytosis-like process. FASEB J 19, 923–933, https://doi.org/10.1096/fj.04-3260com (2005). 64. Bruewer, M. et al. Interferon-gamma induces internalization of epithelial tight junction proteins via a macropinocytosis-like process. FASEB J 19, 923–933, https://doi.org/10.1096/fj.04-3260com (2005). p p g fj 65. Sridharan, G. & Shankar, A. A. Toluidine blue: A review of its chemistry and clinical utility. J Oral Maxillofac Pathol 16, 251–255 https://doi.org/10.4103/0973-029X.99081 (2012).h p g 66. Van Itallie, C. M. et al. The density of small tight junction pores varies among cell types and is increased by expression of claudin-2 J Cell Sci 121, 298–305, https://doi.org/10.1242/jcs.021485 (2008). p g j 67. Rodgers, L. S., Beam, M. T., Anderson, J. M. & Fanning, A. S. Epithelial barrier assembly requires coordinated activity of multiple domains of the tight junction protein ZO-1. J Cell Sci 126, 1565–1575, https://doi.org/10.1242/jcs.113399 (2013). 68. Rao, R. K. Acetaldehyde-induced barrier disruption and paracellular permeability in Caco-2 cell monolayer. Methods Mol Biol 447 171–183, https://doi.org/10.1007/978-1-59745-242-7_13 (2008). 171 183, https://doi.org/10.1007/978 1 59745 242 7_13 (2008). 69. Hawkins, W. Z., FF. Clinical aspects of crew health in Biomedical Results of Apollo (NASA SP 368). 43-81 (Washington, DC, 1975) 9. Hawkins, W. Z., FF. Clinical aspects of crew health in Biomedical 69. Hawkins, W. Z., FF. Clinical aspects of crew health in Biomedical Results of Apollo (NASA SP 368). 43-81 (Washington, DC, 1975). 70. Sawyer, H. R., Moeller, C. L., Phillips, R. W. & Smirnov, K. L. Proliferation of jejunal mucosal cells in rats flown in space. J Appl Physiol (1985) 73, 148S–150S, https://doi.org/10.1152/jappl.1992.73.2.S148 (1992). y p g j pp 71. Radtke, A. L. & Herbst-Kralovetz, M. M. Culturing and applications of rotating wall vessel bioreactor derived 3D epithelial cel models. J Vis Exp. https://doi.org/10.3791/3868 (2012).h 71. Radtke, A. L. & Herbst-Kralovetz, M. M. Culturing and p p g 72. Barrila, J. et al. Three-dimensional organotypic co-culture model of intestinal epithelial cells and macrophages to study Salmonella enterica colonization patterns. NPJ Microgravity 3, 10, https://doi.org/10.1038/s41526-017-0011-2 (2017).h p g y p g 73. Gottardi, C. Scientific Reports \| (2019) 9:17531 \| https://doi.org/10.1038/s41598-019-53862-3 References Identification of ZO-1: a high molecular weight polypeptide associated with the tight junction (zonula occludens) in a variety of epithelia. J Cell Biol 103, 755–766, https://doi.org/10.1083/ jcb.103.3.755 (1986).f j 56. Fanning, A. S. & Anderson, J. M. Zonula occludens-1 and -2 are cytosolic scaffolds that regulate the assembly of cellular junctions. Ann N Y Acad Sci 1165, 113–120, https://doi.org/10.1111/j.1749-6632.2009.04440.x (2009).i 57. Odenwald, M. A. et al. ZO-1 interactions with F-actin and occludin direct epithelial polarization and single lumen specification in 3D culture. J Cell Sci 130, 243–259, https://doi.org/10.1242/jcs.188185 (2017).h 57. Odenwald, M. A. et al. ZO-1 interactions with F-actin and occludin direct epithelial polarization and single l 3D culture. J Cell Sci 130, 243–259, https://doi.org/10.1242/jcs.188185 (2017).h p g j 58. France, M. M. & Turner, J. R. The mucosal barrier at a glance. J Cell Sci 130, 307–314, https://doi.org/10.1242/jcs.193482 (2017). 59 Van Itallie C M Fanning A S Bridges A & Anderson J M ZO 1 stabilizes the tight junction solute barrier through coupling e, M. M. & Turner, J. R. The mucosal barrier at a glance. J Cell Sci 13 58. France, M. M. & Turner, J. R. The mucosal barrier at a glance. J Cell Sci 130, 307–314, https://doi.org/10.1242/jcs.193482 (2017). 59. Van Itallie, C. M., Fanning, A. S., Bridges, A. & Anderson, J. M. ZO-1 stabilizes the tight junction solute barrier through coupling to the perijunctional cytoskeleton. Mol Biol Cell 20, 3930–3940, https://doi.org/10.1091/mbc.E09-04-0320 (2009). h 59. Van Itallie, C. M., Fanning, A. S., Bridges, A. & Anderson, J. M. ZO-1 stabilizes the tight junction solute barrier through c to the perijunctional cytoskeleton. Mol Biol Cell 20, 3930–3940, https://doi.org/10.1091/mbc.E09-04-0320 (2009). 60. Sanchez-Pulido, L., Martin-Belmonte, F., Valencia, A. & Alonso, M. A. MARVEL: a conserved domain involved in membrane apposition events. Trends Biochem Sci 27, 599–601 (2002). pp 61. Raleigh, D. R. et al. Occludin S408 phosphorylation regulates tight junction protein interactions and barrier function. J Cell Bio 193, 565–582, https://doi.org/10.1083/jcb.201010065 (2011). g j 62. Tash, B. R. et al. The occludin and ZO-1 complex, defined by small angle X-ray scattering and NMR, has implications for modulating tight junction permeability. Proc Natl Acad Sci U S A 109, 10855–10860, https://doi.org/10.1073/pnas.1121390109 (2012). 63. Wang, F. et al. Interferon-gamma and tumor necrosis factor-alpha synergize to induce intestinal epithelial barrier dysfunction by up-regulating myosin light chain kinase expression. Am J Pathol 166, 409–419, https://doi.org/10.1016/s0002-9440(10)62264-x (2005). up-regulating myosin light chain kinase expression. References Space, gravity and the physiology of aging: parallel or conv Gerontology 56, 157–166, https://doi.org/10.1159/000252852 (2010). gy p g ( ) 91. Nicogossian, A. E. N., Huntoon, C. & Pool S. L. (Lea and Feibiger, 1989).il gy p g ( ) 91. Nicogossian, A. E. N., Huntoon, C. & Pool S. L. (Lea and Feibiger, 1989).il g ( g ) 92. Hughes-Fulford, M. To infinity… and beyond! Human spaceflight and life science. FASEB J 25, 2858–2864, https://doi.org/10.1096/ fj.11-0902ufm (2011).l fj 93. Meck, J. V., Dreyer, S. A. & Warren, L. E. Long-duration head-down bed rest: project overview, vital signs, and fluid balance. Avia Space Environ Med 80, A1–8 (2009). p 94. Hargens, A. R. & Vico, L. Long-duration bed rest as an analog to microgravity. J Appl Physiol (1985) 120, 891–903, https://doi. org/10.1152/japplphysiol.00935.2015 (2016). g j pp p y 95. Mullin, J. M., Valenzano, M. C., Verrecchio, J. J. & Kothari, R. Age- and diet-related increase in transepithelial colon permeability of Fischer 344 rats. Dig Dis Sci 47, 2262–2270 (2002). g 96. Meier, J. & Sturm, A. The intestinal epithelial barrier: does it become impaired with age? Dig Dis 27, 240–245, https://doi org/10.1159/000228556 (2009).h g 97. Saweirs, W. M., Andrews, D. J. & Low-Beer, T. S. The double sugar test of intestinal permeability in the elderly. Age Ageing 14, 312–315 (1985). 98. Saltzman, J. R., Kowdley, K. V., Perrone, G. & Russell, R. M. Changes in small-intestine permeability with aging. J Am Geriatr Soc 43, 160–164 (1995). ( ) 99. Tran, L. & Greenwood-Van Meerveld, B. Age-associated remodeling of the intestinal epithelial barrier. J Gerontol A Biol Sci Med Sci 68, 1045–1056, https://doi.org/10.1093/gerona/glt106 (2013). p g g g 00. Nickerson, C. A. et al. Three-dimensional tissue assemblies: novel models for the study of Salmonella enterica serova Typhimurium pathogenesis. Infect Immun 69, 7106–7120, https://doi.org/10.1128/IAI.69.11.7106-7120.2001 (2001). 01. Fan, P. et al. Supplemental lipoic acid relieves post-weaning diarrhoea by decreasing intestinal permeability in rats. J Anim Physio Anim Nutr (Berl) 101, 136–146, https://doi.org/10.1111/jpn.12427 (2017). Acknowledgements g We are grateful to Dr. Cheryl Nickerson and Dr. Jennifer Barrila (Arizona State University) for technical advice on generating epithelial-microcarrier bead cultures in the rotating wall vessel (RWV). We are also grateful to Dr. RK Rao (University of Tennessee Health Science Center) for technical advice on acetaldehyde vapor exposure protocols. Funding support from: NIH-NASA BioMed-ISS (UH2 AA019708) to D.F.M. and G.K.P.; NIH- 2R01DK091281 to D.F.M. Competing interestsh p g The authors declare no competing interests. Author contributions R.A., C.A.S., G.K.P. and D.F.M. conceived and designed the experiments. R.A. and C.A.S. performed the RWV experiments. R.A., R.R.M. and C.A.S. performed immunofluorescence imaging. A.S.B. performed Western blotting and densitometry, and wrote the relevant Methods section. R.A., C.A.S., R.R.M, A.S.B. and D.F.M. analyzed the data. R.A., C.A.S., R.R.M., A.S.B. and D.F.M. prepared the figures. D.F.M. wrote the main manuscript text. G.K.P. provided critical analysis and subject matter expertise. All authors reviewed the manuscript. References & Talbot, J. M. Nutrition and metabolism in spaceflight. J Nutr 117, 421–427 (1987). l h h d h h l l d l g 81. Lane, H. W., LeBlanc, A. D., Putcha, L. & Whitson, P. A. Nutrition and human physiological adaptations to space flight. Am J Clin Nutr 58, 583–588 (1993).f 82. Amidon, G. L., DeBrincat, G. A. & Najib, N. Effects of gravity on gastric emptying, intestinal transit, and drug absorption. J Clin Pharmacol 31, 968–973 (1991). Scientific Reports \| (2019) 9:17531 \| https://doi.org/10.1038/s41598-019-53862-3 www.nature.com/scientificreports/ 83. Groza, P., Boca, A. & Bordeianu, A. Digestive histochemical reactions in rats after space flight of different duration. Physiologist 34 S100–101 (1991).tl gt pl gf y g S100–101 (1991). 84. Rabot, S. et al. Variations in digestive physiology of rats after short duration flights aboard the US space shuttle. Dig Dis Sci 45, S100 101 (1991). 84. Rabot, S. et al. Variations in digestive physiology of rats after short duration flights aboard the US space shuttle. Dig Dis Sci 45, 1687–1695 (2000) 84. Rabot, S. et al. Variations in digestive physiology of rats after short duration flights aboard the US space shuttle. Dig Dis Sci 45 1687–1695 (2000). 85. Shi, J. et al. Intestinal microbiota contributes to colonic epithelial changes in simulated microgravity mouse model. FASEB J 31, 3695–3709, https://doi.org/10.1096/fj.201700034R (2017). p g fj ( ) 86. Wells, J. M. et al. Homeostasis of the gut barrier and potential biomarkers. Am J Physiol Gastrointest Liver Physiol 312, G171–G193 https://doi.org/10.1152/ajpgi.00048.2015 (2017). 87. Hayes, C. L. et al. Commensal microbiota induces colonic barrier structure and functions that contribute to homeostasis. Sci Rep 8, 14184, https://doi.org/10.1038/s41598-018-32366-6 (2018). , , p g ( ) 88. Chassaing, B., Aitken, J. D., Malleshappa, M. & Vijay-Kumar, M. Dextran sulfate sodium (DSS)-induced colitis in mice. Curr Protoc Immunol 104, Unit 15, 25, https://doi.org/10.1002/0471142735.im1525s104 (2014).f , , , p g ( ) 89. Ray, E. K. Introduction: are aging and space effects similar? Exp Gerontol 26, 123–129 (199 ay, E. K. Introduction: are aging and space effects similar? Exp Gero 89. Ray, E. K. Introduction: are aging and space effects similar? Exp Gerontol 26, 123 129 (1991). 90. Vernikos, J. & Schneider, V. S. Space, gravity and the physiology of aging: parallel or convergent disciplines? A mini-review. Gerontology 56, 157–166, https://doi.org/10.1159/000252852 (2010). f 90. Vernikos, J. & Schneider, V. S. Additional information Supplementary information is available for this paper at https://doi.org/10.1038/s41598-019-53862-3. Supplementary information is available for this paper at https://doi.org/10.10 Correspondence and requests for materials should be addressed to D.F.M. Reprints and permissions information is available at www.nature.com/reprints. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Cre- ative Commons license, and indicate if changes were made. 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https://openalex.org/W3117691083	https://europepmc.org/articles/pmc7798619?pdf=render	English	null	Smell, taste and COVID-19: testing is essential	QJM	2,020	public-domain	7,506	TESTING IS ESSENTIAL Christopher H Hawkes1 MD FRCP FAAN Christopher H Hawkes1 MD FRCP FAAN 1. Blizard Institute, Queen Mary University of London, 4 Newark St, London UK E1 2AT Word count excluding references, Table and abstract 3095. Word count excluding references, Table and abstract 3095. Word count excluding references, Table and abstract 3095. Key words: Olfaction. Taste. Covid-19. Chemosensation. Key words: Olfaction. Taste. Covid-19. Chemosensation. 1 1 © The Author(s) 2020. Published by Oxford University Press on behalf of the Association of Physicians. All rights reserved. For Permissions, please email: journals.permissions@oup.com Abstract. During the Covid-19 pandemic is became clear that smell and taste (chemosensory) disturbance is very common in the early stages of disease. This article addresses: 1) why Covid-19 specifically targets the modalities of smell and possibly taste and what is the mechanism 2) what is the frequency of smell and taste loss and, 3) what is the overall prognosis. It is suggested that mouth breathers may be at particular risk of Covid-19. Symptom-based questionnaires are likely to under- estimate the prevalence of chemosensory impairment by as much as 50%. The prevalence of smell loss is so high that a person who has normal olfaction on formal testing is unlikely to be infected significantly with Cov-2. Furthermore, someone without symptoms who has an abnormal smell test could still be infected and liable to spread the disease. Brief, low cost, olfactory tests are available that would permit a high throughput in field stations and airports. A normal result might obviate the need for a nasopharyngeal swab for the Cov-2 virus On March 20th 2020, Claire Hopkins, President of the British Rhinological Society and Nirmal Kumar, President of ENT UK 1 circulated a letter to fellow members that drew attention to the heightened incidence of isolated anosmia in their clinics. In normal circumstances they would see around one case of post-viral anosmia per month whereas in the recent past this had increased to 4 per week and remarkably all were under 40 years old. They questioned whether new onset anosmia in relatively young people might be an early warning of Covid-19 infection and emphasised the presence of similar observations from China, South Korea and Iran. It is unusual for impairment of smell sense to be such a prominent symptom of upper respiratory viral infection. The majority of the latter are recognised in ear nose and throat (ENT) units particularly as a symptom from middle-aged or elderly in the aftermath of upper respiratory infection (URT). Word count excluding references, Table and abstract 3095. Most individuals who experience URT such as the common cold, accept a degree of smell impairment that results typically from a blocked nose, but what is remarkable about Covid-19 is that its occurrence is often early, of acute onset, severe and only occasionally associated with a blocked nose2,3. These thoughts have been given preliminary support from a study of 10 patients with proven Covid-19 infection compared to 10 people with an acute cold and 10 healthy controls4. Using the extended version of Sniffin’ Sticks there were significant differences: those with Covid-19 infection scored lower than the acute cold group, with identification scores affected more than threshold or discrimination. 2 2 Several questions arise: why should Covid-19 specifically target chemosensation (i.e. smell and taste) and what is the mechanism? What is the frequency of smell and taste loss and what is the overall prognosis? 1. Why should Covid-19 specifically target the sense of smell and possibly taste? Indeed MRI-based studies have shown oedema of the olfactory bulb9 (Figure 3) as well as more central changes, namely in the gyrus rectus10 and by CT/PET, in the orbitofrontal cortex11. Figure 3. Transient olfactory bulb oedema as shown in coronal 3D MRI T2-weighted imaging (1.5T) during anosmia (day 7; C) compared to recovery (day 24;D). Olfactory bulb (ob; pink) displays transient volume and signal increase, olfactory cleft oedema (OC; brown), and focal left ethmoid (eth; green) sinusitis (), and normal cranial fossa (grey line) and orbit (orb; yellow). Reproduced from Figure 1 C and D with permission from Laurendon et al9. There is provisional evidence that ACE2 receptors are present in the tongue (Figure 4) particularly taste buds and to lesser degree in the lingual epithelium12,13. Cov-2 can be isolated from saliva14 thus there is a plausible mechanism for such infection to involved taste bud receptors. Less is known about ACE2 expression in the major taste nerves, namely the chorda tympani and glossopharyngeal nerves. Figure 4. Bulk RNA-seq analysis of public datasets. Bar plot of ACE2 expression in normal tissues from FANTOM5 CAGE dataset, coloured by organs. Reproduced from Figure 1b with permission under Open Access from: Xu et al14. 1. Why should Covid-19 specifically target the sense of smell and possibly taste? Preliminary information about the likely mode of nasal invasion is just emerging. The virus, SARS- CoV-2, (shortened here to Cov-2) that causes the illness, Covid-19, infects cells through interactions between its spike protein and the ACE2 protein on target cells (Figure 1). Preliminary information about the likely mode of nasal invasion is just emerging. The virus, SARS- CoV-2, (shortened here to Cov-2) that causes the illness, Covid-19, infects cells through interactions between its spike protein and the ACE2 protein on target cells (Figure 1). Figure 1. Molecular structure of the SARS-Cov-2 virus to show how the virus can attach to a pneumocyte (alveolar cell) that lines the alveoli. Reproduced with permission from David Baker, Blizard Institute, Queen Mary, University of London. This interaction requires cleavage of the spike protein, likely by the cell surface protease, serine (TMPRSS2) and other proteases such as cathepsin B and L. It has been demonstrated that non- olfactory epithelial cells from the human upper airway express high levels of ACE2 and serine proteases5 as shown in Figure 2, a finding that implies they could act as a viral reservoir. Figure 2. Simplified model for CoV-2-induced anosmia/hyposmia in COVID-19 based on results obtained from patients and animal models. Article from Open Access journal reproduced with permission from Bilinska and Butowt6 According to Brann and colleagues7 olfactory epithelial sustentacular cells, horizontal basal cells and Bowman’s gland cells express the receptors required for entry of CoV-2 but there is no ACE2 expression in mature olfactory receptor neurones. In essence they propose that the anosmia of Covid-19 relates to primary infection of non-neuronal cell types and by implication, that smell loss is a consequence of local inflammation in and around the nasal neuro-epithelium. This concept has received preliminary confirmation from MRI-based studies that reveal congestion in the olfactory cleft – the area that houses olfactory neurones8. Although these findings are plausible it is possible, as the authors suggest, that other non-ACE2 dependent receptors may facilitate cellular entry of CoV-2. These observations are preliminary and it is still possible that CoV-2 may involve the olfactory 3 3 bulb. Indeed MRI-based studies have shown oedema of the olfactory bulb9 (Figure 3) as well as more central changes, namely in the gyrus rectus10 and by CT/PET, in the orbitofrontal cortex11. bulb. 2. What is the frequency of chemosensory loss? There have been numerous estimates worldwide, but with a few exceptions detailed below, most have been based on questionnaire surveys without objective measurement and several have not contained a control group. Samples have been varied: some are based predominantly on out- patients others reliant on in-patients with testing at varying stages of illness. The largest investigation15,16 employed a smartphone-based app to retrieve symptomatic data on over 2 million people in UK and USA and found that in those reporting chemosensory impairment, 65% had a subsequent positive PCR for Covid-19. When this was combined with fever, cough, fatigue and loss of appetite the correlation with PCR for Cov-2 was very high. A large meta-analysis totalling 38,198 subjects17documentated an overall prevalence of smell impairment in Caucasians of 49% and 16.7% in Asians. Taste symptoms occurred in 51% Caucasians and 18% Asians. Other studies show wide 4 4 estimates of prevalence – up to 70%18with an approximately equal rate for smell or taste. Sometimes isolated impairment of smell or taste is documented as a presenting symptom. A study from San Diego based on ambulatory individuals with inﬂuenza-like symptoms, noticed that subjective report of smell impairment was associated with a 10-fold lower risk of hospital admission for Covid-1919. This finding is discussed further below Surveys that have relied on patient reports are susceptible to multiple confounders including recall bias20 and a tendency to over-representation of female respondents21. Even more importantly, less than 40% of individuals are actually aware of a proven olfactory defect22. For subjective awareness, the defect needs to be bilateral and of at least moderate severity. Furthermore, smell loss and taste loss are very frequently confused. Most people who complain of impaired taste have reduced olfaction23 whereas it is unusual for someone with primary taste loss to complain of smell impairment. The mechanism of this phenomenon has not been satisfactorily explained. Patient reports of olfactory impairment are therefore intrinsically unreliable and will tend to underestimate the true picture due to lack of awareness and confusion with taste. Furthermore, if taste is really affected in Covid-19 any such deficiency would inflate estimates of smell impairment where based on subjective reports. According to PubMed, at the time of writing there have been 14 articles worldwide where various objective olfactory measurements have been made (Table 1). Case numbers range by centre from 14-345 individuals. 2. What is the frequency of chemosensory loss? In nearly all instances a confirmatory polymerase chain reaction (PCR) test for Cov-2 has been undertaken. In only 9 cases was there a control group and where present the PCR test is not stated in 7 of these. Matching by age and gender was performed in just 4 instances. Subjective awareness of olfactory loss was indicated in 12 studies with a prevalence ranging from 28%-86% (mean 54%). Some authors have used non-standard olfactory measurement e.g. modified ethyl-alcohol threshold test24 or an in-house identification test of 10 odours25 neither containing details of control data. In one article, patients quarantined at home were instructed on how to make up their own smell and taste ingredients26, despite the existence of readily available standardised commercial test kits for home use. The 4-odour Pocket Smell Identification Test used by one group27 or the 3-odour Quick Smell Identification Test (Q-SIT) employed by others28,29 are more appropriate for rapid screening in the clinic, rather than large research projects. For example, a score of 3/3 correct answers on the Q-SIT is likely to indicate normal olfaction30 but as emphasised by the authors, a value of 2/3 could represent either hyposmia or a normal result because of wide variance. Nonetheless, a score of 3/3 would help exclude 5 5 anosmia where a low-cost, high-volume survey is required. The data from Iran31,32 have been criticised unfairly because many of the 40 odours used in the identification kit were allegedly unfamiliar to Iranians33. However, the test used was in fact specifically modified to account for cultural differences34. It is important to be aware of the time of olfactory assessment in calculating the prevalence of Cov-2 related smell impairment, whether based on questionnaires or psychophysical tests. Clearly the closer to acute symptom onset, the more chance of an abnormal result. In four instances this information is not supplied. Where the time of testing is supplied, this ranges from 4-37 days. It is important to be aware of the time of olfactory assessment in calculating the prevalence of Cov-2 related smell impairment, whether based on questionnaires or psychophysical tests. Clearly the closer to acute symptom onset, the more chance of an abnormal result. In four instances this information is not supplied. Where the time of testing is supplied, this ranges from 4-37 days. 2. What is the frequency of chemosensory loss? Taking into account the above reservations, there are just four more robust studies that have used standardised smell tests, have a control group, time of examining 14 days or less and adequate patient numbers, arbitrarily set at 45 or more 28,31,32,35. With this reservation, it may be inferred a) that subjective awareness of smell impairment is highly variable i.e. 28%-49% b) olfactory impairment on objective testing is present in 84%-98%. c) in general, hospitalised patients who are assessed within 14 days of symptom onset have more abnormal smell tests (71%-98%). The picture for outpatients is less clear information is not supplied. Where the time of testing is supplied, this ranges from 4-37 days. Taking into account the above reservations, there are just four more robust studies that have used standardised smell tests, have a control group, time of examining 14 days or less and adequate patient numbers, arbitrarily set at 45 or more 28,31,32,35. With this reservation, it may be inferred a) that subjective awareness of smell impairment is highly variable i.e. 28%-49% b) olfactory impairment on objective testing is present in 84%-98%. c) in general, hospitalised patients who are assessed within 14 days of symptom onset have more abnormal smell tests (71%-98%). The picture for outpatients is less clear Compared to subjective patient reports, smell measurement will therefore uncover a further 40% - 50% of proven Cov-2 infected people, indicating that the olfactory defect is near universal. In practical terms this means that an abnormal smell test may be present in someone with no symptoms and yet be capable of spreading the virus. Conversely, a normal standardised smell test such as the Sniffin’ Sticks or UPSIT should help exclude the presence of Covid-19 and would be valuable for mass population screening. A less clear picture is available for the sense of taste. Only 6 studies report taste measurement (Table 1) and details of a control group are not given in four of these. Just three centres27,34,36. implemented a standardised measurement (taste strips) and documented a normal result in a total of 40 patients from two centres27,36 with an abnormal value from one unit34 (5/72; 7%). The other three25,26, 37 used in-house tests and observed abnormalities ranging from 27%-49%. No reliable conclusion can be drawn from these limited observations. c Table 1. Summary of articles where objective chemosensory testing was undertaken. CCCRC= Connecticut Chemosensory Clinical Research Center orthonasal olfaction test. 3. Prognosis. Some subjective patient reports describe recovery of olfaction in 2-6 weeks. This finding is exemplified by one article with serial longitudinal objective assessment32. Return to normal was shown in nearly two thirds (61%) within 8 weeks (Figure 5). At that point, 35% still had varying degrees of impairment although complete smell loss affected just 4%. Distortion of perceived smells (cacosmia) and smell hallucinations (phantosmia) are recognised in the established and disease recovery phase40. Some subjective patient reports describe recovery of olfaction in 2-6 weeks. This finding is exemplified by one article with serial longitudinal objective assessment32. Return to normal was shown in nearly two thirds (61%) within 8 weeks (Figure 5). At that point, 35% still had varying degrees of impairment although complete smell loss affected just 4%. Distortion of perceived smells (cacosmia) and smell hallucinations (phantosmia) are recognised in the established and disease recovery phase40. The olfactory neuroepithelium has considerable capacity for regeneration, provided the stem cell layer is not damaged41. This process is unlikely to account for the rapid subjective recovery that in some instances appears complete in as little as 2 weeks (Figure 5). Such swift improvement is more in keeping with resolution of inflammation/oedema surrounding the nasal neuro-epithelium as shown on by MRI (Figure 3) . There are insufficient reports relating to the prevalence and recovery of taste impairment. Figure 5. Proportion of patients with varying degrees of recovery according to COVID-19 symptom onset. All initial (n = 100) and follow-up (n = 82) scores are combined. Reproduced from Figure 4 with permission under Open Access from Moein et al32. Potential risk for mouth breathers. There are multiple causes of mouth breathing. It relates usually to nasal obstruction from a displaced septum, congestion, polyps and a variety of developmental abnormalities of the nasal cavity including Down’s syndrome. In some it is just a bad habit. Most snorers breathe through the mouth and there is evidence that people with obstructive sleep apnoea are mouth breathers42. In the San Diego study of olfaction and Covid-1919 it was speculated that milder cases of COVID-19 may present with severe anosmia and higher self-reporting, compared to the undetected or slight hyposmia associated with moderate to severe COVID-19 cases. If correct, this dichotomy may relate in part, to an individual’s pattern of inspiration. 2. What is the frequency of chemosensory loss? PCR=polymerase chain reaction. ID=identification. Q-SIT= Quick Smell Identification Test. UPSIT=University of Pennsylvania Smell Identification Test. SST = Sniffin’ Sticks test). TOT= time of testing. The Taste Strips Test uses four tastants at four different concentrations. Table 1. Summary of articles where objective chemosensory testing was undertaken. CCCRC= Connecticut Chemosensory Clinical Research Center orthonasal olfaction test. PCR=polymerase chain reaction. ID=identification. Q-SIT= Quick Smell Identification Test. UPSIT=University of Pennsylvania Smell Identification Test. SST = Sniffin’ Sticks test). TOT= time of testing. The Taste Strips Test uses four tastants at four different concentrations. 6 6 7 3. Prognosis. Thus, habitual nose- breathers would direct airborne virus into the nasal passages where there are multiple immune- based defence functions that serve as a primary mucosal immune barrier. e.g. the nasopharynx- associated lymphoid tissue43 (NALT) known collectively as Waldeyer’s ring. A mouth-breather would therefore bypass the nasal component i.e. the adenoid and tubal tonsils and have to rely on the laryngeal and lingual tonsils. In theory, those who have had tonsillectomy or adenoidectomy might be more susceptible to subsequent viral infection although the consensus view is against this 8 contention44,45. A further defence mechanism favouring nose breathers relates to increased synthesis of sino-nasal nitric oxide (NO) which is an integral and highly conserved part of the host immune response. It acts as a first-line of defence against micro-organisms and upregulates ciliary motility. At low concentration, NO acts as a signalling molecule that promotes growth and activity of immune cells. At high concentrations it binds DNA, proteins and lipids, thereby inhibiting or killing target pathogens46. In support of this in the clinical setting47, 6 human volunteers were infected with human rhinovirus (HRV-16), a non-enveloped RNA virus. Elevated nitric oxide synthase mRNA was detected in nasal turbinate scrapings from infected individuals and increased levels of exhaled NO from the nose and lower airways. Others48 are exploring the possible benefits of inhaled nitric oxide in acute respiratory distress syndrome. Discussion. Objective evaluation of taste impairment is complex and reviewed elsewhere49. There are clearly some weaknesses in this analysis. Although 14 studies address Covid-19 and hyposmia, patient numbers are relatively small and the tests employed are varied, sometimes unorthodox. Some groups have no control group and do not state clearly the time of testing – or if so, the range in days is wide. Other investigations have been published without peer review or possibly submitted (and published) in haste, given the urgency of the current pandemic. Ethnic differences may account for some of the wide variation in results17. Despite this, the conclusions based primarily on the 4 most thorough investigations, show a consistent relationship between smell impairment and Covid-19 and support the main messages in this paper Discussion. Smell impairment occurs in Covid-19 probably by involvement of ACE2-expressing cells (particularly the sustentacular cells) in the nasal olfactory area rather than the olfactory neurones per se thus resulting in a local inflammatory response in the nasal cleft, thereby impairing olfactory transduction. Involvement of the olfactory bulb and its central connections may occur in more advanced cases. The value of subjective reports is severely limited by low sensitivity to an established smell defect and confusion with taste impairment. Objective testing suggests that there is smell loss in nearly all patients suffering from Covid-19. In theory, a mouth breather would be more at risk of lung infection (and severe Covid-19) than a nose breather. Partial or complete smell recovery takes place in around two thirds subjects over a period of 2- 6 weeks. Hence, anosmia constitutes an important warning symptom and sign of infection by Cov-2 and has been highlighted in the UK public domain since June 2020. Olfactory testing elevates the detection rate of a defect by about 50% i.e. from around 30-40% according to symptoms, to more than 90% where based on measurement. The importance of smell testing as opposed to smell questioning cannot be emphasized more strongly The prevalence of smell symptoms and signs is so high that a person who has normal olfaction on testing by procedures listed below, is unlikely to be infected with Cov-2 or if so, their viral load would be low and unlikely to result in transmission to others. Where resources are limited it is suggested that a rapid screening test of olfaction could be used in field stations or airports as a substitute for or complementary to nasal and throat swabs. False positives i.e. anosmia may result from other rhinotropic viruses but these patients would require formal viral testing in any event. The risk of a false negative result is low with estimates ranging from 2%-28%. If one excludes the small 9 9 study of 41 patients that implemented a 4 odour test27 then the false negative rate drops to 2%- 16%). The position regarding taste impairment is less clear. Subjective complaint of dysgeusia is frequent but in most instances represents confusion with olfactory loss. Objective evaluation of taste impairment is complex and reviewed elsewhere49. The position regarding taste impairment is less clear. Subjective complaint of dysgeusia is frequent but in most instances represents confusion with olfactory loss. The Way Forward. The following are suggested for future Covid-19 chemosensory research 1. Smell measurement should be undertaken by centres with a proven track record of chemosensory research using internationally recognised tests 2. Large numbers of cases at varying stages of disease and healthy controls should be collected in the community and hospital setting. The number required should be determined from power calculations based on the number of test odours used. 3. Any of the following identification procedures would be suitable for large scale studies in walk-in centres or airports:  Sniffin’ Sticks, 12 odour version. This may be re-used multiple times within its shelf life. Given the potential risks of transmission it would be best administered by a trained operator rather than the subjects themselves  B-SIT (Brief Smell Identification Test). This is a ‘scratch and sniff’ procedure for single use only and comprises 12 odours. It is suitable for on-site or home completion  NHANES-8. (US National Health and Nutrition Examination Survey). This is a low cost 8 odour version similar in principle to the B-SIT. It is under evaluation for future field use. 10 The above procedures could be undertaken in Covid-19 walk-in centres or airports as an inexpensive screening procedure. An initial large-scale trial would be required to assess the sensitivity of the chosen test. Based on current data, a normal result would likely avoid the need for nasal/throat swabs whereas an abnormal result would require formal virological analysis. Acknowledgement. The author would like to acknowledge the help and advice in the preparation of this manuscript from: Professor RL Doty, Director, University of Pennsylvania Smell and Taste Center, Philadelphia, USA and Dr Glenis Scadding, Consultant Physician in Allergy and Rhinology at the Royal National Throat, Nose and Ear Hospital, London UK. Footnote: Q-SIT (Quick Smell Identification Test), B-SIT (Brief Smell Identification Test), UPSIT (University of Pennsylvania Smell Identification Test) and the Pocket Smell Test are trademarks of Sensonics International, Haddon Heights, New Jersey, USA. Sniffin’ Sticks and Taste Strips are trademarks of Burghart Messtechnik GmbH, Wedel, Germany. Sniffin’ Sticks and Taste Strips are trademarks of Burghart Messtechnik GmbH, Wedel, Germany. References 1. Hopkins C, Kumar N. Loss of sense of smell as marker of COVID-19 infection. The Royal College of Surgeons of England: British Rhinological Society. 2020 Mar 21. 1. Hopkins C, Kumar N. Loss of sense of smell as marker of COVID-19 infection. The Royal College of Surgeons of England: British Rhinological Society. 2020 Mar 21. 2. Lechien JR, Chiesa-Estomba MD CM, Hans S, Barillari MD MR, Jouffe L, Saussez S. Loss of Smell and Taste in 2013 European Patients With Mild to Moderate COVID-19. Annals of Internal Medicine. 2020 May 26. 2. Lechien JR, Chiesa-Estomba MD CM, Hans S, Barillari MD MR, Jouffe L, Saussez S. Loss of Smell and Taste in 2013 European Patients With Mild to Moderate COVID-19. Annals of Internal Medicine. 2020 May 26. 3. Beltrán-Corbellini Á, Chico-García JL, Martínez-Poles J, Rodríguez-Jorge F, Natera-Villalba E, Gómez-Corral J, et al. Acute-onset smell and taste disorders in the context of COVID-19: a pilot multicentre polymerase chain reaction based case–control study. European journal of neurology. 2020 Apr 22. 3. Beltrán-Corbellini Á, Chico-García JL, Martínez-Poles J, Rodríguez-Jorge F, Natera-Villalba E, Gómez-Corral J, et al. Acute-onset smell and taste disorders in the context of COVID-19: a pilot multicentre polymerase chain reaction based case–control study. European journal of neurology. 2020 Apr 22. 4. Huart C, Philpott C, Konstantinidis I, Altundag A, Trecca EM, Cassano M, et al. Comparison of COVID-19 and common cold chemosensory dysfunction. Rhinology. 2020 Aug 19. 4. Huart C, Philpott C, Konstantinidis I, Altundag A, Trecca EM, Cassano M, et al. Comparison of COVID-19 and common cold chemosensory dysfunction. Rhinology. 2020 Aug 19. 5. Sungnak W, Huang N, Bécavin C, Berg M, Queen R, Litvinukova M et al. SARS-CoV-2 entry factors are highly expressed in nasal epithelial cells together with innate immune ges. Nature medicine. 2020 May;26(5):681-7. 5. Sungnak W, Huang N, Bécavin C, Berg M, Queen R, Litvinukova M et al. SARS-CoV-2 entry factors are highly expressed in nasal epithelial cells together with innate immune ges. Nature medicine. 2020 May;26(5):681-7. 11 11 6. Bilinska K, Butowt R. Anosmia in COVID-19: A Bumpy Road to Establishing a Cellular Mechanism. ACS Chemical Neuroscience. 2020 Jul 16;11(15):2152-5. 7. Brann D, Tsukahara T, Weinreb C, Logan DW, Datta SR. Non-neural expression of SARS-CoV-2 entry genes in the olfactory epithelium suggests mechanisms underlying anosmia in COVID- 19 patients. BioRxiv. 2020 Jan 1. 8. References Eliezer M, Hamel AL, Houdart E, Herman P, Housset J, Jourdaine C et al. Loss of smell in COVID-19 patients: MRI data reveals a transient edema of the olfactory clefts. Neurology. 2020 Sep 11. 9. Laurendon T, Radulesco T, Mugnier J, Gérault M, Chagnaud C, El Ahmadi AA, et al. Bilateral transient olfactory bulb edema during COVID-19–related anosmia. Neurology. 2020 Aug 4;95(5):224-5. 10. Politi LS, Salsano E, Grimaldi M. Magnetic resonance imaging alteration of the brain in a patient with coronavirus disease 2019 (covid-19) and anosmia. JAMA Neurology. 2020 May 29. 11. Galougahi MK, Ghorbani J, Bakhshayeshkaram M, Naeini AS, Haseli S. Olfactory bulb magnetic resonance imaging in SARS-CoV-2-induced anosmia: the first report. Academic Radiology. 2020 Apr 11. 11. Galougahi MK, Ghorbani J, Bakhshayeshkaram M, Naeini AS, Haseli S. Olfactory bulb magnetic resonance imaging in SARS-CoV-2-induced anosmia: the first report. Academic Radiology. 2020 Apr 11. 12. Shigemura N, Takai S, Hirose F, Yoshida R, Sanematsu K, Ninomiya Y. Expression of renin- angiotensin system components in the taste organ of mice. Nutrients. 2019 Sep;11(9):2251. 13. Xu H, Zhong L, Deng J, Peng J, Dan H, Zeng X et al. High expression of ACE2 receptor of 2019- nCoV on the epithelial cells of oral mucosa. International journal of oral science. 2020a Feb 24;12(1):1-5. 14. Xu J, Li Y, Gan F, Du Y, Yao Y. Salivary glands: potential reservoirs for COVID-19 asymptomatic infection. Journal of Dental Research. 2020b Apr 9:0022034520918518. 14. Xu J, Li Y, Gan F, Du Y, Yao Y. Salivary glands: potential reservoirs for COVID-19 asymptomatic infection. Journal of Dental Research. 2020b Apr 9:0022034520918518. 15. Menni C, Valdes A, Freydin MB, Ganesh S, Moustafa JE, Visconti A et al. Loss of smell and taste in combination with other symptoms is a strong predictor of COVID-19 infection. MedRxiv. 2020a Jan 1. 15. Menni C, Valdes A, Freydin MB, Ganesh S, Moustafa JE, Visconti A et al. Loss of smell and taste in combination with other symptoms is a strong predictor of COVID-19 infection. MedRxiv. 2020a Jan 1. 16. Menni C, Valdes AM, Freidin MB, Sudre CH, Nguyen LH, Drew DA, et al. Real-time tracking of self-reported symptoms to predict potential COVID-19. Nature medicine. 2020b May 11:1-4. 17. von Bartheld CS, Hagen MM, Butowt R. Prevalence of chemosensory dysfunction in COVID- 19 patients: a systematic review and meta-analysis reveals significant ethnic differences. ACS h l ( ) 16. References Menni C, Valdes AM, Freidin MB, Sudre CH, Nguyen LH, Drew DA, et al. Real-time tracking of self-reported symptoms to predict potential COVID-19. Nature medicine. 2020b May 11:1-4. 16. Menni C, Valdes AM, Freidin MB, Sudre CH, Nguyen LH, Drew DA, et al. Real-time tracking of self-reported symptoms to predict potential COVID-19. Nature medicine. 2020b May 11:1-4. 17. von Bartheld CS, Hagen MM, Butowt R. Prevalence of chemosensory dysfunction in COVID- 19 patients: a systematic review and meta-analysis reveals significant ethnic differences. ACS chemical neuroscience. 2020 Sep 1;11(19):2944-61. 17. von Bartheld CS, Hagen MM, Butowt R. Prevalence of chemosensory dysfunction in COVID- 19 patients: a systematic review and meta-analysis reveals significant ethnic differences. ACS chemical neuroscience. 2020 Sep 1;11(19):2944-61. 17. von Bartheld CS, Hagen MM, Butowt R. Prevalence of chemosensory dysfunction in COVID- 19 patients: a systematic review and meta-analysis reveals significant ethnic differences. ACS chemical neuroscience. 2020 Sep 1;11(19):2944-61. 12 18. Tong JY, Wong A, Zhu D, Fastenberg JH, Tham T. The prevalence of olfactory and gustatory dysfunction in COVID-19 patients: a systematic review and meta-analysis. Otolaryngology– Head and Neck Surgery. 2020 May 5:0194599820926473. 19. Yan CH, Faraji F, Prajapati DP, Ostrander BT, DeConde AS. Self-reported olfactory loss associates with outpatient clinical course in COVID-19. In: International Forum of Allergy & Rhinology 2020 Apr 24. 20. Coughlin, SS. Recall bias in epidemiologic studies. J Clin Epidemiol 1990; 43: 87-91. 21. Wymer W. The implications of sex differences on volunteer preferences. Voluntas: International Journal of Voluntary and Nonprofit Organizations. 2011 Dec 1;22(4):831-51. 22. Wehling E, Nordin S, Espeseth T, Reinvang I, Lundervold AJ. Unawareness of olfactory dysfunction and its association with cognitive functioning in middle aged and old adults. Arch Clin Neuropsychol 2011; 26(3): 260-9 23. Deems DA, Doty RL, Settle RG, Moore-Gillon V, Shaman P, Mester AF et al. Smell and taste disorders, a study of 750 patients from the University of Pennsylvania Smell and Taste Center. Archives of otolaryngology–head & neck surgery. 1991 May 1;117(5):519-28. 24. Calvo-Henriquez C, Maldonado-Alvarado B, Chiesa-Estomba C, Rivero-Fernández I, Sanz- Rodriguez M, Villarreal IM et al. Ethyl alcohol threshold test: a fast, reliable and affordable olfactory assessment tool for COVID-19 patients. European Archives of Oto-Rhino- Laryngology. 2020 Oct;277(10):2783-92. 25. Vaira LA, Deiana G, Fois AG, Pirina P, Madeddu G, De Vito A et al. Objective evaluation of anosmia and ageusia in COVID-19 patients: Single-center experience on 72 cases. Head & Neck. 2020a Jun;42(6):1252-8. References 26. Vaira LA, Hopkins C, Salzano G, Petrocelli M, Melis A, Cucurullo M et al. Olfactory and gustatory function impairment in COVID-19 patients: Italian objective multicenter-study. Head & Neck. 2020b Jul;42(7):1560-9. 27. Hintschich CA, Wenzel JJ, Hummel T, Hankir MK, Kühnel T, Vielsmeier V et al. Psychophysical tests reveal impaired olfaction but preserved gustation in COVID-19 patients. In: International forum of allergy & rhinology 2020 Sep 1. Wiley-Blackwell. 28. Lima MA, Silva MT, Oliveira RV, Soares CN, Takano CL, Azevedo AE et al.. Smell dysfunction in COVID-19 patients: More than a yes-no question. Journal of the Neurological Sciences. 2020 Nov 15;418:117107. 29. Tsivgoulis G, Fragkou PC, Delides A, Karofylakis E, Dimopoulou D, Sfikakis PP et al. Quantitative evaluation of olfactory dysfunction in hospitalized patients with Coronavirus [2](COVID-19). Journal of Neurology. 2020 May 25:1. 13 13 30. Jackman AH, Doty RL. Utility of a three-item smell identification test in detecting olfactory dysfunction. The Laryngoscope. 2005 Dec;115(12):2209-12. 31. Moein ST, Hashemian SM, Mansourafshar B, Khorram-Tousi A, Tabarsi P, Doty RL. Smell dysfunction: a biomarker for COVID-19. International Forum of Allergy & Rhinology 2020a; 10: 944-950, 2020 32. Moein, S.T., Hashemian, S.M., Khorram-Tousi, A., Tabarsi, P., Doty, R.L. Prevalence and reversibility of smell dysfunction measured psychophysically in a cohort of COVID-19 patients. Int Forum Allergy Rhinol. 2020b Aug 6:10.1002/alr.22680. doi: 10.1002/alr.22680. Online ahead of print. PMID: 32761796 33. Mariño-Sánchez F, Santamaría-Gadea A, de Los Santos G, Alobid I, Mullol J. Psychophysical olfactory testing in COVID-19: is smell function really impaired in nearly all patients? Int Forum Allergy Rhinol. 2020 Aug;10(8):951-952. doi: 10.1002/alr.22639. Epub 2020 Jun 25. PMID: 32497397; PMCID: PMC7300888. 34. Moein ST, Hashemian SM, Mansourafshar B, Khorram-Tousi A, Tabarsi P, Doty RL. Reply to: Psychophysical olfactory testing in COVID-19: is smell function really impaired in nearly all patients?. InInternational forum of allergy & rhinology 2020c Jun 4. Wiley-Blackwell. 35. Hornuss D, Lange B, Schröter N, Rieg S, Kern WV, Wagner D. Anosmia in COVID-19 patients. Clinical Microbiology and Infection. 2020 May 22. 35. Hornuss D, Lange B, Schröter N, Rieg S, Kern WV, Wagner D. Anosmia in COVID-19 patients. Clinical Microbiology and Infection. 2020 May 22. 36. Bocksberger S, Wagner W, Hummel T, Guggemos W, Seilmaier M, Hoelscher M et al. Temporäre Hyposmie bei COVID-19-Patienten. Hno. 2020 May 28:1. 37. Altin F, Cingi C, Uzun T, Bal C. Olfactory and gustatory abnormalities in COVID-19 cases. European Archives of Oto-Rhino-Laryngology. 2020 Oct;277(10):2775-81. 38. References Hawkes CH, Doty RL. Smell and taste disorders. Cambridge University Press; 2018. References Chung TW, Sridhar S, Zhang AJ, Chan KH, Li HL, Wong FK et al. Olfactory dysfunction in COVID-19 patients: observational cohort study and systematic review. InOpen Forum Infectious Diseases 2020 Jun 5. 39. Le Bon SD, Pisarski N, Verbeke J, Prunier L, Cavelier G, Thill MP et al. Psychophysical evaluation of chemosensory functions 5 weeks after olfactory loss due to COVID-19: a prospective cohort study on 72 patients. European Archives of Oto-Rhino-Laryngology. 2020 Aug 4:1-8. 40. Lechien JR, Cabaraux P, Chiesa-Estomba C, Khalife M, Plzak J, Hans S et al. Objective olfactory testing in patients presenting with sudden onset olfactory dysfunction as the first manifestation of confirmed COVID-19 infection. Medrxiv. 2020a Apr. 41. London B, Nabet B, Fisher AR, White B, Sammel MD, Doty RL. Predictors of prognosis in patients with olfactory disturbance. Ann Neurol 2008; 63(2): 159-66 14 14 42. Koutsourelakis I, Keliris A, Minaritzoglou A, Zakynthinos S. Nasal steroids in snorers can decrease snoring frequency: a randomized placebo-controlled crossover trial. Journal of Sleep Research. 2015 Apr;24(2):160-6. 43. Tacchi L, Musharrafieh R, Larragoite ET, Crossey K, Erhardt EB, Martin SA et al. Nasal immunity is an ancient arm of the mucosal immune system of vertebrates. Nature communications. 2014 Oct 22;5(1):1-1. 44. Bitar MA, Dowli A, Mourad M. The effect of tonsillectomy on the immune system: A systematic review and meta-analysis. International journal of pediatric otorhinolaryngology. 2015 Aug 1;79(8):1184-91 45. Altwairqi RG, Aljuaid SM, Alqahtani AS. Effect of tonsillectomy on humeral and cellular immunity: a systematic review of published studies from 2009 to 2019. European Archives of Oto-Rhino-Laryngology. 2020 Jan 1:1-7. 46. Schairer DO, Chouake JS, Nosanchuk JD, Friedman AJ. The potential of nitric oxide releasing therapies as antimicrobial agents. Virulence. 2012 May 1;3(3):271-9. 47. Sanders SP, Proud D, Permutt S, Siekierski ES, Yachechko R, Liu MC. Role of nasal nitric oxide in the resolution of experimental rhinovirus infection. Journal of allergy and clinical immunology. 2004 Apr 1;113(4):697-702. 47. Sanders SP, Proud D, Permutt S, Siekierski ES, Yachechko R, Liu MC. Role of nasal nitric oxide in the resolution of experimental rhinovirus infection. Journal of allergy and clinical immunology. 2004 Apr 1;113(4):697-702. 48. Harris NS and Strickland B. Inhaled Nitric Oxide Therapy for Emergency Room COVID-19 Patients. https://rally.partners.org/study/no_cov_ed?code=ca 48. Harris NS and Strickland B. Inhaled Nitric Oxide Therapy for Emergency Room COVID-19 Patients. https://rally.partners.org/study/no_cov_ed?code=ca 49. Hawkes CH, Doty RL. Smell and taste disorders. Cambridge University Press; 2018. 49. Conflict of interest statement I receive an honorarium from Elsevier in respect of his Chief Editor duties for Multiple Sclerosis and Related Disorders. I receive royalties from Cambridge University Press for two books: Neurology of Olfaction by Hawkes CH and Doty RL 2009 and Smell and Taste Complaints, by Hawkes CH and Doty RL 2018. I receive royalties from Oxford University Press for Instant Neurological Diagnosis. Hawkes CH, Sethi K and Swift TJ 2019 (second edition) Funding: none Ethical approval. Not required Ethical approval. Not required Contributions. This article is the sole work of the author Contributions. This article is the sole work of the author 15 16 Reference and Country of Test Listed alphabetically by lead author Type of tests CASES Number, source, mean age/gender, Cov-2 PCR status. Time of testing CONTROLS Number, source, mean age/gender Covid-19 PCR status. Time of testing CASES. Number aware of smell / or taste impairment CASES Smell test results CONTROLS Smell test results CASES: Taste test results Altin 37 Turkey. 16 odour SST ID test In house taste ID of sucrose, salt, vinegar and coffee. 81 in-patients. 40 female. Mean age: 54y. All PCR positive. TOT not stated. 40 age/gender matched healthy controls. 19 female (47%). Mean age: 55y. Source not stated. All PCR negative. TOT not stated. 50/81 (62%) Median score 6/16 Percent abnormal not stated Median score 10/16 Percent abnormal not stated 22/81 (27%) abnormal Bocksberger36 Germany 12 odour SST ID test Taste Strips Test 14 in-patients for smell tests. Taste test in 10. Mean age 46y. 13 female. Cov-2 status not stated. TOT 4-23 days from symptom onset None 26/63 (41%) complained of loss of smell or taste 10/14 (71%) abnormal Not helped by nasal decongestant None All 10 patients were normal Calvo-Henriquez24 Spain Modified ethyl alcohol threshold test 129 in- or out-patients. Mean age 55y. 67 (52%) female. Severe cases excluded. All PCR positive. TOT not given 146 healthy hospital staff Mean age 55y. 76 female. (52%). PCR: not stated. TOT not given Not stated Abnormal threshold Not supplied directly Not done Chung38 Hong Kong UPSIT and Butanol threshold test (BTT) 18 mildly infected in-patients. Mean age 28y. 11 female (61%). All PCR positive. Median TOT: 14 days. 18 students or healthcare workers. Mean age 31y. 13 (72%) female. PCR not stated. TOT not stated 12/18 (67%) Abnormal BTT in 6/18 (33%). All 6 had abnormal UPSIT. Conflict of interest statement Not given Not done Hintschich27 Germany Pocket Smell Test (4 odours) Taste Strips Test Both self-administered 41 patients under home quarantine. All PCR positive. Median age 37y. 28 (68%) female. TOT: 3 days after positive PCR. Median of 13 days after first symptoms 30 patients. Source: not stated. Median age 33y. 22 (73%) female. All negative for IgG antibodies. TOT not stated. 25 (61%) for smell. 18 (44%) for taste 22 (54%) abnormal Where there was subjective loss of smell, abnormal in 18 (72%) Not stated Not significantly different from controls Hornuss35 Germany 12 odour SST ID test 45 in-patients. 20 female (44%). Median age 56y. All PCR positive. Mean duration of symptoms / time of testing: 10 days. 45 asymptomatic in-patients or health-care workers. Median age 54y. Gender not stated. PCR: not done. TOT not stated Smell: 22/45 (49%) 38/45 (84%) abnormal 12/45 (27%) abnormal Not done Le Bon34 Belgium Extended SST (threshold, discrimination and Identification to 16 odours). Taste Strips Test 72 outpatients. 49 (68%) female. Mean age 38.9y. 25 PCR positive. 47 IgG antibody positive. TOT: mean of 37 days after symptom onset None Smell: 100% as self selected 27/72 (38%) abnormal Main effect on threshold scores. 45 normal (62%). None 5/72 (7%) abnormal. Lechien40 Belgium 16- odour SST ID test 46 out-patients with ‘initial sudden olfactory anosmia’. Mean age: 40.6y. 46 female (59%) PCR positive in 42/46 when tested in <12 days from symptom onset None Smell: 35/41 (86%) had subjective loss as reported from earlier study 35/46 (76%) abnormal overall None Not done Lima28 Brazil QSIT. 3 odour ID test 57 Out-patients. 31 females (54%). Mean age 41.4y. All PCR positive. All but two had mild disease. Mean symptom duration: 4 days. Total: 36. Source not stated. Mean age: 37.2y. 19 female (53%). PCR: not stated. TOT not stated Smell: 34/57 (60%). 20/23 (87%) abnormal in those with subjective smell loss. 11/34 (32%) abnormal in those without subjective smell loss. 4/36 (11%) abnormal Not done Moein31 Iran UPSIT. 40 odour ID test Revised Persian language version Total: 60. All in-patients. 20 female (33%). Mean age: 46y. All PCR positive. TOT: <14 days of symptom onset 60 healthy sex & age-matched controls from prior study. PCR: not stated. Smell: 21/60 (35%) 59/60 (98%) abnormal Mean UPSIT score 21/40. 11/60 (18%) abnormal. Mean score 34/40 (normal) Not done Moein32 Iran UPSIT. Conflict of interest statement 40 odour ID test Revised Persian language version Total: 100 initial inpatients. Mean age 45y. 33 females (33%). TOT: near end of acute disease phase. After symptom onset 82 retested at 1-4 weeks. 51 retested at 6- 8 weeks. All PCR positive 51 healthy age- & sex-matched to 52 COVID patients from prior study. 19 female (37%). Mean age 45.4y. PCR: not stated. Smell: 28/100 (28%) 96/100 (96%) abnormal on initial testing. Mean UPSIT score 22/40 Not stated. Not done Tsivgoulis29 Greece Q-SIT. 3 odour ID test Total: 22 in-patients. Mean age 55y. 10 female (45%). TOT: mean of 12 days after hospital admission. All PCR positive 22 age- & sex-matched controls taken from movement disorders clinic. PCR: not stated. TOT not stated. Not stated 17/22 (77%) abnormal 8/22 (36%) abnormal Not done Vaira25 Sardinia CCCRC. In-house ID of 10 odours and butanol threshold. In-house taste identification for: salt, sugar, lemon & coffee solutions Total: 72 health personnel. 25 In-patients. Others out-patients. 45 female (62%). Mean age 49y. TOT: mean 19 days from symptom onset. All PCR positive None Smell and/or taste symptoms in 53/72 (74%). 60/72 (83%) abnormal for composite olfactory score (threshold and discrimination) None Abnormal: 35/72 (49%) Vaira26 Italian multicentre For quarantined patients: home self- administered and prepared odor discrimination test to 6 odour classes. Also used home self administered and prepared solutions to 4 tastants. For in- patients: CCCRC Total 345 patients. 161 in quarantine (self evaluated at home). 184 in-patients. 199 (58%) female. Mean age 49y. TOT: mean 15 days from symptom onset. All PCR positive None Smell and/or taste symptoms in 256 (74%) Overall percentages not supplied. From sequential graphs: around 70% abnormal for olfaction. 45% overall abnormal on taste test. None Abnormal in 190 cases (45%) Figure 1. Molecular structure of the SARS-Cov-2 virus to show how the virus can attach to a pneumocyte (alveolar cell) that lines the alveoli. Reproduced with permission from David Baker, Blizard Institute, Queen Mary, University of London. 106x62mm (96 x 96 DPI) Figure 1. Molecular structure of the SARS-Cov-2 virus to show how the virus can attach to a pneumocyte (alveolar cell) that lines the alveoli. Reproduced with permission from David Baker, Blizard Institute, Queen Mary, University of London. 106x62mm (96 x 96 DPI) 106x62mm (96 x 96 DPI) Figure 2. Simplified model for CoV-2-induced anosmia/hyposmia in COVID-19 based on results obtained from patients and animal models. Conflict of interest statement Article from Open Access journal reproduced with permission from Bilinska and Butowt6 119x87mm (96 x 96 DPI) Figure 2. Simplified model for CoV-2-induced anosmia/hyposmia in COVID-19 based on results obtained from patients and animal models. Article from Open Access journal reproduced with permission from Bilinska and Butowt6 119x87mm (96 x 96 DPI) Figure 3. Transient olfactory bulb oedema as shown in coronal 3D MRI T2-weighted imaging (1.5T) during anosmia (day 7; C) compared to recovery (day 24;D). Olfactory bulb (ob; pink) displays transient volume and signal increase, olfactory cleft oedema (OC; brown), and focal left ethmoid (eth; green) sinusitis (), and normal cranial fossa (grey line) and orbit (orb; yellow). Reproduced from Figure 1 C and D with permission from Laurendon et al9. Figure 3. Transient olfactory bulb oedema as shown in coronal 3D MRI T2-weighted imaging (1.5T) during anosmia (day 7; C) compared to recovery (day 24;D). Olfactory bulb (ob; pink) displays transient volume and signal increase, olfactory cleft oedema (OC; brown), and focal left ethmoid (eth; green) sinusitis (*), and normal cranial fossa (grey line) and orbit (orb; yellow). Reproduced from Figure 1 C and D with permission from Laurendon et al9. Figure 4. Bulk RNA-seq analysis of public datasets. Bar plot of ACE2 expression in normal tissues from FANTOM5 CAGE dataset, coloured by organs. Reproduced from Figure 1b with permission under Open Access from: Xu et al14. Figure 4. Bulk RNA-seq analysis of public datasets. Bar plot of ACE2 expression in normal tissues from FANTOM5 CAGE dataset, coloured by organs. Reproduced from Figure 1b with permission under Open Access from: Xu et al14. Figure 5. Proportion of patients with varying degrees of recovery according to COVID-19 symptom onset. All initial (n = 100) and follow-up (n = 82) scores are combined. Reproduced from Figure 4 with permission under Open Access from Moein et al32. Figure 5. Proportion of patients with varying degrees of recovery according to COVID-19 symptom onset. All initial (n = 100) and follow-up (n = 82) scores are combined. Reproduced from Figure 4 with permission under Open Access from Moein et al32.
https://openalex.org/W2952375630	https://europepmc.org/articles/pmc6362150?pdf=render	English	null	Consistent Reanalysis of Genome-wide Imprinting Studies in Plants Using Generalized Linear Models Increases Concordance across Datasets	Scientific reports	2,019	cc-by	10,922	Consistent Reanalysis of Genome- wide Imprinting Studies in Plants Using Generalized Linear Models Increases Concordance across Datasets Stefan Wyder 1, Michael T. Raissig1,2 & Ueli Grossniklaus 1 Received: 20 April 2018 Accepted: 27 November 2018 Published: xx xx xxxx Genomic imprinting leads to different expression levels of maternally and paternally derived alleles. Over the last years, major progress has been made in identifying novel imprinted candidate genes in plants, owing to affordable next-generation sequencing technologies. However, reports on sequencing the transcriptome of hybrid F1 seed tissues strongly disagree about how many and which genes are imprinted. This raises questions about the relative impact of biological, environmental, technical, and analytic differences or biases. Here, we adopt a statistical approach, frequently used in RNA-seq data analysis, which properly models count overdispersion and considers replicate information of reciprocal crosses. We show that our statistical pipeline outperforms other methods in identifying imprinted genes in simulated and real data. Accordingly, reanalysis of genome-wide imprinting studies in Arabidopsis and maize shows that, at least for Arabidopsis, an increased agreement across datasets could be observed. For maize, however, consistent reanalysis did not yield a larger overlap between the datasets. This suggests that the discrepancy across publications might be partially due to different analysis pipelines but that technical, biological, and environmental factors underlie much of the discrepancy between datasets. Finally, we show that the set of genes that can be characterized regarding allelic bias by all studies with minimal confidence is small (~8,000/27,416 genes for Arabidopsis and ~12,000/39,469 for maize). In conclusion, we propose to use biologically replicated reciprocal crosses, high sequence coverage, and a generalized linear model approach to identify differentially expressed alleles in developing seeds. In a diploid cell, the maternal and paternal alleles of a given gene usually share the same expression state in a spe- cific tissue, meaning that they are either both expressed or both silent. Important exceptions to this rule are genes regulated by genomic imprinting, where the expression state depends on the parental origin of the alleles, and only one is expressed while the other remains silent or is weakly expressed. The two alleles do not differ in their sequence but rather carry parent-specific, epigenetic imprints that allow the cell to distinguish the two alleles1–8. Genomic imprinting evolved independently in mammals and flowering plants (angiosperms) (reviewed in9–15). In both groups, offspring develop within the mother and depend solely on her to supply nutrients for growth and development. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Received: 20 April 2018 Accepted: 27 November 2018 Published: xx xx xxxx Consistent Reanalysis of Genome- wide Imprinting Studies in Plants Using Generalized Linear Models Increases Concordance across Datasets Stefan Wyder 1, Michael T. Raissig1,2 & Ueli Grossniklaus 1 Accordingly, a number of research groups performed genome-wide, allele-specific transcriptome profiling studies of hybrid seeds in Arabidopsis and maize to identify genes that are preferentially expressed from one parental allele27–38. As a result, the total number of imprinted genes increased from around 206 to over 900 potentially imprinted plant genes28–33,35,36,38.i p p g However, comparisons of the identified imprinted candidate genes revealed little overlap between the stud- ies30,34,39. In general, the analysis of RNA-sequencing (RNA-seq) data to identify allele-specific expression is prone to false positives due to both, biological and technical variation40–42. Thus, even studies with seemingly similar design heavily disagree on the number of imprinted genes in the mouse brain, e.g. ranging from less than 20040 to over a thousand43. To date, although guidelines for the analysis of allele-specific expression have recently become available42, many different methods have been applied to filter, normalize, and statistically assess allelic imbalance from RNA-seq data. For the analysis of allele-specific expression, several analysis methods and software42 have been developed, yet only very few are suitable for an analysis of imprinted expression. Moreover, no specialized method is available for statistical testing of imprinting in the triploid endosperm, where the expected allelic ratio is 2:1 because the mother contributes two genomes to this tissue. In plants, many authors have used count tests (such as Chi-Square, binomial, or Fisher’s exact tests), which heavily underestimate the count dispersion typically observed in RNA-seq data41,42,44, resulting in increased numbers of false positives particularly for large counts. Highly expressed transcripts may appear imprinted with high statistical significance, as count tests are sensitive to very small allelic imbalance at high counts, requiring additional filtering with somewhat arbitrary imbalance cut-offs.i f Here, we present a new statistical approach to call imprinted genes from large allele-specific RNA-seq datasets from endosperm that outperforms other methods in simulated and real data. We propose a generally applicable approach using generalized linear models (GLM) implemented in edgeR45, which is based on the negative bino- mial distribution to deal with potential count overdispersion46 as it is typically seen in RNA-seq data. The pre- sented pipeline outperforms other methods using simulated data. Furthermore, we reanalyze the raw data from seven studies to assess the relative importance of differences in data generation and data analysis. Consistent Reanalysis of Genome- wide Imprinting Studies in Plants Using Generalized Linear Models Increases Concordance across Datasets Stefan Wyder 1, Michael T. Raissig1,2 & Ueli Grossniklaus 1 The consistent reanalysis by the proposed pipeline results in a larger overlap of imprinted candidate genes across Arabidopsis datasets, but showed little improvement across maize datasets. In conclusion, consistent data analysis can improve concordance between datasets but biological and technical variation in data generation contributes most to the differences among datasets. Consistent Reanalysis of Genome- wide Imprinting Studies in Plants Using Generalized Linear Models Increases Concordance across Datasets Stefan Wyder 1, Michael T. Raissig1,2 & Ueli Grossniklaus 1 This common reproductive strategy results in an intragenomic parental conflict over resource allocation, which likely underlies the evolution of genomic imprinting, at least for loci that control growth14,16,17. Accordingly, some imprinted genes in both, mammals and plants, have a role in controlling growth (e.g.18–26). Consistent with this function, many imprinted genes are preferentially expressed in the tissues that support embryonic growth, i.e. the placenta in mammals or the triploid endosperm in the seeds of flowering plants. y g p p pl g p Over the last decade, the advent of Next-Generation Sequencing (NGS) allowed (nearly) genome-wide imprinting studies by sequencing the transcriptome of hybrid F1 seed tissues: Given exonic polymorphisms 1Department of Plant and Microbial Biology & Zurich-Basel Plant Science Center, University of Zurich, Zollikerstrasse 107, CH-8008, Zurich, Switzerland. 2Present address: Centre for Organismal Studies, Heidelberg University, Im Neuenheimer Feld 230, 69120, Heidelberg, Germany. Correspondence and requests for materials should be addressed to U.G. (email: grossnik@botinst.uzh.ch) Scientific Reports \| (2019) 9:1320 \| https://doi.org/10.1038/s41598-018-36768-4 1 www.nature.com/scientificreports/ Figure 1. Power of detecting imprinted candidate genes in reanalyzed datasets. (A) Callable genes assessable for genomic imprinting were required to overlap with at least 1 exonic SNP and to have read counts of at least 10. Number of genes are shown as percentages of the total number of genes. (B) Venn diagrams showing the overlap of callable genes for Arabidopsis and maize datasets. Figure 1. Power of detecting imprinted candidate genes in reanalyzed datasets. (A) Callable genes assessable for genomic imprinting were required to overlap with at least 1 exonic SNP and to have read counts of at least 10. Number of genes are shown as percentages of the total number of genes. (B) Venn diagrams showing the overlap of callable genes for Arabidopsis and maize datasets. between the parents, reads overlapping heterozygous SNPs can be assigned to their parent-of-origin, and recip- rocal crosses allow the discrimination between parent-of-origin-dependent and strain-specific genetic effects. Accordingly, a number of research groups performed genome-wide, allele-specific transcriptome profiling studies of hybrid seeds in Arabidopsis and maize to identify genes that are preferentially expressed from one parental allele27–38. As a result, the total number of imprinted genes increased from around 206 to over 900 potentially imprinted plant genes28–33,35,36,38.i between the parents, reads overlapping heterozygous SNPs can be assigned to their parent-of-origin, and recip- rocal crosses allow the discrimination between parent-of-origin-dependent and strain-specific genetic effects. Scientific Reports \| (2019) 9:1320 \| https://doi.org/10.1038/s41598-018-36768-4 Results C i Numbers in brackets denote the percentage of non-shared genes relative to the full set reported. The “Xin” and “Zhang11” sets comprise genes identified at 10 days after pollination and “Zhang14” comprises genes at 12 days after pollination. Genes with an accession/inbred-specific Figure 2. Venn diagrams showing the number of imprinted genes in hybrid endosperm reported by different studies in Arabidopsis Ler/Col accessions and in maize B73/Mo17 inbreds. Only genes were considered that could be evaluated for imprinting in all datasets. Numbers in brackets denote the percentage of non-shared genes relative to the full set reported. The “Xin” and “Zhang11” sets comprise genes identified at 10 days after pollination and “Zhang14” comprises genes at 12 days after pollination. Genes with an accession/inbred-specific bias in expression were excluded from the analysis. protein-coding Arabidopsis genes, 31% do not overlap with any exonic SNP and their imprinting cannot be assessed. Another 17–39% of the genes are not or only very weakly expressed (<10 allelic counts overall), and were thus discarded from further analysis. Ultimately, only 28–52% of all predicted Arabidopsis genes can be assessed for genomic imprinting in the three different studies, because only those have a sufficient number of allele-specific reads to identify statistically significant biased expression (Fig. 1A). 7,499 genes (27% of all Arabidopsis genes) can be assessed for genomic imprinting in all three datasets (Fig. 1B). p g g p g g Among the 39,469 maize protein-coding genes, 33% do not overlap with any exonic SNP (Fig. 1A) between the B73 and Mo17 inbreds. Another 26–33% were not or very weakly expressed (<10 counts overall) and were discarded (Fig. 1A). In the end, between 34% (Zhang11) and 41% (Waters) of maize genes can be assessed for genomic imprinting per dataset and only 31% of all maize genes (12,354 genes) can be assessed in all datasets (Fig. 1A,B). In conclusion, even after consistent reanalysis, the set of genes that can be assessed for genomic imprinting differs considerably between the datasets. For all the following analyses, we considered only genes that could be evaluated for imprinting in all datasets. p g In Arabidopsis, a total of 185 genes that could be evaluated by all datasets were proposed to be maternally expressed imprinted genes (MEGs) in at least one study using the Landsberg erecta (Ler) and Columbia-0 (Col-0) accessions (Fig. 2). The three studies proposed between 55 and 89 MEGs28,29,38. Results C i Comparison of Genome-wide Imprinting Studies in Plants: Biological, Technical, and Statistical Differences. To shed light onto discrepancies between published studies and potential biases, we compared the genome-wide, allele-specific transcriptome profiling studies of hybrid seeds in Arabidopsis and maize that were designed to identify imprinted candidate genes in the endosperm. All studies were based on reciprocal crosses of two polymorphic inbred strains, and all studies used manually dissected endosperm. For simplicity, we only compared studies using Ler and Col-0 accessions (Arabidopsis) or B73 and Mo17 inbreds (maize). First, we set out to identify the genes that can be evaluated for imprinting by all studies and reanalyzed the raw data from the seven published studies starting from the raw reads. We examined all reads overlapping with previously known exonic SNPs (see Methods), separated and counted maternal and paternal reads overlapping SNPs, and summed up informative (i.e. SNP containing) reads across transcripts. After discarding very lowly expressed genes (<10 counts per gene), median reads per transcript were 95–654 for the different datasets. Th f ll l b l d d d l h d f ll l b h d f The power of allelic imbalance detection depends mainly on the degree of allelic bias, the sequencing read length and coverage (expression strength), as well as the divergence of the crossed strains (number of SNPs per transcript)47. Although the three Arabidopsis studies all use the same accessions, the number of callable genes ranges from 7,792 for the Hsieh dataset to 14,229 for the Pignatta dataset (Fig. 1A): Among the 27,416 Scientific Reports \| (2019) 9:1320 \| https://doi.org/10.1038/s41598-018-36768-4 2 www.nature.com/scientificreports/ Figure 2. Venn diagrams showing the number of imprinted genes in hybrid endosperm reported by different studies in Arabidopsis Ler/Col accessions and in maize B73/Mo17 inbreds. Only genes were considered that could be evaluated for imprinting in all datasets. Numbers in brackets denote the percentage of non-shared genes relative to the full set reported. The “Xin” and “Zhang11” sets comprise genes identified at 10 days after pollination and “Zhang14” comprises genes at 12 days after pollination. Genes with an accession/inbred-specific bias in expression were excluded from the analysis. Figure 2. Venn diagrams showing the number of imprinted genes in hybrid endosperm reported by different studies in Arabidopsis Ler/Col accessions and in maize B73/Mo17 inbreds. Only genes were considered that could be evaluated for imprinting in all datasets. Scientific Reports \| (2019) 9:1320 \| https://doi.org/10.1038/s41598-018-36768-4 Results C i The large majority of genes (81%) were unique to a single study, and only eleven MEGs were identified as imprinted in all three studies (6%; Fig. 2). Thirtyfive genes were proposed to be paternally expressed imprinted genes (PEGs) in at least one study with five PEGs being commonly identified in all three studies (14%; Fig. 2). In maize, the four available studies using the inbreds B73 and Mo1731,33,36,37 listed 182 different MEGs and 182 PEGs (Fig. 2). The majority of genes (65% and 41% for MEGs and PEGs, respectively) was proposed by a single study only. The overlap between studies was also small, with 14 MEGs (8%) and 23 PEGs (13%) being commonly identified by all four studies.hf g yi y The low concordance between studies could be due (i) to intrinsic, biological differences (e.g. developmental stage analyzed), (ii) to technical differences (particularly library preparation and complexity, sequencing depth and batch effects, reviewed in41,42, and/or (iii) to the varying bioinformatics/statistical analysis protocols applied. As summarized in Table 1, all studies differ in terms of developmental stage and some use different accessions cre- ating considerable biological variation. Technical differences like library preparation, sequencing platform, read length, and single-end vs. paired-end sequencing introduce a further level of variation. Particularly, the observed Scientific Reports \| (2019) 9:1320 \| https://doi.org/10.1038/s41598-018-36768-4 3 www.nature.com/scientificreports/ differences in sequencing depth, expected read-mapping biases48, as well as in the completeness and quality of available SNP annotations, present likely technical sources of inconsistency (Table 1). Results C i Regarding statistical analy- Wolff 2011 Gehring 2011 Hsieh 2011 Pignatta 2014 Waters 2011 Zhang 2011 Xin 2013 Waters 2013 Zhang 2014 Organism Arabidopsis Arabidopsis Arabidopsis Arabidopsis Maize Maize Maize Maize Maize Strains Col-0, Bur-0 Col-0, Ler Col-0, Ler Col-0, Ler, Cvi B73, Mo17 B73, Mo17 B73, Mo17 B73,Mo17,Ki11,Oh43 B73, Mo17 Starting Material whole seeds dissected endosperm/ embryo dissected endosperm/ embryo dissected endosperm/ embryo dissected endosperm/ embryo dissected endosperm/ embryo whole kernels (0,3,5 DAP)/ dissected endosperm (7, 10,15 DAP) dissected endosperm dissected endosperm Timepoint 4 DAP 6 – 7 DAP 7- 8 DAP 6 DAP 14 DAP 10 DAP 0,3,5,7,10,15 DAP 14 DAP 12 DAP Biological Replicates per cross 1 1 2 3 1 1 1 1 1 Sequencing Platform Illumina GAII Illumina GAII Illumina GAII Illumina HiSeq Illumina GAII/HiSeq Illumina HiSeq Illumina HiSeq Illumina HiSeq Illumina HiSeq Read Length 36 bp 50/36 bp 76 bp 40/80 bp 2 × 76 bp 2 × 100 bp 2 × 90 bp 2 × 100/2 × 50 bp 2 × 100 NCBI SRA study ID SRP005700 SRP007424 SRP003799 SRP033371 SRP009313 SRP011991 SRP026399 SRP031872 SRP011991 Genome annotation TAIR 8 TAIR 9 TAIR 8 TAIR 10 B73 AGPv2 n/a B73 5b.60 n/a B73 (V2) Total number of raw reads (hybrids) 122 mio 100 mio 165 mio 1,837 mio 245 mio 149 mio 379 mio 1,969 mio 154 mio Total Number of SNPs 569,859 347,928 402,226 384,612 1.6 mio 51,416 exonic 6.5 mio 28,195–142,033 exonic 4.2 mio Mapping Mapping software vmatch TopHat Bowtie Tophat v2.0.8 GSNAP bwa TopHat2 TopHat TopHat Number mismatches allowed 2/36 n/a 3/76 1/40 2/36 n/a n/a 2 n/a Allele-specific mapping bias Alignment to Col and Bur pseudoreference (only SNPs) n/a Alignment to Col and Ler pseudoreference (only SNPs) n/a n/a Mapping to SNP-masked genes Mapping to both genomes (reference- guided assembly of Mo17) Mapping to SNP- masked transcripts (filtered gene set v5b.60) Mapping to SNP-masked genes Counting and Statistics Minimal coverage (allelic reads) ≥10 (≥30) ≥15 n/a n/a ≥10 reads assigned to one allele in both hybrids ≥10 per cross ≥40 (non- stringent: ≥5 per cross) ≥10 per cross n/a Summing Reads across gene gene individual SNPs gene gene gene Statistical Test Binomial Storer-Kim Fisher’s exact test Fisher’s exact test Chi-Square Chi-Square Chi-Square Chi-Square Chi-Square Multiple Testing Correction yes (FDR 5%) no (p < 0.01) no (p < 0.001) yes (FDR 1%) no (p < 0.01) no (p < 0.05) unknown adjustment (p < 0.001) no (p < 0.05 or p < 0.01) no (p < 0.05) Allelic bias filtering n/a only unique expression MEGs: maternal score ≥ 4x paternal score; PEGs: paternal score ≥ 1.5x maternal score MEGs: ≥ 85% maternal reads PEGs: ≥ 50% paternal reads ≥90% reads from one parent in both reciprocal crosses ≥83% reads from one parent MEGs: ≥ 90% maternal reads PEGs: ≥ 70% paternal reads < 6 paternally derived reads at 0 DAP Moderate MEGs: > 90% maternal reads PEGS: > 60% paternal reads. Results C i Fisher’s exact test is the analysis method used in most published plant studies where a Fisher’s exact test is performed with allelic counts of summed reciprocal crosses. From now on this method will be called “Fisher-summed”. Stouffer’s method49 is a method to combine p-values bearing the same null hypothesis. We combined the two p-values per gene calculated by Fisher’s exact tests comparing to expectations for each recip- rocal cross separately, and calculated a combined adjusted p-value using Stouffer’s method. From now on this method will be called “Fisher-combined”. In the simulation setting, our pipeline (edgeR) identified the largest number of observed true positives (spiked-in MEGs and PEGs) (Fig. 3A), identifying 131 true positives (of 200, 66% true positive rate [TPR]) with 8 false positives (falsely detected as imprinted), whereas the second-best method, Fisher-summed, identified only 102 true positives (51% TPR) with 0 false positives (0% FPR). Fisher-combined was close third, identifying 93 true positives (47% TPR) with 0 false positives (0% FPR).hi The Venn diagram (Fig. 3A) shows that 93 genes were identified between all three methods, 9 were shared between edgeR and Fisher-summed, 37 genes were only identified by edgeR, and 61 true positive genes were not identified by any method. Considering the genes that were simulated to be imprinted as the true positive group, and the remaining genes as the true negative group, we computed the false positive rate and the true positive rate for all possible score thresholds and constructed a ROC (Receiver Operating Characteristic) curve for each method (Fig. 3B). edgeR and Fisher-summed had a small performance advantage in detecting the spike-in genes over Fisher-combined. We divided the assessed genes into four equally sized bins according to their number of counts. As expected, the true positive rate (TPR) increased with larger counts per gene for all methods (Fig. 3C; Supplementary Fig. S2). TPR was highest for edgeR in all categories with a large advantage in the two categories with the lowest counts, while the other two methods only passed the 50% TPR for the two quartiles of genes with the largest counts. True positive rates also depended on the degree of allelic imbalance: at a 5% False Discovery Rate (FDR) cut-off, edgeR achieved TPRs of 80% and 54%, respectively, for strong and weak MEGs, and TPRs of 70% and 58%, respectively, for strong and weak PEGs. Results C i Strong MEGs/ PEGs: > 90% maternal/ paternal and p < 0.01 ≥ 83% reads from one parent (reduced criteria: Chi-Square p < 0.05) Filtering out potentially contaminating transcripts derived from the seed coat Expression SLR in endosperm ≥ 3x seed coat and SLRs in vegetative tissues < 5 (less strigent criteria for low expressed genes) MEGs expression in endosperm ≥ 2x higher than in the seed coat Expression levels in endosperm < 4x than LCM- disected endosperm Expression levels < 2x higher in the seed coat than embryo or endosperm n/a n/a n/a B73 expression atlas n/a Table 1. Characteristics of data generation and data analysis of published genome-wide imprinting datasets in Arabidopsis and maize. Abbreviations: DAP, days after pollination; FDR, false discovery rate; LCM, Laser Capture Microdissection; MEG, maternally expressed imprinted gene; PEG, paternally expressed imprinted gene; SNP, single-nucleotide polymorphism; SLR, signal log ratio; SRA, short read archive (http://www.ncbi. nlm.nih.gov/sra). Table 1. Characteristics of data generation and data analysis of published genome-wide imprinting datasets in Arabidopsis and maize. Abbreviations: DAP, days after pollination; FDR, false discovery rate; LCM, Laser Capture Microdissection; MEG, maternally expressed imprinted gene; PEG, paternally expressed imprinted gene; SNP, single-nucleotide polymorphism; SLR, signal log ratio; SRA, short read archive (http://www.ncbi. nlm.nih.gov/sra). Table 1. Characteristics of data generation and data analysis of published genome-wide imprinting datasets in Arabidopsis and maize. Abbreviations: DAP, days after pollination; FDR, false discovery rate; LCM, Laser Capture Microdissection; MEG, maternally expressed imprinted gene; PEG, paternally expressed imprinted gene; SNP, single-nucleotide polymorphism; SLR, signal log ratio; SRA, short read archive (http://www.ncbi. nlm.nih.gov/sra). differences in sequencing depth, expected read-mapping biases48, as well as in the completeness and quality of available SNP annotations, present likely technical sources of inconsistency (Table 1). Regarding statistical analy- sis, all studies applied count statistics (Table 1), which do not properly model count dispersion of RNA-Seq data, differences in sequencing depth, expected read-mapping biases48, as well as in the completeness and quality of available SNP annotations, present likely technical sources of inconsistency (Table 1). Regarding statistical analy- sis, all studies applied count statistics (Table 1), which do not properly model count dispersion of RNA-Seq data, Scientific Reports \| (2019) 9:1320 \| https://doi.org/10.1038/s41598-018-36768-4 4 www.nature.com/scientificreports/ resulting in increased numbers of false positives particularly for large counts41,44. Furthermore, the studies apply very different criteria for filtering potentially imprinted genes according to the allelic bias (Table 1). Results C i The require- ment to call a gene’s expression parentally biased differed tremendously between the studies and ranged from 90% of all reads that have to derive from one parent31, over 5 times more reads from one parent33, to simply assessing deviations from the expected 2:1 ratio in the endosperm28. Lastly, to deal with potential contamination from the seed coat, transcripts that were highly expressed in this maternal tissue were filtered out in silico in most studies (Table 1). The filtering conditions and the expression data used varied considerably across studies. Analysis of Allelic Bias Using edgeR Outperforms other Methods. It was previously noticed that a large part of the differences between publications is owed to different statistical pipelines to call imprinted genes. When the two Arabidopsis datasets that analyzed the same accessions and a similar developmental stage and tis- sue (Gehring and Hsieh datasets) were analyzed in the same way, the overlap increased substantially (from 14 to 56 MEGs and from 6 to 18 PEGs28). Therefore, we created a statistical pipeline to identify genes with statistically significant allelic imbalance from the expected, endosperm-specific 2:1 ratio. Our pipeline is based on edgeR45 and analyzes counts by a generalized linear model (GLM), based on a negative binomial distribution in a paired design (parentals of the same cross) with two or more biological replicates (or reciprocal crosses). Importantly, edgeR models count overdispersion as shown exemplarily for the Pignatta dataset (Supplementary Fig. S1). W h d h f f l l d h h d b d h g y g y g We then tested the performance of our statistical pipeline compared to other methods based on synthetic data, where we could control the settings and the true genomic imprinting status of each gene. We simulated counts using negative binomial distributions, with mean and dispersion parameters estimated from real data (see Material and Methods). Imprinted genes were introduced by adding 200 genes with a parent-of-origin-specific allelic bias: 50 MEGs each with strong (99% maternal reads) or moderate (85% maternal reads) allelic bias, as well as 50 PEGs each with strong (34% maternal reads) or moderate (48% maternal reads) allelic bias. Random allelic read sampling was modeled by sampling from a binomial distribution.f g y g We evaluated the performance of three different methods: our edgeR-based pipeline, Fisher’s exact test, and Stouffer’s method. Results C i Benchmarking of three tested methods to identify imprinted genes using simulated data. (A) Overlap of detected spike-in imprinted genes between different methods (FDR 5%). (B) ROC curves. (C) True positive rates (TPR) and false positive rates (FPR) across four equally sized categories of genes with increasing expression levels (number of counts) at FDR 5%. shared with any other dataset, potentially owing to the fact that (i) the Pignatta dataset had the largest sequencing depth and therefore the largest gene coverage, and (ii) three biological replicates per cross could be analyzed, allowing the identification of high-confidence candidates with a small number of false positives. We also iden- tified an almost three-fold increased number of PEGs (98 instead of 35 genes as originally published), although with an increased proportion of non-shared PEGs in the Hsieh dataset (from 17% [1/6] to 62% [40/65]).i p p ( [ ] [ ]) In maize, we identified a large number of imprinted candidate genes, 218 MEGs and 221 PEGs (Fig. 4). 70 MEGs (32%) and 83 PEGs (38%) were identified from at least two different datasets, leading to an overlap of MEGs similar to the one found in the originally published lists (Fig. 2). The number of candidate imprinted genes varied a lot between datasets, limiting the maximum number of genes shared by all four datasets. The proportion of non-shared MEGs and PEGs decreased for all datasets, except for Waters MEGs and PEGs, which increased from 22% to 48% and from 36% to 62%, respectively, likely due to the significant increase in the number of imprinted candidate genes (Fig. 4). In contrast, the Zhang datasets, as well as the PEGs identified from the Xin dataset, showed almost no exclusive candidate imprinted genes.ii p g Furthermore, we identified the top50 imprinted candidate genes, which are statistically most significant for each dataset and compared the number of shared genes (Supplementary Fig. S4). Between 17–28 candidate genes were common to all compared datasets and the proportion of non-shared candidates decreased or was similar as in the originally published lists except for the Hsieh, Pignatta, and Xin PEGs. In summary, a reanalysis of the datasets using our edgeR-based pipeline produced gene lists with a clearly larger overlap in Arabidopsis, despite identifying a larger number of imprinted candidate genes with less pro- nounced allelic imbalance. Results C i In contrast, the second best method Fisher-summed achieved TPRs of 74% and 44%, respectively, for strong and weak MEGs, and TPRs of 58% and 28%, respectively, for strong and weak PEGs. Simulations with count distributions similar to the largest observed datasets showed that edgeR still performed better than the other methods (data not shown). EdgeR outperformed the other meth- ods when 200 genes with an accession-specific bias in expression were added to the 200 simulated imprinted genes (data not shown). In addition, the performance of edgeR was robust against varying proportions of simu- lated strongly and moderately expressed imprinted genes (Supplementary Table S4) and against varying numbers of imprinted genes, as long as the total number did not drop below 50 genes (Supplementary Table S5). Consistent Reanalysis of Published Imprinting Studies with edgeR Identifies More Common Imprinted Candidate Genes in Arabidopsis but not in Maize. To test our statistical pipeline on real data, we identified genes with statistically significant allelic imbalance from the expected, endosperm-specific 2:1 ratio using edgeR45 with reanalyzed data starting from raw reads. We identified between 165–319 candidate MEGs and 21–65 candidate PEGs for the examined datasets using a 5% FDR cut-off (Fig. 4). Diagnostic plots exemplary for the Pignatta dataset display a good model fit (Supplementary Fig. S3). A list of the called imprinted candidate genes in Arabidopsis and maize can be found in Supplementary Tables S1 and S2, respectively.i In Arabidopsis, we identified a larger number of potentially imprinted genes with clearly increased overlaps: 143 common MEGs were found from all three datasets (87% of the smallest dataset [Pignatta], and 45% of the largest dataset [Gehring]) and, in addition, 60 MEGs (20% of the smaller dataset) were shared by the Gehring and Hsieh datasets (Fig. 4). Now only 31% and 29% of identified MEGs were unique to the Gehring and Hsieh datasets, respectively. In the Pignatta dataset, only a single MEG (1/165, 0%) and a single PEG (1/21, 5%) were not Scientific Reports \| (2019) 9:1320 \| https://doi.org/10.1038/s41598-018-36768-4 5 www.nature.com/scientificreports/ Figure 3. Benchmarking of three tested methods to identify imprinted genes using simulated data. (A) Overlap of detected spike-in imprinted genes between different methods (FDR 5%). (B) ROC curves. (C) True positive rates (TPR) and false positive rates (FPR) across four equally sized categories of genes with increasing expression levels (number of counts) at FDR 5%. Figure 3. Results C i Thus, the Jaccard similarity indices between Arabidopsis datasets were higher for MEGs and identical for PEGs after reanalysis with edgeR compared to the originally published gene lists (Supplementary Table S6). For maize, we saw an increase in dataset concordance after reanalysis for both MEGs and PEGs. Reanalysis Using the edgeR Analysis Pipeline Identifies Novel Imprinted Candidate Genes in Comparison to the Original Analysis. Having identified imprinted candidate genes using our new analysis pipeline, we com- pared the candidate genes pairwise with the lists from the original publications (Fig. 5). When comparing with our Scientific Reports \| (2019) 9:1320 \| https://doi.org/10.1038/s41598-018-36768-4 6 www.nature.com/scientificreports/ Figure 4. Venn diagrams showing the overlap between imprinted candidate genes across datasets when reanalyzing the raw data using the same standardized method with generalized linear models/edgeR at a FDR cut-off of 5%. Only genes were considered that could be evaluated for imprinting in all datasets. Numbers in brackets denote the percentage of non-shared genes relative to the full set detected in the dataset. The “Xin” and “Zhang11” sets comprise genes identified at 10 days after pollination and “Zhang14” comprises genes at 12 days after pollination. Figure 4. Venn diagrams showing the overlap between imprinted candidate genes across datasets when reanalyzing the raw data using the same standardized method with generalized linear models/edgeR at a FDR cut-off of 5%. Only genes were considered that could be evaluated for imprinting in all datasets. Numbers in brackets denote the percentage of non-shared genes relative to the full set detected in the dataset. The “Xin” and “Zhang11” sets comprise genes identified at 10 days after pollination and “Zhang14” comprises genes at 12 days after pollination. candidate MEGs and PEGs at a 5% FDR cut-off, 0–71% of previously published imprinted candidate genes were also identified by our pipeline.i i From the reanalysis we selected the genes with topmost significance to get the equivalent number of the previously published imprinted candidate genes and compared them to the original publications. Arabidopsis PEGs and maize MEGs and PEGs generally overlapped at 44–85% between the reanalysis and the original gene lists (Supplementary Fig. S5), much higher than for Arabidopsis MEGs, where often only negligible overlaps were observed.i In conclusion, the reanalysis with a standardized analytical pipeline identifies a large number of similar can- didates but also additional novel candidate genes. Results C i Furthermore, it fails to identify previously called imprinted candidates, likely owing to the improved statistical analysis, which takes count overdispersion into account and neglects large allelic biases in transcripts covered by a low number of reads. Consistent Reanalysis Using Fisher’s Exact Test Identifies Fewer Overlapping Imprinted Candidate Genes. We also reanalyzed the data from the seven studies using the “Fisher-summed” method in order to assess its performance with real data. We selected the genes with topmost significance to get for each dataset the equivalent number of the previously published imprinted candidate genes (Supplementary Fig. S6). A proportion of maternal reads of ≥85% and ≤50% was required for candidate MEGs and PEGs, respectively. In Arabidopsis, the number of genes identified from all three datasets (27 MEGs and 1 PEGs; Supplementary Fig. S6) was only a fraction of those identified after reanalysis with edgeR (41 MEGs and 5 PEGs; Supplementary Fig. S7). Also, the number of genes shared between at least two different datasets (63 MEGs and 10 PEGs; Supplementary Fig. S6) was smaller than after reanalysis with edgeR (70 MEGs and 19 PEGs; Supplementary Fig. S7). The proportion of non-shared genes was 29–68% per dataset, much higher than after reanalysis with edgeR, where proportions of non-shared genes were 4–32%. A reanalysis of the maize datasets using the “Fisher-summed” method produced a similar concordance between datasets as edgeR (Supplementary Figs S6 and S7). Accordingly, Jaccard similarity indices between datasets were markedly higher in Arabidopsis after reanalysis with edgeR for MEGs compared with the Fisher-summed reanalysis or the originally published gene lists (Supplementary Table S6). For PEGs, Jaccard similarity indices after reanalysis with edgeR were higher than after Fisher-summed reanalysis and iden- tical with the originally published gene lists. Scientific Reports \| (2019) 9:1320 \| https://doi.org/10.1038/s41598-018-36768-4 7 www.nature.com/scientificreports/ Power of Detecting Imprinted Genes Is Relatively Small due to Non-saturating S Th l i l ti hi i ll li i b l d t ti i t ll d t d Figure 5. Pairwise comparison of imprinted genes between the originally published analys using generalized linear models and edgeR with a 5% FDR cut-off. Only genes were conside evaluated for imprinting in all datasets. Numbers in brackets denote the percentage of non- to the full set detected in the dataset. For the “Xin” dataset only one timepoint (10 days after shown. Results C i The “Zhang11” set comprises genes identified at 10 days after pollination and “Zhan genes expressed at 12 days after pollination. Figure 5. Pairwise comparison of imprinted genes between the originally published analysis and the reanalysis using generalized linear models and edgeR with a 5% FDR cut-off. Only genes were considered that could be evaluated for imprinting in all datasets. Numbers in brackets denote the percentage of non-shared genes relative to the full set detected in the dataset. For the “Xin” dataset only one timepoint (10 days after pollination) is shown. The “Zhang11” set comprises genes identified at 10 days after pollination and “Zhang14” comprises genes expressed at 12 days after pollination. Figure 5. Pairwise comparison of imprinted genes between the originally published analysis and the reanalysis using generalized linear models and edgeR with a 5% FDR cut-off. Only genes were considered that could be evaluated for imprinting in all datasets. Numbers in brackets denote the percentage of non-shared genes relativ to the full set detected in the dataset. For the “Xin” dataset only one timepoint (10 days after pollination) is shown. The “Zhang11” set comprises genes identified at 10 days after pollination and “Zhang14” comprises genes expressed at 12 days after pollination. Figure 5. Pairwise comparison of imprinted genes between the originally published analy using generalized linear models and edgeR with a 5% FDR cut-off. Only genes were consid Figure 5. Pairwise comparison of imprinted genes between the originally published analysis and the reanalysis using generalized linear models and edgeR with a 5% FDR cut-off. Only genes were considered that could be evaluated for imprinting in all datasets. Numbers in brackets denote the percentage of non-shared genes relative to the full set detected in the dataset. For the “Xin” dataset only one timepoint (10 days after pollination) is shown. The “Zhang11” set comprises genes identified at 10 days after pollination and “Zhang14” comprises genes expressed at 12 days after pollination. Power of Detecting Imprinted Genes Is Relatively Small due to Non-saturating Sequencing Depth. The power-sample size relationship in allelic imbalance detection is not well understood as it depends mainly on the degree of allelic bias, the sequencing read length and coverage (expression strength), but also on the detection Power of Detecting Imprinted Genes Is Relatively Small due to Non-saturating Sequencing Depth. Discussion R l i f Reanalysis of Imprinting Studies Reveals that Biological and Technical Differences Strongly Contribute to Biases in Identifying Imprinted Genes. Published studies strongly disagree in the extent and composition of imprinted genes in the endosperm (Fig. 2). We reanalyzed both simulated and real data from seven genome-wide imprinting studies in Arabidopsis and maize, using a standardized bioinformatics pipeline based on generalized lin- ear models. In Arabidopsis, our analysis identified an increased number of imprinted genes co-identified by at least two different datasets, particularly for MEGs where the overlap between datasets was strikingly larger (Fig. 4 and Supplementary Fig. S4). In maize, consistent reanalysis identified a slightly increased number of imprinted genes shared between studies both for MEGs and PEGs. For MEGs, it is conceivable that the increased concordance is partially due to the fact that we did not filter out potential seed coat contamination in the reanalysis. However, as it has previously been shown that most imprinted genes in the embryo are also expressed in the seed coat50, such a filter could also elim- inate truly imprinted genes.f y p g It was previously proposed that the discrepancies might at least in part stem from different data analysis pipe- lines to call imprinted genes and, in fact, using similar filtering conditions produced more overlap28,34. However, even after consistent reanalysis, a high number of imprinted candidate genes are unique to a single dataset, and reanalysis is able to increase the overlap across datasets only by a limited degree. This suggests that biological variation (such as different environmental growth condition, different developmental seed stage, stochastic allelic expression differences), technical differences (e.g., library preparation/complexity, batch effects41,42), and differ- ences in sequencing depth inherent to the datasets contribute to these discrepancies and cannot be corrected for in silico. Particularly in maize, reanalysis did not increase the concordance between datasets. Even though the develop- mental seed stages did vary to some extent (Table 1; 10–14 DAP), we cannot fully explain this finding by biolog- ical variation only. All original analyses used the same statistical test and highly similar conditions for allelic bias filtering. Possibly, the published original analyses of maize datasets used filtering conditions close to the optimum, such that edgeR-based reanalysis could not further improve concordance. Results C i The power-sample size relationship in allelic imbalance detection is not well understood as it depends mainly on the degree of allelic bias, the sequencing read length and coverage (expression strength), but also on the detection Scientific Reports \| (2019) 9:1320 \| https://doi.org/10.1038/s41598-018-36768-4 8 www.nature.com/scientificreports/ Figure 6. Saturation curves showing numbers of detected MEGs and PEGs from various datasets. The curves were generated by randomly sampling increasing proportions of each dataset and identifying imprinted candidate genes using the same pipeline. Values are means (and standard errors) of 10 random subsamples. Figure 6. Saturation curves showing numbers of detected MEGs and PEGs from various datasets. The curves were generated by randomly sampling increasing proportions of each dataset and identifying imprinted candidate genes using the same pipeline. Values are means (and standard errors) of 10 random subsamples. procedure. We show that sequencing depth is far from being saturated, as shown by random subsampling of each dataset and detecting MEGs and PEGs by our edgeR-based pipeline (Fig. 6). For most datasets, the number of detected MEGs and PEGs do flatten to some extent with increasing sampling proportions. The low numbers of detectable genes in the Zhang 11 and Zhang 14 are likely due to low mapping rates and low sequencing depth. The relatively flat slopes for the total number of callable genes indicate that a huge increase in sequencing depth would be required to assess the remaining 4–10% genes that were expressed, but did not reach the minimal read coverage of 10 reads. www.nature.com/scientificreports/ In our simulation setting, edgeR’s sensitivity was highest at a minimal cut-off of 10 or 20 reads with TPRs of 0.66 and 0.65, respectively, but dropped markedly at a cut-off of 50 reads with a TPR of 0.54 (Supplementary Table S7). ( pp y ) Importantly, our simulation did not aim to model biological variability predicting biological replicates. All biological systems have inherent biological variation and edgeR can account for it, whereas Fisher’s exact test completely ignores the within-condition variability, as it requires counts from replicates to be summed up for each condition. Therefore, we expect that edgeR would outperform methods using Fisher’s exact tests even more strikingly when biological replicates are available. Higher Coverage, Replicate Samples, and edgeR-based Analysis Could Improve the Identification of Imprinted Genes. The first generation of genome-wide studies identified many new imprinted genes in plants, yet a considerable proportion of genes could not be characterized due to low sequencing depth or insuf- ficient genetic heterogeneity between the parents. Future experiments in genomic imprinting will be performed using paired-end reads with high sequencing coverage. With the rapid development in NGS, higher coverage is now readily achievable, which will notably increase the statistical power to detect imprinted genes. In the studies reanalyzed here, low counts were observed for many genes close to the minimal coverage cut-off of 10, where the variance is large and a large allelic bias is required to reach significance. Importantly, a sufficient number of bio- logical replicates (i.e., at least three per reciprocal cross51) would permit to reliably estimate the variability from the data, enabling the performance of a more robust differential expression analysis and a more reliable estima- tion of the total number of imprinted genes. Biological Perspectives. When comparing datasets from different studies, we assume that the same set of genes is imprinted over the sampling time period. Partial or complete violations of the assumption also decrease the amount of overlap across datasets in addition to technical and analytic biases. Indeed, first studies showed dynamic expression of imprinted genes in maize36. Furthermore, we cannot solely rely on statistical significance in calling a gene imprinted or not. There are several ways to prioritize a gene list after statistically calling genes with an allelic bias in a given tissue. www.nature.com/scientificreports/ expression analysis of RNA-seq data. By interpreting allelic counts of a cross as separate samples and imprinting analysis as a differential gene expression problem, we benefit from the power and flexibility of generalized linear models based on edgeR. The method is highly flexible, i.e. also batch effects can be modeled. The general meth- odology used here is also applicable to imprinting analysis in other tissues, where a deviation from 1:1 is tested. A further advantage of our statistical approach is that it circumvents somewhat arbitrary minimum cut-offs of effect size (allelic imbalance), which would require an individual optimization for each dataset. Most genes are only partially imprinted and we believe that the current knowledge does not support the exclusion of genes with moderate allelic imbalance yet reaching statistical significance. Our approach also allows (nearly) genome-wide ranking of genes according to their likelihood of being imprinted, allowing downstream applications, such as gene set enrichment analysis, with increased statistical power (e.g. for Gene Ontology analysis).i g y p g gy y Prior to read counting, many steps are required to map, filter, and count allelic reads. To assess the importance of the preprocessing/counting relative to statistics, we reanalyzed the original allelic counts from28 using our method. We found a large discrepancy for MEGs and PEGs with original gene lists, whereas MEGs and PEGs largely agreed with the gene lists obtained from complete reanalysis with our analysis pipeline (data not shown). Given the large discrepancy, the statistical approach seems to have a larger relative contribution than the earlier steps required to obtain the variant counts. Simulation Reveals that edgR-based Analysis Outperforms Other Methods. The edgeR-based approach performed well in our simulation setting and clearly outperformed the other methods we tested. Notably, the other methods based on Fisher’s exact test seem overly conservative and missed most spike-in imprinted genes. edgeR was the only method to predict false positives due to a relatively poor FDR control for the lowest quartile of counts (10–23 counts per gene) where 4 of edgeR’s totally 8 false positive imprinted genes were identified. edgeR’s specificity could be further increased by selecting a larger minimal number of counts per gene (e.g., a cut-off of 20 allelic counts per gene). Scientific Reports \| (2019) 9:1320 \| https://doi.org/10.1038/s41598-018-36768-4 Discussion R l i f Furthermore, the co-identification of imprinted genes is additionally hampered by unequal read coverage: 48 of 129 imprinted genes proposed by Zhang and colleagues33 had too few reads to characterize imprinted expression in the data of Waters and colleagues31. The relative importance of biological versus technical biases on the large discrepancy between the datasets from maize is currently difficult to assess. A Statistical Approach that Uses Generalized Linear Models and Takes Replicate Information into Account Largely Increases Sensitivity. Our statistical approach involves modeling of count data and is quite different from the approach chosen by the authors in the original publications. Our approach relies on gen- eralized linear models and edgeR, which is a commonly used, well-established and robust method for differential Scientific Reports \| (2019) 9:1320 \| https://doi.org/10.1038/s41598-018-36768-4 9 www.nature.com/scientificreports/ Methods Read Mapping and Counting. FASTQ-formatted raw reads were downloaded from the NCBI Short Read Archive (SRA) for endosperm experiments for Arabidopsis (GSM674847, GSM674848, GSM756822, GSM756824, GSM607727, GSM607728, GSM607732, GSM607735, GSM1276498, GSM1276500, GSM1276502, GSM1276504, GSM1276505, GSM1276508, GSM1276509, GSM1276512, GSM1276514, GSM1276515) and for maize (SRX105679, SRX105678, SRX114629, SRX114630, SRX047539, SRX047544, SRP031872, GSE48425). Hsieh samples obtained through laser capture microdissection were not included in the analysis as their inclusion reduced the overlap with other datasets (data not shown). Reads were quality-checked with the FastQC applica- tion (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). To reduce the bias in mapping reads towards reference alleles, we aligned the reads to a masked reference genome, in which bases at known polymorphic sites were replaced with “N”. Reads were mapped to the genome using STAR v2.3.0e57. Only reads that mapped to a unique position in the genome were considered for further analysis. q p g y For Arabidopsis the genome annotation TAIR10 (http://www.arabidopsis.org/) was used, and for maize AGPv3 annotated genes were downloaded from Ensembl Plants Release 31 (http://plants.ensembl.org/)58. Arabidopsis SNP annotation files were obtained from the 1001 Genomes project59 http://1001genomes.org/data/ MPI/MPISchneeberger2011/releases/2012_03_14/). Maize hapmap v3.2.1 SNP variants60 in VCF format (Release date 3/3/2016) were obtained from the Panzea database (http://cbsusrv04.tc.cornell.edu/users/panzea/download. aspx?filegroupid=15) and filtered for high-confidence (flag LLD) biallelic SNP variants, which were polymorphic between B73 and Mo17 and homozygous in both inbred strains. Allelic reads were counted at previously identi- fied SNP positions between homozygous parentals, using Python v3.2 with pysam v0.8.461, while positions with low sequencing quality (phred quality <20) were excluded. No more than one SNP was counted per read to pre- vent pseudo-replication. Counts were summed up per gene and variants with less than 10 reads (summed across the two reciprocal crosses) were discarded. Testing for Allele-specific Expression. To assess allele-specific expression, we used edgeR version 3.4.245. It uses an empirical Bayes estimation based on the negative binomial distribution. For library size normaliza- tion and to eliminate composition biases between libraries, we used the TMM (Trimmed Mean of M-values) method. TMM normalization keeps the ratio between maternal and paternal allelic reads in a cross at approx. 2 (data not shown). Experiments were analyzed using a generalized linear model with a paired design (the two allelic counts of the same cross treated as paired samples) and at least two biological replicates (the two reciprocal crosses). When biological replicates were available, they were included in the model. We used tagwise dispersion estimates. www.nature.com/scientificreports/ First, filtering the gene lists by fold difference between the two parental alleles could help to identify genes that can more easily be validated experimentally. Second, filtering the gene lists against tissue-specific expression data from seeds might identify genes relevant to the tissue of interest52,53. However, expression of a given gene in other (e.g. maternal seed coat) tissues does not exclude an allelic bias in the fertilization products50. Third, comparing the gene list against central cell and sperm cell expression data54–56 can inform to what extent the allelic bias is a result of expression in the fertilization products or might at least partially represent carry-over of gametic transcripts that were produced prior to fertilization.f y y g An improved assessment including larger gene sets (by using different sets of strains), as well as imprinted genes with moderate allelic imbalance, will provide further insights into the extent and biological significance of genomic imprinting. Having the full catalog of imprinted genes in several plant species will also allow the tack- ling of evolutionary questions about genomic imprinting, including its origin and fixation, the conservation of imprinted genes, and their gain and loss of imprinting status on the phylogenetic tree.i p g g p g p y g Lastly, we have to stress that without further confirmation, gene lists are not representative with regard to the absolute number of imprinted candidate genes expressed in the endosperm. Considering this, it is indispensable to confirm bioinformatically identified imprinted candidate genes by alternative methods, such as allele-specific expression analysis using RT-PCR and Sanger sequencing, pyrosequencing, and/or reporter gene assays. Scientific Reports \| (2019) 9:1320 \| https://doi.org/10.1038/s41598-018-36768-4 10 www.nature.com/scientificreports/ Methods False Discovery Rate (FDR) was calculated according to Benjamini and Hochberg62. The R software version 3.0.363 was used for statistical analysis and for creating the graphs. Simulation of genomic imprinting. To generate synthetic data we used the function makeExampleDESe- qDataSet of the DESeq2 version 1.2.1046 R/Bioconductor package. Mean and dispersion parameters that were used in the simulation were estimated from real RNA-seq data (interceptMean = 2, interceptSD = 3). Two biolog- ical replicates were simulated to serve as the two reciprocal crosses. No outlier counts and differential expression were introduced. The total number of genes in each simulated dataset was 15,000, and their true proportion of maternal reads was set to 2/3. Then we randomly picked 200 genes and modified their imprinting status, 50 each of strong MEGs (99% maternal reads), weak MEGs (85% maternal reads), strong PEGs (34% maternal reads) and weak PEGs (48% maternal reads). Random allelic read sampling was modeled by sampling from a binomial distribution with the probability of success set to the true proportion of maternal reads.i p y p p We evaluated three methods for identifying genes with parent-of-origin-specific expression: edgeR, Fisher’s exact test, and Stouffer’s method. Fisher’s exact test does two separate statistical tests for the two reciprocal crosses, and the resulting two p-values per gene were combined with Stouffer’s method (calculated by sumz function of R package metap). Benchmarking was performed using Venn diagrams and Receiver Operating Characteristic (ROC) curves with iCOBRA64 https://github.com/markrobinsonuzh/iCOBRA. Comparison with Published Gene Lists. Published gene lists were compiled from the Supplementary Data of the respective publications. For the Waters B73xMo17 comparison, the updated list from35 was used, not the original one31. If lists of imprinted genes were available at various stringencies, we used the least stringent list (e.g. moderately imprinted genes for the Waters dataset). Genes with an accession/inbred-specific bias in expres- sion were omitted. The Zhang14 dataset comprised only the lists of 106 MEGs and 91 PEGs at 12DAP (Prof. Xiaomei Lai, personal communication) described in37 but not the endosperm samples at 10DAP which were already described in33 and contained in the Zhang11 dataset. Genes that could not be evaluated for imprinting in all datasets were filtered out. Saturation Plots. In order to assess the degree of undersampling, we performed random subsampling on the count data and performed the same processing and statistical analysis steps as for the full data. Conclusionsf A new, effective data analysis pipeline is reported that allows for an improved analysis of RNA-seq data from reciprocal F1 crosses. The pipeline allows for the modelling of biological replicates and is applicable to any diploid or triploid tissue. Furthermore, our genome-wide ranking of Arabidopsis and maize imprinted candidate genes, which integrates all available datasets, provides a useful resource to inform future experiments focused on under- standing genomic imprinting in plants. Data Availability d d d y Scripts and data used in this manuscript are available on github (http://www.github.com/swyder/Reanaly plant_imprinting) and as supplementary files. y Scripts and data used in this manuscript are available on github (http://www.github.com/swyder/Reanalysis_ plant_imprinting) and as supplementary files. Methods Scientific Reports \| (2019) 9:1320 \| https://doi.org/10.1038/s41598-018-36768-4 11 www.nature.com/scientificreports/ References 26. Monk, D. Genomic imprinting in the human placenta. Am. J. Obstet. Gynecol. 213, S152–162 (2015). 7. Autran, D. et al. Maternal epigenetic pathways control parental contributions to Arabidopsis early embryogenesis. Cell 145, 707–719 (2011).f 28. Gehring, M., Missirian, V. & Henikoff, S. Genomic analysis of parent-of-origin allelic expression in Arabidopsis thaliana seeds. One 6, e23687 (2011). 29. Hsieh, T.-F. et al. Inaugural Article: Regulation of imprinted gene expression in Arabidopsis endosperm. Proc Natl Acad Sci USA 1755–1762 (2011).ii 30. McKeown, P. C. et al. Identification of imprinted genes subject to parent-of-origin specific expression in Arabidopsis thaliana seeds. BMC Plant Biol. 11, 113, https://doi.org/10.1186/1471-2229-11-113 (2011).f i t Biol. 11, 113, https://doi.org/10.1186/1471-2229-11-113 (2011).f 31. Waters, A. J. et al. Parent-of-origin effects on gene expression and DNA methylation in the maize endosperm. Plant Ce 4221–4233 (2011).f ( ) 32. Wolff, P. et al. High-resolution analysis of parent-of-origin allelic expression in the Arabidopsis endosperm. PLoS Genet. 7, e1002126, https://doi.org/10.1371/journal.pgen.1002126 (2011). p g j pg ( ) 33. Zhang, M. et al. Extensive, clustered parental imprinting of protein-coding and noncoding RNAs in developing maize endosperm. Proc Natl Acad Sci USA 108, 20042–20047 (2011). . Pignatta, D. & Gehring, M. Imprinting meets genomics: new insig 34. Pignatta, D. & Gehring, M. Imprinting meets genomics: new insights and new challenges. Curr Opin Plant Biol. 15, 530–535 (2012). 35. Waters, A. J. et al. Comprehensive analysis of imprinted genes in maize reveals allelic variation for imprinting and limited conservation with other species. Proc Natl Acad Sci USA 110, 19639–19644 (2013). 36. Xin, M. et al. Dynamic expression of imprinted genes associates with maternally controlled nutrient allocation during m endosperm development. Plant Cell 25, 3212–3227 (2013).i 37. Zhang, M. et al. Genome-wide high resolution parental-specific DNA and histone methylation maps uncover patterns of imprinting regulation in maize. Genome Res. 24, 167–176 (2014).i g 38. Pignatta, D. et al. Natural epigenetic polymorphisms lead to intraspecific variation in Arabidopsis gene imprinting. eLife 3, e03198, https://doi.org/10.7554/eLife.03198 (2014).f p g ( ) 9. Köhler, C., Wolff, P. & Spillane, C. Epigenetic mechanisms underlying genomic imprinting in plants. Annu Rev Plant Biol. 63 331–352 (2012). 40. DeVeale, B., van der Kooy, D. & Babak, T. Critical evaluation of imprinted gene expression by RNA-Seq: a new perspective. PLoS Genet. 8, e1002600 (2012). l k d f d d ( ) 41. Wang, X. & Clark, A. G. References References 1. Reik, W. & Walter, J. Genomic imprinting: parental influence on the genome. Nat Rev Genet. 2, 21–32 (2001).f l 2. Grossniklaus, U. Genomic Imprinting in Plants: A Predominantly Maternal Affair in Plant Epigenetics (ed. Meyer, P.) 174–200 (Blackwell Publishing Ltd, 2007). 2. Grossniklaus, U. Genomic Imprinting in Plants: A Predominantly Maternal Affair in Plant Epigenetics (ed. Meyer, P.) 174–200 (Blackwell Publishing Ltd, 2007). 3. Bartolomei, M. S. & Ferguson-Smith, A. C. Mammalian genomic imprinting. Cold Spring Harb Perspect Biol. 3, https://doi.org/10.1101 cshperspect.a002592 (2011). p p 4. Ferguson-Smith, A. C. Genomic imprinting: the emergence of an epigenetic paradigm. Nat Rev Genet. 12, 565–575 (2011). nomic imprinting: a mammalian epigenetic discovery model. Annu p g p g y 6. Raissig, M. T., Baroux, C. & Grossniklaus, U. Regulation and flexibility of genomic imprinting during seed development. Plant Cel 23, 16–26 (2011). 7. Jiang, H. & Köhler, C. Evolution, function, and regulation of genomic imprinting in plant seed development. J Exp Bot. 63 4713–4722 (2012). 8. Gehring, M. Genomic imprinting: insights from plants. Annu Rev Genet. 47, 187–208 (2013).f Grossniklaus, U. Genomic imprinting in plants. Results Probl Cell D essing, J. & Grossniklaus, U. Genomic imprinting in plants. Results P g g ff 10. Baroux, C., Spillane, C. & Grossniklaus, U. Genomic imprinting during seed development. Adv Genet. 46, 165–214 (2002). 11. Feil, R. & Berger, F. Convergent evolution of genomic imprinting in plants and mammals. Trends Genet. 23, 192–199 (2007). 1. Feil, R. & Berger, F. Convergent evolution of genomic imprinting in plants and mammals. Trends Genet. 23, 192–199 (2007). 2 Köhler C & Weinhofer Molisch I Mechanisms and evolution of genomic imprinting in plants Heredity 105 57 63 (2010) 11. Feil, R. & Berger, F. Convergent evolution of genomic imprinting in plants and mammals. Trends Genet. 23, 192–199 (2007) 12. Köhler, C. & Weinhofer-Molisch, I. Mechanisms and evolution of genomic imprinting in plants. Heredity 105, 57–63 (2010) 13. Gutierrez-Marcos, J. F., Constância, M. & Burton, G. J. Maternal to offspring resource allocation in plants and mammals. Pla 33(Suppl. 2), e3–10 (2012).fl 4. Pires, N. D. & Grossniklaus, U. Different yet similar: evolution of imprinting in flowering plants and mammals. F1000prime Rep. 6 63, https://doi.org/10.12703/P6-63 (2014). g 15. Rodrigues, J. A. & Zilberman, D. Evolution and function of g 15. Rodrigues, J. A. & Zilberman, D. Evolution and function of genomic imprinting in plants. Genes Dev. 29, 2517–2531 (2 16. Scientific Reports \| (2019) 9:1320 \| https://doi.org/10.1038/s41598-018-36768-4 References Haig, D. & Westoby, M. Parent-specific gene expression and the triploid endosperm. Am. Nat. 134, 147–155 (1989). g y pi g p p p 17. Moore, T. & Haig, D. Genomic imprinting in mammalian development: a parental tug-of-war. Trends Genet. 7, 45–49 (1991). i 17. Moore, T. & Haig, D. Genomic imprinting in mammalian deve 8. Lau, M. M. et al. Loss of the imprinted IGF2/cation-independent mannose 6-phosphate receptor results in fetal overgrowth and perinatal lethality. Genes Dev. 8, 2953–2963 (1994). perinatal lethality. Genes Dev. 8, 2953–2963 (1994 19. Ludwig, T. et al. Mouse mutants lacking the type 2 IGF receptor (IGF2R) are rescued from perinatal lethality in Igf2 and Igf1r null backgrounds. Dev Biol. 177, 517–535 (1996). g 0. Grossniklaus, U., Vielle-Calzada, J. P., Hoeppner, M. A. & Gagliano, W. B. Maternal control of embryogenesis by MEDEA, a Polycomb group gene in Arabidopsis. Science 280, 446–450 (1998). group gene in Arabidopsis. Science 280, 446–450 (1998 g p g p 1. Kinoshita, T., Yadegari, R., Harada, J. J., Goldberg, R. B. & Fischer, R. L. Imprinting of the MEDEA Polycomb gene in the Arabidopsi endosperm. Plant Cell 11, 1945–1952 (1999). 22. Kiyosue, T. et al. Control of fertilization-independent endosperm development by the MEDEA Polycomb gene in Arabidopsis. Proc Natl Acad Sci USA 96, 4186–4191 (1999).f 3. Luo, M., Bilodeau, P., Dennis, E. S., Peacock, W. J. & Chaudhury, A. Expression and parent-of-origin effects for FIS2, MEA, and FIE in the endosperm and embryo of developing Arabidopsis seeds. Proc Natl Acad Sci USA 97, 10637–10642 (2000).ff 24. Ingouff, M., Haseloff, J. & Berger, F. Polycomb group genes control developmental timing of endosperm. Plant J. 42, 663–674 (2005). 25 T k B & M i I M Ph i l i l f ti f i i t d J C ll Ph i l 192 245 258 (2002) ., Haseloff, J. & Berger, F. Polycomb group genes control developmental timing of endosperm. Plant J. 42, 663–674 (2005). & Morison, I. M. Physiological functions of imprinted genes. J Cell Physiol. 192, 245–258 (2002). 4. Ingouff, M., Haseloff, J. & Berger, F. Polycomb group genes control developmental timing of endosperm. Plant J. 42, 663 674 (20 5. Tycko, B. & Morison, I. M. Physiological functions of imprinted genes. J Cell Physiol. 192, 245–258 (2002). ff 25. Tycko, B. & Morison, I. M. Physiological functions of imprinted genes. J Cell Physiol. 192, 245–258 (2002). www.nature.com/scientificreports/ k k l f h h d h l h b 0. Bukowski, R. et al. Construction of the third generation Zea mays haplotype map. Preprint at, https://www.biorxiv.org/content early/2015/09/16/026963 (2015). 1. Li, H. A statistical framework for SNP calling, mutation discovery, association mapping and population genetical parameter estimation from sequencing data. Bioinformatics 27, 2987–2993 (2011). q g f 2. Benjamini, Y. & Hochberg, Y. Controlling the false discovery rate: A practical and powerful approach to multiple testing. J. R. Stat Soc. Ser. B Methodol. 57, 289–300 (1995). 63. R Core Team. R: A Language and Environment for Statistical Computing, http://www.r-project.org (2012). 4. Soneson, C. & Robinson, M. D. ICOBRA: open, reproducible, standardized and live method benchmarking. Nat. Methods. 13, 283 https://doi.org/10.1038/nmeth.3805 (2016). Acknowledgements g We thank Malgorzata Nowicka and Mark Robinson (Department of Molecular Life Sciences, University of Zurich), as well as Marc W. Schmid (Department of Plant and Microbial Biology, University of Zurich) for helpful discussions. This work was supported by the University Research Priority Program ‘Evolution in Action’ of the University of Zurich and, in part, by an Advanced Grant of the European Research Council (ERC AdG Nr. 243996) to U.G. Author Contributions .W., M.T.R., and U.G. designed the study, U.G. raised money and supervised the study, S.W. performed the nalyses, and all authors wrote the paper. All authors read and approved the final manuscript. www.nature.com/scientificreports/ 8. Fontanillas, P. et al. Key considerations for measuring allelic expression on a genomic scale using high-throughput sequencing. Mol Ecol. 19(Suppl. 1), 212–227 (2010).fi 48. Fontanillas, P. et al. Key considerations for measuring allelic expression on a genomic scale using high-throughput sequencing. Mol. Ecol. 19(Suppl. 1), 212–227 (2010). 49. Degner, J. F. et al. Effect of read-mapping biases on detecting allele-specific expression from RNA-sequencing data. Bioinformatics Ecol. 19(Suppl. 1), 212–227 (2010). 49. Degner, J. F. et al. Effect of read-mapping biases on detecting allele-specific expression from RNA-sequencing data. Bioinformatics 25 3207 3212 (2009) pp 9. Degner, J. F. et al. Effect of read-mapping biases on detecting allele-specific expression from RNA-sequencing data. Bioinformatic 25, 3207–3212 (2009). , ( ) 50. Raissig, M. T., Bemer, M., Baroux, C. & Grossniklaus, U. Genomic imprinting in the Arabidopsis embryo is partly regulated by PRC2. PLoS Genet. 9, e1003862 (2013).f ( ) 1. Liu, Y., Zhou, J. & White, K. P. RNA-seq differential expression studies: more sequence or more replication? Bioinformatics 30 301–304 (2014).i 52. Belmonte, M. F. et al. Comprehensive developmental profiles of gene activity in regions and subregions of the Arabidopsis seed. Proc. Natl. Acad. Sci. USA 110, E435–444 (2013). 53. Schon, M. A. & Nodine, M. D. Widespread Contamination of Arabidopsis Embryo and Endosperm Transcriptome Data Sets. Cell 29, 608–617 (2017). , ( ) 54. Borges, F. et al. Comparative transcriptomics of Arabidopsis sperm cells. Plant Physiol. 148, 1168–1181 (2008). . Borges, F. et al. Comparative transcriptomics of Arabidopsis sperm g p y 55. Wuest, S. E. et al. Arabidopsis female gametophyte gene expression map reveals similarities between plant and animal gametes. Curr Biol. 20, 506–512 (2010).ii 6. Schmid, M. W. et al. A powerful method for transcriptional profiling of specific cell types in eukaryotes: laser-assisted microdissection and RNA sequencing. PLoS One 7, e29685 (2012). q g , ( ) 57. Dobin, A. et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29, 15–21 (2013). q g bin, A. et al. STAR: ultrafast universal RNA-seq aligner. Bioinforma q g f 8. Monaco, M. K. et al. Gramene 2013: comparative plant genomics resources. Nucleic Acids Res. 42, D1193–1199 (2014). q g f 58. Monaco, M. K. et al. Gramene 2013: comparative plant genomics resources. Nucleic Acids Res. 59. Cao, J. et al. Whole-genome sequencing of multiple Arabidopsis thaliana populations. Nat. Genet. 43, 956–963 (2011). References Using next-generation RNA sequencing to identify imprinted genes. Heredity 113, 156–166 (2014). l h h d k l l d b f d 42. Castel, S. E., Levy-Moonshine, A., Mohammadi, P., Banks, E. & Lappalainen, T. Tools and best practices for data processing in allelic expression analysis. Genome Biol. 16, 195 (2015). 43 G C t l Hi h l ti l i f t f i i ll li i i th b i S i 329 643 648 (2010) y 43. Gregg, C. et al. High-resolution analysis of parent-of-origin allelic expression in the mouse brain. Science 329, 643–648 (2010) 44. Zhou, Y.-H., Xia, K. & Wright, F. A. A powerful and flexible approach to the analysis of RNA sequence count data. Bioinformatics 27, 2672–2678 (2011).f 45. Robinson, M. D., McCarthy, D. J. & Smyth, G. K. edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 26, 139–140 (2010).f p f ( ) 6. Anders, S. & Huber, W. Differential expression analysis for sequence count data. Genome Biol. 11, R106 (2010). 7. Stouffer, S. A. The American Soldier: Adjustment During Army Life. (Princeton University Press, 1949). Scientific Reports \| (2019) 9:1320 \| https://doi.org/10.1038/s41598-018-36768-4 12 www.nature.com/scientificreports/ Additional Information Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-018-36768-4.h Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-018-36768-4. Competing Interests: The authors declare no competing interests. Competing Interests: The authors declare no competing interests. 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https://openalex.org/W2792399088	http://ijarcs.info/index.php/Ijarcs/article/download/5328/4513	English	null	DISTRIBUTED AGENT BASED TECHNIQUE FOR DETECTING DISTRIBUTED DENIAL-OF-SERVICE (DDoS) ATTACKS IN WLAN	International journal of advanced research in computer science	2,018	cc-by	4,967	1. INTRODUCTION network traffic during the wormhole. [14, 17, 20]. In this paper an attempt has been made to propose a fully distributed approach for early warning, when pre-attack activities are detected, using trust mechanisms. The remaining paper is organized in the different sections. Section II discuss the research work done in the field. Section III presents the Distributed Agent Based detection Technique for DDoS. Section IV presents the results and discussion. In last Section -V conclusions are presented. 1.2 PREVIOUS WORK In the last few years a large number of studies have been done by various researchers. In the last few years a large number of studies have been done by various researchers. In the previous work, a MAC layer based defense architecture for the Reduction of Quality (RoQ) attacks in Wireless LAN was designed for the detection and prevention of intruders in WLAN [1]. In the second work, a cross-layer based intrusion detection technique for wireless local area networks was designed. In this technique a combined weight value was computed from the RSS and TT. If the weight value is greater than a threshold value, then the corresponding node is considered as an attacker. Significant results were obtained using proposed techniques [2]. A misbehaving Wireless Access Point (WAP) is introduced into the wireless networks 802.11which attacks and collects the sensitive data from the WLAN. Even the users introduce the misbehaving nodes. WLANs can be improved well on combining the advantages of the less expense and trouble- free installation along with the mobility. A backdoor can be created into the network by implementing a WAP on an identified WLAN [1, 18]. As continuation in the previous works, detection technique for the wormhole attacks are proposed in this paper. Many location-based wireless defense techniques & routing protocols are affected by the wormhole attack. The existing wireless network routing protocols are not able to find the routes lengthy than two or more hops if there is no mechanism for defending wormhole attacks which disrupts communication severely. [11, 15, 19]. Keywords: DDoS, WLAN, network traffic during the wormhole. [14, 17, 20]. 1.1 THREATS TO WIRELESS LAN Wireless Local Area Networks (WLANs) have many possible disturbing threats. The security issues which affect the WLAN are the various attacks including Denial of Service (DoS) and mis-configured wireless access point (WAP). In addition to the number of attacks on wired networks, a there are few specific attacks vulnerabilities in the wide array of 802.11 affect the wireless networks. For protecting and detecting these possible threats, an intrusion detection and prevention system must be used by the wireless networks. Intrusion detection system solution is also important for the organizations which do not have a WLAN due to the dangerous wireless threats [1, 18, 22, 24, 25, 26]. Volume 9, No. 1, January-February 2018 International Journal of Advanced Research in Computer Science RESEARCH PAPER Available Online at www.ijarcs.info ISSN No. 0976-5697 DISTRIBUTED AGENT BASED TECHNIQUE FOR DETECTING DISTRIBUTED DENIAL-OF-SERVICE (DDoS) ATTACKS IN WLAN International Journal of Advanced Research in Computer Science RESEARCH PAPER A il bl O li t ij i f © 2015-19, IJARCS All Rights Reserved DISTRIBUTED AGENT BASED TECHNIQUE FOR DETECTING DISTRIBUTED DENIAL-OF-SERVICE (DDoS) ATTACKS IN WLAN Er. Harmeet Singh Research Scholar Department of Computer Science & Engineering, SBBS University, Jalandhar, India Dr.Vijay Dhir Professor Department of Computer Science & Engineering, SBBS University, Jalandhar, India Abstract: By sending large amount of data flows from multiple sites, Distributed Denial-of-Service (DDoS) attacks target the victims. Thus, there is a demand to implement number of DDoS defense techniques all together and collaboratively on many nodes, especially on where there is a need to maintain round-the-clock Internet connectivity. The security mechanism works on a probabilistic basis that is based on the detection of illegitimate traffic and then to discard it, that forced a specific number of legitimate packets to be fallout in the process and reducing the overall quality of service. In this paper a Distributed Agent Based technique for detecting DDoS Attacks in wireless LAN has been proposed. It is fully distributed and provides an early warning when pre-attack activities are detected, using trust mechanisms. From the simulation results it has been found that the proposed distributed agent based architecture achieves high throughput with low packet drop, by detecting and isolating the attack traffic flows. Keywords: DDoS, WLAN, DOI: http://dx.doi.org/10.26483/ijarcs.v9i1.5328 DOI: http://dx.doi.org/10.26483/ijarcs.v9i1.5328 ISSN No. 0976-5697 Volume 9, No. 1, January-February 2018 1.3 DDOS (DISTRIBUTED DENIAL OF SERVICE) ATTACKS In this paper an Intelligent Agent Based Defense (IABD) Architecture for DDos Attacks was proposed, which is fully distributed and provides an early warning when pre-attack activities are detected, using trust mechanisms. Choi et al. [10] developed a secure and effective mechanism without using special support of hardware called Wormhole Attack Prevention (WAP). The mechanism provide detection as well as prevention in wireless networks. They proposed an effective mechanism based on dynamic source routing protocol. The major benefit of this approach is that it can work without time synchronization of the location information. Their future work is to study false negative and false positive rate problems for the detection of wormholes. Znaidi et al. [4] have proposed a mechanism for securing, defending and detecting wormhole attack in wireless networks. This mechanism considering local and neighborhood information and not necessitating the other parameters such as location information, clock synchronization or dedicated hardware. Furthermore, the algorithm is also not dependent on the wireless communication models. The limitation of this mechanism is that, a wormhole link could be detected only if the actual distance between the any two wormhole nodes is greater than four-hop because of the computed coefficients. Phuong et al [12] have presented a transmission time based approach for the detection of wormhole attacks in wireless networks. The proposed approach is used for the detection of wormhole by computing the transmission time (TT) between established path of two successive nodes. Attack is identified by transmission time between two fake nodes created by wormhole and this transmission time is significantly higher between the two real nodes that are within the range. Xu et al. [5] have proposed a distributed wormhole detection algorithm (DWDA) in the basis of network disorder detection initiated by the presence of a wormhole. In this algorithm a hop-counting approach was used as an investigation procedure for the wormhole attack detection and then rebuilds local maps in all node. Later, it uses a “diameter” feature to detect irregularities triggered by wormholes. The key benefit of using DWDA approach is that it can provide the approximate location of a wormhole that helps the researcher for designing further defense approaches. Sriram et al. [13] have proposed a classification for the various possible wormhole attack using an infrastructure based wireless networks. They had proposed a detection approach by using Neighbor Discovery and Link Verification parameters. 1.3 DDOS (DISTRIBUTED DENIAL OF SERVICE) ATTACKS The main advantage of this algorithm is that it works without the time stamp of the packet for detecting a wormhole attack and does not need any geographical information about the nodes. This is an imperative parameter for the resource confined behavior of the sensor nodes. In future work, some modifications can be introduced to our routing protocol in order to get a balanced tree where the load would be fairly distributed among the nodes since this will considerably help in reducing the value of Trefresh. Kaissi et al. [9] have presented a new protocol called DAWWSEN having a defence and detection approach against the wormhole attack, a powerful attack that has serious consequences on sensor routing protocols. The main advantage of this algorithm is that it works without the time stamp of the packet for detecting a wormhole attack and does not need any geographical information about the nodes. Kaissi et al. [9] have presented a new protocol called DAWWSEN having a defence and detection approach against the wormhole attack, a powerful attack that has serious consequences on sensor routing protocols. The main advantage of this algorithm is that it works without the time stamp of the packet for detecting a wormhole attack and does not need any geographical information about the nodes. This is an imperative parameter for the resource confined behavior of the sensor nodes. In future work, some modifications can be introduced to our routing protocol in order to get a balanced tree where the load would be fairly distributed among the nodes since this will considerably help in reducing the value of Trefresh. There is need to implement number of approaches simultaneously and collaboratively on the wireless networks to protect various nodes from the attackers. There are number of approaches introduces by adding digital signatures and encryption features, but these are relative complex, more overhead, and more delay to the network,. The prevention and detection mechanism is need to address for providing the quality of services. This is an imperative parameter for the resource confined behavior of the sensor nodes. In future work, some modifications can be introduced to our routing protocol in order to get a balanced tree where the load would be fairly distributed among the nodes since this will considerably help in reducing the value of Trefresh. © 2015-19, IJARCS All Rights Reserved 1.3 DDOS (DISTRIBUTED DENIAL OF SERVICE) ATTACKS DDoS attacks commonly overthrow the channel by sending enormous packets from various attack sites. As a result the channel wastes its important resources. During extensive attacks, DDoS traffic also generates a intense congestion in the Internet which disturbs the normal communication between all Internet users [1]. Wormhole helps in the networking services as it offers a lengthy network link to the link layer and up which is useful for the attacker to use that link. The flow of data is disrupted by modifying data packets, creating unwanted routing activities, selectively dropping and turning off the wormhole link periodically, when the attackers utilize a high volume of There is only one approach to fully eradicate the DDoS attack is by securing the various nodes on the Internet 375 © 2015-19, IJARCS All Rights Reserved Harmeet Singh et al, International Journal of Advanced Research in Computer Science, 9 (1), Jan-Feb 2018,375-380 following types of leashes (i) geographic leashes and (ii) temporal leashes. Finally, an efficient authentication protocol was designed, called TIK by using temporal leashes. against misuse that is impractical. Generally major spots currently handle the issues with critical approaches. This will creates the barrier for the attacker. The prevention and detection of DDoS is remain open challenges and there is need to develop defenses approach for the detection of attacks and responsive system by falling excess traffic. Ronghui et al. [8] have introduced a simple and effective approach locate and detect wormhole attack. This approach was based on the idea of location discovery in wireless networks using hop counting method. This algorithm also provides an estimation of location of wormhole in wireless networks. Ronghui et al. [8] have introduced a simple and effective approach locate and detect wormhole attack. This approach was based on the idea of location discovery in wireless networks using hop counting method. This algorithm also provides an estimation of location of wormhole in wireless networks. DDoS attacks are triggers by the victim, source and the intermediate networks means the three major components of network. From the destination, it is very easy to detect an attack at the destination as compared to the source because destination generate high volume of traffic that source [2]. Kaissi et al. [9] have presented a new protocol called DAWWSEN having a defence and detection approach against the wormhole attack, a powerful attack that has serious consequences on sensor routing protocols. FOR INCOMING TRAFFIC: Let NPtk be the amount of received packets by an agent Ai at time tk. We can define the historical approximation of average number of packets as: (1) AVGNPtk = (1 – λ) . AVGNPt ( k 1) + λ . NPtk Where 0<λ<1 is sensitivity factor showing the long-term average performance to the current traffic change. Then the change of the input traffic dvitk from the average by time k is given by Where 0<λ<1 is sensitivity factor showing the long-term average performance to the current traffic change. Then the change of the input traffic dvitk from the average by time k is given by dvitk = dvitk 1 + NPtk - AVGNPtk In DDoS attack, the total deviation is significantly more than the random fluctuations. Since the deviation of input traffic is sensitive to the traffic changes, we calculate the abnormal deviation of input traffic AVGdv from historical average as: (2) dvitk = dvitk 1 + NPtk - AVGNPtk In DDoS attack, the total deviation is significantly more than the random fluctuations. Since the deviation of input traffic is sensitive to the traffic changes, we calculate the abnormal deviation of input traffic AVGdv from historical average as: Figure 1: System Architecture (3) AVGdvtk = dvitk / AVGNPtk (3 If AVGdv > δ, where δ is the detection threshold that represents attack traffic. The value of δ can be fixed based on the previous detection results. If AVGdv < δ, then there is no DDoS attack. AVGdvtk = dvitk / AVGNPtk (3 If AVGdv > δ, where δ is the detection threshold that represents attack traffic. The value of δ can be fixed based on the previous detection results. If AVGdv < δ, then there is no DDoS attack. In our proposed architecture, many autonomous agents tend to protect the internal system network. In any possible attack related scenarios, the agents will sent alert messages for the network administrator. Each agent will execute a security monitoring function at a specified node and monitor the network traffic data against any attacks. Figure 1 presents the sample network topology with four attackers (marked as A1, A2, A3 and A4) and two legitimate clients (marked as C1 and C2). The bottom most node (marked as victim) is the victim node. It also consists of one BS (node 18). The attack related alert messages are send to this BS by the agents. FOR INCOMING TRAFFIC: There is an authenticator placed between the victim and the BS in order to verify the alert messages. p If AVGdv < δ, then there is no DDoS attack. p If AVGdv < δ, then there is no DDoS attack. dviotk = dvotk / dvitk (6) The detection approach is proposed at the agent level for the detection of sudden variations in the flows of traffic. When a DDoS attack is triggered, the agents which are deployed initially, monitor variation in spatiotemporal distribution of volumes of traffic. Typically, these variations in traffic flows are towards the direction of the victim node, whereas random fluctuations occurred due to legitimate traffic flows will not be in the direction of the victim. Based on these changes, these agents exchange traffic surge reports with each other, and collaboratively detect the attacker. Then an alert message is send upstream towards the source node. Through monitoring the dvio value, an agent decides whether deviation is due a DDoS attack. If dvio is close to 1 (one), the traffic aggregation pattern is considered suspicious. The agent then triggers an alert message and also intimate the pattern to the source. Otherwise the agent will sends a repeated message regarding status, indicating that there is no any anomaly observed. 2.1 SYSTEM ARCHITECTURE AND OVERVIEW Figure 1: System Architecture Figure 1: System Architecture Let t1, t2,…, tn be the time intervals. 1.3 DDOS (DISTRIBUTED DENIAL OF SERVICE) ATTACKS These parameters are used to observe traffic in and out of its adjoining access point and uses of first-hop and second-hop neighbors’ data structure. This approach decrease the risk of wormhole attacks and does not need any location information and clock synchronization. Win et al. [6] had addressed the various wormhole attack detection methods for wireless networks. In this paper they addressed approach exercised in the DaW security model which combines a defense and detection approach for wormhole attack by using false positive, false negative and precision of alarm performance parameters. The alarms were compared with LF analysis and found to be more specific. The performance of the approach was measured using ns-2 simulations and found that the proposed routing protocol was performed much better in terms to attain low delay. Recently, many researchers have proposed intrusion detection techniques for wireless sensor networks [3,16, 21- 24]. Hu et al. [7] have presented a challenging attack to defend against a wormhole attack in a wireless networks, and propose a new method for defending and detecting wormhole. In this approach a packet leashes method was proposed to detect wormhole attacks using 376 © 2015-19, IJARCS All Rights Reserved Harmeet Singh et al, International Journal of Advanced Research in Computer Science, 9 (1), Jan-Feb 2018,375-380 FOR OUTGOING TRAFFIC Let NPtk be number of packets leaving at time k. Then the deviation of the output traffic dvotk from the average at time k is given by dvotk = dvotk 1 + NPtk - AVGNPtk (5) Where AVGNPtk is the historical average leaving packets determined similar to (1). 2. AGENT BASED DEFENSE ARCHITECTURE Each agent monitors change in traffic and computes the amount of received packets in a particular time window at each I/O port. All packets of a super flow must be directed towards the same destination network. © 2015-19, IJARCS All Rights Reserved Where AVGNPtk is the historical average leaving packets determined similar to (1). Each agent monitors the traffic in a cost effective manner at a superflow level. This level comprises all packets destined from all possible source IP for the same network domain and having the same prefix IP address of the same destination domain. It also operates different protocols such as UDP or TCP etc that detects the cumulative from individual flows. If AVGdv > δ, then calculate dviotk ,which is defined as the ratio of incoming and outgoing traffic deviations, which is calculated using (2) and (5) as, dviotk = dvotk / dvitk (6) 4. SIMULATION RESULTS Figure 2 shows the bandwidth affected or occupied by the attackers when the attack traffic rate is increased. From the figure it is clearly seen that in the distributed scenario, the amount of attack bandwidth is less when compared to the normal scenario, which indicates that the adverse effect of attackers is reduced in the distributed case. In this section, the performance of the proposed algorithm has been evaluated. In order to test the proposed protocol, the NS2 simulator [15] is used. The proposed Distributed detection techniques has been compared with the normal wormhole attack scenario without applying any detection techniques. From Figure 3, it is clearly shown that the number of attack packets dropped when the traffic rate is increased. From the results it has been found that the distributed approach eliminates most of the attack packets when compared to the normal approach. A. BASED ON RATE compromised agent nodes. If any agent finds that the received report from any other agent does not match with the expected results, it can be considered as a compromised agent node. In the first experiment the traffic rate was varied as 200Kb to 500Kb. Fig 2: Rate Vs Bandwidth Fig 3: Rate Vs Packets Dropped Each agent i maintains a trust index T for all the other agents, which is updated for each report it receives from the other agents. Fig 2: Rate Vs Bandwidth If the agent i, receives a report from the agent k , then , trust index of k is calculated as (i) Tik = (MRk – NRk) / n (1) (1) Where MRk is the number of reports that matches with agent i’s report. NRk is the number of reports that does not match with agent i’s report. n is the number of messages received in the time period tn. ) If agent k’s report is different from agent i’s report, then agent i checks Tik. If Tik < (ii) If agent k’s report is different from agent i’s report, then agent i checks Tik. If Tik < Fig 2: Rate Vs Bandwidth Tth, where Tth is the trust threshold, then agent i broadcast a trust request message TReq to other agents. TReq contains the node id suspected agent node. Fig 3: Rate Vs Packets Dropped (iii) On receiving TReq, the agents send their corresponding trust index value of agent k, as a trust reply message TRep. (iv) After collecting the trust reply messages TRep from other agents, the agent i once again checks If Tjk < Tth , where Tjk is the trust index value of agent k, collected from agents j, j=1,2… (j <> k) . If the condition is true for atleast m agents (m < j) , then the agent k is considered as compromised . Any report from that agent will be discarded. (iv) After collecting the trust reply messages TRep from other agents, the agent i once again checks If Tjk < Tth , where Tjk is the trust index value of agent k, collected from agents j, j=1,2… (j <> k) . If the condition is true for atleast m agents (m < j) , then the agent k is considered as compromised . Any report from that agent will be discarded. Fig 3: Rate Vs Packets Dropped 4.1 PERFORMANCE METRICS For evaluating the performance of the proposed technique following metrics have been used:  Attack Bandwidth – Attack bandwidth is the amount of bandwidth affected by the attackers (in Mb/s) 3. TRUST AMONG AGENTS The DDoS attacks are detected by using an agents, initially deployed near the source. Then the process is continued from agent to agent, dynamically towards the victim. In our distributed agent based IDS, we are using the concept of trust mechanism among the agents. In our agent based system, each agent depends on the reports of other agents. As intruders will try to attack any node in the network, agents has to check the trust of other agents, to identify the © 2015-19, IJARCS All Rights Reserved 377 Harmeet Singh et al, International Journal of Advanced Research in Computer Science, 9 (1), Jan-Feb 2018,375-380 REFERENCES Fig 4: Time Vs Attacked Bandwidth [1]. J. Singh, S. Gupta and L. Kaur, “A MAC Layer Based Defense Architecture for Reduction of Quality (RoQ) Attacks in Wireless LAN”, International Journal of Computer Science and Information Security, Vol. 7, Issue 1, pp. 284-291, 2010. [2]. J. Singh, S. Gupta and L. Kaur, “Taxonomy of Attacks in Wireless Local Area Networks”, Journal of Research in Computer Engineeri ng, Vol.4, No. 1, 2010. [3]. J. Mirkovic, M. Robinson, P. Reiher and G. Oikonomou, “Distributed defense against DDoS attacks” University of Delaware, CIS Department Technical Report CIS-TR- 2005-02, pp. 1-12, 2005. pp [4]. A. Challita, M.E. Hassan, S. Maalouf, A. Zouheiry, “A Survey of DDoS Defense Mechanisms”, Department of Electrical and Computer Engineering, American University of Beirut, 2004. Fig 4: Time Vs Attacked Bandwidth Fig 5: Time Vs Attack Packets Dropped [5]. M. Robinson, J. Mirkovic, M. Schnaider, S. Michel and P. Reiher. "Challenges and principles of DDoS defense." In ACM SIGCOMM. 2003. [6]. W. Znaidi, M. Minier and J.P. Babau, “Detecting wormhole attacks in wireless networks using local neighborhood information." In proceedings of IEEE 19th International Symposium on Personal, Indoor and Mobile Radio Communications, pp. 1-5, 2008. [7]. Y. Xu, G. Chen, J. Ford and F. Makedon, “Distributed wormhole attack detection in wireless sensor networks." In Proceedings of the First Annual IFIP Working Group International Conference on Critical Infrastructure Protection, 2007. [8]. K.S. Win, “Analysis of detecting wormhole attack in wireless networks", In World Academy of Science, Engineering and Technology, pp. 422-428, 2008. Fig 5: Time Vs Attack Packets Dropped Figure 4 shows that the bandwidth affected or occupied by the attackers when the time interval is increased. From the graph it is clearly visible that in the distributed scenario, the amount of attack bandwidth is less when compared to the normal scenario, which indicates that the adverse effect of attackers is reduced in the distributed case. From Figure 5 it is clearly shown that the number of attack packets dropped when time interval is increased from 0 to 20 seconds. From the experimental results it has been analyzed that the distributed approach eliminates most of the attack packets when compared to the normal approach. [9]. Y. C. Hu, Y. Chun, A. Perrig and D.B. Johnson, "Packet leashes: a defense against wormhole attacks in wireless networks." In IEEE Twenty-Second Annual Joint Conference of the Computer and Communications, Vol. 3, pp. 1976-1986, 2003. REFERENCES [10]. H. Ronghui, M. Guoqing, W. Chunlei and F. Lan, “Detecting and locating wormhole attacks in wireless sensor networks using beacon nodes”, World Academy of Science, Engineering and Technology, Vol. 3, 2009. [11]. R.Z. El Kaissi, A. Kayssi, A. Chehab and Z. Dawy, “DAWWSEN: A defense mechanism against wormhole attacks in wireless sensor networks”, Ph.D. Dissertation, American University of Beirut, Department of Electrical and Computer Engineering, 2005. B. BASED ON TIME In the second experiment, the simulation time interval was varied from 5 to 25 seconds.  Attack Packets Drop – Attack Packets Drop is the number of packets of the attackers that are detected and eliminated by the monitoring agents. In this experiment, the performance of proposed distributed agent based technique has been evaluated. In this experiment, the performance of proposed distributed agent based technique has been evaluated. In this experiment, the performance of proposed distributed agent based technique has been evaluated. © 2015-19, IJARCS All Rights Reserved 378 Harmeet Singh et al, International Journal of Advanced Research in Computer Science, 9 (1), Jan-Feb 2018,375-380 Harmeet Singh et al, International Journal of Advanced Research in Computer Science, 9 (1), Jan-Feb 2018,375-3 Fig 4: Time Vs Attacked Bandwidth Fig 5: Time Vs Attack Packets Dropped 5. CONCLUSION [12]. S. Choi, D.Y.Kim, D.Y. Lee and J. Jung, "WAP: Wormhole attack prevention algorithm in mobile adhoc networks." In IEEE International Conference on Sensor Networks, Ubiquitous and Trustworthy Computing, pp. 343-348, 2008. In this paper, a Distributed agent Based technique for detecting DDoS Attacks in wireless LAN has been proposed. The main advantage of the proposed technique is that it provides an early warning when pre-attack activities are detected. The agents proposed in the architecture are autonomous, mobile and cooperative entities. Each agent executes a detection algorithm and based on the detection results, it exchanges its report with other agents. Based on the collaborative report, a final alert is sent to the base station. Further a trust mechanism in which each agent checks the trust of other agents, to identify the compromised agent nodes has been designed. If any agent finds that the received report from any other agent does not match with the expected results, it can be considered as a compromised agent node. Through simulations, it has been proved that the proposed architecture achieves high throughput with low packet drop, by detecting and isolating the attack traffic flows. Therefore the proposed technique can be used for the detection of DDoS attacks in wireless LAN. [13]. T.V. Phuong, N.T. Canh, Y.K. Lee, S. Lee and H. Lee, “Transmission time-based mechanism to detect wormhole attacks”, In 2nd IEEE Asia-Pacific Service Computing Conference, pp. 172-178, 2007. pp [14]. V.S.S. Sriram, A.P. Singh and G. Sahoo, "Methodology for Securing Wireless LANs against Wormhole Attack”, International Journal of Recent Trends in Engineering, Vol. 1, no. 1, 2009. International Journal of Recent Trends in Engineering, Vol. 1, no. 1, 2009. [15]. Network Simulator, http://www.isi.edu/nsnam/ns [16]. M. Shojaei, N. Movahhedinia and B.T. Ladani, "DDoS attack detection in IEEE 802.16 based networks”, Wireless Networks, Vol. 20, Issue 8, pp. 2543-2559, 2014. [17]. M. GhasemiGol, A. Ghaemi-Bafghi, M. Moghaddam and H. Sadoghi-Yazdi, “Anomaly detection and foresight response strategy for wireless sensor networks”, Wireless Networks, Vol. 21, Issue 5, pp. 1425-1442, 2014. [18]. T.V.P. Sundararajan, S. M. Ramesh, R. Maheswar and K. © 2015-19, IJARCS All Rights Reserved 379 Harmeet Singh et al, International Journal of Advanced Research in Computer Science, 9 (1), Jan-Feb 2018,375-380 R. Deepak, "Biologically inspired artificial intrusion detection system for detecting wormhole attack in MANET", Wireless Networks, Vol. 20, Issue 4, pp. 563- 578, 2014 R. © 2015-19, IJARCS All Rights Reserved 5. CONCLUSION Deepak, "Biologically inspired artificial intrusion detection system for detecting wormhole attack in MANET", Wireless Networks, Vol. 20, Issue 4, pp. 563- 578, 2014 DoS attacks in VANET”, Wireless Personal Communications, Vol. 73, Issue 1, pp. 95-126, 2013. DoS attacks in VANET”, Wireless Personal Communications, Vol. 73, Issue 1, pp. 95-126, 2013. [23]. G. Chen, Y. Zhang and C. Wang, “A wireless multi-step attack pattern recognition method for WLAN”, Expert Systems with Applications, Vol. 41, Issue 16, pp. 7068- 7076, 2014. [19]. [19]. G. Koutepas, F. Stamatelopoulos and B. Maglaris, "Distributed management architecture for cooperative detection and reaction to DDoS attacks", Journal of Network and Systems Management, Vol. 12, Issue 1, pp. 73-94, 2004. [24]. M. Andreolini, M. Colajanni and M. Marchetti, “A collaborative framework for intrusion detection in mobile networks”, Information Sciences, Vol. 321, pp. 179-192, 2015. [20]. T. Thapngam, S.Yu, W. Zhou and S.K. Makki, "Distributed Denial of Service (DDoS) detection by traffic pattern analysis” Peer-to-Peer Networking and Applications, Vol. 7, Issue 4, pp. 346-358, 2014. [25]. J. Singh and R.Singh “WRHT: A Hybrid Technique for Detection of Wormhole Attack in Wireless Sensor Networks”, Mobile Information Systems, Vol.6, 2016. [21]. G. Lee, W. Kim, K. Kim, S. Oh and D. Kim, “An approach to mitigate DoS attack based on routing misbehavior in wireless ad hoc networks”, Peer-to-Peer Networking and Applications, Vol. 8, Issue 4, pp. 684-693, 2013. [26]. V.Dhir, R.Kumar and V.Joshi “Performance comparison of routing protocols in mobile ad hoc networks” International Journal of Engineering Science and Technology,Vol.2, pp. 3494-3502, 2010. [22]. K. Verma, H. Hasbullah and A. Kumar, “Prevention of © 2015-19, IJARCS All Rights Reserved 380 380
https://openalex.org/W4303457477	https://zenodo.org/record/7181735/files/IPBES_SUA_Chapter3.pdf	English	null	IPBES Sustainable Use of Wild Species Assessment - Chapter 3. Status of and trends in the use of wild species and its implications for wild species, the environment and people	Zenodo (CERN European Organization for Nuclear Research)	2,022	cc-by	243,300	2 Authors are listed with, in parentheses, their country or countries of citizenship, separated by a comma when they have more than one; and, following a slash, their country of affiliation, if different from that or those of their citizenship, or their organization if they belong to an international organization. The countries and organizations having nominated the experts are listed on the IPBES website (except for contributing authors who were not nominated). 1 This is the final text version of Chapter 3 from the Assessment Report on the Sustainable Use of Wild Species. A laid-out version of the full assessment report will be made available in the coming months. Chapter 3. Status of and trends in the use of wild species and its implications for wild species, the environment and people1,2 Coordinating Lead Authors: Elizabeth S. Barron (United States of America, Norway/Norway), Ram Prasad Chaudhary (Nepal) Coordinating Lead Authors: Elizabeth S. Barron (United States of America, Norway/Norway), Ram Prasad Chaudhary (Nepal) Lead Authors: Sonia Carvalho Ribeiro (Portugal/Brazil), Eric Gilman (United States of America), Jaqueline Hess (Germany), Ray Hilborn (United States of America, Canada/United States of America), Esther Katz (France), Ritah Kigonya (Uganda/Norway), Hicham Masski (Morroco, France/Morocco), Prateep Kumar Nayak (Canada), Helder Queiroz (Brazil), Anna Sidorovich (Belarus), Renato Azevedo Matias Silvano (Brazil, Portugal/Brazil), Yan Zeng (China), Chabi Djagoun (Benin) Lead Authors: Sonia Carvalho Ribeiro (Portugal/Brazil), Eric Gilman (United States of America), Jaqueline Hess (Germany), Ray Hilborn (United States of America, Canada/United States of America), Esther Katz (France), Ritah Kigonya (Uganda/Norway), Hicham Masski (Morroco, France/Morocco), Prateep Kumar Nayak (Canada), Helder Queiroz (Brazil), Anna Sidorovich (Belarus), Renato Azevedo Matias Silvano (Brazil, Portugal/Brazil), Yan Zeng (China), Chabi Djagoun (Benin) Fellows: Laura Isabel Mesa Castellanos (Colombia), Penelope Jane Mograbi (South Africa/ United Kingdom) Fellows: Laura Isabel Mesa Castellanos (Colombia), Penelope Jane Mograbi (South Africa/ United Kingdom) Contributing Authors: Hélène Artaud (France), Yishai Barak (United States, Israel), Monica Biondo (Switzerland), David Bray (United States of America), Matthew Brien (Australia), Ariadna Burgos (France), Martina Calovi (Italy), Nicolas Casajus (France), Alejandro Casas (Mexico), Paolo Cerutti (Italy), Brian Child (United States of America), Steven Cooke (Canada), Benjamin Cretois (Norway), Peter Cronkleton (United States of America), Hannah Cunningham (Canada), Georgi Daskalov (Bulgaria), Ahmad Dermawan (Indonesia), Shiva Devkota (Nepal), Shalini Dhyani (India), Amy Dickman (United Kingdom), John Donaldson (South Africa), Nick Dulvy (Canada), Nora Duncritts (United States of America), Filippa Ek (Sweden), Marla R. Emery (United States of America), Ana Luiza Espada (Brazil), Food and Agriculture Organization, Global Forest Resources Assessment, Clément Garineaud (France), Henry Huntington (United States of America), Francis Johnson (Sweden), Vincent Leblan (France), Guillaume Lescuyer (France), John Linnel (Norway), Hong Liu (United States of America), Peigui Liu (China), Irina Lukina (Belarus), Lusine Margaryan (Armenia), Sergey Matveytchuk (Russia), Iliana Monterroso (Guatemala), Daisuke Naito (Japan), Grant Nickes (United States of America), Hemant Ojha (Nepal), Pablo Pacheco (Bolivia), Brenda Parlee (Canada), Ana Parma (Argentina), Benno Pokorny (Germany), Nicolas Pollet (France), Sisir Kanta Pradhan (Canada), Nicolas Puillandre (France), Herry Purnomo (Indonesia), Francis Putz (United States of America), Dilys Roe (United Kingdom), Robin Rachel Sears (United States of America), Hannah Skelding (Canada), Sara Teitelbaum (Canada), Kathleen Thompson (United States of America), Valter Trocchi (Italy), Karen Vanderwolf (Canada), Edson Vidal (Brazil), Grahame Webb (Australia), Kristine Wray (Canada), Stanislas Zanvo (Benin), Seweryn Zielinski (Poland, South Korea) Edson Vidal (Brazil), Grahame Webb (Australia), Kristine Wray (Canada), Stanislas Zanvo (Benin), Seweryn Zielinski (Poland, South Korea) Review Editors: Ryo Kohsaka (Japan), Charlie Shackleton (South Africa) Technical support unit: Marie-Claire Danner, Agnès Hallosserie, Daniel Kieling Technical support unit: Marie-Claire Danner, Agnès Hallosserie, Daniel Kieling This chapter should be cited as: Barron, E.S., Chaudhary, R.P., Carvalho Ribeiro, S., Gilman, E., Hess, J., Hilborn, R., Katz, E., Kigonya, R., Masski, H., Mesa Castellanos, L.I., Mograbi, P.J., Nayak, P.K., Queiroz, H., Sidorovich, A., Silvano, R.A.M., Zeng, Y, Djagoun, C, and Danner, M.C. (2022). Chapter 3: Status of and trends in the use of wild species and its implications for wild species, the environment and people. In: Thematic Assessment Report on the Sustainable Use of Wild Species of the Intergovernmental Science-Policy Platform on Biodiversity and Ecosystem Services. Fromentin, J.M., Emery, M.R., Donaldson, J., Danner, M.C., Hallosserie, A., and Kieling, D. (eds.). Schematic and adapted figures can be found in the following Zenodo repository: https://doi.org/10.5281/zenodo.7009633 Fellows: Laura Isabel Mesa Castellanos (Colombia), Penelope Jane Mograbi (South Africa/ United Kingdom) IPBES Secretariat, Bonn, Germany. https://doi.org/10.5281/zenodo.6451322 This chapter should be cited as: Barron, E.S., Chaudhary, R.P., Carvalho Ribeiro, S., Gilman, E., Hess, J., Hilborn, R., Katz, E., Kigonya, R., Masski, H., Mesa Castellanos, L.I., Mograbi, P.J., Nayak, P.K., Queiroz, H., Sidorovich, A., Silvano, R.A.M., Zeng, Y, Djagoun, C, and Danner, M.C. (2022). Chapter 3: Status of and trends in the use of wild species and its implications for wild species, the environment and people. In: Thematic Assessment Report on the Sustainable Use of Wild Species of the Intergovernmental Science-Policy Platform on Biodiversity and Ecosystem Services. Fromentin, J.M., Emery, M.R., Donaldson, J., Danner, M.C., Hallosserie, A., and Kieling, D. (eds.). IPBES Secretariat, Bonn, Germany. https://doi.org/10.5281/zenodo.6451322 Disclaimer on maps: The designations employed and the presentation of material on the maps used in the assessment do not imply the expression of any opinion whatsoever on the part of IPBES concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. These maps have been prepared or used for the sole purpose of facilitating the assessment of the broad biogeographical areas represented therein. Schematic and adapted figures can be found in the following Zenodo repository: https://doi.org/10.5281/zenodo.7009633 Schematic and adapted figures can be found in the following Zenodo repository: https://doi.org/10.5281/zenodo.7009633 Contents Contents Executive Summary ............................................................................................................... 1 3.1. Introduction ............................................................................................................... 10 3.2. Scale and scope: a global overview........................................................................... 11 3.2.1. Datasets available and global estimates of wild species used ..................................................... 12 3.2.1.1. Fishing ..................................................................................................................................... 18 3.2.1.2. Gathering ................................................................................................................................. 20 3.2.1.3. Terrestrial Animal Harvesting ................................................................................................. 23 3.2.1.4. Logging ................................................................................................................................... 24 3.2.1.5. Non-extractive use................................................................................................................... 25 3.2.2. Global Indicators.......................................................................................................................... 27 3.2.2.1. Indigenous Indicators .............................................................................................................. 38 3.2.3. Temporal scale and use ................................................................................................................ 39 3.2.4. Economic, ecological, and social contexts of sustainable use ..................................................... 44 3.3. Practices and uses ...................................................................................................... 46 3.3.1. Fishing ......................................................................................................................................... 46 3.3.1.1. Introduction ............................................................................................................................. 47 3.3.1.2. Status and trends in global marine capture fisheries ............................................................... 50 3.3.1.3. Status and trends in selected fisheries ..................................................................................... 58 3.3.1.4. Small-scale fisheries ................................................................................................................ 60 3.3.1.4.1. Indicators of small-scale fisheries sustainability ..................................................................... 72 3.3.1.4.2. The role of indigenous and local knowledge in small-scale fisheries ..................................... 75 3.3.1.4.3. Pelagic fisheries for forage fish ............................................................................................... 84 3.3.1.4.4. Pelagic fisheries for billfishes, tuna and tuna-like species ...................................................... 85 3.3.1.4.5. Whaling ................................................................................................................................... 91 3.3.1.4.6. Industrial demersal fisheries in coastal areas .......................................................................... 94 3.3.1.5. Uses of wild caught aquatic organisms ................................................................................. 102 3.3.1.5.1. Food and Feed ....................................................................................................................... 103 3.3.1.5.2. Medicine and hygiene ........................................................................................................... 117 3.3.1.5.3. Recreational fisheries ............................................................................................................ 123 3.3.1.5.4. Decorative and aesthetic........................................................................................................ 128 3.3.1.5.5. Ceremony and cultural expression ........................................................................................ 129 3.3.1.6. “Non-lethal” fishing practices and uses ................................................................................ 129 3.3.1.6.1. Catch and release recreational fishing ................................................................................... 129 3.3.1.6.2. Ornamental or aquarium fish................................................................................................. 131 3.3.2. Gathering ................................................................................................................................... 134 3.3.2.1. Introduction ........................................................................................................................... 134 3.3.2.2. The diversity of contemporary gathering .............................................................................. 137 3.3.2.2.1. Gathering in Western European and Other Group (WEOG) countries ................................. 137 3.3.2.2.2. Urban gathering ..................................................................................................................... 139 3.3.2.2.3. Gender trends ........................................................................................................................ 142 3.3.2.3. Uses of wild plants, algae, and fungi, including the leaves and fruits of trees ..................... 143 3.3.2.3.1. Ceremony and cultural expression ........................................................................................ 144 3.3.2.3.2. Decorative and aesthetic........................................................................................................ 145 3.3.2.3.3. Energy ................................................................................................................................... 151 3.3.2.3.4. Food and beverage................................................................................................................. 152 3.3.2.3.5. Medicine and hygiene ........................................................................................................... 174 3.3.2.3.6. Recreation.............................................................................................................................. 181 3.3.2.3.7. Science and education ........................................................................................................... 181 3.3.2.3.8. Materials and shelter ............................................................................................................. 184 3.3.2.4. Emerging issues in gathering ................................................................................................ 186 3.3.3. Terrestrial animal harvesting ..................................................................................................... 187 3.3.3.1. Introduction ........................................................................................................................... 187 3.3.3.2. Uses ....................................................................................................................................... 189 3.3.3.2.1. Ceremonial and cultural expression ...................................................................................... 189 3.3.3.2.2. Decorative and aesthetic uses ................................................................................................ 190 3.3.3.2.3. Food and beverage................................................................................................................. 191 3.3.3.2.4. Recreational hunting.............................................................................................................. 202 3.3.3.2.5. Science and education ........................................................................................................... 217 3.3.3.2.6. Medicine and hygiene ........................................................................................................... 219 3.3.3.3. “Non-lethal” terrestrial animal harvesting ............................................................................ 223 3.3.3.3.1. Decorative and aesthetic........................................................................................................ 223 3.3.3.3.2. Food and beverage: honey..................................................................................................... Contents 224 3.3.3.3.3. Recreation: green hunting ..................................................................................................... 225 3.3.3.3.4. Pet and zoo trade ................................................................................................................... 225 3.3.3.4. Emerging issues: terrestrial animals harvesting for integrated species and habitat management 227 3.3.4. Logging ...................................................................................................................................... 229 3.3.4.1. Introduction ........................................................................................................................... 229 3.3.4.2. Global trends and overview................................................................................................... 233 3.3.4.3. A stratified typology on sustainable use of wild species in logging ..................................... 238 3.3.4.3.1. Smallholder Logging practice ............................................................................................... 240 3.3.4.3.2. Community Logging practice................................................................................................ 246 3.3.4.3.3. Industrial Logging practice ................................................................................................... 256 3.3.4.4. Uses ....................................................................................................................................... 264 3.3.4.4.1. Decorative and aesthetic........................................................................................................ 264 3.3.4.4.2. Energy ................................................................................................................................... 266 3.3.4.4.3. Material and construction ...................................................................................................... 274 3.3.4.5. Emerging issues in logging and timber management............................................................ 278 3.3.4.5.1. Covid-19 pandemic ............................................................................................................... 278 3.3.5. Non-extractive practices ............................................................................................................ 279 3.3.5.1. Introduction: Significance of non-extractive practices ......................................................... 279 3.3.5.2. Uses ....................................................................................................................................... 280 3.3.5.2.1. Ceremony and cultural expression ........................................................................................ 281 3.3.5.2.2. Medicine and hygiene ........................................................................................................... 283 3.3.5.2.3. Recreation.............................................................................................................................. 288 3.3.5.2.4. Education and learning .......................................................................................................... 303 3.3.5.3. Emerging issues..................................................................................................................... 306 3.4. Trade-offs and synergies ......................................................................................... 307 3.4.1. Introduction................................................................................................................................ 307 3.4.2. Conceptualizing trade-offs and synergies .................................................................................. 308 3.4.3. A framework to analyze trade-offs and synergies in the sustainable use of wild species ......... 308 3.4.3.1. Trade-offs and synergies at intra-practice and intra-use level .............................................. 309 3.4.3.2. Trade-offs and synergies between practices and uses ........................................................... 311 3.4.3.3. Trade-offs and synergies involving the social, economic, environmental and policy aspects of sustainable use ........................................................................................................................................ 314 3.4.4. Selected case studies of trade-offs and synergies in sustainable use ......................................... 315 3.4.4.1. Whaling and whale-watching ................................................................................................ 315 3.4.4.2. Recreational trophy hunting and wildlife watching tourism ................................................. 317 3.4.4.3. Elasmobranch tourism opportunity and shark fishing........................................................... 319 3.4.5. Key attributes necessary to respond to trade-offs and strengthen synergies in sustainable use ........ 321 3.4.5.1. Levels and scales at which trade-offs and synergies occur ................................................... 322 3.4.5.2. Equity and justice considerations in responding to trade-offs and negotiating synergies..... 323 3.4.5.3. Power dynamics and politics of use ...................................................................................... 324 3.4.5.4. Governing trade-offs and synergies for sustainable use........................................................ 325 3.5. Knowledge gaps ...................................................................................................... 326 3.6. Challenges and research priorities........................................................................... 333 3.6.1. Challenges ..................................................................................................................................... 333 3.6.1.1. Global scale and scope ............................................................................................................... 333 3.6.1.2. Informal trade of wild species.................................................................................................... 334 3.6.1.3. Fishing........................................................................................................................................ 334 3.6.1.4. Gathering.................................................................................................................................... 334 3.6.1.5. Terrestrial animal harvesting ..................................................................................................... 334 3.6.1.6. Logging ...................................................................................................................................... 335 3.6.1.7. Non-extractive uses .................................................................................................................... 335 3.6.2. Research priorities ............................................................................................................................. 336 3.6.2.1 Practices and uses ....................................................................................................................... 336 3.6.2.2. Nature’s contributions to people & human well-being .............................................................. 336 3.6.2.3. Documenting under-researched taxa.......................................................................................... 336 3.6.2.4. Social norms that affect uses and practices................................................................................ Executive Summary (1) Monitoring of the ecological and social, including economic aspects of uses of wild species is critical for sustainable use (well established) {3.2.4, 3.3.3.3.4}. Progress towards achieving the Sustainable Development Goals and the Aichi Biodiversity Targets is assessed using global indicators, however to date, there is not a comprehensive set of global indicators able to monitor status and trends of wild species use (well established) {3.2.1}. Scientific monitoring is limited or lacking for many extractive and non- extractive practices (well established) {3.3.1, 3.3.3, 3.3.5} and is identified as a critical knowledge gap for sustainable use {3.5}. The indicators available provide a fragmented view of wild species use in different social-ecological systems across the globe and within each practice. Global indicators on biodiversity status and trends emphasize major fisheries and terrestrial animal harvesting of large mammals, while gathering and non-extractive practices lag behind significantly in global indicator initiatives (established but incomplete) {3.2.1.2, 3.2.1.3, 3.2.1.5}. Monitoring is resource intensive and will require more support and investment in all countries to overcome the capacity, financial, technical and institutional challenges that generate strong limitations to monitoring of wild species, which are more pronounced in developing countries. Monitoring efforts that are inclusive of indigenous peoples and local communities, scientific approaches and equitable participation of all key actors can better inform decision-making (well established) {3.2.4, 3.3.3, 3.3.5}. (2) A conservative estimate of approximately 50,000 wild species are used for food, energy, medicine, material, income generation and other purposes through fishing, gathering, logging and terrestrial animal harvesting globally (well established) {3.2.1, 3.3.1, 3.3.2, 3.3.3, 3.3.4}. People all over the world directly use about 7,500 species of wild fish and aquatic invertebrates, 31,100 wild plants (7,400 of which are tree species) 1,500 species of fungi, 1,700 species of wild terrestrial invertebrates and 7,500 species of wild amphibians, reptiles, birds and mammals (well established) {3.2.1.3, 3.3, 3.3.2.3.4}. Among the wild species that are used, more than 20% (over 10,000 species) are used for human food, making the sustainable use of wild species critical for achieving food security and improving nutrition, in rural and urban areas worldwide (well established) {3.3} Knowledge and skills developed over generations make single species likely to deliver multiple uses. The contribution of wild species to livelihoods is context and situationally specific, ranging from 10% to 80% of household income globally (well established) {3.2.2}. Contents 337 3.6.2.5. Integrating indigenous local knowledge .................................................................................... 337 References .......................................................................................................................... 338 Executive Summary An estimated 70% of the world’s poor depend directly on biodiversity and businesses it fosters (well established) {3.2.1}. Therefore, sustainable use supports subsistence livelihoods, trade, and human well- being, including for indigenous peoples and local communities, and provides options for further economic development linked directly to successful conservation (well established) {3.2.1, 3.3.1, 3.3.2, 3.3.3.2.3, 3.3.3.2.4, 3.3.4.3.1, 3.3.4.3.2, 3.3.4.4.2}. While trade in local markets is important, some wild species products are part of long commodity chains and are global commodities {3.3.1, 3.3.2}. In many cases, wild species are considered superior to cultivated alternatives (well established) {3.3.1.5.1, 3.3.2.3.4, 3.3.3.2.3, 3.3.3.3.2, 3.3.5.2}. Fishing, terrestrial animal harvesting, logging, and nature-based tourism are vital to regional and local employment and economies in many developing and 1 developed countries and further contribute to public infrastructure, development and provisioning of related goods and services (well established) {3.3}. The use of wild species also provides nonmaterial contributions by enriching people's physical and psychological experiences, including their religious and ceremonial lives (well established) {3.3.5.2.1}. (3) Fisheries constitute a major source of food from wild species, with a total annual harvest of 90 million tons over recent decades of which about 60 million tons go to direct human consumption and the rest as feed for aquaculture and livestock (well established) {3.2.1.1}. Recent global estimates indicate that approximately 66% are fished within biological sustainable levels and 34% of marine wild fish stocks are overfished, but this global picture displays strong heterogeneities (well established) {3.2.1.1}. In countries or regions with strong fisheries management, which account for approximately half of the fisheries landings reported by the Food and Agriculture Organization of the United Nations, on average stocks are increasing in abundance and above target levels (well established) {3.3.1}. For countries and regions with low intensity fisheries management of large- and small-scale fisheries, the status of stocks is less well known (well established) {3.3.1.2}, but generally believed to be below the abundance that would maximize sustainable food production (established but incomplete) {3.3.1}. At the same time, small scale fisheries contribute two-thirds of the global fish catch destined for direct human consumption (well established) {3.3.1}. In most fisheries, there are large gaps in understanding of life histories for many marine species. Executive Summary For small-scale fisheries that have been assessed around the world, many have been considered to be unsustainable or only partially sustainable, especially in Africa for both inland and marine fisheries and in Asia, Latin America and Europe for coastal marine fisheries (established but incomplete) {3.3.1.4.1}. Small-scale fisheries are strongly anchored in local communities' ways of life on all continents and it is known that small-scale fisheries support over 90% of the 120 million people engaged in capture fisheries globally. About half of the people involved in small-scale fisheries (e.g., production, marketing) are women (well established) {3.4.3.1}. (4) Unintentional bycatch fishing mortality of vulnerable, endangered, threatened and/or protected marine species, which is beginning to be assessed and managed, is unsustainable for many populations of marine turtles, sea snakes, seabirds, sharks, rays, chimaeras, marine mammals and some bony fishes (well established) {3.3.1.1}. Reducing unintentional bycatch and discards is progressing, but still insufficient (well established) {3.3.1.1}. Some of these species may be unintentionally targeted, but are retained for food as incidental catch (including retention of shark fins and manta and devil ray gill plates and discarding of the remaining carcass), or discarded (well established) {3.3.1.3}. Among the 1,250 shark and ray species identified today, 1,199 have been recently assessed and 449 (37.5%) have been assessed as threatened (well established) {3.3.1.3}. While fishing of target species may be sustainable, the conservation status of bycatch species and other associated and dependent species is often poorly known. Bycatch is a well-known issue for several large-scale fisheries, such as the shrimp or bottom trawl fisheries, but it is also a concern for several small- scale fisheries (well established) {3.3.1.1, 3.3.1.5}. There have been recent advances in monitoring and managing fishing mortality of marketable incidental species and discarded 2 bycatch species, however global uptake of effective bycatch management measures is severely lagging in a majority of marine capture fisheries (well established) {3.3.1.5}. For example, nearly all (99%) shark and ray species are officially declared to be taken unintentionally, but are valuable and are retained for food. Consequently, shark species have been declining steeply since the 1970s, especially in tropical and subtropical coastal shelf waters (well established) {3.3.1.3}. (5) Increases in recreational fishing show it is becoming a significant component of marine capture fisheries (well-established) {3.3.1.5.3} and a potentially significant contributor to fish declines (established but incomplete) {3.3.1.5.3} in combination with the commercial fleet. Executive Summary There have been recent advances in monitoring and managing fishing mortality of marketable incidental species and discarded bycatch species, however global uptake of effective bycatch management measures is severely lagging in a majority of marine capture fisheries. Therefore, stock assessments which do not incorporate recreational fishing do not provide accurate assessments of global uptake and fish mortality. Recreational catch and release fishing can have negative impacts, but can be done sustainably if responsibly practiced (well established) {3.3.1.5.3}. (5) Increases in recreational fishing show it is becoming a significant component of marine capture fisheries (well-established) {3.3.1.5.3} and a potentially significant contributor to fish declines (established but incomplete) {3.3.1.5.3} in combination with the commercial fleet. There have been recent advances in monitoring and managing fishing mortality of marketable incidental species and discarded bycatch species, however global uptake of effective bycatch management measures is severely lagging in a majority of marine capture fisheries. Therefore, stock assessments which do not incorporate recreational fishing do not provide accurate assessments of global uptake and fish mortality. Recreational catch and release fishing can have negative impacts, but can be done sustainably if responsibly practiced (well established) {3.3.1.5.3}. (6) Indigenous peoples and local communities contribute vital knowledge to the sustainable use of wild animals {3.3.3}, wild plants and fungi {3.3.2}, wild timber species {3.3.4.3.1} and small-scale fisheries {3.3.1.4} (well established). Subsistence uses of wild species are important sources of food, medicine, fuel and other livelihood resources for indigenous peoples and local communities in both developed and developing countries. A key to sustainable gathering, terrestrial animal harvesting, fishing, and logging practices is to work with indigenous peoples and local communities in data collection and knowledge production, which is deemed essential to evaluate and reconstruct temporal trends on resource use, establish participatory monitoring programs and develop locally based co-management systems (well established). Many wild foods have nutritional benefits over processed foods and there may be no culturally acceptable alternative for ceremonial and ritual materials (well established) {3.3.1.7.1, 3.3.2.3.4, 3.3.3.3.3, 3.3.3.4.2, 3.3.5.2.1}. Wild species also provide a basis for culturally meaningful employment {3.3.3.2.1, 3.3.5.2.3}. In light of ongoing growth and demand for health and food security, collaboration with indigenous peoples and local communities on wild plants and fungi, genetic resources of crop wild relatives, and small-scale fisheries is an especially urgent need (well established) {3.3.1.4, 3.3.2.3.7}. Executive Summary (7) The gathering and trade of wild fungi, plants and algae for food, medicine and ornamental use is increasing because of public demand (well established) {3.3.2}, and continues to be an economically and culturally important activity worldwide. An estimated one-fifth of the world's population participates in gathering practices, often irrespective of economic status (established but incomplete) {3.3.2}. People in economically disadvantaged urban and rural areas rely on wild plants, algae and fungi as a source of of essential calories, micronutrients and medicine (well established) {3.3.2, 3.3.2.2.2}. Gathering is often assumed to be an activity more prevalent in the Global South. However, estimates of individuals and households participating in gathering in Europe and North America range from (7) The gathering and trade of wild fungi, plants and algae for food, medicine and ornamental use is increasing because of public demand (well established) {3.3.2}, and continues to be an economically and culturally important activity worldwide. An estimated one-fifth of the world's population participates in gathering practices, often irrespective of economic status (established but incomplete) {3.3.2}. People in economically disadvantaged urban and rural areas rely on wild plants, algae and fungi as a source of of essential calories, micronutrients and medicine (well established) {3.3.2, 3.3.2.2.2}. Gathering is often assumed to be an activity more prevalent in the Global South. However, estimates of individuals and households participating in gathering in Europe and North America range from 3 4% to 68%, with the highest rates of gathering by households in Eastern Europe (established but incomplete) {3.3.2.2.1}, often irrespective of economic status (established but incomplete) {3.3.2.2.3}. Nor is gathering is confined to rural areas, with dozens to hundreds of wild plant and fungi species gathered for food, medicine, firewood, decoration, and cultural practices in urban ecosystems worldwide (well established) {3.3.2.2.2}. Gathering is often a gendered activity in many parts of the world, with roles depending on cultural rules, on the type of harvested wild plants, algae or fungi and the places where they are harvested. In many countries, women perform the bulk of gathering and processing of wild plants for food, medicine, fuel and handicrafts for subsistence purposes and sale in local markets (well established) {3.3.2.2.3}. Trade of wild plants, algae and fungi is a billion-dollar industry and establishment of supply chains can fuel economic development and diversification (well established) {3.3.2.1}. Trade in ornamental plants has increased rapidly over the past 40 years. Executive Summary Wild meat is an important source of protein, fat and other micronutrients such as calcium, iron, zinc and fatty acids (well established) {3.3.3.3.3}. Large mammals alone comprised 55 - 75% of total wild meat biomass extracted annually in different regions of the world, although in some traditional small band societies (e.g the San, the Hadza, the Ache, Native American groups) small game as well as wild plant resources are gathered as primary sources of protein and daily nutrition (well established) {3.3.3.2.3}. Estimates of wild meat consumption differ greatly - from more than 5 million tons a year globally to around 4.6 million tons in the Congo Basin and 1.3 million tons a year in the Amazon respectively. In tropical forests, exploitation of wild meat increased drastically during recent decades due to large numbers of urban consumers, individual food preferences, change in hunting technologies, and scarcity of alternative sources of protein (established but incomplete) {3.2.1, 3.3.3.2.3}. Sustainability of hunting for food, especially in tropical areas, has been negatively affected by profound socio-economic changes, which have resulted in shifts from local-level subsistence towards more intensive wild meat trade (well established) {3.3.3.2.3}. The sustainability of wild meat hunting is increasingly driven by socio-economic changes, recreation, entertainment, trade, or trafficking, rather than solely hunting for subsistence (well established) {3.3.3}. (9) Many game species with high intrinsic rates of population increase or high ecological adaptability have been used sustainably and tolerate even high utilization levels (well established) {3.3.3.2.4}. The impacts of hunting on the abundance of wild species vary worldwide depending on the biological characteristics of the animals as well as the management systems but are generally lower for species with high population growth rates, or high ecological adaptability, and where hunting is well managed (well established) {3.3.3.2.4}.Research suggests that hunting can support sustainable use because it increases the economic value of wild species and the habitats they depend on for local people and communities, providing critical benefit flows that can motivate and enable sustainable management approaches (established but incomplete) {3.3.3.3.4}. Hunting can also create major costs for biodiversity, ecosystem functioning, and animal welfare (well established) {3.3.3.2.4}. Executive Summary Although much of the trade is in cultivated plants, poaching of ornamental species from the wild continues to occur, and can threaten the survival of species (well established) {3.3.2.3.2}. There is a growing demand for wild foods in the food and aromatics industries including among fine dining and haute cuisine establishments, and among urban populations (well established) {3.3.2.2.2, 3.3.2.3.4}. There is also a growing demand for products produced at least in part from harvested wild plants and fungi, for example to complement chemical medicines in many developed and developing countries (well established) {3.3.2.3.5}. Unsustainable gathering is one of the main threats for several plant groups, notably cacti, cycads, and orchids (well established) as well as other plants and fungi harvested for medicinal purposes {3.2.2, 3.3.2.3.2}. Harvests that have been sustainable in the past due to smaller markets and sustainable harvesting practices may become unsustainable if, for example, harvesting is undertaken without following established techniques and protocols (well established) {3.3.2.3.4}, or new technologies are employed which increase the volume of harvest or result in damage to or death of the organism, for example when entire trees are felled rather than climbed to harvest ripe fruits (established but incomplete) {3.3.2}. Wild plants, algae, fungi and trees are at risk from land use change, environmental degradation, deforestation, climate change and overharvesting (well established) {3.3.2.3.2}, but long-term systematic research on the relative importance and interplay of these factors is lacking (well established) (3.6.2). Traditional management practices and cultivation / silviculture are promising approaches to increase the sustainable use of wild species (established but incomplete) {3.3.4.3}. (8) Terrestrial animal harvesting takes place in a variety of governance, management, ecological and socio-cultural contexts, which affect the outcomes for sustainable use. Globally, populations of many terrestrial animals are declining due to unsustainable use, but the impacts of use on wild species and society can be neutral or positive in some places (well established) {3.3.3}. Terrestrial animal harvesting contributes to the food security of many people living in rural and urban areas worldwide, especially in developing countries (well established) {3.3.3.2.3}. The most targeted species for subsistence and commercial hunting (a sub-category of terrestrial animal harvesting) are the largest-bodied (> 30 kg), as these animals provide more meat for consumption and sale and generate more economic benefits for hunters’ 4 households (well established) {3.3.3.2.3}. Executive Summary Selective hunting of particular species or of individuals or of populations which have particular attributes (e.g., large-sized or large horns) can impact ecosystem structure and processes through modifying vegetation composition and structure, including forest succession and regeneration patterns, shifts in ecosystem functions, such as nutrient cycling and carbon capture, declines in carnivore densities, changes of the genetic structure of affected populations {3.3.3.2.4}, changes in predator-prey relationships and shifts in distribution of species and biomass across multiple trophic levels (well-established) {3.3.3.4, 3.3.3.2.4}. Unsustainable hunting has been identified as a threat for 1,341 wild mammal species, including 669 species that were assessed as threatened, and declines in largebodied species with low intrinsic rates of population increase have been linked to hunting pressure (well established) {3.3.3}. Negative impacts of hunting have also been reported on bird species (well established) {3.3.3.2.5, 3.3.3.2.6, 3.3.3.3.4}.A long-term holistic approach with consideration of all ecological, evolutionary, economic, and social consequences is required to fully evaluate hunting wild species as a conservation tool and provide appropriate management policies {3.3.3.3.4}. 5 5 (10) Recreational hunting is highly controversial and has been written about extensively in the scientific literature, however only a limited number of these studies contain well- argued, data-driven evidence and even fewer address recreational hunting with regards to sustainable use and its trade-offs (well established) {3.3.3.2.4}. There is considerable variation in the way recreational hunting is governed and administered in different regions, which makes any generalization about its sustainability or unsustainability difficult {3.3.3.2.4}. Some species are recovering from small population sizes under management systems that allow regulated recreational hunting, usually as a way to generate revenue and increase the land area for population expansion (established but incomplete) {3.3.3.2.4}. Sustainable use needs to consider the social (including institutional and economic) and ecological factors and is therefore highly context specific. Operationally, sound biological management is contingent on appropriate institutional, social and economic conditions, which include proper regulation of the hunting system by scientific and/or local control and knowledge. Weak tenure, the centralization of revenues derived from hunting and breakdown of community governance without any effective replacement by state officials can result in unsustainable recreational hunting (well established) {3.3.3.2.4}. Executive Summary Large areas of land that are managed for recreational hunting (e.g., ~1.4 million km² in Africa) could contribute to conservation objectives and spatial conservation targets, but their unique biodiversity values as well as their ecological and social durability have mostly not been evaluated (established but incomplete) {3.3.3.2.4}. Economically, recreational hunting has been considered an important activity and is credited with generating revenues and creating jobs, as well as providing income and other important economic and social benefits to indigenous and local people in rural, remote and/or otherwise marginal areas. Some recreational hunting activities can generate hundreds to hundreds of thousands of United States dollars, and globally create a substantial revenue flow from developed to developing countries, as well as from urban to rural areas within countries (well established) {3.3.3.2.4}. (11) Logging for energy is prevalent globally but reliance on wood for heating and cooking is highest in developing countries (well established) {3.3.4}. Logging is also an important source of subsistence resources and income for millions of people worldwide (well established) {3.3.4.3}. Logging for energy accounts for 50% of all wood consumed globally, and accounts for 90% of timber harvested in Africa. Fuel wood use is declining in most regions but is increasing in sub-Saharan Africa (established but incomplete) {3.3.4.4.2}. Fuel wood demand can be met at global and national scales when comparing supply-demand balances, but localized fuel wood shortages and the associated forest and woodland degradation occur in areas where people have few alternatives for cooking and heating (established but incomplete) {3.3.4.4.2}. Worldwide, 2.4 billion people rely fuel wood for cooking and an estimated 880 million people globally log firewood or produce charcoal, particularly in developing countries (established but incomplete) {3.3.4.4.2}. Sustainable fuel wood logging remains a renewable energy opportunity that provides income, heating and cooking in developing countries where 1.1 billion people do not have access to electricity or alternative energy sources (established 6 but incomplete) {3.3.4.4.2}, provided air pollution (indoor and outdoor) and climate change emissions are mitigated. Logging is carried out by smallholders, communities and industrial entities (established but incomplete) {3.3.4.3}. For example, logging by smallholders provides thousands of jobs in Central African countries (well established) {3.3.4.3.1}. Executive Summary An estimated 15% of global forests are managed as community resources by indigenous peoples and local communities, often with a strong focus on multiple use management (established but incomplete) {3.3.4.3.2}, while industrial logging occurs in over one quarter of the world’s forests (well established) {3.3.4.3.3}. (12) Wild tree species are currently the major sources for wood and wood products and will continue to be in the coming decades (well established) {3.3.4.1}. Globally, wild tree species provide two thirds of industrial roundwood {3.3.4.3.3}. However, destructive logging practices and illegal logging threaten sustainable use of natural forests (established but incomplete) {3.3.4} and an estimated 12% of wild tree species are threatened by unsustainable logging {3.2.1.4}. The outcomes of logging affect forest ecology, as well as other forest-based uses of wild species, such as gathering, terrestrial animal harvesting and observing wild species (well established) {3.3.4}.Although there is an expected increase in production of plantation wood, there is also a projected increase in timber demand, which will not be matched by plantation wood (well established) {3.3.4.1, 3.3.4.2}. Inventory- based management plans and selective logging could reduce the impacts of logging, but its sustainability depends on the planning, techniques and implementation used to minimize damage to the residual forest stand, as well as forest soils, flora and fauna (well established) {3.3.4.2}. About 20% of the world’s tropical forests (3.9 million km²) are currently subject to selective logging (well established) {3.2.1.4, 3.3.4.2}. (13) A geographic shift is observed in illegal logging and related timber trade (established but incomplete) (3.3.4.2). Illegal logging has declined in parts of the tropical Americas, as well as parts of the tropical and mountain regions of Asia due to improved monitoring and collaborative transboundary collaborations. However, illegal logging and trade has increased in other regions, including Southeast Asia, Northeast Asia and parts of Africa (established but incomplete) {3.3.4.2, 3.3.4.3.1} (14) Non-extractive practices using wild species occur widely in all areas by all cultures, although the nature of the practice differs across cultures and locations (well established) {3.3.5}. The benefits of wild species for improving human mental and physical health have been widely documented in both urban and natural settings (well established) {3.3.5.2.2}. Non-extractive practices are core to human identity, support mental and physical well-being, raises awareness and facilitate connection to nature and society (well established) {3.3.5.2.2}. Executive Summary Despite the crucial importance of non-extractive practices for human-nature connections, with the exception of recreational tourism, there is extremely limited knowledge on the use, trends or sustainability of these practices (well established) {3.3.5, 3.5}. (14) Non-extractive practices using wild species occur widely in all areas by all cultures, although the nature of the practice differs across cultures and locations (well established) {3.3.5}. The benefits of wild species for improving human mental and physical health have been widely documented in both urban and natural settings (well established) {3.3.5.2.2}. Non-extractive practices are core to human identity, support mental and physical well-being, raises awareness and facilitate connection to nature and society (well established) {3.3.5.2.2}. Despite the crucial importance of non-extractive practices for human-nature connections, with the exception of recreational tourism, there is extremely limited knowledge on the use, trends or sustainability of these practices (well established) {3.3.5, 3.5}. 7 7 (15) Nature-based tourism is the most prominent non-extractive practice and demand for wild species media (i.e., documentaries) and in situ observing (e.g., wildlife watching tourism) was growing steadily until 2020 and the global COVID-19 pandemic (well- established) {3.3.5.2.3}. Wildlife watching generates substantial revenue, contributing US$120 billion in 2018 to global gross domestic product (five times the estimated value of the illegal wild species trade) and sustaining 21.8 million jobs {3.3.4.2.3}. Prior to the COVID-19 pandemic, protected areas, globally, received approximately 8 billion visitors per year, generating 600 billion United States dollars per year, with wild-species rich countries experiencing bigger increases in tourism visitation (well established ) {3.3.5.2.3}. (15) Nature-based tourism is the most prominent non-extractive practice and demand for wild species media (i.e., documentaries) and in situ observing (e.g., wildlife watching tourism) was growing steadily until 2020 and the global COVID-19 pandemic (well- established) {3.3.5.2.3}. Wildlife watching generates substantial revenue, contributing US$120 billion in 2018 to global gross domestic product (five times the estimated value of the illegal wild species trade) and sustaining 21.8 million jobs {3.3.4.2.3}. Prior to the COVID-19 pandemic, protected areas, globally, received approximately 8 billion visitors per year, generating 600 billion United States dollars per year, with wild-species rich countries experiencing bigger increases in tourism visitation (well established ) {3.3.5.2.3}. Wildlife watching is crucial for local livelihoods, provides employment and promotes development of tourism-related infrastructure, particularly in some remote locations (well established) {3.3.5.2.3, 3.4.4.2}. These and additional benefits make positive contributions to conservation, community development, and livelihoods in underdeveloped and remote regions when well-managed, but may also create vulnerability to shocks such as global recessions or pandemics (well-established) {3.3.5.2.3, 3.3.5.3}. Although non-extractive practices are frequently less directly harmful to wild species and ecosystems than extractive ones, wildlife watching may have unintended detrimental impacts through changes to species behavior, physiology, or damage to habitats (well established) {3.3.5.2.3}. Many of the unsustainable impacts of the tourism industry could be mitigated through context-based understanding, implementation of best practice guidelines for observing, communication, education and public awareness of tourists and tour operators, collaborative engagement with all stakeholders and sector-specific regulation (well-established) {3.3.5.2.3, 3.3.5.2.4}. (16) Effective management systems that promote the sustainable use of wild species can contribute to broader conservation objectives (established but incomplete) {3.3.3.3.4, 3.3.3.4.1, 3.3.4.3.2, 3.3.5.2.3}. (14) Non-extractive practices using wild species occur widely in all areas by all cultures, although the nature of the practice differs across cultures and locations (well established) {3.3.5}. The benefits of wild species for improving human mental and physical health have been widely documented in both urban and natural settings (well established) {3.3.5.2.2}. Non-extractive practices are core to human identity, support mental and physical well-being, raises awareness and facilitate connection to nature and society (well established) {3.3.5.2.2}. Despite the crucial importance of non-extractive practices for human-nature connections, with the exception of recreational tourism, there is extremely limited knowledge on the use, trends or sustainability of these practices (well established) {3.3.5, 3.5}. Based on assessment of 10,098 species from 10 taxonomic groups documented for the International Union for Conservation of Nature Red List of Threatened Species, at least 34% of the wild species assessed are used sustainably (established but incomplete) {3.2.1, 3.2.2, 4.2.4.3.1}. This includes 172 threatened or near-threatened species. Overall, unsustainable harvest contributes towards elevated extinction risk for 28-29% of near-threatened and threatened species from 10 taxonomic groups assessed on the International Union for Conservation of Nature Red List of Threatened Species {3.2.1, 3.2.2}. reconfiguration of trade-offs and synergies within and among practices with negative impacts on sustainable use (well established) {3.4}. 3.1. Introduction People directly benefit from nature by interacting with and using wild species (see 1.3.2) for definition of wild species), which provide provisioning and material contributions, and cultural and spiritual uses for human well-being (Millennium Ecosystem Assessment, 2005). Furthermore, as discussed in greater depth in Chapter 1, the ability to use wild species is crucial for social and economic justice, and to maintain the livelihoods, well-being and cultural diversity of indigenous peoples and local communities. The use of wild species involves three interconnected dimensions: (i) the wild species itself, (ii) the practices undertaken by people to obtain parts of or the whole organism, and (iii) the uses (both extractive and non-extractive) of the organism (Figure 1.1). Identifying and documenting the status of these dimensions, and their interactions and trends, is the subject of this chapter. It is important to note that the scoping document for this assessment calls for "an understanding of sustainable use of wild species that are important elements in the present and future functioning of ecosystem and their contributions to people," (p.3 of the sustainbale use of wild species scoping document). Thus, the systematic literature reviews on which much of the current chapter are based specifically focused on those uses considered to be sustainable, rather than reporting on all uses and determining their sustainability. This has clear implications for the status and trends reported in the following pages in terms of which literatures were reviewed and how status and trends are reported. The scale and scope of the overall use of wild species is needed in order to understand the status and trends of specific uses at a finer scale. This overview is provided in section 3.2, based on an analysis of a subset of global indicators previously used by IPBES and from the Biodiversity Indicators Partnership. Subsistence use includes the use of wild species by individuals or their direct social network, for nutritional, cultural, spiritual and social survival (Emery & Pierce, 2005). Wild species use also includes trade in informal and formal markets. Informal trade is defined as unrecorded trade which may be paid for in currency or in goods and services. Formal trade refers to recorded transactions in legal and illegal markets. These aspects are considered part of sustainable use. This section also provides a global level overview of human-used wild species distributions, practices and purposes. (16) Effective management systems that promote the sustainable use of wild species can contribute to broader conservation objectives (established but incomplete) {3.3.3.3.4, 3.3.3.4.1, 3.3.4.3.2, 3.3.5.2.3}. Based on assessment of 10,098 species from 10 taxonomic groups documented for the International Union for Conservation of Nature Red List of Threatened Species, at least 34% of the wild species assessed are used sustainably (established but incomplete) {3.2.1, 3.2.2, 4.2.4.3.1}. This includes 172 threatened or near-threatened species. Overall, unsustainable harvest contributes towards elevated extinction risk for 28-29% of near-threatened and threatened species from 10 taxonomic groups assessed on the International Union for Conservation of Nature Red List of Threatened Species {3.2.1, 3.2.2}. (17) Trade-offs and synergies among fishing, gathering, terrestrial animal harvesting, logging, and non-extractive practices are inherently linked but often treated exclusively or in isolation from each other (well established) {3.4}. This exclusivity is reflected in the dominant approach of practice specific policies, which leads to significant compartmentalization of rules and regulations. The bifurcation of existing uses alongside the emergence of new uses within a practice area must also be considered; for example, the positioning of capture fisheries vs. aquaculture within fishing practices; or ceremony and cultural expression vs. recreation and nature-based tourism within gathering practices. Considering these uses exclusively has led to an intense reconfiguration of intra-practice trade- offs and synergies with similar effects (well established) {3.4.5}. Intensification of existing uses and/or emergence of new uses for wild species have often led to rapid and substantial 8 reconfiguration of trade-offs and synergies within and among practices with negative impacts on sustainable use (well established) {3.4}. reconfiguration of trade-offs and synergies within and among practices with negative impacts on sustainable use (well established) {3.4}. reconfiguration of trade-offs and synergies within and among practices with negative impacts on sustainable use (well established) {3.4}. 9 9 3.1. Introduction As discussed in detail in Chapter 1, the IPBES conceptual framework recognizes different types of evidence, including but not limited to scientific knowledge. It aims to include different worldviews and associated knowledge systems equally, as much as possible, in the assessment. Therefore, throughout the chapter every effort was made to augment the systematic review of the scientific literature with knowledge from additional sources. This included drawing from experts' own experiences working with indigenous people and local communities, attending the indigenous people and local communities Workshops organized as part of this assessment, and drawing from non-scientific reputable sources when appropriate. Reports on the status and trends separated out by practice and uses (as defined in chapter 1) are provided in section 3.3 ((fishing (3.3.1), gathering (3.3.2), terrestrial animal harvesting (3.3.3), logging (3.3.4), and non-extractive practices (3.3.5)). These analyses were conducted following a common standard, but somewhat independently in order to be consistent with the standard approach in the relevant scientific and policy literature. Throughout section 3.3 all authors made every effort to draw from multiple knowledge systems in tandem. Within each 10 sub-section the information is organized, as much as possible, according to the relevant uses: ceremony and cultural expression, decorative and aesthetic, energy, food and feed, medicine and hygiene, recreation, science and education, and materials and shelter. In order to save space, only those uses relevant for the practice, and being undertaken at significant enough levels as to be appropriate for inclusion in a global assessment, are included in the various sub- sections. Within the fishing and terrestrial animal harvesting sections there are separate sections for non-lethal uses. Each practice sub-section concludes with a brief review of emerging issues to highlight complex and novel topics. These vary by practice but all sections include information on the emerging effects of the COVID-19 pandemic. Nature’s contributions to people are discussed throughout the chapter, as it was felt most effective to include the information in the relevant sections rather than sequestered into its own section. 3.1. Introduction Throughout section 3.3 it becomes abundantly clear that the ability to sustainably use wild species is important for people all over the world, in all countries where people eat meat or fish, eat berries or wild vegetables, use paper, and enjoy nature; and it is absolutely critical to indigenous peoples and local communities worldwide who fundamentally rely on wild species for their own subsistence and livelihoods in terms of food and medicinal provisioning, informal and formal trade, and often also cultural and spiritual practices. Furthermore, several sections in 3.3 point out that certain kinds of uses may create new opportunities for upward social and economic mobility for some but simultaneously exclude others, resulting in differential qualities of life and well-being for groups of people, often exaggerating existing inequalities. A growing trend in the scientific literature is increasing awareness of the trade-offs and synergies among the practices and uses, which is addressed in section 3.4. This includes a discussion of multifunctionality in different sectors. Trade-offs and synergies reflect a host of interactions, connections, relationships and linkages within, between and among practices and uses. This being the case, achieving and maintaining the goal of sustainable use of wild species hinges on the level of understanding of the key trade-offs and possible areas of synergy within and across practice areas. A simple three-pronged approach is used to consider the various trade-offs and synergies by focusing on (i) Trade-offs and synergies at intra-practice and intra- use level; (ii) Trade-offs and synergies between practices and uses; and (iii) Trade-offs and synergies involving the social, economic and environmental aspects of sustainable use. 3.2. Scale and scope: a global overview Use of wild species varies across space and over time. While use of wild species is often addressed based on local case studies, a global overview on status and trends of wild species use is lacking. In order to provide this global overview, a search was conducted across different global organization websites to select available datasets and global estimates on wild species use (3.2.1) (see the data management report for Chapter 3 systematic literature review at https://doi.org/10.5281/zenodo.6452651). Indicators for sustainable use of wild species were selected based on criteria in the scoping document and Chapter 1; this process is outlined in section 3.2.2. From a high diversity of indicators available (see chapter 2), this section focuses on (i) indicators selected by IPBES experts in the context of the global and regional assessments, (ii) Sustainable Development Goals indicators by the United Nations and (iii) the 11 Aichi Biodiversity Target indicators by the Convention on Biological Diversity, particularly focusing on indicators within the theme “sustainable use” and “species”. In addition to searching data and indicators across different institution websites, a literature search was completed in “Google” and “Science Direct” using the following keywords: “wild species” AND “use” AND “indicators” OR “indices” OR “indexes” (accessed in June 2020). Data sources and indicators suggested by reviewers during internal and external reviews were also reviewed and considered. Following the section indicators which focuses on spatial scales and distribution, we include a special section on the importance of the temporal scale in relation to use of wild species (3.2.3). Finally, this we explore the relative importance of different contexts in which wild species are used both for subsistence and trade (3.2.4). 3.2.1. Datasets available and global estimates of wild species used Sources: Flora of China (FOC), The Plant List (TPL), World Flora Online (World Flora Online), State of the World's Plants 2016 (SOTWP-2016), State of the World's Plants and Fungi 2020 (SOTWPF-2020), State of the World’s Fungi 2018 (Willis, 2018), Food and Agriculture Organization (FAO), Butchart, (2008), Global Tree Assessment (BGCI, 2021; Global Tree Assessment, 2020, 2021), (Balmford et al., 2015; WTTC, 2019a). ng on 3.3.1) Gathering (section 3.3.2) Terrestrial animal harvesting (section 3.3.3) Logging (section 3.3.4) Non extractive (section 3.3.5) oximately wild fish es (Chordata) and traded shima, mola, & oso, 2020) of crustacea es and 38 % of usca species ed by humans , 2020d) % of cone snail es are used by ns (Marsh et 021) Approximately 31,100 wild plant and 1,500 wild fungi species have documented uses (SOTWP, 2016; TPL, 2020; WFO, 2020) -Approximately 5,600 terrestrial bird, mammal, amphibian, and squamate reptile species are used and traded globally (Scheffers, Oliveira, Lamb, & Edwards, 2019) -Approximately 2,000 species of invertebrates, amphibians, fish, reptiles, birds and mammals are used as wild meat across the world (Coad et al., 2019) -4,561 birds are used for food or as pets (Butchart, 2008) -Over 300 mammal species are used for hunting (William J. Ripple et al., 2016), -Vertebrates (Chordata) are the most traded organisms (Fukushima et al., 2020) ~11% amphibians are used by people (Marsh et al., 2021) -Logging is reported to be a threat to approximately 7,400 tree species (27%) (Global Tree Assessment, 2021; IUCN, 2020b:3) -Approximately 34,000 tree species are used on a regular basis but not only for logging, (State of the World's Forest Genetic Resources (FAO, 2014c) -One in five tree species are recorded as having a specified human use and many have a variety of different uses (Global Tree Assessment, 2020) ~6,000 tree species (10%) have medicinal or aromatic use (Global Tree Assessment, 2020) ~3,716 tree species have timber use (IUCN, 2020) ~2,500 wild species are documented sources of fuel or bioenergy (SOTWP, 2020) -most common uses for trees as recorded by the International Union for Conservation of Nature Red list (IUCN, 2020b:3): Non-extractive uses tend to be based in the whole ecosystem instead of species. For example, worship in sacred groves includes all the species in the grove and its vicinity. Recreational tourism may focus on charismatic species or taxonomic group (e.g. butterfly-watching) but encompasses the whole park/ coral reef experience. 3.2.1. Datasets available and global estimates of wild species used Estimates on the number of wild species used by humans across the different regions of the globe are scarce and scattered amongst different datasets and organizations. The review of datasets presented below show that there is an uneven distribution of data available across the world documenting the number of wild species and their direct uses by humans. Most of the global datasets reviewed predominantly register and document use of wild species in the Northern Hemisphere (Figures 3.1 to 3.3). However, evidence suggests that the greater part of global biodiversity occurs in the tropical and subtropical regions, and in many of these countries local communities depend heavily on direct use of natural resources. Uses are dynamic and change over time. Traditional knowledge and skills as well as science and technology continue to develop novel techniques and adapt to changing uses (Kersey et al., 2020). The evolving relationships between wild species use and associated knowledge/skills, together with the development of science and technology, drives the creation of novel economies surrounding to and associated with the use of wild species. Unfortunately, the review shows that although traditional and scientific knowledge often highlight that one wild species can have many uses (e.g., food, raw material, cultural expression, etc.), and provide a range of nature’s contributions to people (NCP) benefits, the datasets reviewed generally focus on a single use category for a single species. Table 3.1 summarizes key estimates in order to provide an overview of the total number of wild species and their uses across different taxa and practices of use. About 50,000 wild species are used for food, energy, medicine, material and other purposes through fishing, gathering, logging and terrestrial animal harvesting globally. People all over the world directly use about 7,500 species of wild fish and aquatic invertebrates, 31,100 wild plants, of which 7,400 ON 5 species are trees, 1,500 species of fungi, 1,700 species of wild terrestrial invertebrates and 7,500 species of wild amphibians, reptiles, birds and mammals. Among the wild species that are used, more than 20% (over 10,000 species) are used for human food. The practices are further analyzed in the following sections (3.3.1 to 3.3.5). 12 Table 3.1 Number of species and their uses by practice. The table shows estimates from different sources. Only estimates corroborated by two or more sources are included. 3.2.1. Datasets available and global estimates of wild species used Forest therapy uses the whole forest, not single species. ~6,000 tree species (10%) have medicinal or aromatic use (Global Tree Assessment, 2020) ~3,716 tree species have timber use (IUCN, 2020) ~2,500 wild species are documented sources of fuel or bioenergy (SOTWP, 2020) 13 cons medi 1646 hum good Uses (average annual consumption OR trade volume) -Average consumption of 90 million tons/year (FAO, 2020d) -Food fish consumption grew form 9.0 kg per capita in 1961 to 20.2kg per capita in 2015 at an average rate of about 1.5 percent per year -There are between 3.5 and 5.76 billion users of Alagae, fungi and plants globally (Charlie M. Shackleton & de Vos, 2022) -Sales of BioTrade beneficiary companies reached €4.3 billion (2015). -International trade volume for wild edible fungi was estimated at 1.23 million tons in 2017 (de Frutos, 2020) -Around 5 million people worldwide from collectors/fishers/ hunters, workers, among others are involved in BioTrade (UNCTAD, 2017) -70% of the world's poor depend directly on biodiversity (UNCTAD, 2017) -The number of companies that report on biodiversity in their annual reporting is growing. 36 of the top 100 cosmetic companies and 60 of the top 100 food companies now mention biodiversity. -Medicinal wild plants: 60–90% of medicinal and aromatic plants in trade are wild collected -14–15 billion United States dollars estimated value of trade in essential oils by 2025 (TRAFIC, 2018) -Global value of wild algae, fungi, plants and animal origin was estimated by the Food and Agriculture Organization of the United -Very different estimates of use, ranging from 5 million tons/year globally to 4.6 million tons/year in Congo Basin alone) -Central Africa: 1.6 to 11.8 million tons/year Brazilian Amazon: 0.07 to 1.3 million tons/year (Coad et al., 2019) -Tim over (TRA -880 colle ~1.2 com urba -Glo 2011 & U -2.4 cook -Ove inter tree prod Wor 2014 -Sele glob Cock -Ove fores prac 2018 constructuction:3,716 wild species, medicine:1,951 wild species, horticulture: 1646 wild species, fuels: 1444 wild species, human food 1,382 wild species, household goods:1,302 wild species on me) -Average consumption of 90 million tons/year (FAO, 2020d) -Food fish consumption grew form 9.0 kg per capita in 1961 to 20.2kg per capita in 2015 at an average rate of about 1.5 percent per year -There are between 3.5 and 5.76 billion users of Alagae, fungi and plants globally (Charlie M. 3.2.1. Datasets available and global estimates of wild species used Shackleton & de Vos, 2022) -Sales of BioTrade beneficiary companies reached €4.3 billion (2015). -International trade volume for wild edible fungi was estimated at 1.23 million tons in 2017 (de Frutos, 2020) -Around 5 million people worldwide from collectors/fishers/ hunters, workers, among others are involved in BioTrade (UNCTAD, 2017) -70% of the world's poor depend directly on biodiversity (UNCTAD, 2017) -The number of companies that report on biodiversity in their annual reporting is growing. 36 of the top 100 cosmetic companies and 60 of the top 100 food companies now mention biodiversity. -Medicinal wild plants: 60–90% of medicinal and aromatic plants in trade are wild collected -14–15 billion United States dollars estimated value of trade in essential oils by 2025 (TRAFIC, 2018) -Global value of wild algae, fungi, plants and animal origin was estimated by the Food and Agriculture Organization of the United -Very different estimates of use, ranging from 5 million tons/year globally to 4.6 million tons/year in Congo Basin alone) -Central Africa: 1.6 to 11.8 million tons/year Brazilian Amazon: 0.07 to 1.3 million tons/year (Coad et al., 2019) -Timber trade as a whole (including wild) at over 200 billion United States dollars (TRAFIC, 2018) -880 million people spending time collecting firewood or producing charcoal ~1.2% global workforce is engaged in commercial fuel wood activities to supply urban centers (FAO & UNEP, 2020) -Global fuel wood production revenue in 2011: 33 billion United States dollars (FAO & UNEP, 2020) -2.4 billion people use fuel wood for cooking (FAO & UNEP, 2020) -Over 1,500 tree species are traded internationally (BGCI, 2021) and ~2,400 tree species are actively managed for their products and/or services (State of the World's Forest Genetic Resources (FAO, 2014c) -Selective logging represents 15 % of the global timber supply (Poudyal, Maraseni, & Cockfield, 2018) -Over 400 million ha, about 10% of global forests, are subject to selective logging practices (Poudyal, Maraseni, & Cockfield, 2018) - 120.1 billion United States dollars in gross domestic product to the global economy (2018), including multiplier effects: 343.6 billion United States dollars sustaining 21.8 million jobs (WTTC, 2019a) -8 billion visits to protected areas annually 600 billion United States dollars per year. constructuction:3,716 wild species, medicine:1,951 wild species, horticulture: 1646 wild species, fuels: 1444 wild species, human food 1,382 wild species, household goods:1,302 wild species 3.2.1. Datasets available and global estimates of wild species used (Balmford et al., 2015) constructuction:3,716 wild species, medicine:1,951 wild species, horticulture: 1646 wild species, fuels: 1444 wild species, human food 1,382 wild species, household goods:1,302 wild species es of million 4.6 Congo 11.8 zilian million 19) -Timber trade as a whole (including wild) at over 200 billion United States dollars (TRAFIC, 2018) -880 million people spending time collecting firewood or producing charcoal ~1.2% global workforce is engaged in commercial fuel wood activities to supply urban centers (FAO & UNEP, 2020) -Global fuel wood production revenue in 2011: 33 billion United States dollars (FAO & UNEP, 2020) -2.4 billion people use fuel wood for cooking (FAO & UNEP, 2020) -Over 1,500 tree species are traded internationally (BGCI, 2021) and ~2,400 tree species are actively managed for their products and/or services (State of the World's Forest Genetic Resources (FAO, 2014c) -Selective logging represents 15 % of the global timber supply (Poudyal, Maraseni, & Cockfield, 2018) -Over 400 million ha, about 10% of global forests, are subject to selective logging practices (Poudyal, Maraseni, & Cockfield, 2018) - 120.1 billion United States dollars in gross domestic product to the global economy (2018), including multiplier effects: 343.6 billion United States dollars sustaining 21.8 million jobs (WTTC, 2019a) -8 billion visits to protected areas annually 600 billion United States dollars per year. (Balmford et al., 2015) -Timber trade as a whole (including wild) at over 200 billion United States dollars (TRAFIC, 2018) -880 million people spending time collecting firewood or producing charcoal -Over 1,500 tree species are traded internationally (BGCI, 2021) and ~2,400 tree species are actively managed for their products and/or services (State of the World's Forest Genetic Resources (FAO, 2014c) 14 Nations as 20.6 billion United States dollars in 2010 (TRAFIC, 2018) -Global value of organic wild collected products to be between EUR 630 to 830 million (base year 2005 (IFOAM/ITC, 2007) 15 The International Union for Conservation of Nature (IUCN) Red List of Threatened Species (https://www.iucnredlist.org/) is one of the most widely used datasets to determine status and trends of wild species use. The list includes assessments of 128,918 species of vertebrates, invertebrates, wild plants, fungi and protists; its major focus is to report their threat categories. In the November (IUCN, 2020b:4) update of the list, the total number of species assessed was: animal: 78,126, wild plants: 50,369, fungi: 408 and Chromista: 15. 3.2.1. Datasets available and global estimates of wild species used The use of wild species is captured by the International Union for Conservation of Nature Red List in two ways: as a threat (under the threats classification scheme) and as a form of use or trade (under the use and trade classification scheme). While the coding of major threats is required (except for species of least concern), the coding of use and trade is only recommended, and is therefore less consistently coded across listed species, including the comprehensively assessed groups. To qualify as a comprehensively assessed group, the taxonomic group must include at least 150 species, of which more than 80% have been assessed (Marsh et al., 2021). The 2020 July (IUCN, 2020b:3) report shows that around 35,765 species (28%) are considered threatened to minor or major degrees. Of these, 20,935 species of animals (26.8% of the total assessed animals), 13,142 species of wild plants (26.1%) and 162 species of fungi (39.7%) were reported as threatened. Using Red List data, Marsh et al. (2021) analyzed species-level data for 30,923 species from 13 taxonomic groups which have been comprehensively assessed. Results of this study demonstrate widespread use across taxa, of approximately 40% of species (10,098 of 25,009 from 10 taxonomic groups with adequate data). This estimate is an important reminder of the relevance of the current assessment. According to this data source, wild plant groups tend to be used for more purposes than animal groups, including for food and animal feed, medicinal use, household goods and handicrafts /jewelry, fuels and chemicals. For aquatic animals, the top uses were human food (bony fishes and crustaceans), specimen harvest (cone snails), and pets and display animals (corals and bony fishes). Additional uses included handicrafts and jewelry (cone snails and corals) and medicine (cone snails). It should be noted that the majority of the taxa have multiple uses. McRae et al. (2022) using the Living Planet index data (https://livingplanetindex.org/) to show the locations of populations which were coded as utilized (black diamonds) and non- utilized (white diamonds) across the globe for practices such as fishing, gathering and hunting (non-extractive uses were not included) (Figure 3.1). Threat information from the International Union for Conservation of Nature Red List was available for 3,195 populations analyzed (1,694 utilized and 1,501 not utilized) (McRae et al., 2022). Nearly three quarters of overexploitation threats of coded utilized populations were as result of practices such as hunting and gathering. 3.2.1. Datasets available and global estimates of wild species used The results show that the global trends for those wild populations analyzed were negative, and in populations where there is no management, decline was more rapid, especially in Africa and the Americas (section 3.2.1.2). 16 Figure 3.1 Locations of utilized (black diamonds) and non-utilized populations (white diamonds). This map is directly copied from its original source (McRae et al., 2022) and was not modified by the assessment authors. The map is copyrighted under license CC BY-NC-ND 4.0. The designations employed and the presentation of material on the maps used in the assessment do not imply the expression of any opinion whatsoever on the part of IPBES concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. These maps have been prepared or used for the sole purpose of facilitating the assessment of the broad biogeographical areas represented therein and for purposes of representing scientific data spatially. Figure 3.1 Locations of utilized (black diamonds) and non-utilized populations (white diamonds) This map is directly copied from its original source (McRae et al 2022) and was Figure 3.1 Locations of utilized (black diamonds) and non-utilized populations (white diamonds). This map is directly copied from its original source (McRae et al., 2022) and was not modified by the assessment authors. The map is copyrighted under license CC BY-NC-ND 4 0 Th d i ti l d d th t ti f t i l th d i th Figure 3.1 Locations of utilized (black diamonds) and non-utilized populations (white diamonds). This map is directly copied from its original source (McRae et al., 2022) and was not modified by the assessment authors. The map is copyrighted under license CC BY-NC-ND 4.0. The designations employed and the presentation of material on the maps used in the assessment do not imply the expression of any opinion whatsoever on the part of IPBES concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. These maps have been prepared or d f th l f f ilit ti th t f th b d bi hi l 3.1 Locations of utilized (black diamonds) and non-utilized populations (white Figure 3.1 Locations of utilized (black diamonds) and non utilized populations (white diamonds). 3.2.1. Datasets available and global estimates of wild species used This map is directly copied from its original source (McRae et al., 2022) and was not modified by the assessment authors. The map is copyrighted under license CC BY-NC-ND 4.0. The designations employed and the presentation of material on the maps used in the assessment do not imply the expression of any opinion whatsoever on the part of IPBES concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. These maps have been prepared or used for the sole purpose of facilitating the assessment of the broad biogeographical areas represented therein and for purposes of representing scientific data spatially. In reports from 91 countries submitted to the Food and Agriculture Organization of the United Nations (FAO) reports on the use of wild species (wild plant, animals and fungi) for food across different taxa, over 60% of responses in Africa, Asia, Latin America and the Caribbean and Near East and North Africa Caribbean (FAO, 2019a) refer to use of wild plants as food sources. The use of wild fish as food is most reported in North America and the Pacific, while the use of wild birds as food sources is most reported in Latin America and the Caribbean (Figure 3.2). 17 Figure 3.2 Response on use of wild species for food reported by type and region. A “response” is the report of a given wild food species by a given country. Analysis based on 91 country reports. Source: (FAO, 2019a) under license CC BY-NC-SA 3.0. Figure 3.2 Response on use of wild species for food reported by type and region. A “response” is the report of a given wild food species by a given country. Analysis based on 91 country reports. Source: (FAO, 2019a) under license CC BY-NC-SA 3.0. Figure 3.2 Response on use of wild species for food reported by type and region. A “response” is the report of a given wild food species by a given country. Analysis based on 91 country reports. Source: (FAO, 2019a) under license CC BY-NC-SA 3.0. In summary, no comprehensive global dataset was found for reporting status and trends of wild species that are directly used by humans. Furthermore, aggregating estimates of species used across taxa would only serve to aggregate the levels of uncertainty within each data set. Therefore, information on use is presented by practice type. 3.2.1. Datasets available and global estimates of wild species used The following section details, in the context of these data constraints, the indicators available for assessing the sustainability of use of wild species across different practices. The practices are further analyzed in section 3.3. 3.2.1.1. Fishing Fish are a valued food source throughout the world contributing both culturally and economically to food security, especially in coastal areas (Figure 3.3). Capture fisheries constitute the largest wild food consumed by humans, with estimates from the FAO of a total capture fisheries harvest of 90 million metric tons per year over recent decades, of which about 60 million metric tons goes to direct human consumption and most of the rest as feed for aquaculture and livestock (FAO, 2020d). The most widely used data on global fisheries is on fisheries landings from 1950 to present maintained by the FAO. Reporting includes landings by country, region, and taxonomic group (http://www.fao.org/fishery/statistics/ global-capture-production/3/en). These data are widely accepted and used, while it is recognized that the landings of small-scale fisheries are almost certainly underestimated. The FAO also presents a bi-annual report estimating what fraction of these fish stocks are underfished, sustainably fished, and overexploited. As of 2017, 34.2% of global fish stocks were overfished, 59.6% were fished in accordance with maximum sustainable yield guidelines and 6.2% were underfished (FAO, 2020d). The share of fish stocks within biologically sustainable levels (maximally sustainably fished or underfished) declined from 90% in 1974 to 18 65.8% in 2015 (FAO, 2020d). Figure 3.3 below shows FAO estimates of capture production and aquaculture. These data are reported by individual countries and include only estimates of landing so do not include non-retained catch that are discarded at sea. Landings estimates for small scale fisheries are widely regarded to be significantly underestimated. 65.8% in 2015 (FAO, 2020d). Figure 3.3 below shows FAO estimates of capture production and aquaculture. These data are reported by individual countries and include only estimates of landing so do not include non-retained catch that are discarded at sea. Landings estimates for small scale fisheries are widely regarded to be significantly underestimated. 65.8% in 2015 (FAO, 2020d). Figure 3.3 below shows FAO estimates of capture production and aquaculture. These data are reported by individual countries and include only estimates of landing so do not include non-retained catch that are discarded at sea. Landings estimates for small scale fisheries are widely regarded to be significantly underestimated. Figure 3.3 Global trends in world capture fisheries and aquaculture production (excluding aquatic mammals, crocodiles, alligators and caimans, seaweeds and other aquatic plants). Source: (FAO, 2020d) under license CC BY-NC-SA 3.0 IGO. 3.2.1.1. Fishing Figure 3.3 Global trends in world capture fisheries and aquaculture production (excluding aquatic mammals, crocodiles, alligators and caimans, seaweeds and other aquatic plants). Source: (FAO, 2020d) under license CC BY-NC-SA 3.0 IGO. So far, there is no available global estimate of total number of wild fish species (marine and freshwater) used or how this varies across the globe (list of species across regions are incomplete to give an estimate). There are, however, reports that ~7,500 Chordata species traded globally are fish (Fukushima et al., 2020). A wide range of countries and regional fisheries management organizations report the status and trends of individual fish stocks, and the University of Washington maintains a database (www.ramlegacy.org) of these giving abundance trends and status for about 1,200 individual marine species stocks constituting roughly half of global fish landings (Figure 3.4). While there is data for IPBES regions such as Europe (e.g., https://www.eumofa.eu/) and North America, data for other IPBES regions are missing. 19 Figure 3.4 Map showing the amount of total marine fish landings (MMT: millions of metric tons) in a country or region covered by stocks in the RAM Legacy Database. The area of circles is proportional to the total landings from the country or region, and the dark green portion represents the fraction of landings from stocks in the RAM Legacy Database. Green-shading of countries (or regions) on the map is applied for the top 50 countries (or regions) in terms of landings in the Food and Agriculture Organization of the United Nations’ Capture Fisheries Landings Database. This map is directly copied from its original source (RAM Legacy Stock Assessment Database, 2018) and was not modified by the assessment authors. The map is copyrighted under license CC-BY 4.0. The designations employed and the presentation of material on the maps used in the assessment do not imply the expression of any opinion whatsoever on the part of IPBES concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. These maps have been prepared or used for the sole purpose of facilitating the assessment of the broad biogeographical areas represented therein and for purposes of representing scientific data spatially. Figure 3.4 Map showing the amount of total marine fish landings (MMT: millions of metric tons) in a country or region covered by stocks in the RAM Legacy Database. 3.2.1.1. Fishing The area of circles is proportional to the total landings from the country or region, and the dark green portion represents the fraction of landings from stocks in the RAM Legacy Database. Green-shading of countries (or regions) on the map is applied for the top 50 countries (or regions) in terms of landings in the Food and Agriculture Organization of the United Nations’ Capture Fisheries Landings Database. This map is directly copied from its original source (RAM Legacy Stock Assessment Database, 2018) and was not modified by the assessment authors. The map is copyrighted under license CC-BY 4.0. The designations employed and the presentation of material on the maps used in the assessment do not imply the expression of any opinion whatsoever on the part of IPBES concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. These maps have been prepared or used for the sole purpose of facilitating the assessment of the broad biogeographical areas represented therein and for purposes of representing scientific data spatially. Figure 3.4 Map showing the amount of total marine fish landings (MMT: millions of metric tons) in a country or region covered by stocks in the RAM Legacy Database. The area of circles is proportional to the total landings from the country or region, and the dark green portion represents the fraction of landings from stocks in the RAM Legacy Database. Green-shading of countries (or regions) on the map is applied for the top 50 countries (or regions) in terms of landings in the Food and Agriculture Organization of the United Nations’ Capture Fisheries Landings Database. This map is directly copied from its original source (RAM Legacy Stock Assessment Database, 2018) and was not modified by the assessment authors. The map is copyrighted under license CC-BY 4.0. The designations employed and the presentation of material on the maps used in the assessment do not imply the expression of any opinion whatsoever on the part of IPBES concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. These maps have been prepared or used for the sole purpose of facilitating the assessment of the broad biogeographical areas represented therein and for purposes of representing scientific data spatially. 3.2.1.2. Gathering 3.2.1.2. Gathering Both the State of the World’s Plants (KEW, 2020) and Flora of China (FOC, 2020) estimate that there are approximately 31,100 wild plants with documented use. Although both datasets agree on the number of plant species used, they differ on the typologies of use. Kew royal botanical garden (2020) defines “useful plants” as plant species that have been described as fulfilling a particular need for humans, animals or the environment. This definition of use differs from that produced as parts of this assessment (see Chapter 1), however since it is the definition tied to the Kew data it is used for the remainder of this discussion. According to Kew (2020), the total number of wild plant species used for human food is of 5,538, and 3,649 species are used as animal food. Medicinal use is made of 21,695 plants for medicines (20,313) and social uses (1,321). Wild plants are also used as sources of fuel (1,621) and raw 20 materials (11,365). The flora of China (FOC, 2020) reports economic use of species from 301 plant families: 1,068 species used as food, 3,815 species of medicinal plants, 713 plants for feed (grass/honey source), 531 plant species used for fiber, 1,318 timber species, 1,296 species used for ornamental purposes and 989 species used for oil (essential oils, gums, gels) (accessed June 2020). Reviews of additional datasets such as “The Plant List “(http://www.theplantlist.org/) (TPL, 2020), “World Flora Online” (http://www.worldfloraonline.org/) (WFO, 2020) and the United States of America “Plant Germplasm System” (https://npgsweb.arsgrin.gov/gringlobal/taxon/ taxonomysearch) (GRIN-WEP, 2020) were not possible in the same way because those databases are not searchable in a way that allows identification of wild species and uses across different regions of the globe. Reviews of additional datasets such as “The Plant List “(http://www.theplantlist.org/) (TPL, 2020), “World Flora Online” (http://www.worldfloraonline.org/) (WFO, 2020) and the United States of America “Plant Germplasm System” (https://npgsweb.arsgrin.gov/gringlobal/taxon/ taxonomysearch) (GRIN-WEP, 2020) were (https://npgsweb.arsgrin.gov/gringlobal/taxon/ taxonomysearch) (GRIN WEP, 2020) were not possible in the same way because those databases are not searchable in a way that allows identification of wild species and uses across different regions of the globe. Despite the documentation from Kew (2020) and the Flora of China (2020), it remains challenging to provide an estimate on the number of wild plant species that are used across different regions. 3.2.1.2. Gathering There are estimates showing that around 7,000 wild plant species are traded globally (Khoury et al., 2019; UNCTAD, 2017), suggesting that approximately 22% (7,000 out of 31,000) of those collected are destined for formal markets (see section 3.2.3). Other global estimates on wild plant use and associated gathering practices are from certification bodies such as the International Federation of Organic Agriculture Movements (https://www.ifoam.bio/) and International Trade Centre (https://www.intracen.org/). These provide an overview of organic and other standards that deal with wild gathering (mostly for certified organic) and wild harvested products worldwide. Acknowledging that these datasets likely underestimate gathering areas that are not certified, they are the best that were found at the time of the assessment. The study used certification bodies data (base year 2005) to estimate gathering areas, wild harvested products, harvest quantities, processing, collector households and sustainability across the globe. Results suggest that certified wild products are gathered across approximately 62 million hectares of land worldwide. The total global gathering area is estimated to be much larger than reported, as not all existing organic wild gathering projects were identified. According to this report, the global figure may in fact be between 78 and 104 million hectares. For comparison, the total land area of the planet is estimated at 13,003 million hectares, 4,889 million hectares of which are classified as 'agricultural area' by the FAO (this is 37.6% of the land area) (F.A.O., 2017). The largest gathering areas were reported to be in Africa (26.8 million ha) and Europe (26.7 million ha). The ten countries with largest registered areas where wild products are gathered include Romania, Kenya, Zambia, Finland, Azerbaijan, China, South Africa, Uganda, Namibia and Bolivia. These countries cover nearly 92% of the total reported registered wild gathering area. In Europe, Finland and Romania were reported to have the largest gathering areas followed by Bulgaria, Iceland and Albania. The two countries in Africa with the largest reported gathering areas (Kenya and Zambia) have only few gathering activities officially recorded. Globally, the ten products which are harvested in largest quantity are bamboo shoots, Brazil nut, lingonberry, rosehip, tea seed for oil, blueberry, iron walnut, green laver, coconut and white mushroom. These products make up 136,411 of the 223,754 tons (61%) of globally reported harvests (IFOAM/ITC, 2007). The highest quantity (138,426 tons) was reported harvested in Asia, from a relatively small area (6.2 million ha). 3.2.1.2. Gathering Approximately 200 21 different wild plant products were reported harvested in Europe. Wild berries and mushrooms were reported to be the dominant wild harvested products there. The highest amounts were harvested in Romania, Russia and Bulgaria as well as Serbia and Montenegro, Bosnia and Herzegovina and Albania. In Africa, the most important products, in terms of quantity, were reported to be rosehip, argan oil, gum Arabic, shea butter and honeybush (International Finance Corporation (IFC), 2018). The most important wild harvested products in North America are wild rice, maple syrup, wild blueberries and blue green algae. Unlike Canada, organic wild gathering in the United States of America is of less significance. Brazil nuts were reported to be the most important wild harvested product in Latin America, harvested mostly in Bolivia, Peru and Brazil. Other important products are coconut, heart of palm and rosehip. In terms of gathering area Bolivia was reported to be the leading country, followed by Brazil, Peru and Guatemala. Asia shows the widest variety of harvested products (approximately 241). Products such as bamboo shoots, walnuts, tea seeds, seaweed, berries and mushrooms are harvested in large quantities (International Finance Corporation (IFC), 2018). These products make up more than 80% of the total harvest. China is the leading country in Asia in terms of registered gathering areas (International Finance Corporation (IFC), 2018). An even larger area was reported in Azerbaijan, but the certification status was not clear. China is also the country with largest reported harvesting of organic wild harvested products in terms of weight. In Australia and Oceania, organic wild gathering has little commercial importance. Products include noni, sandalwood, sea weed, kangaroo grass and honey. There was almost no data provided on registered areas or quantities. Estimates of wild useful fungi, including those presented in the Kew reports (Willis, 2018), are largely based on a 2004 report from FAO (Boa, 2004) which is now somewhat out of date. The sustainable use assessment presents a comprehensive review of more recent literature on the various uses of wild fungi in section 3.3.2.3.4. A bit of information here demonstrates the complexities and rapid changes in this area. For example, 282 species of wild fungi are listed on official governmental legislation or guidelines as ‘fit for commercialization’ in Europe alone (Peintner et al., 2013). 3.2.1.2. Gathering Moreover, taxonomic description of fungi is far from complete and an estimated 2 million species are yet to be described (Willis, 2018). In 2019 alone 1,886 species of fungi were scientifically named (SOTWP, 2020). This knowledge gap includes widely-used and internationally traded species. For example, a study of a packet of dried porcini mushrooms purchased at a supermarket contained three species of porcini relatives previously unknown to science (Dentinger & Suz, 2014). Use of fungi as a food source is particularly important in IPBES regions such as Central Asia and Europe. One of the most recent global datasets on fungi is presented in Figure 3.5. The global fungi database, from which this figure was generated, contains over 600 million observations of fungal sequences across the world and over 17,000 samples with geographical locations (Větrovský et al., 2020). 22 Fig re 3 5 Locations of samples in the global ild f ngi database Thi i di tl Figure 3.5 Locations of samples in the global wild fungi database. This map is directly copied from its original source (Větrovský et al., 2020) and was not modified by the assessment authors. The map is copyrighted under license CC-BY 4.0 and copyright © 2019 Esri. The designations employed and the presentation of material on the maps used in the assessment do not imply the expression of any opinion whatsoever on the part of IPBES concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. These maps have been prepared or used for the sole purpose of facilitating the assessment of the broad biogeographical areas represented therein and for purposes of representing scientific data spatially. Figure 3.5 Locations of samples in the global wild fungi database. This map is directly copied from its original source (Větrovský et al., 2020) and was not modified by the assessment authors. The map is copyrighted under license CC-BY 4.0 and copyright © 2019 Esri. The designations employed and the presentation of material on the maps used in the assessment do not imply the expression of any opinion whatsoever on the part of IPBES concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. 3.2.1.2. Gathering These maps have been prepared or used for the sole purpose of facilitating the assessment of the broad biogeographical areas represented therein and for purposes of representing scientific data spatially. 3.2.1.3. Terrestrial Animal Harvesting Humans use terrestrial animals for very different purposes, such as food-feed and pets. In 2013, the United Nations Environment Programme-Worl Conservation Monitoring Centre (UNEP- WCMC), in partnership with the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) secretariat, brought various data-holdings together into one comprehensive data portal to assist Parties to implement biodiversity Multilevel Environmental 23 Agreements using available data. This global dataset documenting use of terrestrial animals is called Species+ (https://speciesplus.net). Species+ contains information on all species listed in the Appendices of the Convention on International Trade in Endangered Species of Wild Fauna and Flora, and other family listings and species included in the annexes to the European Union wildlife trade regulations. A recent global estimate shows that of 31,500 terrestrial bird, mammal, amphibian, and squamate reptile species, 5,579 species (18%) are used and traded globally (Scheffers et al., 2019). Reptiles, for example freshwater and marine terrestrial turtles, lizards, snakes, and crocodiles are widely used by indigenous people. There are very different estimates on the number of wild animals used as food sources. Estimates suggest that globally, as many as 2,000 species of invertebrates, amphibians, reptiles, birds and mammals are used for food and considered as wild meat (Ingram et al., 2015a; Redmond, 2006; Stafford, Preziosi, & Sellers, 2017). However, Marsh et al (2021) (after Butchart (2008)) reported over 4,500 wild bird species alone are used for food and pets (Butchart, 2008). Use of mammals for food is reported from North America, Africa, Europe and central Asia (Figure 3.2). The International Union for Conservation of Nature Red List (version 2021.1) has coded 1,248 mammal and 250 reptile species for use as food. Global estimates of hunting (section 3.3.3) highlight regional differences and again challenges with data collection. Estimates of annual offtake rates from forests in Central Africa, for example, range between 1.6 and 11.8 million tons of meat per year. Estimates in the Brazilian Amazon range between 0.07 and 1.3 million tons per year. No similar reviews were found for Asia, where there are still insufficient site-level hunting data to make adequate comparisons. Off-take data are similarly scarce for animal communities in savanna habitats in Africa and South America (Coad et al., 2019). 3.2.1.3. Terrestrial Animal Harvesting A meta-analysis of 78 hunting studies from sites located in Central America, Amazonia and the Guiana Shield, recorded a total of 90 hunted mammal species including 12 primates, 6 ungulate and 8 rodent genera. As in Africa, ungulates and rodents make up the majority of the wild meat offtake in neotropical communities. In the Amazon Basin, with regional variations, much of the wild meat offtake is medium-sized ungulates such as white-lipped peccary (Tayassu peccari), collared peccary (Pecari tajacu), white-tailed deer (Odocoileus virginianus) and various brocket deer (Mazama species) and tapir (Tapirus species). 3.2.1.4. Logging Estimates for the total number of tree species used vary somewhat. Both the global tree assessment (Global Tree Assessment, 2020) and estimates from the world flora online (WFO, 2020) list around 60,000 tree species across the world. Estimates differ on the number of wild tree species that are used. The FAO has previously reported 34,000 species, including fruit- and nut-trees and their wild relatives, are used on a regular basis for a range of uses, including logging, environmental, social and scientific purposes, and food (FAO, 2014c). The global tree assessment estimates 12,000 species as having at least one use, and many have a variety of uses (BGCI, 2021). According to the International Union for Conservation of Nature red list (IUCN, 2020b:3), 17,510 tree species (29.9% of all tree species), are considered threatened, 7,400 species (12%) from logging. The 2021 state of the world’s trees report also state that “the 24 second major threat to tree species, is direct exploitation, especially for timber, impacting over 7,400 tree species” (Global Tree Assessment, 2021). Although the amount of timber harvested (volume) from wild and plantation forests is recorded in several global datasets, there is little or no information available about the ways in which those trees were felled and removed from the landscape. In other words, the practice is not recorded, only the result. It is well established that clear felling is prevalent in boreal and temperate forests, and selective logging is the dominant timber harvesting practice in natural tropical forests. Over 20% of the world's tropical forests have been selectively logged (Bicknell, Struebig, & Davies, 2015). Furthermore, in the majority of the cases the data on selective logging refers only to the minimum felling diameter (normally between 45 and 60 cm diameter at breast height (DBH) only. However, selective logging practices also include other management actions such as type of cutting and regeneration planning. Therefore, the International Tropical Timber Organization estimates that less than 10% of the total permanent forest estate of tropical countries is managed sustainably (Poudyal, Maraseni, & Cockfield, 2018). 3.2.1.5. Non-extractive use Global estimates on wild species use for non-extractive practices (section 3.3.5) tend to be undocumented, and therefore there are very limited global statistics available. In contrast with fishing, gathering, hunting and logging, non-extractive uses tend to be based in experiencing the whole ecosystem. For example, worship in sacred groves includes all the species in the grove and its vicinity. Recreational tourism may focus on charismatic species but encompasses the whole park / coral reef experience. Forest therapy uses the whole forest. Species-specific tourists, such as bird- / butterfly-watchers, aim to view every species in the taxonomic group and making these observations in their native habitats in part of the experience. Global estimates on wild species use for non-extractive practices (section 3.3.5) tend to be undocumented, and therefore there are very limited global statistics available. In contrast with fishing, gathering, hunting and logging, non-extractive uses tend to be based in experiencing the whole ecosystem. For example, worship in sacred groves includes all the species in the grove and its vicinity. Recreational tourism may focus on charismatic species but encompasses the whole park / coral reef experience. Forest therapy uses the whole forest. Species-specific tourists, such as bird- / butterfly-watchers, aim to view every species in the taxonomic group and making these observations in their native habitats in part of the experience. While non-extractive uses are not always directly tied to specific species, they are an important part of wild species use and generate significant amounts of revenue worldwide. Balmford et al. (2015) report 8 billion tourist visits per year to protected areas around the world, generating approximately 600 billion United States dollars. It is estimated that tourism revenues generate 120.1 billion United States dollars in gross domestic product to the global economy (WTTC, 2019a). Cultural and economic values from the United Nations Educational, Scientific and Cultural Organization (UNESCO) Word Heritage Sites are also linked to wild species (Figure 3.6). For example, analysis of the data from United Nations on cultural landscapes (http://whc.unesco.org/en/, accessed February 2021) reveals that 23% (25 out of 113) of world heritage sites can be associated directly or indirectly with use of wild species (with different degrees of domestication). However, with the exception of recreational use of wild species, there is limited to no global data on the status and trends of other non-extractive uses such as ceremonial and cultural use, medicinal, and educational use (see section 3.3.5). 3.2.1.5. Non-extractive use 25 Figure 3.6 Locations of UNESCO cultural and natural landscapes around the world based on (UNESCO, 2021) © 1992 - 2022 UNESCO/World Heritage Centre. See data management report for the figure at https://doi.org/10.5281/zenodo.6462960. Figure 3.6 Locations of UNESCO cultural and natural landscapes around the world based on (UNESCO, 2021) © 1992 - 2022 UNESCO/World Heritage Centre. See data management report for the figure at https://doi.org/10.5281/zenodo.6462960. In addition of the datasets available for the different practices there are also worldwide repositories such as the Global Biodiversity Information Facility (GBIF) that gather data for different taxa. The Global Biodiversity Information Facility platform (https://www.gbif.org/), which currently houses 1,4 billion records (accessed 15th June 2020), documents the occurrence of a species at a given time and place, however data on wild species use is not reported systematically. The results of this review show that while there is a vast legacy of available data on species taxonomy and ecology for different taxa, most datasets do not distinguish wild from domesticated species, making this assessment on the use of wild species (as defined in the sustainable use assessment scoping document) very challenging. Although there is available data on taxonomy and ecology for different taxa, particularly in germplasms/herbariums across the world, lists of wild species available for some taxa are very incomplete. Even for the taxa where there are lists of wild species available, the focus is on biological conservation or economic value related to trade and markets rather than specifically on use as defined here. These reports are framed under different perspectives and goals (see Chapter 2) and on a practice by practice or use by use basis. Another concern regarding the available datasets is that while the reporting focuses on a use-by-use basis, a single wild species is often used for a variety of purposes. As shown in table 3.1, single species of wild plants, animals and fungi often are used for a variety of reasons (as food source, raw materials, rituals, culture and community identity). Successful and sustainable use is often associated with specialized knowledge and skills of the multifunctional use (Carvalho Ribeiro et al., 2018). Throughout generations indigenous peoples and local 26 communities often cultivate specialized knowledge and maintain skills in ways that support community well-being and maintain nature’s contributions to people. These comprehensive uses of single species are not yet captured in global datasets. 3.2.2. Global Indicators IPBES, in the context of the global and regional assessments, reviewed and systematized a list of 345 global indicators (IPBES Technical Support Unit on Knowledge and Data, 2021). From the list of indicators reviewed by experts in the context of the IPBES global assessment of biodiversity and ecosystem services, the ones likely suitable for measuring status and trends in the sustainable use assessment were selected. In order to update this list, additional indicators from the Biodiversity Indicators Partnership (https://www.bipindicators.net/; accessed October 2020) were included. The Biodiversity Indicators Partnership provides the best available information on status and trends of biodiversity, which helps to monitor progress towards the Convention on Biological Diversity and other multilateral environmental agreements. At the moment, the Biodiversity Indicators Partnership integrates indicators grouped into 8 themes. The themes on sustainable use (22 indicators) and species (42 indicators) are those most likely to apply to the sustainable use assessment. Most of the indicators reviewed by IPBES are from the Sustainable Development Goals and the Aichi Biodiversity Targets. There are 241 indicators to assess progress towards the achievement of Sustainable Development Goals (https://unstats.un.org/sdgs/indicators/; accessed October 2020). There are 22 indicators as part of the Biodiversity Indicators Partnership. For the current assessment, indicators were selected based on a three-stage process from these three sets (IPBES, SDG – Sustainable Development Goals, and BIP – Biodiversity Indicators Partnership). Indicators were chosen through a set of recurrent stages (initial selection, narrowing, assigning usefulness): 3.2.1.5. Non-extractive use In sum, although there have been advances in recent decades, there is not yet a global, harmonized observation system for delivering regular, timely data on species status and trends of biodiversity change, particularly on species that are used by humans. Core elements of this developing data infrastructure have been prototyped. For example, the “Map of Life” website (http://mol.org/) couples raw data on species biology (but not on use) with modelling approaches to inform policy making (Jetz et al., 2019) . Stage 1 - Initial selection g 1. Covers those boxes and arrows of the IPBES conceptual framework that are particularly relevant to sustainable use of wild species, 1. Covers those boxes and arrows of the IPBES conceptual framework that are particularly relevant to sustainable use of wild species, 2. Relevant for different stakeholders and end users (i.e., policy- and/or decision- relevant), 3. Reflects various knowledge systems, diverse worldviews and multiple conceptualizations of values, 4. Relevant at different spatial and temporal scales. Stage 2 - Narrow the set of indicators (IPBES global assessment used ~30 indicators) taking the following into consideration which indicators 27 1. Contributes best to the socio-ecological narrative for sustainable use (i.e., reflects both ecological and social aspects), 2. Provides the most useful information on the sustainability of the use of wild specie 3. Need to be developed to reflect the multi-dimensionality of sustainable use of wild species, 4. Provide the most relevance for future monitoring of sustainable use of wild species, 5. Reflect interdependencies and trade-offs (indicators more connected to others provide more nuanced information), 6. Apply across taxa and practices (generic indicators). If not possible, selections should include specific indicators for the major taxa and practices, 7. Are most useful for ongoing assessments. Stage 3 - Further considerations on the usefulness of the indicator for the sustainable use assessment 1. Are data already available (X) or under active development (Y)? 2. Is the indicator suitable for communication? 2. Is the indicator suitable for communication? 3. Is there a possibility for aggregation or disaggregation of data used elsewhere (e.g., National data aggregated to form a global indicator)? 3. Is there a possibility for aggregation or disaggregation of data used elsewhere (e.g 3. Is there a possibility for aggregation or disaggregatio National data aggregated to form a global indicator)? National data aggregated to form a global indicator)? 4. Is it an indicator for the Sustainable Development Goals? This review resulted in a total of 47 meaningfully useful indicators for the assessment as defined in this assessment. Fifteen indicators were likely to meaningfully contribute to estimating status and trends of (sustainable) use of wild species (Table 3.2). Thirty-two indicators relate specifically to the sustainable use assessment practices/uses (Box 3.1). Stage 1 - Initial selection This is a notably small number of indicators given that we reviewed approximately 1000 possible indicators against these criteria (including 245 indicators used for the Sustainable Development Goals, 345 from IPBES, and 300 from the Biodiversity Indicators Partnership). The analysis of the selected indicators started by associating each indicator to the list of principles of sustainable use reviewed in Chapter 2 (section 2.2.6): 16-Additional community benefits 28 17-Raise understanding and awareness 17-Raise understanding and awareness These principles were included into broad categories such as governance (1 to 5), management and monitoring (6 to 9), ecological impacts (10 to 12), socio economic benefits (13 to 16) and education (17) (see Chapter 2). Although these global indicators tend to fill in one (or in the maximum two) principles there are no indicators that cover all principles: governance, management & monitoring, ecological impacts, socioeconomic benefits and education. This suggests a need to adapt these global indicators sets to better represent the holistic dimensions of sustainable use of wild species. Our review of the global indicator sets also shows that most of the indicators developed and used by the Sustainable Development Goals, the Convention on Biological Diversity (Biodiversity Indicators Partnership) and IPBES would need to be adapted in order to target wild species that are used. Table 3.2 lists indicators that can currently be used to assess status and trends in the use of wild species. The ones in bold are described in more detail below the table. The additional sources are included here for reference, but are not included in the more detailed analysis. Table 3.2 Selected status and trends indicators. Abbreviations: SDGs: Sustainable Development Goals. Source Name and brief description of the indicator 1 McRae et al., (2022) A global indicator of utilized wild species populations: regional trends and the impact of management 2 Marsh et al., (2021) Prevalence of sustainable and unsustainable use of wild species inferred from the International Union for Conservation of Nature’s Red List 3 Biodiversity Indicator Partnership, Tierney et al., (2014) “Use it or lose it” 4 Biodiversity Indicator Partnership, Khoury et al., (2019) Comprehensiveness of conservation of socioeconomically as well as culturally valuable species 5 IPBES-SES Species Habitat Index (wild species) Species Status Information Index 6 Aichi Biodiversity Target 13 Red List Index (impacts of utilization/wild relatives of domesticated animals) 7 Biodiversity Indicator Partnership, IPBES Proportion of local breeds (wild species) classified as being at risk, not-at-risk or at unknown level of risk of extinction 8 Biodiversity Indicator Partnership Biodiversity Intactness Index Table 3.2 Selected status and trends indicators. Abbreviations: SDGs: Sustainable Development Goals. Table 3.2 Selected status and trends indicators. Abbreviations: SDGs: Sustainable Development Goals. 17-Raise understanding and awareness 29 9 Sustainable Development Goal 2 Indicator 2.5.1 Number of plant and animal genetic resources for food and agriculture secured in either medium or long-term conservation facilities (wild species) 10 Biodiversity Indicator Partnership Number of species extinctions (birds and mammals) (used species) 11 Faitrade International Trade volume in Fairtrade certified goods (wild) 12 Sustainable Developemt Goals/Organization for Economic Cooperation and Development Number of intangible cultural heritage practices - under the category of 'knowledge and practices concerning nature' per country (sustainable use) 13 Sustainable Developemt Goals /Organization for Economic Cooperation and Development Number of countries with national instruments on biodiversity- relevant taxes, charges and fees 14 IPBES Biodiversity Engagement Indicator 15 Biodiversity Indicator Partnership Living Planet Index (utilized/non utilized species) The following text provides details regarding the key sources listed in table 3.2. Those presented in more detail are those that report on the most recent global indicators from the peer- reviewed literature. Source 1: The global indicator developed by McRae et al. (2022) follows the method used to calculate the Living Planet Index (https://www.bipindicators.net/indicators/living-planet- index). McRae et al., (2022) used a global data set of over 11,000 time-series to derive indices of ‘utilized’ and ‘not utilized’ wild species and assess global and regional changes, principally for mammals, birds and fish . Their work also explored the role that targeted management has in predicting population trends in utilized populations. The results of this work show that from 1970 – 2016 wild species population trends globally, both used and non-used, are negative (Figure 3.7) for both terrestrial and freshwater (TFW) and marine (M) species for all IPBES regions. Note that the trends being shown here are for populations, not for sustainable use. 30 Figure 3.7 Index of utilized populations for IPBES Regions. Abbreviations: TFW: Terrestrial and Freshwater, M: Marine Source (McRae et al., 2022) under license CC BY-NC- ND 4.0. Figure 3.7 Index of utilized populations for IPBES Regions. Abbreviations: TFW: Terrestrial and Freshwater, M: Marine Source (McRae et al., 2022) under license CC BY-NC- ND 4.0. Figure 3.7 Index of utilized populations for IPBES Regions. Abbreviations: TFW: Terrestrial and Freshwater, M: Marine Source (McRae et al., 2022) under license CC BY-NC- ND 4.0. On average, utilized populations declined by 50% over the 46-year period (0.41 - 0.62) and non-utilized populations declined by 3% (0.80 - 1.18). 17-Raise understanding and awareness Figures 3.7 and 3.8 show the estimated total change from the best linear mixed-effect model (binomial and location as random effects). Coefficients show the estimated overall change (log10) for mammals, fish and birds. This work found no significant interaction between taxonomic group and utilization; however, it does show utilized populations tend to decline more rapidly, especially in Africa and the Americas (McRae et al., 2022). However, where utilized populations are managed, there is a positive impact on the trend. This work corroborates that use of species can either be a driver of negative population trends, or a driver of species recovery, with numerous species and population specific case examples making up these broader trends (see section 3.3 for more details and case studies). The role of management, especially with regards to trade, has been controversial. A considerable body of research on vertebrate species in international trade reports an overall perverse trend in use, with management having a limited mitigating effect sustainability species (Morton, Scheffers, Haugaasen, & Edwards, 2021). International trade databases such as the Convention on International Trade in Endangered Species of Wild Fauna and Flora 31 (https://trade.cites.org/) can shed light on this by reporting both negative and positive effects on population status. In some cases, economic incentives to use a listed species can be directly linked to facilitating recovery and demonstrating non-detrimental use. The role of the Convention on International Trade in Endangered Species of Wild Fauna and Flora can therefore be pivotal for linking use of species with its management and recovery plans. Figure 3.8 Global trends in utilized vs non utilized species for species of bird, mammal and fish. Source (McRae et al., 2022) under license CC BY-NC-ND 4.0. Figure 3.8 Global trends in utilized vs non utilized species for species of bird, mammal and fish. Source (McRae et al., 2022) under license CC BY-NC-ND 4.0. Source 2: the International Union for Conservation of Nature red list data assess species risk of extinction in relation to threat categories: Least Concern (LC), Near Threatened (NT), Vulnerable (VU), Endangered (EN), Critically Endangered (CE). Red list data show that while use is considered a threat for some species, for others use is not associated with red list threat categories. For example, the work by Marsh et al. (2021) shows that for the 10,000 wild species where use and trade data are reported, use is likely unsustainable for 16% of species. 17-Raise understanding and awareness However, the majority (72%) of species that are used are not threatened, with 34% of used species having stable or improving population trends. Marsh and colleagues (2021) suggest that use is likely to be sustainable for the majority of the species analyzed. Across Near Threatened (NT) and threatened species, a higher overall proportion of aquatic species than terrestrial species have intentional biological resource use coded as a threat. Among aquatic groups, the taxa with highest prevalence are corals (388 species) and almost all cartilaginous fishes (314 out of 318 species), with fishing the predominant threat. In the terrestrial groups, cycads appear most affected (147 –152 of 255 species), largely due to gathering (147 species) (Figure 3.9). For 48% of the total number of species assessed it was 32 not possible to determine the associations between use and status and trends of the species (Marsh et al., 2021).). Figure 3.9 Percentage species by the International Union for Conservation of Nature Red List Category in A) aquatic and B) terrestrial groups that are subject to use and trade. Legend: LC(-) = Least Concern species with declining population trend; LC(?) = Least Concern species with unknown population trend; LC () = Least Concern species with stable or increasing population trend. Note that being LC and having a declining population trend, or being threatened and being subject to use and trade, does not imply that use is a major threat. NT =Near Threatened, Vu =Vulnerable, EN= Endangered, CR=Critically Endangered. Source: (Marsh et al., 2021) under license CC-BY. Figure 3.9 Percentage species by the International Union for Conservation of Nature Red List Category in A) aquatic and B) terrestrial groups that are subject to use and trade. Legend: LC(-) = Least Concern species with declining population trend; LC(?) = Least Concern species with unknown population trend; LC () = Least Concern species with stable or increasing population trend. Note that being LC and having a declining population trend, or being threatened and being subject to use and trade, does not imply that use is a major threat. NT =Near Threatened, Vu =Vulnerable, EN= Endangered, CR=Critically Endangered. Source: (Marsh et al., 2021) under license CC-BY. Source 3: The “use or lose it” by Tierney et al. 17-Raise understanding and awareness (2014) measures trends in the use of wild species, with a focus on both terrestrial and aquatic vertebrate arctic species using two indicators which they developed: the Utilized Species Index (applied at the global scale based on the Living Planet Index) and the Harvest Index (applied in the Arctic region only). The examined data is on amphibian, bird, fish, mammal and reptile species from freshwater, marine and terrestrial realms. Source 3: The “use or lose it” by Tierney et al. (2014) measures trends in the use of wild species, with a focus on both terrestrial and aquatic vertebrate arctic species using two indicators which they developed: the Utilized Species Index (applied at the global scale based on the Living Planet Index) and the Harvest Index (applied in the Arctic region only). The examined data is on amphibian, bird, fish, mammal and reptile species from freshwater, marine and terrestrial realms. The results of the utilized species index show that between 1970 and 2007 populations of utilized freshwater species declined by 3% and utilized marine species declined by 17%. The populations of utilized terrestrial species decreased 21% over the same period. However, according to this study since the early 2000s the rate of decline of utilized marine and terrestrial species indices has slowed or stabilized. The utilized freshwater species index has been 33 increasing steadily since 2000. The index for species used reports that from 1997 to 2007 wild species population used for food and species used as pets declined by 17% and 9%, respectively. However, like with terrestrial species, after 2000 this trend inverted (Figure 3.10). increasing steadily since 2000. The index for species used reports that from 1997 to 2007 wild species population used for food and species used as pets declined by 17% and 9%, respectively. However, like with terrestrial species, after 2000 this trend inverted (Figure 3.10). Figure 3.10. Trends (-/+ 95% CI) in (a) utilized Artic species compared to the Artic Species Trends Index between 1970 and 2007 and (b) Harvest Index of Artic Species between 1970 and 2006, with zones of unsustainable, cautionary and sustainable harvest levels shown. Source (Tierney et al., 2014), © Cambridge University Press, license number 5146950749639 CC-BY NC. Figure 3.10. 17-Raise understanding and awareness Trends (-/+ 95% CI) in (a) utilized Artic species compared to the Artic Species Trends Index between 1970 and 2007 and (b) Harvest Index of Artic Species between 1970 and 2006, with zones of unsustainable, cautionary and sustainable harvest levels shown. Source (Tierney et al., 2014), © Cambridge University Press, license number 5146950749639 CC-BY NC. Despite these initial reviews, it remains challenging to undertake comparisons of population trends between utilized and non-utilized species. Most of the datasets available lack the detail needed to do meaningful comparisons amongst utilized vs non utilized species. In this context it is difficult to account for the range of influential factors that could be influencing these trends. Without the ability to account for additional factors and correlated them with these datasets, it is incorrect to assume that use is the primary driving factor of decline or increase in population size. 34 Source 4: The “Comprehensiveness of conservation of useful wild plants” by Khoury et al., (2019) was included in the Biodiversity Indicators Partnership. In developing the indicator, 6,941 wild plants native to different countries were selected from the United States of Amercia dataset “GRIN-WEP” (2020). The resulting in-situ indicator shows the extent to which wild species economically used across the world are conserved in situ (through conservation areas). This indicator ranges from 1 (Andorra, Falkland Islands, Gibraltar, Kiribati, Niue, Palestinian Territory, St. Helena, Timor-Leste, and United States of America minor outlying islands) to 642 (Turkey). The mean number of species used across countries is 141; the median is 86. An interactive version of this indicator is available at https://ciat.cgiar.org/usefulplants- indicator/. Areas where in-situ conservation is likely low are concentrated in Asia and to a lesser extent in Sub-Saharan Africa (Figure 3.11). Source 4: The “Comprehensiveness of conservation of useful wild plants” by Khoury et al., (2019) was included in the Biodiversity Indicators Partnership. In developing the indicator, 6,941 wild plants native to different countries were selected from the United States of Amercia dataset “GRIN-WEP” (2020). The resulting in-situ indicator shows the extent to which wild species economically used across the world are conserved in situ (through conservation areas). This indicator ranges from 1 (Andorra, Falkland Islands, Gibraltar, Kiribati, Niue, Palestinian Territory, St. Helena, Timor-Leste, and United States of America minor outlying islands) to 642 (Turkey). The mean number of species used across countries is 141; the median is 86. FISHING (13) 1. Sustainable Development Goals indicator 14.4.1 Proportion of fish stocks within biologically sustainable levels 2. Sustainable Development Goals indicator 14.6.1 Degree of implementation of international instruments aiming to combat illegal, unreported and unregulated fishing 3. Sustainable Development Goal indicator 14.b.1 Degree of application of a legal/regulatory/policy/institutional framework which recognizes and protects access rights for small‐scale fisheries 4. Number of Marine Stewardship Council’s (MSC) chain of custody certification holders by distribution country 5. Number and volume of Marine Stewardship Council certified, consumer-facing products by distribution country 6. Marine Stewardship Council certified catch, Ocean Health Index 7. Red List Index (impacts of fisheries) & number of species listed in the Appendices of the Convention on International Trade in Endangered Species of Wild Fauna and Flora 9. Living Planet Index (trends in target and bycatch species) 10. Large reef Fish, policies make adequate provisions to minimize impacts of fisheries on threatened species 11. Illegal, unreported and unregulated fishing 12. Full access to marine resources 13. Inland fishery production 17-Raise understanding and awareness 35 Box 3.1 List of possible indicators by practice (selected from indicators developed for the Sustainable Development Goals, Biodiversity Indicators Partnership & IPBES) FISHING (13) Box 3.1 List of possible indicators by practice (selected from indicators developed for the Sustainable Development Goals, Biodiversity Indicators Partnership & IPBES) 17-Raise understanding and awareness An interactive version of this indicator is available at https://ciat.cgiar.org/usefulplants- indicator/. Areas where in-situ conservation is likely low are concentrated in Asia and to a lesser extent in Sub-Saharan Africa (Figure 3.11). Figure 3.11 In situ conservation indicator. This map is directly copied from its original source (Khoury et al., 2019) and was not modified by the assessment authors. The map is copyrighted under license CC-BY 4.0. The designations employed and the presentation of material on the maps used in the assessment do not imply the expression of any opinion whatsoever on the part of IPBES concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. These maps have been prepared or used for the sole purpose of facilitating the assessment of the broad biogeographical areas represented therein and for purposes of representing scientific data spatially. Figure 3.11 In situ conservation indicator. This map is directly copied from its original source (Khoury et al., 2019) and was not modified by the assessment authors. The map is copyrighted under license CC-BY 4.0. The designations employed and the presentation of material on the maps used in the assessment do not imply the expression of any opinion whatsoever on the part of IPBES concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. These maps have been prepared or used for the sole purpose of facilitating the assessment of the broad biogeographical areas represented therein and for purposes of representing scientific data spatially. In addition to the 16 indicators listed above (Table 3.2) there are indicators which can be used to characterize the specific practices detailed in section 3.3 (Box 3.1). The review of indicators highlights that while information on harvesting of terrestrial species is limited, there is a diversity of indicators tracking the off-take and use of marine species. Although there are several indicators used for fisheries, they do not capture the specificities of small-scale fisheries and inland fisheries. These topics are discussed in greater detail in section 3.3. LOGGING (5) 1. Sustainable Development Goal indicator 15.2.1 Progress towards sustainable forest management (wild species) 2. Area of forest under sustainable management (wild species): total forest area under management certification (Forest Stewardship Council and Programme for the Endorsement of Forest Certification) 3. Red List Index (forest tree specialist species) & number of species listed in the Appendices of the Convention on International Trade in Endangered Species of Wild Fauna and Flora 4. Timber trade volume in Fairtrade certified goods 4. Timber trade volume in Fairtrade certified goods 5. Total wood removals 5. Total wood removals 5. Total wood removals GATHERING (5) 1. Quantity of mushrooms and truffles, yield (hectogram/hectare) per country 2. Species richness of medicinal plants per country 3. Indigenous and local knowledge trends associated with medicinal plants 4. Number of contracting Parties to the International Treaty on Plant Genetic Resources for Food and Agriculture (adapted for wild species) 5. Red List Index (wild species used for food and medicine) & number of species listed in the Appendices of the Convention on International Trade in Endangered Species of Wild Fauna and Flora TERRESTRIAL ANIMAL HARVESTING (6) 1. Agreement on International Humane Trapping Standards (AIHT) database 36 2. Red List Index (internationally traded wild species) & number of species listed in the Appendices of the Convention on International Trade in Endangered Species of Wild Fauna and Flora 3. The Living Planet Index (a measure of the state of global biological diversity based on population trends of vertebrate species from around the world) 4. The species abundance per country (for selected species) 5. Animal individuals hunted yearly per country (for selected species) 6. Proportion of traded wild species that was poached or illicitly traded 3.2.2.1. Indigenous Indicators The importance of wild species in a diversity of livelihood strategies is well recognized, particularly for indigenous peoples and local communities. However little attempt has been made in the available global indicator sets to comprehensively quantify the spatial and temporal scales of sustainable use of wild species occurring specifically in indigenous and local communities across the globe. The United Nations are aware of this gap. The permanent forum requested the inter-agency support group on indigenous peoples’ issues, specifically those agencies working on land tenure and changes in land use, to step up cooperation in order to operationalize indicators on these topics as they pertain to traditional territories (lands and waters) of indigenous peoples. The goal was to create a global multipurpose indicator in order to report on status and trends, in line with the Convention on Biological Diversity, the 2030 Agenda for Sustainable Development and the United Nations Declaration on the Rights of Indigenous Peoples (UNDRIP). The permanent forum recommended that the inter-agency and expert group on Sustainable Development Goal indicators provide support for the inclusion and methodological development of core indicators for indigenous peoples in the global indicator framework (https://www.un.org/development/desa/indigenouspeoples/mandated-areas1/data-and- indicators/recs-data.html). In particular, the inclusion of an indicator on the legal recognition of land rights of indigenous peoples for the targets under Sustainable Development Goals 1 and 2 was requested (United Nations Department of Economic and Social Affairs, 2020). indicators/recs-data.html). In particular, the inclusion of an indicator on the legal recognition of land rights of indigenous peoples for the targets under Sustainable Development Goals 1 and 2 was requested (United Nations Department of Economic and Social Affairs, 2020). A key data point for indigenous indicators is regarding spatial patterns of occupancy of indigenous communities around the globe, including estimates for total area and sizes of land plots for habitation and a range of traditional livelihood practices. A recent effort to map the occupancy patterns of indigenous lands at the global scale was undertaken by Garnett et al. (2018). In this study, the authors show that indigenous lands seem to have the appropriate scale to support the implementation of several global conservation and climate agreements while also maintaining sustainable local use and local governance institutions. However, details on the scale of sustainable use (both spatial and temporal) were not explicitly presented in this study. NON-EXTRACTIVE PRACTICES (3) 1. Sustainable Development Goal indicator 12.b. Number of sustainable tourism strategies or policies and implemented action plans with agreed monitoring and evaluation tools 2. Importance of protected areas for stimulating eco-tourism and nature-related leisure activities 3 Proportion of jobs in sustainable tourism industries out of total tourism jobs 2. Importance of protected areas for stimulating eco-tourism and nature-related leisure activities 3. Proportion of jobs in sustainable tourism industries out of total tourism jobs These global indicators cover different dimensions associated with sustainability and sustainable use (Box 3.1). For example, while most of the indicators for gathering focus on the extent of harvest as a function of area per country, indicators for terrestrial animal harvesting tend to focus on trends in use (overall use increasing or decreasing). Terrestrial animal harvesting indicators also tend to emphasize trade data sources such as the Convention on International Trade in Endangered Species of Wild Fauna and Flora, which may exclude a large number of species which are harvested but not formally traded. In summary, despite the importance of wild species to economies and livelihoods, relatively few global datasets and indicators have been developed speciﬁcally to monitor the status and trends of wild species that people use, except for economically valuable fish species reported on by the biannual reports “State of world fisheries and aquaculture” prepared by the FAO. Particularly lacking are attempts to examine how indicators of species use and sustainable harvest could be linked to provide a broader picture of what, where and how people are using wild species (Tierney et al., 2014). 37 3.2.3. Temporal scale and use Use of wild species varies over time. Although there is evidence that temporal scale influences sustainability of use of wild species, based on the review above the temporal dimension has been overlooked in global datasets (section 3.2.1.1) and the global indicator system (section 3.2.1.2). The dedicated attempt here to introduce longer-term temporal indicator dimensions to sustainable use indicators is therefore very much a step forward. There are many insights to be gathered from longer term perspectives, many of which directly challenge more temporally constrained conclusions. Correlative reasoning is sometimes entirely displaced through longer term trend reviews. Another important reason to consider temporal scale for assessing sustainable use is that harvesting intervals vary widely across species. Some species may be subject to seasonal, periodic or annual harvests, others may be biennial or triannual. Timber is often harvested according to a decadal cycle. Other species, such as some wild edible fungi, may be harvested sporadically when they fruit abundantly. Perception and organization of time is very basic to the internal ordering of all cultures and there are strong evidences of such calendars from all continents across the globe (Dhyani, 2018; Dhyani, Maikhuri, & Dhyani, 2011). Seasonal calendars reveal a body of knowledge about the relationship between people and the environment and underpin local Natural Resource Management (NRM) strategies. These knowledge systems have been built through strong observational, practice-based methods that have been used for centuries. They continue to be enacted and tested, and have sustained consecutive generations by adapting continually, if incrementally, to the local context over time (Woodward & Marrfurra McTaggart, 2019). Seasonal calendars have been used by indigenous peoples and local communities for generations for monitoring and adaptive management of natural resources, agricultural systems (Bhagawati, Sen, & Shukla, 2017; Jiao et al., 2012; Saylor, Alsharif, & Torres, 2017), climate change (Balehegn, Balehey, Fu, & Liang, 2019; Cochran et al., 2016; Fu et al., 2012; D. Yang & Pomeroy, 2017), water (Woodward, Jackson, Finn, & McTaggart, 2012), and to guide eco-health decision making (SantoDomingo, Castro-Díaz, González-Uribe, Wayúu Community of Marbacella and El Horno, & Barí Community of Karikachaboquira, 2016).The temporal scale of use is also important for measuring the nutritional value and food availability across the "seasonal evenness": when there are many species in the system, the likelihood is increased that one species or another is “in season” at all times (FAO, 2017a; Powell et al., 2015). 3.2.2.1. Indigenous Indicators Contrary to the large size of most indigenous lands (large extents that can be mapped at coarse resolutions), identifying the spatial patterns of occupancy of “other” traditional livelihoods, (plots with smaller sizes than can only be mapped at finer grained resolutions) is very challenging. Yet, actors at smaller scales are active natural resource users within many social-ecological systems. The failure to so far comprehensively map and measure the multi- scaled and interwoven distributions of traditional communities’ and livelihoods’ diverse spatial occupancy patterns likely make these users of wild species invisible to policy makers. For example, in order to estimate scale of use of wild species supporting different types of livelihoods one can, to some extent, explore the spatial scale (grain and extent) of the land consigned by law to different communities and map their rights of use and land tenure regimes. Indeed, traditional communities and their rights are defined by law (including international agreements). Recognizing and identifying these diverse legal frameworks and the associated spatial occupancy patterns of their territories can be a way forward to estimate the spatial scale of use 38 of wild species globally. However, territoriality and tenure clarification are highly complex, politically driven and often a very slow process. Moreover, while de jure standards may be defined, the de facto realities might show evidence of positive long-term care and stewardship or negative effects such as failed law enforcement, denied constitutional protections, and in some cases a weak and indiscriminate rule of law. Other data/indicators can then be used, that can complement land ownership datasets in order to provide the best estimate available for different types of uses of wild species. The next section provided a brief review of key aspects of the temporal scale of use (3.2.3) and economic, ecological and social contexts for sustainable use across the globe (3.2.4). Section 3.3 goes into detail on a practice-by-practice basis. 3.2.3. Temporal scale and use Seasonal calendars have been relevant in cross-cultural interpretation of indigenous 39 ecological knowledge and a relevant communication tool. While seasonal knowledge for temporal scale of use of wild species has not been sufficiently utilized in sustainable use of wild species or natural resource management (Franco, 2015; Prober, O’Connor, & Walsh, 2011; Woodward et al., 2012), this is beginning to change. One of the best-known representations of indigenous seasonal calendars in Australia is the poster series developed by multiple researchers and indigenous communities, supported and collated by the Commonwealth Scientific and Industrial Research Organization (CSIRO https://www.csiro.au/en/). This series started with the Ngan’gi seasons calendar in 2009 (on the Daly River, Northern Territory, see Figure 3.13) and includes the Tiwi seasons calendar (Prober et al., 2011) (see Figure 3.14). The main focus is ecological knowledge and customary activities of resource use. Examples: Examples: 1. Fodder and leaf litter removal calendars. Fuelwood and fodder gathering from nearby forests is a frequent and year-round activity, but leaf litter removal is a seasonal practice occurring only during dry winter months (November-March) when there is a lot of leaf litter available on the forest floor (Dhyani, 2018; Dhyani et al., 2011) (Figure 3.12). Seasonal harvesting is enforced by local village forest management committees to ensure sustainable harvest of fodder, fuelwood, litter and other wild plants and fungi (Misra, Maikhuri, Kala, Rao, & Saxena, 2008). 1. Fodder and leaf litter removal calendars. Fuelwood and fodder gathering from nearby forests is a frequent and year-round activity, but leaf litter removal is a seasonal practice occurring only during dry winter months (November-March) when there is a lot of leaf litter available on the forest floor (Dhyani, 2018; Dhyani et al., 2011) (Figure 3.12). Seasonal harvesting is enforced by local village forest management committees to ensure sustainable harvest of fodder, fuelwood, litter and other wild plants and fungi (Misra, Maikhuri, Kala, Rao, & Saxena, 2008). Figure 3.12 Annual local biomass removal calendar for Western Himalayas. A: Fuelwood, B: Fodder, C: Leaf litter, D:Crop support and other wild algae, fungi and Figure 3.12 Annual local biomass removal calendar for Western Himalayas. A: Fuelwood, B: Fodder, C: Leaf litter, D:Crop support and other wild algae, fungi and 40 plants, E: Timber. Source: (Dhyani, 2018) © International Society for Tropical Ecology CC- BY NC. plants, E: Timber. Source: (Dhyani, 2018) © International Society for Tropical Ecology CC- BY NC. 2. Seasonal migratory calendars of Tibetan pastoralist communities in Tibet, (an autonomous region of China) and Western Himalayas, India. Pastoralists in high mountains of Tibet and Indo-Mongoloid Bhotiya tribal sub-communities (Tolchha, Marcha and Jad) primarily adopt centuries old ancestral seasonal migratory livestock raising as a key mechanism for enhancing their ecological sustainability and use first hand observations as ecological indicators to decide the timing of seasonal activities (Fu et al., 2012; Maikhuri et al., 2011; H. Yang et al., 2019). 3. Calendar of Tajik community, Xinjiang, China. Tajik people perceive indicators, including the appearance of migratory birds (Motacilla alba and Motacilla citreola), the height of grass and the conditions of farmland for conducting their activities for the aims of food production, livestock keeping, and fodder and gathering medicinal plants. They have also developed strategies to keep themselves protected from firewood shortage due to high elevations. Examples: These indicators are recognized by local people, associated with their seasonal activities, and passed down through generations. 4. The Ngan’gi seasons calendar. This is an indigenous temporal management approach practiced by remote indigenous communities of Pine Creek and Naiuyu Nambiyu in the Daly River catchment, Australia. The Ngan’gi Seasons calendar has informed the scientific understanding of patterns of resource use and relationships between people, subsistence use and river flows in the Daly River catchment (Woodward et al., 2012) (Figure 3.13). The calendar is a relevant guidance approach for sustainable and rotational gathering, hunting and fishing of wild resources. Hunting and gathering of resources start towards the end of the Wet season, known as Wudupuntyurrutu in the calendar, with the harvest of fruits. Saltwater crocodile (Crocodylus porosus), echidna (Tachyglossus aculeatus) and rock python (Liasis olivaceus) are also actively hunted during this period. The dry season, known as Wurr wirribem filgarri, brings active hunting for freshwater prawn (Macrobrachium rosenbergii) in the river and creeks. Indicators of the start of dry season are wind flow from the east and presence of dragonflies that indicates fishing time for barramundi (Lates calcarifer). Wurr bengin derripal, a late wet/early dry season, is a good time to harvest the eggs of magpie goose (Anseranas semipalmata) and catfish (Arius spp.), but is not yet time for hunting other fish. Resource gathering increases in Wirirr marrgu with hunting for turtles (Carettochelys insculpta; Chelodina rugosa; Emydura spp.; Elseya spp.) and also fish (black bream, Hephaestus fuliginosus; archer fish, Toxotes chatereus; mullet Liza spp.; and freshwater species). During the beginning of the wet season a range of lilies and other water‐dependent plants are gathered from swampy areas that include waterlily, red lotus lily, and water chestnut. At this time, native peanut, and bush banana are also harvested. With lower water levels it is easier to harvest mussels, and crabs from creeks and springs. 41 Figure 3.13 The Ngan’gi Seasons calendar. Source: (CSIRO, 2021). © Tiwi Land Council and CSIRO. CC-BY NC. Figure 3.13 The Ngan’gi Seasons calendar. Source: (CSIRO, 2021). © Tiwi Land Council and CSIRO. CC-BY NC. 5. Urban foraging calendars. Urban foraging as modern gathering practice has received attention around the world (Friedlander, Stamoulis, Kittinger, Drazen, & Tissot, 2014). Urban foragers make and share foraging calendars that guide them on what to gather in urban landscapes, where and in what seasons. Examples: National Geographic developed a guide for the United Kingdom (https://www.nationalgeographic.co.uk/travel/2020/07/a-year- round-foraging-calendar-what-to-pick-and-where-in-the-uk).This not only informs foragers about better foraging approaches but also promotes more sustainable harvesting of wild species from urban spaces that already have a lot of pressure on natural urban green spaces. 6. Tiwi seasons calendar. Traditional owners from the Tiwi Islands and the Tiwi land council collaborated with the Commonwealth Scientific and Industrial Research Organization to develop two calendars, a calendar of Tiwi seasonal ecological knowledge and a calendar of wild plants and animals of Tiwi significance (Figure 3.14). The development of the calendars came from a desire to document seasonal-specific knowledge and ecological knowledge of the Tiwi Islands in an appealing format accessible to both students and the broader community, as well as a strong concern about the loss of knowledge as older people pass away. 6. Tiwi seasons calendar. Traditional owners from the Tiwi Islands and the Tiwi land council collaborated with the Commonwealth Scientific and Industrial Research Organization to develop two calendars, a calendar of Tiwi seasonal ecological knowledge and a calendar of wild plants and animals of Tiwi significance (Figure 3.14). The development of the calendars came from a desire to document seasonal-specific knowledge and ecological knowledge of the Tiwi Islands in an appealing format accessible to both students and the broader community, as well as a strong concern about the loss of knowledge as older people pass away. 42 Figure 3.14 Tiwi seasons calendar. This calendar show month of year in the outermost ring, then three “major” Tiwi seasons recognized by weather. Note that Kuwunupunari does not have a shar boundary with Tiyari. Within this ring are smaller seasons recognized by weather or ecological and associated with particular activities. Source: (CSIRO, 2014) © Tiwi Land Council and CSIRO. CC-BY NC. Figure 3.14 Tiwi seasons calendar. This calendar show month of year in the outermost ring, then three “major” Tiwi seasons recognized by weather. Note that Kuwunupunari does not have a shar boundary with Tiyari. Within this ring are smaller seasons recognized by weather or ecological and associated with particular activities. Source: (CSIRO, 2014) © Tiwi Land Council and CSIRO. CC-BY NC. 7. Seasonal round of harvest activities in Fort Yukon. The Gwich'in Athabaskans of Fort Yukon, Alaska, follow a strict seasonal round established by their ancestors over centuries. Examples: Their calendar of activities has evolved in response to northern environmental conditions such as animal migrations which make them seasonally abundant or absent, ice and snow cover which affect travel and access to resources, and preferences for certain qualities found in resources at specific times of the year (https://www.culturalsurvival.org/publications/cultural-survival-quarterly/wild-food- its-season-seasonal-round-harvest-activities). 8. Hawaiian moon calendar for responsible fishing practices. The community in the Ho'olehu Hawaiian Homesteads on the island of Moloka'i is strengthening community influence and accountability for the health and long-term sustainability of their marine resources through revitalization of local traditions and resource knowledge. The traditional system in Hawai'i emphasized social and cultural controls on fishing with a code of conduct that was strictly enforced. Local resource monitors, in conjunction 43 with visiting scientists, are creating a predictive management tool based loosely on the Hawaiian moon calendar to guide responsible fishing practices. Community- sanctioned norms for fishing conduct are being reinforced through continual feedback based on local resource monitoring, education, and peer pressure. Hawaiian community building and proper cultural protocols are essential to understand and revitalize marine conservation traditions (Friedlander et al., 2014). with visiting scientists, are creating a predictive management tool based loosely on the Hawaiian moon calendar to guide responsible fishing practices. Community- sanctioned norms for fishing conduct are being reinforced through continual feedback based on local resource monitoring, education, and peer pressure. Hawaiian community building and proper cultural protocols are essential to understand and revitalize marine conservation traditions (Friedlander et al., 2014). 9. Seasonal calendar of Manangis in the Trans-Himalayas, Nepal. The Manangis, a group of indigenous peoples and local communities, have maintained a dynamic cultural landscape of the trans-Himalayas, Nepal through different socio-economic activities that are reflected in the seasonal calendar of Manang. The seasonal calendar clearly exhibits the typical lifestyle of people influenced by the cold climate: longer photoperiod for agricultural crops, inadequate food materials, important forest and water resources, high tourism activities, skilled trading activities, and topographic obstacles. The Managis sustainably collect the wild resources from common lands only during specified periods. Species include vegetables (Allium species), mushrooms including caterpillar fungus (Ophiocordyceps sinensis), and winter fodder grass (Chaudhary, Aase, Vetaas, & Subedi, 2007). The seasonal calendar including harvest of wild species is regulated by traditional knowledge of the indigenous peoples and local communities and social norms monitored by community leaders. Examples: These calendars also reflect seasonal circumstances of access to different areas to hunt and gather. In some areas, the wet season results in tall, matted grasses, which need to be burned when the dry season arrives before people can walk to different areas to hunt and gather. It is a selective rotational system associated with discrete wet (flooding, rain, long grass) and dry seasons (drying, floodwaters abate, grasses are burned, isolated billabongs reappear) in both Day and Tiwi areas – across the whole of the wet-dry tropics – like Llanos and Pantanal in Latin America. These and other seasonal calendars (e.g celtic tree calendar) are well known amongst indigenous indicators. Indigenous indicators have been recently evolving in the literature, challenging more technocratic views and highlighting that there is an alternative way of including values for guiding indicator development and selection. This work recognizes areas where conventional sustainability indicators cannot be developed for measuring crucial socioecological functions (J. Reid & Rout, 2018, 2020). 3.2.4. Economic, ecological, and social contexts of sustainable use Wild species are used by billions of people in very different socioecological systems and circumstances around the world. Subsistence gathering, hunting and fishing occur worldwide, as documented in previous IPBES assessments for Africa (IPBES, 2018d), the Americas (IPBES, 2018c); Asia and the Pacific (IPBES, 2018a), and Europe and Central Asia (IPBES, 2018b). Estimates on the number of people who use nontimber forest products, for example, range from 3.5 billion to 5.76 billion globally (Charlie M. Shackleton & de Vos, 2022). FAO also estimated 18% of respondent countries (65% of nation-members of the Organization for Economic Cooperation and Development (OECD) and 4% of countries outside the 44 Organization for Economic Cooperation and Development) are engaged in recreational harvesting of wild foods. Activities commonly undertaken include hunting, angling, mushroom gathering and berry picking (FAO, 2019b). One of the reasons these and the following data range so widely is that many products are used by the harvester themselves or informally traded in small quantities in small village markets, neighborhood exchanges, or amongst kin (see section 3.1 for explanation of informal vs. formal grade). Individuals, groups, and even companies engage in informal trade. The state of world's forests (FAO, 2014) is one of the few sources available for estimating the value of informal markets across the globe. For the year 2011, FAO estimated the value of global informal trade to be 88,013 million United States dollars. Estimates of informal trade value were higher for Asia and Oceania (FAO, 2014b). Wild species contributions to household income are highly variable ranging from 17% in Acre and Amazonas states in Brazil (Carvalho Ribeiro et al., 2018) to 28.6% of average household income across Latin America, whereas in Asia and Africa forest income shares are 20.1% and 21.4%, respectively (Angelsen et al., 2014). In general, roughly 25-30% of household income in tropical forest countries was from wild forest products in the early 2000s, a percentage almost as high as agriculture (Wunder, Angelsen, & Belcher, 2014). The same level of market informality is also present in fisheries; especially in developing countries where there are informal markets for small-scale coastal and freshwater fisheries. Although informal and largely unreported, the catch from small-scale fisheries may be large and this informal trade is important to local economies (e.g., in villages or small cities) and to the food security and nutrition of impoverished peoples living in remote areas. 3.2.4. Economic, ecological, and social contexts of sustainable use Small scale fishing is discussed extensively in section 3.3.1. The lack of monitoring may render the importance of these activities to local communities and some of their environmental impacts, invisible to decision makers (Bartley, De Graaf, Valbo‐Jørgensen, & Marmulla, 2015; Doria, Athayde, Lima, Carvajal-Vallejos, & Dutka-Gianelli, 2020). While subsistence uses often occur somewhat "under the radar" in the informal economy, there is a very large formal economy surrounding wild species. This formal economic activity is collectively referred to by the United Nations as BioTrade (UNCTAD, 2017): the collection, production, transformation and commercialization of goods and services derived from native biodiversity (species and ecosystems) under environmental, social and economic sustainability criteria. Under the Nagoya Protocol on Access to Genetic Resources and the Fair and Equitable Sharing of Benefits Arising from their Utilization, parties are to issue internationally recognized certificates (IRCC) of compliance evidencing that access to genetic resources was based on prior informed consent and that mutually agreed upon terms were established between local communities and research and industry stakeholders. India leads by far in the number of internationally recognized certificates of compliance worldwide (accessed June 2020). BioTrade subscribes to the objectives of biodiversity-related multilateral environmental agreements including the context of sustainable development and responsible business. It stresses that 70% of the world’s poor depend directly on biodiversity and businesses it fosters. BioTrade partners estimate that 86% of species (and their potential uses) are still unknown (UNCTAD, 2017). There are seven established BioTrade Principles and Criteria (BT P&C) as follows: (P1) Conservation of biodiversity, (P2) Sustainable use of biodiversity, (P3) Equitable benefit-sharing, (P4) Socioeconomic sustainability, (P5) Compliance with 45 international legislation and agreements, (P6) Respect for actors’ rights, and (P7) Clear land tenure and resources access. These, combined with the four distinctive approaches described within BioTrade (value chain, sustainable livelihoods, ecosystem and adaptive management), greatly contribute to the sustainability of trade in wild species. While only 20 countries officially participate in BioTrade partnerships, over 12,000 companies worldwide in more than 70 countries have signed up to the United Nations Global Compact, committing to greater environmental responsibility. The number of companies that report on biodiversity in their annual reporting is growing. For example in 2015, thirty-six of the top 100 cosmetic companies and 60 of the top 100 food companies mentioned biodiversity. Sales of BioTrade beneficiary companies reached 5.1 billion United States dollars (UNCTAD, 2017). 3.2.4. Economic, ecological, and social contexts of sustainable use Approximately 5 million people worldwide from collectors/fishers/ hunters to workers, among others are involved (UNCTAD, 2017). 3.3. Practices and uses The use of wild species includes three interacting systems: the wild species themselves, the human practices by which they are obtained from nature, and the uses for which they are intended (Chapter 1, Figure 1.6). Here the status and trends of the use of wild species are reported, organized according to the practices defined at length in Chapter 1: fishing (including lethal and non-lethal use), gathering, terrestrial animal harvesting (including lethal and non- lethal use), logging, and non-extractive practices. These practices are somewhat intuitive, but not always. Thus, readers should be attentive to the definitions and explanations of the practices and why certain organisms (e.g., living shellfish vs. shells) or certain parts of organisms (e.g., tree branches vs. tree fruits, leaves and sap) are discussed in a particular practice category. Each section begins with an overview presented in a format consistent with ways of thinking most prevalent in that field. This is followed by specific information relevant for the practice. The following section reviews uses according to the structure detailed in Chapter 1: ceremonial/cultural, decorative/aesthetic, energy, food/beverage, medicine/hygiene, recreation, science/education, shelter/construction, and other (see Chapter 1, Figure 1.6). Only the relevant uses are reported upon in each practice section. These categories are not exclusive, and many species have more than one use depending on a range of variables including their biology, habitat, life cycle, knowledge on utilization, existing rules, and regulations. There may thus be some overlap in the reporting. A selection of cases of multiple and complex use systems is discussed in section 3.4 to demonstrate some of the complexities of reporting on status and trends at national and international scales. When possible, the use categories have structured the reporting in this section. However, in many cases the knowledge about the sustainability of use is not organized according to these use categories. Therefore, in order to increase accessibility to policy makers, in sections where the bulk of knowledge is reported using a different system, hybrid organizing structures were created as an attempt to be attentive both to the organizing structure of this assessment, and the expectations of the readers. 3.3.1. Fishing 3.3.1.1. Introduction Prior to 1950 large-scale motorized fishing was mainly confined to the North Atlantic and Japan. Marine capture fishery has substantially expanded in the last 70 years in terms of geospatial and vertical distribution, and intensity of catch effort. Automatic identification systems data indicates that industrial fishing currently occurs in over 55% of the global ocean (Kroodsma et al., 2018) although a much smaller footprint is estimated from the same data when a spatial grid of finer resolution is used in the calculation (Amoroso, Parma, Pitcher, McConnaughey, & Jennings, 2018). Relative to coastal ecosystems, high seas ecosystems are much less affected (Halpern et al., 2008; Jackson, 2001). However, reported landings from the high seas has been accelerating since the mid- 20th century with under two million tons in 1950 to over ten million tons in 2008 (FAO, 2010c). The history of sustainable use of capture fisheries is closely tied with several key events and international agreements. Prominent among those is the United Nations Convention on the Law of the Sea ratified in 1982 by 157 parties. One of its most significant provisions was the establishment of 200-mile exclusive economic zones and introducing the concept of maximum sustainable yield as the default goal of fisheries management. The 200-mile exclusive economic zone allowed countries to exclude wide ranging foreign fishing fleets that earlier were able to legally fish within 12 miles of the national coastline. As a result, several countries established fisheries management systems (e.g., scientific assessment, regulation of harvest) for the newly expanded waters under their jurisdiction. This also led to expansion of many domestic fishing fleets. The legal framework of the United Nations Convention on the Law of the Sea did not include several fish stocks across multiple exclusive economic zones or in the high seas. The United Nations fish stocks Agreement from 2001 provided international protocols for managing these “straddling stocks” (G. R. Munro, 2000). It mandated the formation of Regional Fisheries Management Organizations (RFMO) to sustainably manage high seas and the straddling stocks. Following the Agreement, there are now 17 Regional Fisheries Management Organizations that cover almost all the high seas fisheries and associated straddling stocks outside national exclusive economic zones. Regional Fisheries Management Organizations are competent and mandated to establish binding conservation and management measures. 3.3.1. Fishing 46 3.3.1.1. Introduction Abbreviations: ICCAT: International Commission for the Conservation of Atlantic Tunas; IATTC: Inter-American Tropical Tuna Commission, IPHC: International Pacific Halibut Commission; NPAFC: North Pacific Anadromous Fish Commission; WCPFC: Western and Central Pacific Fisheries Commission; CCSBT: Commission for the Conservation of Southern Bluefin Tuna; IOTC: Indian Ocean Tuna Commission; NASCO: North Atlantic Salmon Conservation Organization; 48 PSC: Pacific Salmon Commission; IWC: International Whaling Commission; CACFISH: Central Asian and Caucuses Regional Fisheries and Aquaculture Commission; CTMFM: Joint Technical Commission of the Maritime Front; NAFO: Northwest Atlantic Fisheries Organization; NPFC: North Pacific Fisheries Commission; SEAFO: South East Atlantic Fisheries Organization; SPRFMO: South Pacific Regional Fisheries Management Organization; CCAMLR: Commission for the Conservation of Antarctic Marine Living Resources; GFCM: General Fisheries Commission for the Mediterranean; NEAFC: North-East Atlantic Fisheries Commission; RECOFI: Regional Commission for Fisheries; SIOFA: Southern Indian Ocean Fisheries Agreement; LVFO: Lake Victoria Fisheries Organization. These maps are directly copied from its original source (Løbach, Petersson, Haberkon, & Mannini, 2020) and was not modified by the assessment authors. The maps are copyrighted under license CC BY-NC-SA 3.0 IGO. The designations employed and the presentation of material on the maps used in the assessment do not imply the expression of any opinion whatsoever on the part of IPBES concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. These maps have been prepared or used for the sole purpose of facilitating the assessment of the broad biogeographical areas represented therein and for purposes of representing scientific data spatially. Fishing has impacts on marine ecosystems other than the target species. A range of international agreements have evolved to provide guidance on managing non-target species and vulnerable marine ecosystems (VMEs). Legal instruments establishing international responsibility to conserve associated and dependent species are relatively recent, which first became an obligation under the 1982 Law of the Sea Convention, and was reiterated and clarified further in subsequent United Nations resolutions (United Nations 1982 [Article 119], 1995 [Article 5(f), Article 10(d), and Annex 1]; 2006a, b). These provisions were elaborated further in subsequent instruments and guidance from other multilateral organizations. This includes the 1995 code of conduct for responsible fisheries of the FAO, which calls for the sustainable use of aquatic ecosystems and promotes the conservation of biodiversity and ecosystems by minimizing fisheries impacts on non-target species and the ecosystem in general (FAO, 1995a). 3.3.1.1. Introduction They provide a formal mechanism for fishing states and states in whose jurisdiction fishery resources occur to meet their international obligation to cooperate to sustainably govern shared living marine resources throughout their distributions (the United Nations Convention on the Law of the Sea Articles 63, 66(5), 118; Code Articles 7.1.5, 6.12 (FAO, 1995a); Agreement on Port State Measure (PSMA) Article 4(1)(b) (FAO, 2010a). Regional Fisheries Management Organizations have played a critical role in multilateral fisheries governance of stocks that straddle or occur beyond national jurisdictions and are highly migratory. While spatial and taxonomic gaps remain, a large proportion of global marine fisheries are now managed by one or multiple Regional Fisheries Management Organizations, and they cover most areas of the high seas (Figure 3.15). 47 48 Figure 3.15 Species-specific regional fisheries management organizations (top) and othe regional fisheries management organizations (bottom). Abbreviations: ICCAT International Commission for the Conservation of Atlantic Tunas; IATTC: Inter-America Tropical Tuna Commission, IPHC: International Pacific Halibut Commission; NPAFC: Nort Pacific Anadromous Fish Commission; WCPFC: Western and Central Pacific Fisherie Commission; CCSBT: Commission for the Conservation of Southern Bluefin Tuna; IOTC Indian Ocean Tuna Commission; NASCO: North Atlantic Salmon Conservation Organization 48 Figure 3.15 Species-specific regional fisheries management organizations (top) and other regional fisheries management organizations (bottom). Abbreviations: ICCAT: International Commission for the Conservation of Atlantic Tunas; IATTC: Inter-American Tropical Tuna Commission, IPHC: International Pacific Halibut Commission; NPAFC: North Pacific Anadromous Fish Commission; WCPFC: Western and Central Pacific Fisheries Commission; CCSBT: Commission for the Conservation of Southern Bluefin Tuna; IOTC: Indian Ocean Tuna Commission; NASCO: North Atlantic Salmon Conservation Organization; Figure 3.15 Species-specific regional fisheries management organizations (top) and other regional fisheries management organizations (bottom). 3.3.1.1. Introduction FAO has also produced a voluntary international plan of action on reducing the incidental capture of seabirds in longline fisheries (FAO, 1999), an international plan of action on the conservation and management of sharks (FAO, 1999b), international guidelines on reducing marine turtle fishing mortality (FAO, 2009), and broad guidelines on managing fisheries bycatch (FAO, 2011). These new instruments and international guidelines broadened the mandate of pre-existing Regional Fisheries Management Organizations, expanding their mandates from one target species to meet newer expectations for ecosystem-based management and precautionary approaches, i.e., establishing explicit limits of acceptable impacts on fish and non-fish bycatch species, associated or dependent and threatened species (Fisheries Agency of Japan, 2007; Lodge, Anderson, & Lobach, 2007; United Nations, 2006b, 2006a). Fisheries targeting relatively fecund species can have profound impacts on co-occurring incidentally caught or bycatch species with delayed maturation, low fecundity and other life history traits that make them vulnerable to anthropogenic causes of mortality. While target stocks may be sustainable, the conservation status of bycatch species and other associated and 49 dependent species is often not known. For instance, 47 of 68 fisheries that catch marine resources managed by Regional Fisheries Management Organizations have no observer coverage (Gilman, Passfield, & Nakamura, 2014); for the vast majority of the ca. 4.6 million fishing vessels globally, information on non-retained catch is absent. In most fisheries, there are large gaps in understanding of life histories for many marine species. Information on total cumulative anthropogenic levels of fishery removals from an individual population, knowledge of the conservation status of individual populations, and deficits in monitoring are all unknown. Data collection protocols, observer coverage rates, and sufficient time-series to detect the response in absolute population abundance of long-lived species to this anthropogenic mortality source are also knowledge gaps in various global fisheries (Gilman et al., 2020; Lewison, Crowder, Read, & Freeman, 2004; Musick, 1999a; Pérez Roda et al., 2019). United Nations Resolution 61/105 (UNGA, 2006) provides for responsible management of vulnerable marine ecosystems and non-target species as a legally binding instrument. It provides for collection of data on the impacts of fishing on vulnerable marine ecosystems and specific actions to protect them. 3.3.1.1. Introduction Another important international protocol is the Agreement on Port State Measures (FAO, 2016a) aimed at preventing, deterring and eliminating illegal, unreported and unregulated fishing by preventing vessels engaged in illegal, unreported and unregulated fishing from using ports and landing their catches (FAO, 2021a). Outside of formal international agreements, there have been many efforts to improve management both by non-governmental organizations and national governments. The 1990s were an era of greatly expanding concerns about overfishing, in many ways stimulated by the highly publicized collapse of the northern cod fishery in Canada (Finlayson, 1994; Kurlansky, 1997; Rice, Shelton, Rivard, Chouinard, & Fréchet, 2003). The Marine Stewardship Council was formed in 1997 with the goal to use market pressure to improve fisheries sustainability, and now is a major force in market access, particularly in Europe (MSC, 2021). Many environmental non-governmental organizations formed marine conservation divisions, and entirely new non-governmental organizations appeared with a focus on marine ecosystems. These were, to a great extent, funded by United States of America foundations with amounts up to 500 million United States dollars per year spent by environmental non-governmental organizations and foundations on marine conservation (Hilborn & Hilborn, 2019). Since the 1990s national governments have expanded the science and management efforts through changes in legislations such as the United States of America Magnuson-Stevens act and revisions, and the creation of the Common Fisheries Policy in the European Union. Finally, there has been increasing attention paid to consider impacts on fishing dependent coastal communities in almost all countries. As examples, Canada guarantees the first 90,000 tons of cod quota to small-scale inshore fishers, the United States of America allocates 8% of the allowable catch in the large industrial fisheries of the Bering Sea to local communities, and in Chile fishing cooperatives can apply for and be granted exclusive ownership of local inshore resources. 3.3.1.2. Status and trends in global marine capture fisheries For the purposes of this assessment, in accordance with Chapter 1, fishing is defined as the harvest of entire organisms or parts of organisms that result in mortality of the aquatic animals, 50 for example commercial fisheries or shark finning. Non-lethal fishing is defined as harvesting of entire or parts or products of organisms without intended mortality. Examples of non-lethal fishing include harvesting fish for the aquarium trade, catch and release fishing, or the extraction of blood from horseshoe crabs. The status and trends of wild fish are estimated by a range of methods including scientific surveys, size or age distribution, catch per boat day and other estimates based on catch rate / fishing gear. A sophisticated method, known as “stock assessments”, combines all these types of data to provide scientific estimates of the trend in abundance and harvest rate for fish stocks. The most robust approaches now involve multispecies and ecosystem-level assessments, an improvement over conventional single stock assessments, even though single stock assessments remain the dominant approach. Produced by national fisheries agencies and international regional fisheries management organizations, these scientific assessments are publicly available for roughly half of the global fish catch. Considerable effort in recent years has been towards increasing understanding of the status of stocks that produce the other half of global marine catches. This effort is ongoing. The most cited stock status assessment comes from the state of world fisheries and aquaculture of the FAO(2020d), which uses a sample of roughly 500 fish stocks from around the world to describe the status of stocks. When scientific assessments are not available, expert knowledge is often used to make some sort of assessment. The material presented below follows this approach. The status of fish stocks can be described in many ways. The most common approach is to compare the current abundance of the fish stock to target abundance, usually a target based on maximizing the long-term harvest, often called “maximum sustainable yield”. In FAO terminology, stocks that are above this target level are called “underfished”, stocks below the target are “overfished” and stocks with abundance close to the target are called “maximally sustainably fished." FAO uses the range 0.8 to 1.2 of the abundance as an indicator of maximum sustainable yield. 3.3.1.2. Status and trends in global marine capture fisheries Because fish stocks fluctuate naturally, sometimes over orders of magnitude of abundance, a better evaluation of the status of the fishery is to look at the fishing pressure relative to the targets. Fishing harder than the target rate is called “overfishing”. Some assessments of stock status are based solely on the trends in catch. When catch declines it is assumed that the stock is in poor shape. Comparisons may also be made between the current abundance of fish stocks to estimates from before significant fishing began, which is most commonly done using various kinds of ecosystem models (Figure 3.16). 51 Figure 3.16 Global trends in the state of the world’s marine fish stocks, 1974-2017. Source: (FAO, 2020d) under license CC BY-NC-SA 3.0 IGO. Figure 3.16 Global trends in the state of the world’s marine fish stocks, 1974-2017. Source: (FAO, 2020d) under license CC BY-NC-SA 3.0 IGO. A common misinterpretation of the above data is that stocks that are “maximally sustainably fished” are somehow being pushed to the limit and this is an undesirable state. In fact, “maximally sustainably fished” means that stocks are at an abundance level that will provide long-term maximum sustainable yield. Another misinterpretation is that stocks that are overfished are headed towards extinction or necessarily declining. “Overfished” simply means an abundance lower than would produce maximum sustainable yield, and many stocks remain at this level for decades; if fishing pressure is reduced these stocks can rebuild. Despite this common understanding, there is no agreed upon definition of what is overfished. The FAO defines overfished as the stock biomass being below 80% of the abundance that would produce maximum sustainable yield; the United States of America and New Zealand use a 50% cutoff, while many tunas’ Regional Fisheries Management Organizations define overfished as being below the target level. From a conservation perspective, stocks that are fished to very low abundance, where recovery is often very slow, are a concern due to lack of knowledge of potential recovery. Neubauer et al. (2013) conclude that "prolonged intense overexploitation, especially for collapsed stocks, not only delays rebuilding but also substantially increases the uncertainty in recovery times. Furthermore, when stocks become depleted, catch rates are lower and therefore the effort needed to catch a given volume of fish is higher and so is its environmental footprint. 3.3.1.2. Status and trends in global marine capture fisheries For those fisheries that produce half of the world's marine catch for which good data is available, on average fish stocks are increasing because fishing pressure is lower than levels that would produce maximum long-term yield, and abundance is above target levels (Figure 3.17) (Hilborn et al., 2020). 52 53 53 Figure 3.17 Estimated abundance of global fish stocks 1970 – 2016 (A), European Union non-Mediterranean fish stocks (B), Mediterranean-Black Sea fish stocks (C), and Atlantic (D) and Pacific (E) ocean tuna fish stocks. Estimated abundance (B/BMSY -Biomass relative to the biomass that produces Maximum Sustainable Yield- in orange) and fishing pressure (U/UMSY -Fishing pressure or mortality relative to the fraction of the population harvested- in green) are shown for the stocks that are scientifically assessed around the world from 1970 to 2016 - shaded area is the confidence intervals. The biomass and fishing pressure are scaled to the level that would produce maximum sustainable yield. See data management report for the figure at https://doi.org/10.5281/zenodo.6452917. Figure 3.17 Estimated abundance of global fish stocks 1970 – 2016 (A), European Union non-Mediterranean fish stocks (B), Mediterranean-Black Sea fish stocks (C), and Atlantic (D) and Pacific (E) ocean tuna fish stocks. Estimated abundance (B/BMSY -Biomass relative to the biomass that produces Maximum Sustainable Yield- in orange) and fishing pressure (U/UMSY -Fishing pressure or mortality relative to the fraction of the population harvested- in green) are shown for the stocks that are scientifically assessed around the world from 1970 to 2016 - shaded area is the confidence intervals. The biomass and fishing pressure are scaled to the level that would produce maximum sustainable yield. See data management report for the figure at https://doi.org/10.5281/zenodo.6452917. Figure 3.17A shows the estimated abundance (B/BMSY -Biomass relative to the biomass that produces Maximum Sustainable Yield- in orange), fishing pressure (U/UMSY - Fishing pressure or mortality relative to the fraction of the population harvested- in green), and catch (in blue) for the stocks that are scientifically assessed around the world from 1970 to 2016. The biomass and fishing pressure are scaled to the level that would produce maximum sustainable yield. Abundance declined from 1970 to 1995, then leveled off for 10 years and about 2005 began to increase. This is consistent with increased fishing pressure from 1970 to the mid 1990s, then declining pressure since that time (Figure 3.17). 3.3.1.2. Status and trends in global marine capture fisheries When looking at different regions where there is good scientific understanding of stock status, one notes contrasting trends (Figure 3.17 B-E). The European Union (Figure 3.17B), Atlantic and Baltic stocks were already fished hard in 1970 and harvest rates increased up to about 1995, and then declined. Stocks were above target levels in 1970, declined to about 2005 and then began to increase. Mediterranean stocks (Figure 3.17C) have seen increasing fishing pressure since 1970 and declining abundance. Fishing pressure is far above target levels and abundance well below. One species specific estimate is included here (Figure 3.17 D & E). Global tuna fisheries were not fully developed in 1970 and saw generally increasing fishing pressure and declining abundance until recent years when abundance leveled off at or above target levels. Atlantic tuna fisheries were fished harder and earlier than Pacific (Figure 3.17) that would produce maximum sustainable yield. In the FAO’s state of the world fisheries and aquaculture annual reports there are many stocks that are evaluated using expert knowledge because there is no scientific stock assessment. Melnychuk et al., (2017) used an expert opinion survey of the 28 countries landing the most fish to determine the status of stocks and found that generally temperate stocks were considered to be in good shape while tropical stocks where not (Figure 3.18). 54 Figure 3.18 Global abundance by coastline based on expert estimates. Green indicates experts believe that most stocks are at abundance consistent with long term maximum sustainable yield, red indicates few stocks are at that level. Data from (Melnychuk et al., 2017). See data management report for the figure at https://doi.org/10.5281/zenodo.6452953. Figure 3.18 Global abundance by coastline based on expert estimates. Green indicates experts believe that most stocks are at abundance consistent with long term maximum sustainable yield, red indicates few stocks are at that level. Data from (Melnychuk et al., 2017). See data management report for the figure at https://doi.org/10.5281/zenodo.6452953. Costello et al. (2012) attempted to estimate the status of the half of the world’s fisheries that are not scientifically assessed and combine this with the data from assessed stocks to provide a global estimate of status. They grouped stocks into four classes; (i) large assessed (large industrial fisheries of the world where a scientific assessment of status and trends is performed); (ii) large unassessed, (iii) small assessed and (iv) small unassessed stocks. 3.3.1.2. Status and trends in global marine capture fisheries The trends estimate showed that the large stocks, both assessed and unassessed, are on average about target levels, but small assessed stocks were declining and small unassessed stocks were well below target levels (Figure 3.19). 55 Figure 3.19 Trend estimates for global large and small stocks. The black lines are for stocks scientifically assessed and are generally the same stocks as used in Hilborn et al., 2020. The red lines are estimates of the trends for stocks not scientifically assessed. Source: (Costello et al., 2012) © 2012, American Association for the Advancement of Science. CC-BY NC. Figure 3.19 Trend estimates for global large and small stocks. The black lines are for stocks scientifically assessed and are generally the same stocks as used in Hilborn et al., 2020. The red lines are estimates of the trends for stocks not scientifically assessed. Source: (Costello et al., 2012) © 2012, American Association for the Advancement of Science. CC-BY NC. Rosenberg et al. (2018) combined four different methods (one being the Costello et al. (2012)) to estimate the status of unassessed stocks using an approach called ensemble modelling (Figure 3.20). However, when the stock status was compared to the status for stocks that were scientifically assessed, the performance was rather poor and the ensemble method provided roughly similar status estimates both in regions where scientific assessment show stocks are in very poor shape such as the Mediterranean Sea, and also in regions where stocks are in very good shape such as the Northeast Pacific. Thus, we know the status of fish stocks which provide half of the world’s catch – largely from the temperate North, and do not know the status of the other half of the global catch - largely from Southeast Asia. Christensen et al. (2014) examined 200 marine food web models covering the period 1880 to 2007 and compared the change in abundance of different trophic levels of fish. They estimated that high trophic level fish had declined by 2/3 (to roughly the level that would produce maximum sustainable yield) while the far more numerous low trophic level species would have more than doubled. 56 Figure 3.20 Estimation of the status of unassessed stocks by several data poor methods. Abbreviations: BMSY: Biomass that would support Maximum Sustainable Yield, CMSY:Catch Biomass that would support Maximum Sustainable Yield, COMSIR: catch-only-model with sampling-importance resampling, SSCOM: state-space catch-only model. 3.3.1.2. Status and trends in global marine capture fisheries Source: (Rosenberg et al., 2018) under license CC BY 4.0. Figure 3.20 Estimation of the status of unassessed stocks by several data poor methods. Abbreviations: BMSY: Biomass that would support Maximum Sustainable Yield, CMSY:Catch Biomass that would support Maximum Sustainable Yield, COMSIR: catch-only-model with sampling-importance resampling, SSCOM: state-space catch-only model. Source: (Rosenberg et al., 2018) under license CC BY 4.0. The performance of marine fisheries in terms of providing food security can be measured by comparing levels of sustainable yield at the current fishing pressure and if people fished at rates that would provide maximum sustainable yield. This is only available for the assessed fish stocks of the world. The status of assessed stocks is maintained on-line in the RAM Legacy Stock Assessment Database (Ricard, Minto, Jensen, & Baum, 2012). Using the data from assessed stocks and calculation of lost yield (Hilborn, 2018) the figure below shows the amount of potential yield that is lost by fishing too hard (red), or too little (blue) and how much of the potential yield is achieved at current fishing pressures (blue). It is estimated that in 1950 when the data began, roughly half of the potential yield was lost by low fishing pressure and there was little loss from fishing too hard (overfishing). The loss from overfishing rose to between 10% and 20% during the 1980s and 1990s and has now declined to about 5%. Potential increase in yield by fishing harder is now about 17%, and across these stocks the current fishing pattern is achieving about 73% of potential yield (Figure 3.21). These calculations are based on the assumption that parameters that determine the productivity of fish stocks will remain unchanged at current estimated values. Note that fish production is not just a function of how hard people fish, but it depends on variable environmental conditions (temperature, food, ocean currents, etc.), including conditions affected by climate change. 57 Figure 3.21 The fraction of potential yield lost in each year by overfishing (red), and by fishing less than the Maximum Sustainable Yield (blue). Green shows the fraction of potential yield achieved at the fishing pressure for that year. See data management report for the figure at https://doi.org/10.5281/zenodo.6453019. Figure 3.21 The fraction of potential yield lost in each year by overfishing (red), and by fishing less than the Maximum Sustainable Yield (blue). 3.3.1.2. Status and trends in global marine capture fisheries Green shows the fraction of potential yield achieved at the fishing pressure for that year. See data management report for the figure at https://doi.org/10.5281/zenodo.6453019. 3.3.1.3. Status and trends in selected fisheries As no satisfactory global reviews were found in the literature, significant effort was invested in a systematic review of small-scale fisheries because of their importance for local communities. Due to high variability, the review of marine and inland small-scale fisheries was made by geographic region (see the data management report for Chapter 3 systematic literature review at https://doi.org/10.5281/zenodo.6452651). Three other sections dedicated to distinct fisheries were also developed: (i) small to medium pelagic or forage fish fisheries that are mainly coastal and provide about 25% of world capture fisheries; (ii) tuna and tuna-like fisheries, which are of high economic value and are widely spatially distributed from coastal regions to the high seas; (iii) industrial demersal fisheries in coastal areas, which are a complex set of heterogeneous fishing fleets using diverse fishing gears active within the exclusive economic zones of coastal countries. When necessary, for taxonomic groups of special concern, we added information on their status and trends in dedicated boxes (e.g., Box 3.2). Box 3.2 Status and trends of sharks, rays, and chimaeras: implications for species, the environment, and people There are approximately 1,250 species of sharks and rays found throughout the world’s marine, and some freshwater, habitats. Sharks and rays are relatively large-bodied predators Box 3.2 Status and trends of sharks, rays, and chimaeras: implications for species, the environment, and people There are approximately 1,250 species of sharks and rays found throughout the world’s marine, and some freshwater, habitats. Sharks and rays are relatively large-bodied predators 58 and, hence, are both highly susceptible to a wide range of fishing gears (predominantly trawls, longlines, gill and tangle nets) and highly sensitive to fishing mortality because of their long generation lengths and low fecundity resulting in very low maximum population growth rates and low density-dependent compensation (Forrest & Walters, 2009; Eric Gilman et al., 2008; Pardo, Kindsvater, Reynolds, & Dulvy, 2016). Consequently, they are highly vulnerable to overfishing compared to the teleost fishes they are caught alongside and are particularly prone to disappearing prior to adequate monitoring (Myers & Worm, 2005; Yan et al., 2021). Global shark and ray catches reported to FAO rose to a peak in 2003 and declined at least 17% thereafter, likely due to overfishing (Davidson, Krawchuk, & Dulvy, 2016; Dent & Clarke, 2015). However, the global catch is underestimated and is likely to be two-to-four times greater (Clarke et al., 2006). 3.3.1.3. Status and trends in selected fisheries Based on these FAO data and accounting for discards and illegal, unreported, and unregulated fishing, it is possible that 63–273 million individuals were captured in the early 2000s (Boris Worm et al., 2013). Only 4% of the global estimated catch is managed sustainably, based on 65 fisheries stock assessments from 47 species from Canada, United States of America, Australia, and New Zealand (Simpfendorfer & Dulvy, 2017). Catch estimates of unassessed data-poor fisheries show that large coastal sharks have been very unsustainably fished since 1975 (B/Bmsy - Biomass relative to the biomass that produces Maximum Sustainable Yield < 0.5) (Costello et al., 2012). Consequently, steep regional declines of coastal sharks have been documented (Ferretti, Osio, Jenkins, Rosenberg, & Lotze, 2013; MacNeil et al., 2020). Oceanic sharks and rays have limited spatial refuge from fisheries (Queiroz et al., 2019) and declined by 71% since 1970 due to an 18-fold increase in relative fishing pressure (Pacoureau et al., 2021). Sharks and rays from the tropical and subtropical coastal seas are currently at higher risk (Dulvy et al. 2021). The International Union for Conservation of Nature Red List provides a framework for integrating disparate data sources ranging from historical ecology, to catch data and stock assessments (International Union for Conservation of Nature Standards and Petitions Committee, 2019; Mace et al., 2008; Sherley et al., 2020, p. 20). These comprehensive global assessments of sharks and rays offer a unique opportunity to calculate Living Plant and Red List indices to track progress toward the Convention on Biological Diversity and Sustainable Development Goals (Pacoureau et al., 2021; Walls & Dulvy, 2021). Shark and ray extinction risk has been rising over the past half century (Pacoureau et al., 2021, Walls and Dulvy, 2021). Now, one one-third (391 of 1,199; 32.5%) of sharks and rays are classified as threatened (vulnerable, endangered, or critically endangered) (Dulvy et al., 2021). Assuming the 155 data deficient species are threatened in the same proportion to the other species then an estimated 449 species are threatened (37.5%, range 32.6–45.5%). Three species are critically endangered (possibly extinct), because they have not been recorded for over 80 years but there have been insufficient surveys to confirm their extinction (Dulvy et al., 2021). A further eight species are regionally extinct in one or more countries and there have been at least 28 local extinctions (Dulvy et al., 2014, 2021). 3.3.1.3. Status and trends in selected fisheries The shark and ray extinction rate of 25 E/MSY (extinction per million spesies-year) is 25–250 times greater than the background fossil record extinction rate and 2.5 times greater than the proposed target rate of 10 E/MSY (extinction per million spesies-year) over the next century 59 (Rounsevell et al., 2020). Nearly all (99.6%) species are taken incidentally, but are valuable and are retained for food: half (51.5%) for human consumption of the meat only, with remaining species used for food in combination with the production of animal feed, skins, and liver oil (Dulvy et al., 2021). The International Union for Conservation of Nature classification scheme does not record shark and ray fins or devil ray gill plates (Mobulidae), but these significant trades are subject to increasing international regulation (Cardeñosa, Quinlan, Shea, & Chapman, 2018; Friedman et al., 2018). The global value of the shark and ray trade is worth 4.1 billion United States dollars, with the meat trade (2.6 billion United States dollars) exceeding the value of the global fin trade (1.5 billion United States dollars) (Niedermüller et al., 2021). (Rounsevell et al., 2020). Nearly all (99.6%) species are taken incidentally, but are valuable and are retained for food: half (51.5%) for human consumption of the meat only, with remaining species used for food in combination with the production of animal feed, skins, and liver oil (Dulvy et al., 2021). The International Union for Conservation of Nature classification scheme does not record shark and ray fins or devil ray gill plates (Mobulidae), but these significant trades are subject to increasing international regulation (Cardeñosa, Quinlan, Shea, & Chapman, 2018; Friedman et al., 2018). The global value of the shark and ray trade is worth 4.1 billion United States dollars, with the meat trade (2.6 billion United States dollars) exceeding the value of the global fin trade (1.5 billion United States dollars) (Niedermüller et al., 2021). Widespread overfishing of sharks and rays will likely have profound consequences for the environment and people. The depletion and loss of sharks and rays, particularly in the tropics, does not bode well for the livelihoods of many coastal human populations, dependent on their meat and products for food and income (Booth, Squires, & Milner- Gulland, 2019; Seidu et al., 2022). 3.3.1.3. Status and trends in selected fisheries Indeed, the depletion of sharks and rays reflects increasing evidence that the target teleost fisheries are overfished in South America, Africa, and Southeast Asia (Dyhia Belhabib, Greer, & Pauly, 2018; Lam & Pauly, 2019). 3.3.1.4. Small-scale fisheries Small-scale fisheries are strongly anchored in local communities where fisheries represent a way of life (FAO, 2015). Despite their importance, small-scale fisheries around the world are facing major challenges from the effects of global change, e.g., climate change, urbanization, industrialization, aquaculture intensification, and large-scale fisheries (Berkes, 2015; Chuenpagdee, 2011). Ongoing threats to small-scale fisheries affect entire production systems (harvest, processing, retail and transport) and create vulnerabilities that have no easy solution (Chuenpagdee, 2011; Jentoft & Chuenpagdee, 2009). In many cases, these challenges have placed the livelihoods, economy, food security, values and identity, and the viability of small- scale fisheries communities at risk (Bavinck, Jentoft, & Scholtens, 2018; Bundy et al., 2016; Jentoft & Chuenpagdee, 2015; Jentoft & Eide, 2011; Nayak & Armitage, 2018). An estimated 5.8 million fishers in the world who earn less than 1 United States dollars per day (FAO, 2014d). Ommer et al. (2007) characterize these large-scale, globalized processes as a crisis in social-ecological ‘health’, with dire consequences on small-scale fisheries communities. The COVID-19 pandemic will affect many small-scale fisheries and coastal communities worldwide, especially those more vulnerable, mainly through reduced (or closure of) markets, decreases in revenues from tourism, increases in health risks to fishers and traders and increased occurrence of illegal fishing due to lack of enforcement. Mitigation of these factors would likely require institutions to provide short- and long-term responses (N. J. Bennett et al., 2020). There can be some positive outcomes from the pandemic crisis, including enhanced local cooperation among fishing communities and other institutions, valorization of local markets, food sharing and some recovery of fishing resources (N. J. Bennett et al., 2020). The state of inland capture-fishery resources that includes small-scale inland fisheries is more difficult to monitor (Welcomme, 2011) for a number of reasons, including the diffused character of the practice due to: (i) large numbers of people being involved in the seasonal and 60 subsistence nature of fisheries activities; (ii) much of the catch being consumed locally or traded informally; and (iii) fisher populations being greatly affected by activities other than fishing, including stocking from aquaculture and diversion of water for other uses such as agriculture and hydroelectric development (FAO, 2012c). This section is based on a comprehensive review of 350 studies on small-scale fisheries from 107 countries worldwide (Figure 3.22). With regard to ecological sustainability, 39 studies indicate sustainable fisheries but almost half the studies (#165) indicate unsustainability. 3.3.1.4. Small-scale fisheries Whereas fisheries reported by 129 studies were considered to be partially sustainable; a few studies (#16) do not assess ecological sustainability but include some accounts on economic or social sustainability. Most of the reviewed literature on small-scale fisheries addresses the use of fish as food and feed, and is presented in detail below by major world regions. Other uses for fish are also mentioned in some regions (see the data management report for Chapter 3 systematic literature review at https://doi.org/10.5281/zenodo.6452651). This review supports the text below, considering the available evidence from most of the revised studies (for details on the reviewed studies, see the data management report for Chapter 3 systematic literature review at https://doi.org/10.5281/zenodo.6452651). Figure 3.22 Global distribution of the 350 reviewed studies on small-scale fisheries among 107 countries (countries in gray means that no study was included). See data management report for the figure at https://doi.org/10.5281/zenodo.6453056. Figure 3.22 Global distribution of the 350 reviewed studies on small-scale fisheries among 107 countries (countries in gray means that no study was included). See data management report for the figure at https://doi.org/10.5281/zenodo.6453056. 61 A systematic literature review on small-scale fisheries was undertaken based on literature obtained through various combinations of a set of keywords: fisheries, sustainable, sustainability, small-scale, coastal, freshwater, catch, trend, success, local knowledge, use, fishers, co-management, increasing, review, catches, fish, and ecological. These keywords were selected to get a manageable number of hits (literature) to assess and to direct the search results to those articles analyzing sustainable fisheries, or at least to those showing trends of an increase in catches. The database SCOPUS was used for articles from the last 20 years (since 2000), which initially retrieved a total of 1635 articles. A complementary search was made on Google Scholar using a subset of these keywords. However, due to the large amount of literature retrieved (34300 hits), only the first 200 hits were reviewed on Google Scholar, including some of the more recent articles from the last 10 years. A total of 447 articles on small-scale fisheries were selected after an initial screening, including only articles that reported some data on fisheries, preferably trends and some kind of indicator, such as abundance, size or catch per unit of effort, or fishing effort among others. 3.3.1.4. Small-scale fisheries Articles addressing details of management or policy options which did not include data, or theoretical approaches and effects from drivers, such as climate change, pollution, or development projects, were not included. These 447 articles were sorted by major regions and the case studies on small-scale fisheries were selected from these (see the data management report for Chapter 3 systematic literature review at https://doi.org/10.5281/zenodo.6452651). This literature review was complemented with relevant articles inserted by the authors from their personal libraries, by suggested articles from internal and external reviewers, and through cross-reference from the selected articles. Our review did not retrieve a large number of articles dealing with uses other than food (ornamental, medicinal, etc.) and those addressing social and economic dimensions of sustainability in small-scale fisheries. The selected studies were sorted across a gradient of ecological sustainability, ranging from fully sustainable (exploited populations stable, no habitat damage, no ecological filters or shifts in the composition of exploited species) to unsustainable (exploited populations declining or overfished). Intermediate or partial sustainability included situations in which current exploited populations are stable, but some higher valued species were depleted or extinct, which are considered here as ecological filters (see also section 3.3.1.4.2), or the fishing practice has caused habitat damage or bycatch. Fisheries lacking data on temporal trends to clearly indicate sustainable catches were also allocated to these partially sustainable categories (for details on the reviewed studies, see the data management report for Chapter 3 systematic literature review at https://doi.org/10.5281/zenodo.6452651). A major challenge in evaluating the sustainability of small-scale fisheries is the lack of data on catches and measures of exploited stocks (size, proportion of juveniles caught, etc.), especially over broader spatial or temporal scales. Nevertheless, participatory research in collaboration with fishers and analyses of the fishers’ knowledge about fishing resources have contributed evidence to assess patterns of sustainability, catches and fishing effort. Relatively few studies have evaluated the economic sustainability of small-scale fisheries. A review on global marine fisheries indicates that well-managed and locally supported small-scale fisheries could be a more sustainable option to provide employment and food than the current subsidy-driven industrial fisheries, which may increase effort in spite of 62 declining fishing resources (Zeller & Pauly, 2019). 3.3.1.4. Small-scale fisheries However, conventional economic models that have been applied to assess fisheries economic viability may not be appropriate to small- scale fisheries, which need inclusion of social and environmental variables to conduct economic viability analyses that go beyond profit maximization (Schuhbauer & Sumaila, 2016). The literature search retrieved 49 studies of global scope, which encompass multiple countries from more than one of the broad regions defined here, of which 18 studies were included in this review (see the data management report for Chapter 3 systematic literature review at https://doi.org/10.5281/zenodo.6452651). Among these, studies 15 address coastal fisheries, two address inland fisheries and two include both coastal and inland. These studies usually have a broad coverage in space or time, grouping data from many regions and communities and sometimes showing long time series of 50 up to 600 years (see the data management report for Chapter 3 systematic literature review at https://doi.org/10.5281/zenodo.6452651). One of these studies, which brings data for over 1,900 coastal indigenous communities around the world, representing 27 million people across 87 countries, claims that sustainability depends on increased recognition and directed research regarding the marine knowledge and resource needs of indigenous peoples, whose needs must be explicitly incorporated into management policies (Cisneros-Montemayor, Pauly, Weatherdon, & Ota, 2016). Other studies point to the potential overfishing of marine invertebrates (including cephalopods, shellfish, lobsters, crabs, sea cucumbers) estimating that, in 2004, 34% of invertebrate fisheries were over-exploited, collapsed, or closed, as global invertebrate catches have increased 6-fold (Anderson, Flemming, Watson, & Lotze, 2011). This problem is especially severe for sea cucumber fisheries, 81% of which show population declines from overfishing, and 35% had declines in the average harvested body size. Harvesters moved from near- to off-shore regions in 51% of cases and from high- to low-value species in 76% of these fisheries (Anderson, Flemming, Watson, & Lotze, 2011). Similarly, a global survey indicates that sawfishes (family Pristidae) have been heavily affected by intense harvesting and habitat degradation and these sawfish are now extinct in 55 of the 90 nations where they originally occurred (Yan et al., 2021). A study comparing the fisheries in Florida (Atlantic) and Hawaii (Pacific) over a period of 600 years indicated that, although fishing had been sustainable in Hawaii for 400 years, landings have declined and some species are recorded as overexploited in both the study regions (Mcclenachan & Kittinger, 2013). 3.3.1.4. Small-scale fisheries A study reviewing context and attributes of co- management initiatives in small-scale fisheries concludes that more research is needed to discern when co-management initiatives can transform pre-existing conflicts, challenge power asymmetries and distribute benefits more equitably (d’Armengol, Prieto Castillo, Ruiz-Mallén, & Corbera, 2018). However, another study indicates that fishers perceived improved livelihoods and compliance in co-managed sites, thus evidencing contributions of co- management to improve social sustainability (Cinner et al., 2012).  Europe Out of the 56 papers reviewed for Europe (see the data management report for Chapter 3 systematic literature review at https://doi.org/10.5281/zenodo.6452651). The vast majority 63 cover mainly the coastal and marine/oceanic fisheries in Europe or in European archipelagos in the Atlantic Ocean, the Mediterranean Sea (and its internal seas, like the Adriatic, the Aegean, the Marmara) or in the Black Sea. Ocean or marine small-scale fisheries is discussed in 48 papers, whereas only a small number of those (eight) investigated the European inland small-scale fisheries. A majority of these papers focused on Iberian freshwater fishing (Antunes, Cobo, & Araújo, 2015; Braga, Pereira, Morgado, Soares, & Azeiteiro, 2019; Marcos, Torres, López-Capel, & Pérez-Ruzafa, 2015; Maynou, Martínez-Baños, Demestre, & Franquesa, 2014), although there are other very important fishing practices, such as the trout fisheries, taking place in many different countries of the region (Shephard et al., 2019). The vast majority of the papers discuss the exploitation of fish species, but other organisms are also discussed, including a large diversity of targets in single fishing systems such as crustaceans and mollusks (Alonso-Fernández et al., 2019; Antunes et al., 2015; Azzurro et al., 2019; Battaglia et al., 2017; Carrà, Monaco, & Peri, 2017; Colloca, Scarcella, & Libralato, 2017; Corral & Manrique de Lara, 2017; Fabio, Silvia, Paolo, & Anelli Monti, 2016; Grati et al., 2018; Guyader et al., 2013; Palmer et al., 2017; Quetglas et al., 2017). A small number of papers also cover exploitation of crustaceans (Carvalho, Vasconcelos, Piló, Pereira, & Gaspar, 2017; Rivera et al., 2016; Rivera et al., 2017), mollusks (Baeta, Breton, Ubach, & Ariza, 2018; Duncan, Brand, Strand, & Foucher, 2016; Öndes, Kaiser, & Güçlüsoy, 2020; Pereira, Vasconcelos, Moreno, & Gaspar, 2019; Silva et al., 2019; Szostek, Murray, Bell, & Kaiser, 2017), benthic invertebrates (Bastari, Beccacece, Ferretti, Micheli, & Cerrano, 2017; Fourt, Faget, Dailianis, Koutsoubas, & Pérez, 2020; Pita et al., 2019) and even sea mammals (Maynou et al., 2011). The diversity of topics is a sign of the high diversity of fishing practices, technologies and techniques present in the European small-scale fishing. Contrary to the pattern observed in other regions, the literature on fishing rarely mentions lack of data on European small-scale fisheries. Still, lack of data does remain a concern in a number of cases including inaccuracy, large underestimation of parameters, undeclared information, and lack of stock assessment analysis for some fishing systems.  Europe Contrary to what is observed in the literature about the small-scale fisheries in other regions, no major cases of illegal, unreported and unregulated(Colloca et al., 2017; Ulman et al., 2013, 2015a) activities are focused upon in these studies (Colloca et al., 2017; Dinesen et al., 2019; Hornborg & Främberg, 2019; Marcos et al., 2015). Small scale fishing is an economically, socially, and culturally significant practice throughout Europe. It is well established that small-scale fishing plays an important role in many national economies (Guyader et al., 2013; Lloret et al., 2018), and almost 80% of the European fishing fleet belongs to small-scale fisheries (Quetglas et al., 2016). Sometimes, in general terms, this fishing is more profitable than the large-scale fishing industry since costs are lower and catches are similar (Almeida, Vaz, Cabral, & Ziegler, 2014). In some parts, the increase in the tourism industry and, less conspicuously, the increase in recreational fishing, led to a slight expansion in local economies (Marengo, Culioli, Santoni, Marchand, & Durieux, 2015) and generated new incomes and additional revenues in the form of concessions and permits (Antunes et al., 2015). On the other hand, it is also well established in the literature that small-scale European fishing is threatened by the competition among different uses of 64 aquatic resources and by decreasing profitability, detected in almost all systems evaluated (Maynou et al., 2014). When European small-scale fishing systems are analyzed, the majority of the papers describe activities that are still profitable (Roditi & Vafidis, 2019; Ünal & Franquesa, 2010), but that these profits dropped consistently in recent decades (Maynou et al., 2014; Pita et al., 2019; Quetglas et al., 2016). The reduction in market values and revenues is causing a marked change in local economies and in employment rates (Ünal & Franquesa, 2010), with serious impacts on traditional fishing communities. It is estimated that the European small-scale fisheries dropped from 30-50% in terms of income over this time period (Lloret et al., 2018). But in most of these cases small-scale fisheries continues as an important source of employment (Baeta et al., 2018) even if fishers have to work additional jobs to maintain their livelihoods (Braga et al., 2019; Pereira, Vasconcelos, Moreno, & Gaspar, 2019b). The drop in profits, revenues and wages are not only due to overexploitation of stocks, the decrease in market values or to climate change.  Europe Competition is also increasing due to the introduction of industrial and recreational fishing, which have caused major reductions to commercial small- scale fisheries landings and profits (Marengo et al., 2015; Maynou et al., 2013). European small-scale fishing the literature also highlights the exploitation of economically important and profitable high-valued stocks (Grati et al., 2018), with particular emphasis on scallops (Duncan et al., 2016; Szostek, Murray, Bell, & Kaiser, 2017), large demersal fish species (Quetglas et al., 2017), octopuses (Silva et al., 2019), carps (Hornborg & Främberg, 2019), cod (Dinesen et al., 2019), barnacles (Carvalho et al., 2017) and salmon (Antunes et al., 2015). Some of the additional profits can also come with the opportunity or possibility to exploit “labels of topicality” (Dinesen et al., 2019; Sartor et al., 2019). There are increasing trends in the demand of international market for these items, and their market values may pose a threat to their stocks (Antunes et al., 2015; Lloret et al., 2018). Most of these high- valued stocks were severely overexploited for a long time, and some of them are only now recovering after the introduction of more careful management measures (Rivera et al., 2016; Rivera et al., 2017). The strong economic and technological changes experienced in the last 60 or 80 years are accompanied by consistent social and cultural importance of these practices (Carvalho et al., 2017). Most of the local populations show a marked dependence on small-scale fisheries, in terms of food security, for the maintenance of local employment and for the resilience of cultural heritage (Braga et al., 2019; Colloca et al., 2017; Grati et al., 2018; Pereira et al., 2019; Ünal & Franquesa, 2010). In some European countries, more than 50% of the fishers are linked to one of the small-scale fishing systems in place (Antunes et al., 2015; Quetglas et al., 2016; Sartor et al., 2019; Silva et al., 2019). Small-scale fisheries employ twenty-four times more fishers than large-scale fishing (Leleu et al., 2014). The history of more traditional fishing systems goes back thousands of years (Antunes et al., 2015; Marcos et al., 2015). This strengthens cultural and historical bonds, and provides ongoing social meaning for indigenous people and local communities(Guyader et al., 2013).  Europe With the technological changes in the last 50 to 60 years, the efficiency of the fishing systems has (Alonso-Fernández et al., 2019; Pita et al., 2019; Quetglas et al., 2017; Ünal & Franquesa, 2010). Besides unemployment, other problems such as mechanization (Lloret et al., 2018). 65 While some unemployed fishers searched for new jobs, better wages or other sources of income (Maynou et al., 2013), many families had to close down business and sell their fishing equipment and boats to larger companies (Dinesen et al., 2019). The collapse of fishing systems and the overexploitation of stocks created new social contexts which demanded new and stricter management rules and improved governance, also seen as means to avoid social conflict (Marengo et al., 2015). These needs were partially met with the official management measures adopted in many areas, with distinct levels of success. Apparently, the recovery of social recognition of those engaged in this practice and the relevance of the small-scale fisheries was also an outcome of successful management initiatives at some places (Carvalho et al., 2017).  Africa From the initial selection of 63 papers, this evaluation on African small-scale fisheries is based on 51 papers covering mainly the coastal and marine/oceanic small-scale fishing, which was the subject of approximately 40 papers (see the data management report for Chapter 3 systematic literature review at https://doi.org/10.5281/zenodo.6452651). Despite the importance of established fisheries in tropical and subtropical African rivers, the reviewed literature focused on inland small-scale fishing in the great African lakes and small rivers. The fishing practices in African great lakes was studied in eight papers (Bulengela, Onyango, Brehm, Staehr, & Sweke, 2019; Hara & Njaya, 2015; Jamu, Banda, Njaya, & Hecky, 2011; Kolding, Béné, & Bavinck, 2014; Mgana et al., 2019; Mkuna & Baiyegunhi, 2019a, 2019b; van der Knaap & Ligtvoet, 2010). Similar analysis for fishing practices in some African smaller lakes was published in three studies (Kininmonth et al., 2017; Obegi et al., 2020; Tefera, Zerihun, & Wolde-Meskel, 2019), and there were a few examples of small river fishing in South Africa and Egypt (McCafferty, Ellender, Weyl, & Britz, 2012; Samy-Kamal, 2015). The majority of the papers describe fishing for fish species, but a small number also include fishing for crustaceans (Bush et al., 2017; Cochrane, Eggers, & Sauer, 2020; Fulanda, Ohtomi, Mueni, & Kimani, 2011; Le Manach et al., 2012; Le Manacha, Goughb, Humberb, Harperc, & Zellerc, 2011; Mirera, Ochiewo, Munyi, & Muriuki, 2013). There is scarce published data about African small-scale fishing. However, it is well established that many peoples rely on small-scale fishing for their subsistence and livelihoods throughout Africa (Musembi, Fulanda, Kairo, & Githaiga, 2019). Absence or inadequacy of data, under-estimates, and lack of stock assessment analysis were consistently mentioned by almost all papers reviewed. Those data sets supported by the FAO in many countries are usually underestimates since they are based only on landings, not considering data from illegal, unreported and unregulated fishing. Some papers present a reconstruction of data series, which attempted to include illegal, unreported and unregulated catch (Barnes-Mauthe, Oleson, & Zafindrasilivonona, 2013; Jacquet, Fox, Motta, Ngusaru, & Zeller, 2010; Le Manach et al., 2012; Seto et al., 2017). Only one third of the papers reviewed presented any socioeconomic evaluation of fishing sustainability across the continent, and only two papers were focused on this topic. All other social evaluations demonstrated the high level of dependence of local communities on fishing practices (Belhabib, Greer, & Pauly, 2018; Bush et al., 2017).  Latin America (South and Central America, Mexico) Latin America (South and Central America, Mexico) For the purpose of this assessment, Latin America includes the countries in South and Central America, Mexico and Caribbean Islands based on the similarities in their small-scale fisheries and social-ecological characteristics. This review is based on 107 articles (see the data management report for Chapter 3 systematic literature review at https://doi.org/10.5281/zenodo.6452651) from the review sources and those added by the assessment authors. These studies address coastal and inland small-scale fisheries in 15 countries with large numbers of studies from Brazil (55) and Mexico (20), which may reflect a larger number of fisheries scientists working in these countries rather than greater small-scale fisheries activity there. A selection of the studies provides international comparisons (Defeo et al., 2016; Maldonado, Lopes, Fernández, Alcala, & Sumalia, 2017) or continental level comparisons (Brotz et al., 2017). Most studies (78) addressed the use of finfish but also reported on sharks, shellfish, lobsters, octopus, crabs and jellyfish. More than two thirds of the studies (78) deal with coastal fisheries with fewer (29) studies addressing inland fisheries, and most of these (25) were in the Amazon region (for details on the reviewed studies, (see the data management report for Chapter 3 systematic literature review at https://doi.org/10.5281/zenodo.6452651). More than half of the studies (56) had short time series ranging from 1 to 15 years of data collection. A few studies (17) included long range data series of 50 years or more, some of which included indigenous and local knowledge through interviews with seniors. As expected, those well managed and ecologically sustainable fisheries were also considered to be economically sustainable and showed improved economic indicators such as increased prices or profits from sales of managed resources. These offset eventual decreases in total catches due to management measures, as observed among coastal invertebrate fisheries under territorial rights in Chile and Mexico (Álvarez, Espejel, Bocco, Cariño, & Seingier, 2018; De la Cruz-González, Patiño-Valencia, Luna-Raya, & Cisneros-Montemayor, 2018; Defeo et al., 2016; Gelcich et al., 2010). Nevertheless, the territorial users’ rights fisheries management in Chile also caused economic shortages through the collapse of a clam fishery and reduced economic opportunities to fishers not engaged in territorial users’ rights fisheries management, who relied on depleted open access areas (Aburto & Stotz, 2013; Garmendia, Subida, Aguilar, & Fernández, 2021).  Africa 66 Formal economic review shows that market prices either kept stable or increased in the last 60 years. This is an important factor to explain the increase in fishing effort and overexploitation of most stocks. Pressure from international markets for some high value species for exportation also added pressure on the stocks. This increased pressure led to increased competition between international fleets and local boats and sometimes conflict (Belhabib et al., 2016; Seto et al., 2017).  Latin America (South and Central America, Mexico) The positive economic effects observed in coastal shellfish fisheries were also observed in the pirarucu co-managed fishery in the Brazilian Amazonian rivers (Campos-Silva & Peres, 2016; Castello, Viana, Watkins, Pinedo-Vasquez, & Luzadis, 2009), where increased revenues from co-management led to further social benefits, through gender equality and improved income for women (Freitas, Espírito-Santo, Campos-Silva, Peres, & Lopes, 2020). Other studies on coastal small-scale fisheries employed economic modelling, which indicate that a 67 fishery of octopus (Octopus maya) in Mexico would be more sustainable under current management, as economic performance does not improve under alternative management scenarios (Duarte, Hernández-Flores, Salas, & Seijo, 2018a). Similarly, the recovery of shellfish through co-management in a Mexican community was shown to be profitable under two of four estimated future economic scenarios (Palacios-Abrantes, Herrera-Correal, Rodríguez, Brunkow, & Molina, 2018). One study on fisheries in French Guiana evaluated various sustainability indicators, which suggested average sustainability for ecological, economic and social dimensions. Smaller fishing fleets were considered to be more sustainable (Cissé, Blanchard, & Guyader, 2014). Several coastal small-scale fisheries considered to be less economically sustainable were the fishing of spawning aggregations of reef fish in Mexico (Erisman et al., 2010) and the shark fishing in Mexico (Martínez-Candelas, Pérez-Jiménez, Espinoza-Tenorio, McClenachan, & Méndez-Loeza, 2020) and Brazil (Martins et al., 2018). The decline in the economic sustainability of shark fishing is attributed to decreases in shark fishing activity, revenues and profits from shark fins. Other economic problems refer to inequalities in the distribution of profits among crew members and boat owners (De Figueiredo Silva, Camargo, & Estupiñán, 2012), low prices paid to fishers by the middlemen, the concentration of profits in large private companies (Gamboa- Álvarez, López-Rocha, Poot-López, Aguilar-Perera, & Villegas-Hernández, 2020a; Jimenez, Barboza, Amaral, & Lucena Frédou, 2019) and increasing costs related to fishing operations such as fuel to reach more distant fishing grounds (Daw, 2008). A study with crab gatherers in the Brazilian Amazonian coast considers the fishery ecologically sustainable (catches and sizes of crabs did not change), but not economically and socially sustainable. The relative revenue for fishers also declined, which sometimes led to social conflicts (Glaser & Diele, 2004). The social aspects of small-scale fisheries were addressed by only 19 of 78 studies on coastal small-scale fisheries and 5 of 29 studies on inland small-scale fisheries (see the data management report for Chapter 3 systematic literature review at https://doi.org/10.5281/zenodo.6452651).  Latin America (South and Central America, Mexico) Some of the territorial co-management coastal fisheries of invertebrates, mainly in Chile and Mexico, show social benefits such as improved perceptions among fishers about the fishery, more time available to dedicate to other activities, decreased conflicts over resources, reinforced property rights over resources, improved institutional collaboration, community organization and capacity building (Álvarez et al., 2018; Defeo et al., 2016; Gelcich et al., 2017, 2010; Palacios-Abrantes, Herrera-Correal, Rodríguez, Brunkow, & Molina, 2018). Similarly, the co-managed pirarucu fisheries in the Brazilian Amazon have improved social sustainability through more equalitarian distribution of income, sense of pride, stronger culture and indigenous and local knowledge (Campos-Silva & Peres, 2016; Freitas et al., 2020). In coastal small-scale fisheries some problems undermining social sustainability are ongoing conflicts between fishers and managers of protected areas (De Figueiredo Silva et al., 2012; Jimenez et al., 2019; Lopes, Rosa, Salyvonchyk, Nora, & Begossi, 2013; Lopes, Silvano, Nora, & Begossi, 2013). These include increased theft of fishing gear and potential competition for space with industrial vessels (Daw, 2008), high risk practices, such as diving, which can involve accidents (Gamboa-Álvarez et al., 2020; Guebert-Bartholo, Barletta, Costa, Lucena, & Da Silva, 2011) and disruption of fishing cooperatives (Rubio-Cisneros, Aburto-Oropeza, 68 Jackson, & Ezcurra, 2017). Even in the relatively successful co-managed Chilean shellfish. Other social problems at the Brazilian coast include increased commercialization and price of shark meat, which decreases the availability of shark meat for local people and threatens their food security (Barbosa-Filho et al., 2019). Scientific and indigenous and local knowledge informed assessments have at times differed about the sustainable use of certain fisheries. For example, in a Colombian lagoon community social conflict arose between fishers and researchers due to differences in how they conceptualize sustainability, (Torres-Guevara, Lopez, & Schlüter, 2016). A similar situation was observed in the Dominican Republic where fishers, based on their indigenous and local knowledge, considered the fisheries as more depleted through catches of juvenile fish of most species but scientists believed the fisheries targeted mostly adult fish and would thus be in a better state (Mclean & Forrester, 2018). Both cases draw attention to the need for better dialogue and cooperation between fishers and scientists.  Latin America (South and Central America, Mexico) The main social problems related to the inland ornamental fisheries in the Brazilian Amazon are the negative effects of a reduced trade in the Negro River and a potential collapse of exploited species in the Xingu River, which will drastically reduce income and negatively affect the livelihoods of many impoverished riverine people, most of whom lack employment alternatives (Evers, Pinnegar, & Taylor, 2019a). Another social problem of this fishery is the health issues related to the labor-intensive fishing performed mostly by aged fishers. Younger people are less and less involved in these activities. Not only does this have negative impacts on the labor distribution, but may also disrupt knowledge transmission of indigenous and local knowledge (Ladislau et al., 2020).  North America From a total of 28 sources on coastal and inland small-scale fisheries in temperate North America retrieved, 22 are included in this review (see the data management report for Chapter 3 systematic literature review at https://doi.org/10.5281/zenodo.6452651), which are evenly distributed between the United States of America (12) and Canada (9). One study addressed both countries, which is also the only study on inland fisheries (Cooke & Murchie, 2015). The reviewed studies include a variety of fishing resources, such as coastal and reef fishes, crabs, lobster, shellfish and sea cucumbers (see the data management report for Chapter 3 systematic literature review at https://doi.org/10.5281/zenodo.6452651). We also include a case study on the sustainability of small-scale whaling activities in the north (see Box 3.4). Six studies focus on economic and 12 studies highlight social considerations in small- scale fisheries of Canada and the United States of America (see the data management report for Chapter 3 systematic literature review at https://doi.org/10.5281/zenodo.6452651). Some of these studies underscore the high economic value of the recreational fisheries practice in Florida, which provides jobs and revenues (Ault, Bohnsack, Smith, & Luo, 2005). For example, the catch-and-release fishery in South Florida (and the Caribbean) has an estimated value of at least half a billion dollars per year (Kroloff et al., 2019). Similarly, the lobster (Homarus americanus) fishery is very important to the region of the Gulf of Maine in Canada (Boudreau & Worm, 2010). Some studies indicate potential negative interactions among economic activities. For example, commercial fishing coupled with the expansion of sports (recreational) fishing in the last decades may had affected yelloweye rockfish (Sebastes ruberrimus) 69 populations (Eckert et al., 2018). Similarly, food security in Alaska has been negatively affected by the development of export-oriented commercial fisheries and tourism-oriented sport fisheries (Harrison & Loring, 2016). Another study reports changes in fishing area or practices in response to changing market infrastructure (e.g., switch to frozen from salt cod), besides changes in economic factors external to the fishery, such as loss of other income generating activities, which can affect the economic sustainability of cod (Gadus morhua) in Newfoundland, Canada (Murray, Neis, & Schneider, 2008). Some of the studies that mention social characteristics of small-scale fisheries comment on the relevance of fishing resources to local peoples’ livelihoods and food security (see the data management report for Chapter 3 systematic literature review at https://doi.org/10.5281/zenodo.6452651).  North America For example, fishing of Arctic char (Salvelinus alpinus) has high value for food security, cultural identity and local economic development among Arctic communities (Roux et al., 2019). Conversely, the observed decline in the catches of the Dungeness crab may compromise the ability of indigenous fishers to access traditional foods in Canada (Ban et al., 2017). Other studies emphasize the relevance and benefits of integrating multiple knowledge sources in fisheries assessments, including fishers’ indigenous and local knowledge, which may improve dialogue, cooperation and social relations between fishers and scientists (Ambrose et al., 2014; Ban et al., 2017; Murray, Neis, Palmer, et al., 2008; Murray, Neis, & Schneider, 2008; Rehage et al., 2019). The study on inland fisheries mentions that food security and the move towards eating locally may create new markets for freshwater fish, as long as they have low contaminant loads and are considered healthy (Cooke & Murchie, 2015).  Asia-Pacific The Asia-Pacific region includes countries from Asia, Oceania and the South Pacific Island countries. From a total of 119 sources originally retrieved for this region, 96 studies were included in the review, in conjunction with literature from assessment authors. These studies cover small-scale fisheries in more than 36 countries (see the data management report for Chapter 3 systematic literature review at https://doi.org/10.5281/zenodo.6452651) from Southeast Asia (Mattson, 2006), Western Asia (Al-Abdulrazzak, Zeller, Belhabib, Tesfamichael, & Pauly, 2015) and the Pacific (Cohen & Foale, 2013; Cruz-Trinidad, Aliño, Geronimo, & Cabral, 2014; Eriksson et al., 2018; Kronen, Magron, McArdle, & Vunisea, 2010; D. Zeller et al., 2015). Several countries appeared in only one or two studies; more studies addressed small-scale fisheries in Indonesia (18), the Philippines (10), Australia (7), India (9), Bangladesh (5) and the Solomon Islands (5). The overwhelming majority (82 %) of studies addressed coastal or marine and only 10 studies focused on inland small-scale fisheries, whereas three recent studies in Southeast Asia included both coastal and inland fisheries (Jahan, Ahsan, & Farque, 2017; Liao et al., 2019; Millar et al., 2019). Most studies report the uses of finfish, while fewer studies focus on other organisms (sharks, invertebrates). Several studies included many species (finfish and other organisms), evidencing the multi-species characteristic of these small-scale fisheries (see the data management report for Chapter 3 systematic literature review at https://doi.org/10.5281/zenodo.6452651). Although some studies had short time series of up to one year, several studies analyzed time series of 10 years or more (see the data management report for Chapter 3 systematic 70 literature review at https://doi.org/10.5281/zenodo.6452651) and at least one study included indigenous and local knowledge and archeological data to analyze a time series of 3,000 years in American Samoa (P. Craig, Green, & Tuilagi, 2008). Among the studies analyzing long time series of 50 to 60 years, some include indigenous and local knowledge on temporal trends (Lavides et al., 2016; Muallil, Mamauag, Cababaro, Arceo, & Aliño, 2014; Selgrath, Gergel, & Vincent, 2018a, 2018b; Thurstan, Buckley, Ortiz, & Pandolfi, 2016a), while others apply a methodology to reconstruct catches along time series with missing data (Al-Abdulrazzak et al., 2015; Léopold et al., 2017; D. Zeller et al., 2015).  Asia-Pacific Considerations or analyses related to economic sustainability were included in 45 and 11 of the reviewed studies on coastal and inland (or coastal and inland) small-scale fisheries, respectively (see the data management report for Chapter 3 systematic literature review at https://doi.org/10.5281/zenodo.6452651). Some of the ecologically sustainable or partially sustainable coastal fisheries also show net economic benefits due to improved or maintained catches, as observed for the shrimp fisheries in Indonesia (Anna, 2017) and abalone fisheries in Australia (Mayfield, Mundy, Gorfine, Hart, & Worthington, 2012). Fishing is an important economic activity for the Pacific Island countries located in the coral triangle area (Cruz-Trinidad et al., 2014). Some of the co-managed reef fisheries in Pacific Island countries can deliver tangible economic benefits to local communities in the form of increased catches (Tilley, Hunnam, et al., 2019; Webster et al., 2017; Yang & Pomeroy, 2017), for example, through periodic harvesting in protected areas, which can provide a needed boost to local economies (Cohen, Cinner, & Foale, 2013). However, some highly valued economic resources, such as sea cucumbers or lobsters (Panulirus ornatus), have been overfished, particularly in the Philippines and Indonesia, due to increased market demands (Hair, Foale, Kinch, Yaman, & Southgate, 2016; Macusi, Laya-og, & Abreo, 2019; Prescott, Riwu, Prasetyo, & Stacey, 2017). The sea cucumbers fishery has high export value and provides an economic insurance for island populations of Pacific Island countries, but some of these fisheries had to be closed to recover, which compromised the economic benefits (Eriksson et al., 2018; Hair et al., 2016). The economic sustainability of coastal fisheries could also be negatively affected by long market chains with strong inequalities in the distribution of profits between fishers and final retailers (Ferse, Glaser, Neil, & Schwerdtner Máñez, 2014). The low price paid to fishers can interact with increased costs of fuel and other components of the fishing activity, prompting fishers to intensify their fishing effort to cover fishing trips to more distant fishing grounds (Sebastian Ferse, Knittweis, Krause, Maddusila, & Glaser, 2012; G. M. N. Islam, Noh, Sidique, & Noh, 2014; Muallil, Mamauag, Cababaro, et al., 2014; Rhodes, Tupper, & Wichilmel, 2008). Aspects related to the social and cultural sustainability were presented in 32 and 8 of the reviewed studies on coastal and inland small-scale fisheries, respectively (see the data management report for Chapter 3 systematic literature review at https://doi.org/10.5281/zenodo.6452651).  Asia-Pacific Several studies highlighted the social benefits of these fisheries in the form of food provision and sustaining livelihoods of local communities (Al-Abdulrazzak et al., 2015; Butler, Tawake, Skewes, Tawake, & McGrath, 2012; Cruz- Trinidad et al., 2014; Friedlander et al., 2014; Golden, Naisilsisili, Ligairi, & Drew, 2014; Rassweiler et al., 2020). Fishing is also an important cultural and social activity among many of the coastal fishing communities, reinforcing cultural identity and social practices, such as The economic sustainability of coastal fisheries could also be negatively affected by long market chains with strong inequalities in the distribution of profits between fishers and final retailers (Ferse, Glaser, Neil, & Schwerdtner Máñez, 2014). The low price paid to fishers can interact with increased costs of fuel and other components of the fishing activity, prompting fishers to intensify their fishing effort to cover fishing trips to more distant fishing grounds (Sebastian Ferse, Knittweis, Krause, Maddusila, & Glaser, 2012; G. M. N. Islam, Noh, Sidique, & Noh, 2014; Muallil, Mamauag, Cababaro, et al., 2014; Rhodes, Tupper, & Wichilmel, 2008). Aspects related to the social and cultural sustainability were presented in 32 and 8 of the reviewed studies on coastal and inland small-scale fisheries, respectively (see the data management report for Chapter 3 systematic literature review at https://doi.org/10.5281/zenodo.6452651). Several studies highlighted the social benefits of these fisheries in the form of food provision and sustaining livelihoods of local communities (Al-Abdulrazzak et al., 2015; Butler, Tawake, Skewes, Tawake, & McGrath, 2012; Cruz- Trinidad et al., 2014; Friedlander et al., 2014; Golden, Naisilsisili, Ligairi, & Drew, 2014; Rassweiler et al., 2020). Fishing is also an important cultural and social activity among many of the coastal fishing communities, reinforcing cultural identity and social practices, such as 71 sharing fish, in the Pacific Island countries (Golden et al., 2014; Rassweiler et al., 2020). Indeed, Maori coastal fishers in New Zealand have perceived declines in culturally important nearshore resources (fish and invertebrates), which has negative cultural effects on communal activities, social connections, traditions, connections to nature and loss of pride of being able to feed themselves and guests by using seafood (Mccarthy et al., 2014).  Asia-Pacific Besides improving catches and increasing the abundance of fishing resources, the commons-based management systems implemented in Pacific Islands can promote social sustainability through empowerment of local communities, increased compliance with management rules and the development of a sense of ownership of fishing resources (Butler et al., 2012; Cinner et al., 2012; Friedlander et al., 2014; Webster et al., 2017; Yang & Pomeroy, 2017). These co-management systems often include community rules and beliefs, sometimes resulting in social benefits by participating communities even before perceived improvements on fisheries (Tilley, Hunnam, et al., 2019). Fishery closures imposed by co-management may exclude some social groups, such as women or immigrants, from access to fishing grounds, besides imposing social costs in the form of restricted harvestings (Ayunda, Sapota, & Pawelec, 2018; Cohen & Foale, 2013). The relationship between fishers and middlemen can either improve or undermine social sustainability. For example, in Indonesia, some of the middlemen (locally called patrons) may have social ties with fishers and contribute to social welfare by providing social security for impoverished fishers in need, whereas other, wealthier patrons (big patrons), may not have these social ties. This may result in provision of credit and loans to fishers to buy fishing gear (including illegal and high impact types) which may result in unsustainable fishing practices and further exploit fishers by making them sell catches at low prices (Ferse et al., 2014; Ferse et al., 2012). 3.3.1.4.1. Indicators of small-scale fisheries sustainability Across the 350 small-scale fisheries studies the main indicators adopted were: (i) catch biomass or composition (landings’ data) in 214 studies, (ii) measures of catch per unit of effort, in 78 studies), (iii) abundance estimates and trends (72 studies), (iv) based on either fishers’ knowledge or biological sampling, fishing effort, such as number of boats and other measures (73 studies), (v) size of harvested species (57 studies) and varied measures of stock assessment (51 studies). The majority (214) of reviewed studies included indigenous or local knowledge from fishers to inform the indicators outlined here, so fishers’ knowledge can be also considered an important indicator and information source for small-scale fisheries. Some studies have also included economic related indicators, such as market prices, costs, revenues (83 studies) or social indicators, such as culture, governance or management (46 studies), see the data management report for Chapter 3 systematic literature review at https://doi.org/10.5281/zenodo.6452651 for more detailed data.  Europe A very diverse set of indicators using three perspectives (i.e., ecological, economic and social) was employed to assess sustainability in the papers reviewed. The use of parameters from stock assessments analysis as indicators for ecological sustainability assessments is not very common 72 in the literature and only a few studies use them in conjunction with other indicators, such as maximum sustainable yield (Colloca et al., 2017; Dinesen et al., 2019; Hornborg & Främberg, 2019; Marcos et al., 2015), or different measurements of stock abundance and distribution (Bastari et al., 2017; Braga, Pardal, & Azeiteiro, 2018; Damalas et al., 2015; Lloret et al., 2015; Macdonald, Angus, Cleasby, & Marshall, 2014; Shephard et al., 2019; Szostek, Murray, Bell, & Kaiser, 2017). The use of fish biometry and size distributions in cohort analysis is not usual, but is present (Grati et al., 2018; Shephard et al., 2019; Vasconcelos et al., 2020), also in association with other methods and indicators. However, as expected, most of the ecological assessments reviewed (41 out of 63 papers) support their conclusions with landing statistics (production/catch biomass, catch composition) and related parameters to measure fishing effort and catch-per-unit-of-effort. Catch biomass or biomass landed (58.2% of reviewed studies), catch-per-unit-of-effort (40.3%) and catch composition or species landed (13.4%) were the indicators used more frequently in the ecological evaluations of small-scale fishing in Europe (see the data management report for Chapter 3 systematic literature review at https://doi.org/10.5281/zenodo.6452651). In addition, the use of indicators of local ecological knowledge from local fishers in association with other indicators, is notable (Azzurro et al., 2019; Braga et al., 2017, 2019; Coll et al., 2014; Corral & Manrique de Lara, 2017; Damalas et al., 2015; Dinesen et al., 2019; Figus et al., 2017; Lloret et al., 2015; Maynou et al., 2011; Öndes, Kaiser, & Güçlüsoy, 2020). Socioeconomic assessment alone was a rare approach in the literature of fishing sustainability (Ünal & Franquesa, 2010).  Europe Nevertheless, the assessment of economic and social aspects of European small-scale fisheries as part of ecological assessments was not that unusual, and made use of a set of related indicators such as values of landings, market values, market prices, revenue and income generation, both by the fleets and by the individual fishers (Carvalho et al., 2017; Grati et al., 2018; Guyader et al., 2013; Lloret et al., 2018; Maynou et al., 2014, 2013; Pita et al., 2019; Quetglas et al., 2017; Rivera et al., 2016; Rivera et al., 2017; Roditi & Vafidis, 2019; Sartor et al., 2019; Silva et al., 2019b; Tzanatos et al., 2013; Ulman et al., 2013). Despite the fact that a very limited number of assessments based on the social perspective was found in the reviewed literature, these studies applied a diverse set of indicators. Those indicators were based on tradition (cultural, historic values) and on the level of dependence of the local communities on the fishing practices for their livelihoods (Guyader et al., 2013; Ünal & Franquesa, 2010). The more frequent approach for social assessments was the use of the indicators of governance efficiency and effectiveness of fishers’ organizations in charge of co-management, or participatory management systems of aquatic resources (Baeta et al., 2018; Morales-Nin et al., 2017; Silva et al., 2019b). These may represent the main critical issues that are discussed by experts on the social perspectives of the European small-scale fishing and fishers.  Africa Since proper stock assessments are not very common (due to high costs, lack of personnel, time and other means) the authors used a diverse set of indicators. Only a small number of studies 73 used stock assessments to produce estimates of maximum sustainable yield, yield per recruit, or cohort analysis and species-specific life table parameters (Fulanda et al., 2011; Hara & Njaya, 2015; Jamu et al., 2011; Meissa, Gascuel, & Rivot, 2013; Rehren, Wolff, & Jiddawi, 2018). Most of the assessments support their conclusions based on series of catch/production, such as landing statistics. Catch biomass (biomass landed) and catch composition (species landed) are the more frequent parameters in the ecological evaluations. Nevertheless, catch- per-unit-of-effort and size distribution of fish landed are also frequently used indicators. More than 45 papers use fish landings and/or catch-per-unit-of-effort as indicators to support their analysis (see the data management report for Chapter 3 systematic literature review at https://doi.org/10.5281/zenodo.6452651). Additional indicators were used for the assessment of economic and social aspects. Indicators for economic evaluation were revenue and market prices (Blythe, Murray, & Flaherty, 2013), relevance of foreign markets for exportations, and added costs and values (Baker-Médard & Faber, 2020). Indicators for social evaluation were level of dependence for livelihoods, employment, number of people involved (Belhabib et al., 2015), influence of indigenous and local knowledge and the persistence/resilience of these last two (Bulengela et al., 2019; Gaspare, Bryceson, & Kulindwa, 2015). In some cases, the persistence of cultural traits, like traditional knowledge, was seen as an indicator of social sustainability (Mirera et al., 2013).  Latin America This review evidenced the limitations imposed by the lack of continuous monitoring to provide fisheries and biological data to evaluate sustainable use. Only a few studies included more detailed population analyses and measured conventional stock parameters, such as maximum sustainable yield, natural mortality, fishing mortality, among others (Aburto & Stotz, 2013; Baigún, Minotti, & Oldani, 2013; Catarino, Kahn, & Freitas, 2019; Cavieses Núñez, Ojeda Ruiz De La Penã, Flores Irigollen, Rodríguez Rodríguez, & Jardim, 2018; Duarte et al., 2018a; Martínez-Candelas et al., 2020; Mesquita, Cruz, Hallwass, & Isaac, 2019). The reviewed studies applied a varied set of indicators, often in combination, including total catches or landings (58), catch-per-unit-of-effort (22), and size of exploited fishing resources (37). Catch composition and its variation through time, measures of fishing effort, such as number of fishers, vessels and the distribution of effort in space and time, economic indicators (revenues and costs), and overall abundance trends estimated from indigenous and local knowledge were also used as indicators of sustainable use (see the data management report for Chapter 3 systematic literature review at https://doi.org/10.5281/zenodo.6452651). A few studies calculated and compared sustainability indicators based on ecological, economic and social data (Cissé et al., 2014; Robotham et al., 2019; Torres-Guevara et al., 2016). However, most of the reported trends are based on total catches only. The lack of effort or catch-per-unit-of-effort data makes it more difficult to properly assess the sustainability of these fisheries. Furthermore, while some species are preferred, most of these fisheries are multi-species and multi-gear (see the data management report for Chapter 3 systematic literature review at https://doi.org/10.5281/zenodo.6452651). This imposes further challenges to sustainability assessments, as exploited species may differ regarding their resilience to fishing pressure and stock status. These challenges were addressed by most of the reviewed 74 studies through two main, non-mutually exclusive, approaches. First, to rely on a variety of the indicators described above and second, to include fishers’ knowledge about catches, trends, details of fishing effort in combination with fisheries data, biological surveys or modelling. Indeed, indigenous and local knowledge was included in the majority (69) of studies reviewed (see the data management report for Chapter 3 systematic literature review at https://doi.org/10.5281/zenodo.6452651).  North America The indicators adopted in the reviewed studies include catch (landings data), population and stock parameters, environmental or ecological indicators, productivity susceptibility analysis and various modelling approaches (see the data management report for Chapter 3 systematic literature review at https://doi.org/10.5281/zenodo.6452651). Even considering that both countries have a well-developed fisheries science and management with strong financial and technical capacity, the majority of the reviewed studies (17) include fishers’ knowledge or indigenous and local knowledge, usually in combination with the above-mentioned fisheries and ecological indicators (see the data management report for Chapter 3 systematic literature review at https://doi.org/10.5281/zenodo.6452651). Moreover, fishers’ knowledge has been included in these studies on various forms or manifestations, from traditional knowledge of indigenous people, usually from the Artic (Ambrose et al., 2014; Ban et al., 2017; Eckert et al., 2018; Roux et al., 2019) to local knowledge held by recreational fishers or commercial harvesters (Frezza & Clem, 2015; Kroloff et al., 2019; Murray, Neis, Palmer, et al., 2008; O’Regan, 2015).  Latin America Many studies stressed the important economic role of both coastal and inland small- scale fisheries in the studied regions, but relatively few studies included economic indicators (profits, revenues), analyzed market chains, or evaluated the economic sustainability of the studied fisheries (see the data management report for Chapter 3 systematic literature review at https://doi.org/10.5281/zenodo.6452651). Economic considerations were mentioned by 42% of the 78 studies on coastal small-scale fisheries and by 45% of the 29 studies on inland small- scale fisheries, sometimes linked to the analysis of catch trends and ecological sustainability.  Asia-Pacific The reviewed studies employed a wide range of indicators, most commonly catches (landings data), catch-per-unit-of-effort, fishing effort, abundance (density) and size of exploited fishing resources, besides socioeconomic indicators (see the data management report for Chapter 3 systematic literature review at https://doi.org/10.5281/zenodo.6452651). Almost two thirds (66) of the reviewed studies included indigenous and local knowledge-based indicators to inform fish abundance trends, catches, catch-per-unit-of-effort, sizes, fishing effort, perceptions on management or socioeconomic status, thus indicating the relevance of indigenous and local knowledge and collaboration with fishers for research on these small- scale fisheries. 3.3.1.4.2. The role of indigenous and local knowledge in small-scale fisheries 75 Despite the review provided here, it is also widely acknowledged that most small-scale fisheries remain unreported and unmonitored, resulting in the lack of longer time series data to evaluate their sustainability. This is especially the case in tropical countries and the Arctic, where small-scale fisheries are widespread. These data gaps can be overcome through collaborative research to record and analyze fishers’ local ecological knowledge, a form of indigenous and local knowledge based on an experiential understanding of one’s environment coupled with communal and historical use. Fishers’ local ecological knowledge contributes to estimates on temporal trends in abundance of fisheries resources, and can extend the time series available for the analysis to periods before scientific monitoring (Giglio, Luiz, & Gerhardinger, 2015; Hallwass et al., 2020; Jahan, Ahsan, & Farque, 2017; Maia et al., 2018; Stocks, Foster, Bat, Ha, & Vincent, 2019a) or data (Sáenz–Arroyo, Roberts, Torre, & Cariño‐Olvera, 2005) were available. Indeed, in many cases worldwide fishers’ knowledge is the only available knowledge source. In the last 20 years, several studies have recorded fisher indigenous and local knowledge and local ecological knowledge through using qualitative methods, such as interviews with fishers, to reconstruct temporal trends in fisheries resources. This was the case in 56 of the studies reviewed here. Through these studies data were collected from an aggregated total of 13,565 fishers (through interviews), on approximately 454 fish species in 32 countries worldwide (Table 3.3). All the studies further quantitatively analyzed fishers’ local ecological knowledge to identify trends in abundance, size and composition of fisheries resources through a series of indicators such as estimated abundance categories (declined, same, increased), catch per unit of effort, amounts of regular, poor and best catches, and size (length or weight) of largest ever caught (Table 3.3).  Asia-Pacific The time span covered by these studies varies from 5 to 10 years (Daw, Robinson, & Graham, 2011; Liao et al., 2019; Lima, Begossi, Hallwass, & Silvano, 2016; O’Donnell, Molloy, & Vincent, 2012) to several decades, with some going back to the 1950s and 1960s (Ainsworth, 2011; Lavides et al., 2016; Lozano-Montes, Pitcher, & Haggan, 2008). The influence of time on fishing parameters has been analyzed either as a continuous variable (for example, year of the best catch) or as an interval categorical variable (for example, discrete years or decades according to fishers’ age groups, specific events, etc.) (Table 3.3). Most of the studies reported declining trends in abundance, catch-per-unit-of-effort or size of fishing resources (Table 3.3). Reported declines were usually focused on threatened species, some of which had been intensely exploited, such as reef fishes from the genus Epinephelus and Mycteroperca (groupers) (Bender, Floeter, & Hanazaki, 2013; Bender et al., 2014; Bunce, Rodwell, Gibb, & Mee, 2008; Castellanos-Galindo et al., 2018; Giglio et al., 2015; Ribeiro, Damasio, & Silvano, 2021a; Zapelini, Bender, Giglio, & Schiavetti, 2019), the large catfish (Pangasius sanitwongsei) in the Mekong River (Gray, Phommachak, Vannachomchan, & Guegan, 2017), seahorses (Hippocampus spp.) (Stocks, Foster, Bat, Ha, & Vincent, 2019b), the angel shark (Squatina squatina) in the Mediterranean (Fortibuoni, Borme, Franceschini, Giovanardi, & Raicevich, 2016), sawfish species (Pristis spp.) in coastal ecosystems (Jabado et al., 2017; Leeney & Poncelet, 2015), and the paddlefish (Psephurus gladius) in Yangtze River (Turvey et al., 2010), among others (Table 3.3). g ) g ( y ) g ( ) A phenomenon sometimes related to studies based on fishers’ memories to reconstruct past events is known as shifting baseline syndrome, i.e., environmental changes may be 76 recognized only by older fishers and underestimated or not recognized by younger ones (Papworth, Rist, Coad, & Milner-Gulland, 2009; Pauly, 1995). Shifting baseline has been observed by many studies worldwide, which reported an influence of age on fishers’ perceptions about changes in the abundance of fisheries resources (Bender et al., 2013, 2014; Katikiro, 2014; Lozano-Montes et al., 2008; Maia et al., 2018; Turvey et al., 2010; Ulman & Pauly, 2016). However, further studies show that this is not always the case, and both older and younger fishers may hold similar perceptions (Barbosa-Filho et al., 2020; Hallwass, Lopes, Juras, & Silvano, 2013; Ribeiro et al., 2021; Thurstan, Buckley, Ortiz, & Pandolfi, 2016b).  Asia-Pacific Furthermore, fishers also report stable catches or sizes of at least some fish species (Ribeiro et al., 2021; Silvano & Hallwass, 2020). Limitations related to the application of fishers’ local ecological knowledge to estimate abundance trends include heavy reliance on fishers’ memories, which at time may be inaccurate or biased due to memory illusion or shifting baseline syndrome (Daw et al., 2011; O’Donnell, Molloy, & Vincent, 2012; Papworth et al., 2009). However, it should be noted that the ways in which local ecological knowledge and indigenous and local knowledge data are collected include methods for minimizing bias such as data triangulation amongst community members, data comparisons with archival and spatial data, and sampling techniques intended to identify the most robust knowledge holders. It is also quite common to search for points of comparison between indigenous and local knowledge/ local ecological knowledge and scientific knowledge. For example, more than half (29) of the reviewed studies included conventional scientific databases, such as biological sampling, fish catches, or governmental monitoring, which were compared with data gathered from fishers (Table 3.3). Although disagreements or partial agreements were observed in eight studies, most studies (21) showed high levels of agreement between trends based on local ecological knowledge and those based on scientific data (Table 3.3). This further reinforces the usefulness and reliability of fishers’ local ecological knowledge to evaluate temporal trends in fisheries. Other studies integrated fishers’ local ecological knowledge and conventional scientific data in models to show fisheries trends (Ainsworth, 2011; Ban et al., 2017). A few studies also analyzed and observed temporal changes in the composition of fishing resources (Table 3.3), usually indicating a shift from the exploitation of more valuable large fish to less valuable smaller fish (Coll et al., 2014; Godoy, Gelcich, Vasquez, & Castilla, 2010; G. Hallwass et al., 2019; Jaiteh, Hordyk, Braccini, Warren, & Loneragan, 2017; Strieder Philippsen, Minte-Vera, Okada, Carvalho, & Angelini, 2017) or the disappearance of some species altogether (Damasio, Lopes, Guariento, & Carvalho, 2015; Katikiro, 2014; Lavides et al., 2016). These temporal changes in catch composition (Table 3.3) suggest that fisheries may have experienced ‘ecological filters’ in some freshwater and marine ecosystems, indicating genetic selection through specific forms of harvesting activities. The extent to which species diversity or specific species characteristics are affected in this way is uncertain.  Asia-Pacific Some of the reviewed studies have also provided useful information based on fishers’ local ecological knowledge related to drivers or consequences of observed trends, including protected areas (Hallwass et al., 2020), environmental impacts including dams or pollution (S. Dey, Choudhary, Dey, Deshpande, & Kelkar, 2019; Frezza & Clem, 2015; Gustavo Hallwass, Lopes, Juras, & Silvano, 2013; Jahan, Ahsan, & Farque, 2017; Strieder Philippsen et al., 2017), climate change (Ernesto Azzurro, Moschella, & Maynou, 2011; Eckert et al., 2018), 77 distribution and ecology of invasive species (Araujo Catelani, Petry, Mayer Pelicice, & Azevedo Matias Silvano, 2021; Ernesto Azzurro & Cerri, 2021; Boughedir et al., 2015; van Putten et al., 2016), or trophic cascades associated with fishing (Boudreau & Worm, 2010; Ulman & Pauly, 2016). Literature based on fishers’ local ecological knowledge provides relevant and new data about many ecological parameters of fisheries including reproduction (season, sizes, sites), migratory behavior, spatial distribution, conditions, and trophic relationships (Aswani & Hamilton, 2004; Begossi, Salivonchyk, Lopes, & Silvano, 2016; Begossi et al., 2011, 2019; Figus et al., 2017; Gaspare et al., 2015; Gerhardinger, Marenzi, Bertoncini, Medeiros, & Hostim-Silva, 2006; Hamilton, Giningele, Aswani, & Ecochard, 2012; Johannes, Freeman, & Hamilton, 2000; Le Fur, Guilavogui, & Teitelbaum, 2011; Leite & Gasalla, 2013; Lopes, Verba, Begossi, & Pennino, 2019; Mclean & Forrester, 2018; Nunes, Cardoso, Soeth, Silvano, & Fávaro, 2021; Nunes, Hallwass, & Silvano, 2019; Silva et al., 2019b; Silvano & Begossi, 2012; Silvano, MacCord, Lima, & Begossi, 2006). Fishers’ knowledge has also contributed to participatory spatial planning to map bycatch potential of endangered species, such as sea turtles by artisanal fisheries in the coast of Mexico (Cuevas, Guzmán-Hernández, Uribe- Martínez, Raymundo-Sánchez, & Herrera-Pavon, 2018) or to assess bycatch rates and mortality of the Ganges River dolphins (Platanista gangetica gangetica) (Dewhurst‐Richman et al., 2020). These ecological data provided by fishers could also be useful to assess sustainability of small-scale fisheries and improve their management. A promising way forward to better integrate fishers’ local ecological knowledge and provide needed data about poorly known small-scale fisheries includes collaborations with fishers.  Asia-Pacific This could include participatory monitoring that facilitates fisher involvement in abundance surveys and recording catch, size and information on reproduction of fisheries resources, and occurrence of bycatch (Begossi, Salivonchyk, & Silvano, 2016; Cuevas et al., 2018; Dias, Cinti, Parma, & Seixas, 2020; Keppeler, Hallwass, Santos, da Silva, & Silvano, 2020; Keppeler, Hallwass, & Silvano, 2017; Obura, Wells, Church, & Horrill, 2002; O’Donnell et al., 2012; Schemmel et al., 2016; Silvano, 2020; Silvano & Hallwass, 2020; Webster et al., 2017). 78 Table 3.3 Study cases applying fishers’ local or indigenous ecological knowledge for quantitative analyses of temporal trends on small- 1 scale fisheries. N: number of interviewed fishers; Trend: time trends (C: continuous; I: interval (categories)); Abundance: overall trends in 2 abundance estimated by fishers (D: declined; S: stable, IC: increased); CPUE: catch per unit of effort; Catches: include estimates of either regular 3 or best catches; Size: usually the largest individual ever caught, in length or weight; Composition: relative abundance and number of species in 4 the catch; Scientific data: besides data from fishers’ knowledge (AG: agreement between fishers’ knowledge and scientific data; DA: 5 disagreement; PA: partial agreement); () Taxonomic level: species groups. 6 Table 3.3 Study cases applying fishers’ local or indigenous ecological knowledge for quantitative analyses of temporal trends on small- 1 scale fisheries. N: number of interviewed fishers; Trend: time trends (C: continuous; I: interval (categories)); Abundance: overall trends in 2 abundance estimated by fishers (D: declined; S: stable, IC: increased); CPUE: catch per unit of effort; Catches: include estimates of either regular 3 or best catches; Size: usually the largest individual ever caught, in length or weight; Composition: relative abundance and number of species in 4 the catch; Scientific data: besides data from fishers’ knowledge (AG: agreement between fishers’ knowledge and scientific data; DA: 5 disagreement; PA: partial agreement); () Taxonomic level: species groups.  Asia-Pacific 6 Country Ecosystem N Trend Abundance CPUE Catches Size Composition # Species Taxon Scientific data Source AFRICA Guinea Coastal 178 C / I D / S D Changed 06 Fish No [1] Guinea-Bissau Coastal 274 I D 01 Fish (sawfish) No [2] Mauritius Islands Coastal 093 I D D Changed 25 Fish No [3] Red Sea (Eritrea, Sudan, Yemen) Coastal 423 C D D Several Non specified No [4] Seychelles Coastal 040 I D D Several Fish Yes / DA [5] Tanzania Coastal 350 I D D Changed 17 Fish, shrimp, squid, octopus() No [6] ASIA Bangladesh Freshwater 200 I D 01 Fish No [7] China Coastal 400 I D D 02 Horseshoe, crabs Yes / AG [8] China Freshwater 599 C D 03 Fish Yes / AG [9] India Freshwater 100 I D 58 Fish, shrimp No [10] Ecosystem N Trend Abundance CPUE Catches Size Composition Speci 79 Lao Freshwater 120 I D / S 08 Fish No [11] UAE Coastal 082 I D 01 Fish (sawfish) No [12] Vietnam Coastal 077 C D S 06 Fish No [13] Country Ecosystem N Trend Abundance CPUE Catches Size Composition # Species Taxon Scientific data Source EUROPE Adriatic (Italy, Slovenia, Croatia) Coastal 052 C / I D D 01 Fish (shark) Yes / AG [14] Italy Coastal 032 C / I D / S / IC Changed 59 Fish No [15] Mediterranean (Spain, Italy, Greece) Coastal 091 I D / S / IC D D / S / IC 42 Fish, invertebrates (shrimp, lobster, mollusks) No [16] Poland Coastal 031 I D D 01 Fish Yes / AG [17] Scotland Coastal 062 I IC IC IC 01 Fish Yes / AG [18] Spain Coastal 064 C D D Changed 06 Fish Yes / AG [19] Turkey Coastal 176 C D D Several Non specified Yes / AG [20] Turkey Coastal 155 I D 01 Shellfish Yes / AG [21] NORTH AMERICA EUROPE Adriatic (Italy, Slovenia, Croatia) Coastal 052 C / I D D 01 Fish (shark) Yes / AG [14] Italy Coastal 032 C / I D / S / IC Changed 59 Fish No [15] Mediterranean (Spain, Italy, Greece) Coastal 091 I D / S / IC D D / S / IC 42 Fish, invertebrates (shrimp, lobster, mollusks) No [16] Poland Coastal 031 I D D 01 Fish Yes / AG [17] Scotland Coastal 062 I IC IC IC 01 Fish Yes / AG [18] Spain Coastal 064 C D D Changed 06 Fish Yes / AG [19] Turkey Coastal 176 C D D Several Non specified Yes / AG [20] Turkey Coastal 155 I D 01 Shellfish Yes / AG [21] NORTH AMERICA D NORTH AMERICA 80 Canada Coastal 020 I D D D 1 Sea cucumber No [22] Canada Coastal 042 C D / IC 16 Fish Yes / AG [23] Canada Coastal 038 C / I D D D / S 1 Crab Yes / AG [24] Canada Coastal 042 I D D 1 Fish Yes / AG [25] Mexico Coastal 108 C D D 1 Fish Yes / DA [26] Mexico Coastal 127 C D 2 Shellfish Yes / AG [27] Mexico Coastal 049 I D 3 Fish Yes / AG [28] Mexico Coastal 081 I D 22 Fish, shrimp, lobster, mollusks() Yes / AG [29] Country Ecosystem N Trend Abundance CPUE Catches Size Composition # Species Taxon Scientific data Source NORTH AMERICA (continued) United States of America Coastal 0219 C D D 01 Fish No [30] United States of America Coastal 0064 C / I D D D 01 Fish No [31] United States of America Coastal 0101 I D / S 19 Fish, crab Yes / AG [32] PACIFIC Australia Coastal 0141 C S 05 Fish, shrimp Yes / PA [33] Indonesia Coastal 0186 I D Changed 16 Fish (shark) Yes / AG [34] 1 Country Ecosystem N Trend Abundance CPUE Catches Size Composition # Species Taxon Scientific data Source NORTH AMERICA (continued) United States of America Coastal 0219 C D D 01 Fish No [30] United States of America Coastal 0064 C / I D D D 01 Fish No [31] United States of America Coastal 0101 I D / S 19 Fish, crab Yes / AG [32] PACIFIC Australia Coastal 0141 C S 05 Fish, shrimp Yes / PA [33] Indonesia Coastal 0186 I D Changed 16 Fish (shark) Yes / AG [34] 81 Philippines Coastal 2655 I D D Changed 05 Fish No [35] Philippines Coastal 3446 I D D Stable Several Fish No [36] Philippines Coastal 0025 C S 01 Fish Yes / PA [37] Tonga Coastal 0029 I IC Several Fish Yes / DA [38] SOUTH AMERICA Brazil Coastal 0081 C / I D / IC D / IC D / S Changed 08 Fish, crab, shrimp No [39] Brazil Freshwater 0203 C / I D Changed 15 Fish No [40] Brazil Coastal 0082 I S Changed 22 Fish Yes / DA [41] Brazil Freshwater 0041 I D / IC D / S Changed 16 Fish Yes / PA [42] Brazil Coastal 0222 I D S 01 Fish No [43] Brazil Coastal 0240 C D 01 Fish No [44] Brazil Coastal 0079 I D 01 Fish (shark) No [45] Brazil Coastal 0034 C D 04 Fish No [46] Country Ecosystem N Trend Abundance CPUE Catches Size Composition # Species Taxon Scientific data Source SOUTH AMERICA (continued) Brazil Coastal 0359 I D 04 Fish No [47] Brazil Coastal 102 C / I D D 02 Fish No [48] Brazil Coastal 053 C D / S 09 Fish No [49] 82 Brazil Coastal 210 I D / S D D 08 Fish No [50] Brazil Coastal 214 C D D 09 Fish Yes / AG [51] Brazil Coastal 022 C D 01 Fish Yes / AG [52] Brazil Freshwater 182 I D 01 Fish Yes / AG [53] Brazil Freshwater 300 I D / IC 14 Fish, shrimp Yes / AG [54] Chile Coastal 123 C / I D D Changed 03 Fish Yes / AG [55] Colombia Coastal 046 C D S 01 Fish Yes / DA [56] 7 8 Sources: [1] (Maia et al., 2018); [2] (Leeney & Poncelet, 2015); [3] (Bunce et al., 2008); [4] (Tesfamichael, Pitcher, & Pauly, 2014); [5] (Tim M Daw et al., 9 2011); [6] (Katikiro, 2014); [7] (Jahan et al., 2017); [8] (Liao et al., 2019); [9] (Turvey et al., 2010); [10] (S.  Asia-Pacific Dey et al., 2019); [11] (T. N. E. Gray et al., 2017); 0 [12] (Jabado et al., 2017); [13] (Stocks et al., 2019); [14] (Fortibuoni et al., 2016); [15] (Ernesto Azzurro et al., 2011); [16](Damalas et al., 2015); [17] (Figus 1 et al., 2017); [18] (P. Macdonald et al., 2014); [19] (Coll et al., 2014); [20] (Ulman & Pauly, 2016); [21] (Öndes, Kaiser, & Güçlüsoy, 2020); [22] (O’Regan, 2 2015); [23] (Boudreau & Worm, 2010); [24] (Ban et al., 2017); [25] (Eckert et al., 2018); [26] (Sáenz–Arroyo et al., 2005); [27] (Sáenz-Arroyo & Revollo- 3 Fernández, 2016); [28] (Lozano-Montes et al., 2008); [29] (Ainsworth, 2011); [30] (Rehage et al., 2019); [31] (Frezza & Clem, 2015); [32] (Beaudreau & Levin, 4 2014); [33] (Thurstan et al., 2016a); [34] (Jaiteh et al., 2017); [35] (Lavides et al., 2016); [36] (Muallil, Mamauag, Cababaro, et al., 2014); [37] (O’Donnell et 5 al., 2012); [38] (Webster et al., 2017); [39] (Santos Thykjaer, dos Santos Rodrigues, Haimovici, & Cardoso, 2019); [40] (G. Hallwass et al., 2019); [41] (Damasio 6 et al., 2015); [42] (Strieder Philippsen et al., 2017); [43] (Barbosa-Filho et al., 2020); [44] (de Souza Junior, Nunes, & Silvano, 2020); [45] (Giglio & 7 Bornatowski, 2016); [46] (I. M. Martins, Medeiros, Di Domenico, & Hanazaki, 2018); [47] (Jimenez et al., 2019); [48] (Giglio et al., 2015); [49] (Bender et al., 8 2013); [50] (C. Zapelini et al., 2019); [51] (Bender et al., 2014); [52] (Cleverson Zapelini, Giglio, Carvalho, Bender, & Gerhardinger, 2017); [53] (Leandro 9 Castello, Arantes, Mcgrath, Stewart, & Sousa, 2015); [54] (Gustavo Hallwass et al., 2013); [55] (Godoy et al., 2010); [56] (Castellanos-Galindo et al., 2018). 0 8 Sources: [1] (Maia et al., 2018); [2] (Leeney & Poncelet, 2015); [3] (Bunce et al., 2008); [4] (Tesfamichael, Pitcher, & Pauly, 2014); [5] (Tim M Daw et al., 9 2011); [6] (Katikiro, 2014); [7] (Jahan et al., 2017); [8] (Liao et al., 2019); [9] (Turvey et al., 2010); [10] (S. Dey et al., 2019); [11] (T. N. E. Gray et al., 2017); 10 [12] (Jabado et al., 2017); [13] (Stocks et al., 2019); [14] (Fortibuoni et al., 2016); [15] (Ernesto Azzurro et al., 2011); [16](Damalas et al., 2015); [17] (Figus 11 et al., 2017); [18] (P. Castello, Arantes, Mcgrath, Stewart, & Sousa, 2015); [54] (Gustavo Hallwass et al., 2013) 0  Asia-Pacific Macdonald et al., 2014); [19] (Coll et al., 2014); [20] (Ulman & Pauly, 2016); [21] (Öndes, Kaiser, & Güçlüsoy, 2020); [22] (O’Regan, 12 2015); [23] (Boudreau & Worm, 2010); [24] (Ban et al., 2017); [25] (Eckert et al., 2018); [26] (Sáenz–Arroyo et al., 2005); [27] (Sáenz-Arroyo & Revollo- 13 Fernández, 2016); [28] (Lozano-Montes et al., 2008); [29] (Ainsworth, 2011); [30] (Rehage et al., 2019); [31] (Frezza & Clem, 2015); [32] (Beaudreau & Levin, 14 2014); [33] (Thurstan et al., 2016a); [34] (Jaiteh et al., 2017); [35] (Lavides et al., 2016); [36] (Muallil, Mamauag, Cababaro, et al., 2014); [37] (O’Donnell et 15 al., 2012); [38] (Webster et al., 2017); [39] (Santos Thykjaer, dos Santos Rodrigues, Haimovici, & Cardoso, 2019); [40] (G. Hallwass et al., 2019); [41] (Damasio 16 et al., 2015); [42] (Strieder Philippsen et al., 2017); [43] (Barbosa-Filho et al., 2020); [44] (de Souza Junior, Nunes, & Silvano, 2020); [45] (Giglio & 17 Bornatowski, 2016); [46] (I. M. Martins, Medeiros, Di Domenico, & Hanazaki, 2018); [47] (Jimenez et al., 2019); [48] (Giglio et al., 2015); [49] (Bender et al., 18 2013); [50] (C. Zapelini et al., 2019); [51] (Bender et al., 2014); [52] (Cleverson Zapelini, Giglio, Carvalho, Bender, & Gerhardinger, 2017); [53] (Leandro 19 Castello, Arantes, Mcgrath, Stewart, & Sousa, 2015); [54] (Gustavo Hallwass et al., 2013); [55] (Godoy et al., 2010); [56] (Castellanos-Galindo et al., 2018). 20 83 3.3.1.4.3. Pelagic fisheries for forage fish Small pelagic fish populations, also called forage fish, such as sardine, capelin, anchovy, herring and mackerel, provide about 25% of the total annual production of capture fisheries worldwide (FAO, 2020d). These resources contribute significantly to the well-being of coastal communities around the world, particularly in developing countries. Small pelagic fish are plankton feeders and represent the main prey items for several predators (piscivorous fish including sharks, mammals and birds), and play a key role in marine ecosystems by sustaining numerous higher trophic level species, many of which are commercially targeted (Alder, Campbell, Karpouzi, Kaschner, & Pauly, 2008; Bakun, Babcock, Lluch-Cota, Santora, & Salvadeo, 2010; Essington et al., 2015; Smith et al., 2011). Fisheries for small pelagic fish have a high economic value because of their use for human consumption and for the production of fish meal and fish oil. These fisheries are not only critically important in terms of future global food security but are also pivotal to the economies of small-scale fisheries communities (Pikitch et al., 2014). It has been estimated that fisheries supported by forage fish are actually more than twice as valuable as forage fisheries themselves, providing a strong economic argument for their conservation (Pikitch, 2015). Populations of small pelagic fish exhibit extreme fluctuations in abundance and geographic distribution due to the impact of environmental factors, which are often amplified by anthropogenic influences (Essington et al., 2015; Izquierdo-Peña, Lluch-Cota, Hernandez- Rivas, & Martínez-Rincón, 2019; Stephenson & Smedbol, 2019). The exploitation of many stocks of pelagic fishes has exhibited a pattern of sharply increasing catches followed by an even more rapid decline (Figure 3.23), leading in several cases to closure of the fishery (Stephenson & Smedbol, 2019). Nonetheless, Froehlich et al. (2018) calculated the maximum sustainable yield for 401 stocks that comprise 99% of global forage fish catch, and estimated that the average small pelagic fish catch could increase by 30% from 2012 levels, which would correspond to raising the average (post-1980) small pelagic fish limit by 1.8 million tons per year. 84 84 Figure 3.23 Standardized catch time series for sardines and anchovies from the four largest small pelagics fisheries: Japan, Humboldt, Benguela, and California ecosystems. Data from the Food and Agriculture Organization of the United Nations’ fishstat data base. Source: (Izquierdo-Peña et al., 2019) © 2018 Elsevier Ltd., license number 5153140108259. CC-BY NC. 3.3.1.4.3. Pelagic fisheries for forage fish Figure 3.23 Standardized catch time series for sardines and anchovies from the four largest small pelagics fisheries: Japan, Humboldt, Benguela, and California ecosystems. Data from the Food and Agriculture Organization of the United Nations’ fishstat data base. Source: (Izquierdo-Peña et al., 2019) © 2018 Elsevier Ltd., license number 5153140108259. CC-BY NC. 3.3.1.4.4. Pelagic fisheries for billfishes, tuna and tuna-like species Fisheries targeting tuna and tuna-like species and billfishes are of great socioeconomic importance due to high economic value and extensive international trade and are therefore highlighted in the sustainable use assessment. Tuna accounts for over 9% of total marine fisheries catch, is the fourth most valuable globally traded fishery product, and is about 8% of 85 the 129 billion United States dollars value of internationally traded fishery products (FAO, 2014d, 2018d). Fisheries targeting these species provide substantial economic revenue, employment and food security to fishing and coastal states (Bell & Secretariat of the Pacific Community, 2011; FAO, 2018d; Gillett, 2009). Tunas and billfishes have been an important food source since ancient times, and are target species of fisheries worldwide (Majkowski, 2007; Miyake, Guillotreau, & Sun, 2010). In the 19th century, most tuna fisheries were coastal, conducted by locally-based fleets (Majkowski, 2005, 2007). Industrial tuna fisheries began in the 1940s. Over the next few decades, fishing grounds quickly expanded as did the number of countries with large-scale coastal and distant-water tuna fleets. About 82% of world tuna is consumed as canned product, and 18% as fresh product (including as sashimi) (Miyake et al., 2010). Japan consumes an estimated 78% of the fresh tuna (Miyake et al., 2010). Demand for both canned and fresh tuna has increased rapidly, with reported landings of principal market tunas increasing from about 700 thousand tons in 1960 to almost 4.8 million tons in 2014 (SPC, 2015) (Figure 3.24). Since 2006, over half of principal market tunas have come from the western and central Pacific Ocean (SPC, 2015). Several Pacific Island countries and territories obtain a large proportion of their gross domestic product through revenue from tuna fisheries, as high as 63% of total government revenue in some cases (Aqorau, 2009; Bell et al., 2015; FFA, 2015; Gillett, 2009). This includes licensing, fees, and granting access to foreign purse seine and longline tuna fisheries to fish in their exclusive economic zones. Capture and processing practices generate additional revenue and substantial employment in the Pacific Islands (Bell et al., 2015; FFA, 2015; Gillett, 2009). In 2014, the Pacific sslands forum fisheries agency (15 Pacific small islands developing states, Australia and New Zealand) obtained an estimated 556 million United States dollars of their combined gross domestic product from the tuna fisheries, and employed over 22,000 people in processing and various other tuna-practice related positions (FFA, 2015). 3.3.1.4.4. Pelagic fisheries for billfishes, tuna and tuna-like species Some locally-based tuna fisheries supply largely low-value fishes (smaller tunas, incidental tuna-like species) to local markets in Pacific Island countries and territories, contributing to local food security and tourism industries (Bell et al., 2015; Gillett, 2009). 86 Figure 3.24 Global reported landings of principal market species of tunas by region, 1960- 2014. Source: (Gilman, Allain, Collette, Hampton, & Lehodey, 2016; SPC, 2015) © 2016 International Union for Conservation of Nature and Natural Resources, CC-BY. Figure 3.24 Global reported landings of principal market species of tunas by region, 1960- 2014. Source: (Gilman, Allain, Collette, Hampton, & Lehodey, 2016; SPC, 2015) © 2016 International Union for Conservation of Nature and Natural Resources, CC-BY. Single-stock assessment models are the most common approach used by fisheries management authorities to assess the sustainability of stocks of principal market species of tuna, tuna-like species and billfishes. The four regional fisheries management organizations for tropical tunas have recently adopted and implemented single-stock harvest strategies. The main elements of harvest strategies are outlined in the following literature: (Sainsbury, 2000; WCPFC, 2014). The status of most but not all stocks of principal market tunas and billfishes is relatively certain (ISSF, 2020; Juan-Jordá, Mosqueira, Freire, & Dulvy, 2013; Pons et al., 2017). Direct mortality caused by pelagic marine fisheries is the main driver of reductions in the size and abundance of pelagic apex predators, including target stocks and incidentally caught species. Many target species are considered to be above limit thresholds and near targets. However, as discussed earlier in this chapter, the fisheries that catch these principal market species also intentionally or accidentally capture species that are highly vulnerable to anthropogenic mortality sources. There is extremely high uncertainty of the status of stocks and populations of these other species. Fisheries that target tuna and tuna-like species, billfishes and other relatively fecund species can have large impacts on incidentally caught species that, due to their lower reproduction rates and other life history traits, are relatively vulnerable to increased mortality. This includes seabirds, sea turtles, marine mammals, elasmobranchs and some teleosts (Branch, Lobo, & Purcell, 2013; E. L. Gilman, 2011; M. A. Hall, Alverson, & Metuzals, 2000). 3.3.1.4.4. Pelagic fisheries for billfishes, tuna and tuna-like species Pelagic fisheries selectively remove individuals based on certain traits (e.g., behavioral traits for 87 boldness; life-history traits for size-at-age; physiological traits for visual acuity; morphological traits for mouth dimensions), reducing intraspecific genetic diversity and altering fitness and evolutionary processes (Heino, Díaz Pauli, & Dieckmann, 2015; Hollins et al., 2018). Fishing gear can alter and damage habitat (Dagorn, Holland, Restrepo, & Moreno, 2013; Escalle, Brouwer, Phillips, Pilling, & PNA, 2017)). Thus, fisheries targeting large, highly migratory pelagic predators of high trophic levels (total lenght > 4.0) indirectly modify trophic food web structure and processes and functionally-linked systems (J. A. Estes et al., 2011; Pace, Cole, Carpenter, & Kitchell, 1999; Polovina, Abecassis, Howell, & Woodworth, 2009; J. Stevens, 2000; Ward & Myers, 2005). At this latter broad level, there is limited understanding of what magnitudes of interacting natural (e.g., large scale climate variability) and anthropogenic pressures (including from fishing) cause pelagic ecosystems to reach a tipping point where they undergo a protracted or permanent regime shift, and how altered components of the state of pelagic ecosystems affect functionally-linked systems (Box 3.3; (Ortuño Crespo & Dunn, 2017; Pace et al., 1999). Box 3.3 Ecosystem effects resulting from combined natural and anthropogenic impacts and their influence on the fisheries Although populated and exploited since the Neolithic, the Black Sea has undergone dramatic ecosystem changes in the last half century, mainly related to anthropogenic impacts such as uncontrolled fishing, cultural eutrophication and invasions by alien species. Fisheries collapses, harmful algal and jellyfish blooms, benthic community loss, and upper shelf hypoxia have had dire consequences for ecosystems and human livelihood depending on them. Recent research studies (G. Daskalov, 2003; Daskalov et al., 2017; Oguz & Gilbert, 2007) have demonstrated that these major changes resulted from synergistic effects of climate forcing, trophic interactions and anthropogenic pressures (overfishing, eutrophication and introduction of invasive species). Driving factors and mechanisms of trophic cascades and regime shifts Driving factors and mechanisms of trophic cascades and regime shifts Ecosystem shifts cascading down from top-predators to primary producers and affecting water quality were registered along the 1970s and 1990s (Daskalov et al., 2008). The first shift followed the depletion of top predators from the 1950-1970, after which the ecosystem stabilized at low abundance of top predators, high abundance of planktivores, low zooplankton biomass and high phytoplankton biomasses during the 1970s and 1980s. The second shift was associated with the collapse of planktivorous fish and outburst of M. leidyi in the early 1990s, which resulted in a second system-wide trophic cascade, with similar alternating effects on zoo- and phytoplankton, and on water chemistry. Overfishing was recognised as the structuring factor affecting not only fish stocks, but the whole ecosystem and held responsible for the system shifts to unhealthy states (Daskalov et al., 2008). Overfishing also contributed to hypoxia by cascading increase of phytoplankton and subsequently bacteria activity. Regional and global climate change, eutrophication, and invasive species were also reported to synergistically contribute to ecosystem shifts (Daskalov et al., 2017; Oguz & Gilbert, 2007). Historical trends in fishing and environmental change in the Black Sea Historical trends in fishing and environmental change in the Black Sea Following the development of the fisheries, the pelagic top-predators have declined by the early 1970s in the Black Sea. For instance, the large population of dolphins diminished about tenfold through overexploitation (Özturk, 1996; Sirotenko, Danilevskiy, & Shlyakhov, 1979). Before 1970, the fishery targeted mainly large, valuable migratory species, such as bonito, mackerel, bluefin tuna and swordfish. All of these important fisheries collapsed mainly due to heavy and unregulated fishing (Daskalov, Demirel, Ulman, Georgieva, & Zengin, 2020; Daskalov, Prodanov, & Zengin, 2008). In the early 1970s, the stocks of planktivorous fishes (sprat, anchovy and horse mackerel) increased considerably and became a target for the industrial fishery (Barange et al., 2009). Their increase in biomass and catch promoted the expansion of powerful trawl and purse seine fishing fleets and a steady increase in fishing effort (Gucu, 1997). The highest catch and fishing mortality were recorded in the late 1980s, but biomasses of exploited populations were declining due to recruitment failures in the previous years. Sharp reductions in biomass and catch in the early 1990s were described as stock collapses (Daskalov et al., 2008). After 1990, the fishing effort decreased 88 and a slow recovery of small pelagic fishes occurred during the 2000s (Daskalov et al., 2017). Starting in the 1970s several human activities further induced a deterioration of the environmental conditions. Intensive bottom trawling on the shelf provoked dispersal of sediment, which severely decreased water transparency, and its re-sedimentation buried demersal life under thick silt layer (Samyshev & Rubinstein, 1988). Increased nutrient loading from rivers and coastal sources (Zaitsev & Mamaev, 1998) favoured frequent plankton blooms, equally contributing decreasing transparency and ventilation leading to benthic life kills. The degradation of massive phytoplankton blooms by aerobic bacteria that pump oxygen from the water further promoted hypoxia, especially near the bottom. By the 1990s, biological invasion of the ctenophore Mnemiopsis leidyi (brought in ship ballast water) has contributed to depletion of zooplankton and collapses of small pelagic fisheries (Knowler, 2005). Effects of trophic cascades and regime shifts on fisheries Effects of trophic cascades and regime shifts on fisheries The cascading shifts have affected the whole food web from top-predators to primary producers, with repercussions on water chemistry (Daskalov et al., 2008). The environmental degradation has naturally affected fish stocks and fisheries relying on them (Daskalov et al., 2008; Zaitsev & Mamaev, 1998). The effect of 1970s trophic cascade on fisheries catches has been positive as small pelagic stocks boomed after being released from predation. The 1990s shift however entrained small pelagic stock and fisheries collapses and substantial socio-economic losses (Knowler, 2005). Although recovery of previous states is unlikely, some components of the ecosystem have been subject to partial recoveries (Daskalov et al., 2017). The overall state on the marine environment has improved with the reduction of the nutrient load, partial control over M. leidyi, and more intense turnover rates related to warmer sea water. Following reduction in the fishing pressure, stocks and catches of small pelagic species recovered to intermediate levels, but large valuable species such as turbot, bonito and bluefish remain scarce according to historical abundances. Current single-species based 89 management practices seem insufficient to deal with consequences of ecosystem regime shifts. At present the existing management bodies at national and international levels fail to implement ecosystem-based management. Recovery of resilient ecosystems should mean restoring all important components (including top-predators) into a desirable state with reduced anthropogenic impacts, normalized species interactions, buffered trophic cascades, increased biodiversity and improved environmental quality. This ecosystem state would provide strategic benefits, such as a clean marine environment, abundant and diverse fish stocks and sustainable economic activities (e.g., fishing, tourism), to a range of stakeholders and society as a whole. Of the 23 stocks of the seven principal market tuna species, 9 have biomass levels that are below a level estimated to produce maximum sustainable yields or similar thresholds. The fishing mortality rate exceeds a maximum sustainable yield-based or similar reference point, indicating that the stock is not rebuilding its biomass, or both (ISSF, 2016). Most tuna stocks are either under-exploited or fully-exploited, dominated by skipjack, albacore and yellowfin tunas. As discussed above, while the use of some of these principal market species is considered sustainable when assessed against certain metrics such as the FAO’s definition of overexploited (3.3.1), vulnerable species bycatch accompanies the fishing activity. Effects of trophic cascades and regime shifts on fisheries As political attention to problematic bycatch in marine capture fisheries has increased over recent decades, more resources have been allocated to assess the status of incidentally captured stocks and populations that are of relatively high risk, including, for example, silky and oceanic whitetip sharks, false killer whales, leatherback and loggerhead sea turtles, and several pelagic seabirds including albatrosses and large petrels. These assessments have included semi-quantitative ecological risk assessments using productivity-susceptibility analysis that informs the relative risk of affected stocks and populations, and quantitative, model-based and data-intensive stock assessments and population models that provide information on the absolute risks to affected stocks and populations. The International Union for Conservation of Nature Red List global species-level categorizations do not provide information on the status of individual populations/stocks. Of the 61 species belonging to Suborder Scombroidei, species assessed against the International Union for Conservation of Nature Red List criteria, 13% were listed as Threatened and 7% as Near Threatened (Collette et al., 2011; IUCN, 2014). Of the Scombroidei, Pacific bluefin, Southern bluefin, Atlantic bluefin and bigeye tuna were categorized as Threatened. The characteristics that these four species of threatened tunas have in common are long generational lengths, longer-lived and later maturity. When combined these traits results in longer time to recover from population declines (Collette et al., 2011). These threatened tuna species also have higher economic values per unit of weight relative to the other market tunas (Miyake et al., 2010). While there were some early concerns over their application to exploited fishes this largely reflects a misunderstanding of how the criteria work (Mace & Hudson, 1999; Reynolds & Mace, 1999). The International Union for Conservation of Nature Red List assessments are global in scale whereas fisheries assessments are regional in scale and hence these different assessment processes are used for different purposes. However residual concerns about the applicability of the International Union for Conservation of Nature criteria have been refuted 90 by extensive empirical evidence that consistently show strong alignment and harmony with fisheries management reference points, based on simulations using data from the global population dynamics database (Connors, Cooper, Peterman, & Dulvy, 2014) and multiple global meta-analyses of all fisheries stock assessments (e.g. Davies & Baum, 2012; d’Eon- Eggertson, Dulvy, & Peterman, 2015; P. G. Fernandes et al., 2017; Porszt, Peterman, Dulvy, Cooper, & Irvine, 2012). Effects of trophic cascades and regime shifts on fisheries The greatest concerns were raised for the highly fecund broad cast- spawning fishes, yet the International Union for Conservation of Nature Red List categories and criteria have been shown to highly aligned with fisheries assessment. As a result, marine fishes assessed by the International Union for Conservation of Nature as being Endangered or Critically Endangered are consistently fished beyond target and limit reference points (Dulvy, Jennings, Goodwin, Grant, & Reynolds, 2005; Simpfendorfer & Dulvy, 2017). For species that are not subject to fisheries assessments, the International Union for Conservation of Nature assessments offer valuable information on the need for fisheries management (ICES, 2018). 3.3.1.4.5. Whaling Aquatic mammals are an important hunting target species for subsistence, culture and identity of some indigenous and local communities (IWC, 2021; S. L. Newell & Doubleday, 2020) (Box 3.4). Across South America and West Africa hunted aquatic mammals includes 33 small cetaceans and all three manatee species (Cosentino & Fisher, 2016; Porter & Lai, 2017). The vast majority of whales hunted for aboriginal subsistence in the United Kingdom of Denmark, Norway and Iceland (Figure 3.25A, (International Whaling Commission., 2021)) are common minkes. Besides, Greenland (United Kingdom of Denmark) has been conducting aboriginal subsistence whaling targeting fin, bowhead and humpback whales as well as commercial whaling targeting narwhal and other small cetaceans. Faroe Islands (United Kingdom of Denmark) has been conducting the drive fishery targeting pilot whales. Norway and Iceland have been conducting commercial whaling on fin whales. In these countries local hunters often sell whale meat to foreign tourists or in European Union markets (Eklund T., 2017). Indigenous communities in the Russian Federation mostly hunt the gray whale (IWC, 2019a) and in the United States of America, the bowhead whale (IWC, 2019b). In India, Pakistan and Sri Lanka cetaceans are also hunted (often illegally) for use as bait in other fisheries (Porter & Lai, 2017). Aquatic wild species can also be utilized on a commercial basis (Figure 3.25). Since 1982, the International Whaling Commission which regulates commercial whaling has maintained a "zero quota" on commercial whaling (with the exception of catches set by countries under objection or reservation) because of historical overexploitation and the challenge of managing whaling sustainably. The organization currently has 88 members. Japan suspended commercial whaling in 1988 and began whaling for scientific research in 1987 to gather population data in accordance with the paragraph 10e of the Schedule of the International Convention for the Regulation of Whaling (Cosentino & Fisher, 2016). In accordance with the provisions of the International Convention for the Regulation of Whaling (Article 8), all meat taken from whales caught for scientific whaling was processed and sold in stores and restaurants, and the proceeds obtained from the sales were used for the research activities in the following years in accordance with the direction by the Government of Japan. 3.3.1.4.5. Whaling See data management report for the figure at https://doi.org/10.5281/zenodo.6453071. Box 3.4 Small-scale indigenous whaling in the North Figure 3.25 (A, B & C). Global catches of whales according to the International Whaling Commission, 1985-2018 (a) Aboriginal subsistence catches (b) special permit catches (c) commercial catches taken under objection or reservation. CC-BY-NC. See data management report for the figure at https://doi.org/10.5281/zenodo.6453071. Figure 3.25 (A, B & C). Global catches of whales according to the International Whaling Commission, 1985-2018 (a) Aboriginal subsistence catches (b) special permit catches (c) commercial catches taken under objection or reservation. CC-BY-NC. See data management report for the figure at https://doi.org/10.5281/zenodo.6453071. Box 3.4 Small-scale indigenous whaling in the North Many northern Indigenous peoples continue traditional whale hunting, a practice dating back centuries or more (Stoker & Krupnik, 1993). Whaling provides substantial quantities of food, is a central part of community activities and culture, and a source of fulfillment and identity (Sakakibara, 2020). Collaborative hunts and sharing of the products promote social cohesion, an essential component of thriving in a challenging environment (Huntington et al., 2021). In some places whale products are sold in local markets, which occasionally creates conflicts among users (Sejersen, 2001), but rarely leading to excessive exploitation. The legacy of large-scale commercial whaling continues to affect some whale populations in the Arctic, 3.3.1.4.5. Whaling The International Court of Justice, using various criteria, ruled that Japan׳s whaling was not 91 “for purposes of scientific research” as required by Article VIII of the International Convention for the Regulation of Whaling, and ordered Japan to immediately cease its JARPA II whaling program (JARPA II: second phase of Japan's whale research program under special permit in the Antarctic) (Clapham, 2015). In 2019, Japan withdrew from the International Convention for the Regulation of Whaling, in line with Japan’s basic policy of promoting sustainable use of aquatic living resources based on scientific evidence, and resumed commercial whaling after 31 years of suspension (Holm, 2019). Norway and Iceland are members of the International Whaling Commission, but have continued to commercially hunt whales either under objection to the moratorium decision or under reservation to it (IWC, 2021). The Russian Federation has also objected to the moratorium, but has not resumed whaling. Countries members of the Illegal Whaling Commission that take whales are obliged to provide statistical, scientific and other pertinent information to the International Whaling Commission. While the Western North Pacific stock of common minke and Bryde’s whales are confirmed by the Illegal Whaling Commission Scientific Committee to be relatively abundant, abundance estimate of North Pacific stock of sei whale is still under examination by the Illegal Whaling Commission Scientific Committee although it has been substantially recovered. Thus, sei whales as a whole are still classified as endangered by the International Union for Conservation of Nature. In 2018, despite a substantial number of opposition, the International Whaling Commission adopted a resolution which reaffirms "that the moratorium on commercial whaling, which has been in effect since 1986, has contributed to the recovery of some cetacean populations, and aware of the cumulative effects of multiple, existing and emerging threats to cetacean populations such as entanglement, bycatch, underwater noise, ship strikes, marine debris and climate change" and "agrees that the role of the International Whaling Commission in the 21st century includes inter-alia its responsibility to ensure the recovery of cetacean populations to their pre-industrial levels, and in this context reaffirms the importance in maintaining the moratorium on commercial whaling" (Figure 3.25A, B and C). 92 92 Figure 3.25 (A, B & C). Global catches of whales according to the International Whaling Commission, 1985-2018 (a) Aboriginal subsistence catches (b) special permit catches (c) commercial catches taken under objection or reservation. CC-BY-NC. Box 3.4 Small-scale indigenous whaling in the North Many northern Indigenous peoples continue traditional whale hunting, a practice dating back centuries or more (Stoker & Krupnik, 1993). Whaling provides substantial quantities of food, is a central part of community activities and culture, and a source of fulfillment and identity (Sakakibara, 2020). Collaborative hunts and sharing of the products promote social cohesion, an essential component of thriving in a challenging environment (Huntington et al., 2021). In some places whale products are sold in local markets, which occasionally creates conflicts among users (Sejersen, 2001), but rarely leading to excessive exploitation. The legacy of large-scale commercial whaling continues to affect some whale populations in the Arctic, 93 but most stocks appear to have recovered and concerns about unsustainable takes at present are limited (Givens & Heide-Jørgensen, 2021; NAMMCO, 2018). The bowhead whale (Balaena mysticetus; (Huntington et al., 2021; Suydam & George, 2021) is hunted primarily by Iñupiaq and Yupik whalers in Alaska under a quota of 67 whales per year established by the International Whaling Commission based on population status and also cultural need. An annual hunt is conducted by Inuit in Nunavut of about one whale per year, with the hunt rotating among communities. The bowhead is also occasionally hunted in Chukotka, Russia, and in Greenland. The beluga whale (Delphinapterus leucas) is hunted in Greenland, Canada, Alaska, and Chukotka by Inuit, Inuvialuit, Gwichin, Iñupiat, Yupik, Yup’ik, and Chukchi. Worldwide, over 1000 beluga are taken per year on average, and the hunt is regarded as sustainable in nearly all locations (Hobbs et al., 2019; NAMMCO, 2018). The narwhal (Monodon monoceros) is hunted in Canada and Greenland by Inuit (Lee, 2017). The worldwide annual harvest is similar to that for beluga whales and is considered sustainable for most populations currently hunted (Hobbs et al., 2019; NAMMCO, 2018). Chukchi and Yupik whalers in Chukotka hunt about 125 gray whales per year (Eschrichtius robustus; (IWC, 2019a)) under an Illegal Whaling Commission quota. The harvest is considered sustainable. In 1999, Makah whalers in the American state of Washington resumed a cultural tradition of gray whale hunting that had been interrupted by colonization and its disruptions, but since 2002 domestic regulations have prevented the hunt from taking place (IWC, 2019b). In Greenland (IWC, n.d.), hunters take approximately 150 minke whales (Balaenoptera acutorostrata), 11 fin whales (Balaena physalus), and 7 humpback whales (Megaptera novaeangliae) per year. Box 3.4 Small-scale indigenous whaling in the North All of these Greenland large whale harvests are under an Illegal Whaling Commission quota and are considered sustainable. In addition to larger cetaceans, some dolphins and porpoises are taken in Arctic communities. Although not indigenous, Faroe Islanders in the North Atlantic hunt long-finned pilot whales (Globicephala melas) each year (Statbank, 2020), a small- scale traditional harvest dating back centuries, which has averaged around 650 whales per year over the last decade. 3.3.1.4.6. Industrial demersal fisheries in coastal areas The status of demersal fisheries in temperate countries is well documented in the RAM Legacy Stock Assessment Database (Christopher Costello et al., 2016; RAM Legacy Stock Assessment Database, 2018; Ricard et al., 2012), where approximately 53% of global reported catch is counted. Those consist of three dominant taxonomic groups, gadids (cod, haddock, pollock and hake), pleuronectids (flatfish), and sebastids (rockfish). While many of these fisheries underwent a historical phase of overexploitation, recent evidence suggests that many of these fisheries have been managed since the 1990s and 2000s in ways that reduced fishing mortality rates (Christopher Costello & Ovando, 2019). In many cases these measures improved stock status (Hilborn & Ovando, 2014; B. Worm et al., 2006) and increased biomass to the point that some authors now focus on underfishing of some key stocks (Hilborn, 2019). The figure below shows the trend in abundance and fishing mortality for these species in temperate areas (Europe, North America, Japan, Chile, New Zealand and Australia). Stock abundance tends to 94 be above the level that would produce long-term maximum sustainable yield and fishing pressure is lower. This has resulted in increasing general stock abundance (Figure 3.26). Figure 3.26 Stock abundance trends. These figures describe the trend in ratios of B/Bmsy (Biomass relative to the biomass that produces Maximum Sustainable Yield), U/Umsy (Fishing pressure or mortality relative to the fraction of the population harvested), and catch/(mean catch) across assessed stocks in the taxonomic group. Source: (Hilborn et al., 2021) under license CC-BY_NC-ND 4.0. Figure 3.26 Stock abundance trends. These figures describe the trend in ratios of B/Bmsy (Biomass relative to the biomass that produces Maximum Sustainable Yield), U/Umsy (Fishing pressure or mortality relative to the fraction of the population harvested), and catch/(mean catch) across assessed stocks in the taxonomic group. Source: (Hilborn et al., 2021) under license CC-BY_NC-ND 4.0. Figure 3.26 Stock abundance trends. These figures describe the trend in ratios of B/Bmsy (Bi l i h bi h d M i S i bl Yi ld) U/U (Fi hi 3.26 Stock abundance trends. These figures describe the trend in ratios of B/Bmsy (Biomass relative to the biomass that produces Maximum Sustainable Yield), U/Umsy (Fishing pressure or mortality relative to the fraction of the population harvested), and catch/(mean catch) across assessed stocks in the taxonomic group. Source: (Hilborn et al., 2021) under license CC-BY_NC-ND 4.0. 3.3.1.4.6. Industrial demersal fisheries in coastal areas The status of demersal fisheries in the rest of the world is much less documented. A quarter of the remaining global reported catch has undergone some form of data-limited stock assessment (FAO, 2016b) while 22% remains unassessed, with little information about population status or risk of over-fishing (Christopher Costello & Ovando, 2019). These data limited stocks make up an increasing proportion of globally reported catch over time, from 20% to 47% in the last 60 years (Vasconcellos & Cochrane, 2005). From two areas for which information is available, the Mediterranean and Western Africa, the evidence is that these stocks are very heavily exploited and almost certainly over-fished and subject to over-fishing (Hilborn et al., 2020). The demersal species from the regions not well covered in the RAM Legacy Stock Assessment database, along with the small pelagic fisheries of the same regions, constitute the dominant component of the unassessed fish stocks of the world. Most of those demersal stocks belong to coastal fisheries which contribute much, if not most, of global catches, but quantitative estimates of the extent of their contribution depend on how coastal fisheries are defined, especially in relation to small scale fisheries. Palomares and 95 Pauly (Palomares & Pauly, 2019) used the “Sea Around Us” reconstructed catch database (Zeller et al., 2016) to estimate the catch in an area at most 50 km from inhabited coastlines or down to a depth of 200 m (Figure 3.27), considered to be the area in which small scale fisheries (artisanal, subsistence, and recreational) are located. Coastal fisheries made up an average of 55% of global marine fisheries in the 5-year period from 2010 to 2014, while small-scale fisheries in the same period contributed 36% of the marine catches consumed directly by people (Figure 3.28). Pauly (Palomares & Pauly, 2019) used the “Sea Around Us” reconstructed catch database (Zeller et al., 2016) to estimate the catch in an area at most 50 km from inhabited coastlines or down to a depth of 200 m (Figure 3.27), considered to be the area in which small scale fisheries (artisanal, subsistence, and recreational) are located. Coastal fisheries made up an average of 55% of global marine fisheries in the 5-year period from 2010 to 2014, while small-scale fisheries in the same period contributed 36% of the marine catches consumed directly by people (Figure 3.28). 3.3.1.4.6. Industrial demersal fisheries in coastal areas Figure 3.27 Catches from 1950-2014 of (A) the world’s marine fisheries (B) coastal fisheries (areas <=50 km from inhabited coastlines or to a depth of <=200 m). Based on (Palomares & Pauly, 2019) under license number 5278770226780. CC-BY NC. Figure 3.27 Catches from 1950-2014 of (A) the world’s marine fisheries (B) coastal fisheries (areas <=50 km from inhabited coastlines or to a depth of <=200 m). Based on (Palomares & Pauly, 2019) under license number 5278770226780. CC-BY NC. 96 Figure 3.28 Global catch data as reported to the Food and Agriculture Organization of the United Nations by fishing countries (reported catch: black line) (1950-2016). Source:(Dirk Zeller, Cashion, Palomares, & Pauly, 2018) under license CC-BY NC. Figure 3.28 Global catch data as reported to the Food and Agriculture Organization of the United Nations by fishing countries (reported catch: black line) (1950-2016). Source:(Dirk Zeller, Cashion, Palomares, & Pauly, 2018) under license CC-BY NC. Figure 3.28 Global catch data as reported to the Food and Agriculture Organization of the United Nations by fishing countries (reported catch: black line) (1950-2016). Source:(Dirk Zeller, Cashion, Palomares, & Pauly, 2018) under license CC-BY NC. Lower-income countries lack the capacity to industrially harvest fish populations off their shores, and thus frequently host foreign fishing fleets through fishing access agreements or joint venture operations (Belhabib et al., 2015; Kaczynski & Fluharty, 2002). The higher capacity and improved technology of higher-income nations has enabled these countries to build and operate distant water fishing fleets, and often to subsidize those fleets heavily (Sala, Aburto-Oropeza, Reza, Paredes, & López-Lemus, 2004; Dirk Zeller & Pauly, 2019). Describing fishing patterns of those industrial fleets in comprehensive and quantitative terms is challenging due to the lack of open access to detailed records on the behavior of fishing vessels. However, McCauley et al., (McCauley et al., 2018) produced fishing patterns of industrial fishing vessels (>24m) based on high-resolution fishing vessel activity information derived from automatic identification systems data (Figures 3.29 and 3.30). Such patterns address one of the fundamental issues of fisheries sustainability, namely direct and collateral impacts by fishing gear on habitats, target and non-target species (Amoroso et al., 2018, 2018; Lewison et al., 2004a; Palomares & Pauly, 2019), directly related to the amount of gear deployed rather than to the amount of target yield extracted coming from catch data (Stewart et al., 2010). 3.3.1.4.6. Industrial demersal fisheries in coastal areas 97 Figure 3.29 Distribution of industrial fishing effort by vessels flagged to nations from different income classes as measured using automatic identification systems data and convolutional neural network models. (A) The percent of fishing effort (measured in fishing hours) detected globally on the high seas and in all exclusive economic zones for vessels flagged to nations from four different World Bank income groups. (B) The percent of automatic identification systems-detected industrial fishing effort in all exclusive economic zones, grouped by the World Bank income groups of the exclusive economic zones. Here, the category Domestic fishing is included, which refers to instances when a fishing country was fishing in its own exclusive economic zone. Other categories represent foreign fishing effort conducted within an exclusive economic zone by a nation flagged to one of the four World Bank income classes. “Invalid identity” refers to vessels with a Maritime Mobile Service Identity (MMSI) number that did not accurately refer to an individual country. “Unclassified” refers to fishing entities that were fishing in an exclusive economic zone but did not have a World Bank income group. All data presented here are summarized from the year 2016. Source: (McCauley et al., 2018) under license CC BY 4.0. 3.29 Distribution of industrial fishing effort by vessels flagged to nations from Figure 3.29 Distribution of industrial fishing effort by vessels flagged to nations from different income classes as measured using automatic identification systems data and convolutional neural network models. (A) The percent of fishing effort (measured in fishing hours) detected globally on the high seas and in all exclusive economic zones for vessels flagged to nations from four different World Bank income groups. (B) The percent of automatic identification systems-detected industrial fishing effort in all exclusive economic zones, grouped by the World Bank income groups of the exclusive economic zones. Here, the category Domestic fishing is included, which refers to instances when a fishing country was fishing in its own exclusive economic zone. Other categories represent foreign fishing effort conducted within an exclusive economic zone by a nation flagged to one of the four World Bank income classes. “Invalid identity” refers to vessels with a Maritime Mobile Service Identity (MMSI) number that did not accurately refer to an individual country. “Unclassified” refers to fishing entities that were fishing in an exclusive economic zone but did not have a World Bank income group. 3.3.1.4.6. Industrial demersal fisheries in coastal areas All data presented here are summarized from the year 2016. Source: (McCauley et al., 2018) under license CC BY 4.0. Figure 3.29 Distribution of industrial fishing effort by vessels flagged to nations from different income classes as measured using automatic identification systems data and convolutional neural network models. (A) The percent of fishing effort (measured in fishing hours) detected globally on the high seas and in all exclusive economic zones for vessels flagged to nations from four different World Bank income groups. (B) The percent of automatic identification systems-detected industrial fishing effort in all exclusive economic zones, grouped by the World Bank income groups of the exclusive economic zones. Here, the category Domestic fishing is included, which refers to instances when a fishing country was fishing in its own exclusive economic zone. Other categories represent foreign fishing effort conducted within an exclusive economic zone by a nation flagged to one of the four World Bank income classes. “Invalid identity” refers to vessels with a Maritime Mobile Service Identity (MMSI) number that did not accurately refer to an individual country. “Unclassified” refers to fishing entities that were fishing in an exclusive economic zone but did not have a World Bank income group. All data presented here are summarized from the year 2016. Source: (McCauley et al., 2018) under license CC BY 4.0. 98 Figure 3.30 Density distribution of global industrial fishing effort, derived automatic identification systems data. (A) Vessels flagged to higher-income countr (B) vessels flagged to lower--income countries. Industrial fishing effort is estimated convolutional neural network models and plotted as the log10 number of fishing hour map is directly copied from its original source (McCauley et al., 2018) and was not m by the assessment authors. The map is copyrighted under license CC BY 4.0. The desig employed and the presentation of material on the maps used in the assessment do not im expression of any opinion whatsoever on the part of IPBES concerning the legal status Figure 3.30 Density distribution of global industrial fishing effort, derived using automatic identification systems data. (A) Vessels flagged to higher-income countries and (B) vessels flagged to lower--income countries. Industrial fishing effort is estimated using convolutional neural network models and plotted as the log10 number of fishing hours. This map is directly copied from its original source (McCauley et al., 2018) and was not modified by the assessment authors. Box 3.5 Bottom trawling: assessing seabed habitat and biota impacts Box 3.5 Bottom trawling: assessing seabed habitat and biota impacts The recognition that sustainability of fisheries not only involves maintaining target stocks at productive levels, but also minimizing wider ecosystem impacts of fishing has turned increasing attention to the evaluation of the environmental footprint of different fishing methods. In particular, the use of bottom-contact mobile gears as a means of catching fish has sparked heated debates in fishery and conservation sciences. On the one hand, bottom trawling contributes close to 20 million tons of fish and invertebrates per year to the global food supply and provides food and livelihoods for millions of people as well as significant export revenues to many countries (Amoroso et al., 2018). On the other hand, bottom trawling impacts seabed habitats, damaging biogenic structures and altering sediment composition and its biogeochemical dynamics, kills benthic organisms and alters ecosystem functions (Clark et al., 2016; De Borger, Tiano, Braeckman, Rijnsdorp, & Soetaert, 2021; Hiddink et al., 2017; O’Neill & Ivanović, 2016; Pusceddu et al., 2014) (Pusceddu et al. 2014, Clark et al. 2016, O'Neill and Ivanović 2016; Hiddink et al. 2017, De Borger et al. 2021). Concerns about environmental impacts of bottom trawling have fueled strong public campaigns and resulted to its ban in some countries and regions. Less extreme approaches for reducing the negative impacts of trawling have been pursued, including changes in gear design and fishing operations, prevention of further expansion of trawled area, ocean zoning, bycatch and habitat quotas and the closure of large areas to protect sensitive habitats (McConnaughey et al., 2020; Williams et al., 2020). United Nations General Assembly Resolutions 61/105 (2007) and 64/72 (2010) required Regional Fisheries Management Organizations to identify vulnerable marine ecosystems on the seabed within their jurisdictions and ensure that fisheries did not cause serious adverse impacts to them. Of particular concern has been the expansion of trawling into deeper areas, leading for example to the ban on bottom trawling in deep waters (below 800 m) and in areas with vulnerable marine ecosystems (below 400 m) adopted by the European Union in 2016. Assessments of the global and regional seabed impacts of bottom trawling require information on the distribution and intensity of trawling, the direct impact of the gear on the swept habitats and communities, and their capacity to recover from trawling disturbances (Mazor et al., 2021; McConnaughey et al., 2020; Pitcher et al., 2017). 3.3.1.4.6. Industrial demersal fisheries in coastal areas The map is copyrighted under license CC BY 4.0. The designations employed and the presentation of material on the maps used in the assessment do not imply the expression of any opinion whatsoever on the part of IPBES concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. These maps have been prepared or used for the sole purpose of facilitating the assessment of the broad biogeographical areas represented therein and for purposes of representing scientific data spatially. 99 The density distribution of global industrial fishing effort reveals global dominance of industrial fishing by wealthy nations (www.worldbank.org; using 2016 classifications). Vessels flagged to higher-income nations are responsible for 97% of the trackable industrial fishing on the high seas and 78% of such effort within the national waters of lower-income countries (McCauley et al., 2018). While legal, these arrangements raise many challenges regarding their sustainability and equity. For instance, the expected benefits of these partnerships, such as revenues and investments in local infrastructure and technologies, have not always materialized (Antonova, 2016; Crona et al., 2016). Distant water fleets are also involved in illegal, unreported and unregulated fishing (Pauly et al., 2014), which are considered as a serious threat to fisheries and fisheries-dependent communities, marine ecosystems and societies at large (Hutniczak, Delpeuch, & Leroy, 2019). Agnew et al. (2009) estimated that 11–26 million tons (from exclusive economic zones and high seas), or roughly one-quarter of the world catch of fish goes to illegal, unreported and unregulated fishing every year. The same authors found a correspondence between their regional estimates of illegal and unreported fishing and the number of depleted stocks in those regions. As exemplified in the case study in Box 3.5, the relationship between industrial fisheries, small scale fisheries, population status, food security, and livelihoods is a complex one indeed. 100 Box 3.5 Bottom trawling: assessing seabed habitat and biota impacts A study using high- resolution satellite vessel monitoring system and logbook data on 24 continental shelves and slopes to 1,000-m depth (covering 7.8 million-km2 in total) showed that 14% of the overall study area was trawled and 86% was not trawled (Amoroso et al., 2018). However, the seabed proportion impacted by trawling varied markedly among and within regions, from less than 1% in southern Chile to a maximum of 80% in the Adriatic Sea and from areas (within region) trawled several times per year and others only disturbed sporadically. Trawling activity was aggregated; the most intensively trawled areas accounting for 90% of activity comprised 77% of footprint on average trawled (R. Amoroso et al., 2018). In most heavily trawled areas of Europe a large fraction of the area (e.g., North Sea, West Iberia and Skagerrak and Kattegat) was trawled at least once per year, while more than half of the seabed was not trawled during the 2-6-year study period in 20 of 24 regions examined. 101 Trawling footprints were also smaller in regions where fishing rates met sustainability benchmarks trawled (Amoroso et al., 2018). To evaluate biotic impacts, the frequency of trawling events further needs to be compared to the rate of recovery of the different types of organisms inhabiting seabeds. Recent meta-analyses of more than three decades of published results for sedimentary habitats have shown that the immediate mortality of animals in the path of the trawl is correlated with the penetration depth of the gear in the sediment, which vary with the type of gear (Hiddink et al., 2017; Sciberras et al., 2018). The most commonly used trawl gear (otter trawls) kills 6% of the biomass per pass, whereas the most destructive gear (hydraulic dredges) kills 41% of the seabed biota present. Estimated recovery rates after trawling ranged from 1.9 to 6.4 years on average, depending on the type of sediment, trawl gear and benthic species longevity (with longer-lived animals showing larger depletion effects in comparative studies (Hiddink et al., 2019, 2017)). Repeated trawling would thus induce a shift toward species with faster life histories in communities exposed to frequent trawl events (Hiddink et al., 2017; Jennings & Cotter, 1999). A reduction in median longevity of the community of close to 20% on average was estimated for the relatively heavily trawled North Sea (McConnaughey et al., 2020). 3.3.1.5. Uses of wild caught aquatic organisms 3.3.1.5.1. Food and Feed Fish and seafood products are important for human diet, providing about 3.1 billion people with almost 20 percent of their average daily animal protein intake (Sunderland et al., 2019). Human consumption of fish in 2018 totaled 96.4 M tons (FAO, 2020d). Of the landed catch of industrial fisheries, about 80% is used for direct human consumption, and close to 100% of the retained catch of small-scale fisheries is eaten by people (FAO, 2018d). It is important to note that some of these estimates include wild fish and farmed fish from aquaculture. For different indicators available at a global scale, especially fish consumption, much of the available literature does not clearly distinguish between farmed and wild caught fish. Further, in several data sets the information on both wild and farmed fish is so intricately mixed up that it is impossible to distinguish between the two. Indeed, this lack of clarity makes proper assessment of the sustainable use of wild fish species extremely challenging and presents a serious issue for accurate reporting and tracking. This issue is discussed in more detail in the knowledge gaps section (3.5). Thus, despite the focus of this assessment on wild species, assessment experts consistently evaluated the various material they reviewed in relation to two options: (i) select not to use available data and literature on farmed fish and exclude the state of the knowledge on this topic or (ic) include available data on farmed fish in combination with wild fish. Since 1973, the global consumption of fish has doubled, due to increased demand in developed and developing countries (Delgado, International Food Policy Research Institute, & WorldFish Center, 2003; FAO, 2018d). Consumption grew from approximately 9.0 kg per capita in 1961 to 20.2 kg per capita in 2015, at an average rate of about 1.5 percent per year (FAO, 2018d). A higher rate of 2.4 percent is observed in developing countries for the same period. The growth of world per capita fish consumption from 18 kg in 2008 to 20 kg in 2013 was due to an increase in per capita consumption of freshwater & diadromous fish (migrate between freshwater and saltwater, example: salmon, eel, etc.), crustaceans and shell molluscs, whereas that of marine fish and cephalopods declined (Cai & Leung, 2017). Box 3.5 Bottom trawling: assessing seabed habitat and biota impacts Regarding fishing practices, the following uses are well-documented in the literature and available data sources: food and feed (3.3.1.5.1), medicine and hygiene (3.3.1.5.2), recreational fishing (3.3.1.5.3), decorative and aesthetic (3.3.1.5.4), and ceremony and cultural uses (3.3.1.5.5). The following uses are not relevant to this practice or were not documented: energy, education and learning, and materials and shelter. With regards to non-lethal uses of wild aquatic organisms, a review of catch and release recreational fishing (3.3.1.6.1) and ornamental and aquarium fisheries (3.3.1.6.2) are included. Box 3.5 Bottom trawling: assessing seabed habitat and biota impacts Selective effects linked to chronic trawling are likely to be much stronger for long-lived sessile epifauna, such as sponges and corals (Hiddink et al., 2017). By combining known distribution of trawl intensity from Amoroso et al. (Amoroso et al., 2018) with predicted abundance distributions of different benthos groups for 13 diverse regions of the globe, Mazor et al. (2021) found that expected benthic community status ranged between 86% and 100% of untrawled status (mean 99%), with more than three- quarters of benthic groups predicted to be at 95% or more of their benchmarks. Mean benthos status was lowest in regions of Europe and Africa and for taxonomic classes Bivalvia and Gastropoda. Communities prevalent in sedimentary habitats of the continental shelves could thus sustain moderate levels of trawling, provided that target fishing mortalities are maintained within accepted sustainability benchmarks. Biogenic habitats, such as coral reefs, maerl beds and sea mounts habitats (not covered by (Hiddink et al., 2017)) are nonetheless expected to be the much more sensitive to trawling impacts due to their long recovery times. The limited data available for long-lived habitat-forming species indicate that post-trawling recovery may take decades (Kaiser, Hormbrey, Booth, Hinz, & Hiddink, 2018; Williams et al., 2010) and be unachievable within acceptable timeframes; spatial closures are therefore essential (Clark et al., 2016). The studies discussed above highlight the importance for policy analysis and implementation of collecting local data on the intensity and distribution of trawling, and the distribution of sediment types and vulnerable marine habitats. These data are needed to identify local best practices and most effective approaches to reduce habitat impacts of fishing, and to allow quantification of trade-offs between fish production for food and the environmental costs associated with different fishing methods and marine policies. 102 Regarding fishing practices, the following uses are well-documented in the literature and available data sources: food and feed (3.3.1.5.1), medicine and hygiene (3.3.1.5.2), recreational fishing (3.3.1.5.3), decorative and aesthetic (3.3.1.5.4), and ceremony and cultural uses (3.3.1.5.5). The following uses are not relevant to this practice or were not documented: energy, education and learning, and materials and shelter. With regards to non-lethal uses of wild aquatic organisms, a review of catch and release recreational fishing (3.3.1.6.1) and ornamental and aquarium fisheries (3.3.1.6.2) are included. 3.3.1.5.1. Food and Feed The importance of global fish production for nutrition and food security varies geographically across regions, countries, and communities dependent on fish at rates far above the global average (Box 3.6). Some of the most fish-dependent populations are located in countries in which the contribution of fish is relatively low at the national level (Bennett et al., 2018). At sub-national scales, individual communities can be almost entirely dependent on seafood for protein. Fish is crucial for coastal indigenous groups, who on average consume fish at a rate that is 15 times higher than the global average (Figure 3.31). 103 Figure 3.31 Fish dependency around the world. This map is directly copied from its original source (Bennett et al., 2018) and was not modified by the assessment authors. The map is copyrighted under license CC BY 4.0. The designations employed and the presentation of material on the maps used in the assessment do not imply the expression of any opinion whatsoever on the part of IPBES concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. These maps have been prepared or used for the sole purpose of facilitating the assessment of the broad biogeographical areas represented therein and for purposes of representing scientific data spatially. Figure 3.31 Fish dependency around the world. This map is directly copied from its original source (Bennett et al., 2018) and was not modified by the assessment authors. The map is copyrighted under license CC BY 4.0. The designations employed and the presentation of material on the maps used in the assessment do not imply the expression of any opinion whatsoever on the part of IPBES concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. These maps have been prepared or used for the sole purpose of facilitating the assessment of the broad biogeographical areas represented therein and for purposes of representing scientific data spatially. Box 3.6 Dried fish in Asian countries Dried fish is an important part of small-scale fisheries (FAO, 2018c; Kawarazuka & Béné, 2010) and includes fish that has been cured, dried, salted, brined, fermented, or smoked fish (see Supplementary material Table S3.1). These are often small and low market value fish from capture fisheries. Approximately 12% of fisheries are prepared and preserved, and 12% are cured. In some countries dried fish consumption is significantly higher (FAO, 2018c). The voluntary guidelines for securing sustainable small-scale fisheries considers fishing and fish processing as important drivers of food security and poverty eradication (FAO, 2015). In Asia and Africa wide varieties of species are dried (Ruddle & Ishige, 2010), including many small pelagic species (Doe, 2017, see Supplementary material Table S3.1). In Bangladesh, dried fish are eaten more frequently than any other type of fish. The contribution of dried products to total fish consumption is disproportionately important for low-income consumers (Belton & Thilsted, 2014). Although dry fish is not cheap, the quantity needed for a meal is less and therefore economical and may explain popularity in rural areas (Samaranayaka, Perera, & Warnasuriya, 2013). 104 Dried fish contribute to food and nutrition security in both coastal and arid mountainous regions of low-income countries as they are a concentrated source of animal protein, rich in calcium and other micronutrients and fats, easily transportable and have a long self-life (Belton, Hossain, & Thilsted, 2018; Thilsted, James, Toppe, Subasinghe, & Karunasagar, 2014). For example, in Malawi, a serving of 24 g of small dried fish twice a day provides an intake of calcium, zinc and iron which is 327%, 152% and 22% higher, respectively than a daily diet without fish (R. S. Gibson & Hotz, 2001; Kawarazuka & Béné, 2010). Since low end processing activities are mostly done by women (Samanta, Bhaumik, & Patra, 2016), their control over family income directly affects household food security and nutritional outcomes (Kawarazuka & Béné, 2010). Women have been involved in the dried fish sector in developed countries and regions as well. Historically, and for centuries (until the 1960s) dried fish processing was a major activity in places such as Newfoundland and other Eastern North American locales and was undertaken significantly by women (Doe, 2017; Neis, 1999). Men were engaged in the pursuit and capture of fish; women in the spreading, turning and drying of fish. This produced food and income security for workers in this profession. Box 3.6 Dried fish in Asian countries Subsistence and artisanal fisherfolk communities in Asia mostly belong to socially and economically marginalized groups (Hapke, 2001), with those engaged in dried fish activity even more marginalized among fisher communities. Hence, the importance of wild marine species in life and livelihoods of the poorest of the poor is immense. At the same time the fisher communities draw life satisfaction by engaging in fishing activities they find challenging and skillfully providing them with a different identity (Nayak, Dias, & Pradhan, 2021). Marine fish used to be the largest species group in world fish consumption, but its share declined from 53% in 1993 to 37% in 2013. Marine fish are still the dominant species consumed in many countries. Indeed, in 2013 marine fish accounted for more than half of fish consumption in more than 170 countries. Over the same period freshwater & diadromous fish consumption grew rapidly, increasing from 3.2 kg in 1993 to 7.5 kg in 2013 (A. Bennett et al., 2018). Crustaceans accounted for nearly 10% of world fish consumption in 2013, increasing from 8% in 1993. Shell molluscs accounted for 13% of world fish consumption in 2013; nearly the same as in 1993. Cephalopods accounted for 2.6% in 2013; down from 3.5% in 1993 (Cai & Leung, 2017) (Figure 3.32). 105 Figure 3.32 Species composition of world per capita fish consumption. Source: (Cai & Leung, 2017) © FAO, 2017. CC-BY NC. Figure 3.32 Species composition of world per capita fish consumption. Source: (Cai & Leung, 2017) © FAO, 2017. CC-BY NC. Figure 3.32 Species composition of world per capita fish consumption. Source: (Cai & Leung, 2017) © FAO, 2017. CC-BY NC. Hatchery-based aquaculture relies on the use of wild fish as feed. The share of fed species in total aquaculture production accounts for the majority (69.5%) of “food fish” production from aquaculture (Clavelle, Lester, Gentry, & Froehlich, 2019; FAO, 2018d). Capture-based mariculture depends on wild-caught juveniles for “seed,” which are then raised and fattened in captivity (Boyd et al., 2020; Ottolenghi et al., 2004). This practice, sometimes referred to as “ranching,” is widespread and an important source of production for many species, including tuna, shrimp, lobster, grouper and eels (Lorenzen, Leber, & Blankenship, 2010). However, no current estimates of the extent of capture--based mariculture exist. Box 3.6 Dried fish in Asian countries (d) Difference in proportion of inclusion in total animal feed over time compared to 1961 for crops, forage fish (also depicted in panel c), other aquatic inputs and terrestrial meat-sourced contributions (by-products) in livestock and aquaculture feed, combined. Source: (Froehlich et al., 2018) © 2018, Springer Nature under license CC BY-NC-SA 4.0. To date, two factors have helped avoid resource limitations of forage fish affecting aquaculture growth. First, forage fish have become an increasingly smaller fraction of fish feed inputs over the decades, driven in part by price (Figure 3.33c). Most aquaculture (and agricultural) feed is now largely crop-based (for example, soy), and this trend continues to increase (Figure 3.33d). Additionally, some countries use trimmings (fish by-products) from aquaculture and fisheries, as well as other aquatic species, as forage fish alternatives (Figure 3.33d). Second, aquaculture of selected species is continuously becoming more efficient, as measured by feed conversion ratios. Together, these factors contribute to lower fish-in-fish-out ratios (weight of forage fish used relative to fed cultured species produced). The issue of fishmeal and oil use from aquaculture is continuing to raise diverging views and the sustainability of such practices remains dispersed in the literature (Natale, Hofherr, Fiore, & Virtanen, 2013). Cashion et al. (2017) underscore the concerns around directing ~20 million tons of wild fish every year towards feeding farmed fish, pigs and chickens instead of humans (Belton & Thilsted, 2014). Importantly, 90% of fish destined for uses other than direct human consumption are food--grade or prime food-grade fish (Cashion et al., 2017). Tacon & Metian (2013) indicate that feed use of small pelagic fish competes with its use for food especially in developing countries. Much of the literature warns against aquaculture’s reliance on forage fish, citing the fully exploited, over-exploited or recovering status of many forage fisheries (Rosamond L. Naylor et al., 2000), though the current global amount being extracted appears below maximum sustainable levels (Froehlich et al., 2018; Hilborn & Costello, 2018). Switching from feed fish to direct human food would depend upon affordability and development of low-cost conserved products. A regional approach is needed to assess the consequences of using more feed fish for human consumption. Box 3.6 Dried fish in Asian countries Production of fed species depends on feeds containing high concentrations of proteins and lipids traditionally sourced from fishmeal and oil rendered from wild-caught forage fish, such as herring, sardines and menhaden (Tacon, Hasan, & Metian, 2011; Tacon & Metian, 2008b, 2008a, 2015). The total annual production of fish meal was 4.5 million tons, and the total annual production of fish oils was 0.9 million tons in 2016, of which 69% and 75%, 106 respectively, were used in aquafeeds (Hua et al., 2019). An additional 23% and 5% of this fish meal is used in pig and chicken feeds. The aquaculture industry is making important gains in improving feed conversion ratios, reducing the inclusion of fishmeal in feed and developing substitutes (FAO, 2016b; Klinger & Naylor, 2012; R. L. Naylor et al., 2009). Nonetheless, the use of wild fish for feed by the aquaculture sector is increasing as a result of overall growth, intensification of farming practices, and from the rising share of higher trophic level species in total production menhaden (Tacon, Hasan, & Metian, 2011; Tacon & Metian, 2008b, 2008a, 2015) (Figure 3.33a). Forage fish have been captured and reduced into fishmeal and oil for decades (reduction fisheries), supporting production of terrestrially farmed species, such as pigs and poultry. Aquaculture did not become the dominant user of rendered forage fish until the 2000s, well after global catches of forage fish had plateaued (Shepherd & Jackson, 2013). These pelagic species now help support over 70% of aquaculture production (FAO, 2016b; Tacon & Metian, 2015) acting as feed for carnivorous species (for example, salmon, tuna) and increasingly non- obligate carnivores (for example, carps, shrimp) alike (Tacon & Metian, 2008b, 2015). The added demand from the rapid growth of aquaculture resulted in terrestrial husbandry substituting forage fish with alternative feed sources, reducing fishmeal and oil use by pigs and poultry to roughly 25% of total forage fish use (Figure 3.33b). 107 Figure 3.33 Animal production (livestock, poultry and fed aquaculture species) and forage fish use trends. (a) Time-series of biomass of primary farmed animal groups (poultry and pig, fed aquaculture) that consume forage fish. (b) Proportion of fishmeal use by the primary consumer groups over time. (c) Proportion (mean ± 95% confidence interval) of forage fish in global feed relative to maximum fishmeal price per year (see color scale).  Europe Europe Small-scale fishing is still the most important component of commercial fishing in the European Union with special relevance in Southern Europe (Lloret et al., 2018). It is a highly diversified fishery, involving fishing systems of many forms and sizes, and targeting a wide range of taxonomic groups. Small-scale fisheries in Europe are responsible for a catch equivalent to one large-scale fishery when it comes to human consumption (Leleu et al., 2014). Traditional European small-scale fisheries are more than 2,000 years old (C. Antunes et al., 2015), and data on some fisheries can be found as far back as 400 years (Marcos et al., 2015) . Stocks of small pelagic fish species and of larger demersal fish species have been exploited since the Middle Ages and are still important today (Almeida et al., 2014; Bastari et al., 2017; Battaglia et al., 2017; Braga, Azeiteiro, Oliveira, & Pardal, 2017b). A number of local fish species exploited by European small-scale fisheries are famous worldwide, such as trout (Shephard et al., 2019), cod (Dinesen et al., 2019), anchovies and sardines (Sartor et al., 2019). As indicated by 47 out of the 63 papers reviewed, small-scale fisheries systems are often unsustainable, although varying levels of sustainability were observed in 30% of the papers (see the data management report for Chapter 3 systematic literature review at https://doi.org/10.5281/zenodo.6452651). This applies equally to inland, coastal and marine/oceanic fishing. A number of studies described multi-faceted fishing systems, where some stocks were sustainably exploited while others were not. In 74% of the papers, focusing on broader analysis, and using long and consistent series of data (a set comprising 19 papers), unsustainability elements were still observed. Approximately 10% of the reviewed papers report cases of partial sustainability and 16% report sustainable cases of small-scale fishing (see the data management report for Chapter 3 systematic literature review at https://doi.org/10.5281/zenodo.6452651). The cases of sustainable fishing reported are mainly supported by enabling factors that reflect the adoption of sound management decisions and measures. These are systems that successfully controlled the fishing effort (Fourt et al., 2020), and enforced the regulation of zones of use and no-use (no-take), determined by officially protected marine areas or estuaries (Antunes et al., 2015; Guidetti & Claudet, 2010; Marengo et al., 2015; Morales-Nin et al., 2017). Box 3.6 Dried fish in Asian countries While there are possible benefits of switching at least part of the catches of forage fish to food in South American countries, in Asia this is a less clearly understood, since cheap fish and trash fish contribute to the development of small-scale aquaculture, which reportedly has positive effects on livelihood and human consumption. In sub-Saharan Africa the effects would be limited since feed fisheries are an exception and aquaculture is not yet widespread or dependent on compound feed (Hasan & Halwart, 2009). Nonetheless, the use of wild fish for feed by the aquaculture sector is increasing as a result of overall growth, intensification of farming practices, and the rising share of fed, higher trophic level species in total production (Tacon et al., 2011; Tacon & Metian, 2008b, 2008a, 2009, 2015). Furthermore, fishmeal and oil are also used in terrestrial livestock feed, and their 108 demand is increasing for pet food, and human food and medicine. Given current trends in aquaculture and demand for seafood and terrestrial meat, estimates suggest that ecological limits of forage fish could be reached as soon as 2037, or even sooner if precautionary measures do not further limit access to the wild resource (e.g., Atlantic herring, Clupea harengus, Clupeidae) (Froehlich et al., 2018). Small Scale Fisheries contributing to Food and feed uses This section was written following the methods used for the systematic review described in 3.3.1.3. (see the data management report for Chapter 3 systematic literature review at https://doi.org/10.5281/zenodo.6452651).  Europe The reported cases of unsustainability of small-scale fishing for food and feed are due to a larger and more diverse array of inter-related causes. Fishing pressure above the capacity of the stocks was mentioned by 41% of the papers. This leads to overfishing (either of the targeted species or of their prey species), catches above the maximum sustainable yield, or to 109 the reduction in catch-per-unit-of-effort (Azzurro et al., 2019; Corral & Manrique de Lara, 2017; Duncan et al., 2016; Figus et al., 2017; Lloret et al., 2018; Quetglas et al., 2016). Overfishing may be a consequence of bad management practices, which was the second most frequently reported cause in the literature, mentioned in 22% of the reviewed papers. This includes ineffective control of fishing effort, incongruence between different management measures adopted simultaneously (Baeta et al., 2018), adoption of dubious measures (Corral & Manrique de Lara, 2017), slow implementation of management measures (Colloca et al., 2017), bad communication with the local fishers (Morales-Nin et al., 2017), adoption of weak governance systems (Pita et al., 2019) and competition between fishing modalities (Battaglia et al., 2017; Carvalho et al., 2017; Das & Afonso, 2017; Lloret et al., 2018; Öndes, Kaiser, & Güçlüsoy, 2020). Environmental disturbances (14% of the papers) leading to the reduction of stocks, either by pollution, inadequate use of fishing gears or climate change were mentioned (Azzurro et al., 2019; Braga et al., 2018; Dinesen et al., 2019; Pita et al., 2019). Excessive discards or bycatch were also mentioned by a smaller number of papers (Öndes, Kaiser, & Güçlüsoy, 2020).  Latin America In Latin America, almost all studies (99) analyze the use of fisheries resources as food, either for subsistence, commerce or both. Overall, 15% of these studies report sustainable use, 48% report unsustainable use (exploited populations declining and other sustainability problems), and 37% indicated partially sustainable use (see the data management report for Chapter 3 systematic literature review at https://doi.org/10.5281/zenodo.6452651). Considering 76 studies of coastal small-scale fisheries (including oceanic islands, bays and estuaries), about 13% (10 studies) mention sustainable use, while 53% indicate unsustainable fishing (see the data management report for Chapter 3 systematic literature review at https://doi.org/10.5281/zenodo.6452651). Among 23 studies on inland or freshwater small- scale fisheries, 22% indicate sustainable and 30% unsustainable uses, whereas the remaining majority of studies point to partially sustainable use, suggesting less data availability for inland fisheries compared to coastal cases (see the data management report for Chapter 3 systematic literature review at https://doi.org/10.5281/zenodo.6452651). Sustainable coastal small-scale fisheries examples include co-management systems through territorial rights granted to fishing communities and well-established rules to exploit mainly shellfish, oysters and lobsters in Chile and Mexico (Álvarez et al., 2018; Castilla, Espinosa, Yamashiro, Melo, & Gelcich, 2016; De la Cruz-González et al., 2018; Defeo et al., 2016; Gelcich et al., 2017, 2010). Some of these co-management systems were effective in supporting recovery of resources after a fishery collapse, such as the shellfish Concholepas concholepas in Chile (Castilla et al., 2016; Defeo et al., 2016; Gelcich et al., 2010), Atrina maura, A. tuberculosa, Pinna rugosa, oyster (Crassostrea iridescens), lobster and fish in Mexico (Álvarez et al., 2018; De la Cruz-González et al., 2018; (Palacios-Abrantes, Herrera-Correal, Rodríguez, Brunkow, & Molina, 2018). In association with small-scale fisheries, a management strategy called “Territorial Users’ Rights Fisheries management” (TURF) has been implemented with varying success in Chile (Defeo et al., 2016; Gelcich et al., 2010). For example, population and catches of the clam (Mesodesma donacium) declined over time after the establishment of a territorial users’ rights fisheries system, causing the collapse of the clam fishery. However, this was at least in part due to management restrictions preventing fishers from moving fishing grounds to cope with natural variability of clam abundance (Aburto & Stotz, 2013).  Africa It is well established that most of the non-artisanal small-scale fishing in Africa is unsustainable (see the data management report for Chapter 3 systematic literature review at https://doi.org/10.5281/zenodo.6452651). This general statement applies equally to inland, coastal and marine/oceanic fishing. Small species dominate the African fishing practice for both food and feed that involves the inland and coastal fisheries driven by tradition, species displacement, and substitution (Jamu et al., 2011). A transition from large piscivorous species to small omnivorous species took place during the last half century, when larger fish were almost extinguished from the catch as a result of increasing numbers of fishers, fishing fleets and gear efficiency. A limited number of high value species continue to be targeted, often for export and for higher prices in international markets. There are many cases in the literature reporting on local problems with foreign industrial fleets competing with small-scale fleets, and both affect the artisanal fishing systems. Consequently, these systems show many indicators of unsustainability, such as declining stocks, catches, average size of fishes, catch-per-unit-of-effort, and so on (see the data management report for Chapter 3 systematic literature review at https://doi.org/10.5281/zenodo.6452651). Many official fish landing data series available are underestimated, and important attempts for reconstruction are taking place. Researchers have tried to uncover records of unregulated artisanal catches to produce realistic series of data. These reconstructed series also show the same declining trends. In comparison with commercial fleet fisheries, artisanal fisheries present more diverse scenarios and different trends (see the data management report for Chapter 3 systematic literature review at https://doi.org/10.5281/zenodo.6452651). Some artisanal fishery systems show signs of reduction of catch volume and size over the decades due to excessive fishing pressures (Dyhia Belhabib et al., 2018; Tuda & Wolff, 2015), mainly because fishing pressure has stayed high. Control of fishing pressure is, sometimes, an inherent trait of a system. Systems based on indigenous and local knowledge use the available habitats and fishing grounds (Mirera et al., 2013) to distribute fishing pressure among a large number of species, 110 or to focus the pressure on specific cohorts or in specific times (Musembi et al., 2019). All of these are effective measures of controlling effort and off-take. or to focus the pressure on specific cohorts or in specific times (Musembi et al., 2019). All of these are effective measures of controlling effort and off-take.  Latin America A study across 500 km of the Chilean coast indicates effects of displacement caused by Territorial Users’ Rights Fisheries, which intensify fishing efforts and thereby reduce shellfish abundance in open access areas which have been reduced in size compared to surrounding areas which have entered into government management. This creates conflict and resource shortages for fishers not engaged Territorial Users’ Rights Fisheries management (Garmendia et al., 2021). Other cases of sustainable coastal small-scale fisheries include two fish species (Paralabrax nebulifer, Caulolatilus princeps) that have been fished sustainably mostly because they are only occasionally fished, when preferable resources are unavailable in Baja California Sur, Mexico (Cavieses Núñez et al., 2018). The sustainability of the important fishery of octopus (Octopus maya) in Yucatan (Mexico) is unresolved (Duarte, Hernández-Flores, Salas, & Seijo, 2018b; Raya & Berdugo, 2019). From one hand, the regulations, including fishing 111 season, applied over 30 years and the interaction of fishing gear (baits) with the reproductive behavior of parental care without feeding performed by females, of may have contributed to maintain stocks of this octopus, even in face of intense fishing pressure (Duarte et al., 2018a). However, an increased market demand and search for profit maximization may have pushed fishers to adopt a combination of legal and illegal, furtive, and undeclared fishing tacticts (including diving), which may undermine sustainability and threatens the long-term viability of the octopus' fishery in the Yucatan Peninsula (Raya & Berdugo, 2019). Most of the studies identifying partially sustainable coastal small-scale fisheries include those lacking temporal series of data to estimate species declines, showing distinct trends among exploited species (i.e., some declining, others stable or increasing) or other indicators (e.g., catch volume and size), besides some studies indicating shifts in composition of fished species (see the data management report for Chapter 3 systematic literature review at https://doi.org/10.5281/zenodo.6452651). Several studies on coastal finfish fisheries fall in this partially sustainable category in Brazil (Barbosa-Filho et al., 2020; Damasio et al., 2015; Lima et al., 2016; Silvano, Nora, Andreoli, Lopes, & Begossi, 2017), Mexico (Erisman et al., 2010; Rife et al., 2013) and Colombia (López-Angarita et al., 2018), besides the fishery for king crab (Lithodes santolla) in Chile (Bozzeda, Marín, & Nahuelhual, 2019).  Latin America Second, some studies were perhaps not retrieved in a review using search words as ‘sustainable, sustainability, success and increasing’. The sustainable cases of inland small-scale fisheries (see the data management report for Chapter 3 systematic literature review at https://doi.org/10.5281/zenodo.6452651) are mostly related to the successful co-management of the large and valuable commercial fish pirarucu (Arapaima gigas) in the Brazilian Amazon (Campos-Silva & Peres, 2016; Castello et al., 2009; Petersen, Brum, Rossoni, Silveira, & Castello, 2016), (see also box 6.5 on community-based fishery of pirarucu in the Amazon in Chapter 6). Other mechanisms that could lead to sustainable fisheries are the exploitation of fish resilient to either fishing pressure or environmental change (e.g., dams). For example, the fishing of Plagioscion squamosissimus, especially in communities within extractive reserves or other kinds of protected areas, as observed in several rivers in the Brazilian Amazon (Gustavo Hallwass, Luís Henrique Tomazoni da Silva, Paula Nagl, Mariana Clauzet, & Alpina Begossi, 2020; Gustavo Hallwass et al., 2011;Hallwass et al., 2020; Gustavo Hallwass & Silvano, 2016; Keppeler et al., 2017; Mesquita et al., 2019; Silvano et al., 2014). Small-scale fisheries have cultural and socioeconomic relevance to indigenous Tacana people in the Beni River, Bolivian Amazon, where a participatory survey indicated that this fishery, which exploits 43 species for food and income, has been ecologically and economically sustainable as catches and sizes of exploited fish remained unchanged for a period of seven years, providing a regular source of revenues to local communities (Salinas et al., 2017). Similarly, a study conducted 20 years ago indicates that the large frugivorous fish Colossoma macropomum in the Bolivian Amazon supports a sustainable fishery partly due to its linkages with a small population and a well-preserved floodplain forest habitat (Reinert & Winter, 2002). Unsustainable cases include migratory fish, such as Prochilodus species among others, which have suffered intense fishing pressure, sometimes aggravated by environmental impacts (e.g., dams in rivers in Brazil) (Catarino et al., 2019; Santos, Pinto-Coelho, Fonseca, Simões, & Zanchi, 2018; Philippsen et al., 2017) and Argentina (Baigún et al., 2013). Another unsustainable pattern in the Brazilian Amazon refers to the decrease in catches and size of some of the larger and most valuable commercial fishes, such as Colossoma macropomum and large catfish (Pimelodidae), among others (Castello, McGrath, & Beck, 2011; Garcez Costa Sousa & de Carvalho Freitas, 2011; Hallwass et al., 2019; Tregidgo, Barlow, Pompeu, de Almeida Rocha, & Parry, 2017).  Latin America Some of these partially sustainable cases involve a temporal shift in the exploited fishing resources, in the form of a decline (or even disappearance) in catches of large, slow growing and high valued fish, such as reef predators, coupled with an increase in catches of smaller, fast growing and usually less valued fishery resources, such as shrimp, reef herbivores, or pelagic fish, as indicated in Brazil (Damasio et al., 2015; Ribeiro, Damasio, & Silvano, 2021; Zapelini, Bender, Giglio, & Schiavetti, 2019), Ecuador (Schiller, Alava, Grove, Reck, & Pauly, 2015), Costa Rica (Sánchez-Jiménez, Fujitani, MacMillan, Schlüter, & Wolff, 2019) and Mexico (Erisman et al., 2010; Rubio-Cisneros, Aburto-Oropeza, & Ezcurra, 2016; Rubio-Cisneros et al., 2017). This pattern was also observed in fisheries of elasmobranchs (sharks and rays) in Mexico, where catch of large and threatened species has declined, whereas smaller and more resilient species have increased and tend to have sustained an intense fishing pressure (Ramírez-Amaro & Galván-Magaña, 2019; Saldaña-Ruiz, Sosa-Nishizaki, & Cartamil, 2017). Although benthic invertebrates have been usually among the more sustainable fisheries (see the data management report for Chapter 3 systematic literature review at https://doi.org/10.5281/zenodo.6452651), there are some cases of overexploitation and even fisheries collapses of high valued and easy to catch invertebrates, such as the abalones (Haliotis spp.) or sea cucumbers (Isostichopus badionotus, among other species), in Chile (Sáenz- Arroyo & Revollo-Fernández, 2016), Ecuador (Schiller, Alava, Grove, Reck, & Pauly, 2015) and Mexico (Gamboa-Álvarez et al., 2020). The size and density of the shellfish Queen conch (Lobatus gigas) had declined over a 15-year period in Belize, raising concerns of recruitment or overfishing, but deep water and protected areas may provide a refuge from fishing pressure (Tewfik, Babcock, Appeldoorn, & Gibson, 2019). Only a few studies were considered to be partially sustainable or unsustainable because of side effects from some fishing practices that would cause habitat damage or by-catch, for example, trawling to catch shrimp (Martins et al., 2018; Rosa et al., 2011; Sánchez-Jiménez et al., 2019), pufferfish (Eduardo et al., 2020) or jellyfish (Brotz et al., 2017). This may be partially due to two factors that may have reduced the number of articles on trawling in this 112 review: first, some or most of these trawling fisheries may be considered to be large-scale to have made it into the small-scale fisheries review.  North America In North America, most of the reviewed studies focus fish and invertebrates as food (15) were from the Artic and Alaska (see the data management report for Chapter 3 systematic literature review at https://doi.org/10.5281/zenodo.6452651), whereas fewer studies address recreational (3), or both uses (4). Among the studies on use of fishing resources as food, only one reported a sustainable lobster fishery in California (United States of America), where a collaborative approach improved the ecological assessment and feedback with human dimensions of the system (Kay et al., 2012). Similarly, a study including fishers’ knowledge indicated that the depletion of Atlantic cod caused an increase in the population of lobsters, thus improving the sustainability of lobster fisheries (Boudreau & Worm, 2010). Another study indicated that Pacific salmon species have been increasing in recent years (1990s and early 2000s) in the Beaufort Sea (Carothers, Sformo, Cotton, George, & Westley, 2019). Some problems affecting the sustainability of coastal small-scale fisheries are the potential serial depletion and regional overfishing of the rock crab fishery in California (Fitzgerald, Wilson, & Lenihan, 2018), severe declines in the abundance and catches of the Dungeness crab (Cancer magister) in Canada (Ban et al., 2017), and overfishing and declines of stocks of salmon in Alaska (H. L. Harrison & Loring, 2016; Loring, Harrison, & Gerlach, 2014) and sea cucumber in Canada (O’Regan, 2015). One study that combines traditional and scientific ecological knowledge showed that two exploited shellfish species were also impacted by local and regional environmental factors (Ambrose et al., 2014). However, other studies have shown that the involvement of indigenous peoples and local communities were critical to the reversion of a declining trend in local populations of lake sturgeons (Acipenser fulvescens) in Wisconsin and the Great Lakes region (United States of America), a very relevant social and economic traditional small-scale fishery (Kline, Bruch, & Binkowski, 2012; Runstrom, Bruch, Reiter, & Cox, 2002). The previous population decline of this species was due to unsustainable practices, such as overfishing and habitat loss or transformation, trends also observed in other parts of the world, including the large-scale fishing of sturgeons (Tavakoli et al., 2021).  North America In the Great Lakes region, the local community-built co-management rules across the last six decades for the sturgeon fishery, including fishing festivals and competitions (Kline et al., 2012), and this fishery is also important for the Menominee Nation (an indigenous tribe in upper Midwestern in the United States of America). Together with local authorities and researchers, the local community build a successful restoration program to reintroduce lake sturgeon larvae to areas where they could no longer be found (Runstrom et al., 2002). In North America, most of the reviewed studies focus fish and invertebrates as food (15) were from the Artic and Alaska (see the data management report for Chapter 3 systematic literature review at https://doi.org/10.5281/zenodo.6452651), whereas fewer studies address recreational (3), or both uses (4). Among the studies on use of fishing resources as food, only one reported a sustainable lobster fishery in California (United States of America), where a collaborative approach improved the ecological assessment and feedback with human dimensions of the system (Kay et al., 2012). Similarly, a study including fishers’ knowledge indicated that the depletion of Atlantic cod caused an increase in the population of lobsters, thus improving the sustainability of lobster fisheries (Boudreau & Worm, 2010). Another study indicated that Pacific salmon species have been increasing in recent years (1990s and early 2000s) in the Beaufort Sea (Carothers, Sformo, Cotton, George, & Westley, 2019). Some problems ff i h i bili f l ll l fi h i h i l i l d l i d  Latin America Even the pirarucu (Arapaima gigas) that increased in co-managed small-scale fisheries is considered to be unsustainably exploited in non-managed Amazonian rivers, where the abundance of this fish has reportedly reduced (Leandro Castello et al., 2015; G. Hallwass et al., 2019), mainly due to widespread illegal fishing (Cavole, Arantes, & Castello, 2015). An interesting exception of this pattern is the fishing of some large and migratory catfish (Brachyplatystoma spp.) in the Brazilian Amazon, as catches of some of these species have increased in some rivers either due to successful regulations, improved fishing technologies (larger nets, motorized boats) or to market opportunities (Cruz et al., 2020; Gustavo Hallwass et al., 2020; G. Hallwass et al., 2019). However, these catfish fisheries are difficult to manage due to the long migrations (more than 1,000 km) that these fish perform along the main Amazon River and its tributaries (Barthem et al., 2017; Nunes et al., 2019; 113 Petrere, Barthem, Córdoba, & Gómez, 2004), which make these fishes especially susceptible to impacts caused by dams (Santos et al., 2018) and may thus require basin wide or even transboundary international management approaches(Doria et al., 2020; Goulding et al., 2019). Petrere, Barthem, Córdoba, & Gómez, 2004), which make these fishes especially susceptible to impacts caused by dams (Santos et al., 2018) and may thus require basin wide or even transboundary international management approaches(Doria et al., 2020; Goulding et al., 2019).  Asia-Pacific In Asia-Pacific, the majority of studies (77) address the use of fishing resources as food, either as subsistence, commercial or to support livelihoods. Only 6 studies report sustainable use of fishing resources for food in coastal small-scale fisheries, including reef fish and invertebrates in the American Samoa (Craig et al., 2008), Solomon Islands ( Cohen et al., 2013), the Torres Strait Islands in Australia (Busilacchi, Russ, Williams, Begg, & Sutton, 2013), besides fisheries 114 of shrimp in Indonesia (Anna, 2017), abalone (Haliotis spp.) in Australia (Mayfield et al., 2012) and co-managed finfish fisheries in Bangladesh (Mazumder et al., 2016). The analysis of a long time series of 3,000 years involving both indigenous and local knowledge from fishers and archaeological data indicates no major changes in catch composition of fish and invertebrates exploited in the American Samoa, where catches are at lower levels than the estimated stock sizes of reef fish and fishing yields (kg/ha) correspond to those of less fished Pacific Islands (Craig et al., 2008). This sustainable pattern may be related to a relatively small population of fishers who fish primarily for subsistence and, even considering that sales increased over time, other economic opportunities may have reduced reliance on fishing and hence fishing pressure (Craig et al., 2008). The observed fisheries’ sustainability in the Torres Strait Islands could also be partially related to more subsistence-oriented fisheries (Busilacchi et al., 2013). Similarly, in French Polynesia catches of reef fish have been stable for nine years, even after major natural disturbances including a cyclone. This could be partially due to government subsidy that reduced poverty among fishers, who are mostly part time (Rassweiler et al., 2020). Nevertheless, these are exceptions among the Pacific Island countries, where most cases of sustainable or potentially sustainable fisheries are usually linked to some form of co- management or customary management system, such as periodic harvest closures (Cohen & Alexander, 2013;Cohen et al., 2013; Cohen & Foale, 2013). Although promising, these co- management systems have shown variable results depending on the life history of exploited species, the size of managed area and the regime of opening and closing the area to fishing, which regulates the fishing pressure (Cohen & Foale, 2013b). Therefore, some of these co- management systems improved fisheries yields for fast-growing exploited species in a context of moderate or low fishing intensity.  Asia-Pacific Others have shown a decline of larger and slow growing species (reef fish), usually associated with smaller closed areas or more intense fishing promoted by shorter closed intervals (less than one year) and longer opening periods (Cohen & Foale, 2013; Goetze, Langlois, Claudet, Januchowski-Hartley, & Jupiter, 2016; Hamilton, Hughes, Brown, Leve, & Kama, 2019; Rhodes et al., 2008; Yang & Pomeroy, 2017). Even the more sustainable reef fisheries observed in American Samoa and French Polynesia show a lack of larger piscivorous reef fish, suggesting these larger predators may have been intensively fished in the past (Craig et al., 2008; Rassweiler et al., 2020). In some Pacific Island countries, fisheries for small pelagic fish could be a sustainable alternative for food production, as these fish seem to be more resilient to fishing pressure compared to larger reef fish, as observed in the Solomon Islands (Roeger, Foale, & Sheaves, 2016) and Timor Leste, where fishing aggregation devices and co-management has helped to improve sustainability of coastal and reef fisheries (Tilley, Hunnam, et al., 2019; Tilley, Wilkinson, et al., 2019). There are examples of co-management measures that helped to recover the abundance and hence to improve sustainability of fisheries resources, such as shellfish in Fiji (Thaman, Thaman, Balawa, & Veitayaki, 2017) and reef fish in Hawaii (Friedlander, Shackeroff, & Kittinger, 2013; Friedlander et al., 2014). However, some studies also indicated declines in catches of Hawaiian fisheries for octopus and reef fish (Delaney et al., 2017; Kittinger et al., 2015). An analysis of bioeconomic modelling, which included stock parameters in addition to data on catch, effort, revenues and costs, indicated that shrimp fisheries could be sustainable in 115 Indonesia by showing increased catches over a period of 27 years and surplus stocks (Anna, 2017). However, potential side effects or impacts from shrimp fisheries, such as by-catch or habitat damage, were not included in this study (Anna, 2017), which could compromise the overall sustainability of this fishery. A moratorium of one year imposed on large scale fisheries for tuna in Indonesia had mixed effects on catches of the small-scale pole and line tuna fisheries, but fishers considered that the moratorium was positive and increased their catches, indicating the potential conflicts and competition between large- and small-scale fisheries (Khan, Gray, Mill, & Polunin, 2018).  Asia-Pacific Some cases of unsustainable coastal small-scale fisheries include sharks in Indonesia (Ainsworth, Pitcher, & Rotinsulu, 2008; Ferse et al., 2014; Jaiteh et al., 2017) and China (Lam & Sadovy De Mitcheson, 2011a), and sawfish in Bangladesh (Hossain et al., 2015). In China, a comprehensive study including market surveys and interviews with fishers in Hong Kong and mainland southern China indicates an overall depletion of sharks in the South China sea, where shark fisheries collapsed between 1970s and 1990s, (Lam & Sadovy De Mitcheson, 2011b). Notwithstanding management measures implemented by the Chinese government, a recent study analyzing landings’ data and fishing effort from China for the period of 1955– 2019, shows variable decadal trends with an overall adverse effect from increased fishing intensity on piscivorous fishes, including sharks and rays (Liu et al., 2021). Effects from overfishing can interact synergistically with effects from climate change (Liu et al., 2021). Moreover, China, especially Hong Kong, is the world largest market for shark fins, thus driving exploitation and trade of sharks worldwide, usually at unsustainable levels (Eriksson & Clarke, 2015; Fields et al., 2018). However, shark fisheries can be at least partially sustainable in the Great Barrier Reef (Australia), where susceptibility to fisheries vary among species and their life histories, as smaller species are caught mainly as adults (less vulnerable), whereas larger ones are regularly caught as juveniles, and thus more vulnerable to fishing (Harry et al., 2011). Furthermore, in Eastern Indonesia the whale shark (Rhincodon typus) is not commercially exploited due to customary beliefs among Bajao people that prohibit harvesting this fish, low market values of shark meat and skin, and a lack of technology to harvest such a large fish. No catch data is provided for other regions of Indonesia where this shark could be commercially fished (Stacey, Karam, Meekan, Pickering, & Ninef, 2012). Unsustainable patterns have been also observed in several countries for fisheries of sea cucumbers (Holoturidae), which usually shows a typical cycle of boom and bust typically ending in sharp declines (Eriksson et al., 2018; Hair et al., 2016; Prescott et al., 2017).  Asia-Pacific The large reef fish that form predictable spawning or feeding aggregations, such as groupers or large herbivores, may be negatively affected by unsustainable practices, such as night spearfishing and catches of juveniles, as these slow growing fish are vulnerable to intense fishing during aggregation periods, even in regions under co-management systems (Hamilton et al., 2019; Hamilton et al., 2012; Rhodes et al., 2008; Robinson, Cinner, & Graham, 2014). Unsustainable nearshore coastal and reef fisheries have been observed in Southeast Asian countries located in biodiversity hotspots, such as Indonesia and Philippines, among others (see the data management report for Chapter 3 systematic literature review at https://doi.org/10.5281/zenodo.6452651). Multiple factors, including increased population, poverty, lack of economic alternatives other than fishing, pressure from domestic and international markets, open access and illegal fishing by using destructive practices (bombs, 116 cyanide) lead to unsustainable levels of fishing effort and overall declines in catches of many fishing resources, such as reef and coastal fish, sharks, rays, sea cucumbers and lobsters in these biodiversity rich countries (Acebes, Barr, Pereda, & Santos, 2016; Ainsworth et al., 2008; Ferse et al., 2014; Jaiteh et al., 2017; Khasanah, Nurdin, Sadovy de Mitcheson, & Jompa, 2020; Macusi et al., 2019; Muallil, Mamauag, Cababaro, et al., 2014; Muallil, Mamauag, Cabral, Celeste-Dizon, & Aliño, 2014; Prescott et al., 2017; Selgrath et al., 2018a, 2018b). None of the 10 studies addressing use of fish or invertebrates for food in coastal and inland small-scale fisheries in Asia Pacific indicate sustainable fisheries (see the data management report for Chapter 3 systematic literature review at https://doi.org/10.5281/zenodo.6452651). These fisheries are usually considered unsustainable due to multiple and interacting effects of overfishing, lack of proper management, illegal or destructive fishing practices, coupled with habitat alteration by river dams, deforestation, pollution and increased water temperature, as observed in Bangladesh (Ahmed, Rahman, Bunting, & Brugere, 2013; Jahan, Ahsan, & Farque, 2017), Laos (Gray et al., 2017; Millar et al., 2019) and India (Dey et al., 2019; Keskar, Raghavan, Kumkar, Padhye, & Dahanukar, 2017). Nevertheless, an increase in low value fish has been observed in Cambodia (Enomoto et al., 2011) and co-management initiatives including fishers’ indigenous and local knowledge could be strategic and promising for recovery of fish stocks in the Mekong River Basin (Baird & Flaherty, 2005) and through community-based freshwater reserves in Thailand (Koning, Perales, Fluet-Chouinard, & McIntyre, 2020).  Asia-Pacific The widespread small-scale coastal fisheries in Japan have a long history of a strong bottom-up, co-management system of governance, actively including local fishers through the fishery cooperative associations, which cooperate with scientists and government to regulate fishing activity and allocate fishing grounds among coastal fishers, among other management activities (Ganseforth, 2021; Makino, Matsuda, & Sakurai, 2009; Matsuda, Makino, & Sakurai, 2009; Teh, Teh, Abe, Ishimura, & Roman, 2020). This co-management system can contribute to the sustainability of small-scale fisheries and to marine conservation in Japan, to the extent that local communities can implement fishery regulations to cope with declining fishing resources. This may include protected areas, gear modifications and restrictions on fishing effort (number of boats), as observed in the Shiretoko World Natural Heritage Site where the management plan considers fishers as part of the ecosystem (Makino et al., 2009; Matsuda et al., 2009). Nevertheless, the social and economic sustainability of the Japanese small-scale fisheries face some challenges, such as limited workforce due to an ageing population and lower incomes from fishing compared to other activities (Teh et al., 2020), besides institutional changes that may reduce participation of local fishing association in fisheries management (Ganseforth, 2021). 3.3.1.5.2. Medicine and hygiene Aquatic organisms provide diverse sources of bioactive compounds of interest for nutraceutical, pharmaceutical, and cosmeceutical industries (Table 3.4). Fish, crustaceans and molluscs produce a variety of biologically active compounds that have been characterized by their antimicrobial, antiviral, anti-inflammatory, antioxidant, anti-cancer/antitumor, antihypertensive, anti-atherosclerotic, anticoagulant, and immunomodulatory properties and other medicinal functions (Chbel, Asmaa, Delgado, Aurelio Serrano, Soukri, Abdelaziz, & El 117 Khalfi, Bouchra, 2021; Nisticò, 2017; Šimat et al., 2020). Fish oil, chitin, peptides, polysaccharides, gelatin, pigments, polyphenols, vitamins and minerals are examples of the compounds that have been used as functional food ingredients (Venugopal, 2018) with health benefits. For a number of countries, especially in the tropics, nutrients such as zinc, calcium and iron available from marine fish are essential to the health of local populations, especially for children under five years old (Hicks et al., 2019). Biological properties of fish have also been used to treat or prevent different kinds of health disorders. The food industry introduced several components to improve the properties of foods (i.e., emulsifier, stabilizer, texture modifier, coating or thickening agent) or to enrich foods with functional components and allow their application in health-promoting foods for direct consumption (Šimat et al., 2020). There are many papers promoting the benefits of biologically active components from wild caught animals, but little data were found on the number of wild animals caught and used in pharmaceuticals, nutraceuticals and hygiene products. Thus, the below review focuses on selected uses for which there is enough information to provide an overall assessment. Table 3.4 Major nutraceuticals and bioactive components from seafood. Source: (Venugopal, 2018) © 2018, Springer International Publishing A, license number 5153531358540. CC-BY-NC. Finfish Bioactive peptides Biological calcium cartenoids Ensymes includinf cold-adapted enzymes Glycosaminoglycans inclusing chondroitin sulfate, dermatan sulfate and hyaluronic acid Long-chain omega-3 polyunsaturated fatty acids (PUFAs) Phosphopeptide from fish bone Protein hormones such as calcitonin Protein isolates including collagen and gelatin Suqlene and squalamine Shellfish (crustaceans and mollusks) Bioactive peptides Carotenoids Chitn, chitosan and chitosan derivatives Enzymes Glucosamine Long-chain omega-3 polyunsaturated fatty acids (PUFAs) Mussel polysaccharides, lipids and other prodcuts Protein isolates including collagen and gelatin  Fish oil as a source of omega-3 long chain polyunsaturated fatty acids Table 3.4 Major nutraceuticals and bioactive components from seafood. Source: (Venugopal, 2018) © 2018, Springer International Publishing A, license number 5153531358540. CC-BY-NC. Table 3.4 Major nutraceuticals and bioactive components from seafood. 3.3.1.5.2. Medicine and hygiene Source: (Venugopal, 2018) © 2018, Springer International Publishing A, license number 5153531358540. CC-BY-NC. Finfish Bioactive peptides Biological calcium cartenoids Ensymes includinf cold-adapted enzymes Glycosaminoglycans inclusing chondroitin sulfate, dermatan sulfate and hyaluronic acid Long-chain omega-3 polyunsaturated fatty acids (PUFAs) Phosphopeptide from fish bone Protein hormones such as calcitonin Protein isolates including collagen and gelatin Suqlene and squalamine Shellfish (crustaceans and mollusks) Bioactive peptides Carotenoids Chitn, chitosan and chitosan derivatives Enzymes Glucosamine Long-chain omega-3 polyunsaturated fatty acids (PUFAs) Mussel polysaccharides, lipids and other prodcuts Protein isolates including collagen and gelatin  Fish oil as a source of omega-3 long chain polyunsaturated fatty acids Table 3.4 Major nutraceuticals and bioactive components from seafood. Source: (Venugopal, 2018) © 2018, Springer International Publishing A, license number 5153531358540. CC-BY-NC. Table 3.4 Major nutraceuticals and bioactive components from seafood. Source: (Venugopal, 2018) © 2018, Springer International Publishing A, license number 5153531358540. CC-BY-NC.  Fish oil as a source of omega-3 long chain polyunsaturated fatty acids 118 Fish oils contain high levels of omega-3 long chain polyunsaturated fatty acids (n-3 LC- PUFA), including those known as EPA and DHA (eicosapentaenoic acid [20:5n-3] and docosahexaenoic acid [22:6n-3]). Those components are well accepted as being essential for a healthy and balanced diet, and a large number of studies demonstrate the positive effects of food supplementation with fish oil on human health and the prevention of certain diseases (see (Ghasemi Fard, Wang, Sinclair, Elliott, & Turchini, 2019) for a review). The vast majority of n-3 LC-PUFA is produced by marine micro-organisms, predominantly microalgae (Harwood & Guschina, 2009), whereas terrestrial wild plants do not produce EPA or DHA (eicosapentaenoic acid [20:5n-3] and docosahexaenoic acid [22:6n-3]). (Harwood, 1996). Therefore, the supply of these components for humans come from the ocean, and predominantly from capture fisheries (almost 90%), whether as food fish or via fish oil and fishmeal, with relatively small additional amounts estimated from seafood by-products and recycling, unfed aquaculture and traditional macroalgal sources (Tocher et al., 2006). The global supply of fish oil remains relatively stable (FAO, 2020d; J. Shepherd & Bachis, 2014), constrained largely by natural supply constraints in the fisheries (Misund, Oglend, & Pincinato, 2017) (Figure 3.34). Supplements in the food industry use 20 to 25 percent of globally available fish oil (2017), up from only 5% in 1990 (Figure 3.35). 3.3.1.5.2. Medicine and hygiene While Fish oil is currently the only economically viable source of n-3 LC-PUFA for feed purposes (Misund et al., 2017), the growing demand from the human nutritional supplement industry has tightened the competition noticeably (J. Shepherd & Bachis, 2014). Based on the recommended dose for cardiac health, the total demand for n−3 LC-PUFA is over 1.25 million metric tonnes (mt) whereas total supply is optimistically estimated at just over 0.8 million mt indicating a shortfall of over 0.4 million mt (Tocher, 2015). Figure 3.34 World fish oil market use by sector 2006-2016 (000Mt). Source: (Seafish, 2018) under license CC-BY. Figure 3.34 World fish oil market use by sector 2006-2016 (000Mt). Source: (Seafish, 2018) under license CC-BY. 119 Figure 3.35 Global fish oil use per destination in 2017 (volume in tonnes). Source: (EUMOFA, 2019) under license CC-BY. Figure 3.35 Global fish oil use per destination in 2017 (volume in tonnes). Source: (EUMOFA, 2019) under license CC-BY.  Squalene, squalane, and related compounds from shark’s liver Livers of deep-sea shark species contain high contents of squalene and other hydrocarbons like pristine, which are of interest for cosmetics and medical uses (Macdonald & Soll, 2020). Many shark species, particularly from the deep-sea >200 m, have relatively large livers (up to 20% of animal weight) (Abel & Grubbs, 2020; Vannuccini, 1999). The proportion of liver oil varies between species from 10 to 70% of liver weight (Nichols, Rayner, & Stevens, 2001), and 15 to 82% of liver oil is squalene (Bakes & Nichols, 1995; Deprez, Volkman, & Davenport, 1990). The preferred commercial source of squalene remains shark liver oil, although produced by different animals and plants, presumably due to availability and high yields relative to most plant-derived sources.  Squalene, squalane, and related compounds from shark’s liver Livers of deep-sea shark species contain high contents of squalene and other hydrocarbons like pristine, which are of interest for cosmetics and medical uses (Macdonald & Soll, 2020). Many shark species, particularly from the deep-sea >200 m, have relatively large livers (up to 20% of animal weight) (Abel & Grubbs, 2020; Vannuccini, 1999). The proportion of liver oil varies between species from 10 to 70% of liver weight (Nichols, Rayner, & Stevens, 2001), and 15 to 82% of liver oil is squalene (Bakes & Nichols, 1995; Deprez, Volkman, & Davenport, 1990). 3.3.1.5.2. Medicine and hygiene The preferred commercial source of squalene remains shark liver oil, although produced by different animals and plants, presumably due to availability and high yields relative to most plant-derived sources. Squalene is a skin rejuvenating agent and together with its hydrogenated product squalane (produced from squalene), there is huge potential in nutraceutical, pharmaceutical, and cosmeceutical industries (Venugopal, 2018). Squalene is also used as an adjuvant in vaccines (Brito & O’Hagan, 2014) especially in influenza vaccines (Panatto et al., 2020; Schultze et al., 2008). Shark liver oil also contains Pristane, a natural saturated terpenoid alkane, and squalamine, an amino sterol antibiotic with antiviral, antitubercular, anti- angiogenic properties (Venugopal, 2018). Recent data shows an increase in reported import and processed production of shark liver oil, with trade volumes reaching 752 tons as the largest reported volume in decades (Figure 38) (FAO, 2020d). A review of scientific and management literature by Macdonald and Soll (C. Macdonald & Soll, 2020) identified 133 shark species which are known to be involved in the liver oil trade. One-third of identified species are classified as threatened (vulnerable, endangered, or critically endangered) according to the International Union for Conservation of Nature Red List criteria (Figure 3.36). Population trends for 56% of these species are unknown, and 34% are assessed as showing a decreasing trend (Figure 3.37). 120 Figure 3.36 The International Union for Conservation of Nature Red List conservation status of elasmobranch species reported in the liver oil trade. Source: (Macdonald & Soll, 2020) under license CC BY-NC-ND 4.0. Figure 3.36 The International Union for Conservation of Nature Red List conservation status of elasmobranch species reported in the liver oil trade. Source: (Macdonald & Soll, 2020) under license CC BY-NC-ND 4.0. Figure 3.37 The International Union for Conservation of Nature Red List population trends of all elasmobranch species reported in the liver oil trade. Source: (Macdonald & Soll, 2020) under license CC BY-NC-ND 4.0. Deep-sea sharks offer larger volumes of liver oil compared to other shark species and are therefore of greater interest to the shark liver oil trade (Figure 3.38). The knowledge on these species remains relatively poor due to low research priority added to the difficulties to conduct research in the deep sea (Kyne & Simpfendorfer 2007; Neiva Coelho & Erzini 2006; Figure 3.36 The International Union for Conservation of Nature Red List conservation status of elasmobranch species reported in the liver oil trade. 3.3.1.5.2. Medicine and hygiene Source: (Macdonald & Soll, 2020) under license CC BY-NC-ND 4.0. Figure 3.37 The International Union for Conservation of Nature Red List population trends of all elasmobranch species reported in the liver oil trade. Source: (Macdonald & Soll, 2020) under license CC BY-NC-ND 4.0. Figure 3.37 The International Union for Conservation of Nature Red List population trends of all elasmobranch species reported in the liver oil trade. Source: (Macdonald & Soll, 2020) under license CC BY-NC-ND 4.0. Figure 3.37 The International Union for Conservation of Nature Red List population trends of all elasmobranch species reported in the liver oil trade. Source: (Macdonald & Soll, 2020) under license CC BY-NC-ND 4.0. Deep-sea sharks offer larger volumes of liver oil compared to other shark species and are therefore of greater interest to the shark liver oil trade (Figure 3.38). The knowledge on these species remains relatively poor due to low research priority added to the difficulties to conduct research in the deep sea (Kyne & Simpfendorfer, 2007; Neiva, Coelho, & Erzini, 2006; 121 Verissimo, MacMillan, & Smith, 2011). Therefore, little is known about population structure, habitat use and reproduction of many of these species. Nevertheless, shark reproductive rates and recovery potential are known to decline when depth increases, and population depletion risks exist even when exploitation (targeted or incidental) rates are low (Simpfendorfer & Kyne, 2009). For these reasons, deep-sea sharks have been identified as a conservation priority (Dulvy et al., 2014). Verissimo, MacMillan, & Smith, 2011). Therefore, little is known about population structure, habitat use and reproduction of many of these species. Nevertheless, shark reproductive rates and recovery potential are known to decline when depth increases, and population depletion risks exist even when exploitation (targeted or incidental) rates are low (Simpfendorfer & Kyne, 2009). For these reasons, deep-sea sharks have been identified as a conservation priority (Dulvy et al., 2014). Figure 3.38 The total reported net weight (tons) of annual trade in shark liver oil reported to the Food and Agriculture Organization of the United Nations. Source: (Macdonald & Soll, 2020) under license CC BY-NC-ND 4.0. Figure 3.38 The total reported net weight (tons) of annual trade in shark liver oil reported to the Food and Agriculture Organization of the United Nations. Source: (Macdonald & Soll, 2020) under license CC BY-NC-ND 4.0. 3.3.1.5.2. Medicine and hygiene The cosmetics industry in Europe and the United States of America has decreased its use of shark-based squalene in recent years, under pressure from non-profit organizations and consumers. Independent tests conducted by the French organization “Bloom” determined that most cosmetics (>90% of products tested) sold in Europe or the United States of America no longer contain shark-derived ingredients, although shark-derived squalene is still commonly used in cosmetics elsewhere (Ducos, Guillonneau, Le Manach, & Nouvian, 2015). The Covid- 19 pandemic has reinvigorated the debate on using shark squalene-derived products in the production of potential SARS-CoV-2 vaccines (C. Macdonald & Soll, 2020). Box 3.7 The promising potential of cone snails Molluscs have long been used in traditional medicine and scientists often rely on local knowledge to identify bioactive compounds with potential therapeutic applications (Benkendorff et al., 2015). In this context, one of the most studied groups of organisms are the cone snails, renowned for their capacity to produce venoms used to capture their prey or deter predators (Dutertre et al., 2014). Cone snails are only the tip of the iceberg: order Neogastropoda, has at least 15,000 recorded species, most of which are suspected to be venomous (Puillandre et al., 2011). Venoms produced by cone snails (termed “conotoxins”) have been studied since the end of the 1970s, and constitute an inexhaustible reservoir of toxins, with more than 1,000 species and up to 200 unique toxins produced by each of them (Olivera, 2006). One toxin of cone snail has been approved to be used as an analgesic to treat chronic pain (PRIALT®). Several others are engaged at various steps of the process of drug approval, with applications such as epilepsy, cardioprotection and diabetes (Bjørn-Yoshimoto et al., 2020). Such promising applications make the cone snails (and relatives) an attractive group of organisms for pharmacological companies. However, the only source of toxins is natural populations (cone snails are highly difficult to reproduce in captivity (Perron, 1981). Researchers are now looking for sustainable solutions to preserve the biodiversity. The Nagoya protocol regulates access to genetic resources to guarantee fair benefit sharing with local populations. This is the case, for example, with cone snails that mostly live in tropical shallow waters of emerging countries. Indeed, the highest diversity of cone snails is encountered in the Indo-Pacific (Puillandre et al., 2014), specifically in the Southwest Pacific (e.g., Philippines, Indonesia, Papua New Guinea, New Caledonia), and the most studied species, such as Conus textile or Conus geographus, the latter being the only deadly species for humans, with a fatality rate of 50% (Kohn, 2018), live in these regions. There, cone snails are harvested for aesthetic reasons, and if local populations harvest common species to sell them to tourists, rare species are subject to an active international market reserved to specialists. Restrictions are applied regardless of intent. Strict application of the Nagoya protocol in a growing number of countries also affects scientific study of biodiversity.  Bioactive compounds from wild caught species and seafood processing by- products Fish and shellfish, including crustaceans, are sources of a wide range of bioactive compounds (Box 3.7) that can be recovered from commercial fish processing waste (scales, shells, frames, backbones, viscera, head, liver, skin, belly flaps, dark muscle, roe, and others) and bycatch (unwanted fish and fish of poor economic value). A large corpus of grey literature promotes the use of such material for the production of nutrients, nutraceuticals and pharmaceuticals (Venugopal, 2018), however most processed fish by-products are reduced to fish meal, fish oil and fish silage (A. Jackson & Newton, 2016; Venugopal, 2018). 122 The potential of using these byproducts is important. Jackson and Newton (A. Jackson & Newton, 2016) estimate that the collection and processing of all byproducts not currently used for fish oil extraction would yield around 50,000 tons of EPA and DHA (eicosapentaenoic acid [20:5n-3] and docosahexaenoic acid [22:6n-3]) with around 80% coming from wild capture fisheries. This additional tonnage of EPA and DHA would increase the global supply by around 25%. 3.3.1.5.3. Recreational fisheries 3.3.1.5.3. Recreational fisheries Box 3.7 The promising potential of cone snails The impact of sampling in the field for scientific purpose has been claimed to be negligible compared to the impact of tourists and collectors (Duda et al., 2004), the latter being itself considered to be negligible in regard to the impact of human- mediated environmental changes (Peters, O’Leary, Hawkins, & Roberts, 2016). 3.3.1.5.3. Recreational fisheries 3.3.1.5.3. Recreational fisheries 123 Recreational fisheries are defined as the fishing of aquatic animals that do not constitute the individual’s primary source of nutrition and are not sold or traded on any market (FAO, 2012b). Recreational fishing is one of the most popular leisure activities in inland waters and coastal zones worldwide, with about 11.5% of the world’s population involved (Arlinghaus, Tillner, & Bork, 2015; Steven J. Cooke & Cowx, 2004; Kelleher et al., 2012). In industrialized countries, this proportion can be much higher, exceeding 30% (e.g., Norway) (Arlinghaus et al., 2015). Benefits derived from recreational fisheries include substantial economic benefits in the form of expenditures and related infrastructure (Cisneros-Montemayor, Sumaila, Kaschner, & Pauly, 2010; Potts, Childs, Sauer, & Duarte, 2009), an increase in the stability of the employment buffer through increased year-round or seasonal tourism employment (Smith, Khoa, & Lorenzen, 2005), psycho-social benefits (Floyd, Nicholas, Lee, Lee, & Scott, 2006; Parkkila et al., 2010), and recreational fisher involvement in conservation efforts such as habitat restoration, citizen science, and research (Copeland, Baker, Koehn, Morris, & Cowx, 2017; Tufts, Holden, & DeMille, 2015). While commercial fisheries catch by country are documented since 1950 by the FAO, data for global marine recreational catches remains scarce. (Freire et al., 2020) reported three published estimates, one of 0.5 million tons per year from FAO approximated recreational catches (marine and inland) based on a questionnaire answered by people in 30 mostly developed countries. A second estimate reached 10.9 million tons per year was derived from an extrapolation of Canadian recreational participation and catch rates, and included both marine and inland areas (Steven J. Cooke & Cowx, 2004). Freire et al. (2020) describe estimates of likely marine recreational catches for 1950-2014, based on independent reconstructions for 125 countries. Those estimates of marine recreational fisheries show that catches grew globally until the early 1980s, stabilized during the 1990s, and began increasing again thereafter, amounting to around 900,000 tons in 2014. Marine recreational catches account therefore for slightly less than 1% of total global marine catches (Figure 3.39). Box 3.7 The promising potential of cone snails The available information shows that the majority of this practice is not necessarily linked with the tourism industry (Cillari et al., 2012). Instead, it is usually carried out by locals as cultural practices that maintain important connections between communities and nature. This allows for some territorial overlap, and consequently for some level of competition with other fishing practices, mainly the commercial fishing for food and feed (Carvalho et al., 2017; Das & Afonso, 2017; Marengo et al., 2015). Although the European regulation of fisheries in general tends to be very widespread, most of the recreational fishing practices are not formal, and are therefore unregulated (Lloret et al., 2018). On the other hand, sustainable recreational fishing practices in Europe are probably due to the use of more selective gears (Cillari et al., 2012), and those that are carried out in marine protected areas or other specific areas designated by local management arrangements (Marengo et al., 2015). In Africa, despite the small number of studies on small-scale recreation and tourism fisheries, the reviewed scientific literature suggested that this type of fishing is unsustainable (Belhabib et al., 2016; Leeney, 2016, 2017; Leeney & Poncelet, 2015; McCafferty et al., 2012). This unsustainability is assumed due to strong fishing pressure, and lack of regulation and monitoring which means there is a relative lack of data available. The assessments cover most of the West Coast, encompassing many fish species and also a good part of the East Coast for the recreational fishing industry, mainly targeting the sawfish (see the data management report for Chapter 3 systematic literature review at https://doi.org/10.5281/zenodo.6452651). This later fishing practice is experiencing a strong decline in the last decades and in some areas the sawfish is now rarely detected. In Latin America only two studies evaluated recreational fisheries, but in both cases these fisheries co-occur with artisanal commercial fisheries that exploit fish for food and none were considered as being fully sustainable (see the data management report for Chapter 3 systematic literature review at https://doi.org/10.5281/zenodo.6452651). The increase in recreational spear fishing caused an unsustainable decline on catches and sizes of three reef fish species in Chile (Godoy et al., 2010). Box 3.7 The promising potential of cone snails Trends vary regionally, decreasing strongly in North America, slightly decreasing in Europe and Oceania, while increasing in Asia, South America and Africa. The derived taxonomic composition indicates that recent catches were dominated by Sparidae (12% of total catches), followed by Scombridae (10%), Carangidae (6%), Gadidae (5%), and Sciaenidae (4%). The importance of Elasmobranchii (sharks and rays) in recreational fisheries in some regions is of concern, given the life-history traits of these taxa. Preliminary catch reconstruction, despite high data uncertainty, should encourage efforts to improve national data reporting of recreational catches (Figure 3.40). 124 Figure 3.39 Global marine catches from recreational fisheries by major geographic region for 1950–2014 for all countries with marine recreational fisheries. Source: (Freire et al., 2020) under license CC BY 4.0. Figure 3.39 Global marine catches from recreational fisheries by major geographic region for 1950–2014 for all countries with marine recreational fisheries. Source: (Freire et al., 2020) under license CC BY 4.0. Figure 3.40 Taxonomic composition of global recreational catches by the nine most represented families or higher groupings. ‘Marine fishes nei’ (nei, not elsewhere included) comprises a large contribution of taxonomically unidentified catches; while ‘Others’ comprises all additional taxa with minor contributions pooled. Source: (Freire et al., 2020) under license CC BY 4.0 Figure 3.40 Taxonomic composition of global recreational catches by the nine most represented families or higher groupings. ‘Marine fishes nei’ (nei, not elsewhere included) comprises a large contribution of taxonomically unidentified catches; while ‘Others’ comprises all additional taxa with minor contributions pooled. Source: (Freire et al., 2020) under license CC BY 4.0 In Europe, the majority of recreational and tourism fishing is carried out in the Mediterranean Sea (Antunes et al., 2015; Cillari et al., 2012; Lloret et al., 2018; Marengo et al., 2015; Mavruk, Saygu, Bengil, Alan, & Azzurro, 2018; Ulman et al., 2015b; Ulman & Pauly, 2016), although some of these fishing practices take place in the Atlantic coast or its islands and archipelagos (Carvalho et al., 2017; Das & Afonso, 2017). It is well established in the literature reviewed that the recreational small-scale fisheries performed in Europe is not sustainable, and only 30% of the studies reviewed show any level of sustainable exploitation of recreational small-scale fishing activities (see the data management report for Chapter 3 125 systematic literature review at https://doi.org/10.5281/zenodo.6452651). Unsustainability is assumed due to the lack of regulation of the recreational fishing activity in general. Box 3.7 The promising potential of cone snails The tourism related to fishing, either in the form of tourists fishing for recreation or eating fish in hotels and restaurants, has increased over time and is an important economic activity in the Bahamas and other Caribbean Island countries (Smith & Zeller, 2016). However, the recreational catches related to tourism, about half of total catches in the Bahamas, have been unreported and poorly regulated, which is again assumed to compromise sustainability over time (Smith & Zeller, 2016). In North America, most of the reviewed studies address the recreational coastal fisheries in the United States of America, especially in Florida (see the data management report for Chapter 3 systematic literature review at https://doi.org/10.5281/zenodo.6452651). Some of these studies on recreational fisheries of the bonefish (Albula vulpes) indicate an unsustainable pattern of decline in abundance and size of this fish species, which has suffered increased fishing effort and post-release mortality, leading to an overexploited catch-and- release fishery with negative population effects (Frezza & Clem, 2015; Rehage et al., 2019; R. O. Santos, Rehage, Kroloff, Heinen & Adams, 2019). Another study showed anglers and guides are environmentally conscientious and self-aware of potential anthropogenic drivers of bonefish decline, which may have also been influenced by climate and water quality (Kroloff 126 et al., 2019). A study analyzing 22 fish species of the snapper-grouper reef fish complex in the Florida Keys reported that the majority of these species have been fished unsustainably, though overfishing appears most severe for those long-lived, slow-growing fish (Ault et al., 2005). The only inland study that provided a comprehensive review of recreational and other fisheries in the region of Great Lakes and Mississippi River (United States of America and Canada). It describes internal threats such as overexploitation and bycatch/release mortality, as well as external threats such as inter-sectoral conflicts, environmental change (e.g., habitat alteration and fragmentation), water availability, and introduction of non-native species and pollution (Cooke & Murchie, 2015). In Asia-Pacific, only four reviewed studies address recreational coastal or inland fisheries (see the data management report for Chapter 3 systematic literature review at https://doi.org/10.5281/zenodo.6452651). Although accounting for only a small fraction of the total reconstructed fisheries catches in 25 Pacific Island countries, recreational fisheries have economic relevance to local coastal communities, as these fisheries are related to tourism (Zeller et al., 2015). 3.3.1.5.4. Decorative and aesthetic Some animal parts are used to make perfumes, mainly as a fixative substance that includes musk, ambergris, civet and castoreum. Of these four animal products, ambergris is jetsam coprolite which originates from the sperm whale (Macleod, Sinding, Olsen, Collins, & Rowland, 2020). It has been found rarely but this is in practice for centuries all over the world. It is difficult to estimate the sustainability of the ambergris gathering, as some samples have been present in the environment for about a thousand years (Rowland, Sutton, & Knowles, 2019). The rest of this section was written following the methods used for the systematic review described in 3.3.1.3 (see the data management report for Chapter 3 systematic literature review at https://doi.org/10.5281/zenodo.6452651). The rest of this section was written following the methods used for the systematic review described in 3.3.1.3 (see the data management report for Chapter 3 systematic literature review at https://doi.org/10.5281/zenodo.6452651). In Europe, only a fraction of the small-scale fisheries exploits aquatic animals for uses other than food. These organisms are usually benthic invertebrate species, which are not only fished for food and feed (Duncan et al., 2016; Pita et al., 2019), but also for a limited number of other uses. Some Porifera are traditionally used and sold as sponges for baths, for instance (Fourt et al., 2020). The literature is unresolved on the sustainability of these practices. In recent decades, traditional gear was replaced by modern technologies, such as trawls (Pita et al., 2019), which in combination with increased demand, led to overfishing (Fourt et al., 2020). Some stocks collapsed, although when this happened is unclear. However, more recently strong control of the catch along with other introduced management measures have resulted in the sustainability of this fishing practice being slowly rebuilt (Fourt et al., 2020). However, in most places the uncontrolled use of trawls is still a severe threat to the sustainability of megabenthic fauna, either for the exploited stocks or for other species of demersal fish, which are equally important for the European economy (Duncan et al., 2016). In Latin America, few studies address other uses than food, including ornamental fish to aquarium trade, decorative (handcrafts) or medicinal uses, and often these alternative uses can be made of the same organisms, some of which are also used as food (see the data management report for Chapter 3 systematic literature review at https://doi.org/10.5281/zenodo.6452651). Box 3.7 The promising potential of cone snails The occurrence of whale sharks, which are not commercially exploited and are regularly sighted by fishers, indicates opportunities for the development of non- extractive tourism activities based on observation of whale sharks and promoting collaboration and use of fisher indigenous and local knowledge in Eastern Indonesia (Stacey et al., 2012). The two cases of inland recreational fisheries indicate potentially overfished populations of crayfish (Euastacus armatus) in Australia (Zukowski, Curtis, & Watts, 2011) and more sustainable fisheries of migratory fish in the lower Mekong River basin (Mattson, 2006). Manta rays (Manta alfredi) have been exploited possibly at unsustainable levels for food and medicinal use in the Philippines (Acebes et al., 2016). Recreational fisheries are of concern as fishers concentrate their effort on specific areas, times, species and sizes, leading to greater impacts on targeted stocks. For instance, the nearshore zones more intensively exploited by marine recreational fishers are often critical habitats for multiple life stages of many fish (e.g., spawning, nursery), and immature life stages may be targeted in these areas (Steven J. Cooke & Cowx, 2004). Recreational fishers also selectively target larger and older “trophy” fish, often of keystone, top-predatory species, with life-history characteristics that make them vulnerable to exploitation (late age-at-maturity, low fecundity), which can lead to demographic or evolutionary effects on fish populations (Robert Arlinghaus & Cooke, 2009; Lewin, Arlinghaus, & Mehner, 2006; J Lloret et al., 2020; Prato et al., 2016) and community changes (e.g., successful invasion by non-native species) (FAO, 2012b). Recreational fishers can be regarded as keystone top-predators (Hilborn & Walters, 1992) with increasing efficiency, as knowledge (techniques, areas, seasons, species, etc.) is becoming more accessible and technology (GPS, sounders, braided lines, etc.) more affordable (Griffiths et al., 2010). Hence, recreational fisheries are now widely recognized as a significant component of marine capture fisheries and a potentially significant contributor to fish declines along with the commercial fleets (Agius Darmanin & Vella, 2019; Robert Arlinghaus & Cooke, 2009; Herfaut, Levrel, Thébaud, & Véron, 2013; Pawson, Glenn, & Padda, 2008). To achieve sustainable fisheries management, it appears essential to incorporate recreational fisheries stock assessments (Gordoa, Dedeu, & Boada, 2019). 127 3.3.1.5.4. Decorative and aesthetic None of the 14 studies that focused on these alternative uses addressed the sustainability of the practices. Some studies indicated partially sustainable uses of medicinal or decorative fish species on the Brazilian coast, which may occur at a local scale (low fishing pressure), but may sometimes include threatened species or be linked to trawling and by-catch (Eduardo et al., 2020; Pinto, Mourão, & Alves, 2015; Rosa et al., 2011; C. A. B. Santos & Nóbrea Alves, 2016). The medicinal or decorative use of parts of sharks (mostly by finning) and sawfish are regarded as unsustainable, leading to declines in the exploited species (Barbosa-Filho et al., 2019; Bonfil et al., 2018). Fisheries exploiting jellyfish mostly for food, but including many occasional uses as food for livestock or aquaculture, bait, medicine or aesthetic (collagen) have developed at different stages in several South American countries (Brotz et al., 2017). These fisheries may be considered as partially sustainable, or potentially sustainable, given limited data on landings, potential problems of bycatch and habitat damage (depending on fishing technique) and coastal pollution from jellyfish processing (Brotz et al., 2017). 128 In North America, no uses other than food and recreation were observed among the reviewed studies in this region (see the data management report for Chapter 3 systematic literature review at https://doi.org/10.5281/zenodo.6452651). In Asia-Pacific, only a few studies (8) from the reviewed coastal and inland small-scale fisheries mention uses other than food, such as ornamental or decorative (see the data management report for Chapter 3 systematic literature review at https://doi.org/10.5281/zenodo.6452651). The use of marine shellfish shells as ornaments and handicrafts in Fiji is likely partially sustainable due to a recovery of exploited shellfish populations as a result of co-management (Thaman et al., 2017). 3.3.1.5.5. Ceremony and cultural expression For many small-scale fishing societies, successful fishing does not depend only on technical procedures, but rather on religious and cultural rituals and practices. Good fishing implies that propitiatory practices, such as fasting, specific diets (Teiwaki, 1988) or sex avoidance (Deb, Haque, & Thompson, 2015; Hoeppe, 2007), accompany the various stages of the technical process, including the manufacture of canoes used in fishing practice (Foale, Cohen, Januchowski‐Hartley, Wenger, & Macintyre, 2011). Rituals may also be led by shamans (Ivanoff, 1992; Laugrand, 2015) or marabouts (Artaud, 2016). Thus, many taboos are meant to favour 'luck' or prevent the breach of rules and ward off the ontological imbalances resulting from a non-respect of the rules (Artaud, 2016, 2020). In fishing societies these rituals play an important part because marine species are perceived as 'partners' (Astuti, 1995; Bataille- Benguigui, 1981; D’Arcy, 2008) rather than simply as 'prey' or 'resources'. Bonds of seduction or alliances (Robert Earle Johannes, 1981; Zerner, 2003), fraternity (Grimble, 1989; Lewis, 1994), co-substantiality (Laugrand, 2015; N. Peterson & Rigsby, 2014) or consanguinity (Ivanoff, 1992) unite human and aquatic communities. Beyond these relationships, fishing rituals aim at strengthening the ties between people, social groups or clans. For instance, for the Tao people, fishing flying fish (Family Exocoetidae) is an opportunity to renew a set of cultural and identity principles and values (Berger, 2019; Fan, 2019; Gaffric, 2013). The same is true of whale hunting, which for several indigenous communities constitutes a means of regulating group relations or asserting their singularity within a State (see section 3.3.1.4.6) (Adamson, 2012; Deutsch, 2017). It is also the case for salmon fishing among the Ainu (Iwasaki-Goodman & Nomoto, 2001). Items from aquatic species, such as sea-shells, are used in some rituals, for instance in the candomblé, an Afro-Brazilian religion (Neto, Voeks, Dias, & Alves, 2012). 3.3.1.6.1. Catch and release recreational fishing Recreational fishing can involve a variety of gear types but catch-and-release fishing is most typically focused on fish caught by hook and line (FAO, 2012b). Therefore, this discussion is focused on angled fish. 129 With respect to recreational catch-and-release fishing, it is difficult to disentangle the socio-economic benefits of harvest versus release-oriented recreational fishing, which collectively generates over 100 billion United States dollars annually while creating opportunities for anglers to connect with nature and spend time with friends and family (Robert Arlinghaus & Cooke, 2009). Recreational fisheries certainly can and do involve harvest for personal consumption (Steven J Cooke et al., 2018), but harvest rates vary markedly among regions, species, and angler typologies. To emphasize that variation, recreational harvest rates of species like muskellunge (Esox masquinongy) and bonefish (Albula spp) are around 1% while species like walleye (Sander vitreus) and Atlantic cod (Gadus morhua) have harvest rates that typically exceed 60% (Robert Arlinghaus et al., 2007). In some cases, release of fish is dictated by regulations (e.g., closed seasons, bag limits, size limits) but it can also be voluntary. Where there are long term trend data available, there is evidence that fish release rates have crept up slowly over time (e.g., Brownscombe et al., 2014). The release of angled fish requires proper handling and not all fish survive (Cooke & Schramm, 2007). From a sustainability perspective, it is irrelevant whether fishing mortality arises from harvest (i.e., from an extractive fishery) or from release mortality (i.e., in a non- extractive fishery). Catch and release mortality rates are highly variable and can range from near total mortality to near total survival (recognizing that zero mortality is never attainable). Several syntheses suggest that the bulk of recreational fisheries exhibit release mortality rates that are less than 10% (Arlinghaus et al., 2007; Bartholomew & Bohnsack, 2005; Muoneke & Childress, 1994). Although mortality rates are informative, alone they provide little information on the population-level consequences of release mortality (Kerns, Allen, & Harris, 2012). Information on fishing effort, life history characteristics, population status, and the role of other fisheries practices dictate whether catch and release mortality threatens the sustainability of fish populations. Mortality arising from catch and release is often cryptic and has been implicated in fisheries collapse (Post et al., 2002; Schroeder & Love, 2002). There are many factors that determine whether an individual fish will survive a catch and release event. 3.3.1.6.2. Ornamental or aquarium fish 3.3.1.6.2. Ornamental or aquarium fish Ornamental fish trade is a global, multibillion-dollar industry, involving over 125 countries (Evers et al., 2019a) and worth billions of United States Dollars. Ornamental fisheries are divided into marine and freshwater fisheries. Some of the original representative sustainable gathering projects of ornamental fishes are losing their competitiveness due to the rise of off- site aquaculture. The freshwater ornamental fish trade involves about 125 countries worldwide, is worth approximately 15-30 billion United States dollars (Evers, Pinnegar, & Taylor, 2019b; Penning et al., 2009) and trading around 1.5 billion specimens per year (C. H. Stevens, Croft, Paull, & Tyler, 2017). Roughly 1,000 of the over 5,300 freshwater fish species traded are widely available in commercial numbers (Evers et al., 2019b). A big difference is that around 90% of freshwater ornamental fishes are farmed, usually in Asia or South America, but also in Israel, the United States of America and Europe. Although a smaller portion of freshwater ornamental fishes are still sourced from the wild, in comparison to marine ornamental fishes, it is still a challenge to determine the volume due to lack of reliable data. The marine aquarium trade supplies public and private aquariums with a large diversity of organisms (Dey, 2016; Wabnitz, 2003). A review found that an estimated 15-30 million specimens of coral reef fishes are extracted each year from tropical coral reefs (Biondo & Burki, 2020). The review did not assess mortality rates (Stevens et al., 2017), making proper harvest estimates more challenging since they cannot be based on trade data (Cohen, Valenti, & Calado, 2013; Militz, Kinch, Foale, & Southgate, 2016; Monticini, 2010; Olivier, 2001; C. H. Stevens et al., 2017; Thornhill, 2012). Most marine ornamental species are being collected from the wild (Biondo, 2017, 2018; Biondo & Burki, 2019; V. Dey, 2016; Rhyne et al., 2012; Rhyne, Tlusty, Szczebak, & Holmberg, 2017; Wabnitz, 2003) including species that are listed as endangered by the International Union for Conservation of Nature Red List, such as the Banggai cardinalfish (Pterapogon kauderni). Of the approximately 4,000 marine ornamental fishes known to date (R. Froese & D. Pauly, 2019), about 2,500 species are in trade (Rhyne et al., 2012, 2017). Of all these species only around 25 species (1%), can be bred in commercial numbers and about 300 have been bred successfully in research stages (Pouil, Tlusty, Rhyne, & Metian, 2020). 3.3.1.6.1. Catch and release recreational fishing The single biggest driver of mortality is anatomical hooking location with fish hooked in the jaw region having comparatively low mortality relative to fish hooked more deeply in areas such as the gills or esophagus (Arlinghaus et al., 2007). Recreational catch and release fishing can have consequences for aquatic and coastal habitats. Issues include tackle loss (e.g., lead sinkers, hooks, line), littering, trampling of shoreline vegetation and in-water habitats (e.g., coral, gravel spawning sites), erosion, noise pollution, and hydrocarbon release from boats, and accidental or intentional release of exotic species (e.g., bait bucket transfers, stocking), among others (reviewed in Cooke & Cowx, 2006; Lewin et al., 2006; McPhee et al., 2002; Venohr et al., 2018). Many fishing guides and outfitters pride themselves on their operations being catch and release focused and use that in marketing. A number of non-governmental organizations focus on educating anglers on how to engage in responsible catch and release. Moreover, governments routinely apply harvest regulations as part of their fisheries management initiatives in an effort to create sustainable fisheries that benefit aquatic ecosystems and the humans that use them. Thus, catch and release activities and the associated tour operators contribute to creating responsible and sustainable recreational fisheries (Cooke et al., 2019). 130 3.3.1.6.2. Ornamental or aquarium fish The International Union for Conservation of Nature Red List category is a starting point to warrant protection of a species, but many species of reef fishes are currently labelled ‘not evaluated’ and ‘data deficient’: 73.3% in 2014 and 44.8% in 2018, meaning that the conservation states for almost half of the species is still unknown (Biondo, 2018). Protection from international trade would come through the Convention on International Trade in Endangered Species but only few species are listed on its appendices (e.g., Hippocampus spp. Cheilinus undulatus, Holacanthus clarionensis) thus very little specific trade data is collected (https://cites.org/eng/app/appendices.php, (CITES, 2012). It is estimated that over 50 countries are actively involved in the marine aquarium industry (Biondo & Burki, 2020; Rhyne et al., 2012, 2017). However, this trade lacks sufficient monitoring, and the specific geographic origin of most specimens uncertain (Biondo & Burki, 2019, 2020; Biondo & Calado, 2021; Cohen et al., 2013; Ploeg, 2007). The largest exporting markets are Indonesia, the Philippines and Sri Lanka (Rhyne et al., 2012, 2017; Wabnitz, 131 2003). While some analyses have tried to estimate trading figures for large importing markets, such as the United States of America (Rhyne et al., 2012, 2017), Australia (Trujillo-González & Militz, 2019), and Europe (Biondo, 2017, 2018; Biondo & Burki, 2019; Leal et al., 2016), they all represent approximations and the figures presented are most likely underestimates. Japan is mentioned in the literature as a large importer, but with no recent trade figures available (Biondo, 2017, 2018; Biondo & Burki, 2019, 2020; Rhyne et al., 2012, 2017; Wabnitz, 2003). Furthermore, there is no information at all for growing markets, such as those located in Southeast Asia, Africa, and South America (Biondo & Calado, 2021). With regards to the literature focused on small scale fishing, in Latin America the nine studies addressing small-scale fisheries of ornamental fish for aquarium trade included only three studies in the Brazilian coast (Eduardo et al., 2020; Monteiro-Neto et al., 2003; Rosa et al., 2011), while all others focus on freshwater fisheries in the Peruvian and Brazilian Amazon (Araújo et al., 2020; Evers et al., 2019a; Gerstner, Ortega, Sanchez, & Graham, 2006; Guzmán Maldonado et al., 2017; Ladislau et al., 2020; Moreau & Coomes, 2007). 3.3.1.6.2. Ornamental or aquarium fish A study in the Brazilian coast shows an increasing trend in the number of fish (mainly native reef species) caught and traded, mostly for export, but there are no data on fishing effort or population status of exploited fish to check for the sustainability of such large trade (Monteiro-Neto et al., 2003). Other studies in the Peruvian and Brazilian Amazon indicate that this activity may be unsustainable due to illegal fishing, rapid expansion of fishing effort, reduced fish abundance in more heavily fished regions compared to protected areas and synergic effects of intense exploitation, market pressure (increased sale prices) and habitat change caused by dams (Evers et al., 2019a; Gerstner et al., 2006; Guzmán Maldonado et al., 2017). Studies addressing either coastal or inland ornamental small-scale fisheries expressed concerns on unreported and unknown fish mortality during collection and transportation (Monteiro-Neto et al., 2003; Moreau & Coomes, 2007). In Africa, only a small number of papers dealt with small-scale fisheries for ornamental trade, both in the coral reefs off the coast of Kenya. Some of the many species studied proved to be at low risk of overexploitation, mainly because there is large and disseminated use of very selective gears to capture the fish in this type of fishing. This selectivity allows the removal of mature, large (and colorful) individuals above the maturation size (Gomes, Erzini, & Mcclanahan, 2014). On the other hand, some other species are vulnerable to overfishing and other species are probably already overfished (Okemwa, Kaunda-Arara, Kimani, & Ogutu, 2016). This overfishing is due to low natural abundance and long-term intense fishing pressure. In those cases, more active management measures could mitigate threats to vulnerable species. In Asia-Pacific, data from both fishers’ knowledge and recordings of fish landings (logbooks) of seahorses (Hippocampus comes), which are exploited as ornamental and medicinal fish in the Philippines, indicate that catch-per-unit-of-effort did not change over a period of nine years (O’Donnell et al., 2012). Fishers’ logbooks that included zero catches (fishing trips on which no seahorse was caught) showed the lowest catch-per-unit-of-effort values, and a previous study based on fishers’ indigenous and local knowledge indicated declines of seahorse catches from 1970 to 2005 (O’Donnell, Pajaro, & Vincent, 2010). Some studies point to unsustainable rates of exploitation of sea horses (Hippocampus spp.) on the coast of Vietnam (Stocks et al., 2019; Stocks, Foster, Bat, & Vincent, 2017). 3.3.1.6.2. Ornamental or aquarium fish In India, nearly 50% of marine aquarium fish and corals, considered highly financially valuable species, have 132 not been assessed for their extinction risk (Prakash et al., 2017). The ornamental fisheries of corals (many species) and the coastal fish (Pterapogon kauderni) in Indonesia are considered to be unsustainable, due to intensive fishing pressure, habitat damage, or overestimated quotas beyond ecological capacity (Ferse et al., 2012; Kolm & Berglund, 2003). A recent monitoring survey of Banggai Cardinalfish populations shows mixed trends from 2004 to 2018 among seven sites in Indonesia: recovery or partial recovery in three sites, stable in one, increase in one and decline in two sites, indicating potential effects from conservation measures in some sites and the relevance of microhabitats (sea urchins and sea anemones) to juveniles and adults of this reef fish (Wiadnyana et al., 2020). Morevoer, some marine protected areas, which were created to protect reef fish for ornamental aquarium trade in Hawaii, have increased abundance of some exploited species and thus possibly improved the sustainability of these commercially valuable ornamental fisheries (Friedlander et al., 2014). The few studies on inland ornamental fisheries indicate potential unsustainable harvest, due mostly to intense fishing effort and weak regulations (see the data management report for Chapter 3 systematic literature review at https://doi.org/10.5281/zenodo.6452651). The increase on fishing effort had caused overexploitation of wild caught populations of the clown loach or tiger botia fish Chromobotia macracanthus in Indonesia, which directed fishers to catch fish larvae to be reared in captivity (Evers et al., 2019a). In India, thousands of individuals of threatened and endemic freshwater fish species have been regularly caught and sold for high values in the export market, stimulating an intense fishing pressure (Raghavan et al., 2013). The aquarium trade of ornamental fishes is usually considered as a profitable, rapidly developing, but somewhat unpredictable economic activity, which is subjected to sudden fluctuations in the international market and may involve high operational costs, either for coastal (Monteiro-Neto et al., 2003) or inland fisheries (Araújo et al., 2020; Moreau & Coomes, 2007). Trade includes many dealers with large differences in prices paid between fishers and final retailers (Rosa et al., 2011). 3.3.2.1. Introduction 3.3.2.1. Introduction Wild algae, fungi and plants provide food, income and nutritional diversity for an estimated one in five people around the world, in particular women, children, landless farmers and others in vulnerable situations (Sorrenti & Food and Agriculture Organization of the United Nations, 2017). The Plant List (http://www.theplantlist.org/) and the World Flora Online (WFO, http://www.worldfloraonline.org/) list around 360,000 species with accepted names (accessed January 2021). The world checklist of vascular plants includes approximately 350,000 accepted species. With regards to fungi, 148,000 species have been scientifically identified, but it is believed that more than 90% of species remain unknown to science (Antonelli et al., 2020). Gathering is defined in the sustainable use assessment as the removal of terrestrial and aquatic algae, fungi, and wild plants or parts thereof from their habitats. This definition includes leaves and fruits of trees. Whole tree or excessive branch removal of trees is discussed under logging (see Chapter 1 for complete definitions of all practices). Gathering may, but often does not, result in the death of the organism. All wild plants, fungi, and parts of plant and fungal bodies harvested in forests, savannas, and grasslands that are not wood harvested for timber are broadly categorized as algae, fungi and plants (Sorrenti & Food and Agriculture Organization of the United Nations, 2017). Exploitation of wild algae, fungi and plants often involves the systematic removal of biological, units or parts of units, from a population, but the level of mortality in the exploited population depends on methods of extraction and the vital parts that are removed (Ticktin, 2004). Local communities and indigenous peoples harvest wild algae, fungi and plants for primary health care, basic livelihood needs, to provide social safety nets, and subsistence income. Traditional algae, fungi and plants gathering, for either subsistence or commercial purposes, is often considered a desirable, low-impact economic activity from wild habitats, compared to alternative forms of land use that involve structural disturbance such as selective logging (Plotkin, Famolare, Conservation International, & Asociación Nacional para la Conservación de la Naturaleza, 1992). Gathering is also an important cultural and recreational activity for many, pursued by individuals and family groups even where there is no pressing financial need (Emery, 2001). A majority of wild algae, fungi and plants gathering was considered ecologically and economically sustainable in a recent review (de Mello, Gulinck, Van den Broeck, & Parra, 2020; Stanley, Voeks, & Short, 2012). 3.3.1.6.2. Ornamental or aquarium fish For example, the well-established aquarium trade in the Negro River (Brazilian Amazon), which exploits mainly the small cardinal tetra fish Paracheirodon axelrodi for the international market, has experienced problems related to the productive chain, such as competition with international producers, absence of local buyers, decrease on sales and lower profits, making some fishers abandon this activity (Evers et al., 2019a; Ladislau et al., 2020). The global trade of marine ornamental fishes has always lagged behind in terms of transparency, as there is a multitude of stakeholders involved from the fishers at location of capture to the (many) intermediaries and traders, the exporters and importers and the intermediaries in the importing countries (Amos & Claussen, 2009). Some attempts have been made to increase transparency in the marine ornamental fish industry. The Global Marine Data Base (GMAD) was introduced in 2002 and collected importer and exporter data but with only 41 contributing companies and unfortunately, only for one year (Green, 2003). Another attempt was the Marine Aquarium Council label that was established in 1998 to ensure traceability, good practice, and sustainable schemes of ecologically and socially responsible fishing, but has been inactive since 2008 (Dee, Horii, & Thornhill, 2014). 133 3.3.2. Gathering 3.3.2.1. Introduction Therefore, exploitation of wild algae, fungi and plants, as such, is usually assumed to be sustainable and is viewed as a best compromise between the requirements of biodiversity conservation and those of extractive communities under varying degrees of market integration. However, commercial harvesting of wild plants has increased in recent years, for food, the pharmaceutical and cosmetic industries, as well as for artisanal herbal teas, natural dyes, and decoration. Due to the wide variation in the nature of wild algae, fungi and plants and the way they are harvested and traded, the sustainability of intensive harvesting for trade is debatable (Isabel B. Schmidt, Mandle, Ticktin, & Gaoue, 2011). The number of people who participate in gathering provides one measure of the significance of this practice to nature’s contributions to people. Data on numbers of people who 134 gather globally are incomplete and differences in methodologies vary such that direct comparison of results across studies is difficult. The challenge of assessing numbers of people who gather are compounded by inter-annual variation in gathering, by individuals and households in response to changing needs and opportunities, and as availability of individuals with the desired characteristics ebbs and flows (Lovrić et al., 2020; Watson et al., 2018). With those caveats, available data suggest globally, numbers of people who engage in gathering are likely higher than those for other extractive practices. Gathering is one of the practices most closely associated with traditional lifeways, subsistence practices, and indigenous and local knowledge in both high and low-income countries worldwide. Which wild species are edible and how they are processed, are essential elements of local and traditional knowledge. Most ethnobiological studies on gathering wild species for food consumption have documented edible species, parts, or processing methods. It is widely agreed upon in the available scientific literature that older women are the primary holders and stewards of indigenous and local knowledge, and pass on their knowledge through mother-child nexus and community sharing. Children from indigenous peoples and local communities have specialized access to specific wild resources, ones which are generally of lesser importance for adults and complement their diet. As almost exclusive harvesters of these resources, children retain their own sphere of knowledge and know-how. They are often neglected in considerations of gathering stakedholders, in spite of being full social actors in these societies and being engaged in transmission and exchange networks (Dounias & Aumeeruddy-Thomas, 2017). 3.3.2.1. Introduction Regarding trade in wild algae, fungi and plants, the International Trade Centre estimated that approximately 440 different organic wild products were identified as of 2005. Nearly all of them are wild plants, seaweed and mushrooms; more than half (253/440) of them are medicinal and aromatic plants. A total of 223,754 tons (t) of organic wild harvested products were harvested in 2005. The largest gathering areas were reported in Africa and Europe, while the highest quantity was reported harvested in Asia from a relatively small area (International Trade Centre UNCTAD/WTO, 2007). There is a large amount of trade in wild algae, fungi and plants in the informal economy with little or no records. However, formal markets for resins, tannins, pine nuts, wild mushrooms and other wild algae, fungi and plants in Europe are developing rapidly. In China formal markets around tea seed oil (Camellia oleifera), Chinese chestnut (Castanea mollissima), Persian walnut (Juglans regia), Eucommia (Eucommia ulmoides) and purpleblow maple (Acer truncatum) are expanding (Sheppard et al., 2020). Regarding conservation and sustainable use of wild algae, fungi and plants, the International Union for Conservation of Nature Red List of Threatened Species contains over 9,600 wild food species of which 20% are considered threatened. Ironically, agriculture is the greatest threat to plants, followed by logging and gathering, which is only slightly more threatening than land use for residential and commercial development (Antonelli et al., 2020). What part of the organism is gathered, its phenology, and life form, affects how susceptible the species is to over-harvesting (Table 3.5). Gathering the flowers and fruits of annual-biennial plants shows the greatest susceptibility to overharvesting. Gathering bark and roots also has a high probability of leading to overharvesting. 135 Table 3.5 Susceptibility of wild plants to overharvesting. Note: +represents a high probability, ++ higher, +++ highest. Source: (modified from Lange, 2006) © 2017 Springer. Life form Plant part used Tree Shrub Perennial herb Annual-biennal Wood ++ ++ Not applicable Not applicable Bark ++ ++ Not applicable Not applicable Root ++ ++ +++ +++ Leaf - - - + Flower - - +(++) +++ Fruit/seed - - +(++) +++ .5 Susceptibility of wild plants to overharvesting. Note: +represents a high lity, ++ higher, +++ highest. Source: (modified from Lange, 2006) © 2017 Springer. 3.3.2.1. Introduction Removing the bark may threaten the survival of plant individual, for example when gathering the medicinal part of the African cherry (Prunus africana) (Fashing, 2004; K. M. Stewart, 2003), Julbernardia paniculata, Isoberlinia angolensis (Chungu, Muimba- Kankolongo, Roux, & Malambo, 2007), Himalayan yew (Taxus wallichiana) (Lanker, Malik, Gupta, & Butola, 2010) and Pepper-Bark Tree (Warburgia salutaris) (Senkoro, Shackleton, Voeks, & Ribeiro, 2019). There are many wild plants whose roots are harvested for medicinal use. Some of the most well-known are ginseng (Panax sp.), Nardostachys grandiflora (S. Ghimire, McKey, & Aumeeruddy-Thomas, 2005), Oshá (Ligusticum porteri) (Kindscher, Martin, & Long, 2019), Black Cohosh (Actaea racemosa L.) (Small, Chamberlain, & Mathews, 2011), Cryptolepis sanguinolenta (Amissah et al., 2016), Stemona tuberosa (G. Chen, Sun, Wang, Kongkiatpaiboon, & Cai, 2019) and Eurycoma longifolia (Susilowati, Rachmat, Elfiati, & Hasibuan, 2019). The gathering of major parts of wild plants such as stems and bulbs is also common in herbaceous plants like orchids. These types of gathering activities may kill the plant and are therefore a focus for species conservation and sustainable management efforts. Sustainable harvest programs for gathering flowers, fruit and leaves for medicinal use have secondary benefits for habitat protection. To study the sustainability of the use and gathering of wild plants a literature review was conducted and studies on the ecological aspect of specific species were collected, based on the parts gathered and the life form of plants used. Note that separate literature reviews were conducted on the sustainable use of wild fungi and for urban gathering (see the data management report for Chapter 3 systematic literature review at https://doi.org/10.5281/zenodo.6452651). For the literature review on wild plants, in accordance with the requirements of the systematic literature review, key word combinations were used such as: #gather/pick/collect# + #plant# + #wild# + #terms of the aim of uses + sustainable# and searched primarily in google scholar, Web of Science SCI (Science Citation Index Expanded) and CNKI (China National Knowledge Infrastructure). A total of 89,400 materials were identified, but most only described how the wild plants were used. Eight hundred and fourteen (814) relevant articles and reports went through the initial screening. Fifty-one (51) cases of specific plant species or groups met the search criteria for inclusion in the study of sustainable use by gathering. 3.3.2.1. Introduction The relevant papers were carefully reviewed to 136 determine the credibility of the conclusions of each set of research (see the data management report for Chapter 3 systematic literature review at https://doi.org/10.5281/zenodo.6452651). Cases of sustainable use by gathering wild plants are from all IPBES regions, including Africa (13), America (21), Asia and Pacific (11) and Europe and central Asia (6). Of the 51 cases of the use and gathering of wild plants retrieved, more than two-third of tree/shrub gathering were sustainably managed, while more than half of the gathered herbs assessed were considered unsustainable. For trees, the existing cases show that the gathering of bark for uses, mainly medicinal aims, are not sustainable due to the lack of management and regulatory systems. For herbs, gathering root for medicines from perennial herbs led to more unsustainability concerns (Table 3.6). Table 3.6 Number of cases of sustainable use and gathering of wild plants through literature review. Note: the cases reported here represent those captured through systematic literature review. Additional material is included in the chapter text from contributing authors, personal experiences and expertise. Af.: Africa, Am.: Americas; AP.: Asia and Pacific; EC.: Europe and Central Asia. NA: Not applicable. Uns/Sus: number of unsustainable cases need solutions versus number of sustainable cases under specific management or regulations. Life form Plant's part used Tree/shrub (Uns/Sus) Herb (Uns/Sus) IPBES Regions Af Am AP EC All Af Am AP EC All Barks 3/1 0/1 1/1 4/3 NA NA NA NA NA Sap/gum/resin 1/1 0/1 1/0 0/1 2/3 NA NA NA NA NA Root/tuber/bulbs 2/0 2/2 5/1 1/1 10/4 Leaves 1/1 1/5 2/7 Flowers 1/0 1/0 Fruits/seeds 1/1 0/5 0/1 0/2 1/9 0/1 0/1 Whole 1/0 1/1 1/0 3/1 Sum 5/3 0/7 2/2 0/3 1/7 4/1 5/9 6/1 1/1 16/13 3.3.2.2. The diversity of contemporary gathering 3.3.2.2. The diversity of contemporary gathering 3.3.2.2. The diversity of contemporary gathering 3.3.2.2.1. Gathering in Western European and Other Group (WEOG) countries 3.3.2.2.1. Gathering in Western European and Other Group (WEOG) countries Gathering wild algae, fungi and plants is often assumed to be an activity more prevalent in developing countries and the Global South, and less so in post-industrial countries. However, results of surveys conducted in Europe, North America, and the United Kingdom over the last 20 years suggest high rates of participation in gathering by individuals and households in many of the countries in these regions (Table 3.7). In Scotland, a 2003 random sample general population survey found 18% of individuals had gathered fungi and tree or plant-derived 137 materials in the previous 12 months, including residents of both urban and rural areas (Emery et al., 2006). The Northeastern United States of America includes both the largest urban concentrations of that nation and substantial rural lands. Eighteen percent of respondents to a 2004 survey in that subnational region reported that they had gathered “tree or plant materials around woodlands: e.g., mushrooms, berries, cones, or moss” in the previous 12 months, while 26% had done so in the previous 5 years. Also in the Northeastern United States of America, 36% of respondents to a survey conducted over the five-year period 2005-2009 indicated they had picked mushrooms and/or berries in the previous 12 months (Cordell, Betz, Mou, & Gormanson, 2012; K. Watson et al., 2018). A 2016 survey of households in 28 European countries found that Europe-wide, 26% of households had gathered in the previous 12 months, ranging from 4% of households in the Netherlands to 68% of households in Latvia (B. Wolfslehner, Prokofieva, & Mavsar, 2019). This study noted a general pattern of highest rates of gathering by households in Eastern Europe (Lovrić et al., 2020). Unlike the surveys in Scotland and the United States of America, the European study documented gathering by households, suggesting that the percentage of individuals gathering in the region may be higher. Table 3.7 Percent of population who gather in three Western European and Other Group (WEOG) subregions. Sources: (M. Emery et al., 2006; Lovrić et al., 2020; K. Watson et al., 2018). 3.3.2.2.1. Gathering in Western European and Other Group (WEOG) countries Survey location Survey years Unit of analysis % Gathering (previous 12 months) Scotland1 2003 Individual 18 US Northeast2 2004 Individual 18 US Northeast2 2005-2009 Individual 36 Europe 2016 Household 26 In Europe, changing patterns in wild plant and fungi use vary by country and region, associated with changing lifestyles, urbanization, large-scale farming, less periods of famine and economic hardship in recent years and changing outdoor recreation patterns. At the same time, large increases in immigrant populations are affecting what is harvested, by whom and for what purposes (Łuczaj et al., 2012). In France, 728 algae, fungi and plants species are extracted from the wild, of which 100 are commonly used (Lescure, Thévenin, Garreta, & Morisson, 2015). Recent research in Norway found a total of 273 wild edible plants from 67 botanical families were identified by collectors, with the majority of harvested material coming from seven families and ten taxa. Fruits and berries, leaves and flowers were the most popular and important plant parts that were foraged by study respondents (Giraud, 2020). Research suggests dozens to hundreds of wild algae, fungi and plants are gathered in urban, rural, and wilderness ecosystems throughout the continental United States of America, Alaska, Hawai'i and United States of America territories. Of these, a small subset enters into large-scale trade with maple syrup (Acer sp.), wild blueberries (Vaccinium sp.), and medicinal species such as American ginseng (Panax quinquefolius) noteworthy among them. Estimates of the number of United States of America residents who gather at least occasionally range 138 from 18% to 36%, with the vast majority (>80%) gathering for personal use only. It seems likely, then, that a majority of United States of America residents who gather do so for personal use, while a few species gathered for commercial purposes account for the majority of biomass removed. Wild algae, fungi and plants gathering plays an important cultural role for many indigenous peoples and local communities in the United States of America including, but not confined to, those formally recognized as indigenous. Rights of access to wild algae, fungi and plants for subsistence purposes are provided for by law in the United States of America’s state of Alaska (for rural residents of that state), Hawai’i (for Native Hawaiians), and under the terms of many treaties between tribes and the federal government (Chamberlain, Emery, & Patel- Weynand, 2018; Cordell et al., 2012; M. R. 3.3.2.2.1. Gathering in Western European and Other Group (WEOG) countries Emery & Pierce, 2005; M. R. Emery, Pierce, & Schroeder, 2004; Hurley, Grabbatin, Goetcheus, & Halfacre, 2013; Robbins, Emery, & Rice, 2008). Gatherers have different identities and sources of knowledge in gathering networks. For example, in Austria, organic certification for wild plants has been issued to three types of gatherers: regular, diversified and single-plant gatherers. Among them, regular gatherers are the principal knowledge sources of traditional and local knowledge, and the diversified gatherers who are less common and learning knowledge from formal courses or self-learning, may be more worried by the loss of traditional knowledge (Schunko & Vogl, 2018). In France, present professional gatherers are of multiple origins, urban or rural, and hold their knowledge from different sources. They care for the sustainability of the plants and ecosystems more than occasional opportunistic gatherers. Through their associations or cooperatives, they establish rules of good gathering practices (Lescure et al., 2015) (Julliand, Pinton, Garreta, & Lescure, 2019). 3.3.2.2.2. Urban gathering Urban gathering is an activity which supports biodiversity and sustainable human-nature interactions, but it is under-recognized as a global activity (McLain et al., 2012; A. Russo, Escobedo, Cirella, & Zerbe, 2017; Tiwary, Vilhar, Zhiyanski, Stojanovski, & Dinca, 2020). Urban gathering promotes positive cultural, ecological, economic, and health outcomes (Shackleton, Hurley, Dahlberg, Emery, & Nagendra, 2017; Synk et al., 2017). As a global phenomenon, it provides three categories of provisioning (woody biomass, food/fibre, and non- timber forest products), and it supports a ‘green economy’ (Shackleton, Chinyimba, Hebinck, Shackleton, & Kaoma, 2015; Tiwary et al., 2020). Of the 43 studies related to urban gathering retrieved for this assessment, 70% are from the Americas, Europe and Central Asia, 20% are from Africa, and the remaining are from Asia and the Pacific. Common characteristics of gathering, such as health risks, ecological conditions, and pressures on wild algae, fungi and plants species are likely not the same between in rural and urban contexts, making further research on urban gathering a knowledge gap on the sustainable use of wild species for nature’s contributions to people (Fischer & Kowarik, 2020; Rupprecht, Byrne, Garden, & Hero, 2015; Shackleton et al., 2017; Short Gianotti & Hurley, 2016). The use of Geographic Information Systems (GIS) and spatial modelling in digital platforms and apps shows promise in quantifying urban natures as baselines for this additional research (Arrington, 2021; Moss, Voigt, & Becker, 2021). 139 Dozens to hundreds of feral and wild plant and fungi species are gathered for food, medicine, firewood, decoration, and cultural practices in urban ecosystems (Kaoma & Shackleton, 2015; Landor-Yamagata, Kowarik, & Fischer, 2018; Łuczaj, Wilde, & Townsend, 2021; McLain et al., 2012; McLain, Poe, Urgenson, Blahna, & Buttolph, 2017; Palliwoda, Kowarik, & von der Lippe, 2017; Poe, LeCompte, McLain, & Hurley, 2014; Shackleton et al., 2017; Shackleton et al., 2015; Somesh, Rao, Murali, & Nagendra, 2021). In some cases, for example in Uganda, New Zealand, French Guiana, Haiti and India, wild plants are primarily gathered for medicinal purposes (Dejouhanet & de Bercegol, 2019; Mollee, Pouliot, & McDonald, 2017; Tareau, Dejouhanet, Odonne, Palisse, & Ansoe, 2019; Wehi & Wehi, 2010). However, in major urban spaces in these countries gathering wild edible plants and fungi was most commonly for food, followed by medicinal uses and personal enjoyment (Amato- Lourenco et al., 2020; Garekae & Shackleton, 2020a; Landor-Yamagata et al., 2018). 3.3.2.2.2. Urban gathering However, the potential of urban 140 gathering to affect food sovereignty and security is not evenly distributed across socioeconomic strata (Bunge, Diemont, Bunge, & Harris, 2019). Most gatherers acquire and pass on knowledge about gathering practices through family and friends or gathering trips (Garekae & Shackleton, 2020b, 2020a; McLain et al., 2014). Oral transmission, amateur society outings, professional scientists, books, and field guides help counteract the decline in more traditional outdoor gathering activities (Łuczaj et al., 2021; McLain et al., 2014; Palliwoda et al., 2017). Stakeholders exchange information on the nature of green spaces, species and ecosystems and allied activities. City managers can make use of gatherers' extensive local ecological knowledge to inform more formal management practices and support the overall management of urban natural areas (McLain et al., 2017; Sardeshpande & Shackleton, 2020b). Voluntary codes of conduct may be the best way to manage urban gathering to prevent over-harvesting (Charnley et al., 2018; McLain et al., 2017). Urban gatherers usually select common wild plant species and plant parts that have little impact on the reproduction of plants (Schunko, Wild, & Brandner, 2021). Many gatherers have adopted the “principles of practice” and appropriate techniques for preventing or limiting negative ecological impacts; meanwhile, they teach and promulgate sustainable and responsible harvesting (Łuczaj et al., 2021; Schunko et al., 2021). Despite these benefits, urban gathering is not extensive enough to be considered as a solution to multiple challenges within the food system (Nyman, 2019). With some exceptions (e.g., cities in the Pacific region (Borelli et al., 2020)), the average contribution of wild algae, fungi and plants to diets is low (Shackleton et al., 2017) due to lower tree density in urban spaces, the relatively low proportion of edible parts, or both (Bunge et al., 2019; Estela, Ghermandi, & Margutti, 1995). There are also concerns and potentially physical health risks from eating wild plants or fungi grown on contaminated urban land (McLain et al., 2012; A. Russo et al., 2017), the spraying of chemical herbicides and pesticides (McLain et al., 2014), and mistaking potentially toxic species with edible species (Fischer & Kowarik, 2020). For example, the wild edible food gathered along freeways and arterial roads often have concentrations of lead exceeding safety levels for human consumption (Amato-Lourenco et al., 2020; von Hoffen & Säumel, 2014). 3.3.2.2.2. Urban gathering Wild edibles, including berries, fruits, nuts, greens, and young shoots, were by far the most frequently mentioned type of product, contributing to diversifying urban diets (Garekae & Shackleton, 2020a; McLain, Hurley, Emery, & Poe, 2014; Sardeshpande & Shackleton, 2020a; Shackleton et al., 2017; Somesh et al., 2021). Urban green spaces where gathering happens are promising pathways towards biodiversity conservation in cities because they facilitate interactions between people and nature which support physical and mental health (Palliwoda et al., 2017). Equitable access to cultural ecosystem services from urban green space helps overcome sociocultural barriers, strengthens social relationships, maintains knowledge and traditions of families and communities, increases shares in the management of goods and services, and increases healthy food intake and personal participation in healthy behaviors (Askerlund & Almers, 2016; Jennings, Larson, & Yun, 2016; Landor-Yamagata et al., 2018; McLain et al., 2012; Šiftová, 2020; Tiwary et al., 2020). Urban gathering can also support identity, place attachment, or mobility and agency of people and communities in the city (Poe, LeCompte, McLain, & Hurley, 2014). Gender and income level affect urban gathering activities differently in different regions. They may be evenly distributed along gender or income categories in the United States of America, Germany and the United Kingdom of Great Britain and Norhtern Ireland (Fischer & Kowarik, 2020; Łuczaj et al., 2021; McLain et al., 2012; McLain et al., 2014). Urban gathering in developing countries tends to be more female-dominated in some countries (Garekae & Shackleton, 2020a; Somesh et al., 2021) and male-dominated in other countries (Garekae & Shackleton, 2020b). Residents with lower income and predominantly living or growing up in rural areas or peri-urban areas are more likely to be urban foragers (Garekae & Shackleton, 2020b, 2020a; Mollee et al., 2017; Short Gianotti & Hurley, 2016). Most urban gathering in the developed world is not commercially oriented; products are mainly for personal consumption and gifting (Charnley, McLain, & Poe, 2018; Rebecca J McLain et al., 2014). In countries in the Global South, rapid urbanization, unplanned settlements, and poor service delivery mean that it remains vital to gather for self-provisioning and income. A substantial contribution of total household income can be generated from urban gathering, particularly in poorer households (Borelli et al., 2020; Dejouhanet & de Bercegol, 2019; Kaoma & Shackleton, 2015; Somesh et al., 2021). 3.3.2.2.2. Urban gathering Tensions sometimes exist between urban gatherers and land managers, and between gatherers and other citizens over gathering, particularly in public spaces (McLain et al., 2012). This varies by region. Gathering in many African cities, for example, is permissible in open urban areas, with tacit support from policy and land managers (Sardeshpande & Shackleton, 2020b). However, in many cities in Europe and North America urban gathering is not widely recognized or encouraged, although it is happening. Many cities have some form of regulations that prohibit or discourage urban foraging (Landor-Yamagata et al., 2018; Ortez, 2021; Shackleton et al., 2017). Urban gathering is growing in popularity. Many scholars agree that more people would like to gather wild algae, fungi and plants (Fischer & Kowarik, 2020), but safety concerns, lack of knowledge, perceived social stigma, and lack of access remain significant barriers to urban gathering for many (Ortez, 2021; Somesh et al., 2021). Conservation practitioners had a negative or ambivalent view about the desirability of allowing or encouraging more foraging, particularly in parks or natural areas (Wehi & Wehi, 2010). Risks to biodiversity seem 141 manageable as overharvesting has not been documented (Landor-Yamagata et al., 2018), and in fact many urban greenspaces conserve considerable biodiversity (Rupprecht et al., 2015). Fruit gathering was likely to be least damaging (Sardeshpande & Shackleton, 2020b), and more abundant species are collected more frequently (Fischer & Kowarik, 2020). Even among those favoring gathering, sustainability assessment and adoption of appropriate rules was a precondition (Sardeshpande & Shackleton, 2020b). Gathering may support invasive species management in urban ecosystems (Arrington, 2021; McLain et al., 2017). Although most utilized species are native (Charnley et al., 2018; Palliwoda et al., 2017), a species’ status as invasive or non-invasive can influence gathering practice (McLain et al., 2017). Since many invasive wild plants have a history of cultivation as food, medicine, and materials, providing some socio-economic values, the gathering and use of edible weeds as a complementary resource has promising possibilities. For example, bracken fern (Pteridium aquilinum), a native plant in the Pacific Northwest region of the United States of America, has been classified and gathered as an edible ‘weed’ Poe, LeCompte, McLain, & Hurley, 2014). An emerging approach is to consider urban forests as nature-based solutions in the urban environment and include them in city management and planning (Roeland et al., 2019). 3.3.2.2.2. Urban gathering Trees are welcomed for their products and regulating services like shade and windbreaks, also their less tangible aesthetic and cultural values (Shackleton et al., 2015). Urban gathering creates ties between people and the surrounding nature, in fact encouraging people to see urban vegetation and green space as natural (Landor-Yamagata et al., 2018). Urban planners may consider these benefits of green spaces and issues of access to nature in the city (Charnley et al., 2018; Shackleton, Drescher, & Schlesinger, 2020). In summary, the combination of edible green infrastructure and urban beautification contributes to urban food production, as well as co-benefits nutrition, socioeconomics, and environment (Russo et al., 2017). Ecosystem services provided by urban green space create urban gardening and gathering opportunities that contribute to healthy lifestyles ( Jennings et al., 2016). Traditional tropical home gardens serve as a model for biocultural diversity in small- scale urban green spaces (Hemmelgarn & Munsell, 2021; Sardeshpande & Shackleton, 2020a). The forest garden helps urban children develop environmental, scientific, and possibly other values (Askerlund & Almers, 2016). The use of edible green infrastructure areas and gardens are playing an important role in the COVID-19 pandemic and post-lockdown period as people have spent more time at home and demonstrated an increased awareness of the need for self- reliance and resilience to emerging threats (A. Russo & Cirella, 2020). 3.3.2.2.3. Gender trends Gathering wild products is a gendered activity in many parts of the world, depending on cultural rules, on the type of harvested wild algae, fungi and plants and the places where they are harvested. In many countries, women perform the bulk of the labor for gathering and processing wild plants for food, medicine, fuel and handicrafts, as well as for other subsistence purposes, and often sell wild products at local markets (Howard, 2003). Some gathering activities are specific to men, some others are conducted equally by men or women, as well as children, or involve the whole family. Today, commercial gathering is done by men and women 142 who make it their primary profession (Julliand et al., 2019). In low-income households, women are often responsible for gathering for self-consumption and to sell (Sabater, 2020). A range of examples show a variety of gender dynamics in gathering around the world. In the 1980s, farmers in the mountains of central France, men and women, harvested wild plants and mushrooms for their own consumption, to share with family and for commercial purposes. Children, teenagers and elders dedicated more time to gathering than adults, the latter being busy with agricultural activities (Larrère & La Soudière, 1985). In Turkey, gathering practices between men and women differ in that woman prefer to gather in social groups, and distribute some of the wild plants such as edible greens that they have gathered as gifts to friends and neighbors (Ertug, 2003). In the tropical forests of French Guiana, the Maroon Ndjuka women gather wild plants close to the village and the fields, while men gather wild plants in the deep forest (Tareau et al., 2019). In the savannas of central Brazil, the Xavante women gather wild plants while the men hunt, but men sometimes join them (Flowers, 2014). Extractivism with long expeditions in the forest is usually practiced by men, for instance rubber or piassava collectors in the Amazon (Schmink & García, 2015), eaglewood (Aquilaria sp.) collectors in Borneo or Papua; in these last regions, some traders even organize expeditions where they drop a group of several men in the middle of the forest from a helicopter (Mittelman, Lai, Byron, Michon, & Katz, 1997). In the dry and semi-dry areas of Africa, gums and resins such as gum arabic (Acacia senegal, A. 3.3.2.3.1. Ceremony and cultural expression The world’s major cultures and ritual practices observe conservation of species and nature as essentials for human well-being. Cultural expression may take the form of song, stories, dances, art, designs, crafts, rituals, ceremonies, and more. Many wild species, especially wild plants and fungi, perform critical roles in ceremonies of various cultures around the world. They are harvested for use in spiritual observances and practices, and are highly valued for their role in maintaining cultural identity in formal ways (Hamilton, 2004). The Millennium Ecosystem Assessment highlighted that impeding religious and social ceremonies by denying people access to required wild plants or fungi could harm social relations as “many cultures attach spiritual and religious values to ecosystems or their components” (Millennium Ecosystem Assessment, 2005). Research on gathering for ceremony and cultural expression focus more on the cultural dimensions, such as the types of rituals (e.g., marriage, birth, death, important memorial points and specific religious rituals) than they do on the sustainable use of the species per se. Wild species are sometimes mixed with horticultural plants, and used for decoration, smoking, dyeing, as non-pharmacological medicine or for energy and nutrition. Flowers and incenses made out of dried plants or resins such as frankincense or myrrh are often used in rituals. It is difficult to make a complete list of species used for ceremonies, as many ethnobotanists make inventories of dozens to hundreds of wild plants from local surveys (Barceló, Butí, Gras, Orriols, & Vallès, 2019; Des, Rizki, & Fitri, 2019; Yanfei Geng et al., 2017; Rangel-Landa, Casas, García-Frapolli, & Lira, 2017). Some of the wild species used for rituals are unusual and rare (Naegel, 2004; Rangel- Landa et al., 2017). Gatherers give them as presents to the organizers of the ceremony or communitarian feast, and commercialization is uncommon (Barceló et al., 2019; Rangel-Landa et al., 2017). However, because of important traditional culture, there is often concern about the disappearance of these particular species in studies of national culture. Rare species are harvested at levels just enough to satisfy the needs of the community (Rangel-Landa et al., 2017), and in some cases substitutions are developed (Des et al., 2019). The ritual practices of Naxi people in Yunnan, China for example, pay high respect to conserving natural resources, although these beliefs and cultural expressions receive less attention from younger generations (Yanfei Geng et al., 2017). 3.3.2.2.3. Gender trends seyal), myrrh (Commiphora myrrha) and frankincense (Boswellia spp.) are usually gathered by men, pastoralists who fulfil this activity while taking their cattle to graze (Mugah, Chikamai, Mbiru, & Casadei, 1997). Tapping resins in general is a male task, especially when it is necessary to climb on trees. Batak benzoin tappers in North Sumatra, Indonesia, describe the benzoin tree (Styrax paralleloneurum) as a woman who gets pregnant of the resin after the tapping, a symbolic sexual act (Esther Katz, García, & Goloubinoff, 2002). 3.3.2.3. Uses of wild plants, algae, and fungi, including the leaves and fruits of trees Unlike the case for some of the other practices, where only selected uses are relevant, all of the uses outlined in Chapter 1 of the assessment are relevant for gathering practices. In fact, in several subsections of 3.3.2.3 the diversity of species gathered for the various uses are so extensive that additional subdivisions have been created. The sections are as follows: Ceremony and cultural expression (3.3.2.3.1); decorative and aesthetic (3.3.2.3.2) with subsections on ornamental, natural cloth and dyes, handicrafts, and perfume and incense; energy (3.3.2.3.3); food (3.3.2.3.4) with subsections on nuts & seeds, starchy fruits, juicy fruits, beverages, syrups, gums, and resins, wild edible mushrooms, and wild vegetables; medicine and hygiene (3.3.2.3.5); recreation (3.3.2.3.6); science and education (3.3.2.3.7); and materials and shelter (3.3.2.3.8). Importantly, the text is not an inventory of all species gathered for various practices. Rather, the focus is on those species of particular interest in relation to sustainable use which emerged through the systematic literature review and those that were highlighted through various rounds of expert discussion and review. 143 3.3.2.3.1. Ceremony and cultural expression Hallucinogenic plants and fungi harvested in the wild are used by shamans or mediums, in religious or curing ceremonies, in particular on the American continent, but also in Siberia (Amanita muscaria) and Africa (iboga, Tabernante iboga, in Gabon). For the Jotï, an indigenous group in the Venezuelan Amazon, mushrooms play a central role in their religious and spiritual beliefs and are fundamental to their cosmology (Zent, 2008). Among the most known in the Americas are ayahuasca (Banisteropsis sp.) from the Amazon, and Psylocybe mushrooms and peyote (Lophophora williamsii) from Mexico and the United States of America Southwest (Furst, 1972; Heim & Wasson, 1958; Schultes & Hofmann, 1979). In the 1960s, after Wasson’s discovery of hallucinogenic Psylocybe mushrooms in Mexico, “hippies” rushed to that country to experience these fungi. There has also been a development of shamanistic tourism among the Mazatecs in Mexico (Demanget, 2010) and in the Peruvian Amazon (Fotiou, 2016). Peyote is still traded in the United States of America (Feeney, 2017). 144 Overall, 216 species of fungi are thought to be hallucinogenic, and of these 116 species belong to the Genus Psilocybe (Willis, 2018). Since rituals are not a daily need, there are few relevant management measures that are directly applied to species specifically relating to ceremonial use, and it is recommended that maintaining traditional and cultural practices can complement management strategies (Kideghesho, 2009). In fact, conserving biodiversity based on cultural and religious faiths may be often more efficient and sustainable than government legislation or regulations given peoples’ long-term relationships with the particular species. 3.3.2.3.2. Decorative and aesthetic Wild species are harvested for crafts and decorative use for personal consumption, as gifts, and for sale as raw or value-added items (M. R. Emery, 1999). The gathering of wild species like orchids, Bromeliads, succulents, and wild fungi are important sources of money and livelihood for collectors at local and regional scales and may also enter into global trade. Hence the sustainability of their wild populations, habitat, economies and communities is a subject of concern. Many wild species harvested for crafts are usually listed in inventories as parts of general ethnobotanical research. It can be challenging to distinguish among uses at the local level, as one collection may result in the gathering one species for food, medicine, ritual decoration, and transplant into the home garden. There is a lack of research on the sustainability of this kind of mixed use.  Ornamental wild plants When the acquisition is part of the organism and managed well, gathering wild plants for the use of decoration may not have too many negative effects. For example, although there is lack of conservation assessment of the 80% of wild harvested Indian plants to make potpourri, the gathering of such 455 species provides a supplementary income to rural poor and is considered as a sustainable use (Cook, Leon, & Nesbitt, 2015). In Minas Gerais, central Brazil, the gatherers of everlasting (sempre-vivas) flowers of the Serra do Espinhaço Meridional enrich the native pastures where the flowers grow with the seeds fallen from the collected flowers and stimulate their growth by fire management (Monteiro et al., 2019), demonstrating a form of traditional management and care which supports sustainable use. Their agro-extrative system was recognized by FAO in 2020 as a Globally Important Agricultural Heritage System (GIAHS) (GIAHS, 2020). Trade in exotic wild plants increased in North America and Europe after the Second World War and demand for wild plants increased pressure on wild populations and even drove the extinction of some rare species in the late 1970s (Lavorgna, Rutherford, Vaglica, Smith, & Sajeva, 2018). Twenty-two European countries reported the total value of “ornamental plants” at almost 1,400 million euros, which amounts to 49.6% and the highest of the marketed plant products from forests (Forest Europe, 2020). The Convention on International Trade in Endangered Species of Wild Fauna and Flora has listed more than 32,000 species of ornamental plants in its Appendices, most in Appendix II (Table 3.8). The gathering for sale of cut flower or foliage of bromeliads, or ornamental plants like aloe and orchids are considered to negatively affect species survival (Flores-Palacios, Bustamante-Molina, Corona-López, & Valencia-Díaz, 2015; Mondragón Chaparro & Ticktin, 145 2011; Mondragón, Méndez-García, & Morillo, 2016; Negrelle & Anacleto, 2012; Phelps & Webb, 2015; Sakai et al., 2016). Many of these species are also cultivated, but no data was available at the time of this assessment on the share of global market sales from wild versus cultivated plants. Sale prices vary between species (Mondragón et al., 2016), but the origin of the plants (wild vs farmed) did not affect price, since cultured plants have better physical variables than wild-harvested plants (Elps, Carrasco, & Webb, 2014). Some researchers believe that the supply-side measures to ensure the sustainable use may lack effectiveness.  Ornamental wild plants Consumer preferences may help to reduce the market driven push to overharvest (Elps et al., 2014). Table 3.8 Ornamental wild plants listed under the Convention on International Trade in Endangered Species of Wild Fauna and Flora. Source: Species+ data (UNEP, 2021 ) (The Species+ Website. Nairobi, Kenya. Compiled by UNEP-WCMC, Cambridge, UK. Available at: www.speciesplus.net. [Accessed 01/March/2021]) Table 3.8 Ornamental wild plants listed under the Convention on International Trade in Endangered Species of Wild Fauna and Flora. Source: Species+ data (UNEP, 2021 ) (The Species+ Website. Nairobi, Kenya. Compiled by UNEP-WCMC, Cambridge, UK. Available at: www.speciesplus.net. [Accessed 01/March/2021]) Common name Family/Genus Appendix Number of listed species in the taxa Agaves Agavaceae I and II 4 Snowdrops Galanthus spp. and Sternbergia spp. II 21 + 9 Cashews Operculicarya spp. II 3 Elephant trunks Pachypodium spp. I and II 23 Ponytail palms Beaucarnea spp. II 11 Bromelias Tillandsia II 3 Cacti Cactaceae I and II 1532 Zygosicyos Zygosicyos II 2 Tree-ferns Cyathea spp. and Dicksonia spp. II 686 + 46 Cycads Cycadaceae spp. and Zamiaceae spp. I and II 109 + 228 Alluaudias Didiereaceae spp. II 12 Elephant’s foot, Dioscorea deltoidea II 1 Venus’ flytrap Dionaea muscipula II 1 Succulent spurges Euphorbia spp. I and II 709 Ocotillos Fouquieria I and II 3 Aloes Aloe spp. I and II 483 Pitcher-plants Nepenthes spp. and Sarracenia spp. I and II 112 + 29 Orchids Orchidaceae spp. I and II 27,924 Palms Palmae I and II 13 Poppy Meconopsis regia III 1 Passion-flowers Adenia sp. II 3 Sesames Uncarina II 2 Table 3.8 Ornamental wild plants listed under the Convention on International Trade in Endangered Species of Wild Fauna and Flora. Source: Species+ data (UNEP, 2021 ) (The Species+ Website. Nairobi, Kenya. Compiled by UNEP-WCMC, Cambridge, UK. Available at: www.speciesplus.net. [Accessed 01/March/2021]) 3.8 Ornamental wild plants listed under the Convention on International Trade in S i f i 146 Lewisias, portulacas, and purslanes Anacampseros spp., Avonia spp. and Lewisia serrata II 25 + 11 + 1 Cyclamens Cyclamen spp. II 27 Stangerias Stangeria eriopus and Bowenia spp. I and II 3 Grapes Cyphostemma spp. II 3 More than a half of all cactus species (57%) are used by people. Cacti are prized for their aesthetic qualities. The most common use is for ornamental horticulture (674 species), which in most cases is related to gathering wild plants and seeds for specialized collections.  Ornamental wild plants Cacti comprise about 130 genera and 1,500 species distributed mainly in North and South America; however, several species of Rhipsalis (mistletoe cactus) occur in tropical Africa. Some species of Opuntia (prickly pear) have been introduced in Africa, Australia and South Asia (India). Nearly all genera are cultivated as ornamentals; some of the more common are Opuntia and Carnegiea (giant saguaro), Cereus (hedge cactus, cereus), Echinopsis (sea-urchin cactus), Epiphyllum (orchid cactus), Hylocereus (night-blooming cereus), Mammilaria (pincushion cactus), Melocactus (Turk’s cap cactus), Rhipsalis, and Schlumbergera (Christmas cactus) (Judd, 1999). Native people of the Americas propagate branches, seeds or transplant complete individuals from the wild to their agroforestry systems and home gardens (Casas & Barbera, 2002). People occasionally harvest useful parts of several species of cacti for use in traditional medicine. Cactus pears from Opuntia stricta are also considered as a potential source of natural colorants (Casas & Barbera, 2002; Goettsch et al., 2015). Due to their popularity and the commercialization of so many wild species, poaching entire plants from the wild is a growing problem. Most species are regulated by the Convention on International Trade in Endangered Species of Wild Fauna and Flora (Table 3.8). Among the threatened cacti species, 64% are utilized by humans in some form and 57% (236 species) are used in horticulture (Goettsch et al., 2015). There is growing concern that a high proportion of cactus species may be threatened with extinction in the near future, mainly due to growing illegal trade. Orchids are a prominent group of the global horticultural trade. While large numbers of orchids are grown commercially, there are still large numbers taken directly from the wild. Over-harvesting of wild orchids associated with floral and medicinal trade is a serious concern for their long-term survival (Hinsley et al., 2018). Cross-border trade of orchids is well recognized as a threat to orchid conservation and regulated by the Convention on International Trade in Endangered Species of Wild Fauna and Flora. However, domestic trade may not be regulated or poorly enforced in some orchid-rich countries (Phelps & Webb, 2015; Tamara Ticktin et al., 2020; Wong & Liu, 2019). This legal and illegal domestic trade of wild orchids can be larger than cross-border trade and can also pose serious threats to species survival, but receive far less attention from orchid conservationists (Phelps & Webb, 2015; Tamara Ticktin et al., 2020; Wong & Liu, 2019).  Ornamental wild plants Snowdrops (Galanthus sp.) is a relatively small genus of perennial herbaceous plants distributed throughout Europe and central Asia, threatened in the wild due to habitat 147 destruction, illegal gathering and climate change. A cherished garden plant with beautiful flowers blooming in winter and early spring, Galanthus is the world’s most traded wild-sourced ornamental bulb genus. To implement the Convention on International Trade in Endangered Species of Wild Fauna and Flora regulations, Turkey sets annual export quotas of wild bulbs at 2.5-5.0 million for G. elwesii and 2-4 million G. woronowii. Georgia sets an export quota of wild G. woronowii at 15 million a year to ensure the trade and gathering do not endanger the survival of wild populations (Rønsted, Zubov, Bruun-Lund, & Davis, 2013; UNEP, 2021, p. 2021).  Natural cloth and dye Numerous wild plants, lichens, and mushrooms have been used as natural dyes for centuries. Some of them, such as Brazil wood (Caesalpinia echinata), were traded across continents. Most natural dyes were substituted by chemical dyes from the 19th century on, but some remained in use in local arts and crafts, and have been revived recently. Some species are not only on textiles but also in the cosmetic and food industries. For instance, the lichen Rocella canariensis is used as a food coloring known as E121 (Cardon, 2007). Cotton, linen, silk, wool and artificial fiber and dyes have replaced many wild sources. Uganda bark cloth was derived from the wild fig or mutuba tree (Ficus natalensis) and has been recognized by UNESCO as a masterpiece of the 'Intangible Cultural Heritage of Humanity'. The production process requires collaboration among local laborers, specific skills and specially designed tools. In recent years bark cloth has been explored as a sustainable fashion luxury textile, providing jobs to local communities (Venkatraman, Scott, & Liauw, 2020). The use of bark facilitates scattered planting of mutuba trees in agroforestry systems, which in turn protects crops and soil from erosion on windy hill slopes. World Overview on Conservation Approaches and Technologies (WOCAT) has developed a guide on the use and propagation of the tree. This example highlights the value of this specialized knowledge. However, traditional knowledge on unique dying sources and processes is vanishing fast, and represents a knowledge gap which may become impossible to address in the near future. Many wild fungi and lichens are also harvested for use in dye making. For example, Emery, Martin and Dyke (2006) found that of the over 200 species harvested from the wild in Scotland, 76 of them were non-vascular species. Of these, 16 were harvested for crafting purposes such as the production of dyes for homespun wool. A group of lichens known collectively as ‘orchil’ has been used as a dyestuff since the Bronze Age in Europe. Trade in orchil declined as manufactured, synthetic and cheaper alternatives were found. It continues at low levels for artisanal use (Wolfslehner et al., 2019). Some firms specialized in plant dyes aim at meeting standards of environmentally and socially responsible manufacturing and have applied to a certification, but as of 2010 this issue remained unresolved (Cardon, 2010).  Handicrafts It is used traditionally to craft or decorate baskets and bowls (Leif, 2010). In South Carolina, gulfhairawn muhly (Muhlenbergia filipes) is also called Sweetgrass. Its leaves are gathered by the Gullah community, descendants of enslaved Africans, to make a form of coiled basketry. The Gullah basket is now recognized as an artform and a major source of income for the local people (USDA & NRCS, 2009). This native coastal grass on which the basket makers depend has become increasingly scarce due to urbanization and limited access to the resource. Basket makers have to develop social-economical strategies, such as purchasing raw materials from other states, or negotiating access to the grass to maintain the traditional artform and their livelihood (Grabbatin, Hurley, & Halfacre, 2011; Hurley et al., 2013; USDA & NRCS, 2009). Many species of wild fungi are harvested for craft purposes. Turkey tail mushrooms (Trametes versicolor) grow throughout North American forests, and also across Europe and Asia. Turkey tail is a very colorful bracket fungus that grows throughout the year on dead or rotting wood. Pieces of the fruiting body are often harvested for use by artists and jewelry makers, who most commonly use them in earrings and necklaces (Spahr, 2009). Ganoderma applanatum (commonly known as the artist’s conk) is also a bracket fungus with a cosmopolitan distribution. It is sometimes used as a medicinal tea, but it is most commonly known in North America for its use as an artist’s canvas of sorts, where burning or carving into the underflesh of a dried polypore leaves behind brown markings to create images (Wetzel, Duchesne, & Laporte, 2006). In this case, while some mycelia live on in the dying or decaying wood medium, polypores take so long to grow that when the fruiting body is harvested, functionally almost the entire the organism is harvested. y y Many species of wild fungi are harvested for craft purposes. Turkey tail mushrooms (Trametes versicolor) grow throughout North American forests, and also across Europe and Asia. Turkey tail is a very colorful bracket fungus that grows throughout the year on dead or rotting wood. Pieces of the fruiting body are often harvested for use by artists and jewelry makers, who most commonly use them in earrings and necklaces (Spahr, 2009). Ganoderma applanatum (commonly known as the artist’s conk) is also a bracket fungus with a cosmopolitan distribution.  Handicrafts The following is not meant to be an exhaustive inventory of all wild algae, fungi and plants used for handicrafts. Rather, it is a review of the wild species of interest with regards to sustainable use which appeared in the systematic literature searches. A wild plant material called golden grass (Syngonanthus nitens) is used to produce golden handicraft articles in Brazil. Rural communities harvest, process and knit the scapes of 148 Syngonanthus nitens, which has been an important source of income for them since the late 1990s. The survival of plant populations was once affected by the increase in community demand for scapes. The Brazilian federal environmental agency (Ibama) has proposed management techniques to prevent overexploitation of the species. For example, the harvest time was set precisely to ensure the removal of inflorescences after seed production or full maturation. Furthermore, returning the capitula of inflorescences used in handicraft to the field represents another important tool for the sustainable management of golden grass (Oliveira, Cruz, Sousa, Moreira, & Tanaka, 2014; I. B. Schmidt, Figueiredo, & Scariot, 2007; I. B. Schmidt & Ticktin, 2012). There are several types of wild plants in the United States of America called Sweetgrass, that can be used to make handcrafts. Hierochloe odorata is native to Northern North America and is commonly used as incense and fragrance by Native Americans. It is used traditionally to craft or decorate baskets and bowls (Leif, 2010). In South Carolina, gulfhairawn muhly (Muhlenbergia filipes) is also called Sweetgrass. Its leaves are gathered by the Gullah community, descendants of enslaved Africans, to make a form of coiled basketry. The Gullah basket is now recognized as an artform and a major source of income for the local people (USDA & NRCS, 2009). This native coastal grass on which the basket makers depend has become increasingly scarce due to urbanization and limited access to the resource. Basket makers have to develop social-economical strategies, such as purchasing raw materials from other states, or negotiating access to the grass to maintain the traditional artform and their livelihood (Grabbatin, Hurley, & Halfacre, 2011; Hurley et al., 2013; USDA & NRCS, 2009). There are several types of wild plants in the United States of America called Sweetgrass, that can be used to make handcrafts. Hierochloe odorata is native to Northern North America and is commonly used as incense and fragrance by Native Americans.  Handicrafts It is sometimes used as a medicinal tea, but it is most commonly known in North America for its use as an artist’s canvas of sorts, where burning or carving into the underflesh of a dried polypore leaves behind brown markings to create images (Wetzel, Duchesne, & Laporte, 2006). In this case, while some mycelia live on in the dying or decaying wood medium, polypores take so long to grow that when the fruiting body is harvested, functionally almost the entire the organism is harvested. The long-term sustainability of wild mushroom, wild fungi and wild lichen gathering varies depending on several factors. First, how much of the organism is harvested is paramount. In most cases, it is actually only the fruiting body that is taken, leaving the mycelium behind in its substrate. However, if the fruiting body is harvested before the spores are released the reproductive potential is essentially removed. Despite variation across species and regions in what is harvested and how, there is general agreement that most fungi harvested for crafts purposes are harvested at sustainable levels. Bark is a popular handicraft item. Otomi people in Mexico use barks of Trema micrantha and several Ficus species for handmaking paper crafts. With the color paintings by the Nahua people, Amate bark paper has been traded nationally and internationally. Bark harvesters include indigenous and non-indigenous peoples, often of low-income. From the 149 1980s to 1995, the bark supply increased dramatically from only 4 main harvesters to around 200 people in an area of 1500 km2. As the main source and preferred species of bark paper, Trema micrantha are fast growing, occur within all vegetation and can be harvested throughout the year. The species is recommended for amelioration of degraded lands. It is planted as a shade tree in coffee plantations. When it reaches five to eight years of age it is removed as part of the management of the coffee plantation. With the expansion of the harvest area, including the above factors, this use of bark to make Amate handicrafts is considered to be growing and sustainable (López, 2005). Birch bark is also harvested throughout central North America and northern Europe and used for a variety of handicrafts including baskets and ornaments. According to Emery et al.  Handicrafts (2014), “Paper birch (Betula papyrifera) is a cultural keystone species for the Anishinaabe in the United States of America Great Lakes region” specifically because of its bark. 3.3.2.3.3. Energy As renewable sources of bioenergy, wild plants and fungi have a huge contribution to make to reducing both carbon emissions and energy poverty. Many African countries have high proportions of fuel species. In East Africa, the indigenous tree species Croton megalocarpus supports a sustainable seed oil industry that provides biofuel for electricity. One microenterprise, EcoFuels Kenya, sources more than 3,000 tonnes of wild-harvested nuts each year. Fungi, in particular, have much unexplored potential within the bioenergy sector. Microbial fuel cells can be run on fungal enzymes, such as those from baker’s yeast (Saccharomyces cerevisiae), to generate electricity from plant biomass (Antonelli et al., 2020). Switchgrass (Panicum virgatum) is native to North and Central America, can grow in many different soils, has low fertilizer requirements and can in some cases promote biodiversity depending on the land use being displaced (Cheng & Timilsina, 2011). It can be used as a biofuel source and has potential economic benefits especially in the United States of America. Despite this potential, the environmental consequences of converting to crop grasslands and large land use needs must be addressed (Barney & DiTomaso, 2010; R. A. Brown, Rosenberg, Hays, Easterling, & Mearns, 2000). Switchgrass has been shown to have the potential to decrease soil erosion rates 30 times during the first year of growth, and up to 600 times during the second and third years when the root system has been established (McLaughlin et al., 2002; Williams, Inman, Aden, & Heath, 2009). Werling et al. (2014) found that perennial grasslands that contained switchgrass and prairie plantings have significantly higher biodiversity than maize lands, as arthropods, grassland birds, soil-living methanotrophic bacteria and pollination-insects were found, among others. Two other interesting wild plant species are Miscanthus spp., which is native to Southeast Asia, and Bermudagrass (Cynodon dactylon), native along the United States of America coast. All three grass species are very interesting as biofuel plants, as they grow in the wild but can also be cultivated (Cheng & Timilsina, 2011). The grass genus Miscanthus is among the first crops for which bilateral agreements have been developed under the Convention on Biological Diversity to guide breeding of new varieties from wild germplasm collections from Asia (Antonelli et al., 2020; Grace et al., 2020).  Perfume and incense The sensitivity 150 is higher in outcrop than in meadow habitats. Positive effects are possible with low harvesting levels under strict management conditions (Ghimire, Gimenez, Pradel, McKey, & Aumeeruddy‐Thomas, 2008; Ghimire et al., 2005; Kamini & Raina, 2013; Larsen, 2005). is higher in outcrop than in meadow habitats. Positive effects are possible with low harvesting levels under strict management conditions (Ghimire, Gimenez, Pradel, McKey, & Aumeeruddy‐Thomas, 2008; Ghimire et al., 2005; Kamini & Raina, 2013; Larsen, 2005). is higher in outcrop than in meadow habitats. Positive effects are possible with low harvesting levels under strict management conditions (Ghimire, Gimenez, Pradel, McKey, & Aumeeruddy‐Thomas, 2008; Ghimire et al., 2005; Kamini & Raina, 2013; Larsen, 2005).  Perfume and incense Aromatic plants often have medicinal values and face the same stress and sustainability problems as medicinal plants. Numerous resins are used as incense around the world, either for local use and small-scale trade or for international trade, such as frankincense (Boswellia spp.) or myrrh (Commiphora spp.). Frankincense and myrrh products also have wide ranges of other industrial uses such as for food and beverages, and are used as traditional medicines in China. The first two quality grades of final products are sold in international markets and the least quality graded items are for domestic use like in churches, coffee ceremonies, etc. Tapping and gathering of frankincense is carried out around the dry season. It follows a specific pattern including shaving a thin layer of the bark, the moderate widening of the wound one month later, and then the gathering the gum. An average of 500 g of frankincense is obtained from each tree each season after three to four months of continued tapping (W. Tadesse, Desalegn, & Alia, 2007). Total world export demand is estimated at around 2500-10,000 tons/year with much uncertainty, since the European Union and the United States of America have a broader classification of natural gums and resins in the harmonized system code. The principal exporters are Ethiopia, Kenya, Somalia and Eritrea (Coppen, 2020b; Wubalem Tadesse, Dejene, Zeleke, & Desalegn, 2020). Boswellia papyrifera which is the main source (70% of the Ethiopia's natural gum and resins production) is declining at alarming rates, due to expansion of agricultural lands, overgrazing, population increase, growing demand for construction and fuel wood, forest fires, and pests and diseases. Recent increases in demand of frankincense have also led overharvesting. The lack of traceability in the supply chain and the ineffectiveness of organic certification also affects populations of substitute frankincense species. Studies suggested cultivation and substitution to mitigate the impact and sustain this historical activity (Brendler, Brinckmann, & Schippmann, 2018; S. Johnson et al., 2019; Wubalem Tadesse et al., 2020). The Spikenard, also called Jatamansi, is made from the rhizomes of Nardostachys jatamansi distributed in the Qinhai-tibet Plateau and Himalayas in Asia. It is vulnerable to harvesting and on the verge of extinction due to overexploitation and habitat destruction in some areas. It was evaluated as critically endangered in India but is common in Himalayas of China and Nepal. Sustainability of harvest is related to the harvesting practices. 3.3.2.3.3. Energy Certain natural grasslands are found in some climate zones and it may be beneficial for future biofuel production to come from grassland as the root system in the soil can prevent erosion. Jatropha is a group of non-edible plants found mostly in America that includes 66 species (Dehgan, 1984; Goel, Makkar, Francis, & Becker, 2007). The most common species, Jatropha curcas, is a multipurpose plant species useful to control soil erosion, improve soil infiltration, reclaim wasteland and phytoremediation of contaminated soil, and prepare green manure (Subedi, Chaudhary, Kunwar, Bussmann, & Oaniagua-Zambrana, 2021). The species has a high core nonvolatile oil content, between 25 and 35% (Díaz et al., 2017; R. S. Kumar, Parthiban, Hemalatha, Kalaiselvi, & Rao, 2009), and is the most domesticated species of Jatropha used today. It was created through a combination of systematic selection, inter- 151 hybridization (between J. curcas and J. integerrima) and breeding programs and has a higher oil content (Sujatha & Prabakaran, 2003), but Jatropha is still a wild plant grown as live fence around agricultural fields (Becker & Makkar, 2008; R. S. Kumar et al., 2009) and is regularly used by indigenous people Subedi et al., 2021). The other plant with oil content—Croton megalocarpus— is native to eastern Africa and can have a seed oil content of 30-45% on a mass basis (Aliyu, Agnew, & Douglas, 2010; Hines & Eckman, 1993). Another interesting wild plant rich with oil is the Beauty Leaf Tree (Calophyllum inophyllum), which can carry 10,000 fruits per tree a year and the seeds contain up to 60-70% useful oil (Friday & Okano, 2006; Jahirul et al., 2013). The tree is native to Australia but has been introduced to Southeast Asia and India and started to use as biofuel plant at small-scale (Friday & Okano, 2006). Brock et al. (2018) noted the gold-of-pleasure (Camelina sativa), which is an old-world oilseed crop that went out of use in the mid-20th century but has now gained renewed interest as a biofuel source. There are various studies about wild-living plants and crops and even Yang et al. (2013) have studied possible wild plants for biofuel production and to avoid competition of using of edible plants for food industry. They studied wild plants from salt-alkali wastelands, which often occur in many arid and semi-arid regions of the world. 3.3.2.3.3. Energy They note that “[…] the direct competition with food production should be avoided and a much wider range of plants possible sources of biomass should be made or screened so that they are able to be grown on marginal lands. The non-edible biofuel plant species with fewer inputs, higher tolerant are required so that the diesel plants can be planted in the desert or on the saline-alkali land.” They listed several wild herbaceous plants rich in oil from stems and leaves in China: Euphorbia heyneana (15.01%), Ricinus communis (13.9%), Cirsium setosum (12.5%), Euphorbia nutans (11.02%), Cirsiu japonicum (9.27%), Metaplexis japonica (8.27%), Taraxacum officnala (7.75%), Lactuca raddeana (7.63%), Euphorbia humifusa (6.88%), Euphorbia thymifolia (6.81%), Euphorbia esula (6.57%) and Aster tataricus (5.64%). It is possible to develop a method to extract biofuel from these herbaceous plants and at the same time use the semi-alkali wasteland as possible cultivation land and avoid competition with crops for food production. 3.3.2.3.4. Food and beverage Food consumption is the most common form of use for gathering wild species. Foraging is the oldest productive activity of people, but it keeps being practiced, in rural as in urban environments (Svizzero, 2016). Information on wild species used for food historically came from ethnobiological/ethnobotanical inventories. It is more recently increasing in the scientific literature due to renewed interest in gathering and sustainable use. The most important sources of human food are almost all vascular plants (flowering plants, conifers and other gymnosperms, ferns, horsetails and clubmosses), accounting for 7,014 species of the 7,039 included in the reviews cited. The remainder are bryophytes (mosses, liverworts and hornworts), and green and red algae (Antonelli et al., 2020; Ulian et al., 2020). In agricultural and forager communities in Asian and African countries, the mean use of wild foods is 90-100 species per location, and in indigenous communities there are an estimated 120 wild species used as food in communities in both industrialized and developing countries (Bharucha & Pretty, 2010). 152 With economic and social development, the acquisition of wild food through gathering has been gradually marginalized. In some places, the harvest and consumption of wild foods is considered antiquated behavior and may even be denigrated and abandoned (Garcia, 2006; Łuczaj et al., 2012). For example, islands of Western Oceania are particularly rich in native fruit and nut trees; in Vanuatu, out of 40 of these native species, 30 are not cultivated; they used to play an important part in local diets but presently are often substituted by industrial food (Walter & Sam, 1999). In places where gathering persists, it has been suggested that some people consider it an optimal alternative to farming. This may include trading foraged goods with farmers. This is recognized to be the case in places where gatherers refrain from practicing agriculture for cultural, social, or institutional reasons. (C. Tisdell & Svizzero, 2015). Nevertheless, it is now valued again in some countries as health food and in haute cuisine (Łuczaj et al., 2012; Doyon, 2019). There is also a growing demand for wild plants in the food and aromatics industry (Lescure et al., 2015). In addition to being a food source, evidence shows that for some indigenous peoples and local communities, during times of food shortage wild foods provide nutritional supplements of important vitamins and minerals (Harris & Mohammed, 2003). This finding extends to urban dwellers in developed countries. 3.3.2.3.4. Food and beverage Gathering wild foods sustains dietary traditions and supports community livelihoods. Trends in consumption of wild foods in Europe vary according to regions and countries, and according to categories of species. One study found that across European Union countries at least 27 species of mushrooms and 81 species of vascular plants are harvested and consumed as wild food (Schulp, Thuiller, & Verburg, 2014). Gathering for food is not a static process; some wild plants are consistently gathered, others are forgotten or re-emerge after periods of unpopularity (Łuczaj et al., 2012). Scientific studies have focused on the analysis of dietary conditions related to human health, such as the nutritional content of wild species, toxic side effects, heavy metal concentration (mainly fungi) and health risk assessment. Recorded indigenous and local knowledge combined with scientific analysis, is promoting new resources for crop development, the protection of crop wild relatives, and the provision of new solutions or ideas to address global hunger and protein sources. Because the number of wild plants (and fungi) gathered for food is so extensive, we have further divided this section into sub-sections. Wild fruits are important source of nutrition, medicine, materials for cosmetics, crafts, fiber, and fuel and are the most widely used wild algae, fungi and plants. Clement (2006) distinguishes three types of fruits: (i) nuts and seeds, which contain oil and are rich in proteins and so can play an important part in the diet, (ii) starchy fruits rich in oil and starch (such as palm fruits) and (iii) juicy fruits, such as berries, rich in vitamins. In the United States of America alone, permitted harvest volumes of edible fruits, nuts, and berries were as follows: 303, 748 gallons and 670,726 pounds ( Chamberlain, Emery, & Patel-Weynand, 2018). While these figures represent the best available data, they likely do not represent total harvest of popular species black walnut (Juglans negra L.), pine nuts, and low-bush blueberries. A recent literature review on wild edible fruits found that studies have increased over the last three decades, a majority of it reports ethnobotanical and taxonomic descriptions with relatively few studies on their landscape ecology, economics, and conservation. Among them, a third of retrieved articles were based on studies in Africa and a quarter were from South America. (Sardeshpande & Shackleton, 2019). 153 Different fruit species respond differently to harvesting and other disturbances, such as fire and herbivory. 3.3.2.3.4. Food and beverage Although the review by Stanley et al. (2012) concludes that the majority of case studies surmise that wild algae, fungi and plants harvests are ecologically sustainable, Sardeshpande and Shackleton (2019) found 14 of the 25 studies explicitly addressing harvest sustainability illustrated overexploitation beyond recovery to optimal vitality. In some cases, extraction in a commercial scale is the attempt to make benefits to avoid tree logging and deforestation, such as the marula (Sclerocarya birrea) fruit, the Brazil nut (Bertholletia excelsa, Lecythidaceae) and the bush mango (Irvingia gabonensis Baill. ex Lanen.). When harvest is lethal to the plant or market demand is high which drives to intensive production, the species of wild edible fruits is domesticated and cultivated as tree crops. Certification is considered to ensure the sustainability of gathering under the influence of the trade chain and to promote socio-economic conditions for harvesters and forest communities (Sardeshpande & Shackleton, 2019). The following examples collate several species of wild edible fruits are a complement to the aforementioned review that are mainly gathered from the wild and also support a certain scale of trade in a medium term.  Nuts and Seeds In the United States of America, pine nuts (Pinus monophyla Torr. & Frém.) are highly prized nuts harvested primarily from natural stands on public lands in the western half of the country. These forests are usually not actively managed for pine nut production and in fact are a forest complex (pinyon-juniper) that was historically seen as without much value and eradicated in favor of range lands. However, pine nuts have a long history of use among indigenous peoples and local communities in the southwestern United States of America, where pine nuts continue to be harvested for local markets and for export. While the United States of America exported approximately 20,000 United States Dollars worth of pine nuts in 2007, it imported about 54 million United States Dollars worth (Chamberlain, Emery, & Patel-Weynand, 2018), suggesting that the majority of pine nuts harvested are for personal and local use. Also in the United States of America, approximately 25 million pounds of black walnut (Juglans nigra L.) were harvested from natural populations in 1998, although it is unknown if this constitutes a sustainable harvest amount (Chamberlain, Emery, & Patel-Weynand, 2018) . The Brazil nut tree is an iconic tree occurring in terra firme (non‐flooded) forests throughout the Amazon basin. It can reach up to 50 meters tall and live for hundreds of years. Brazil nut seed harvesting from natural forests is a cornerstone algae, fungi and plants economies in Amazonia. Brazil nuts are the only globally traded seed gathered from the wild by tens of thousands of rural households and are an integral component of the extractivist culture of many indigenous peoples and local communities in the area. In the Brazilian Amazon alone, over 45,000 tons of Brazil nuts are gathered annually, with sales of over 33 million United States dollars (Guariguata, Cronkleton, Duchelle, & Zuidema, 2017; Peres et al., 2003; Wadt, Kainer, Staudhammer, & Serrano, 2008). Brazil nut is organized in a concession system and the supply chain includes three certification schemes: (i) organic certification; (ii) Fairtrade certification; and (iii) Forest Stewardship Council certification. It is considered a model of the use of wild species for promoting “conservation-through-use”. Extensive research suggests the Brazil nut tree reacts 154 robustly to the type and level of extraction currently practiced in the medium term (Guariguata et al., 2017).  Starchy Fruits  Starchy Fruits At least 30 Amazonian palm species are used as food, most of them for their fruits (Attalea spp., Euterpe spp., Mauritia flexuosa, Oenocarpus ssp.), consumed raw, cooked or processed into drinks (Kahn, 1997). Oenocarpus bataua is the seventh most abundant tree in the Amazon and one of the most used palms in neotropical forests in the Americas. Once, felling adult palms was the most common technique used to harvest fruits, which negatively affected the demography of its population. Inconsistent regulations on O. bataua harvesting across different countries contributes to confusion and threatens sustainable use of this species. Colombia has a harvest quota; Ecuador requires management plans; Peru and Bolivia forbid killing the tree. However, in all cases enforcement is difficult. To support sustainable use, in some villages, adult palms are climbed when they are not too tall to cut racemes with ripe fruits, and such non-destructive harvest techniques may meet the increasing demand and maintain the populations. Pequi (Caryocar brasiliense) is a native fruit from Brazil, found in the Amazon, Caatinga, Cerrado, and Atlantic rainforest regions, and has high potential for sustainable use (Guedes, Antoniassi, & de Faria-Machado, 2017). Pequi was harvested in 265 municipalities in the Cerrado ecoregion, which produced approximately 76 thousand tons. Finally, 42 thousand tons of pequi were harvested from 2012 to 2017.  Nuts and Seeds Given the importance of this species to the local economy, this assessment highlights some specific concerns that may affect future status and trends of Brazil nut production. Without active management, in the past extensive and intensive exploitation led to insufficient juvenile recruitment to maintain populations, and harvested populations went into a process of senescence and demographic collapse. Rainfall is also a key factor in determining tree performance and demography and the forecasted declines in pollinator diversity may threaten the long‐term resilience of the Brazil nut trees. Climate change therefore could potentially negatively impact B. excelsa populations (Peres et al., 2003; Thomas et al., 2017). Changes in human use of the forested landscape are also an immediate concern. Brazil nut extraction is accompanied by unsustainable forestry activities outside the gathering seasons in a given year. Due to development pressures, Brazil nut forests have been gradually destroyed and transformed into market-oriented agricultural areas to support global beef markets. Land conversion in the basin has also sparked violent conflicts and led to decreased sustainable management of Brazil nut producing areas. Some of these challenges are being addressed in Brazil, Bolivia and Peru (Bertwell, Kainer, Cropper Jr, Staudhammer, & de Oliveira Wadt, 2018; Escobal & Aldana, 2003; C. S. Simmons et al., 2019; Wadt et al., 2008).  Juicy Fruits Berries and juicy tree fruits are harvested all over the world for personal, informal economic and formal economic use. In the United States of America people commonly harvest wild low- bush blueberries, wild raspberries, wild strawberries, and less commonly serviceberries (Amelanchier spp.), chokecherries (Prunus virginiana), and other species of wild cherry (Chamberlain, Emery, & Patel-Weynand, 2018). 155 Lingonberry or cowberry (Vaccinium vitis-idaea) is one of the most popular berries in American and European Nordic countries, and it is widely used in the human diet. It is a perennial evergreen shrub distributed in circumboreal regions of northern Eurasia and North America. Lingonberries are most commonly harvested by hand with berry rakes. Lingonberry is an important element of coniferous forests understories in terms of nature’s contributions to people, and it also has cultural and economic importance, linked to a rural lifestyle. Major lingonberry-exporting countries are Sweden, Finland, and the Russian Federation (Padmanabhan, Correa-Betanzo, & Paliyath, 2016; Pouta, Sievänen, & Neuvonen, 2006; Woziwoda, Dyderski, & Jagodziński, 2020). A set of criteria and indicators were involved in assessing the commercial supply chain of bilberry in Finland, and suggested a lack of social sustainability due to decreasing involvement and consultations with forest owners and the local communities (Hamunen, Kurttila, Miina, Peltola, & Tikkanen, 2019). Lingonberries are most commonly harvested by hand with berry rakes. In Finland, 11- 26 million kg of bilberries and lingonberries were gathered in the 1990s. It is estimated that over half of the population still participates in berry picking based on the Nordic allemannsretten or “everyman’s right”, which is a long-standing right to move through and share resources on both private and public lands, including the right to pick berries and mushrooms in communal areas. Some estimates suggest utilization rates of the two most common berries, bilberry (Vaccinium myrtillus L.) and lingonberry are low (4–15% of the total annual yield of wild berries), making this a very sustainable activity. One study found that approximately 32% of the total harvested of berries were for commercial sale (Turtiainen, Salo, & Saastamoinen, 2011). However, the demand for so called “super foods” has accelerated exports for global markets, and the volume of the Nordic wild berry harvest has doubled during the past two decades.  Juicy Fruits The Saguaro cactus (Carnegiea gigantea) grows in desert of Arizona, California, and Mexico, and can reach up to height of 15 meters (50 feet) with a life span of about 200 years (Yetman et al., 2020). The fruits are harvested by O’odham Indians, who cook the pulp to make jam, candy, syrup, and wine, but the wild plant also plays an important role in the lives of many other organisms in these environments of which humans are a part. It takes 50 years for this cactus to bear flowers and fruits. The cactus provides shelter and food for numerous organisms throughout its life span. Carpenter birds and elf owls make nests in the fleshy body of the cactus, and Harris’s hawks build nests in the branches. Bats, doves, butterflies and bees enjoy the nectar when the cactus blooms during May. Many animals such as curved bill thrashers, horned lizards, coyotes, and javelin pigs also eat the fruits. As the cactus nears the end of its lifespan, aquatic beetles swim through the decomposing plant flesh. When the cactus is dead, it is home to termites, spiders, giant centipedes, banded geckos, cactus mice, and spotted night snakes. Box 3.8 The many lives of a single plant. Based on (Paye, 2000, p. 142; Yetman et al., 2020, p. 69). Box 3.8 The many lives of a single plant. Based on (Paye, 2000, p. 142; Yetman et al., 2020, p. 69). Box 3.8 The many lives of a single plant. Based on (Paye, 2000, p. 142; Yetman et al., 2020, p. 69). The Saguaro cactus (Carnegiea gigantea) grows in desert of Arizona, California, and Mexico, and can reach up to height of 15 meters (50 feet) with a life span of about 200 years (Yetman et al., 2020). The fruits are harvested by O’odham Indians, who cook the pulp to make jam, candy, syrup, and wine, but the wild plant also plays an important role in the lives of many other organisms in these environments of which humans are a part. It takes 50 years for this cactus to bear flowers and fruits. The cactus provides shelter and food for numerous organisms throughout its life span. Carpenter birds and elf owls make nests in the fleshy body of the cactus, and Harris’s hawks build nests in the branches. Bats, doves, butterflies and bees enjoy the nectar when the cactus blooms during May.  Juicy Fruits Many animals such as curved bill thrashers, horned lizards, coyotes, and javelin pigs also eat the fruits. As the cactus nears the end of its lifespan, aquatic beetles swim through the decomposing plant flesh. When the cactus is dead, it is home to termites, spiders, giant centipedes, banded geckos, cactus mice, and spotted night snakes.  Juicy Fruits Along with an increase in the market demand, lingonberry has been domesticated and commercially cultivated in several locations across Europe, Scandinavia, and also recently in the United States of America (Forest Europe, 2020; Padmanabhan et al., 2016). The land area covered in bilberry (Vaccinium myrtillus) bushes and locally harvested berries have declined in Central Italian Apennine regions in recent last decades (Nin, Petrucci, Del Bubba, Ancillotti, & Giordani, 2017). Regular gatherers of bilberry in Estonia use clearly delineated picking areas, and typically do not share their areas outside close family relations (Remm, Runkla, & Lohmus, 2018). Bilberries are also a popular wild food in the Czech Republic, where the number of households involved in the gathering of wild fruits has increased in recent years. The ratio of participants and yield of bilberries are the highest in wild fruit (Wolfslehner et al., 2019). There is also a high demand on bilberries in France. The boom started in the late 1960s. At that time some gatherers in the Massif Central area increased the quantity of berries they were gathering by 500%. This gathering is regulated for non-residents (Larrère, 1982). Most cacti produce edible fruit for humans but prickly pears of Opuntia species and fruits from Stenocereus, Cereus, Carnegiea and Pachycereus species are the most important for numerous peoples of the arid Americas (Box 3.8). Fruits are gathered from the wild, semi- cultivated and cultivated stands. People commonly make use of a tool called “chicole” which is a long stick with a kind of basket in the top. With “chicole” people reach and pull fruits without damaging them or injuring themselves or the wild plant. The fruits harvested are stored 156 in a basket or bucket for transporting them to homes and markets. In agroforestry systems, people sometimes leave fruit-producing cacti because they favor their propagation and take special care of these valued plants. Some species, mainly Stenocereus, Cereus, Lemairocereus, are cultivated, and processes of domestication and generation of varieties associated to human selection have been documented in Mexico (Casas & Barbera, 2002; Casas, Otero-Arnaiz, Pérez-Negrón, & Valiente-Banuet, 2007). Box 3.8 The many lives of a single plant. Based on (Paye, 2000, p. 142; Yetman et al., 2020, p. 69).  Beverages Mead, Hyssop, Salep, teas, and wild coffees from dandelion greens and chicory are some of the many beverages people make from wild plants. The English term “tea” refers the infusion made of the leaves of Camellia sinensis but there are kinds of aromatic and refreshing beverages around the world. In Europe, 142 taxa of plants belonging to 99 genera and 40 families are reported the use of recreational tea (Sõukand et al., 2013). In China, 759 plant species have documented for use as teas, and a market survey identified an additional 23 species used as herbal tea (Fu et al., 2018). The majority of wild plants used are perceived as medicinal plants in local folk medicine or “folk functional foods”. The status of the use of herbal tea is dependent on access to the natural resources, cultural and social contexts, and the habit of its use in the region and personal preferences of the consumer. Salep is a beverage made from orchid tubers in Europe and central Asia. Harvesting wild orchid tubers for this purpose dates back to the medieval period. Six species of orchids are named as components of Salep. Tuber gathering for Salep has been cited as a cause of orchid population decline and causes conservation concern in Turkey and neighboring countries (Charitonidou, Stara, Kougioumoutzis, & Halley, 2019; Ghorbani, Gravendeel, Naghibi, & de Boer, 2014; Kreziou, de Boer, & Gravendeel, 2016; Masters, van Andel, de Boer, Heijungs, & Gravendeel, 2020). Scientists and conservationists recommend cessation of wild orchids harvest for this purpose (Ghorbani et al., 2014). 157  Syrups, Gums and Resins Indigenous tribes in Eastern North America know the sap of maples (Acer spp.) and call it “sweet water.” When the first European explorers and colonists arrived, they learned of maple sap and boiling the sap down to produce syrup or sugar. Sugar maple (Acer saccharum) is the species most frequently tapped for sap production. Under the best conditions, sugar maples reach a tappable size in about 40 years and can continue to produce sap for a century (Ciesla, 2002). During the maple sugaring season, which lasts about six weeks in spring, an average maple tree will yield between 35 and 50 liters of sap, which will produce between 1 and 1.5 liter of pure maple syrup (Ciesla, 2002). Maple syrup is produced only in the Eastern United States of America and Canada. Maple syrup production is a hobby that connects people to nature, provides supplementary income for farmers, and is an important cultural practice for indigenous peoples (Weiss et al., 2019). As a large-scale commodity, maple syrup is a luxury item consumed worldwide (Figure 3.41). The largest market for syrup is in the United States of America. Since the late 19th century, maple production in the United States of America has declined while that in Canada has increased. With sugar maple (Acer saccharum Marshall) often distributed throughout the region’s forests, only a small percentage of potentially tappable trees are in use for maple syrup production (an estimated 0.4% in the United States of America; Ciesla 2002, Farrell and Chabot 2012). Maple syrup production is weather dependent and expected to be heavily affected by climate change, with the potential for it to be eliminated in southern reaches of its current distribution peaked in the 19th century, reaching a record 25,032,928 liters of maple syrup in 1860. (Iverson & Matthews, 2018). 158 158 Figure 3.41 Maple syrup production in the United States of America and Canada 1860- 2010. Source: (Farrell & Chabot, 2012) under license CC BY 4.0. Figure 3.41 Maple syrup production in the United States of America and Canada 1860- 2010. Source: (Farrell & Chabot, 2012) under license CC BY 4.0. Figure 3.41 Maple syrup production in the United States of America and Canada 1860- 2010. Source: (Farrell & Chabot, 2012) under license CC BY 4.0. In Europe, the main sources of tree sap are silver and downy birch trees (Betula pendula Roth and Betula pubescens Ehrh).  Syrups, Gums and Resins Birch sap is colorless or slightly opalescent. It is used as a traditional drink, in traditional medicine, in veterinary medicine and as a cosmetic product. Gathering sap from birch and other trees was more widespread in earlier times. In Russia, Ukraine, Belarus, Estonia, Latvia and Lithuania it remains a more common practice. The most productive silver birch trees for sap gathering are those taller than 28 m. A birch tree can produce 36l gallons of sap in nine days. Experiments conducted in Estonia in the 1970s showed that the profit gained from the sap was six times the profit gained from timber. More recently birch sap is becoming a more commercial product, and is of interest to pharmaceutical companies (Grabek-Lejko, Kasprzyk, Zagu\la, & Puchalski, 2017; Mingaila et al., 2020; Svanberg et al., 2012). Gum Arabic or acacia gum is a tree gum exudate gathered from a number of Acacia species and has been an important part of commerce since ancient times. Gum Arabic is used in food and drink industries, in pharmaceuticals and in printing and textile industries as thickening, stabilizing, binding and sizing agents. Gum resin products are harvested from natural exudates by herdsmen, women and children while herding and doing other activities. Yields of gum Arabic from individual trees are very variable. A tree yields an average of 250g of gum per season. Very small proportions of gum enter the local market, but some is directly sold as a road-side snack in some West African countries, in Niger for example (E.S. Barron 159 personal observation). African countries export about 100,000 tons of gum Arabic annually, and demand was previously projected to reach 150,000 tons by 2020. The European Union is responsible for 80% of global trade in gum Arabic, worth around 125 million euros. Sudan is one of the biggest gum Arabic producers in the world and produces more than 80% of the total world gum Arabic (Wubalem Tadesse et al., 2020; B. Wolfslehner et al., 2019) (Table 3.9). Table 3.9 Exports of gum Arabic (tons) from different African countries 2001-2010. Source: (Wubalem Tadesse et al., 2020) under license CC-BY 4.0.  Syrups, Gums and Resins Year Country 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 Sudan 7949 34382 13217 27444 33079 23149 n/a 37860 36636 48598 Nigeria 0 0 0 n/a n/a 1314 14463 14124 40862 34780 Chad 12891 9161 9672 12044 14188 17816 11860 16219 9417 9509 Ethiopia 830 875 381 234 111 317 956 614 622 909 Tanzania 843 693 1252 1361 1169 965 1031 935 631 824 Cameroon 571 592 338 264 371 413 310 151 520 510 Senegal 121 0 0 213 323 475 610 836 935 330 Mali 482 750 704 52 28 17 29 1308 703 275 BurkinaFaso 2 0 21 18 81 n/a 90 57 63 83 Kenya 23 0 92 23 32 28 75 165 41 75 Eritrea n/a n/a 116 49 495 38 688 419 350 51 Somalia 26 12 4 70 714 92 473 513 50 47 Niger 2 20 38 43 42 73 67 66 44 44 Table 3.9 Exports of gum Arabic (tons) from different African countries 2001-2010. Source: (Wubalem Tadesse et al., 2020) under license CC-BY 4.0. e 3.9 Exports of gum Arabic (tons) from different African countries 2001-2010 ce: (Wubalem Tadesse et al., 2020) under license CC-BY 4.0. Karaya gum is produced as an exudate from the genus Sterculia including Sterculia urens tree found in India and Sterculia setigera found in Africa and is used for many industries. World demand for karaya gum is about 7,000 tons, and Senegal is the leading exporter in Africa. The population of karaya trees once markedly declined due to crude traditional tapping methods which lead to the death of the tapped trees and over exploitation. Scientific tapping and proper harvesting methods are now priorities (Nair, 2004; Wubalem Tadesse et al., 2020).  Wild edible mushrooms More than 350 species coming from 18 orders of fungi are commonly eaten as food (Willis, 2018). The number of used wild edible mushrooms is likely much higher than that based on lists and assessments from individual countries, e.g., over 1000 species of edible mushrooms are listed in China (Wu et al., 2019), 371 in Mexico (Moreno Fuentes, 2014) and 268 species are traded in Europe (Peintner et al., 2013). The last comprehensive global assessment was conducted in 2004 (Boa, 2004), and given the high rates of taxonomic discovery among fungi, including of useful species (Dentinger & Suz, 2014; Willis, 2018; F. Wu et al., 2019), a re- evaluation is overdue. Wild mushrooms are harvested for food in over 80 countries worldwide (Pieroni, Nebel, Santoro, & Heinrick, 2005a). Among wild-harvested fungi, most commonly 160 consumed and traded are Chanterelles (Cantharellus spp.), Porcini (Boletus spp.). Truffles (Tuber spp.) Morels (Morchella spp.), Brittlegills (Russula spp.), Milkcaps (Lactarius spp.), Button mushroom (Agaricus spp.), and Matsutake (Tricoloma spp.). Wild edible mushrooms can be found in over 200 genera, and grow in a wide variety of habitats (Boa, 2004). Many of the most popular used species form symbiotic relationships, making them difficult if not impossible to cultivate. For example, all of the above-listed genera (with the exception of button mushrooms) form ectomycorrhizal symbioses with trees, while Termitomyces spp. which are widely consumed across Africa and Asia are symbionts of termites (Boa, 2004). Popular saprotrophic species include button mushrooms, straw mushrooms (Volvariella spp.), shitake (Letinula edodes) and oyster mushrooms (Pleurotus spp.), although these species are cultivated at large scale (Boa, 2004), they are also frequently harvested in the wild, for example in Malaysia (Fui, Saikim, Kulip, & Seelan, 2018), Benin (Codjia & Yorou, 2014), Mexico (Haro-Luna, Ruan-Soto, & Guzmán-Dávalos, 2019) or Italy (Pieroni, Nebel, Santoro, & Heinrick, 2005b). To assess status and trends of wild useful fungi, literature searches were conducted via a variety of search engines (Google Scholar, EBSCO Host and SCOPUS). To this end a Google Scholar search with the terms “(gathering OR collecting OR picking OR hunting OR foraging) AND (mushroom OR lichen OR fungi) AND sustainable AND wild” served as the basis and variations in the combinations of these terms, as well as supplementation with the different use categories (e.g., ceremonial, medicinal, food) were used until 50% saturation of articles already in the database were reached.  Wild edible mushrooms In total, 112 sources were reviewed (see the data management report for Chapter 3 systematic literature review for the gathering of fungi at https://doi.org/10.5281/zenodo.4659811). The extent of usage of different species varies widely. Typically, ethnomycological studies report the use of tens to hundreds of species where a majority is harvested for personal use, gifting or barter (based on 20 articles from the literature review). A smaller number of popular species is sold at local and regional markets, while select species, often global commodities, are sold on to middlemen and traders to enter national and international markets (based on 9 articles from the literature review). This phenomenon is particularly well- documented in Mexico. For example, the Mazahua people use 31 species of wild mushrooms, of which 18 are sold in local or regional markets (Farfán, Casas, Ibarra-Manríquez, & Pérez- Negrón, 2007). The less popular species are also sometimes sold in mixed species bags, while a handful of highly-prized species including Amanita caesarea complex, porcini, morels, chanterelles and matsutake are targeted for export (Montoya, Hernández, Mapes, Kong, & Estrada–Torres, 2008; Pérez-Moreno, Martínez-Reyes, Yescas-Pérez, Delgado-Alvarado, & Xoconostle-Cázares, 2008). A similar imbalance in usage among taxa also exists at larger geographical scales as indicated by a comparison among European guidelines and legislations, where on lists from 24 countries with an average length of 55 taxa and a total of 268, only two taxa were listed in all countries: porcinis (Boletus edulis complex) and chanterelles (Cantharellus cibarius). A further five (Lactarius deliciosus, Morchella esculenta, Boletus badius, Agaricus campestris and Craterellus cornucopioides) were listed in more than 70% of countries, while 134 (about 50%) were listed in only one or two countries (Peintner et al., 2013). Finally, species preferences and use may shift over time as is highlighted by Russula virescens which was highly appreciated in the Southwest of France in the 18th century but is 161 no longer consumed nowadays, while the chanterelle increased in popularity in this region (Duhart, 2012). The use and appreciation of different mushroom species is deeply cultural, and whether a species is used and what for is often due to a multitude of factors, including language, geography, cultural and culinary traditions (Comandini & Rinaldi, 2020).  Wild edible mushrooms For example, regions in Europe with similar occurrence of mushroom species (e.g., Southeast Europe versus Southwest Europe, or Eastern Europe versus the nordic countries) favor different species and the use of species is more strongly influenced by local tastes, traditions and commerce with neighbors than climatic variables or vegetation (Peintner et al., 2013). In line with this, usage frequently reflects cultural interactions, for example in Finland, gatherers in Eastern parts of the country with stronger cultural influence from Russia prefer milk caps (Lactarius spp.), while those in Southwestern regions where French cuisine permeated through Swedish influence prefer porcinis and chanterelles (Comandini & Rinaldi, 2020). Immigrant populations often bring culinary traditions and preferences to their new homes, nicely illustrated in the Western United States of America, where a culture and tradition of gathering, along with different species preferences, was established by early immigrants from Europe, Asia and Russia (Arora, 2008a; Parks & Schmitt, 1997). Another salient example illustrating, fine-grained, context dependence of use are the false morels (Gyromitra esculenta), which are consumed at quantity in Finland (Turtiainen, Saastamoinen, Kangas, & Vaara, 2012), and Gyromitra infula, which is harvested both in Nepal (M. Christensen, Bhattarai, Devkota, & Larsen, 2008) and Mexico (Pérez-Moreno et al., 2008). These species are largely considered toxic and safe consumption rests on the knowledge of correct preparation (Peintner et al., 2013), highlighting the importance of indigenous and local knowledge in shaping use of individual species. The trade of edible fungi has been valued at 42 billion United States dollars in 2018 (Willis, 2018). However, this estimate includes mostly cultivable species and only two (porcinis and morels) out of the nine species evaluated are exclusively gathered in the wild, while other economically important wild taxa such as truffles, chanterelles and matsutake are omitted. Data on trade volumes also is often aggregated at higher levels that include both taxa from cultivation and wild gathering. For example (de Frutos, 2020) estimated international trade for edible fungi at 1.2 million tons for 2017 based on United Nations Comtrade data (https://comtrade.un.org/), using harmonized customs codes that include all species, except the genus Agaricus. Agaricus spp. constitute approximately 30% of the cultivated mushroom trade volume, so this figure is likely still influenced by the other four taxa cultivated at large scale [Pleurotus, Lentinula, Auricularia and Flammulina; (Royse, 2014)].  Wild edible mushrooms FAOSTAT aggregates data for all fungi into a “mushrooms and truffles” category, yielding a production of 10.9 million tons for 2017 (http://www.fao.org/faostat/en/#data/QC; Accessed 27.02.2021). Comparison with the international trade figure suggests that as much as 90% of trade volume may be based on cultivated fungi, although again it is unclear what proportion can be attributed to wild species. The FAO estimates also include Agaricus data and other cultivated species. The heterogeneity in taxonomic granularity of data accumulation and aggregated reporting for both cultivated and wild species makes it challenging to produce meaningful estimates of production and trade volumes of wild edible fungi. This problem constitutes an active area of work within the FAO, as reflected by the introduction of new harmonized system codes for 162 widely traded wild plants algae and fungi coming into effect in January 2022 (World Customs Organization, 2019). Nevertheless, a body of literature focusing on specific regions or target species clearly highlights the economic importance and development potential of wild mushroom trade, especially for rural areas. Our literature review yielded 24 studies that highlight a contribution of gathering and selling wild fungi to incomes of rural populations worldwide (3 Africa, 5 Americas, 8 Europe and Central Asia, 7 Asia Pacific). China, and Yunnan in particular, provides an excellent example of how the gathering of wild edible fungi can fuel economic development in rural areas. Yunnan harbors a large diversity of edible fungi and is the center of the wild edible mushroom industry in China (R. Hua, Chen, & Fu, 2017; Dongyang Liu et al., 2018). Especially in the more remote areas of Yunnan, the contribution from gathering of wild fungi can reach up to 90% of annual household income (Arora, 2008b; R. Hua et al., 2017; Huber, Ineichen, Yang, & Weckerle, 2010). In Nanhua county alone, the yearly production of wild fungi amounted to 7677 tons, valued at 80 million United States dollars (Dongyang Liu et al., 2018). In 2015, the total yield of wild edible fungi for the whole province amounted to 0.17 million tons, with Yunnan being a major supplier of porcini (Boletus spp.) which are primarily exported to Europe and matsutake (Tricholoma matsutake) which are exported to Japan (R. Hua et al., 2017; Huber et al., 2010).  Wild edible mushrooms Although often considered the “meat of the poor” or emergency foods that can cover protein nutritional needs (Christensen et al., 2008; Guissou, Lykke, Sankara, & Guinko, 2008; Oyetayo, 2011; Redzic, Barudanovic, & Pilipovic, 2010), this view diminishes the cultural importance of wild edible fungi. In some communities in Mexico, for example, mushrooms are considered delicacies with great flavor that are superior to meat (Farfán-Heredia et al., 2018; Haro-Luna et al., 2019). The strong appreciation and deep cultural traditions associated with gathering and consumption of fungi are reflected in the fact that many papers explicitly mention recreation, social bonding and stress release as a major reason why people gathered wild mushrooms (9 sources). Gifting and exchange of gathered fungi or products prepared from them among friends, family and members of the community were also mentioned several times (Garibay-Orijel, Cifuentes, & Estrada-Torres, 2006; Haro-Luna et al., 2019; Pieroni et al., 2005b). Gathering and eating wild foods and engaging in culinary traditions provides a sense of place, identity and a connection with nature that is celebrated at festivals, e.g., in Spain (Fusté- Forné, 2019) or China (Dongyang Liu et al., 2018) and has developed into a sizeable foraging tourism industry worth 800,000 euros per year in Spain (Fusté-Forné, 2019). Finally, a study comparing rural populations in Sweden, Ukraine and Russia showed that a high proportion of people engaged in gathering, irrespective of economic status (Stryamets, Elbakidze, Ceuterick, Angelstam, & Axelsson, 2015). Instead, the importance of commercial harvesting to supplement income increased or decreased inversely proportional to the standard of living and employment opportunities, highlighting that the social importance of gathering wild fungi may often be masked by economic necessity and reasons for gathering can shift over time. In the International Union for Conservation of Nature Red list, 116 of 545 species of evaluated Fungi are used as human food, and 16 species of edible fungi are evaluated as threatened. With the exception of Africa, the distribution of the species assessed is relatively balanced in the other three IPBES regions (IUCN, 2020b) (Figure 3.42; Figure 3.43; Table 3.10). Less than 14% of edible fungi are threatened, which is lower compared to the overall level of the species assessed by the International Union for Conservation of Nature red lists (28%). However, due to the limited number of fungi assessed, the figure may not be representative of the global status.  Wild edible mushrooms In all papers surveyed, gathering wild fungi was a supplemental activity to other forms of subsistence, primarily agriculture due to the seasonality of mushroom fruiting and year to year fluctuations in abundance and price. However, due to the highly perishable nature of the product that requires fast processing, the establishment of mushroom supply chains has led to lasting economic diversification in rural areas with the involvement of middlemen, mushroom traders and processing facilities (Arora, 2008b; Huber et al., 2010; Dongyang Liu et al., 2018). In Shangri-la, Diqing Tibetan Autonomous Prefecture, matsutake are often bought and sold several times before leaving the city, spreading the income not only to middlemen, who often do not have access to matsutake habitats themselves, but also to shops, restaurants and other facilities that were established near the mushroom markets (Arora, 2008b). In Mexico mushrooms are also sold to traders and middlemen, although here there was a greater emphasis on sale at local and regional markets for generation of income (Farfán-Heredia, Casas, Moreno- Calles, García-Frapolli, & Castilleja, 2018; Pérez-Moreno et al., 2008). None of the papers reviewed mentioned commercial export of fungi from Africa, but highlighted informal, local sale as the main form of generating income (e.g., Osarenkhoe, John, & Theophilus, 2014; Wendiro, Wacoo, & Wise, 2019; Yorou et al., 2014). However, small scale export of porcini to Italy and the United States of America, primarily from Southern Africa, were indicated based on personal communication (Boa, 2004; Sitta & Floriani, 2008). Besides direct contributions to household income, wild mushrooms provide a rich source of protein and can help to bridge periods of food scarcity which often fall into the rainy season, e.g., in Ethiopia (Dejene, Oria-de-Rueda, & Martín-Pinto, 2017), West Africa (Yorou et al., 2014) and Mexico (Farfán et al., 2007). In addition to the 27 sources mentioned above, a further ten studies were found where wild mushrooms were important contributors to a healthy diet and subsistence of people in economically marginalized positions. The use of wild mushrooms was also reported among several indigenous groups in the Amazon, most prominently the Jotï (Zent, Zent, & Iturriaga, 163 2004; Zent, 2008) and the Yanomami people (Fidalgo & Prance, 1976; Sanuma et al., 2016). Recently, the Yanomami in Brazil started trading some mushrooms as a niche market (Sanuma et al., 2016).  Wild edible mushrooms With regards to figures 3.42 and 3.43, it is important to note that the majority of fungal conservation related inventory and monitoring has historically been based in Europe, hence the density of data from that region (Barron, 2011). 164 Figure 3.42 The threatened status and threats of all assessed fungal species. At the top, the threatened status of all assessed 545 species and at the bottom, the threats of all assessed 545 species. Source: (IUCN, 2020b) © IUCN Red List Data. This figure was made using the International Union for Conservation of Nature website https://www.iucnredlist.org/search/stats, by selecting “Fungi” in the tab “Taxonomy”. Table 3.10 Distribution of edible fungi assessed by the International Union for Conservation of Nature Red List in each IPBES region. Abbreviations: CR: Critically Figure 3.42 The threatened status and threats of all assessed fungal species. At the top, the threatened status of all assessed 545 species and at the bottom, the threats of all assessed 545 species. Source: (IUCN, 2020b) © IUCN Red List Data. This figure was made using the International Union for Conservation of Nature website https://www.iucnredlist.org/search/stats, by selecting “Fungi” in the tab “Taxonomy”. Figure 3.42 The threatened status and threats of all assessed fungal species. At the top, the threatened status of all assessed 545 species and at the bottom, the threats of all assessed 545 species. Source: (IUCN, 2020b) © IUCN Red List Data. This figure was made using the International Union for Conservation of Nature website https://www.iucnredlist.org/search/stats, by selecting “Fungi” in the tab “Taxonomy”. Figure 3.42 The threatened status and threats of all assessed fungal species. At the top, the threatened status of all assessed 545 species and at the bottom, the threats of all assessed 545 species. Source: (IUCN, 2020b) © IUCN Red List Data. This figure was made using the International Union for Conservation of Nature website https://www.iucnredlist.org/search/stats, by selecting “Fungi” in the tab “Taxonomy”. Table 3.10 Distribution of edible fungi assessed by the International Union for Conservation of Nature Red List in each IPBES region. Abbreviations: CR: Critically Endangered, EN: Endangered, VU: Vulnerable, NT: Near Threatened, LC: Least Concern. Source: (IUCN, 2020b) © IUCN Red List Data. Table 3.10 Distribution of edible fungi assessed by the International Union for Conservation of Nature Red List in each IPBES region. Abbreviations: CR: Critically Endangered, EN: Endangered, VU: Vulnerable, NT: Near Threatened, LC: Least Concern. Table 3.10 Distribution of edible fungi assessed by the International Union for Conservation of Nature Red List in each IPBES region. Abbreviations: CR: Critically Endangered, EN: Endangered, VU: Vulnerable, NT: Near Threatened, LC: Least Concern. Source: (IUCN, 2020b) © IUCN Red List Data. Figure 3.43 The threatened status and threats of edible fungal species. At the top, the threatened status of all assessed 116 species and at the bottom, the threats of all assessed 116 species. Source: (IUCN, 2020b) © IUCN Red List Data. This figure was made using the International Union for Conservation of Nature website  Wild edible mushrooms Source: (IUCN, 2020b) © IUCN Red List Data. 165 166 IUCN status IPBES regions CR EN VU NT LC Total Americas 1 3 3 3 35 45 Asia Pacific 1 5 5 37 48 Africa 1 1 7 9 Europe and central Asia 7 5 36 48 All 1 4 9 5 41 60 Figure 3.43 The threatened status and threats of edible fungal species. At the top, the threatened status of all assessed 116 species and at the bottom, the threats of all assessed 116 species. Source: (IUCN, 2020b) © IUCN Red List Data. This figure was made using the International Union for Conservation of Nature website IUCN status IPBES regions CR EN VU NT LC Total Americas 1 3 3 3 35 45 Asia Pacific 1 5 5 37 48 Africa 1 1 7 9 Europe and central Asia 7 5 36 48 All 1 4 9 5 41 60 IUCN status IPBES regions CR EN VU NT LC Total Americas 1 3 3 3 35 45 Asia Pacific 1 5 5 37 48 Africa 1 1 7 9 Europe and central Asia 7 5 36 48 All 1 4 9 5 41 60 Figure 3.43 The threatened status and threats of edible fungal species. At the top, the threatened status of all assessed 116 species and at the bottom, the threats of all assessed 116 species. Source: (IUCN, 2020b) © IUCN Red List Data. This figure was made using the International Union for Conservation of Nature website Figure 3.43 The threatened status and threats of edible fungal species. At the top, the threatened status of all assessed 116 species and at the bottom, the threats of all assessed 116 species. Source: (IUCN, 2020b) © IUCN Red List Data. This figure was made using the International Union for Conservation of Nature website 166 https://www.iucnredlist.org/search/stats, by selecting “Fungi” in the tab “Taxonomy” and then by selecting “Food-human” in the tab “Use and Trade”. https://www.iucnredlist.org/search/stats, by selecting “Fungi” in the tab “Taxonomy” and then by selecting “Food-human” in the tab “Use and Trade”. https://www.iucnredlist.org/search/stats, by selecting “Fungi” in the tab “Taxonomy” and then by selecting “Food-human” in the tab “Use and Trade”. In a regional assessment, take China as an example, the threatened species list of China’s macrofungi assesses the overall threat status of 9302 species and 1.04% of the total number of species (97 species) is assessed as threatened (Yijian et al., 2020). Among the 97 threatened fungi, there are 13 species used as food, 8 species are medicinal use, and other 8 species are used both for food and medicine (Figure 3.44). Figure 3.44 China threatened fungi used as food and medicine. Based on (Ministry of Ecology and Environment of the People’s Republic of China & Chinese Academy of Sciences, 2018). Abbreviations: EN: Endangered, VU: Vulnerable. See data management report for the figure at https://doi.org/10.5281/zenodo.6453079. 3.44 China threatened fungi used as food and medicine. Based on (Ministry of Figure 3.44 China threatened fungi used as food and medicine. Based on (Ministry of Ecology and Environment of the People’s Republic of China & Chinese Academy of Sciences, 2018). Abbreviations: EN: Endangered, VU: Vulnerable. See data management report for the figure at https://doi.org/10.5281/zenodo.6453079. Based on the literature survey, land use change (10 sources), timber harvesting, deforestation (8 sources) and climate change (8 sources) were listed as the most common ecological threats that likely affect a broad range of edible fungi irrespective of their economic importance. Overharvesting was primarily reported on in the context of species gathered for commercial purposes (8 sources), with a particular focus on matsutake (Martínez Carrera, 2002; J. S. Brooks & Tshering, 2010; Dongyang Liu et al., 2018) and truffles (Garcia-Barreda et al., 2018; Radomir, Mesud, & Žaklina, 2018). Long-term scientific studies monitoring the effect of different harvesting techniques (picking versus cutting) showed no adverse effects of gathering fruitbodies on future production of epigeous (aboveground) fruitbodies using either technique, but instead identified trampling associated with gathering activities as reducing the number of fruitbodies (Egli, Peter, Buser, Stahel, & Ayer, 2006). Another study focused on 167 harvesting techniques of the American matsutake (Tricholoma magnivelare) and also found no adverse effects of gathering on the number and weight of fruitbodies produced when mushrooms were picked using best practice methods (no soil removal, careful plucking of fruitbodies using a small tool) over the course of ten years (Luoma et al., 2006) (Box 3.9). https://www.iucnredlist.org/search/stats, by selecting “Fungi” in the tab “Taxonomy” and then by selecting “Food-human” in the tab “Use and Trade”. Box 3.9 Matsutake and sustainable management Matsutake (Tricholoma matsutake) and the closely-related species T. magnivelare and T. caligatum) are subject to some of the richest literature available with regards to management practices for wild edible fungi (Tsing, Satsuka, & for the Matsutake Worlds Research Group, 2008). Matsutake are highly appreciated in Japan where productivity has been in decline since the 1940s. T. matsutake grows as an ectomycorrhizal symbiont of Japanese red pine, a pioneer species that is commonly found around settlements (Saito & Mitsumata, 2008). For centuries people have been coppicing the satoyama (village forests) to harvest wood for fuel https://www.iucnredlist.org/search/stats, by selecting “Fungi” in the tab “Taxonomy” and then by selecting “Food-human” in the tab “Use and Trade”. However, more disruptive harvesting methods using raking and soil removal resulted in fewer and lighter fruitbodies in the nine years following treatment, especially if soil was not replaced. This is in line with reports by Yi gatherers who expressed concerns about younger gatherers uprooting entire fruitbodies instead of using the more careful traditional gathering techniques (Dongyang Liu et al., 2018). Overall, however, the long-term studies reconcile reports of overharvesting with those where no influence was reported despite commercial scale gathering (Arora, 2008b; Christensen et al., 2008; Huber et al., 2010), indicating that a good balance of commercial development and sustainable use is achievable with appropriate management practices. Species most at risk appear to be those subject to disruptive gathering practices such as matsutake and truffles, both of which are developing belowground, although structured research for a larger variety of species is currently lacking. Indigenous peoples and local communities are both the main sources of knowledge with respect to status and management approaches (7 out of 8 articles reporting overharvesting) and key stakeholders in the use of wild edible fungi. Integrative research articulating local scale indigenous and local knowledge with other sources of knowledge that can incorporate the large year to year fluctuations in fruiting due to climatic variables and the impact of other environmental factors are required to better understand the multidimensional drivers influencing sustainable use. Consequently, the erosion and loss of indigenous and local knowledge also presents a major threat to sustainable use of wild fungi. This was reported in twelve studies reviewed and across all IPBES regions (5 Africa, 2 Americas, 2 Europe and Central Asia and 3 Asia Pacific). Indigenous and local knowledge is usually transmitted orally within families, often while engaging in gathering activities, so factors such as increased urbanization and associated cultural changes can decrease interest in gathering and the opportunity to do so (M. R. Emery & Barron, 2010). In three cases societal changes coincided with decline of wild edible fungi through deforestation and land use change and scarcity was one of the major reasons cited why people did not engage in gathering, e.g., in Burkina Faso (Guissou et al., 2008) or Nigeria (Oyetayo, 2011; Uzoebo et al., 2019). One of the latter also cited social stigmas associated with gathering, which all together can lead to a situation of rapidly declining indigenous and local knowledge. Box 3.9 Matsutake and sustainable management Matsutake (Tricholoma matsutake) and the closely-related species T. magnivelare and T. caligatum) are subject to some of the richest literature available with regards to management practices for wild edible fungi (Tsing, Satsuka, & for the Matsutake Worlds Research Group, 2008). Matsutake are highly appreciated in Japan where productivity has been in decline since the 1940s. T. matsutake grows as an ectomycorrhizal symbiont of Japanese red pine, a pioneer species that is commonly found around settlements (Saito & Mitsumata, 2008). For centuries people have been coppicing the satoyama (village forests) to harvest wood for fuel 168 and other uses which created a favorable habitat for matsutake. A low point in the matsutake production was reached in the 1970s when many households switched to propane gas and oil fuels, which was considered the main reason for the decline in productivity (Saito & Mitsumata, 2008). Due to the high market prices, especially for Japanese matsutake which can reach over 400 United States dollars per kg (2006), research has focused on silvicultural approaches for habitat improvement to increase matsutake yields (Saito & Mitsumata, 2008). In a comparison of different land management practices, the most successful one was rooted in the traditional irai system, where wild algae, fungi and plants are considered a communal resource of the village. This management practice involves joint habitat improvement sessions and days where everyone can gather which not only improved matsutake production, but also provided community-building social activities and ultimately a virtuous cycle where increased matsutake production and the social aspects leads to increased interest in participating in management activities (Saito & Mitsumata, 2008). Similar community- based management practices have proven successful in Shangri-la, Diqing Tibetan Autonomous Prefecture, China where matsutake harvest rights are organized by village and overharvesting and competition between gatherers are mitigated by instituting “rest days” of 3 to 5 consecutive days where gathering is prohibited once the quantity of matsutake sales declines (Arora, 2008b). Although the measure was implemented as a means to maximize profit, it also prevents harvest of very young specimens and may thereby benefit the reproductive potential of the fungus. Conflict among gatherers was further minimized by charging high fees for gathering permits for outsiders. Despite matsutake contributing the majority of household income in the region, there were no concerns voiced about declining numbers of mushrooms (Arora, 2008b). Box 3.9 Matsutake and sustainable management Case studies from the United States of America and Europe highlight policies that are rooted in different philosophies of nature and perspectives on resource management (Tsing et al., 2008). When market demand for wild edible mushrooms increased in the United States of America in the 1980s, restrictive gathering laws were put into place, either requiring permits for gathering, selling and buying mushrooms (Rebecca J. McLain, 2008), or forbidding gathering outright (Arora, 2008a). This led to de facto criminalization of gathering and gatherers that often had a long history of engaging in this activity but were not involved in the process of developing meaningful regulation. Consequences were increased volumes of mushrooms sold via black and grey markets (Arora, 2008a; Parks & Schmitt, 1997) and a reframing of gathering from a family activity as work or an outright illegal activity, threatening transmission of indigenous and local knowledge (Arora, 2008a; Rebecca J. McLain, 2008). Similarly, mushroom gatherers and traders were not included as stakeholders in the development of the Forest Development Strategy in Serbia, where only commercial entities can apply for permits to gather wild plants, algae and, fungi (Radomir et al., 2018). Serbia houses a rich variety of popular edible mushrooms, most prominently black truffles (Tuber melanosporum) which can fetch a market price of up to 4000 euros per kg. Due to high taxes levied on gathering and selling truffles, and above-mentioned restrictions on permitting, there is a flourishing black market and the majority of truffle export is purportedly going through 169 illegal routes (Radomir et al., 2018), a situation that neither benefits stakeholders nor allows for a realistic assessment of how to balance gathering activities with a healthy forest ecosystem. In Southern Europe, wild truffle populations have been in decline, largely due to habitat degradation and climate change (Büntgen et al., 2012; Garcia-Barreda et al., 2018; Pieroni, 2016). However, an assessment of policies and regulations relating to truffle gathering in Spain suggest that lack of appropriate management strategies may further exacerbate this trend (Garcia-Barreda et al., 2018). In Spain, gathering rights in public forests are auctioned off for terms of two to six years to private gatherers, which creates little incentive to invest in long- term strategies to maintain harvests. The situation becomes more acute as productivity declines and bidding becomes economically unattractive to commercial entities. Box 3.9 Matsutake and sustainable management In this case harvesting rights are purchased by municipalities which often leads to overexploitation, excessive trampling and damaging picking techniques as a larger number of gatherers face stiff competition with each other (Garcia-Barreda et al., 2018). Nevertheless, overall truffle production in Spain has increased in recent years due to the contribution of truffles grown in plantations, whose share rose from 10% in 1998 to 60% in 2012 (Garcia-Barreda et al., 2018), indicating that cultivation and silvicultural approaches are important avenues towards sustainable use for highly prized and highly commercialized species. 299 277 151 158 98 76 Taxa 299 Taxa 76 Wild vegetables can be important local commodities and are sold at high prices in local and regional markets. However, if they are not gathered as wild vegetables, they are often considered weeds because they need little attention or management and are gathered from the wild, agricultural or disturbed spaces. Wild vegetables are often associated with traditional production systems and a long history of local selection and usage. In France, at least until the 1980s, people in some rural areas ate wild vegetables at the turn of the seasons, bitter salads in spring to purify the blood, and diuretic vegetables in autumn to prevent winter rheumatism (Fédensieu, 1988; Schaal, 1993). Local and traditional knowledge is an important factor in maintaining the sustainability of wild vegetable gathering, cooking and consumption. This knowledge of wild vegetables may serve as baseline data for sustainable use (Ahmad, Ahmad, & Weckerle, 2013; Konsam, Thongam, & Handique, 2016; Maroyi, 2013; Wujisguleng & Khasbagen, 2010). Knowledge on food plants is, however, eroding in various parts of the world. In Mexico, rural indigenous and mestizo populations commonly eat wild greens called quelites, mainly gathered when weeding the fields; the most common species are Amaranthus hybridus, Chenopodium berlandieri, Anoda cristata, Porophyllum ruderale (Bye, 1981). Perceived as poor people’s food, they are disappearing from peoples’ diets, but there are actions to promote them (Mera Ovando, Castro Lara, & Bye Boettler, 2011). Weeds from rice fields are especially consumed in Asia and still play an important part in the diet in Northern Thailand (Cruz-Garcia & Price, 2011) and Laos (Kosaka et al., 2013). There was little evidence of wild greens consumption in South America. In the Amazon, most people are not keen on greens; the few wild species occasionally consumed are Phytolacca rivinoides and Talinum spp. (Katz et al., 2012). In Africa, a large number of indigenous or naturalized vegetables, such as baobab leaves or spider plant (Cleome gynandra), contribute to dietary diversity and food security, but have been neglected in some areas (Towns & Shackleton, 2018). Two widely consumed and popular wild vegetables in the United States of America are fiddlehead ferns and ramps (wild onions). Fiddleheads are newly emerging and immature fronds of the ostrich fern (Matteuccia struthiopteris (L.) Todaro), which occurs throughout temperate areas of the country with high soil moisture.  Wild vegetables Wild vegetables are an important part of the human diet. The gathering and consumption of wild vegetables are of great value in three ways. (i) They contribute to food security and famine, (ii) they are playing an increasingly important role as health food, and (iii) diets including wild vegetables pass on traditional flavors and cultural influences. Wild fruits and mushrooms are more frequently gathered than wild vegetables, and many wild vegetables have been forgotten. Herbicides have also contributed to their disappearance. Some species are regaining popularity as gourmet or health foods (Łuczaj et al., 2012). Wild vegetables were commonly eaten in the past, especially in times of scarcity. In non- famine times, they diversified monotonous diets. Children ate some wild vegetables with an acidic taste (Rumex, Oxalis) as snacks (Łuczaj et al., 2012). Some species are still gathered, such as Asparagus acutifolius or Scolymus hispanicus, as a part of traditional diets (Table 3.11). The Mediterranean diet is a model of healthy dietary patterns and has been recognized on the UNESCO Representative List of the Intangible Cultural Heritage of Humanity for Italy, Spain, Portugal, Morocco, Greece, Cyprus, and Croatia. Cultural and historical factors diversify the use of wild vegetables (Łuczaj, Zovko Končić, Miličević, Dolina, & Pandža, 2013; Łuczaj & Dolina, 2015; Łuczaj, Łukasz, Jug-Dujaković, Dolina, Jeričević, & Vitasović-Kosić, 2019; Geraci, Amato, Di Noto, Bazan, & Schicchi, 2018). Table 3.11 Comparison of the use of wild vegetables among Mediterranean countries. Source: (Geraci et al., 2018) under license CC-BY 4.0. Country Numbers Italy Spain Turkey Morocco Croatia / Herzegovina Cyprus / Greece Families 40 53 36 37 32 23 Genera 162 158 97 98 74 57 Table 3.11 Comparison of the use of wild vegetables among Mediterranean countries. Source: (Geraci et al., 2018) under license CC-BY 4.0. Country Numbers Italy Spain Turkey Morocco Croatia / Herzegovina Cyprus / Greece Families 40 53 36 37 32 23 Genera 162 158 97 98 74 57 le 3.11 Comparison of the use of wild vegetables among Mediterranean countrie ce: (Geraci et al., 2018) under license CC-BY 4.0. 170 299 277 151 158 98 76 For many they are an early food of spring, are also part of rural, local economies and can sometimes be found in larger grocery store chains in regions where they are popular. Total yields are estimated at 100,000 pounds annually, which is believed to be a sustainable yield. Ramps (Allium tricoccum) are a spring ephemeral species popular in the Eastern and central northern United States of America. Like fiddleheads, they are an early spring wild crop that is highly prized and celebrated as part of the return of spring. There is a long history of local and subsistence use of this species, which became nationally recognized in the 1990s due to a growing interest in it as a specialty food product. Now sold nationally in restaurants and health food stores, the accompanying market expansion has led to concerns regarding sustainable harvesting. Total quantities harvested are undocumented (Chamberlain, Emery, & Patel-Weynand, 2018). Seaweeds, or “ocean vegetables”, are collected throughout coastal areas all over the world. Historically, coastal people have been gathering and using seaweeds and seagrasses for 171 a variety of purposes, including food, feed, fertilizer, medicine, fibers, biofuel and materials; they are included here as food is a primary reason for collection. Globally, total macroalgal production has increased by approximately 5.7% per annum (including harvest of wild species and cultivation) (FAO, 2014c; Rebours, Friis Pedersen, Øvsthus, & Roleda, 2014). By volume, production is dominated by aquaculture (>96%), which resulted in 27.3 million tons of annual global production in 2014 (Lotze, Milewski, Fast, Kay, & Worm, 2019; Mac Monagail, Cornish, Morrison, Araujo, & Critchley, 2017). Despite the large scale of production from aquaculture, wild seaweed harvesting still plays an important role in many cultures. Thirty-two countries report active harvesting of seaweeds from the wild, with over 800,000 tons harvested annually from natural beds. Methods, regulations and management regimes vary widely across species and countries. European, Canadian and Latin American seaweed production still comes from harvesting wild populations (Buschmann et al., 2017; Rebours et al., 2014). Chile, China and Norway lead in exploitation of wild seaweed stocks. The Chilean harvest by artisanal fishers has been around 400,000 tons over the last 10 years, and there is concern about the environmental impacts of kelp removals. The marine license vetting committee of Ireland grants licenses to mechanically harvest seaweed and considers the potential negative impact on the marine environment (Mac Monagail et al., 2017). 299 277 151 158 98 76 Seaweed has been harvested in Brittany for several centuries, where this activity became industrial in the 18th century (Arzel, 1987) (Box 3.10). In Hawai’i seaweeds (Limu) are used for food, medicine, and ceremony as a traditional wild green. In recent years, more young Hawaiian men than women reported gathering wild seaweeds, indicating a cultural shift from pre-Contact Hawai’i, when women were the predominant gatherers and consumers of limu. Knowledgeable adults report a decline in the abundance of wild seaweeds driven by over-picking and pollution (Hart, Ticktin, Kelman, Wright, & Tabandera, 2014). Box 3.10 Seaweeds harvest in Brittany (Western France) The tip of the Brittany peninsula is particularly rich in seaweed, where over 330 species of macroalgae have been reported. There are two types of seaweed harvesting (Garineaud, 2017). Kelp is harvested from the sub-tidal sea in the archipelago of Molène-Ouessant (off the tip) and on the northern coast of the tip (from Le Conquet to Roscoff). This activity is locally considered as part of small-scale fisheries, with environmental knowledge transmitted within the family (Garineaud, 2015). Two types of kelp are harvested: Laminaria digitata (40 000 tons/year) and Laminaria hyperborea (25 000 tons/year) (Mesnildrey, Jacob, Frangoudes, Reunavot, & Lesueur, 2012). They are used to produce alginate, a gelling-thickening agent used in industry, especially the food industry. Two companies buy 95% of the harvest. This exploitation is considered sustainable (Frangoudes & Garineaud, 2015) because it is followed and controlled by a scientific institution, IFREMER (Institut Français de Recherche pour l'Exploitation de la Mer), in collaboration with the kelp collectors and industrial companies. However, the economic dependence on two companies and the lack of diversification of trade makes the practice vulnerable. Box 3.10 Seaweeds harvest in Brittany (Western France) The tip of the Brittany peninsula is particularly rich in seaweed, where over 330 species of macroalgae have been reported. There are two types of seaweed harvesting (Garineaud, 2017). Kelp is harvested from the sub-tidal sea in the archipelago of Molène-Ouessant (off the tip) and on the northern coast of the tip (from Le Conquet to Roscoff). This activity is locally considered as part of small-scale fisheries, with environmental knowledge transmitted within the family (Garineaud, 2015). Two types of kelp are harvested: Laminaria digitata (40 000 tons/year) and Laminaria hyperborea (25 000 tons/year) (Mesnildrey, Jacob, Frangoudes, Reunavot, & Lesueur, 2012). They are used to produce alginate, a gelling-thickening agent used in industry, especially the food industry. Two companies buy 95% of the harvest. This exploitation is considered sustainable (Frangoudes & Garineaud, 2015) because it is followed and controlled by a scientific institution, IFREMER (Institut Français de Recherche pour l'Exploitation de la Mer), in collaboration with the kelp collectors and industrial companies. However, the economic dependence on two companies and the lack of diversification of trade makes the practice vulnerable. 172 About 30 species are harvested on shore, in quantities of a few kilograms to several tons per year, reaching a total of about 10,000 tons. Around 300 collectors are involved in this activity, with different status, from seasonal workers to small processing companies (Garineaud, 2017). The most harvested species are Fucales and edible seaweeds such as Palmaria palmata, Himanthalia elongata or “pioka” (Chondrus crispus and Mastocarpus stellatus). The seaweeds are harvested by hand, or with scissors when clinging to a rock. Then they are dried, either preserved in salt or processed and sold fresh, depending on the species, the use and the collector. They are mainly used in food, industrial and pharmaceutical products. It is difficult to analyze this exploitation because of the diversity of harvested species, outlets and stakeholders. The lack of scientific knowledge, follow up, and control of this activity makes it vulnerable to changing conditions. It is difficult to establish administrative frameworks, exploitation regulations and labels matching with the stakeholders and their practices. The main risk with regards to sustainable use would be to turn this small-scale exploitation into a more intensive, more industrial and less diversified trade. Climate change is also likely to have an impact on seaweed harvesting and increase variability of the resource. Box 3.10 Seaweeds harvest in Brittany (Western France) Some species have already been displaced (Gallon et al., 2014; Raybaud et al., 2013). It is unclear how companies will adapt to variability and changing environmental and social conditions (Garineaud, 2017). Finally, the lack of information, transparency and accessible data makes understanding the social dimensions more difficult. 3.3.2.3.5. Medicine and hygiene Humans use wild plants and fungi for medicinal purposes all over the world. Gathering wild species for medicines is motivated by a range of factors. These include poverty or difficulty accessing medical assistance, traditional knowledge and beliefs, cultural heritage, or for profit due to commercialization. There is also a growing demand for products produced at least in part from wild harvested plants and fungi, to complement chemical medicines in many high- income countries (Lamrani-Alaoui & Hassikou, 2018; Lanker et al., 2010; H. Liu, Luo, Heinen, Bhat, & Liu, 2014; Nekratova & Shurupova, 2016; L. Petersen, Reid, Moll, & Hockings, 2017; K. M. Stewart, 2003). A large number of ethnobotanical studies have generated inventories and analysis of medicinal and hygienic uses of wild plants. Online databases summarize information on medicinal plants. For example, the Kew royal botanical garden has established the Medicinal Plant Names Service (https://mpns.science.kew.org), the Africa Museum in Brussels runs the Prelude Medicinal Plants Database (https://www.africamuseum.be), and databases like Native American Ethnobotany (http://naeb.brit.org/), the Indian Medicinal Plants Database (http://www.medicinalplants.in/) and the China National Genebank (https://db.cngb.org) all include information on medicinal uses. These inventories of medicinal plants outline the threat level to the species, conservation status, or priority of conservation for further actions. In South Africa for example, 2,062 indigenous plant species (10% of the total national flora) have been documented for use as traditional medicine. Of these, 82 wild medicinal plant species (0.4% of the total national flora) are considered threatened with extinction at a national level (V. L. Williams, Victor, & Crouch, 2013). Thirteen percent of Myanmar medicinal plant species are considered threatened in the International Union for Conservation of Nature Red List of Threatened Species (DeFilipps, Krupnick, & Krupnick, 2018). These data suggest possibilities for future research, conservation programs, sustainable harvesting projects, management and regulations. When and how wild plants or plant parts are gathered have important effects on their medicinal value. Each category of medicinal plant has its specific collection time to maintain not only efficacy, but also sustainability. In this regard, Kletter and Kriechbaum (2001, p. 12) remarks “a plant has medicinal value when it is harvested at the right time, but is mere grass when harvested during the wrong season”. Local and traditional knowledge is a key to the sustainable gathering of wild medicinal plants.  Protista and blue-green algae The terrestrial cyanobacterial species Nostoc flagelliforme, commonly called Fai-Cai (Fat Choy), lives in desert or semi-arid grasslands in the Asia Pacific region, and is used as a vegetable in Chinese cuisine (Dai, 1992; Gao, 1998; YL Geng & Jiang, 1991). Herders scrape the vegetable with rakes. Indigenous and local knowledge suggests one must forage over approximately 10 acres of grassland to harvest 100g of dry fat choy. The raking can destroy the delicate grasslands and accelerate desertification. Therefore, the species was up-listed into the Class I of state key protected wild plants (even though it is not a plant) in 2000 and harvest and trade were banned at that time (But, Cheng, Chan, Lau, & But, 2002). Nostoc commune or Ge-Xian-Mi (Rice of Immortal Ge) is the second edible species of Nostoc, originally listed for use in the The Compendium of Materia Medica (S. Li, 1596) by Shi-Zhen Li (1518–93?) of the Ming Dynasty. The name of Ge-Xian-Mi is related to Ge Hong (AD 284–364), a Taoist theoretician of the Eastern Jin Dynasty, who used N. commune as food during periods of famine and later introduced it to the emperor. It is used for health food and herbal medicine however the wild type of N. commune has been decreasing as a result of recent increases in market demand and environmental pollution. Artificial culture of the blue green algae generates economic benefits (Diao & Yang, 2014; Nazih & Bard, 2018). Nostoc species are still consumed, not only in China, but also in various countries such as the Philippines, Thailand, Japan, Fiji, Peru, Ecuador, Mexico, Mongolia, and Siberia (Borowitzka, 2018). High-quality agar and agarose for bacteriology and pharmaceuticals originated from wild harvested Pterocladiella capillacea. A report reveals a decline in biomass coupled with a peak in wholesale prices, which have resulted in overharvesting in some countries in due to this increased economic exploitation (Patarra, Iha, Pereira, & Neto, 2019). Ongoing 173 unsustainable commercial harvest of the algae could result in further marine ecological damage; thus, the future of the industry is uncertain. 3.3.2.3.5. Medicine and hygiene Of the articles retrieved in the Web of Science published between 2000-2020, more than one third (n=117/349) mentioned “traditional knowledge”. By its very nature, traditional knowledge is holistic in nature, thus in these articles it was not always distinguished as being specifically for medical use, and could also be related consumption for food or aromatic uses. This is consistent with the fact that many wild medicinal plants have multiple uses at the same time. Like in Angola, 35% of the 127 Leguminosae plants are only used medicinally by the local communities, while the remaining species were reported to have many other uses (S. Catarino, Duarte, Costa, Carrero, & Romeiras, 2019). 174 Wild plant species are chosen for pharmaceutical studies through different methods. One method what has come to be known as bioprospecting: the investigation of indigenous uses of wild plant species based on indigenous local knowledge that can offer strong clues to the biological activities of those plants. There are many examples of this knowledge being used by companies who either do not financially compensate local people at all, or do not do so in proportion to the value of their resultant profits. This is commonly known as biopiracy, and is a major issue is many developing countries and with indigenous communities around the world (Benjaminsen & Svarstad, 2021; Shiva, 2007). In relation to the definitions of sustainable use reviewed in Chapter 2, this form of exploitation is considered as a form of unsustainable use. Scientific experimentation is another method through which medicinal knowledge of natural products has over the last few centuries (D. A. Dias, Urban, & Roessner, 2012). About a quarter of all Food and Drug Administration and/or the European Medical Agency approved drugs are plant based (Thomford et al., 2018). From 1981 to 2002, around 49% of the small-molecule new chemical entities that were introduced were from natural products or based on natural- products. The utilization of natural products in order to discover and develop new drugs is an active area of research (Koehn & Carter, 2005; Newman & Cragg, 2007, 2007).  Medicinal Fungi Fungi are also widely used for medical purposes, especially in the Asia Pacific Region. Our literature review yielded 33 studies that detailed the use of medicinal fungi from all IPBES regions (Africa 8, Americas 7, Europe and Central Asia 8 and Asia Pacific 8). Of these, 90% also reported on species used for food, so many of the aspects pertaining to sustainable use are shared with wild edible mushrooms (see section 3.3.2.3). All studies reporting on wild species and their uses (12 in total) reported fewer medicinal species than species used for food and often species were used both as food and medicine. The largest number of medicinal species was reported from China, with 692 species with medicinal properties with 277 species considered as both food and medicine (Wu et al., 2019). Mexico also hosts a large variety of medicinal fungi with a survey reporting the use of 70 species to treat over 40 different conditions, again many with dual use as food and medicine (Guzmán, 2008). Medicinal fungi also have a long history of use in Europe, where interest in traditional medicines has been increasing again recently after a decline in use in the 20th century (Comandini & Rinaldi, 2020). The most common medicinal fungi include Ophiocordyceps sinensis (caterpillar fungus), Ganoderma lucidum (lingzhi or reishi), Lariciformis officinalis, Lentinula edidodes (shitake), Trametes versicolor (turkey tail), Schizophyllum commune (the split gill) and Pleurotus spp., especially Pleurotus tuber-regium which is used medicinally across Africa (Milenge Kamalebo, Nshimba Seya Wa Malale, Masumbuko Ndabaga, Degreef, & De Kesel, 2018; Oyetayo, 2011). Medicinal fungi produce a range of active compounds, many of which have been shown to have anti-oxidant, anti-tumor or anti-microbial properties (Wu et al., 2019). To this end, G. lucidum is probably the most intensively studied species. It produces over 400 bioactive compounds and has been dubbed “the mushroom of immortality” in China where it has been used for over 2,400 years (Cör, Knez, & Knez Hrnčič, 2018). Nowadays it is widely used to supplement cancer treatment both in China and Western countries. Several records indicating the medicinal use of lichens in Spain and Nepal were also found (Shiva Devkota, Chaudhary, 175 Werth, & Scheidegger, 2017; González-Tejero, Martínez-Lirola, Casares-Porcel, & Molero- Mesa, 1995). Perhaps the most valuable species globally is the illusive caterpillar fungus (Ophiocordyceps sinensis), which grows only in the Himalayan mountains (Box 3.11).  Medicinal Fungi Gathering of caterpillar fungus (Ophiocordyceps sinensis) has dramatically increased over the last 20 years. A short seasonal and rotational approach for gathering is useful for its sustainability. Caterpillar fungus extraction provides up to 72% of household income in the area, and estimates of households involved in the short seasonal gathering range from 52% to 98%. Understanding of local commercial harvest and trade supports sustainable management (J. He, 2018; Kuniyal & Sundriyal, 2013; Woodhouse, McGowan, & Milner-Gulland, 2014). Box 3.11 Status and trends of caterpillar fungus in the Nepalese Himalayas Ophiocordyceps sinensis (Berk.) G.H.Sung, J.M.Sung, Hywel-Jones & Spatafora, (Hypocreales, Ophiocordycipitaceae) is a high-altitude fungus reported only from the alpine meadows in Nepal, India, Bhutan and China. Locally called Yar-tsa-gunbu (summer grass, winter insect), it occurs from 3,540 m to 5,050 m above sea level across 24 different northern districts in Nepal (S. Devkota, 2008) and up to 5,200 m in Bhutan (Cannon et al., 2009). It is an entomopathogenic fungus that parasitizes over 50 species of Thitarodes (Hepialidae) moth larvae (X.-L. Wang & Yao, 2011). In the gathering season (May – July) and particularly when the snow melts, gathering is extensive. As many as 70,000 collectors (men, women, and children) have been reported across 25 principal gathering pastures in a single district (Dolpa of Nepal), living in temporary tent camps for about two months (S Devkota, 2009). The fungus provides a substantial source of cash income for many households: 21.1% contribution to the total household income and 53.3% to the total cash income among rural inhabitants and helping to fund childrens’ education, food purchasing, household construction and debt repayments (Pouliot, Pyakurel, & Smith-Hall, 2018; Shrestha & Bawa, 2014). Apart from this, subsidiary incomes in mountain communities come from farming, animal husbandry, collection and trade of other medicinal and aromatic plants (Olsen & Larsen, 2003). The global annual collection of caterpillar fungus is roughly estimated at 85-185 metric tons (Winkler, 2008). Indigenous peoples and local communities living in the Nepalese Himalayas use it for the treatment of different diseases like diarrhea, headache, cough, rheumatism, liver disease, and also as an aphrodisiac and tonic (S. Devkota, 2006). However, the main market is China, where there are several reasons behind increasing demand. Many consider the species as valuable medicinal fungi in accordance with traditional Chinese medicine. It is traded as the “Himalayan Viagra” and prices have exceeded 140,000 United States dollars per kg for the best quality in Chinese markets, depending upon size, color, aroma, and region of origin (Shrestha & Bawa, 2014). The high number of collectors, their trampling effects on fragile subalpine and alpine landscapes, wild species poaching, improper garbage disposal and annual large harvested volumes have raised several sustainability concerns (Byers, Byers, Shrestha, Thapa, & Sharma, 2020; S Devkota, 2009; Pouliot et al., 2018). Box 3.11 Status and trends of caterpillar fungus in the Nepalese Himalayas 176 The Chinese government has supported been thoroughly making efforts to reduce dependence on wild Ophiocordyceps sinensis through cultivation and fermentation technologies (Yue, Ye, Lin, & Zhou, 2013). Advanced biotechnology is being applied to cultivate Paecilomyces hepialid (fermentation mycelium) with active ingredients from the natural caterpillar fungus as well as compounds of its equivalent medicinal value (Ji et al., 2020). There has been intensive focus on the artificial cultivation of the caterpillar fungus which has yielded successful approaches for its propagation and breeding (X. Li et al., 2019). The emergence and application of culture-based techniques as a substitute for wild caterpillar fungus and the development of artificially bred varieties are a promising path towards protection and sustainable use of wild caterpillar fungus resources.  Seeds, Leaves and fruits for medicinal use The gathering of seeds, leaves and fruits for medicinal use is usually non-lethal and seasonal. In some cases, the average annual harvest is high but the population size is consistent, such as with Aloe ferox in South Africa and Euphorbia antisyphilitica in Mexico (Martinez-Balleste & Mandujano, 2013). These species were once included in the Convention on International Trade in Endangered Species of Wild Fauna and Flora, but after assessing the sustainability of harvest and trade, their products have been exempted from strict control. In order to sustain the trade, improving the techniques of wax extraction and promoting fair trade pricing structures that benefit local harvesters have been suggested (Martinez-Balleste & Mandujano, 2013). Certification schemes can support management for sustainable use. For example, harvesting of the fruits of Schisandra sphenanthera in Chinese forests meet sustainable wild harvesting standards, with an incentive to maintain habitat outside formal protected areas based on FairWild Standards (2010) and Giant Panda Friendly Products Standards (2012) (Brinckmann et al., 2018). In the absence of effective management, gathering of leaves, seeds and fruit is stressful for some sensitive species such as Aloe peglerae, Cola nitida and C. millenii which are endemic to Africa and currently endangered. Studies suggest developing silvicultural techniques to improve domestication through ex situ cultivation in gardens and orchards (Chungu et al., 2007; Lawin et al., 2019; Pfab & Scholes, 2004; Savi et al., 2019).  Barks and stems Bark harvesting for medicinal purposes is widespread in Africa as a form of local and free medicine. Julbernardia paniculate and Isoberlinia angolensis are two species severely negatively affected by bark removal. Traditionally, there have been measures to reduce injuries to the tree. One form of local tree protection is to cover the wound site with mud, which protects the tree from wood deterioration and insect damage (Chungu et al., 2007). In addition to practical measures, domestic legislation can also offer local protection. For example, Warburgia salutaris is endangered and overexploited in many regions and deemed threatened throughout its range. South Africa's environmental legislation now prohibits the harvesting of protected wild plants or plant parts (e.g., the bark and leaves of Warburgia salutaris) and recommends the use of alternative species (Rasethe, Semenya, & Maroyi, 2019; Senkoro et al., 2019). 177 Harvesting bark to meet medicinal demands is becoming less sustainable for some species due to increasing demand. Prunus africana in Africa and The Himalayan yew (Taxus wallichiana) are greatly threatened with unsustainable harvest. Wild-gathering of barks of Prunus africana is no longer sustainable. The population has been declining over much of its geographical range in sub-Saharan Africa in recent decades. Only recently have existing standing crop inventories and scientifically based annual quotas being determined (Fashing, 2004; K. Stewart, 2009; K. M. Stewart, 2003). The Himalayan yew (Taxus wallichiana ) is very slow growing species with poor natural regeneration. Most wild populations in Asia Pacific forests are threatened with extinction and are endangered in the Himalaya due to over- harvesting of their barks and leaves in combination with low seed production and germination. In situ conservation and management and artificial regeneration using efficient biotechnological tools have been proposed (Lanker et al., 2010). Both of these species are included in the Convention on International Trade in Endangered Species of Wild Fauna and Flora to restrict international trade, with the intention to develop tools and methods for sustainable gathering or promote alternative source including cultivation.  Roots, Rhizome, Tuber and Bulbils  Roots, Rhizome, Tuber and Bulbils Gathering roots, rhizome, tuber and bulbils usually does harm to plants. It is fatal to dig out all the roots and tuber. Such gathering, if not managed properly, is unsustainable. Destructive overharvesting is the key threat to Stemona tuberosa, Gymnadenia conopsea in Asia and Siphonochilus aethiopicus and Dioscorea bulbifera in Africa driven by high market demand (G. Chen et al., 2019; Ikiriza et al., 2019; Kala, 2009; Shao et al., 2017; Xego, Kambizi, & Nchu, 2016). The increasing demand on stems and roots coupled with non-sustainable harvesting methods has resulted in a substantial decline of Cryptolepis sanguinolenta in its wild populations in Africa forests. The development of domestication protocols has been suggested as one way to protect the species and decrease rates of decline (J. He, 2018; Kuniyal & Sundriyal, 2013; Woodhouse et al., 2014). Panax quinquefolius (American wild ginseng) is a highly valued wild root collected extensively in the United State of America s. Populations have declined significantly over time in six northern states in the United States of America and harvest pressure is now restricting harvestable stocks. Annual wild ginseng harvests decreased from the high point in the late 1980s to early 1990s, but subsequently increased after 2005. Natural rates of population recovery are slow. Market prices for this species do seem to operate on a supply and demand logic such that quantities supplied are negatively related to prices, theoretically providing economic incentives for forest retention. A federal regulation has banned exports of roots from plants under five years old in effect since 1999. Management includes stewardship-oriented harvest restrictions such as delays in the opening of the permitted harvest season by two weeks, self-limits on harvest intensity, and planting ginseng seeds at the time of harvest (Burkhart & Jacobson, 2009; Burkhart, Jacobson, & Finley, 2012; Case, Flinn, Jancaitis, Alley, & Paxton, 2007; Frey, Chamberlain, & Prestemon, 2018; J. Schmidt, Cruse-Sanders, Chamberlain, Ferreira, & Young, 2019). Some perennial wild plants can tolerate a certain degree of gathering activities. Populations of Neopicrorhiza scrophulariiflora were heavily exploited in one area of the alpine Himalayas but appear more resilient to extraction than other commercially exploited 178 populations (S. Ghimire et al., 2005; Poudeyal, Meilby, Shrestha, & Ghimire, 2019). In the American highlands, the local risk index for conservation status of Oxalis adenophylla was medium, driven by changes in its environment and not directly related to gathering.  Roots, Rhizome, Tuber and Bulbils In fact, gathering of leaves and roots of this species is thought to promote its conservation through the understanding of its sensitivity to harvesting (Ochoa & Ladio, 2014). The rhizome of Hydrastis canadensis (goldenseal) is widely harvested in America's woodlands and has been used for traditional medicine by native peoples. Regeneration time varies between populations, making it difficult to predict overall abundance. Late-summer and fall are the ideal periods when goldenseal rhizomes are traditionally gathered (Albrecht & McCarthy, 2006; Burkhart & Jacobson, 2009; D. L. Christensen & Gorchov, 2010). In the majority of cases, proper management and predictable harvest volumes are required to ensure that root gathering meets the need of regeneration and renewal, but habitat conditions are also critical. For example, black cohosh (Actaea racemose) is highly responsive to harvest intensity in the United States of America. Low to moderate harvest intensities and/or longer recovery periods will be necessary for prolonged and sustainable harvests (Small et al., 2011). A low harvest rate, for example 50% of mature plants every 10 years, may be sustainable for the harvest of osha or wild parsnip (Ligusticum porter) in America's highlands (Kindscher et al., 2019). Harvesting of Rheum acuminatum R. australe and Rhaponticum carthamoides in central Nepal can be considered sustainable under optimal management. Predictable exploitable reserves and volume of harvesting, however, partly differ between species and strongly depends on habitat conditions (Nekratova & Shurupova, 2016; Rokaya, Münzbergová, & Dostálek, 2017). Management including wild cultivation can also protect habitats. Micro- propagation can aid in re-establishing plants in their natural habitats (Ikiriza et al., 2019; Kala, 2009). Overexploitation for traditional medicine and health food sipplements, combined with habitat destruction, has resulted in the rapid decrease of Dendrobium sp. in Asia. However, epiphytic orchids planted in natural forests as part of in situ cultivation are facilitating more sustainable harvesting (H. Liu et al., 2014, 2014; Shao et al., 2017) (Box 3.12). Box 3.12 The sustainable use of wild orchids in traditional Chinese medicine In China, orchids are traded for both ornamental and medicinal purposes (Hong Liu et al., 2020). About a quarter of all Chinese wild orchid species are considered traditional Chinese medicine and market demands for some of them have been extremely high (H. Liu et al., 2014). Wild populations of some traditional Chinese medicinal orchids, such as those in the genus of Dendrobium, have either been extirpated or reduced to small, isolated populations. Augmentations or reintroductions are required to bring these populations back to a healthy state. Recognizing the issue of high demand on exhausted natural resources, the Chinese government has embraced the conservation intervention to increase supply by farming (Hong Liu, Gale, Cheuk, & Fischer, 2019) and has been very successful in encouraging massive shade house commercial cultivation of threatened traditional Chinese medicinal orchids. The total shadehouse products of Dendrobium officinale, one of the most used medicinal orchid species in China, was more than 6.4 billion United States dollars in 2011 (H. Liu et al., 2014). 179 However, it appears as though large commercial shadehouse cultivations have not alleviated pressure on wild populations. One reason for this is related to the public perception that cultivated products are considered to be less potent than wild harvested orchids, and so wild harvested products are considered to be of higher quality and are sold at premium prices (H. Liu et al., 2014). In addition, orchids growing in industrial shade houses are subject to synthetic fertilizers and pesticides, which also make the cultivated product less desirable. A semi-wild cultivation approach in which specimens are outplanted into native wooded areas specifically for harvesting has been implemented in a few places in southern China, such as Renhua County in northern Guangdong province, Xingyi County in Southwestern Guizhou province, and Leye County in northwestern Guangxi province. These areas are relatively undeveloped compared to the Pearl River Delta area in southern China and are within the native ranges of several medicinal Dendrobium orchids. These cultivation operations are a hybrid between commercial cultivation and population restoration because farmers can harvest certain number of stems (pseudobulbs) without killing the plants, and allow some plants to flower and fruit. Seeds produced from these plants are potential sources of population recovery and thus this form of outplanting is called “restoration-friendly cultivation” (H. Liu et al., 2014). The market share of these semi-wild products is unknown. 3.3.2.3.6. Recreation Many of the other uses covered throughout section 3.3.2 include some sort of recreational component. Only a few examples are provided here that stand out in terms of their recreational value. A trend has been observed in recent years to promote forest management and sustainable use by combining gathering of wild algae, fungi and plants with non-extractive practices such as tourism. For example, mycological tourism is growing in popularity, often associated with amateur societies in North America and Europe, where people go mushroom gathering, harvest wild mushrooms and then identify them with the help of professionals (Barron, 2010). On one hand it is considered professional exploitation of wild resources, o the other hand it is a form of forest management (Jiménez-Ruiz, Thomé-Ortiz, Espinoza-Ortega, & Vizcarra Bordi, 2017). In fact, amateur mycological associations continue to grow and are considered a valuable resource by mycologists for everything from taxonomic assistance to data collection (Barron, 2011). Many cultural services and values support recreational gathering of wild species. In the Northeastern United States of America, gathering wild edible huckleberries has been related to maintaining social relations, recreational use and commercial purposes (Carroll, Blatner, & Cohn, 2003). In Spain, while the gathering and consumption of wild edible plants is generally decreasing, there is an increase in the harvest of foods with high cultural value (Reyes-Garcia et al., 2015). In Austria, interviews in 2008-2009 reveal the multiple motivations for gathering wild plants; women, older respondents and home gardeners gather wild plants more often for fun (Schunko, Grasser, & Vogl, 2015). Box 3.12 The sustainable use of wild orchids in traditional Chinese medicine This semi-wild or restoration-friendly cultivation approach has been suggested for other epiphytic orchids that are harvested for cultural and religious festivals and ceremonies in Latin American Countries (Tamara Ticktin et al., 2020). For example, Mexico has more than 1,300 species of orchids (Hágsater et al., 2015) and among these more than 300 orchid species in 90 genera were used for religious and cultural celebrations (Menchaca García, Lozano Rodríguez, & Sánchez Morales, 2012; Tamara Ticktin et al., 2020). About a dozen of these species (e.g., Laelia speciosa, Euchile karwinskii, Barkeria vanneriana) are traded in high volumes legally and illegally (Tamara Ticktin et al., 2020). There have been attempts to establish a rural community nursery system in the relevant areas, in which farmers were encouraged to plant these orchids in their backyard and adjacent community forests. The nurseries were then registered as Environmental Management Units (UMA, for their acronym in Spanish) (Menchaca García et al., 2012). The nursery system was intended to promote sustainable harvesting in rural communities, as non-lethal harvesting can be done sustainably from the nurseries which then in turn allow wild populations to recover, as shown in population viability simulation models in Ticktin et al. (2020). Semi-wild or restoration-friendly cultivation operations have positive impacts on sustainable use, but are not widespread in comparison with harvest quantities. Ecological and socioecological infrastructure needs to be developed and supported to achieve orchid conservation and support livelihoods (H. Liu et al., 2014). For example, mass reproduction centers coordinated with farmers to deliver enough plants for semi-wild planting requires support (Menchaca García et al., 2012). These centers can also provide technical support on growing and harvesting and marketing support. Restoration-friendly cultivation can directly facilitate the recovery of threatened species, encourage protection of natural forests, and benefit marginalized rural communities. However, it is unclear exactly what ecological growth conditions, harvesting regimes, and market conditions are suitable to achieve 180 3.3.2.3.7. Science and education However, African countries, Central, South and Southeast Asian countries and East European countries have been poorly represented in harvest of vascular plants species aggregated in the Global Biodiversity Information Facility and data in the World Checklist of Vascular Plants are also poor (Antonelli et al., 2020; Paton et al., 2020). Rocha et al. (2014) have argued that halting the collection of voucher specimens by scientists would be detrimental. Scientists believe that in order to describe the earth's biodiversity and understand wild species, museum collections should increase by 600%, while still being collected responsibly following best practices (Henen, 2016). Continued gathering would also support herbarium-based publications, which have dramatically increased in the past century (Heberling et al., 2019). To that end, regulatory authorities could develop quotas for specimen harvest that are based on scientific guidelines (Maya & Gómez, 2016). Scientific gathering practices also face a series of economic and social pressures, including budget cuts and shortfalls in university and museum settings (Suarez & Tsutsui, 2004), high gathering costs (Enrique, Daniela, & Fernando, 2020), ethical considerations, and effects of regulations like the Convention on International Trade in Endangered Species of Wild Fauna and Flora for the cross-border exchange of specimens (Roberts & Solow, 2008). To promote scientific research on species conservation and materials sharing between scientists, the Convention on International Trade in Endangered Species of Wild Fauna and Flora established the registered scientific institute scheme, and encourages Parties to register scientific institutes. So far, 74 Parties have registered a total of 857 scientific institutions and individuals with the Secretariat of the Convention on International Trade in Endangered Species of Wild Fauna and Flora (Antonelli et al., 2020; C. Williams et al., 2020). From Kew’s dataset, there are more than 7,039 known species of edible wild plants, but only 417 (5.9%) are considered food crops by the FAO (Antonelli et al., 2020; Ulian et al., 2020). Crop wild relatives are sources of genetic diversity useful for developing more productive, nutritious and resilient crop varieties, and thus contribute to global food security. In 2016, the most important discovered species with potential for new food sources were 11 new Brazilian species of Manihot which are relatives of the highly valued food plant Manihot esculenta (cassava). Manihot esculenta is the third most important food after maize and rice, and it offers more food security than cereals. 3.3.2.3.7. Science and education Around the world, gathering wild specimens continues to generate information of scientific value. This includes dried plants and fungi for herbaria and fungaria, living plants and fungal cultures grown by botanical gardens and mycological institutes, and seeds stored in seed banks (Antonelli et al., 2020; Paton et al., 2020). The world’s preserved botanical and mycological collections mostly date back to the late 1800s and early 1900s. There are 3,324 active herbaria in the world, containing 392,353,689 specimens. Norther America, Europe and temperate Asia (including Russia and China) have the highest number of herbaria (Antonelli et al., 2020; Paton et al., 2020; Pearce et al., 2020). The Millennium Seed Bank Partnership conserves high-quality propagules. It has involved 96 countries and territories, and 32% of taxa (representing half of the collections) have at least one identified use for humans (U. Liu, Breman, Cossu, & Kenney, 2018). Regarding live wild plants gathering, analysis of the PlantSearch database hosted by Botanic Gardens Conservation International indicates that 107,340 accepted species grow in botanic garden collections, representing 31% of vascular plant species. However, 93% of these species are held in temperate parts of the world. As a result, a temperate species has a 60% 181 chance of being cultivated within the botanic garden network, whereas a tropical species has only a 25% chance. Collection for scientific purposes, however, is on the decline (Heberling, Prather, & Tonsor, 2019), and there have been recent calls for more “holistic sampling” to maximize the usefulness of collections to protect individuals in the wild (Heberling et al., 2019; U. Liu et al., 2018). Good photographs, non-lethal harvest techniques, and the sharing of specimen information or molecular methods (Minteer, Collins, Love, & Puschendorf, 2014; D. Russo, Ancillotto, Hughes, Galimberti, & Mori, 2017) all represent good alternatives to lethal harvesting for scientific use. The Global Biodiversity Information Facility (GBIF) provides access to more than 1.4 billion records (including observations, preserved samples, fossils and living specimens) of all types of life on Earth in nearly 53,000 datasets supported by 1,600 institutions. The data of observation-based occurrences is surpassing the harvest of specimen- based occurrences in the Global Biodiversity Information Facility (Troudet, Vignes-Lebbe, Grandcolas, & Legendre, 2018). 3.3.2.3.7. Science and education (Antonelli et al., 2020; Castañeda-Álvarez et al., 2016; Vincent et al., 2013). 182 The Crop Wild Relatives Project (cwrdiversity.org) used the Harlan and de Wet (1971) gene pool concept to set up an inventory of globally important crop wild relatives' taxa for 173 priority crops. It contains 1667 taxa, divided between 37 families and 108 genera. The region with the highest number of priority crop wild relatives is Western Asia with 262 taxa, followed by China with 222 and Southeastern Europe with 181 (Vincent et al., 2013). However, the diversity of crop wild relatives is poorly represented in gene banks. Kew’s Millennium Seed Bank includes 688 crop wild relatives among its over 78,000 accessions. Over 70% of taxa are identified as high priority for further gathering in order to improve their representation in gene banks. The most critical gathering gaps occur in the Mediterranean and the Near East, Western and Southern Europe, Southeast and East Asia, and South America (Antonelli et al., 2020; Castañeda-Álvarez et al., 2016; Vincent et al., 2013) (Figure 3.45). A discussion of crop wild relatives is relevant for this assessment in relation to the sustainable use of collecting specimens. However, analysis of the role of crop wild relatives in supporting crop diversity and providing genetic resources is beyond the scope of the current assessment. Figure 3.45 Gathering priorities for crop wild relatives and the importance of associated crops. Crop types which occur in the upper portion of the graph are major global good stuffs; those on the far right are in greater need of the genetic diversity that crop wild relatives can provide. For example, of the three major global grain crops, wheat, rice, and corn, corn is in greatest need of genetic diversification from wild relatives in order to enhance food security Figure 3.45 Gathering priorities for crop wild relatives and the importance of associated crops. Crop types which occur in the upper portion of the graph are major global good stuffs; 3.45 Gathering priorities for crop wild relatives and the importance of associated crops. Crop types which occur in the upper portion of the graph are major global good stuffs; those on the far right are in greater need of the genetic diversity that crop wild relatives can provide. 3.3.2.3.7. Science and education For example, of the three major global grain crops, wheat, rice, and corn, corn is in greatest need of genetic diversification from wild relatives in order to enhance food security 183 Source: (Castañeda-Álvarez et al., 2016) © 2016, Macmillan Publishers Limited under licencse CC BY-NC-SA 4.0. 3.3.2.3.8. Materials and shelter Artificial materials have replaced many wild sources, but in some remote areas materials from wild species are more readily available and commonly used (Box 3.13; Box 3.14). Other than wood and bamboo, organic materials in tropical areas used for material and shelter include natural fibers, thatch, grass, reeds, sisal fiber, coir waste, elephant grass and straw (Bengtsson & Whitaker, 1988). Sisal fibers are long natural fibers derived from Agave (Agave sisalana) leaves native to Mexico. In the 1960s the global production was 640 (metric) kt/year (UNIDO/CFC, 2005), but has since declined due to the rise of synthetic fibers. Sisal is grown mainly in Brazil, East Africa and China and has low requirements for fiber production and thus high potential for environmental sustainability (Broeren et al., 2017). The FAO recommends natural fibers as future fibers, such as coir waste derived from coconut palm (Cocos nucifera), Abaca extracted from the leaf sheath around the trunk of the abaca plant (Musa textilis) and Jute extracted from the bark of the white jute plant (Corchorus capsularis) (http://www.fao.org/economic/futurefibres). Similar to elephant grass (Pennisetum purpureum), the source of these natural materials is shifting due to agriculture. Box 3.13 Bamboo, a plant of many virtues There are over 1,400 species in the world, and they can thrive at high altitudes and low plains. Bamboo is one of the fastest-growing plants on the planet, and its influence has been widely felt: aside from rice, no other plant has played such as important role in the history as bamboo. Besides being edible, it has medicinal, commercial and practical values: taken together, they yield more than 1,000 different products from their stems and leaves. Many uses of bamboo include preparation of waterproof coat and hat, each wrought out of leaves; agricultural implements; the fishing net, baskets of diverse shapes, arrows, paper and pens, grain-measures, wine-cups, water-ladles, chopsticks, tobacco-pipes, etc. In Asia, the bamboo symbolizes virtues, humanity, and resistance to hardship, and it has played an important role in Asian arts, including in ink drawing and painting. Use of bamboo is the most common by indigenous and local communities of the world and every year people use over three billion cubic meters of wood worldwide to construct buildings, boats, furniture, and fences. Wood and steel have been the main materials for production in the modern construction industry. As deforestation intensifies, fast-growing bamboo is considered as an alternative to wood, easily used as an alternative in flooring, roofing, and even steel-reinforced buildings in Africa. Sources: (Laws, 2010; Paye, 2000). Palm leaves are an important source of roof thatch for rural communities in many parts of the tropics (Svenning & Macı́a, 2002). A total of 194 useful palm species and 2,395 different 184 uses throughout northwest South America, including Amazonia, Andes and Chocó have been documented (Macía et al., 2011). In the Yucatan peninsula, leaves of xa'an palm trees (Sabal yapa, and Sabal mexicana) have been widely used for family homes. The palm is managed by Maya farmers through indigenous and local knowledge. When they clear a forest patch to grow maize, they spare palm trees, introduce them into home gardens and improve their growth. There are one or two harvest events per year and locals recommend leaving one or two leaves in each event. This traditional practice can stimulate palms to compensate for the effects of defoliation by producing new leaves (Martinez-Balleste, Martorell, & Caballero, 2008). Although the harvest of S. Species or group Palms are one of the critical elements in the floristic composition of tropical rainforests (Abensperg-Traun, 2009; Montufar & Pintaud, 2006). The Family includes 181 genera and c. 2,450 species distributed in the tropical region worldwide, with some species that extend into subtropical areas in both hemispheres (Baker & Dransfield, 2016). The South American continent hosts a wealth and diversity of palms and the Amazon contains 70% of the genus of palms of this region (Pintaud et al., 2008) Box 3.13 Bamboo, a plant of many virtues yapa in natural systems has been sustainable for the last 90 years, the availability and quality of mature palm leaves is decreasing as agriculture becomes more intensive (Pulido & Caballero, 2006). In dry forests of northwest Mexico, the recruitment of Brahea aculeata may be threatened by the harvesting and livestock grazing. Therefore management, conservation and restoration of palms require careful consideration related to human and environmental factors (Lopez-Toledo, Horn, & Endress, 2011). Box 3.14 Case study: neotropical palms Species or group Palms are one of the critical elements in the floristic composition of tropical rainforests (Abensperg-Traun, 2009; Montufar & Pintaud, 2006). The Family includes 181 genera and c. 2,450 species distributed in the tropical region worldwide, with some species that extend into subtropical areas in both hemispheres (Baker & Dransfield, 2016). The South American continent hosts a wealth and diversity of palms and the Amazon contains 70% of the genus of palms of this region (Pintaud et al., 2008) Human uses and practices Palms are renowned for their extraordinary usefulness for human communities (Borchsenius, n.d.), providing basic sustenance, construction materials, tools, and medicines. Palms are also often part of symbolic activities of indigenous communities (Macía et al., 2011). They provide valuable income for rural inhabitants (Bernal et al., 2011),(Kahn & Arana, 2008). However, at times unfavorable conditions and lack of oversight may lead to overexploitation, and possibly subsequent degradation of the local culture, the habitat, and the ecosystem. In South America, (Bernal et al., 2011) documented harvesting and management practices for 96 palm species suggest that overexploitation is common without adequate management. Non-destructive management techniques include the harvest of fruits, leaves, fibers and other parts of the plant (in high palms, users climbing the stems, and a tool is used to cut the desired part), and the destructive ones involve cutting down the palms, which is necessary, for instance, for using stems in the manufacture of building materials or for extracting palm hearts (Bernal et al., 2011). Ecological responses across manifestations of biodiversity The impacts of leaf harvesting for roofing purposes of houses and other buildings have been studied for the species Lepidocaryum tenue (Navarro, Galeano, & Bernal, 2011) and Sabal Human uses and practices Palms are renowned for their extraordinary usefulness for human communities (Borchsenius, n.d.), providing basic sustenance, construction materials, tools, and medicines. Palms are also often part of symbolic activities of indigenous communities (Macía et al., 2011). They provide valuable income for rural inhabitants (Bernal et al., 2011),(Kahn & Arana, 2008). However, at times unfavorable conditions and lack of oversight may lead to overexploitation, and possibly subsequent degradation of the local culture, the habitat, and the ecosystem. In South America, (Bernal et al., 2011) documented harvesting and management practices for 96 palm species suggest that overexploitation is common without adequate management. Non-destructive management techniques include the harvest of fruits, leaves, fibers and other parts of the plant (in high palms, users climbing the stems, and a tool is used to cut the desired part), and the destructive ones involve cutting down the palms, which is necessary, for instance, for using stems in the manufacture of building materials or for extracting palm hearts (Bernal et al., 2011). Ecological responses across manifestations of biodiversity The impacts of leaf harvesting for roofing purposes of houses and other buildings have been studied for the species Lepidocaryum tenue (Navarro, Galeano, & Bernal, 2011) and Sabal 185 mauritiiformis (Andrade-Erazo & Galeano, 2015). The impacts due to the extraction of buds for the elaboration of handicrafts and other artifacts have been assessed for populations of Astrocaryum standleyanum (García, Galeano, Bernal, & Balslev, 2013), Astrocaryum malybo (García et al., 2011), Astrocaryum chambira (García et al., 2015) and Copernicia tectorum (Torres Romero, Galeano Garces, & Bernal, 2016). Studies on the effects of the palm heart crop have been made for Euterpe oleracea (Vallejo, Galeano, Bernal, & Zuidema, 2014) (Vallejo et al., 2011). There is some research about harvesting of Euterpe precatoria fruits (Isaza, Galeano, & Bernal, 2014) and Mauritia flexuosa fruits (Sampaio, Schmidt, & Figueiredo, 2008). Socioeconomic effects Trade statistics are only well documented for species that are traded internationally, such as Euterpe oleraceae (açaí) of which Brazil is the leading supplier of palmetto and palm oil from this species (Brokamp et al., 2011). However, for local communities, personal use and informal trade of palm products are part of their primary livelihoods, allowing income creation through the commercialization of raw materials or products traded in local and regional markets. The most commercialized palms in northwestern Amazon (Bolivia, Ecuador, Peru, and Colombia) are Iriartea deltoidea (timber), Mauritia flexuosa and Oenocarpus bataua (fruit, oil), Lepidocaryum tenue (thatch), Ceroxylon spp. (religious ornaments), Phytelephas spp. (Vegetable Ivory), Astrocaryum spp. (fiber, fruit) and Euterpe spp. (Palm hearts, fruit) (Brokamp et al., 2011). Palm fruits and oils have high nutritional value, and high economic value in international markets, however competitive technologies for the extraction and processing of raw materials must be developed (Brokamp et al., 2011). Additionally, increasing economic sustainability would require strengthening value chains and the implementation of existing international and national legislation. This can be quite complicated in countries of South America where there are contradictions between national legislation and the rights of indigenous peoples, as well as the lack of technical and operational capacity of public institutions to control and verify compliance with the rules (de la Torre, Valencia, Altamirano, & Ravnborg, 2011). While implementation of standards that regulate the extraction of forest products is inconsistent, successful examples do exist (Ceroxylon spp.) and sustainable harvest is encouraged (Lepidocaryum tenue) (Brokamp et al., 2011). 3.3.2.4. Emerging issues in gathering The COVID-19 pandemic has had an enormous economic impact on people in many parts of the world, especially jeopardizing livelihoods among already economically marginalized communities. The Center for People and Forests (RECOFTC, 2020). Restrictions imposed due to COVID-19, such as limiting or prohibiting access to forests and the inability to manage land are having a noticeable (RECOFTC, 2020). In Nepal, commercial gathering of the highly- prized medicinal fungus species Ophiocordyceps sinensis (Box 3.11) was officially halted due to COVID-19. However, collectors, including many returning from India after losing their jobs, were forced to disobey orders issued by the District Disaster Management Committees and District Forest Offices to overcome humanitarian crises due to the sale of the fungus being the 186 only source of household income (Singh, 2020). In other instances, locals returned to fallow land to cultivate seasonal crops to compensate for the lack of income from fungus harvest (Samiti, 2020). Overall, the pandemic not only reduced the livelihood opportunities for mountainous communities but also substantially affected generation of revenue due to the sale of the fungus for the Nepalese (NRB, 2015) has estimated that Nepal had generated about 4.7 million United States dollars in revenue from the fungus in 2014, presenting a significant source of income for residents. The loss of income from fungus harvesting during the pandemic has therefore most likely had negative financial effects that are as of yet undocumented. During the pandemic, the biggest flow of “wildlife” in trade has involved wild plants, not animals. The volume of trade in herbal medicines is likely to increase across the world as an impact of the long-term economic crisis due to COVID-19. There have been reports around the use of herbal products as part of the COVID-19 response in Africa, Asia, Europe, South America, and the United States of America (Timoshyna, Ke, Yang, Liang, & Leaman, 2020). In the Asia-Pacific region, there has been an increase in the volume of trade in herbal products, such as those used in traditional Chinese medicine in China and neighboring countries, and Ayurveda in India and neighboring countries. It is anticipated that the number of gatherers of wild species for a variety of uses may increase as the long-term economic impacts due to COVID-19 continue to develop, especially in areas where wild harvesting correlates with high unemployment and poverty rates (Luo et al., 2020; Timoshyna et al., 2020). 3.3.2.4. Emerging issues in gathering Communities where indigenous and local knowledge is well-maintained were able to quickly pivot towards gathering wild algae, fungi and plants to cover their food and medical needs as other sources of income fell away (Walters et al., 2021). This underlines the importance of protecting indigenous and local knowledge and wild algae, fungi and plants as a social safety net (Pierce & Emery, 2005). Increased engagement in gathering to meet subsistence needs and for recreational proposes has also observed in many locations worldwide, for example in Canada, Ukraine and in the United Kingdom (Deutsche Welle, 2020; SickKids, 2020; The New York Times, 2020). Along with increased gathering there have also been reports of increasing incidence of mushroom poisonings, as more people who had not previously engaged in gathering are taking to the forests. 3.3.3.1. Introduction Terrestrial animal harvesting is defined in Chapter 1 as the temporary or permanent removal from their habitat of animals (vertebrates and invertebrates) that spend some or all of their life cycle in terrestrial environments. This definition provides a higher-level classification for several practices relating to the direct use of wild animals, most notably hunting, which results in the death of the animal being harvested, but also live capture and removal from the habitat (e.g., for the pet trade), capture and release back into the environment such as can occur with harvest of animal fiber, and practices in which products of animals are removed without intended mortality (e.g., wild honey). 187 The conceptual framework for this assessment (Chapter 1) recognizes that practices such as terrestrial animal harvesting are influenced by the end use and the associated relational values with wild species. For example, hunting is an ancient practice and continues in many contemporary societies where people hunt to meet a range of nutritional, economic, medicinal, scientific, cultural and recreational needs (A. Fischer et al., 2013; Storaas, Gundersen, Henriksen, & Andreassen, 2001). It is therefore not always possible or meaningful to assess terrestrial animal harvesting according to separate types of use, for example distinguishing the recreational aspect of hunting from other components that may be critical to an assessment of sustainable use. Even the taking of some part of the hunted animal as a memory, or 'trophy', almost never occurs on its own and needs to be considered in the context of all the other uses. Nevertheless, this section presents the evidence according to uses in order to maintain consistency throughout the chapter. Studies relating to terrestrial animal harvesting often focus on activities for particular species, referring to a wide variety of animal species that are harvested under circumstances that range from abundant to threatened and for populations that are defined as wild, introduced or feral in this assessment (Chapter 1). Ungulates are commonly hunted in many countries and are the subject of the most scientific studies but a wide range of other species are also frequently harvested, such as birds, reptiles and invertebrates (Alves & van Vliet, 2018; Barboza, Lopes, Souto, Fernandes-Ferreira, & Alves, 2016; Coad et al., 2019). For this reason, the assessment provides a summary of evidence for sustainable use of different taxonomic groups. 3.3.3.1. Introduction The assessment of sustainable use for terrestrial animals often needs to occur at a landscape level and should consider the spatial distribution and size of areas with and without use, as well as the population dynamics and dispersal behavior of the hunted species (Novaro, Redford, & Bodmer, 2000; Ohl-Schacherer et al., 2007). For example, if hunting occurs only in some parts of the landscape but not in others (due to protected status, regulation, land-use practices, or placement of human settlements) it can result in heterogeneous hunting pressure across the landscape. In these circumstances, source-sink dynamics can mean that declining population productivity in hunted areas can be compensated by constant dispersal and replenishment from areas where hunting does not occur (Koster, 2008; Novaro et al., 2000; Ohl-Schacherer et al., 2007; Peres & Nascimento, 2006; van Vliet & Nasi, 2008). In addition, economic aspects of terrestrial animal harvest seldom involve the use of just one species but more typically involve the use of a variety of species occurring in the same landscape. This means that the assessment of sustainable use should include a more integrated approach, which considers not just the taxa that are being used or the reasons they are being used, but also the social-ecological systems in which the use of animals occurs (Di Minin et al., 2021). These broader landscape and land-use aspects of use require the inclusion of additional dimensions in the assessment of sustainable use, such as governance systems and issues of land ownership, which have been identified as critical factors affecting sustainable use (Fargeot, Drouet- Hoguet, & Le Bel, 2017; Van Schuylenbergh, 2009). The evidence relating to these broader social-ecological issues is often lacking (Di Minin et al., 2021) making it difficult to assess sustainable use across multiple dimensions. In the rest of this section different types of terrestrial animal harvesting are explored. The sections are not meant to be mutually exclusive but are designed to consider sustainable use for different aspects of contemporary terrestrial animal harvesting. Scientific literature for 188 this section was obtained through a systematic literature review following the IPBES protocol. Search results are available in the data management report for Chapter 3 systematic literature review at https://doi.org/10.5281/zenodo.6452651. Perish software (https://harzing.com/resources/publish-or-perish) and Google Scholar were used for the literature search with keywords: "sustainability" or "sustainable" + hunting type (for example, "trophy hunting" or "commercial hunting"). Or "trends" or "status" + hunting type. 3.3.3.1. Introduction This search returned over 20,000 literature sources. To reduce this number a ranking/citation rate of publications was applied, and the first 50 publications were selected (as recommended in IPBES methodological guide). It should be noted that the obtained results were (1) geographically imbalanced (covering mostly certain regions of Africa or the United States of America); (2) sometimes quite old (an artifact of the methodology because older publications often have higher citation ranking); (3) Neither Perish or Google Scholar search for thematic reports. To overcome these imbalances experts supplemented the literature search from their own collections and those recommended during the external review processes. Invited contributing authors added literature for their sections following literatures searches and relying on their professional experience. 3.3.3.2. Uses 3.3.3.2.1. Cultural and religious factors influence hunting practices in all regions of the world. Wild species are an important part of cultural life since various animal parts are used as adornments in ceremonies or as ornaments (e.g., feathers and fur) and tools (e.g., bones and teeth) in daily life (Pangau-Adam, Noske, & Muehlenberg, 2012). Hunting as a ‘socio-cultural’ phenomenon involves non-market values, symbolic and social capital, social status and impacts on good quality of life (see Chapter 4). Hunting supports social interaction and community, especially in terms of creating and maintaining bonds within one’s social group, as well as benefits to physiological and psychological welfare for hunters (Bioeconomy.fi, 2017; A. Fischer et al., 2013). In many cultures across the world, hunting is associated with power, prestige and success, especially when the animals are killed in wild conditions. Europe has a diverse and complex legislative and regulatory hunting environment which includes many traditional elements (Higginbottom, 2004). In Scotland, deer stalking is part of a 150-year-old hunting culture, and continues to be one of the main activities of upland estates. Even where stalking is not commercially viable, it is a culturally important activity and has important bonding functions that help develop and reassure one’s social status (MacMillan & Leitch, 2008). Similar functions are also observed in Sweden, where moose hunting teams are organized on a voluntary basis by local hunters’ groups and landowners (Gunnarsdotter, 2007). There are long traditions of bird hunting throughout Europe. However, the only readily available data on numbers of birds legally killed across the European Union are for derogations issued under the Birds Directive. This applies to four countries: France, Italy, Malta and Spain, in which 1.39 million individual birds (11,000 doves, 448,850 finches, 430,000 larks, 3,200 plovers, 200,000 starlings and 297,200 thrushes) are legally hunted each year under these 189 derogations relating to “traditional practices” (Brochet et al., 2016) (these statistics are reported again under the section on recreational hunting). In addition, very restricted derogations are allowed for capture of living finches in some countries, such as Malta and Spain. Whether directly related or not, numbers of migratory birds in the Mediterranean region have declined substantially, with one study estimating that there are 300 million fewer farmland birds in Europe today than in 1980, primarily as a result of agricultural intensification (BirdLife International, 2008). 3.3.3.2.1. Consuming meat of wild animals may also demonstrate wealth, prestige, and social standing in some cultures, whereas in others it may be a matter of choice, taste and options. In some urban areas, wild meat is a luxury good which is marketed to and adopted by young men to boost their professional and social status (Gangale, 2016). In Papua-New Guinea, it is a tradition among the Genyem that certain animals could be only hunted by clan leaders, while others could not be killed by hunters at certain times (e.g., when their wives were pregnant) (Pangau-Adam et al., 2012). However, there is some evidence that traditional Genyem beliefs are breaking down as some species that were once considered taboo (e.g., cassowaries, certain birds-of-paradise) are now hunted (Pangau-Adam & Noske, 2010). Wild animals, mainly wild boars, are still occasionally killed for community festivals and religious ceremonies. When a large amount of meat is required for a cultural event, hunting is performed in groups and in more rural areas (Pattiselanno, 2006). Many hunters (91%) also target wild boars because the number of boar jaws they harvested was traditionally a sign of their social status. Cultural values are considered to be important drivers of biodiversity conservation and sustainable use of wild animals (see Chapter 4). Taboos represent social norms and beliefs that protect species or places because of their cultural values. Taboos have had an important implication, for example, in relation to primate conservation (Infield et al., 2018; Baker et al., 2018). However, the protection of culturally valuable species rarely extends to other species or habitats (Schneider, 2018). 3.3.3.2.2. Decorative and aesthetic uses The text in this section refers primarily to decorative and aesthetic use of wild animals, documented through formal trade data. Data are not available on informal or subsistence use of wild terrestrial animal species for these purposes. The skin of mammals is used commonly for gloves, shoes, belts, and watchbands. Over 4.6 million mammal skins from wild species were exported for commercial purposes over the period 1996-2010 and vast majority (>99%) were harvested in the wild (CITES, 2012). In Australia, the kangaroo skin industry generates 133 million United States dollars a year. In Peru, total annual value of the peccary-leather trade was estimated at 4,868,500 United States dollars of which only 1.5% was attributed to the rural sector (hunters), 11.1% to the urban sector (the national leather industry), and the majority went to the international leather industry (Bodmer & Lozano, 2001). Since 2007, the skin trade has been decreasing with the decrease in exports of fox (Lycalopex spp.) skins by Argentina (CITES, 2012). Different parts of animals can be legally exported, and legal international trade has contributed to the recovery of some species. For example, skin of peccaries from Peru (Bodmer & Lozano, 2001), kangaroo meat and animal skin from Australia (Boom et al., 2012), skin of 190 foxes (Lycalopex spp.) from South American countries (CITES, 2012), and crocodilian skins (Caldwell, 2017). Legal programs include economic incentives for people to tolerate the recovery of large predators (Fukuda, Webb, Edwards, Saalfeld, & Whitehead, 2020). Conversely, illegal international trade has contributed to the decline of many wild animal species worldwide (Pires & Moreto, 2016; ROUTES, 2020; TRAFFIC, 2008). In Amazonia, from 1904 to 1969, an average 23.3 million wild mammals and reptiles representing at least 20 species were commercially hunted for their hides; averages of 13.9 million terrestrial mammals, 1.9 million aquatic and semiaquatic mammals, and 7.5 million reptiles (Antunes et al., 2016). Hunted species included the manatee (Trichechus inunguis); capybara (Hydrochoerus hydrochaeris), ocelot (Leopardus pardalis), margay (Leopardus wiedii), jaguar (Panthera onca), neotropical otter (Lontra longicaudis), giant otter (Pteronura brasiliensis), collared peccary (Pecari tajacu), white-lipped peccary (Tayassu pecari), red brocket deer (Mazama americana), black caiman (Melanosuchus niger), common agouti (Dasyprocta spp.), Amazonian brocket deer (Mazama nemorivaga), tapir (Tapirus terrestris), iguana (Iguana iguana), tegu lizard (Tupinambis teguixin), caiman lizard (Dracaena guianensis), boa (Boa constrictor), anaconda (Eunectes murinus), and spectacled caiman (Caiman crocodilus). 3.3.3.2.2. Decorative and aesthetic uses The commercial exploitation of animal hides in the 20th century had led to population collapse for the large-bodied aquatic wild species, signaling the possibility of an “empty river” phenomenon. At the same time, various sustainability indices have shown different results suggesting that drivers other than hunting and complexity of applied models must be taken into account in assessing sustainability (Chapters 2 and 4). Several species of crocodile are harvested for the leather and fashion industry, with over 5.2 million crocodilian skins reported in trade between 2013-2015 (Caldwell, 2017). The majority of crocodilian skins in trade are from captive bred stock, although many were originally sourced from legal wild egg ranching programs. In many countries, indigenous and local people benefit through the payment of royalties for eggs, and/or employment through the farm supply chain (Fukuda et al., 2020; Joanen, Merchant, Griffith, Linscombe, & Guidry, 2021). As a result, species such as the saltwater crocodile (Crocodylus porosus) and American alligator (Alligator mississippiensis) have recovered from unregulated hunting in the 1960s and 1970s, back to pre-exploitation levels. The economic value generated through the leather industry has enabled tolerance of this recovery and protection of habitat. The sustainable use of alligators in the United States of America generates more than 100 million United States dollars annually at the raw product level (R Elsey, Woodward, & Balaguera-Reina, 2019). Feathers are used as ornaments in many cultures. Amazonian indigenous people have a very deep knowledge of birds. They invented the technique of tapirage, which is making the feathers change color on a live bird. They often tame birds that they keep as pets or for their feathers. In some countries of Amazonia there are conflicts with conservation laws that do not allow people to kill birds. In Guiana Amazonian Park (Guyane) a program is being developed to harvest feathers in zoos instead of killing birds to make headdresses. 3.3.3.2.3. Food and beverage Millions of animals are killed every year in Africa, Asia, and the Amazon for subsistence hunting and the wild meat trade (Table 3.12). The most frequently hunted taxonomic groups in 191 most studies are ungulates, followed by rodents. Large mammals alone comprised 55 - 75% of total wild meat biomass extracted annually (Table 3.12). Note that given these figures, table 3.12 focuses primarily on wild meat from mammals. Wild meat from bird, amphibians and reptiles are discussed in detail elsewhere. 192 reptiles are discussed in detail elsewhere. Table 3.12 Domestic consumption rates of wild meat from subsistence hunting. Regions and countries reported based on available literature. Region/country Harvest Main target species or taxonomic group Share of large animals Reference Tropical regions of Africa (Cameroon, Central African Republic, Republic of Congo, Democratic Republic of Congo, Equatorial Guinea, Gabon, Ghana) 340 – 84,093 kg/year per site, 16,000 kg per site, on average ungulates (47%), rodents (37%) 22% of carcasses to total kills, but 55% of total wild meat biomass extracted per year (Fa, Peres, & Meeuwig, 2002) Peru 54-255 inds/100 km2, 1605 – 4581 kg/100 km2 White-lipped peccary (Tayassu pecari), collared peccary (Tayassu tajacu), lowland tapir (Tapirus terrestris), brown capuchin (Cebus albifrons), howler monkey (Alouatta seniculus), paca (Agouti paca), agouti (Dasyprocta fuliginosa) Large mammals comprised 78% of the estimated biommass of all hunting animals (Bodmer & Lozano, 2001) Eastern half of Papua-New Guinea, Indonesia Between 4 and 8 million individuals Wild pig, cassowaries, cuscus, and bandicoots Large mammals comprised 58% of the estimated biomass of all harvested animals (Cuthbert, 2010; Mack & West, 2005; Richards, S. J. & Suryadi, S., 2000) Papua (the western half of Papua-New Guinea), Indonesia Wild meat comprised 62.2% of the total animal protein consumed by families Wild pig, rusa deer, bandicoots Large mammals comprised 75% of estimated biomass of all harvested animals (Pangau-Adam et al., 2012) India India population ate an average of 0.158kg of meat per month Barking deer, Wild pig, Asiatic black bear, Sambar, Serow, Assamese macaque, Goral Large mammals comprised 70 % of the estimated biomass of all harvested animals (Karanth, Nichols, Karanth, Hines, & Christensen, 2010) Vietnam More than 58% of Vietnam population ate 1kg of meat per month Wild Pig, soft-shelled turtle, Bear, Snake, Civet Large mammals comprised 50 % of the estimated biomass of all harvested animals (E.L. 3.3.3.2.3. Food and beverage Bennett & Rao, 2002) Amazonian forests 10,691 tons of wild meat might be consumed annually in Mammals, reptiles, and birds 38% of species more than 1 kg (Bahuchet & de Garine, 1990; H. El Bizri et e 3.12 Domestic consumption rates of wild meat from subsistence hunting. Region countries reported based on available literature. 192 Amazonia, the equivalent of 6.49 kg per person per year. al., 2020; Fa & Peres, 2001; Noss, 1998) Tropical forests 177-358.4 kg/km2/year on average The main taxa represented are primates (ungulates, rodents, and carnivores High harvest rates of largebodied diurnal animals (Fa et al., 2002) In West and Central Africa, wild meat consumption has increased drastically in recent decades (Wilkie, Bennett, Peres, & Cunningham, 2011). Wild meat comprised 62.2% of the total animal protein consumed by families in Papua New Guinea (Pangau-Adam et al., 2012). Estimates of wild meat consumption differs greatly – with global estimates of more than 5 million tons a year (Kanagavel, Parvathy, Nameer, & Raghavan, 2016) to separate regional estimates of 4.6 million tons in the Congo Basin and 1.3 million tons a year in the Amazon (Rosie Cooney, Roe, Dublin, & Booker, 2018). Wild meat comprised 62.2% of the total animal protein consumed by families in Papua New Guinea (Pangau-Adam et al., 2012). For scale of comparison, it is worth noting that if global wild meat consumption is roughly 5 million tons a year, this is only equivalent to approximately half of the European Union's beef production (Fa et al., 2002; Nasi, Taber, & Van Vliet, 2011). In semi-arid regions (South America, Africa, Asia), mammal meat is crucial for the nutritional well-being of many human communities especially because the availability of fish or other sources of protein are limited (Barboza et al., 2016; da Silva Santos et al., 2019). In this ecoregion, wild meat can be especially important during the frequent drought periods, a typical phenomenon in these areas, when crops are scarce and domestic animals may die because of starvation and dehydration (Barboza et al., 2016). Within a vast savanna ecosystem, about 50 million people depend to varying extents on wild species for their food security and daily subsistence (Olivero et al., 2016). A significant part of the population, often poor and rural, hunts for their own consumption and as a primary source of income by supplying food to more or less distant consumption centers. 3.3.3.2.3. Food and beverage Crocodile and alligator meat is considered a delicacy (Huchzermeyer, 2003a, 2003b), and it is particularly consumed in Australia, South Africa, Thailand, Ethiopia, Cuba, and in some regions of the United States of America (Hoffman & Cawthorn, 2012). The consumption of snakes is generally opportunistic, but in Asian countries and West Africa, these animals are important sources of meat (S. E. Brooks, Allison, Gill, & Reynolds, 2010; Hoffman & Cawthorn, 2012). Although amphibians are consumed on a smaller scale than vertebrates, Mohneke et al. (2009) highlight that at least 32 amphibians (3 Urodela spp., 29 Anura spp.) are used as food. Many investigations showed that the crucial factor explaining target species preferences is the anticipated benefits from hunting the largest-bodied animals. Usually opportunistically hunted small and medium-sized game are consumed by the hunters themselves, especially in low-income countries throughout Africa, Asia, South America and Eastern Europe (Fischer et al., 2013). In contrast, big game provides a greater return for the energy invested in hunting, more meat for consumption, and significant revenue for hunters’ households (Coad et al., 2013; Constantino, 2016; de Albuquerque et al., 2012; D. J. Ingram et al., 2015; P. Lindsey, Balme, Booth, & Midlane, 2012; Maisels, Keming, Kemei, & Toh, 2001; Nasi et al., 2008; Pangau- Adam et al., 2012; Redmond, 2006) with a few exceptions due to underdeveloped markets (MacMillan & Leitch, 2008). It should be noted that while it may seem that hunting larger animals is energetically more efficient, large game are infrequently acquired (there are higher number of unsuccessful days) and storage is often a problem. Furthermore, it is typically a riskier activity. In some traditional small band societies (e.g., the San, the Hadza, the Ache, various Native American and First Nation peoples) small game and plant resources are more regularly gathered as primary sources of protein and daily nutrition (Hawkes, O’Connell, & Blurton Jones, 2001). Over-harvesting may take place due to the lack of knowledge or monitoring, lack of sufficient regulation, or lack of political will and prioritization of conservation. 3.3.3.2.3. Food and beverage Even before commercial sale of meat, heads, legs and intestines of harvested animals are typically removed (~1–5 kg per animal) for family consumption prior to transporting the prime meat cuts to the market (Pangau-Adam et al., 2012). Profound social-economic changes (the introduction of a cash market economy through globalization, combined with rapid urban and infrastructure development) have resulted in marked shifts in hunting practices of many indigenous and local communities. The nature of hunting has changed from local-level subsistence hunting towards more intensive commercial hunting for wild meat trade (Pangau-Adam et al., 2012). For many rural families, wild meat trade is the main source of cash income, providing access to modern services and basic necessities such as medicines, energy and education (Abernethy, Maisels, & White, 2016). However, increases in commercial harvesting of wild species threatens the traditional lifestyles of indigenous populations through the weakening or loss of traditional laws and taboos, which may push hunting activities towards becoming unsustainable (Pangau-Adam et al., 2012). In tropical forests, harvesting of wild meat by forest dwellers has drastically increased recently due to large numbers of urban consumers, advances in hunting technology, scarcity of alternative sources of protein, and individual food preferences (Fa & Brown, 2009; Groom, 193 Meffe, & Carroll, 2006). In competition with these families, professional hunters and/or traders organize illegal networks to transport and sell the products (Van Schuylenbergh, 2009). Reptiles and amphibians also serve as an important source of protein for human populations (Coad et al., 2019). Of all reptiles, turtles and tortoise species (chelonians) are most heavily harvested for human consumption (Alves, Gonçalves, & Vieira, 2012; Pezzuti, Lima, da Silva, & Begossi, 2010). Live animals (e.g., turtles, tortoises, and lizards) as well as processed, dried, and frozen meat (e.g., pangolin) are commonly traded into food markets for consumption (see routespartnership.org). In South America, the giant Amazon River turtle (Podocnemis expansa), the largest South American river turtle, is one of the most consumed species. Caiman meat (as other crocodilians) is a product that is increasing in acceptance in the world food market. Currently there is a supply of meat from many managed areas in Argentina, Bolivia, Brazil and the United States of America (Piña, Lucero, Simoncini, Peterson, & Tavella, 2017). 3.3.3.2.3. Food and beverage In these cases varying degrees of hunting pressure often result in faunal biomass collapses, mainly through declines of large-bodied species with low intrinsic rates of population increase, as was the case in Oceania (Pangau-Adam et al., 2012), Africa (Coad et al., 2019; Gill, Fa, Rowcliffe, & Kümpel, 2012; Groom et al., 2006; Ingram et al., 2015; Milner-Gulland & Bennett, 2003; Van Vliet, Milner-Gulland, Bousquet, Saqalli, & Nasi, 2010; van Vliet, Muhindo, Kambale Nyumu, Mushagalusa, & Nasi, 2018; van Vliet & Nasi, 2008; Weinbaum, Brashares, Golden, & Getz, 194 2013), and Asia (Bennett & Rao, 2002; Karanth, Jain, & Mariyam, 2017). As populations of larger animal decline, the time and effort required to catch these large species will eventually outweigh the potential gain, leading hunters to shift to target mid-size and small species (Jerozolimski & Peres, 2003). Throughout this process, the largest species of a multispecies hunt will continue to be opportunistically captured whenever possible, preventing large species recovery even when the primary target is now a smaller species (Robinson & Bennett, 2004). Unsustainable hunting for human consumption is only one factor affecting declines in mammalian species (Figure 3.46), (Alves, 2012; Chapman & Peres, 2001; Dirzo et al., 2014; IUCN, 2016; Ripple et al., 2014; Ripple et al., 2016), but is especially prevalent in tropical environments (Coad et al., 2019; Fa et al., 2002; Fa, Ryan, & Bell, 2005; Weinbaum et al., 2013). Overall extraction rates in the Congo Basin, for example, were calculated on the basis of extraction–production models to be as much as six times greater than the maximum sustainable rate (Fa et al., 2002). Fossil evidence suggests that hunting has contributed to the local extinction of several species of larger mammals in New Guinea in the past (Flannery, 2000). Ripple et al. (2016) found that 301 mammals are threatened by hunting globally: 113 species in Southeast Asia (13% of all threatened mammals are east of India and south of China) and 61 in the rest of Asia (7%); 91 in Africa (8%); 38 in Latin America (3%); and 32 in Oceania (7%). . Unsustainable hunting has been identified as a threat for 1,341 wild mammal species assessed by the International Union for Conservation of Nature, including 669 species that were assessed as threatened. 3.3.3.2.3. Food and beverage In the absence of effective governance, many experts continue to focus primarily on total offtake from an area, suggesting that as long as hunting is profitable, the largest animals will be driven to local extinction by hunters (Branch et al., 2013; Harrison et al., 2016; Lindsey, Alexander, Balme, Midlane, & Craig, 2012). These drivers are discussed in detail in Chapter 4. In the absence of effective governance, many experts continue to focus primarily on total offtake from an area, suggesting that as long as hunting is profitable, the largest animals will be driven to local extinction by hunters (Branch et al., 2013; Harrison et al., 2016; Lindsey, Alexander, Balme, Midlane, & Craig, 2012). Wild meat consumption (Table 3.12) and trade carry health risks related to the transmission of zoonotic diseases to humans through handling (e.g., hunters, middle market distributors, and sellers) or consumption of wild meat. This is especially of concern at traditional food markets when wild animals are caged, and then slaughtered and dressed in close proximity to the public (OIE, WHO, & UNEP, 2021). The emergence of new infectious diseases, particularly zoonoses (derived from animals), is increasing. With regards to commercial demand for wild meat, there is growing demand in cities stimulated by migration of rural peoples to urban landscapes (Bennett et al., 2007). There is evidence that the commercial trade of wild meat has heavily increased offtakes in West and Central Africa because of the higher prices likely to be paid by urban dwellers, with the situation anticipated to worsen as populations continue to rise and become more urbanized. A similar trend is apparent in Eastern and Southern Africa, where increasing urbanization is associated with a growing consumption of wild meat resources (Barnett, 2000; Cowlishaw, Mendelson, & Rowcliffe, 2004; Peter Lindsey & Bento, 2012). The demand of game meat in many European Union countries is also growing due to beliefs that it is a more ecological and ethical choice consistent with ideas of the green transition. The demand for wild meat in many developed countries among the diaspora communities from developing countries has also created new demand for international trade in wild meat (Chaber, Allebone-Webb, Lignereux, Cunningham, & Rowcliffe, 2010). Economic incentives and unclear rules and regulations may be leading to additional commercial hunting on indigenous lands (Fischer et al., 2013; Pangau-Adam et al., 2012). 3.3.3.2.3. Food and beverage Nearly 20% of the International Union for Conservation of Nature Red List’s threatened and near threatened species are directly linked to hunting (Maxwell, Fuller, Brooks, & Watson, 2016). Eleven of the 14 species of tree-kangaroos (Dendrolagus spp.), most of them endemic to New Guinea, and two of the three cassowaries (Casuariidae; 25–60 kg), are now considered threatened by, or vulnerable to extinction, principally due to hunting (IUCN, 2016; Stattersfield, Crosby, Long, Wege, & Rayner, 1998). Increasing commercial demand, availability of sales markets (Pangau-Adam et al., 2012), lack of adequate monitoring and enforcement by the government (Pangau-Adam et al., 2012), poaching and illegal trade (Pangau-Adam & Noske, 2010) further complicate this process. 195 Figure 3.46 The percentage of species threatened by hunting for human consumption and other threatened species in each mammalian order. Values on the x-axis refer to the percentage of species out of all mammal species in each order. The category “Other threatened species” consists of the other threatened mammal species where hunting for consumption is not a primary or major threat. Horizontal bars are sorted from highest to lowest total percentage of threatened species in each order. Numbers on the y-axis after the order names are the number of species threatened by hunting followed by the total number of species in the order. The order Notoryctemorphia (marsupial moles) was omitted as it contains only data-deficient species. Source: (Ripple et al., 2016) under license CC BY 4.0. Figure 3.46 The percentage of species threatened by hunting for human consumption and other threatened species in each mammalian order. Values on the x-axis refer to the percentage of species out of all mammal species in each order. The category “Other threatened species” consists of the other threatened mammal species where hunting for consumption is not a primary or major threat. Horizontal bars are sorted from highest to lowest total percentage of threatened species in each order. Numbers on the y-axis after the order names are the number of species threatened by hunting followed by the total number of species in the order. The order Notoryctemorphia (marsupial moles) was omitted as it contains only data-deficient species. Source: (Ripple et al., 2016) under license CC BY 4.0. The sustainability of wild meat hunting is increasingly driven by social-economic changes, recreation, entertainment, trade, and trafficking, rather than take-off for subsistence. 196 These drivers are discussed in detail in Chapter 4. 3.3.3.2.3. Food and beverage In Papua, Indonesia, the anticipated financial gain for a hunter from the sale of three individual wild animals (35–50 United States dollars each) is approximately equivalent to the monthly salary of a locally employed permanent worker (Pangau-Adam et al., 2012). In Central Amazon, Brazil, wild species hunting and consumption are driven by many factors such as source of income, taste preference, culture, lack of alternative meat, meat price, and wealth. The relative importance of these factors varies from place to place (Chaves, Valle, Tavares, Morcatty, & Wilcove, 2021).  Amphibians and Reptiles p p Amphibians and reptiles were historically harvested and traded for different reasons. For example, tortoises, large freshwater turtles, sea turtles, and crocodilians were used as an important source of protein for human populations around the world (Klemens & Thorbjarnarson, 1995; Pritchard, P.C.H., 1996; Schlaepfer, Hoover, & Dodd, 2005). Exploitation of these species for food is heaviest in the tropical and sub-tropical regions, but also occurs in temperate areas also. Amazonian markets, for example, include the domestic consumption of wild meat and turtle eggs and the use of crocodile parts and products in the international leather industry. In examples such as these the mixed-use nature of terrestrial animal harvesting is apparent: where meat consumption is a by-product of the commercial skin 197 harvest of crocodilians, snakes, and lizards (Gorzula, 1996; Schlaepfer, Hoover, & Dodd, 2005). The United States of America plays a major role in the international trade of wild amphibians and reptiles. During 1998–2002 in the United States of America alone, 14.7 million wild-caught whole amphibians, 5.2 million kg of wild-caught amphibians and 18.4 million wild-caught reptile parts and products were imported, and 26 million wild-caught whole reptiles were exported ( Schlaepfer, Hoover, & Dodd, 2005). The crocodilian harvest programs in the United States of America (alligator) and Australia (saltwater crocodile) are highly regulated and monitored, with a coordinated system of permits, licenses, and rigorous tagging and export requirements (Elsey et al., 2019; Fukuda et al., 2020; Joanen et al., 2021). More than 50% of all traded individuals of reptiles had no species-specific identification, making species-based regulation especially difficult without extensive genetic testing, which is temporally and financially unrealistic. Crocodilian meat is particularly favoured in Southeast Asia. The top species traded for meat are C. niloticus and C. siamensis, with trade peaking annually in 2006 at 1000 tonnes (Caldwell, 2017). The most commonly traded species of amphibians and reptiles are abundant, widely distributed, and have long histories of sustaining use and trade, with varying degrees of regulation matched to their life history parameters. A species with a large range, high density, and high reproduction rate, for example, may be able to sustain a relatively large harvest. In contrast, species with restricted ranges, high levels of endemism (e.g., small island species), or life-history strategies that depend on high adult survivorship like many turtle and tortoise species (e.g. Heppell, 1998), could be detrimentally affected by relatively low harvest rates.  Amphibians and Reptiles Many amphibian and reptile species aggregate in small areas during breeding or hibernation, making them particularly vulnerable to intensive harvest efforts during that period (Klemens & Thorbjarnarson, 1995; Schlaepfer, Hoover, & Dodd, 2005). Frog meat is considered a delicacy in many countries. The FAO has estimated the worldwide production of frog legs at 80,000 metric tons annually (FAO, 2012a). In Europe, there are 4600 tons of frog meat imported per year, corresponding to c.a. 46 million frogs, mainly coming from Indonesia and Vietnam, where they are predominantly harvested from the wild (Warkentin, Bickford, Sodhi, & Bradshaw, 2009). Human populations from Southeast Asia are estimated to be the largest producers and consumers of amphibians worldwide, even if there is a lack of proper evaluation for comparative purposes (Warkentin et al., 2009). In his book “The culinary herpetologist”, Liner (2005) cites cooking recipes based on 26 salamander and 193 frog species; only a few of these edible species are consumed in large quantities. At the same time, edible amphibian populations are declining worldwide and humans have already faced the risk of losing this food source due to the overexploitation of animals harvested from nature (Carpenter et al., 2007; Carpenter, Andreone, Moore, & Griffiths, 2014). India, followed by Pakistan and Bangladesh, banned the export of frogs in the early 1980s (Fugler, 1985). More recently, Turkish authorities have banned frog hunting in some provinces and advocated the promotion of frog farming (Şereflişan & Alkaya, 2016). Frog farming is already quite extensive in Indonesia, where many exotic and invasive species are harvested for international trade. Indonesian exports of frogs were 28 tons per year in 1969 and increased to 5600 tons in 1992 before decreasing to about 3800 tons in the early 2000s 198 (Kusrini & Alford, 2006). Sustainable frog farming is lagging behind in major consumer countries, the first frog farm in France opened only in 2009. Unlike in Indonesia, in Africa frogs are mainly used for local consumption and local trading. A long-standing tradition of frog hunting exists in the Lake Chad basin that relies on large populations of grassland frogs (Ptychadena trinodis), edible bullfrogs (Pyxicephalus adspersus), African tiger frogs (Hoplobatrachus occipitalis) and the marbled shovelnose frog (Hemisus marmoratus) (Seignobos, 2014). In West African countries, six species of frogs are among the most consumed and sold frog species (Mohneke, 2011; Mohneke, Onadeko, Petersen, & Rödel, 2010).  Amphibians and Reptiles Studies carried out in Benin and Nigeria showed that between both countries and over 2.7 million frogs are harvested annually for cross-border trade (Mohneke, 2011). In Central Africa, goliath frog (Conraua goliath) and slippery frog (Conraua robusta) are heavily harvested from the wild and sold in local wild meat markets in Cameroon (Gonwouo & Rödel, 2008; Herrmann, Babbitt, Baber, & Congalton, 2005). Similarly, frog species harvested from the wild contribute to the local supply chain including markets and restaurants in the Democratic Republic of Congo (Sandra Altherr, Goyenechea, & Schubert, 2011). Large tadpoles of endemic species such as Conraua sp., Trichobatrachus sp. and Astylosternus sp. are also harvested and traded for consumption (Gonwouo & Rödel, 2008; Mohneke, 2011). Despite the importance of these small wild animals, assessments of the value chains of which they are a part are scant, especially in many Central Africa regions.  Edible Insects There are a considerable number of reports on the need for forest conservation using edible insects. They are important sources of food in arid and semi-arid areas of Africa and in the great sandy deserts of Australia (Yen, 2009). Traditional consumption of edible insects and small terrestrial invertebrates is common in one third of the world's population, mainly in Asian, African, Central American and South American cultures. Globally, more than two thousand identified arthropods are eaten (Arnold van Huis, 2018). Over 500 species of edible insects are reported for Mexico (Ramos-Elorduy, Pino-Moreno, & Martínez-Camacho, 2012) and 324 species of insects from 11 orders are documented as either edible or associated with entomophagy in China. People also feed insects to livestock and indirectly consume them (Feng et al., 2018). People throughout higher income countries in Europe and North America are contemplating using of edible insects as an alternative, more sustainable source of protein than animals (Mlcek, Rop, Borkovcova, & Bednarova, 2014) (Figure 3.47). 199 Figure 3.47 Recorded number of edible insects, by country. This map is directly copied from its original source (Tiencheu & Womeni, 2017) and was not modified by the assessment authors. The map is copyrighted under license CC BY 3.0. The designations employed and the presentation of material on the maps used in the assessment do not imply the expression of any opinion whatsoever on the part of IPBES concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. These maps have been prepared or used for the sole purpose of facilitating the assessment of the broad biogeographical areas represented therein and for purposes of representing scientific data spatially. Figure 3.47 Recorded number of edible insects, by country. This map is directly copied from its original source (Tiencheu & Womeni, 2017) and was not modified by the assessment authors. The map is copyrighted under license CC BY 3.0. The designations employed and the presentation of material on the maps used in the assessment do not imply the expression of any opinion whatsoever on the part of IPBES concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries.  Edible Insects These maps have been prepared or used for the sole purpose of facilitating the assessment of the broad biogeographical areas represented therein and for purposes of representing scientific data spatially. Globally 92% of insect species used by people are harvested from the wild (Alan L. Yen, 2015). Edible insects are often harvested by women and minority groups (A. van Huis & Oonincx, 2017; Arnold van Huis, 2018; Arnold van Huis et al., 2013), but not exclusively. Insects are often harvested by hand. Some examples of harvest techniques are included here. The most common technique to harvest swarming termites and edible grasshoppers (Ruspolia differens) is using light sources at night. In Central African countries, women listen to trunks of the palm trees to check whether larvae of the palm weevil (Rhynchophorus sp.) are ready to be harvested (van Huis & Oonincx, 2017; van Huis, 2018). This also happens in Colombia, but it is usually the men who search and find the larvae in palm trees (Oenocarpus bataua, Oenocarpus bacaba in most cases). In many cases they cut down the palm (Mesa & Galeano, 2013) to harvest the insects. In the Venezuelan Amazon, the Jöti people manipulate Oenocarpus bacaba palms in order to increase abundance of their favorite palm weevil, Rhynchophorus palmarum (Choo, Zent, & Simpson, 2009). In the Asia Pacific, tarantulas (Haplopelma sp.) are harvested out of tropical forests. Yen and Ro (2013) observed that skilled spider hunters are able to harvest several hundred spiders a day, although how this is done is 200 undocumented. Only female spiders are cooked and eaten. This may be because the females are larger. The income from selling spiders for food and medicine is substantial enough that this can be considered an important subsistence practice. Although reports suggest a decline in the population around Skun and other provinces is observed, direct causality from human harvesting has not been proven. There is little additional information available on the biology or population status of this species. The life cycle and host plants of edible caterpillars are well understood by local communities and this knowledge is communicated orally over generations. A survey of 39 ethnic groups, covering 21.4% of the all-ethnic groups in the Amazon basin, identified 115 edible insects with 131 local names.  Edible Insects An additional 384 local names of edible small invertebrates could not be identified, indicating that local traditional knowledge was richer than the scientific understanding at the time (Paoletti, Buscardo, & Dufour, 2000). Traditional land owners have, in most cases, developed harvesting protocols and habitat management practices that ensure sustainability (Yen, 2009). Traditional regulation of caterpillar harvesting in northern Zambia involves several aspects. Local people monitor development and abundance of edible caterpillars, changes in caterpillar habitats, protection of host plants and moth eggs against late bush fires and temporary restrictions on harvest of edible caterpillars (Mbata, Chidumayo, & Lwatula, 2002). Local knowledge also involves an understanding of processing to remove toxins that make inedible insects edible. In agricultural systems, chitoumou (Cirina butyrospermi) are harvested. The time of harvesting, eating and selling of these caterpillars (so called ‘chitoumou wakati’) varied greatly in different areas and from year to year. Women consider caterpillars and shea nuts to be their primary income sources (Payne, Badolo, Cox, et al., 2020; Payne, Badolo, Sagnon, et al., 2020). Harvesting caterpillars has increased food security, although this is often from increased income from sale of fresh or dried caterpillars, rather than direct consumption. Insects on the whole are vulnerable to overharvesting, habitat destruction, pesticides and other pollution and to climate change (Arnold van Huis et al., 2013). For instance, habitat destruction has an impact on the availability of edible caterpillars (Eucheira socialis) in mountainous regions of Mexico. Problems can also arise when there are market demands that encourage non-specialist harvesters to harvest. In Australia, the use of edible insects by traditional indigenous owners has decreased significantly since European settlement. This is due in part to the displacement of indigenous people, the loss of traditional knowledge and language, and the adoption of a European diet. The harvest of edible insects, particularly in relation to nature-based tourism, now has implications for overharvesting in Australia ( Yen, 2009). This is also the case of escamoles (Liometopum spp.) in Mexico where edible ant larvae with a high market value were affected when non-local people harvested them for profit (Ramos-Elorduy, 2006). During the last five years the scientific interest and knowledge on insects as food has grown exponentially (van Huis, 2020). The industrial sector is increasingly engaged in rearing, processing and marketing of edible insects. 3.3.3.2.4. Recreational hunting Recreational hunting refers to practices where the purpose of the hunt is for the hunter’s own personal use and enjoyment as opposed to harvesting for commercial or subsistence use (which are dealt with in section 3.3.3.2.3). Hunting is broadly considered as one way in which nature contributes to human wellbeing in a variety of context specific ways (Díaz et al., 2018) and recreational hunting may be associated with a range of values and motivations, including food, social and cultural motivations, sport and exercise. As in all forms of hunting, there is a high degree of multi-functionality (sensu Fischer et al., 2013). For example, a Scandinavian moose hunter may hunt in order to secure a year’s supply of wild meat, in order to enjoy time exercising outdoors in the forest during autumn, to enjoy time spent socializing with family members or friends that make up his hunting team, to maintain the cultural tradition of harvesting natural resources by hunting in a forest, to help regulate the size of the moose population so that damage to commercially harvested tree species and traffic collisions is kept to acceptable levels, and for the possible chance to bring home a “trophy” set of antlers. Depending on if the hunter is a landowner, he/she may also have commercial interests via the sale of meat or hunting licenses (Fischer et al., 2013; Storaas et al., 2001). The range of values associated with recreational hunting is reflected in the many terms linked to recreational hunting in the literature, including inter alia sport hunting, hunting tourism, safari hunting, trophy hunting, and big game hunting, or the use of terms associated with the hunting of particular species like deer hunting or duck hunting. Although these terms are sometimes used as synonyms, there is no agreed typology and the same terms can have different meanings in the literature, which can confound any attempt at synthesizing the evidence on sustainable use. The International Union for Conservation of Nature Red List uses the term ‘sport hunting’ for hunting where the end use is for the “collection and preservation of dead specimens for personal pleasure” (IUCN, 2020a). This definition is close to the definition of trophy hunting (see following paragraph) but differs from other interpretations where the term sport hunting/shooting is meant to differentiate it from market or commercial hunting and therefore covers a broader range of end uses.  Edible Insects The use of insects as human food (or as food supplements) or for feeding poultry and fish can contribute to more energy-efficient food production and promote environmental protection. An assessment conducted by the FAO concluded that insects represent a potential sustainable food source to address global food 201 security concerns (van Huis et al., 2013). However, insects could pose several microbiological and chemical health risks, which must also be considered (Imathiu, 2020). 3.3.3.2.4. Recreational hunting For example, grouse shooting in Scotland and England is regarded as sport shooting (Tharme, Green, Baines, Bainbridge, & O’Brien, 2001). This is an important distinction when trying to identify and interpret data sources for this assessment. The definition of recreational hunting used here encompasses all forms of hunting where the primary purpose is not subsistence or the commercial harvest of animals. The term “trophy” hunting is a non-technical label that has been used for hunting practices where one of the end products is a photograph and/or the preservation of the whole or part of the hunted animal (i.e., a “trophy’). Within the context of recreational hunting, trophy hunting is often used for hunting practices where client hunters pay high prices to shoot particular species or individuals with particular attributes, e.g., large horns. There are therefore certain ecological, social and economic considerations that differ from other forms of recreational hunting. 202 There is a large amount of academic literature on the sustainability of recreational hunting and active management strategies for maintaining this practice. However, only a limited number of these studies contain well-argued, data-driven evidence. A recent assessment of recreational hunting (Di Minin et al., 2021), using a similar protocol to IPBES assessments, identified 1342 relevant references but still concluded that “despite the extensive literature on recreational hunting, the evidence to address some of the most pressing academic and societal questions is still limited”. Crucially, this included a paucity of evidence on critical policy relevant questions about when recreational hunting is sustainable and who benefits from it. One consequence of the limited information is that conclusions often reflect the value system, community status (“outsiders” versus “locals”), and professional background of the authors (Houdt et al., 2021; Mkono, 2019; Nordbø, Turdumambetov, & Gulcan, 2018). Despite the limitations of the available literature and the challenges with assessing recreational hunting as a form of sustainable use, some of the key points raised in the literature are discussed further in this section. The section first outlines differences in approaches across IPBES geographic regions and then examines evidence for various aspects relating to the sustainability of recreational hunting.  An overview of recreational hunting across IPBES regions There is no global database of countries where recreational hunting occurs but several sources indicate that it is widespread. 3.3.3.2.4. Recreational hunting Species assessed for the International Union for Conservation of Nature Red List, and where sport hunting has been identified as a use, come from all major IPBES regions. Academic studies of recreational hunting have been conducted in 147 countries (Di Minin et al., 2021) indicating that the practice takes place in a large number of countries spread across all IPBES regions. There is considerable variation in the way that recreational hunting is governed and administered in different regions, especially relating to whether recreational hunting is allowed, whether it is regulated, who owns the wild species (government or private), who owns the land where the hunt takes place (private, public or communal lands), who can hunt (residents vs foreigners), how the hunt is managed (with an outfitter or community involvement), whether the use is purely personal or the hunted animal can be sold, who issues the licenses, whether there are bags or quotas for target species, what monitoring systems are in place, and whether the revenue from hunting is retained by landowners. These factors all have important implications for assessing sustainable use. It is not possible to provide a detailed analysis for all countries but some of the major aspects relating to each IPBES region are presented below. Americas There are important policy differences regarding recreational hunting across the Americas. The practice is mostly not encouraged in Central and South American countries and legislation to prohibit recreational hunting exists in at least Colombia, Costa Rica and Brazil. Some South American countries allow recreational hunting of introduced animals (Argentina) and recreational hunting has been recorded as a use for at least 39 species of birds and mammals endemic to the region (IUCN Red List 2021). Despite prohibitions on recreational and other forms of hunting in South America, it is regarded as widespread and under-researched 203 (Petriello & Stronza, 2020). An analysis of online videos showed that recreational hunting occurs frequently in Brazil (El Bizri, Morcatty, Lima, & Valsecchi, 2015) and is regarded as a part of local culture (Bragagnolo et al., 2019). In contrast, recreational hunting of wild animals is allowed in Canada, United States of America and Mexico where there also are active communities of hunters. In Canada there are an estimated 1.3 million hunters (Conference Board of Canada, 2018) whereas in the United States of America there were 11.5 million hunters in 2016, down from 37.8 million in 2001 (U.S. Department of the Interior, 2017). Recreational hunting occurs across large parts of Canada and the United States of America where the practice is allowed on private and public lands. The United States of America Department of Interior noted that hunting was permitted in “76 areas managed by the National Park Service, 336 national wild species refuges and 36 wetland management districts managed by the United States of America Fish and Wildlife Service, and over 220 million acres (890 000km2) of managed public lands” (U.S. Department of the Interior, 2017). In the United States of America, regulated recreational hunting has been an integral part of the North American model of wild species conservation, providing social and political support as well as financing for wild species management activities (Arnett & Southwick, 2015; P. Mahoney & Geist, 2019). The early phase of North American wild species management concerned halting and reversing wild species decline, but more latterly the focus has been to manage populations within a ‘social carrying capacity’ (Heffelfinger, Geist, & Wishart, 2013). Wild species in the United States of America “owned” by state governments and hunting, are administered by State Fish and Wildlife Departments on both public and private land. Americas However, it is estimated that >60% of hunting days occur on private land, which can present challenges in prescribing the legal relationships between publicly owned wild species and privately owned land (Freyfogle & Goble, 2019). The financing, management and governance of this land is under-studied (Poudyal, Bowker, Green, & Tarrant, 2012). Hunting is generally open to residents, with low priced hunting tags providing access to most prospective hunters. The sale of wild species meat and other products is illegal, and exchange is usually personal. Hunting revenues form part of a publicly managed and funded system where part of the budgets for all fifty State Fish and Wildlife Agencies is derived from user fees, including hunting and fishing licenses and federal excise taxes on hunting/fishing equipment (Arnett & Southwick, 2015). Through the Pittman-Robertson Act (Federal Aid in Wildlife Restoration Act of 1937), there is an 11 percent excise tax on the sale of firearms and ammunition products. The funding paid into the Wildlife Restoration Trust Fund provided an average of 751 million United States dollars annually from financial year 2012 to 2018 (P. Mahoney & Geist, 2019). Total expenditures on hunting decreased from 36.1 billion United States dollars in 2011 to 26.2 billion United States dollars in 2016, in line with declines in the number of hunters (U.S. Fish and Wildlife Service, 2016). Africa Africa is the only continent that retains its full spectrum of Pleistocene wild species (Ripple et al., 2015) but there are substantial differences across Africa in the abundance of wild species, the way that wild species is managed, whether hunting is allowed, and the conditions regulating recreational hunting. Recreational hunting is not permitted in Kenya whereas it is allowed in 204 many other African countries. Recreational hunting is recorded as a use for at least 90 species of mammals and birds across Africa (IUCN Red List 2021), and recreational hunting opportunities are advertised on the internet for at least nine countries in Southern, East and West Africa. In African countries where recreational hunting is allowed, large areas of land may be managed partially or exclusively for hunting, especially for high paying clients. The area managed for recreational hunting, or where recreational hunting occurs, comprises as much as 26% in some countries, e.g., Tanzania (Di Minin, Leader-Williams, & Bradshaw, 2016) and has been estimated to be 1,394,000 km2 for all of Africa (Lindsey, Roulet, & Romañach, 2007) and separately as 140 000-170 000km2 in South Africa (Taylor, Lindsey, Nicholson, Relton, & Davies-Mostert, 2020) and 288 000 km2 in Namibia ( Lindsey, 2011). The revenues from hunting have been estimated at 217 million United States dollars per year for seven Southern African countries (Di Minin et al., 2016) and these revenues have been credited with funding ‘rewilding’ of commercial and communal farmlands in some Southern African countries where land conversion has been reversed or avoided by allowing regulated hunting and other uses of wild species (Child, 2019; P. Lindsey, 2011; W. A. Taylor et al., 2020). In the case of South Africa, hunting is estimated to contribute 64% of income on lands that are managed for wild species compared to live sales (28%) and nature-basted tourism (8%) (DEA, 2015). Several park agencies in Africa are at least partially funded by hunting revenues, although the percentage of revenues used to fund conservation agencies may be considerably less than what accrues to private companies managing recreational hunting (Di Minin et al., 2016). Europe and Central Asia Hunting is an integral part of the cultures and traditions of European rural society and there are estimated to be over 7 million hunters across the continent (Brainerd, 2007). The governance of hunting is often situated within the broader context of biodiversity conservation and recognizes that Europe is a biocultural system with blurred boundaries between nature and culture and wild and domestic systems (John D. C. Linnell, 2015). Some form of recreational hunting has been recorded as a use for at least 88 species of mammals and birds from across Europe (IUCN Red List 2021). In terms of European Union legislation, 82 species of birds are allowed to be hunted in the European Union (Hirschfeld, Attard, & Scott, 2019) and 13 species of mammals and seven birds have been regularly recorded in hunting bags from Central Europe (Reimoser & Reimoser, 2016). Some species such as Red deer have been valued game species for millennia (John D. C. Linnell, 2015). The recorded volumes of animals hunted every year varies from a few individuals to several million: in the bags from nine countries in Central Europe, six species comprised >100 000 individuals and wild boar exceeded one million per annum (Reimoser & Reimoser, 2016); the estimated bags for birds across Europe was 52 million (Hirschfeld et al., 2019) per year. The trend in some countries is for hunting of fewer species but for an overall increase in the biomass of hunted animals (Figure 3.48). The increase in biomass could be explained by the increase in the number of harvested ungulates which amount to approximatively 7 million every year (Linnell et al., 2020). Hunting of large carnivores may also be allowed with the aim of reducing human-wildlife conflict, maintaining stable populations, and building public support for carnivores. 205 Figure 3.48 Composition of harvested biomass (for nine European countries) in 1000 tons. Source: (Reimoser & Reimoser, 2016) under license CC BY 4.0. Figure 3.48 Composition of harvested biomass (for nine European countries) in 1000 tons. Source: (Reimoser & Reimoser, 2016) under license CC BY 4.0. Despite the apparent increase in hunting bags of some species over the past 120 years (Reimoser 2014) populations of wild ungulates have increased across Europe (Linnell et al., 2020) and this has also facilitated the recovery of large carnivores (Linnell et al., 2020; Popescu, Artelle, Pop, Manolache, & Rozylowicz, 2016). Europe and Central Asia Wildlife populations have tended to increase in Eastern Europe since 1990, especially in countries with reforms on the management of land and wild species (Bragina et al., 2018). Hunters in Europe have been credited with providing monitoring data that supplements other forms of citizen science for wild species monitoring in 32 European countries (Cretois, Linnell, Grainger, Nilsen, & Rød, 2020). There is also a long history of hunting and use of wild animals by people in Central Asia. The professional hunting economy that existed up to the 1950s gradually disappeared and was replaced by a growing number of amateur hunters. During the Soviet period, strict protected areas were imposed and some species were recovered through hunting bans (e.g., the nearly extinct saiga population). Hunting was controlled by central authorities. However, dramatic habitat loss and over-exploitation of wild species outside protected areas increased the threat to ungulates and other wild species, especially when trade liberalization after the Soviet era coincided with economic hardships and the weakening of state controls and capacities (Damm G.R., 2008). Unregulated hunting of species like markhor, combined with widespread and unregulated use of wild species for multiple purposes, has resulted in unsustainable use where poaching and sale of game meat became normal, and ungulates were reduced by poaching and rapidly increasing livestock populations (Blank & Li, 2021). The hunting sector is generally managed by government organizations through a permit system and wild species ownership remains centralized. This has replaced ancient kin-related ownership of hunting grounds, and some of the challenges associated with sustainable use of wild species have been ascribed to the lack of enforcement by state agencies and the loss of local systems of control (Blank & Li, 2021). 206  Recreational hunting and sustainable use Several studies have pointed out that an assessment of sustainable use needs to consider the social (including institutional and economic) and ecological factors affecting sustainable use ( Fischer et al., 2013), and be aware that sustainable use relating to recreational hunting is highly context specific (Di Minin et al., 2021) (Figure 3.49). This section examines evidence relating to the ecological, social and economic dimensions of sustainable use as it relates to recreational hunting. Asia and the Pacific There is limited information on recreational hunting in Asia and the Pacific although it is recorded as a use for at least 100 resident or migratory mammal and bird species across all subregions (IUCN Red List 2021). The number of recorded scientific studies of recreational hunting is very low across the region, particularly for South Asia and Southeast Asia (with fewer than 10 publications) and to a slightly lesser extent for Northeast Asia and Oceania (Di Minin et al., 2021). Recreational hunting in New Zealand and Australia focuses primarily on introduced or feral populations. New Zealand has hunting zones specifically set aside for recreational hunting. Ecological aspects of sustainable use The ecological and biological metrics used to assess sustainable use vary considerably but typically include the impact on population numbers. For bird and mammal species assessed for the International Union for Conservation of Nature Red List, and where sport hunting is identified as a use, 51% (n=620) have a declining population trend (IUCN Red List 2021). This implies that recreational hunting may not be biologically sustainable for these species. However, there are several limitations to the use of Red List data at the species level which would affect this conclusion. First, almost all the assessed species are subjected to multiple threats across different sites of which recreational hunting may only be a minor threat or a threat in only some areas of its range, so it is important to understand the context in which recreational hunting occurs. Second, the same species can be subjected to subsistence, commercial and recreational hunting and it is often not possible to disaggregate the effects of these different types of hunting. An analysis of >1000 publications specifically focusing on recreational hunting (Di Minin et al., 2021), identified 35 species that had been studied across multiple sites and these data provide a better understanding of population trends across sites. The results showed that only one species declined consistently across all sites, 11 (33%) species showed population declines in some sites but not others, and 23 (66%) species showed no decline or the results were inconclusive. These results highlight the extent of variation between sites. The study noted the geographical and taxonomic bias in published results with most of the studies focusing on a small number of mammal species mostly in Africa and North America. The paucity of evidence for many species and across other IPBES regions is important because the International Union for Conservation of Nature Red List indicates that many more species across other IPBES regions are used for recreational hunting but there is no additional information to assess sustainable use of these species. 207 208 Figure 3.49 Impact of recreational hunting on the population abundance of targeted species. Depicted is the proportion of studies that found inconclusive evidence, evidence of population declines, or evidence of no population declines. Number of studies is indicated in parentheses next to species name; only species included in more than one study are included. Abbreviations: LC: Least Concern; VU: Vulnerable; NT: Near Threatened. Small antelope refers to steenbok Raphicerus campestris, oribi Ourebia ourebi, grysbok Raphicerus sharpei, duiker Cephalophus sp. or Sylvicapra grimmia, and dik-dik Madoqua kirkii. Source: (Di Minin et al., 2021) under license CC BY-NC-ND 4.0. Figure 3.49 Impact of recreational hunting on the population abundance of targeted species. Depicted is the proportion of studies that found inconclusive evidence, evidence of population declines, or evidence of no population declines. Number of studies is indicated in parentheses next to species name; only species included in more than one study are included. Abbreviations: LC: Least Concern; VU: Vulnerable; NT: Near Threatened. Small antelope refers to steenbok Raphicerus campestris, oribi Ourebia ourebi, grysbok Raphicerus sharpei, duiker Cephalophus sp. or Sylvicapra grimmia, and dik-dik Madoqua kirkii. Source: (Di Minin et al., 2021) under license CC BY-NC-ND 4.0. For those species that have been more intensively studied, there is evidence that mammalian game species with high reproduction rates, such as roe deer and wild boar, can tolerate more intensive exploitation and still maintain population numbers and structure as well as genetic diversity (Baldus, Damm, & Wollscheid, 2008; Challender & Cooney, 2016; J D C Linnell et al., 2020; Loveridge, Reynolds, & Milner-Gulland, 2006; Tapper & Reynolds, 1996). As an example, populations of roe deer (Europe) and white-tailed deer (North America) have increased their range and density despite the intended use of hunting to reduce density-related human conflicts (Morellet et al., 2007). The evidence also shows that some populations of threatened species and those with low regeneration capacity have increased in numbers in systems where hunting is well managed (Table 3.13). Attempts to combine hunting with the effective management and conservation of such species, has taken place in several IPBES regions. Note that the examples provided in Table 3.13 apply only to the particular populations that were assessed, and not for the species generally. Table 3.13 Examples of populations of wild mammals that have recovered in areas where hunting management is in place even though global trends may be decreasing (this does not mean there is an absence of continued threats). Species name or taxonomic group International Union for Conservation of Nature species status & global trends Region or country References Black rhino (Diceros bicornis) CR -Critically Endangered, increasing Africa (Challender & Cooney, 2016; CITES, 2019; Rosie Cooney et al., 2017; NACSO, 2019) White rhino (Ceratotherium simum) NT – Near Threatened, decreasing Africa (Challender & Cooney, 2016; CITES, 2019; Rosie Cooney et al., 2017; NACSO, 2019) African lion (Panthera leo) VU – Vulnerable, decreasing Africa (P. Lindsey, Balme, et al., 2012; NACSO, 2019; Whitman, Starfield, Quadling, & Packer, 2004) Different deer species (Cervus spp.) LC – least concern, increasing Europe (e.g., Germany), United States of America, Canada, Russia (J D C Linnell et al., 2020; Mustin, Newey, Irvine, Arroyo, & Redpath, 2012; Reimoser & Reimoser, 2016) Table 3.13 Examples of populations of wild mammals that have recovered in areas where hunting management is in place even though global trends may be decreasing (this does not mean there is an absence of continued threats). Table 3.13 Examples of populations of wild mammals that have recovered in areas where hunting management is in place even though global trends may be decreasing (this does not mean there is an absence of continued threats). 209 Bighorn sheep (Ovis canadensis) LC – least concern, increasing North America and Mexico (Challender & Cooney, 2016) Markhor (Capra falconeri) NT – near threatened, increasing Asia (Rosie Cooney et al., 2017) Argali (Ovis ammon) NT – near threatened, decreasing Asia (Rosie Cooney et al., 2017) Urial (Ovis orientalis) VU – vulnerable, decreasing Asia (Rosie Cooney et al., 2017) Grey wolf (Canis lupus) LC – least concern, stable Some European countries (Epstein, 2017) Waterfowl LC – least concern North America (M. G. Anderson & Padding, 2015; Hirschfeld et al., 2019; P. Mahoney & Geist, 2019; Mustin et al., 2012; Reimoser & Reimoser, 2016) Mallard (Anas platyrhynchos) LC – least concern, increasing Europe, North America (P. Mahoney & Geist, 2019; J. D. Table 3.13 Examples of populations of wild mammals that have recovered in areas where hunting management is in place even though global trends may be decreasing (this does not mean there is an absence of continued threats). Nichols, Runge, Johnson, & Williams, 2007) Greater white-fronted goose (Anser albifrons) LC – least concern, unknown Europe (Hirschfeld et al., 2019) Phasianids (e.g., black grouse (Lyrurus tetrix) LC – least concern, decreasing Europe (Hirschfeld et al., 2019) Red-legged partridge (Alectoris rufa) LC – least concern, decreasing Europe (Hirschfeld et al., 2019) Wild turkey (Meleagris gallopavo) LC – least concern, increasing North America (Hirschfeld et al., 2019) European bison (Bison bonasus) VU – vulnerable, increasing Belarus (Артеага В., 2019) White-lipped peccary (Tayassu pecari) VU – vulnerable, decreasing South America (Bodmer & Lozano, 2001) Collared peccary (Pecari tajacu) LC – least concern, stable South America (Bodmer & Lozano, 2001) Paca (Agouti paca) LC – least concern, stable South America (Bodmer & Lozano, 2001) Agouti (Dasyprocta fuliginosa) LC – least concern, stable South America (Bodmer & Lozano, 2001) Polar bear (Ursus maritimus) VU – vulnerable, decreasing Canada (Foote & Wenzel, 2009) American alligator (Alligator mississippiensis) LC - least concern, increasing/stable United States of America (Ruth Elsey, Woodward, & Sergio Balaguera-Reina, 2018) In contrast, there is also evidence for populations where poorly regulated hunting is not sustainable and has contributed to local population declines that have reduced the number of animals that can be harvested sustainably, for example, some populations of lions and elephants in Africa (Fischer et al., 2013; IUCN, 2016; Loveridge et al., 2016; Mweetwa et al., 2018; Packer et al., 2009), brown bears in Northern Europe ( Frank et al., 2017), ungulates and Snow leopards in Asia (Rashid, Shi, Rahim, Dong, & Sultan, 2020). 210 Operationally, sound biological management is contingent on appropriate institutional, social and economic conditions. Scientists argue that biological sustainability of recreational hunting is highly connected with the proper regulation of the hunting system, including regular monitoring and adaptive management responses that adjust offtake to changes in population size (M. G. Anderson & Padding, 2015; Damm G.R., 2008; P. Mahoney & Geist, 2019; Souchay, Besnard, Perrot, Jakob, & Ponce, 2018). While these factors are important, they can also be achieved through local control and knowledge, and simple adaptive management systems (Goredema, Taylor, Bond, & Vermeulen, 2005). Some of the instances of unsustainable use have been associated with weak tenure, the centralization of revenues derived from hunting (Child, 2019) and breakdown of community governance without any effective replacement by state officials (Blank & Li, 2021). Table 3.13 Examples of populations of wild mammals that have recovered in areas where hunting management is in place even though global trends may be decreasing (this does not mean there is an absence of continued threats). Beyond population numbers, scholars have identified other biological and ecological issues that should be considered in the assessment of the sustainable use of recreational hunting. These include the indirect effects of hunting, which are often poorly known and therefore make it difficult or impossible to fully assess biological sustainability (for example Artelle et al., 2018; Frank et al., 2017; Macdonald et al., 2017; M. N. Peterson & Nelson, 2017; Popescu et al., 2016; Swenson et al., 2017). In addition, all forms of hunting can have evolutionary and behavioral consequences for the target species, affect food chains, or alter herbivory, predation and other ecological processes (Fukushima et al., 2020; Leclerc, Frank, Zedrosser, Swenson, & Pelletier, 2017). Selective harvesting of animals with particularly desirable phenotypes can also alter the distribution of traits in a population (Allen, Brent, Motsentwa, Weiss, & Croft, 2020; Coltman et al., 2003; Crosmary et al., 2013; Knell & Martínez-Ruiz, 2017; Milner, Nilsen, & Andreassen, 2007; Russo et al., 2019; Wielgus, Morrison, Cooley, & Maletzke, 2013). Features such as body size or horn shape and size, may be linked to other fitness-related attributes, including physiological tolerances or disease resistance (Crosmary et al., 2013; Knell & Martínez-Ruiz, 2017; Russo et al., 2019). Although these are important issues, the nature of available studies means that is not possible to make any firm conclusions regarding sustainable use based on these parameters (Di Minin et al., 2021). Social sustainability Humans control their use of resources through formal or informal rules or institutions. The literature suggests that the primary variable affecting the sustainability or otherwise of recreational hunting is the governance of hunting systems (Cooney, 2017) and the quality and social legitimacy of relevant institutions (Fischer et al., 2013). Analysis of a global dataset of utilized populations (not just for hunting) showed that utilized species declined more rapidly than unutilized species, but that where management systems were in place there was a positive impact on trends (McRae et al., 2022).This broad analysis did not include institutional quality as an independent variable, and it is not possible to disaggregate the data for utilization under controlled versus open-access conditions, so it is not possible to assess the impact of management in more detail. In an analysis of sustainable use, (AFischer et al., 2013) fix citation format identified two aspects of institutional misfit that affect sustainability of recreational hunting: (i) conflicts between the functions of hunting as defined by the government and functions identified by 211 local communities; and (ii) ecological functions embedded in formal institutions generated by non-local actors that are developed separately from, and in conflict with, the local institutions guiding the social and economic functions of hunting and land use more generally. One of the hypotheses is that hunting and the management of wild species become unsustainable when they are under-policed as open access resources and where wild species-based livelihoods are deinstitutionalized by land-use policies that favor agriculture farming (Bowles & Choi, 2013). These ideas have not been widely tested in hunting systems. Legal, well-regulated recreational hunting has been shown in specific instances to play an important role in delivering benefits for both wild species conservation and for the livelihoods and well-being of indigenous and local communities living with wild species (Baldus et al., 2008; Eklund T., 2017; C. Fischer, 2010). Investments from revenues generated through hunting on community conservancies have been used to improve local services such as water infrastructure, schools and health clinics, as well as providing meat for community members (IUCN, 2016; Naidoo, Weaver, et al., 2016). Nevertheless, the evidence regarding these benefits across all areas where recreational hunting occurs is lacking. Economic aspects of sustainable use Fish and Wildlife Service, 2016) Russia USD 518 million More than 5587 thousand 2.8million (Braden, 2014) South Africa EUR 0,341 billion 17,000 76000 (Saayman, van der Merwe, & Saayman, 2018) South Africa (“trophy” hunting alone) USD 181 million (Snyman et al., 2021) Canada USD 13,2 billion 107 000 More than 50,000 (The Economic Footprint of Angling, Hunting, Trapping and Sport Shooting in Canada, 2019) Sub-Saharan Africa at least USD 201 million per year More than 150000 More than 100000 (Di Minin et al., 2016) Prices paid for hunts vary from hundreds to hundreds of thousands of United States dollars (Table 3.15), and globally create a substantial revenue flow from developed to developing countries, as well as from urban to rural areas within countries (Booth, 2010; Di Minin et al., 2016; IUCN, 2016; Sánchez-García et al., 2021). Besides spending money on hunting equipment, guns, ammunition, transportation, clothing, and meat processing, hunters typically also spend large amounts of money on permits, guide and outfitting services and travel (Lindsey et al., 2007; U.S. Fish and Wildlife Service, 2016), contributing to the economies of the areas where this practice occurs, for example, on communal conservancies in Namibia (NACSO, 2015; NACSO & MET, 2018; Schmitt & Rempel, 2019). Table 3.15 Indicative information on the species hunted, the number of individuals and the costs of trophy hunts in different countries. Abbreviations: DKK: Danish Krone, EUR: euros, USD: United Staes Dollars. Country Main hunted species Individuals hunted/year Cost of the trophy Reference neighborhood of USD 460 million Austria EUR 0,475 billion More than 157 thousand 123,283 OECD.stat 2019 France EUR 3,6 billion 25,800 150-200 (P. A. Lindsey et al., 2007) Germany EUR 1,6 billion More than 617 thousand 368,664 OECD.stat 2019 United Kingdom EUR 3,2 - 5,5 billions 12,000-74,000 600,000 (Mensah & Elofsson, 2017) United States of America USD 7,978,472 million 57,251,937 11,500,000 (U.S. Economic aspects of sustainable use The economic literature on recreational hunting, specifically the generation and allocation of financial flows, tends to concentrate on two separate policy relevant questions. At a national level, total economic value is important because national policy makers are interested in economic growth, jobs and taxes and the revenue from hunting can therefore influence broader policy decisions affecting the sustainable use of wild species. At a local level, the policy question is whether the proportion of the value chain captured by the manager of land on which wild species occur is sufficient to enable reinvestment in the supply and management of wild species. Recreational hunting has been considered an important economic activity by various scholars and stakeholders where it is credited with generating revenues and creating jobs in the land management and hospitality sector, as well as providing income and other important economic and social benefits to indigenous and local people in rural, remote and/or otherwise marginal areas (Conference Board of Canada, 2018; R. Cooney, 2017; Di Minin et al., 2016; Sánchez-García et al., 2021). Economic impacts of recreational hunting can be measured in terms of gross output (revenue), sales, income, employment or value-added benefits, and a summary of the data is provided in table 3.14. While these measures are not always comparable, the table provides an indication of economic values. Table 3.14 Hunting economic output. Abbreviations: OECD: Organization for Economic Cooperation and Development, EUR: euros, USD: United States Dollars. Country Gross revenues People employed Number of hunters Reference Finland EUR 0,23 billion 100 304,245 (Bioeconomy.fi, 2017) Sweden The annual gross hunting value is estimated to be in the More than 12000 300,000 (Mattsson, 2008; Mensah & Elofsson, 2017) 3.14 Hunting economic output. Abbreviations: OECD: Organization for Economic ation and Development, EUR: euros, USD: United States Dollars. 212 neighborhood of USD 460 million Austria EUR 0,475 billion More than 157 thousand 123,283 OECD.stat 2019 France EUR 3,6 billion 25,800 150-200 (P. A. Lindsey et al., 2007) Germany EUR 1,6 billion More than 617 thousand 368,664 OECD.stat 2019 United Kingdom EUR 3,2 - 5,5 billions 12,000-74,000 600,000 (Mensah & Elofsson, 2017) United States of America USD 7,978,472 million 57,251,937 11,500,000 (U.S. Economic aspects of sustainable use Fish and Wildlife Service, 2016) Russia USD 518 million More than 5587 thousand 2.8million (Braden, 2014) South Africa EUR 0,341 billion 17,000 76000 (Saayman, van der Merwe, & Saayman, 2018) South Africa (“trophy” hunting alone) USD 181 million (Snyman et al., 2021) Canada USD 13,2 billion 107 000 More than 50,000 (The Economic Footprint of Angling, Hunting, Trapping and Sport Shooting in Canada, 2019) Sub-Saharan Africa at least USD 201 million per year More than 150000 More than 100000 (Di Minin et al., 2016) Prices paid for hunts vary from hundreds to hundreds of thousands of United States dollars (Table 3.15), and globally create a substantial revenue flow from developed to developing countries, as well as from urban to rural areas within countries (Booth, 2010; Di Minin et al., 2016; IUCN, 2016; Sánchez-García et al., 2021). Besides spending money on hunting equipment, guns, ammunition, transportation, clothing, and meat processing, hunters typically also spend large amounts of money on permits, guide and outfitting services and travel (Lindsey et al., 2007; U.S. Fish and Wildlife Service, 2016), contributing to the economies of the areas where this practice occurs, for example, on communal conservancies in Namibia (NACSO, 2015; NACSO & MET, 2018; Schmitt & Rempel, 2019). Table 3.15 Indicative information on the species hunted, the number of individuals and the costs of trophy hunts in different countries. Abbreviations: DKK: Danish Krone, EUR: euros, USD: United Staes Dollars. Country Main hunted species Individuals hunted/year Cost of the trophy Reference United States of America Deer, wild turkey, elk More than 6 million USD 2,659 (Bergstrom, 2008; Munn, Hussain, Spurlock, & Henderson, 2010) Finland moose 49,667 (Bioeconomy.fi, 2017) 3.15 Indicative information on the species hunted, the number of individuals and ts of trophy hunts in different countries. Abbreviations: DKK: Danish Krone, EUR: USD: United Staes Dollars. 213 Kyrgyzstan Snow leopard Average of 25 EUR 7,000- 10,000 (Eklund T., 2017) Middle Europe Red deer - EUR 10,000- 15,000 (Bioeconomy.fi, 2017) United Kingdom of Denmark Red deer Approx. 25,000 red-deer DKK 7,000- 25,000 (Bioeconomy.fi, 2017) Germany Red deer 9-12 EUR 1,000 without antlers, up to 5,000 with antlers (Bioeconomy.fi, 2017) Zambia Lechwe, Hippopotamus, Leopard 300 Science Direct/Statista Charts, (P. Lindsey, Balme, et al., 2012) Tanzania Leopard, Hippopotamus, Elephant 7034 Science Direct/Statista Charts, (P. A. Lindsey et al., 2007) Botswana Elephant, Leopard, Lechwe 2500 Science Direct/Statista Charts, (P. A. Economic aspects of sustainable use Lindsey et al., 2007) South Africa Impala, Warthog Kudu 53,885 Science Direct/Statista Charts, (P. A. Lindsey et al., 2007) Zimbabwe Elephant, Leopard, Chacma Baboon 11,318 Science Direct/Statista Charts, Lindsey et al., 2012(P. A. Lindsey et al., 2007) Mozambique Crocodile Elephant, 900 Science Direct/Statista Charts International (Sheikh & Bermejo, 2019) Namibia Zebra, Chacma Baboon, Leopard 22,462 (P. Lindsey, Balme, et al., 2012; Sheikh, Bermejo, & Procita, 2019) From a production perspective, recreational hunting is regarded by scholars in this area as different from other forms of harvesting. First, the commodity value is only one of many values which collectively exceed the value of the raw commodity. Second, the process of hunting is considered a benefit to the production system, whereas with harvesting it is a production cost (Child, 2019). This is expected to give the multi-value approach to managing lands for wild species use, including recreational hunting, an economic comparative advantage over simple commodity production, and therefore provides an avenue to keep natural lands intact (Child, 2019). However, these economic advantages may not be realized due to the tendency to associate property rights with domestic species but not wild ones (Bowles and Choi 2013). It should be noted that this perspective also focuses on recreational hunting in comparison with other more commercial activities such as production forestry. It is not meant to be a direct comparison with the wide range of practices reported on in other parts of section 3.3. 214 Large areas of land are managed for the production of recreational hunting. For all of Africa, this was calculated as 1 394 000 km2 (Lindsey et al., 2007), which includes 288 000 km2 of freehold land in Namibia (Lindsey, 2011) and between 170 000 and 205 000 km2 (14- 17% of total land area) in South Africa (Taylor et al., 2020). Figures for other areas comprise at least 890 300 km2 in the United States of America (Bureau of land management areas, US Dept of Interior 2017) and 1 780 km2 managed as Recreational Hunting Areas in New Zealand (Fraser & Department of Conservation, 2000), noting that in New Zealand these areas are designated for hunting introduced land mammals and that larger areas were designated for commercial hunting. Economic aspects of sustainable use The key species that generate the largest proportion of income through recreational hunting tourism in Africa are: elephants in Mozambique, Namibia and Zimbabwe, African buffalo in Tanzania, and sable antelopes (Hippotragus niger) in Zambia (P. A. Lindsey et al., 2007). With the exception of rhinoceroses (Ceratotherium simum and Diceros bicornis) in Namibia and South Africa, and exceptionally large elephant trophies, lions generate the highest revenue per hunt (24,000–71,000 United States dollars) of any species in Africa (Figure 3.50). Prices for lion hunts have been particularly high in Tanzania, and were also high in Botswana prior a hunting moratorium placed in that country (up to 140,000 United States dollars per hunt) (Lindsey et al., 2012). 215 Figure 3.50 Mean price for the cheapest trophy hunting packages (daily rates and trophy fees) for each of four key species. Source: (Lindsey, Balme, et al., 2012) under license CC BY 4.0. Legal, well-regulated recreational hunting can therefore support conservation by contributing to the preservation of the target species and the habitat in which it lives (Baldus et al., 2008; Eklund T., 2017; Fischer et al., 2013) (for discussion of this form of management as a driver of sustainable use, please refer to Chapter 4). For emotional and ideological reasons hunting is often excluded as an option for income generation by international conservation non-governmental organizations and certain international funders. Some species are indeed so rare, endangered or sensitive that they are not suitable for even strictly managed and regulated hunting use. However, if wild species and protected areas are not successful resources and options for alleviating poverty, conservation efforts could be undermined. Total protection and trade bans can lead to a major devaluing of wild species because there are no longer economic incentives to protect them (Baldus et al., 2008; Rosie Cooney et al., 2017; NACSO, 2019). For example, it was estimated that if lion hunting were banned, areas across Southern Africa and outside of national parks (approximately 59,500 km2) currently set aside for lion habitat could be converted to other uses such as agriculture (P. Lindsey, Balme, et al., 2012). It is unlikely these areas would be incorporated into existing protected parks due to lack of funding (P. Lindsey, Balme, et al., 2012). 3.3.3.2.5. Science and education Scientific gathering is a tightly regulated and highly controlled activity. It brings benefits to conservation, management and science, can help diagnose or monitor the health of a population, species, or ecosystem and, as a result, protect certain species of animals from other causes of decline (Remsen, 1995; Sikes & Paul, 2013; Winker et al., 2010). It can also have detrimental effects. Documented cases of decline due to removal of animals for scientific purposes usually involve large vertebrates (Gibbons et al., 2000). For small mammals, responsible specimen gathering and removal have little impact on populations and have several benefits for science (Hope, Sandercock, & Malaney, 2018). However, recent studies indicate that harvest of voucher specimens for bats research is harming fragile populations (Russo et al., 2017). For many invertebrate species, collection for scientific research is fundamentally important for species identification. Currently extraction of wild animals for scientific and educational purposes faces a series of economic and social pressures, including budget cuts and shortfalls (Suarez & Tsutsui, 2004), high harvesting cost (Enrique et al., 2020), ethical considerations and significant and costly compliance procedures like those from the Convention on International Trade in Endangered Species of Wild Fauna and Flora for the cross-border exchange of specimens (Roberts & Solow, 2008). Systematized repositories of life in all of its forms are cornerstones of quality research and education in many areas of science and innovation (National Academies of Sciences, Engineering, and Medicine; Division on Earth and Life Studies; Institute for Laboratory Animal Research; Roundtable on Science and Welfare in Laboratory Animal Use, 2019; Winker et al., 2010). Schools, universities, and research laboratories use biological collections to teach concepts of evolution, ecology, taxonomy, physiology, biogeography, conservation, and more. Museum collections, while historically significant, have been greatly reduced by limiting numbers, even if species are common, as financial costs and ethics of maintaining and building these collections have changed. Larger series collected historically have been profoundly important in establishing both presence of absence, and providing evidence on historical population levels. This has been especially important with amphibians (Mahoney & Rueschemeyer, 2003). Biological collections also connect the public to nature and science, bolstering lifelong learning (Graham, Ferrier, Huettman, Moritz, & Peterson, 2004; Hill et al., 2012; MacFadden, 2019; National Academies of Sciences, 2020; Suarez & Tsutsui, 2004). In some cases, digital technologies are able to successfully replace extractive practices for scientific and educational purposes.  Canned hunting “Canned hunting” is a non-technical label that refers to the practice of placing captive-bred, semi-domesticated, and exotic animals within relatively restricted outdoor enclosures for the sole purpose of having the animals “hunted” and killed by paying clients (Graves, Mosman, & Rogers, 2012). “Canned hunting” represents a very small proportion of world hunting (IUCN, 2016) and is not a conservation strategy (Bilchitz, 2016; Williams, Loveridge, Newton, & Macdonald, 2017; G. C. Young, 2007). The practice has resulted in negative environmental and political consequences relating to recreational hunting and is regarded as a potential source of zoonotic diseases (HSI/HSUS, 2016; P. Lindsey, Alexander, et al., 2012; D. W. Macdonald & Willis, 2013; Organ, Decker, & Lama, 2016; Somers & Hayward, 2012; B. K. Williams, Johnson, & Wilkins, 1996). This is a highly contentious practice but mainly involves animals that are bred in captivity and therefore does not fall under the definition of wild species used in this assessment. In scientific and policy analyses, canned hunting needs to be separated from “ranched” wild species production, which involves the management of wild populations across extensive areas. In 2004 the World Conservation Congress, noting strong opposition to all forms of “canned hunting”, accepted that well-managed recreational hunting has a role in the managed sustainable extractive use of wild species, and condemned the killing of animals in small enclosures where they have little or no chance of escape or where they do not exist as free ranging (IUCN, 2004). The International Union for Conservation of Nature encouraged the media and decision-makers to distinguish between canned hunting of confined animals and trophy hunting of free-ranging animals (IUCN, 2016). 216 3.3.3.2.5. Science and education In many countries, legislation improves animal welfare by setting minimum standards and currently covers all taxonomic groups of vertebrates and cephalopods. In European Union countries the use of wild animals is largely prohibited (Hartung, 2010). In the United States Institutional Animal Care and Use Committees (IACUC) are based at colleges and universities and follow national standards to conduct evaluations of animal care and use, including ethical and properly implemented care of wild animals by researchers. Permit-granting agencies are also in a position to place severe restrictions on the number of specimens that may be taken by scientists (Remsen, 1995; Russow & Theran, 2003; Silverman, Suckow, & Murthy, 2000). Currently, there is a tendency for reducing animals in experimentation and replacing animals 217 for artificial models or digital simulators (Robinson et al., 2019; Soulsbury et al., 2020; Volker D., 2006). The potential harm to animal populations should be balanced with anticipated benefits (Brønstad et al., 2016; Russow & Theran, 2003). Stricter controls can be detrimental to building conservation knowledge (Hochkirch et al., 2021). The impact on wild populations of scientific extraction of specimens is usually, but not always, small relative to other causes of mortality including natural mortality, hunting, collisions (e.g., road kill, bird death due to glass windows and communication towers, etc.), and habitat loss or alteration (Erickson, Johnson, & Young, 2005; Remsen, 1995; Rocha et al., 2014; Winker et al., 2010). For example, the entire vertebrate specimen collection of Museum Victoria (Australia), houses less than 200,000 specimens harvested within Victoria over the past 150 years (Clemann et al., 2014). In comparison, duck and quail hunters in Victoria are estimated to have killed 638,000 native birds as of 2012 (Moloney & Turnbull, 2012). It should be noted that museum collections, unlike game hunting, aim at covering a much broader biodiversity, and hence may also exploit small, rare, or endangered populations unlikely to be targeted by hunters (Donegan, 2009). The use of animals in human biomedical research has been of particular focus, more so for ethical considerations than for whether or not extraction of individuals for medical research is a sustainable form of use. Members of the Callitrichidae primate family (marmosets and tamarins) have been used since 1960s as biomedical research subjects because of their small size, wide availability, and relatively inexpensive costs. They are extracted mostly from wild populations in South American countries such as Brazil, Colombia, Peru, and Venezuela. 3.3.3.2.5. Science and education In the 1960s and 1970s the Oak Ridge Associated Universities had the largest tamarin/marmoset population in the United States of America, housing about 550–650 animals (National Academies of Sciences, Engineering, and Medicine; Division on Earth and Life Studies; Institute for Laboratory Animal Research; Roundtable on Science and Welfare in Laboratory Animal Use, 2019). Due to the Convention on International Trade in Endangered Species of Wild Fauna and Flora, the export of wild primates for biomedical research from Central and South America decreased significantly from 200,000 in the 1950s and 1960s to 5,000 specimens after 1975 when the Convention on International Trade in Endangered Species of Wild Fauna and Flora was enacted (Fialho, Ludwig, & Valença-Montenegro, 2016). High export levels have led to declines of some species, such as the cotton-top tamarin (Saguinus oedipus) (National Academies of Sciences, Engineering, and Medicine; Division on Earth and Life Studies; Institute for Laboratory Animal Research; Roundtable on Science and Welfare in Laboratory Animal Use, 2019). The cotton-top tamarin, as well as most marmosets and tamarins, are listed under Appendix I of the Convention. Thus, current import controls will favor wild populations, even though it does make it harder for researchers to acquire marmosets (National Academies of Sciences, Engineering, and Medicine; Division on Earth and Life Studies; Institute for Laboratory Animal Research; Roundtable on Science and Welfare in Laboratory Animal Use, 2019). Today, marmosets as model organisms are attracting so much research interest that their demand far outstrips the already limited supply. Currently, 10–15 institutions are developing small marmoset colonies (of 20–60 animals each) for neuroscience studies. The growing focus on transgenic work has led to the development of some larger colonies (250– 218 350 animals). If the field continues to grow, some facilities may establish much larger colonies (up to 1,000 animals) for line maintenance and characterization (National Academies of Sciences, Engineering, and Medicine; Division on Earth and Life Studies; Institute for Laboratory Animal Research; Roundtable on Science and Welfare in Laboratory Animal Use, 2019). According to the United Nations World Conservation Monitoring Centre and the Convention on International Trade in Endangered Species of Wild Fauna and Flora trade database, reported exports of live macaques (for example, long-tailed macaques for research purposes) from six Southeastern Asian countries were more than 25,000 in 2019. 3.3.3.2.5. Science and education While many animals are bred in captivity for scientific research, there are still significant extractions from wild populations to provide breeding stock. When the illegal trade is factored in (which often relies on legal trade to launder animals into the trade) coupled with unreliable or absent data on wild population numbers, this overall trade may be unsustainable. The high demand for research animals has resulted in the manipulation of the Convention on International Trade in Endangered Species of Wild Fauna and Flora to ban imports of wild primates and birds into the United States of America and the European Union in order to bolster profit generated through commercial captive breeding programs. This raises all sorts of ethical issues (especially with the Convention on Biological Diversity) about the ability of indigenous and other peoples in range states to use their natural resources for economic development. The ex-situ commercial captive breeding industry economically favors extinction of wild populations in range states that can potentially compete with them (Kasso & Balakrishnan, 2013). The second-largest use of amphibians, after food, is for teaching and medical research. Frogs and salamanders are used as model organisms in medical research and are one of the classics for teaching animal biology at universities all over the world (Smith, Wassersug, & Tyler, 2007). The use of amphibians to aid advancing science has led to a number of significant scientific breakthroughs, and several Nobel prizes in physiology or medicine have benefitted from studies involving frogs, the last of which pertain to stem cells in regenerative medicine (Rossant & Mummery, 2012). According to studies on small lizards in Central America, many reptile populations are resilient to standard herpetological gathering (intensive gathering in short-term) (Poe & Armijo, 2014). Current sustainability efforts can potentially focus on reducing and replacing the use of animals in research and teaching with scientific alternatives emerging from innovative education and medical technologies. Using common and widespread species or animals raised in facilities for such activities would promote sustainability (Coleman, Carpenter, & Dunphy, 1996). The killing of critically endangered birds and reptiles for scientific reference has caused debate and ethical dispute in the last two decades (Collar, 2000; Donegan, 2009). 3.3.3.2.5. Science and education It was argued that the scientific gathering of voucher specimens is linked to the decline or loss of Mexico’s elf owl (Micrathene whitneyi soccorroensis), but others ascribe the extinction to invasive species (Minteer, Collins, Love, & Puschendorf, 2014;Rocha et al., 2014). 3.3.3.2.6. Medicine and hygiene 219 The 2019 version of the International Union for Conservation of Nature Red List reports 1,660 species of animals have medicinal uses. Most known species (~77%) are chordates in terrestrial habitats (~72%). Globally, animals used for medicine comprise a relatively narrow subset of all animals, but they do occur across diverse taxa, habitats, and geographies. At least about 62% (n = 1025) of species have multiple uses. The most common additional use is food for human consumption, which approaches half (~46%, n = 769). Geographic hotspots of medicinal species occur in South America, Southeast Asia, India, and the tropical regions of Africa. Although not previously examined, geographic areas of prominent medicinal use (and threats to their use) likely occur where so-called human development is low (Short & Darimont, 2021). The 2019 version of the International Union for Conservation of Nature Red List reports 1,660 species of animals have medicinal uses. Most known species (~77%) are chordates in terrestrial habitats (~72%). Globally, animals used for medicine comprise a relatively narrow subset of all animals, but they do occur across diverse taxa, habitats, and geographies. At least about 62% (n = 1025) of species have multiple uses. The most common additional use is food for human consumption, which approaches half (~46%, n = 769). Geographic hotspots of medicinal species occur in South America, Southeast Asia, India, and the tropical regions of Africa. Although not previously examined, geographic areas of prominent medicinal use (and threats to their use) likely occur where so-called human development is low (Short & Darimont, 2021). Across varied geographies, threats to medicinal animals are more closely related to overall ecosystem degradation than human use. Among species with known population trends (n = 839), the highest proportion have a decreasing trend (~63%, n = 525), whereas about 30% (n = 254) are stable, and only about 7% (n = 60) have increasing populations. Primary threats are related to agriculture and aquaculture (~45% of species, n = 143) and biological resource use (~44%, n = 142), which includes exploitation for medicine, food, clothing, and other uses (Short & Darimont, 2021). 3.3.3.2.5. Science and education There are many examples of surveys that have documented the diversity of animals used in traditional medicine, some are highlighted in the section below.  Africa In Benin, 87 mammal species have been reported as traded for medicinal purposes including some vulnerable, endangered and threatened species (Djagoun, Akpona, Mensah, Nuttman, & Sinsin, 2013). El-Kamali (2000) identified 23 animal species whose products were commercialized for traditional medicine purposes in Central Sudan. Sodeinde and Soewu (1999) recorded the use of 45 medicinal species in Nigerian markets. Simelane and Kerley (1998) showed that 44 species (eight reptiles, six birds, 30 mammals) were sold in 19 herbalist shops in the Eastern Cape Province of South Africa. Cunningham and Zondi (1991) examined the trade in animals for medicinal uses in KwaZulu-Nata Province and reviewed the literature reports for South Africa, recording at least 79 species of vertebrate (18 reptiles, 16 birds, 45 mammals), excluding domestic mammals and various marine invertebrates and fishes. A total 132 species of vertebrates (21 reptiles, 32 birds, 79 mammals) were reported by Ngwenya (2001) to be traded across KwaZulu-Natal Province, with 50 species highly demanded by the costumers. These were vultures, chacma baboon, green mamba, Southern African python, Nile crocodile, puff adder, striped weasel, and black mamba. Whiting et al. (2013) identified 147 vertebrate species that were traded in all South African traditional medicine markets. This represented around 63% of the total number of documented wild species. Recently William et al. (2014) reported 354 bird species (from 205 genera, 70 families, and 25 orders) used for traditional medicine in 25 African countries. In numerous societies of Western and Central Africa, body parts of great apes (chimpanzee, gorilla, bonobo) are used for medicinal and/or ritual purposes. These practices usually operate according to the principle of analogy: the quality and value of the foodstuff is incorporated by the person who ingests it (Epelboin, 2012; Leblan, 2017). For instance, in Guinea, consuming the right arm of a chimpanzee will protect children from disease and make them good hunters, because monkeys are considered as violent and powerful beings (Leblan, personal observation). Scientists point out the unsustainability of such practices and the need for conservation strategies (Sá, da Silva, Sousa, & Minhós, 2012 on Guinea-Bissau). However, given the widespread interest in these species, it is also a matter of global debate. Williams and Whiting (2016) reported 301 uses of animal parts for 122 broad-use categories (Figure 3.51) across South Africa. They used a word cloud to report their findings for visual impact.  Species of global interest Amphibians and reptiles are used in traditional medicine or as part of cultural beliefs all over the world, resulting in harvest of these animals from the wild (Gorzula, 1996; Hocking & Babbitt, 2014; Schlaepfer, Hoover, & Dodd, 2005; UNODC, 2016). Alves et al. (2013) found that 331 species (284 reptiles and 47 amphibians) are used as part of traditional folk medicines around the world. The use of secretions, especially those of Bufonids that contains numerous active molecules, is one of the reasons they are desirable (Rodríguez, Rollins-Smith, Ibáñez, Durant-Archibold, & Gutiérrez, 2017). Insects are also used as medicinal resources all over the world (Costa-Neto, 2005). Pangolins (four species in Asia and four species in Africa) are the most heavily traded wild mammal in the world (UNODC, 2016). Their various body parts, especially their scales, fetuses, blood, bones, and claws are largely used in traditional medicines (Boakye, Pietersen, Kotzé, Dalton, & Jansen, 2014; Mohapatra, Panda, Nair, Acharjyo, & Challender, 2015; Soewu A Durojaye & Sodeinde A Olufemi, 2015). Harvesting of two Asian species of Pangolins is largely driven by demand from China. This, in combination with additional threats related to habitat decline, are affecting the sustainability of use. These species are listed as critically endangered in the International Union for Conservation of Nature Red List (Heinrich et al., 2016). With declining Asian pangolin populations, a shift in trade from Asian to African pangolin species has been suggested. As a result, the total number of incidents involving Asian species declined since 2000, yet they were still being traded in large volumes (more than 17,500 estimated whole Asian pangolins were traded between 2001 to 2014) despite a zero-export quota for commercially traded wild sourced Asian species (Heinrich et al., 2016). The United States of America is also a significant largest importer of pangolins and their products (UNODC, 2016). 220  Europe p The traditional use of animals as a source of medicine is relatively low in Europe. However, Benitez (2011) reported 26 different animals provided 61 distinct medicinal uses in Western Granada Province, Andalusia (Spain). The high number of uses is due to the fact that some animal species are involved in more than one preparation method, sometimes with different parts used.  Africa Although the study was conducted in South Africa, the categories of uses reported paint a picture of the health needs of the consumers of animal-based medicine elsewhere. ‘Strength’ (e.g., home strength, imbuing physical strength and overcoming fear) stands out as a dominant use, followed by protection to ward off evil spirits from within a person or from their residence. 221 Figure 3.51 Word cloud of the use categories derived from species used in animal-based medicine in South Africa. The size of the words in the figure is proportional to the number of times the uses were mentioned. Source: (Vivienne Linda Williams & Whiting, 2016) © 2015 Elsevier Ireland Ltd., license number 5153130663118. CC-BY NC. Figure 3.51 Word cloud of the use categories derived from species used in animal-based medicine in South Africa. The size of the words in the figure is proportional to the number of times the uses were mentioned. Source: (Vivienne Linda Williams & Whiting, 2016) © 2015 Elsevier Ireland Ltd., license number 5153130663118. CC-BY NC.  Latin America  Latin America A recent literature review on animal-based medicine recorded at least 584 wild species (13 taxonomic categories) as being used in the entire continent of Latin America (R. R. N. Alves, Rosa, Albuquerque, & Cunningham, 2013). The authors even speculated that this number might be underestimated given the limited number of studies on the theme, highlighting the conservation implications of the wild species use in medicine. Surveys carried out in 15 Brazilian cities reported that at least 180 animal species are traded for medicinal purposes (R. R. N. Alves & Rosa, 2010). In the State of Bahia, in Northeast Brazil, 50 insect species were reportedly used for medicinal purposes (Costa-Neto, 2005). A recent literature review on animal-based medicine recorded at least 584 wild species (13 taxonomic categories) as being used in the entire continent of Latin America (R. R. N. Alves, Rosa, Albuquerque, & Cunningham, 2013). The authors even speculated that this number might be underestimated given the limited number of studies on the theme, highlighting the conservation implications of the wild species use in medicine. Surveys carried out in 15 Brazilian cities reported that at least 180 animal species are traded for medicinal purposes (R. R. N. Alves & Rosa, 2010). In the State of Bahia, in Northeast Brazil, 50 insect species were reportedly used for medicinal purposes (Costa-Neto, 2005). Didelphis. marsupialis has an undeniable cultural significance for local communities in the Amazon, both in terms of food and medicine. it is also designated as the best wild meat in the region. It is hunted by men, but the preparation of meat and medicinal oil are tasks mainly performed by women. The current study focused on riverine communities, who reportedly hunt the “common opossum” in morning or at night. They have a variety of techniques including handmade traps called “mundé”, made from locally gathered wood and vines. However, this 222 technique is declining because riverine people themselves believe that “mundé” does not select animals and it is harmful. Based on structured and semi-structured interviews with the local community, Barros and Azevedo (2014) found that this activity has not negatively affected the local populations of D. marsupialis. Some respondents stated that there is a decreased number of animals, other respondents argued that there is an increased number of opossums in the region. 3.3.3.3. “Non-lethal” terrestrial animal harvesting Non-lethal uses of wild animals include all use forms that do not result in the death of animal through killing, contrary to lethal uses which take the life of animals. Non-lethal uses include ornamental use, scientific research, pets, green hunting, and religious and cultural practices and can benefit food security, economy, industry, and result in conservation. Traditional non-lethal uses of wild animals at local scales occur among indigenous communities, although biodiversity conservation and poverty alleviation remain a challenge in tropical biodiversity hotspots (Tranquilli, 2014).  Latin America A third group said that the common opossum is a species that has a good reproductive capacity (it is a “mineral animal”), therefore, they think the population remains stable. Scientific studies suggest consumption of this species should be the subject of further studies, as this marsupial species has been described as a reservoir for parasites that cause severe disease.  Asia Use of wild terrestrial animals for medicinal purposes is widespread throughout Asia. Ashwell and Waltson (2008) recorded at least 47 animal species being traded for medicinal purposes in Cambodian markets, while Van and Tap (2008) recorded 100 different medicinal products from 68 animal species traded in Ho Chi Minh City, mainly sold as dried products (either the whole animal or parts) soaked in rice wine, or as a gel product which remains after boiling animal remains slowly in water. The rhinoceros horn cut from live individuals are used in traditional Chinese medicine to dispel heat, detoxify blood, but were split over other purported medicinal properties, including its ability to treat cancer (Cheung, Mazerolle, Possingham, & Biggs, 2018). In 2018, the import and export of rhinoceros and their products will continue to be strictly prohibited; the sale, purchase, transportation, carrying and mailing of rhinoceros and their products are strictly prohibited; rhino horn and tiger bone are strictly forbidden to be used as medicine (http://www.china.com.cn/news/2018-12/13/content_74271446.htm). 3.3.3.3.1. Decorative and aesthetic Natural fibers have important properties and are used as luxury goods and handicrafts that sell for better prices and generate higher profits for the community. Vicuñas (Vicugna vicugna) are a species which has received considerable attention regarding its sustainable use. Its hair produces one of the finest natural fibers in the world and is highly valued to make luxury fabric and clothing. The vicuña is the most representative wild ungulate of the high Andes of South America. In 1965, at its low point, the population of vicuña was estimated at only 6000, having 223 collapsed from 1 million animals 25 years prior. Current population size is about 460,000 - 520,000 individuals, but they went through a serious and long-term overexploitation for 500 years. The recovery has benefited from a series of conservation actions, including the early prohibition of hunting and trade, establishment of the National Council of South American Camelids (Consejo Nacional de Camélidos Sudamericanos, CONACS), corral programs on community land and the practices of capturing and live shearing wild animals to earn high profits from selling the fiber. The benefit to society and the natural world of these efforts was the survival of a charismatic animal in its historical landscape (Sahley, Vargas, & Valdivia, 2007; Wakild, 2020). The restoration of depleted wild populations of vicuñas has reinstated the species in the ecosystem, and has allowed the development of sustainable use programs that directly assist the livelihoods and well-being of local people, and provides options for further economic development linked directly to successful conservation. 3.3.3.3.4. Pet and zoo trade There are two distinct, but related, aspects to the live animal trade: the pet trade and the zoo trade. An array of live animals, eggs, and taxidermy are targeted by buyers worldwide for private collections and zoos or as exotic pets. Examples include reptiles, such as chameleons and tortoises; birds such as parrots and falcons; and mammals, such as tiger cubs and apes (ROUTES, 2022). Zoological gardens and zoos represent ex-situ conservation of wild animals for research and educational activities, tourism and recreation. The zoological parks basically exchange or buy/sell animals from each other, rarely do they buy specimens coming from the wild. Wild animals are maintained in captivity for visual observations by the public. In an increasingly urban world, the ability of people to have contact with animals through zoos and pets adds significantly to the positive values people attribute to wild species – a prerequisite for their active engagement in conservation (Fukuda et al., 2011). They constitute the ideal sites for environmental education, and therefore may have secondary level benefits for sustainable use due to a better educated public. In these cases, animals may progressively lose their wild instinct through a long-term habituation process. Given relatively few captive-bred animals do get released back into the wild, where they can make a significant contribution to in-situ conservation, the role of encouraging people to value wild species positively is arguably the biggest conservation benefit that flows from zoos and exotic pet ownership. The global pet trade is a large and complex industry. Pets are widely kept in many countries with 46% of United Kingdom of Great Britain and Northern Ireland and 62% of United States of America households estimated to have pets, supporting a multi-billion-dollar industry dedicated to their care and feeding (Human Society of United States, 2014; Pet Food Manufactures Association, 2014). Pet owners not only display more positive attitudes toward animals (Daly & Morton, 2009; N. Taylor & Signal, 2009), but also engage in more animal- related activities such as bird watching and viewing nature documentaries (Bjerke, Østdahl, & Kleiven, 2003). Pet owners are also more inclined to join and support animal welfare and environmental organizations (R. Bennett, 2003). 3.3.3.3.3. Recreation: green hunting Green hunting occurs with tranquilizer dart guns and the animals are released alive. This is typically performed for veterinary procedures or translocation, and has been suggested as an alternative to lethal forms of hunting (Greyling, McCay, & Douglas-Hamilton, 2004). Green hunting is cheaper and less harmful compared to traditional hunting and while immobilized, the animal can be micro-chipped or have tissue sampled (Greyling et al., 2004). However, as green hunting is as of yet not a significant recreational activity, there is insufficient information on the status, trends and/or impact of the activity with regards to its potential impact on sustainable use of wild terrestrial species. 3.3.3.3.2. Food and beverage: honey Wild honey is an important source of nutrition and medicine, and contributes to the income of local communities in many parts of the world. Wild honey harvesting is practiced by men and women belonging to many indigenous peoples and local communities. The harvest, filtration and preservation of wild honey relies in many parts of the world on rich traditional knowledge and its continued transmission across generations. Wild honeybee local knowledge and traditional skills are key to sustainable use. In Lizongole, Mozambique wild honey gathering is typically carried out by groups of five to seven men sometimes called honey hunters (Ribeiro, Snook, Vaz, & Alves, 2019). Honey hunters have a mutualistic interaction with the honey guide bird (Indicator indicator) that directs men to trees containing honey. There they burn dried sticks to initiate fire and the felling of trees. Honey hunters apparently fell up to 560 trees per year. Impacts on tree populations vary among the 12 species killed for honey and are considered as a diminishing resource. Non-destructive traditional practices based on tree climbing are recommended (N. S. Ribeiro et al., 2019). In Asian countries including India, the harvesting of wild honey from tall forest trees is done using bamboo baskets and bamboo ladders, and climbing trees with a smoke torch (Deori, Deb, Singha, & Choudhury, 2017). In the Southeastern United States of America tupelo honey production has been part of rural livelihood practices for several generations, and is carried out according to local ecological knowledge of both the trees (Nyssa ogeche) and the bees (Watson, 2017). This non-lethal use of wild bees is widely proposed by local conservation stakeholders and is generally integrated into most of management plans of worldwide protected areas. It constitutes a sustainable alternative which provides a long-term income source to local people (Syampungani et al., 2020). However, in many areas the required traditional knowledge is threatened due to changes in the related socio-economy, as more young people choose to work in the cash economy. For example, only 24% of the 251 local community members surveyed in Palawan Philippines could correctly identify the giant honeybee (Matias, Borgemeister, & von Wehrden, 2018). 224 3.3.3.3.4. Pet and zoo trade As specified in the 2016 World Wildlife crime report: “In a range of countries, the capture and sale of wild-caught pets can be a way for rural communities to make money and for urban communities to express a link to the natural heritage of their countries. Display of these wild species can also draw tourists – exotic birds or even primates may be strategically positioned in front of restaurants for example, or wild species 225 may be shown for a fee as a roadside attraction. International trade in exotic species has also become big business. Most of this involves relatively common species, but dedicated collectors may pay thousands of dollars for protected specimens, captive bred or supplied from the wild. Much of this trade involves birds, reptiles, and fish - populations that may prove difficult to monitor. The trade of tropical fish for aquaria and freshwater turtles and tortoises for terraria involves millions of individuals annually, and the share of this trade that comes from the wild is not always clear. About one quarter of all commercial live animal exports permitted under the Convention on International Trade in Endangered Species of Wild Fauna and Flora in 2013 were declared as wild sourced, with most involving species of birds, amphibians, or reptiles prized in the pet trade. In terms of total live animals, the most commonly exported were map turtles” (Vereinte Nationen, 2016). From the 1980s to the present, approximately 12 million live internationally protected parrots were reported in international trade, according to the Convention on International Trade in Endangered Species of Wild Fauna and Flora export data. Most were either wild-sourced or of unknown origin (62%). Trade trends have been strongly influenced by national controls in key destination markets (UNODC, 2016). In 1992, the United States of America passed the Wild Bird Conservation Act, which sharply reduced the number of parrots and other wild birds imported to the United States of America. In 2005, the European Union banned the import of wild birds due to concerns about bird flu transmission (Vereinte Nationen, 2016). Both acts radically changed the international live bird market. The pet-trade in wild frogs and amphibians concerns a minority of the recorded importations or exportations compared to other taxonomic groups of vertebrates. 3.3.3.3.4. Pet and zoo trade An analysis of trade data reported in the Convention on International Trade in Endangered Species of Wild Fauna and Flora database showed that trade in amphibians has increased in the last years, with ~ 40,000 animals exported per year globally as part of the trade of captive-sourced live animals (Harfoot et al., 2018). This has resulted in a decrease in wild sourced exports since 2000. At the same time, these numbers may be underestimates due to mislabeling of specimens as captive-bred which may in fact be wild-caught (Auliya et al., 2016). For example, between 2013 and 2018, the United States of America alone imported 3,655,620 live amphibians for the pet trade, belonging to 283 species (Mohanty & Measey, 2019). The Asian houbara bustard (Chlamydotis macqueenii) is listed as Vulnerable by Birdlife International (2004) due to global population decline of 35 per cent over the last 20 years. The principal cause of declines has been hunting by Arab falconers (Collar et al., 2017; Seddon & Launay, 2008; Tourenq et al., 2004), and associated poaching of live birds, especially from Pakistan, for training of falcons in the Arabian Peninsula. However, Saudi Arabia has taken necessary steps to conserve dwindling populations of Houbara Bustards. The goal of houbara conservation in Saudi Arabia is to restore self-sustaining populations of resident breeding birds protected within a network of protected areas, but which may one day support sustainable falconry in hunting areas outside reserves (Gelinaud, Combreau, & Seddon, 1997; Seddon, Knight, & Budd, 2009; van Heezik & Ostrowski, 2001). Unfortunately, not all species can be bred in captivity and some consumer countries do not have access to captive bred animals, so demand for wild animals persists. The harvest of live specimens, in many cases, involves significant mortality during capture, transport and holding. Many wild animal species controlled under current policies remain unsustainably 226 traded to supply the international pet markets, with rare and endemic species most threatened (Auliya et al., 2016; E. G. Frank & Wilcove, 2019; R. O. Martin, 2018; R. O. Martin et al., 2014; Ngo, Nguyen, Phan, van Schingen, & Ziegler, 2019). Even with existing international regulations, the majority of species in exotic pet trade are not protected under the Convention on International Trade in Endangered Species of Wild Fauna and Flora, leaving international trade mostly unregulated and unmonitored (Janssen & Shepherd, 2018). 3.3.3.3.4. Pet and zoo trade In particular, species with small wild populations and/or small areas of occupancy, including island populations, are highly prone to overexploitation and decline due to the exotic pet trade (S. Altherr & Lameter, 2020; Flecks et al., 2012; Lyons & Natusch, 2013). The high demand by specialized collectors for a "new" (i.e., only recently scientifically described) or rare species has caused intense collections in the wild, shortly after type localities were published - which is why an increasing number of scientists warn against publishing type localities (Lindenmayer & Scheele, 2017a; Maron, D.F., 2019). The sustainability of this form of consumer-driven use is unclear. Additional issues related to the pet trade are: (i) some common methods of animal harvest for commercial trade result in destruction of habitats and shelters (see for example, Goode, Horrace, Sredl, & Howland, 2005) and (ii) the exotic pet trade has been identified as a pathway for the spread of invasive alien species (Shivambu, Shivambu, & Downs, 2020; Soule, 1990; Warwick & Steedman, 2021). 3.3.3.4. Emerging issues: terrestrial animals harvesting for integrated species and habitat management Human-wildlife conflicts often include damage to agriculture, disease transmission, traffic collisions, etc. Trends in increasing populations for several popular game species has had detrimental effects on non-game species, both because of competition for resources and they are pursued by hunters as ‘vermin’ that threaten game populations (Denny, Latham-Green T., & Hazenberg R., 2021; Gross, 2008; Linnell et al., 2020; Ripple et al., 2014; Teichman, Cristescu, & Darimont, 2016). In the 20th century, large predators’ populations were almost exterminated in North America and Western Europe (Ripple et al., 2014), which caused multiple cascade effects to ecosystem functioning, such as “mesopredator release” effects (Brashares, Prugh, Stoner, & Epps, 2013; Prugh et al., 2009; Soule et al., 1988). Growing numbers of ungulates and other desirable game species (Grant, Mallard J., Leigh, S., & Thompson, P. S., 2012; Kuijper et al., 2013) resulted in habitat alterations and degradation (Grant et al., 2012; Kuijper et al., 2013; Theuerkauf & Rouys, 2008), increased levels of infanticide among certain species (Swenson et al., 2017) and hybridization (Salvatori et al., 2020). Presently, populations of most large predators are maintained at a socially acceptable maximum and are even decreasing in certain parts of Europe (Fernández-Gil et al., 2016; J D C Linnell & Cretois, 2018; Niedziałkowski, Sidorovich, Kireyeu, & Shkaruba, 2021; Virgós & Travaini, 2005). In the United Kingdom, one of the most criticized management actions of grouse hunting is population control of raptors (Denny et al., 2021). Killing of people and domestic stock by predators is a serious human-wildlife conflict. Most predator populations were historically subject to severe depletion and sometimes eradication to the point of extinction. However, societal perspectives on wild species have changed over time, and now conservation actions are focused on rebuilding populations. If successful this often results in a need for additional management of the conflicts which then arise from larger predator populations. The result is that many wild species require ongoing, active management to negotiate the human-wildlife interface (Arroyo-Quiroz, García-Barrios, Argueta-Villamar, Smith, & Salcido, 2017; Lute, Carter, López-Bao, & Linnell, 2018). Saltwater crocodile populations in Australia have followed this pattern (Saalfeld, Fukuda Y., Duldig T., & Fisher A., 2016; G.J.W. Webb, 2014; Grahame J W Webb, 2021). Recovery of their populations has largely been tolerated due to the economic benefit derived from commercial skin and meat production, egg collection and tourism (Fukuda et al., 2020; Joanen et al., 2021). 3.3.3.4. Emerging issues: terrestrial animals harvesting for integrated species and habitat management Hunting is not only done for food and other products, but can also be an important component of wild species management (Linnell et al., 2020; Winker et al., 2010), and can be an important part of sustainable management practices for the wild species and their habitats. Wild species management is defined as the application of science-based and local knowledge in the stewardship of wild animal populations (including game) and their habitats in a manner that is beneficial to the environment and society (IUFRO, 2017). Wild species are managed for several reasons, such as: (i) to reduce a range of human- wildlife conflicts; (ii) to prevent over-population and thus reduce related socio-economic and ecological threats; (iii) to maintain desired structure of game species populations (e.g., sex, age, morphology, etc.); (iv) and to support ecosystem functioning and resilience, including control of invasive and alien species. There are many ongoing debates within conservation and management science concerning the best models for human-nature interactions (Cornicelli, Fulton, Grund, & Fieberg, 2011; Linnell et al., 2020). Wild species management institutions designed to regulate hunter impacts on wild species and wild species impacts on human interests go back centuries in various forms, although the modern tradition appeared in North America and Europe in the early 20th century (e.g., Leopold, 1933). Management for hunting may involve the introduction of alien species, habitat modification, artificial feeding and the intensive control of predators, all of which can have widespread ecosystem effects. Wild species management institutions motivated and funded by hunting activities have led to the dramatic recovery of many species of game (roe deer, red deer, white-tailed deer, moose, wild boar, brown bears, black bears, mountain lions, wild turkeys, the American 227 Alligator) across North America and Europe to the extent that their populations are today higher than they may have been for centuries (Gross, 2008; Joanen et al., 2021; Linnell et al., 2020; P. Mahoney & Geist, 2019; Ripple et al., 2014). The population levels of game species that are optimal for commercial hunting can at the same time be detrimental for forest regeneration and biodiversity conservation and lead to conflicts between different groups of actors and management goals. These high populations have secondary effects including changes in animal and plant community structure and function and spread of diseases (Gortázar, Acevedo, Ruiz-Fons, & Vicente, 2006; Mustin et al., 2018). 3.3.3.4. Emerging issues: terrestrial animals harvesting for integrated species and habitat management Wolves in Southeastern Norway and the French Alps in Europe, and the Midwestern and Western United States of America have similarly rebounded after strict protection in recent decades, leading to conflicts between pro-wolf and anti-wolf “camps” that highlight different aspects of the wolf recovery history in attempts to influence management (Ruid et al., 2009; Skogen, Mauz, & Krange, 2008; Smith & Peterson, 2021). Although economic valuation through nature’s contributions to people is a popular approach to address 228 such issues, it is likely to fail with regard to wild species management (Linnell et al., 2020) because so many of the costs and benefits of wild species conservation are of an intangible nature, and not conducive to economic valuation. In addition, the distribution of costs and benefits vary widely by spatial scales (Linnell, 2015) and within different value domains (Arias-arévalo, Gómez-baggethun, Martín-lópez, & Pérez-rincón, 2018). These complex trade-offs challenge governance structures. When decisions are likely to be controversial, it is essential that decision making processes maintain broad societal legitimacy by balancing inputs of diverse experts, key stakeholders and the public before making transparent decisions. It is important to consider not only the direct practical and economic impacts of human-wildlife conflicts but the wider social, cultural and political context within which these impacts occur and which co-constitute sustainable use (e.g., Linnell & Cretois, 2018; Linnell et al., 2020; Lüchtrath & Schraml, 2015; Skogen, Krange, & Figari, 2017). In order to attend to the increasing diversity of conflicting interests and objectives, existing management structures would require greater transparency, scientific robustness and social legitimacy. The integration of all these elements is more likely ensure successful co- habitation among humans and wild species can continue (Carter & Linnell, 2016). Finally, eradication of invasive alien species, including invasive wild animals, is globally acknowledged as a key management option for mitigating the impacts they cause to biological diversity, economy and human well-being (Courchamp et al., 2011, p. 2011; Genovesi & Carnevali, 2011; Simberloff, Parker, & Windle, 2005). Most of these eradications have been done on islands and involved vertebrates (Genovesi, 2005), but there are also examples of successful eradications of invertebrates, including fruit flies from Nauru (Allwood, Vueti, Leblanc, & Bull, 2002), mosquito Anopheles gambiae from Brazil (Davis JR & Garcia R, 1989), and the Asian Gypsy Moth in North America (Elkinton & Liebhold, 1990). 3.3.3.4. Emerging issues: terrestrial animals harvesting for integrated species and habitat management In Europe, rats (Rattus spp., 67% of all eradications) and rabbits were the most common target species (Genovesi, 2005). Although effective, possible cascading ecological effects of eradications must be taken into account (Courchamp et al., 2011, p. 2011). 3.3.4.1. Introduction Logging practices differ widely around the world. These include felling of individual wild trees, selective timber-harvesting, clearcutting and variable retention harvesting. The broader the group of forest users, the more likely logging needs to be reconciled with other uses and services, which support very diversified and complex livelihood strategies (Zenteno, Zuidema, de Jong, & Boot, 2013). This section assesses the status and trends of logging in the relation to the sustainable use of wild tree species. Due to the relative complexity of grouping all logging practices together, in this introduction several topics relevant to logging are briefly introduced. This includes the formal definition from Chapter 1, the issue of plantation vs. natural forests, and how forests are classified and forest management defined. A more detailed overview of the global status and trends of forests and forest management is presented in the following section (3.3.4.2). Section 3.3.4.3. is a review of timber products and uses structured similarly to the other uses sections in section 3.3. Finally, emerging issues are discussed in section 229 3.3.4.4. Direct and indirect drivers of use and sustainable use are discussed in detail in Chapter 4. 3.3.4.4. Direct and indirect drivers of use and sustainable use are discussed in detail in Chapter 3.3.4.4. Direct and indirect drivers of use and sustainable use are discussed in detail in Chapter 3.3.4.4. Direct and indirect drivers of use and sustainable use are discussed in detail in Chapter 4. 4. The review on key aspects of sustainable use focusing on logging practices relies heavily on meta-analyses carried out by either independent academic scholars or in affiliation to forest-based research departments or institutions including the FAO, the Center for International Forestry Research and the International Tropical Timber Organization (see the data management report for Chapter 3 systematic literature review at https://doi.org/10.5281/zenodo.6452651). Due to the peculiarities of the forest management and logging practices across and within the same biomes and regions, the analyses are further supplemented with a limited number of country specific case-studies. A review of the available relevant scientific literature is also included. Reports from national forest management departments, case study reports and academic theses at both masters and doctoral levels were also used as appropriate (see the data management report for Chapter 3 systematic literature review at https://doi.org/10.5281/zenodo.6452651). Logging is defined in this assessment as the removal of whole trees or woody parts of trees from their habitat. 3.3.4.1. Introduction Logging generally results in the death of the tree, but also includes cases in which it may not, such as coppicing. Harvesting of non-woody parts of trees, such as fruits, bark or leaves, is considered under gathering (See Chapter 1 for definition, 3.3.2 for gathering). Logging is a key aspect of forest management, guided by site-specific requirements and prescriptions set out in forest management (and harvest) plans or through long-standing practices. It occurs in varying land tenure conditions including private, communal, and public ownership, and in forests ranging from simple (few dominant species) to complex (multiple species). The practice can be carried out formally or informally at small to large scales, for different uses, and for subsistence and commercial benefits (Figure 3.52). 230 230 Figure 3.52 Flow diagram of timber products from natural and plantation forests. Based on Global Forest Products: Facts and Figures 2018 (FAO, 2019a) under license CC BY-NC- SA 3.0 IGO. Figure 3.52 Flow diagram of timber products from natural and plantation forests. Based on Global Forest Products: Facts and Figures 2018 (FAO, 2019a) under license CC BY-NC- SA 3.0 IGO. Timber is obtained from both natural and planted forests (Figure 3.52). Estimates suggest plantations provide one third to one half (500-800 million m3) of global industrial round wood (Jürgensen, Kollert, & Lebedys, 2014; Siry, Cubbage, & Ahmed, 2005), meaning that natural forests are still the major sources of timber globally. Widespread adoption of tree planting for industrial purposes began in the 1960s (Bull et al., 2006; Evans, 2009; McEwan, Marchi, Spinelli, & Brink, 2020; Szulecka, Pretzsch, & Secco, 2014) to generate mainly industrial roundwood and reduce deforestation (FAO, 1967). However, while there are projected increases in the extent and volume of wood that will be produced from plantations (Armesto, Smith-Ramirez, & Rozzi, 1999; C. Brown, 2000; FSC, 2012), their relative contribution is projected to decrease as demand increases (Carle & Homgren, 2008). Thus, the pressure on existing natural forests is expected to greatly increase in the coming decades, starting with the areas with easiest access. Forests are classified under four climatic domains. The largest domain is tropical, constituting 45% (1834 million ha) of the world’s forests, followed by boreal (27%) (1110 million ha), then temperate with 16% (666 million ha) and lastly the subtropical domain that constitutes 11% (449 million ha) of the world’s forests (FAO, 2020a) (Figure 3.53). 3.3.4.1. Introduction For the purposes of this assessment, tropical and subtropical are at times referenced together and temperate and boreal are at times referenced together. Widespread changes in forest types are more evident in tropical forests (Fearnside, 2004; Malhi & Phillips, 2004; Root et al., 2003), which are more sensitive to climate changes 231 (Hughen, Eglinton, Xu, & Makou, 2004) and have been greatly affected by loss of forest cover and forest degradation. These changes affect the ability of species to migrate and can lead to extinction of some species (Pounds et al., 2006; Pounds, Fogden, & Campbell, 1999). The subtropics contain some of the most prominent biodiversity hotspots in Latin America, Australia, and South Africa, however many forest tree species exist in highly fragmented environments and are at particular risk of extinction (Locatelli, Brockhaus, Buck, & Thompson, 2010). Figure 3.53 Global distribution of forests sub-divided by climatic domains. Red: tropical, purple: subtropical, green: temperate, and blue: boreal. This map is adapted from its original source (FAO, 2020a) and is copyrighted under license CC BY-NC-SA 3.0 IGO. The designations employed and the presentation of material on the maps used in the assessment do not imply the expression of any opinion whatsoever on the part of IPBES concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. These maps have been prepared or used for the sole purpose of facilitating the assessment of the broad biogeographical areas represented therein and for purposes of representing scientific data spatially. Figure 3.53 Global distribution of forests sub-divided by climatic domains. Red: tropical, purple: subtropical, green: temperate, and blue: boreal. This map is adapted from its original source (FAO, 2020a) and is copyrighted under license CC BY-NC-SA 3.0 IGO. The designations employed and the presentation of material on the maps used in the assessment do not imply the expression of any opinion whatsoever on the part of IPBES concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. These maps have been prepared or used for the sole purpose of facilitating the assessment of the broad biogeographical areas represented therein and for purposes of representing scientific data spatially. 3.3.4.1. Introduction Temperate forests are the most extensively altered forest biome due to global change factors, with a smaller fraction of original vegetation remaining compared to boreal and tropical forests (Reich & Frelich, 2002). More changes in vegetation type are anticipated over the next 70-100 years (Locatelli et al., 2010), though with a high degree of uncertainty due to interactions among increased fire, invasive species, pathogens, and storms (Virginia H. Dale et al., 2001). It is reported that temperate and boreal forests are expanding northwards, a trend expected to continue due to climate change (Chamberlain, Emery, & Patel-Weynand, 2018; Locatelli et al., 2010). Models suggest boreal forests will also undergo increased fires, increased insect and disease infestations, altered stand composition and structure. Declines in 232 old-growth forests and conversion of southern-central dry forests to grasslands are also predicted due to climate change over the next several decades (Locatelli et al., 2010). 3.3.4.2. Global trends and overview Logging and trade in timber products has increased over the last several decades due to land use change including conversion to agricultural lands, transition to timber plantations and urban development, leading to deforestation and forest degradation (Estrada, Garber, & Chaudhary, 2019; Hosonuma et al., 2012; Kissinger, Herold, & De Sy, 2012; Miller, Mansourian, & Wildburger, 2020; Ngansop, Biye, Fongnzossie, Forbi, & Chimi, 2019). According to the Food and Agriculture Organization of the United Nations (2020a), forests decreased from 32.5 percent to 30.8 percent of the global area between 1990 and 2020, representing a net loss of 178 million hectares (FAO, 2020a) (Figure 3.54). Africa had the highest net loss of forest area between 2010–2020, with a loss of 3.94 million hectares per year, followed by South America with 2.60 million hectares per year. Asia showed the highest net gain in forest area in the period 2010–2020 (FAO & UNEP, 2020), however this is attributed to expanding already extensive plantation forests (Paradis, 2020; Sloan, Meyfroidt, Rudel, Bongers, & Chazdon, 2019; Szulecka et al., 2014) (Figure 3.54). Figure 3.54 Forest area by region, from 1990 to 2020. Data from the Global Forest resource assessment (FAO, 2020a) under license CC BY-NC-SA 3.0 IGO. See data management report for the figure at https://doi.org/10.5281/zenodo.6453095. Figure 3.54 Forest area by region, from 1990 to 2020. Data from the Global Forest resource assessment (FAO, 2020a) under license CC BY-NC-SA 3.0 IGO. See data management report for the figure at https://doi.org/10.5281/zenodo.6453095. Primary forests, which are defined as naturally regenerated forests of native species (FAO, 2018c) have reduced by 81 million ha. since 1990, though the rate of loss decreased by over 50% between 2010-2020. Forests with high ecosystem integrity remain in Canada, Russia, the Amazon, Central Africa, and New Guinea (Grantham et al., 2020). Ecosystem integrity here refers to the degree to which a system is free from 233 anthropogenic modification of its structure, composition, and function (Parrish, Braun, & Unnasch, 2003). The majority of remaining forest areas have moderate to low forest ecosystem integrity as a result of human use of forest systems, affecting the capacity of forests to provide benefits. This degradation can be a precursor to outright deforestation (Grantham et al., 2020; McNicol, Ryan, & Mitchard, 2018). Planted forest cover increased by 123 million ha between 1990 and 2020, although rates of increase have slowed since 2010 (Figure 3.55, also see Supplementary material Table S3.2). 3.3.4.2. Global trends and overview In 2020, plantations and other planted forests equaled 294 million ha (7%) of the world’s forest cover. Asia has the largest proportion of planted forests, 135 million ha, which constitute 22% of the region’s total forest cover. Approximately 44% of plantation forests feature introduced species. Native species are mainly planted in North and Central America (96%) and Asia (68%) while the percentage of plantation forests comprised of native species are 30%, 23%, 22% and 3% in Africa, Europe, Oceania and South America respectively (FAO, 2020a). 234 Figure 3.55 Changes in global planted forest cover between 1990 – 2015. (A) Changes in privately owned forest land. The figure shows an overall four percent (4%) increase in privately owned forest lands, with decreases in Africa and South America, no change in North Figure 3.55 Changes in global planted forest cover between 1990 – 2015. (A) Changes in privately owned forest land. The figure shows an overall four percent (4%) increase in privately owned forest lands, with decreases in Africa and South America, no change in North 234 and Central America, and increases in Asia, Europe and Oceania. (B) Changes in publicly owned forest land. Figure shows an overall decrease of four percent (4%), with decreases in Africa, Asia, Oceania and South America, no change in Europe and a slight increase (1%) in North America. (C) Changes in forest land with unknown ownership. Figure shows an overall decrease of 22% in forest cover on lands with unclear ownership. Large decreases are observed in Asia and South America with lesser decreases in Africa, North and Central America. There was a slight increase in Oceania and a larger increase in Europe. Source: Global Forest resource assessment (FAO, 2020a) under license CC BY-NC-SA 3.0 IGO. See data management report for the figure at https://doi.org/10.5281/zenodo.6453095. Forest management objectives can be categorized under production, protection of soil and water, conservation of biodiversity, social services, multiple use, and other uses (Table 3.16) (FAO, 2020a). Approximately 1.15 billion ha., accounting for 31 percent of the world’s total forest area is managed for production purposes, that is for timber, fiber, bioenergy and/or wild plants and fungal products. The area has however slightly decreased by 1.22 million ha between 1990 and 2020 with some fluctuations. Decreases in production forests occurred in Europe, Asia and most significantly in Africa (from 109 to 91.4 million ha) with a corresponding decrease in forest area. 3.3.4.2. Global trends and overview North America and Oceania had slight increases in forest area under production during the same time period. Concurrently, approximately 749 million ha (22% of total forest area) of forest globally are designated primarily for multiple use. This total decreased by 70.7 million ha between 1990 and 2020 in all regions except Asia and Europe (FAO, 2020a). 235 Table 3.16 Forest area (1000 ha) designated primarily for production, and annual change, 1990-2020. Source: Global Forest resource assessment (FAO, 2020a) under license CC BY- NC-SA 3.0 IGO. Year Annual change 1990 2000 2010 2020 1990-2000 2000-2010 2010-2020 Forest area 4236433 4158050 4106317 4058931 -7838 -5173 -4739 Planted Forests 170061 210662 261958 292587 4060 5130 3063 Forest area with long- management plans 1757831 1855538 1990865 9771 13533 Forests in protected areas 437 821 499 853 600 845 629 139 6 203 10 099 2 829 Forest designated for Production 1 135 826 1 112 657 1 097 126 1 134493 -2 317 -1 553 3 737 Forest designated for multiple use 809 181 780 458 750 728 738 464 -2 872 -2 973 -1 226 Selective harvesting is one of the dominant logging practices that contributes nearly 15 percent of global timber needs (P. A. Martin, Newton, Pfeifer, Khoo, & Bullock, 2015; Poudyal, Maraseni, & Cockfield, 2018). Selective harvesting can be low impact timber- harvesting when it involves harvesting 1-2 species and 1-2 individuals per hectare. It can be moderate when 5-15 species are harvested and 1-3 individuals per hectare (Uhl et al., 1997). The practice is done on a large scale through mechanized tree extraction or on a small scale Table 3.16 Forest area (1000 ha) designated primarily for production, and annual change, 1990-2020. Source: Global Forest resource assessment (FAO, 2020a) under license CC BY- NC-SA 3.0 IGO. Table 3.16 Forest area (1000 ha) designated primarily for production, and annual change, 1990-2020. Source: Global Forest resource assessment (FAO, 2020a) under license CC BY- NC-SA 3.0 IGO. Table 3.16 Forest area (1000 ha) designated primarily for production, and annual change, 1990-2020. Source: Global Forest resource assessment (FAO, 2020a) under license CC BY- NC-SA 3.0 IGO. NC-SA 3.0 IGO. 3.3.4.2. Global trends and overview Year Annual change 1990 2000 2010 2020 1990-2000 2000-2010 2010-2020 Forest area 4236433 4158050 4106317 4058931 -7838 -5173 -4739 Planted Forests 170061 210662 261958 292587 4060 5130 3063 Forest area with long- management plans 1757831 1855538 1990865 9771 13533 Forests in protected areas 437 821 499 853 600 845 629 139 6 203 10 099 2 829 Forest designated for Production 1 135 826 1 112 657 1 097 126 1 134493 -2 317 -1 553 3 737 Forest designated for multiple use 809 181 780 458 750 728 738 464 -2 872 -2 973 -1 226 Selective harvesting is one of the dominant logging practices that contributes nearly 15 percent of global timber needs (P. A. Martin, Newton, Pfeifer, Khoo, & Bullock, 2015; Poudyal, Maraseni, & Cockfield, 2018). Selective harvesting can be low impact timber- harvesting when it involves harvesting 1-2 species and 1-2 individuals per hectare. It can be moderate when 5-15 species are harvested and 1-3 individuals per hectare (Uhl et al., 1997). The practice is done on a large scale through mechanized tree extraction or on a small scale 235 through manual extraction (Rendón-Carmona, Martínez-Yrízar, Balvanera, & Pérez-Salicrup, 2009). The practice can also be carried out by local people who hand harvest wood in exchange for staples, while large distant companies do the wood processing Globally selective logging is practiced on about 20.3% (3.9 million km2) of humid tropical forests (Asner, Rudel, Aide, Defries, & Emerson, 2009). Selective logging is considered unsustainable when it is carried out the conventional way without measures to reduce damage to the residual forest stand. It is considered sustainable when specific planning and techniques are used to minimize damage to the residual stand. Several of these techniques are included in guidelines which are referred to as Reduced Impact Logging (RIL) (Arets et al., 2011; Dykstra & Heinrich, 1996; Pinard, Putz, Tay, & Sullivan, 1995; F. E. Putz, Sist, Fredericksen, & Dykstra, 2008). Reduced impact logging is implemented at the operational level by planning skid trails, practicing carefully controlled felling and skidding, and reducing damage to soils and residual trees (Sist and Ferreira, 2007). Implementation of reduced impact logging is still limited (Arets et al., 2011; P. A. Martin et al., 2015) and conventional logging practices continue to dominate (F. E. Putz, Dykstra, & Heinrich, 2000). 3.3.4.2. Global trends and overview Although illegal timber trade and unsustainable 236 logging that threaten sustainable use are rampant and not well documented, it is promoting large-scale forest destruction, especially in the tropics (Laurance, 2004). The Illegal timber trade is highly international, which may result in substantial loss of large old trees. Owing to the higher prices of timber in India and China, smugglers are motivated to export timber from Nepal to the Tibetan Autonomous Region of China (Chaudhary, Uprety, & Rimal, 2016); and Nepal-India border (Chaudhary et al., 2016). Regarding data on logging, a common understanding is that it is hard to obtain accurate data on the scope of illegal logging. Scientific studies as well as reports present conflicting views on whether illegal logging is declining or not (Kleinschmit, Mansourian, Wildburger, & Purret, 2016). According to Hoare (2015), there has been important progress made in reducing illegality in the forest sector over the last decades. However, another report published three years earlier claims that illegal logging has remained high in many regions, even increased in some areas, and become more advanced with better organized activities also comprising criminal activities (Nellemann, International Criminal Police Organization, & GRID--Arendal, 2012). China (importing more than 50%, of total illegal export value), Vietnam, India, the European Union, Thailand and the United States of America are among the major importers of illegal timber accounting for 84% of the total value of imports. Southeast Asia (mainly Cambodia, Laos, Myanmar Indonesia and Malaysia), the Russian Federation, Papua New Guinea and the Congo Basin (Democratic Republic of Congo, the Republic of Congo and Cameroon are among the main exporters with Southeast Asia accounting for 55% of the exports (Chaudhary et al., 2007). There has also been an observed geographic shift in illegal logging and related timber trade. Illegal logging in Brazil, Indonesia and Malaysia has declined in recent years (Hoare, 2015). After decades of conservation efforts, forests along the China-Russia border have been recolonized (Wang et al., 2016). In recent years the smuggling of timber as well as other forest resources has declined along Nepal-China and Nepal-India borders due to improved monitoring and collaborative transboundary conservation (Chaudhary et al., 2016; personal communication with Bishnu Lama, indigenous people and local community member and chairman of the Namkha rural municipality - Humla District, Nepal, December 2020). 3.3.4.2. Global trends and overview Among the major factors hindering adaptation of reduced impact logging is the expense in comparison with conventional timber-harvesting (F. E. Putz et al., 2000, 2008). In addition, evidence that reduced impact logging achieves the desired objectives is also contradictory. Some studies do suggest a reduction in the negative impacts of logging activities when reduced impact logging guidelines are followed (Bicknell, Struebig, Edwards, & Davies, 2014; R. Pereira, Zweede, Asner, & Keller, 2002; Putz et al., 2012; T. A. P. West, Vidal, & Putz, 2014). However, other studies suggest that positive effects of reduced impact logging are in fact more closely related to differences in harvesting intensity (Griscom, Ellis, & Putz, 2014; Johns, 1992; Picard, Gourlet-Fleury, & Forni, 2012; Sist, 2000; Sist, Fimbel, Sheil, Nasi, & Chevallier, 2003; Sist, Nolan, Bertault, & Dykstra, 1998). Reduced impact logging provides guidelines to reduce environmental impacts of logging, however its lack of specificity regarding intensity can sometimes result in perverse effects. Therefore, more research is recommended to clarify whether reduced impact logging should be practiced in a way which incorporates harvesting intensity (Martin et al., 2015). Low intensity harvesting that is recommended in reduced impact logging may encourage expansion into previously unlogged areas in order to distribute the impact more widely (Martin et al., 2015). In addition, the recovery rate of commercial trees after reduced impact logging is very low. One study in tropical rain forests, revealed that only 50% of the commercial stand was predicted to recover after a period of 30 years, creating a major reduction in stock for the next harvesting cycle in that area. This is not compatible with sustainable yield production on a long-term basis (Sist & Ferreira, 2007). Sist and Ferreira (2007) suggest that more sophisticated silvicultural systems are required to ensure sustainable management of the forests on a long-term basis. There is evidence suggesting that reduced-impact logging practices, if actually employed, could increase future timber yields (Griscom et al., 2014; Johns, 1992; Picard et al., 2012; Sist, 2000; Sist et al., 2003, 1998). There is not much change on the ground in spite of these recommended practices (Putz, 2018). The status of illegal logging and associated timber trade as well as its trends in harvesting practice, constitute complex and serious challenges in the sustainable use of wild species (J. Liu, Yong, Choi, & Gibson, 2020). 3.3.4.2. Global trends and overview However, Russia, other Southeast Asian countries (e.g., Cambodia, Laos and Myanmar), Papua New Guinea and some African countries have also witnessed increases in illegal forest activities (Guan et al., 2016). Russia (primarily in its Far East region) is one among rising timber producer countries and exports timber mainly to China (Guan et al., 2016). China has become the world’s largest importer of tropical timber since a ban on domestic logging was implemented in 1998. It is also a key processing country, for example, it is the leading manufacturer of furniture worldwide, occupying 40 percent of the global market share (Richer, 2016); much is exported to the United States of America and Europe (Tacconi et al., 2016). Commercial logging is illegal in Afghanistan which leaves a massive smuggling industry to satisfy international demand. Local communities have lost control over the resources on which they depend for their survival, and forest resources are now largely used for immediate profit by organized crime syndicates and traders (Milbrandt & Overend, 2011). Additionally, poor forest management, lack of incentives for reforestation, lack of community involvement and awareness, and agricultural and urban encroachments on forest land also contributed to the severe decline of forest cover in Afghanistan (Milbrandt & Overend, 2011). 237 The results have been that rangelands have deteriorated, forests have been felled, and wild species populations have greatly diminished from uncontrolled hunting and habitat degradation (UNDP, 2014). In 2006, an executive order that was issued by then President Hamid Karzai banned illegal timber-harvesting and felling of trees and shrubs in natural forests in Afghanistan. After that, Afghanistan’s Forest Management Law, passed in 2012, declared natural forests and woodlands as public property owned by the national government. The law also has a provision to support community-based forest management, allowing indigenous communities to utilize and manage the forest in collaboration with the Department of Natural Resources. However, the deterioration of overall law and order situation in Afghanistan means that the 2012 forest law has only been partially implemented. Curbing illegal timber extraction and trade poses special challenges because of the need for cooperation among sovereign states. In order to support producer countries, bilateral arrangements have emerged, either between neighboring countries or between primary export and import countries (Kleinschmit et al., 2016). 3.3.4.2. Global trends and overview Imports of illegal tropical hardwood timber in China with the republic of Congo, Ghana, Papua New Guinea, Laos, Brazil and Malaysia; India with Brazil, and Paua New Guinea; Japan with Republic of Congo, Cameroon, Malaysia and Paua New Guinea; and South Korea with Malaysia are considerable (Z. Guan, Chen, Xu, & Liu, 2020). Bilateral actions that also include transboundary cooperation have been initiated at the national level (Tacconi et al., 2016). Besides scientists, transboundary conservation deserves more attention from policymakers too (Liu et al., 2020). Policy in one country can easily have a major impact in other countries. For example, some research suggests that logging bans in Thailand and China have led to increased logging and forest loss in the neighboring countries including Lao People's Democratic Republic, Cambodia, Indonesia, the Russian Far East and Mongolia (Fisher, Maginnis, Jackson, Barrow, & Jeanrenaud, 2008). Hence, there is need to further strengthen international cooperation and domestic legislation in order to control the imports of illegal timber, enhance the protection and cultivation of forest resources and reduce dependence on imported timber (Guan et al., 2020). The spread of illegal logging and other forest crimes into protected areas occurs because valuable timber is still available in commercial volumes (Wardojo, Suhariyanto, & Purnama, 2001). Timber felling in protected areas in Indonesia involve multiple stakeholders, including local people, logging companies, military personal and forestry officials (Barber & Talbott, 2003; Hiller et al., 2004; Laurance, 2004; McCarthy, 2002; Ravenel, 2004; Robertson & van Schaik, 2001). Illegal logging provides immediate income for local communities and may aid in day-to-day survival (Schroeder-Wildberg & Carius, 2005). In some places illegal forestry activity is a function of local livelihood context such as reduced income from farming (Yonariza & Webb, 2007). 3.3.4.3. A stratified typology on sustainable use of wild species in logging Forests are owned either publicly by the state for the benefit of the citizens or privately by individuals, local, tribal and indigenous communities, or business entities and institutions. The proportion of forests under public ownership has declined, while those under private ownership increased between 1990 and 2015. In all regions, public administration holds management 238 rights to most of the publicly owned forests. Globally, individuals own most privately owned forests, followed by local, tribal and indigenous communities and the least are owned by business entities and institutions (FAO, 2020a) (Table 3.17). Table 3.17 Management of forest area under private and public ownership. Source: Global Forest resource assessment (FAO, 2020a) under license CC BY-NC-SA 3.0 IGO. Area of forest in three types of private ownership, by region, 2015 (1000 ha) Holders of management rights to public forests, by region, 2015 (1000 ha) Region/ subregion Individuals Local, tribal and indigenous communities Business entities and institutions Public administration Individuals Local, tribal and indigenous communities Business entities and institutions Unknown/other Africa 824 15599 1978 378849 0 7104 41485 844 Asia 7196 3900 1742 323232 45 30245 1275 40052 Europe 50946 2535 11691 641273 1 1324 244003 809 North and Central America 129468 45579 59723 389302 202 5570 54882 2956 Oceania 160 37551 0 6728 0 0 278 0 South America 0 3491 144 435192 2014 7173 5925 3 World 188592 108655 75279 2174576 2263 51416 347848 44664 le 3.17 Management of forest area under private and public ownership. Source bal Forest resource assessment (FAO, 2020a) under license CC BY-NC-SA 3.0 IGO. Features of logging activities vary depending on the specific contexts in which they develop. Land tenure, the level of access to public infrastructure (e.g., roads, energy, health, education) and proximity to markets are all important structural conditions. Ecological conditions including stand composition, seasonality, and soil types also affect harvesting conditions. In all these cases, logging may apply technologies that range from artisanal, often manual and carried out with or without permits by individual small-scale millers, to industrial operations with highly mechanized large scale tree removal. To try to account for these variables, the status and trends of logging operations have been analyzed using a three-element typology which generally corresponds to the scale of volume harvested and size of harvest area (Table 3.18). Specific actors have also been associated with these categories. 3.3.4.3. A stratified typology on sustainable use of wild species in logging In increasing volume and area, these are identified as: (1) smallholder, (2) community and (3) industrial logging operations: 1. Smallholder forestry, where logging is undertaken by individuals or family groups in lands on which they hold individual private access to forests and timber 1. Smallholder forestry, where logging is undertaken by individuals or family groups in lands on which they hold individual private access to forests and timber 2. Community logging or community forestry, where logging is organized and carried out collectively in forests stocking on community lands, using either artisanal techniques or externally supported reduced impact logging with heavy machinery 3. Industrial Logging, where individual companies holding individual or long-term concession rights conduct either conventional or reduced impact logging. 239 Table 3.18 Typology of logging systems. Actors = Social entities organizing logging operations. Harvest regimes = equipment used, volume harvested, species, age class, size, return interval, regeneration, etc.. Governance = customary and formal norms (including cultural knowledge and principles), rules, and regulations, management plans. Economy = subsistence, informal trade, formal trade; harvest to consumption value chains, distribution of benefits, and capital accumulation. Table 3.18 Typology of logging systems. Actors = Social entities organizing logging operations. Harvest regimes = equipment used, volume harvested, species, age class, size, return interval, regeneration, etc.. Governance = customary and formal norms (including cultural knowledge and principles), rules, and regulations, management plans. Economy = subsistence, informal trade, formal trade; harvest to consumption value chains, distribution of benefits, and capital accumulation. Actors Harvest regime Governance Economy Aggregate All 3.3.4.2 3.3.4.2 3.3.4.2 Smallholder Individual or collective 3.3.4.3.1. 3.3.4.3.1. 3.3.4.3.1. Community Collective 3.3.4.3.2. 3.3.4.3.2. 3.3.4.3.2. Industrial Individual or corporation 3.3.4.3.3. 3.3.4.3.3. 3.3.4.3.3. These logging operations are further differentiated by key aspects of use, identified here as harvest regime, governance, and economy: These logging operations are further differentiated by key aspects of use, identified here as harvest regime, governance, and economy: 1. Harvesting regime refers to the species harvested, species characteristics which affect volume of harvest such as growth and regeneration rates, and the techniques and equipment used. 1. Harvesting regime refers to the species harvested, species characteristics which affect volume of harvest such as growth and regeneration rates, and the techniques and equipment used. 2. Governance refers to different forms of access to forests and timber. 3.3.4.3. A stratified typology on sustainable use of wild species in logging It also refers to individual and collective rights, which range from diffuse and well-defined customary rights to full formal ownership of private lands and long-term usufruct rights in public lands. Legality is also considered a governance issue. 3. Economy refers to ways in which benefits are accumulated by actors. These include subsistence needs, and produce goods for the formal and informal economies. There are differential distributions of benefits depending on the form of capital accumulation and capital distribution. 3.3.4.3.1. Smallholder Logging practice Estimates suggest that 1.3 billion people live in or around the world’s remaining forests (Chao, 2012). These include right holders with individual and collective access to forests, either formal or informal. Rights holders may include individual landowners, indigenous traditional communities, local communities with established land tenure and historical access, and naturalized immigrant communities (for example from settler colonial expansion). In many cases different individuals and communities may co-exist in the same locations. For example, in the Brazilian Amazon, traditional dwellers are comprised by caboclos or local people who descended from immigrants who followed the several waves of resource exploitation into the region and mixed with indigenous residents (Adams, Murrieta, Neves, & Harris, 2009). In the north central United States of America, the indigenous Menominee and Ojibwe peoples manage their tribal forests independently of the surrounding state and federally owned lands 240 (Mausel, Waupochick, & Pecore, 2017; Ronald L. Trosper, 2012; Waller & Reo, 2018). Naturalized communities and migrants are more recent arrivals into forest zones who settled spontaneously or followed government-sponsored programs (B. M. Fernandes, 2004). In Southeast Asia, immigrants followed state-driven immigration programs but also followed the development of plantations that attracted rural labor to forest landscapes (Budidarsono, Susanti, & Zoomers, 2013). All these local groups undertake some type of small-scale logging, along with landless people trying to make a living, a portion of which may carry out logging operations on smallholder lands through different arrangements. Smallholder plot sizes range widely across world regions. Plots in more remote areas tend to have more independent logging activities. For example, 60% of family forest owners in the United States of America have an area ranging between 0.4 - 4.0 ha (Snyder, Butler, & Markowski-Lindsay, 2019). Many smallholder farmers in the Amazon have access to larger pieces of land of up to roughly 100 ha (Siegmund-Schultze, Rischkowsky, da Veiga, & King, 2007) (Budiman, Fujiwara, Sato, & Pamungkas, 2020). In the Amazon, the more remote farms are, the higher the probability that they still have some primary forest remnants stocking their property. These remote farmers often operate more independently regardless of the status of their tenure (Serra, 2020). In the Amazon, most of the forests on the land occupied by immigrant smallholders are already degraded from fires or former harvesting by commercial loggers. 3.3.4.3.1. Smallholder Logging practice Smallholders may harvest trees from their plots, but, due to the immense logistical and legal challenges, this is rarely carried out for commercial purposes. Accordingly, for most farmers, forests are a reserve for agricultural land, or provide materials for subsistence needs (e.g., fences, fuel wood) (Pacheco, 2009). If marketable timber is still available, they may also extract trees for commercial purposes when quick cash is needed (Pokorny, 2013). While smallholder farmers selectively extract high-value timber from remnant forests, they may also sell timber that originated from secondary forests emerging in agricultural fallows, often to local markets. At the same time, growing trees is an essential component of most smallholders' production systems (Hoch, Pokorny, & de Jong, 2012). Accordingly, over time, landscapes occupied by smallholders develop complex land-use mosaics that include swidden fields, fallows, agroforestry plots and forest patches (Denevan & Padoch, 1987; Padoch & Pinedo- Vásquez, 2006). The small-scale logging industry is characterized by stakeholders that may or may not have a felling permit, often use chainsaws (sometimes mobile saw) for felling and processing in the forest, have smaller numbers of trees per operation, often produce lower quality sawnwood for national market and neighboring countries and is largely informal (Cerutti & Lescuyer, 2011). In addition to chainsaws, winches and canoes with outboard motors are often used when water transport is involved. Chain saw milling requires a relatively small investment as the equipment is readily available and inexpensive to buy or rent, is portable and efficient (Pinard et al., 2006). Among the products of chainsaw milling are boards and planks for personal use, those that are sold directly to the market, and blocks or scantlings that are further processed in sawmills (Wit, van Dam, Omar Cerutti, Lescuyer, & Mckeown, 2010). The logging team consists of a few individuals who could be part of an entitled community or recruited from elsewhere, and they may own their own equipment or operate equipment owned by others (Salo, Sirén, & Kalliola, 2013). They harvest significantly smaller volumes of timber. The practice is usually very selective, concentrating only on the most 241 valuable commercial species such as, in the tropics, mahogany, cedar, teak and tornillo (Cedrelinga catanaeformis). Chainsaw systems persist especially in areas with more rugged terrain. Across the United States of America, one-third to over three-quarters of loggers used chainsaw felling (Conrad, Greene, & Hiesl, 2018). 3.3.4.3.1. Smallholder Logging practice This is based on production of 38 m3 ha-1 of sawnwood on a stand of 7 years. Smallholder farmers extract timber from remnant standing forests but also from secondary forests growing from fallows. The main product from the fallow-forestry system is small dimension lumber from Guazuma crinita (Sterculiaceae) and Calycophyllum spruceanum and Rubiaceae (Sears et al., 2014). In tropical regions, forest concessions occupy more than 20% of public forests in west and central Africa and Southeast Asia and about 4% in Latin America. In the tropics 15% of forests are managed by communities (Arts & de Koning, 2017). All these communities manage 3.3.4.3.1. Smallholder Logging practice Wherever possible, small-scale chainsaw millers target large trees to maximize their output (Cerutti & Lescuyer, 2011). Wood production by small-scale chain saw operators can be for personal use (Snyder et al., 2019) and to supply domestic markets (Rozemeijer & Aggrey, 2011). In some instances, the wood is for social or community purposes and not sold or exchanged (Lesniewska & McDermott, 2014). When entering into formal markets, the timber is usually purchased by middlemen at cheaper prices who then sell it to the timber industries (Salo et al., 2013). The industry is rapidly growing in tropical countries (Hoare, 2015), representing approximately 30- 40% (in Guyana, Republic of Congo, Democratic Republic of Congo and Uganda), more than 50% (in Ghana, Cameroon and Peru), and almost 100% (in Liberia) of total timber trade (Wit et al., 2010). However, wood for timber is only a small part of the total domestic market, with most locally traded wood in the tropics being used for fuel or made into charcoal (Wit et al., 2010). In many Amazonian countries (e.g., Bolivia, Peru, Ecuador), smallholders are allowed to extract timber from their properties for commercial purpose, yet they have to obtain permits, often through simplified processes including simpler management plans (Cerutti & Lescuyer, 2011) (Box 3.15). That said, few smallholders, such as those in the Peruvian Amazon, have secured formal property rights (Cronkleton & Larson, 2015). For those with formal usufruct rights to households occupying forest lands, they may be able to register a formal forest management plan to carry out selective timber-harvesting (Robiglio, Acevedo, & Simauchi, 2015). In spite of the options allowing for the use of simplified plans, only a small portion of smallholders formally apply for forest permits (Pacheco, Mejía, Cano, & de Jong, 2016). Box 3.15 Smallholder logging in Ucayali, Peruvian Amazon There are approximately 440,000 smallholder producers (i.e., plots <115 hectares) in Ucayali region in the Peruvian Amazon, with approximately 80% holding less than 20 hectares of land (Robiglio et al., 2015). It is estimated that mosaic production systems of these smallholders cover more the 4.5 million hectares in the Peruvian Amazon, with approximately 90,000 ha under fallow-forestry (Sears, Pinedo-Vasquez, & Padoch, 2014). Farmers in the Ucayali region of Peru produce 950,000 m3 of sawn wood annually (Sears, Cronkleton, Polo Villanueva, Miranda Ruiz, & Pérez-Ojeda del Arco, 2018). Box 3.15 Smallholder logging in Ucayali, Peruvian Amazon There are approximately 440,000 smallholder producers (i.e., plots <115 hectares) in Ucayali region in the Peruvian Amazon, with approximately 80% holding less than 20 hectares of land (Robiglio et al., 2015). It is estimated that mosaic production systems of these smallholders cover more the 4.5 million hectares in the Peruvian Amazon, with approximately 90,000 ha under fallow-forestry (Sears, Pinedo-Vasquez, & Padoch, 2014). Farmers in the Ucayali region of Peru produce 950,000 m3 of sawn wood annually (Sears, Cronkleton, Polo Villanueva, Miranda Ruiz, & Pérez-Ojeda del Arco, 2018). This is based on production of 38 m3 ha-1 of sawnwood on a stand of 7 years. Smallholder farmers extract timber from remnant standing forests but also from secondary forests growing from fallows. The main product from the fallow-forestry system is small dimension lumber from Guazuma crinita (Sterculiaceae) and Calycophyllum spruceanum and Rubiaceae (Sears et al., 2014). In tropical regions, forest concessions occupy more than 20% of public forests in west and central Africa and Southeast Asia and about 4% in Latin America. In the tropics 15% of forests are managed by communities (Arts & de Koning, 2017). All these communities manage 242 forests through different socio-ecological systems with very peculiar characteristics that are associated with traditional knowledge and community identity. Informal logging by smallholders provides thousands of jobs in Central African countries. In the Congo Basin, countries have embraced forest policies that mainly targeted the sustainable management of timber in large-scale timber-harvesting concessions targeting export markets and overlooked small-scale production. Yet small-scale chainsaw milling, which is chiefly informal, has undergone rapid development to meet the domestic demand for cheap timber in Central African countries and other nearby countries, as well as the interests of stakeholders all along the chain of custody (Eba’a Atyi et al., 2016). Over the last decade, in Central Africa, the annual volume of timber from informal chainsaw milling consumed domestically or unofficially exported to nearby countries is greater than that of timber from the industrial sector (Guillaume Lescuyer & Cerutti, 2013). In Cameroon, around 45,000 people find their main employment in this sector (Cerutti & Lescuyer, 2011). In the cities of Congo, the Central African Republic and Gabon, more than 1,000 people have jobs directly linked to the sale of small-scale timber production (Guillaume Lescuyer, Cerutti, & Robiglio, 2013). Box 3.15 Smallholder logging in Ucayali, Peruvian Amazon Second, the industry contributes significantly in terms of foreign exchange. In 2019, Indonesia exported 1.7 billion United States dollars from the furniture exports (Bank Indonesia, 2020). Third, the furniture industry also represents Indonesian identity in international markets since Jepara is a furniture producing district with global recognition as furniture and woodcraft center (Pujiati, 2017). Ironically, the performance of the industry at the national level is not well-known, and national estimates regarding the size of the industry are based on limited data. The most valuable data comes from the Central Statistical Agency, which reported that by 2019, there were 145,000 furniture micro, small and medium enterprises in Indonesia (wooden and nonwooden-based), representing about 3.3% of the total sample of 4.4 million micro, small, and medium enterprises (BPS, 2020). A Ministry of Industry report shows that wooden- based furniture producers represent about 80% of the total furniture producers (Munadi, 2017; Pujiati, 2017). From this information and the Central Statistical Agency data, there was an estimated 116,000 wooden-based furniture producers in Indonesia. p A survey of furniture producers in Jepara and Pasuruan in 2020 by the Center for International Forestry Research (Dermawan, 2020) estimated that one producer uses about 71 m3 of wood annually. Multiplying this number with the estimated total national producers, the wood consumption by the furniture industry in Indonesia could reach approximately 8.2 million m3 of wood. A high segment of wooden furniture in Indonesia uses teak as the primary raw material. Teak is mainly available in Java and some areas in other islands, such as Sulawesi. With the mean annual increment of 10 m3/ha/year (Kallio, Kanninen, & Krisnawati, 2012), meeting the need for 8.2 million m3 of wood would require approximately 820,000 hectares of teak forests. In the Amazon, much timber harvest takes place at the scale of individual households, even within communal properties granted to indigenous communities (Cronkleton & Larson, 2015). The smallholders may harvest the timber themselves with chainsaws and then process the log to produce planks, which are easier to transport, they hire specialized loggers (Pokorny, 2013). Yet, this informal practice is generally penalized by law, except in some countries like Ecuador (Sears et al., 2014). In some cases, smallholders sell standing timber to professional loggers that have better connection with sawmills, which often approach a larger number of farmers so to compensate for the use of heavy machinery (Mejia et al., 2015). Box 3.15 Smallholder logging in Ucayali, Peruvian Amazon Small-scale chainsaw milling is an important source of income for rural stakeholders, and accepted by urban consumers (Guillaume Lescuyer et al., 2017), who gain access to materials at prices three to four times lower than those from industrial timber (Guillaume Lescuyer et al., 2013). In remote areas, smallholders, when in need to harvest and sell timber often face distorted market conditions, mainly for two reasons. They may suffer from elevated transport costs, due to long distances, bad roads, and small quantities, or, to avoid logistical challenges depend on intermediaries or sawmill operators that tend to underprice the timber (Pacheco, 2012). In locations closer to the markets, smallholders who still dispose on forests, are better engaged to extend market networks managed by intermediaries who organize the extraction in response to orders from end-buyers in the cities (Mejia, Pacheco, Muzo, & Torres, 2015). Main markets are for construction such as in Central Africa (Eba’a Atyi et al., 2016), and the furniture industry such as in Jepara district, Indonesia (Box 3.16). In some places in the Amazon, the broader value chain for small-dimension lumber supports hundreds of other actors involved in the harvest, transport, transformation, and wholesale activities within marketing networks stretching from remote areas of the Amazon to major urban centers in Peru’s coast and highlands (Pokorny, 2013; Sears et al., 2018). In the Amazon, for most smallholders, primary forests play only a little role for income generation, whenever wood products such as firewood, poles, or for construction are regularly used by the families (Porro et al., 2014). In addition, forest fallows have a potential to generate income if the production areas are located near to roads and markets. In such conditions, farmers benefit from additional incomes from selling wood ranging from $35 to $1870 United States dollars per hectare (Hoch et al., 2012; Sears et al., 2014), with multiplier income effects associated with the processing. In Central Africa, chainsaw milling also constitutes an important source of employment for rural population, which translates into a relatively regular income stream on the lack of other job opportunities (Eba’a Atyi et al., 2016). Box 3.16 The furniture industry in Indonesia 243 Furniture is an important industry within the forest-based sectors for several reasons. First, micro, small and medium enterprises play a significant role in creating employment. The furniture sector provides direct employment to approximately 500,000 individuals (Munadi, 2017). Box 3.15 Smallholder logging in Ucayali, Peruvian Amazon In the Peruvian Amazon, commercial harvest of fallow timber is done with a chainsaw portable mill with a circular saw set up on the farm for in situ primary transformation. It is often the case that the rough-hewn planks are planed into the finished product in either lumber yards or workshops in the urban centers. Systems for permitting are quite different and greatly vary in many countries in temperate zones, where land tenure systems tend to be more closely regulated with regards to private vs. public ownership. In cases of private ownership, there is variation in the freedom to decide the amount of timber to harvest, approvals required to harvest and freedom of owners 244 to perform the actual harvesting (Nichiforel et al., 2018). Freedom to decide the amount of timber to harvest can be based on a framework of general silvicultural restrictions (e.g., Norway, Austria, United Kingdom of Denmark, Ireland, Latvia, Portugal and Sweden) and size/quantity provided for in the legislation. For example, in France the forest owners maximumly harvest 50% of the standing timber on their property in comparison to Estonia, where one can harvest 20m3 per year and Bulgaria where one can harvest 10m3 per year. These ranges are a result of forest management planning in combination with owners' decisions. In some cases, such as in Finland and Netherlands, more restrictions apply. In other countries owners are generally required to ask for approvals and adhere to the conditions of approval (Bulgaria, Greece, Romania). Approvals may be required when forest management plans do not apply (e.g., France and Czeck Republic) or when there are special circumstances such as exceeding a given size of clear cut. There is little regulation on private forests in the United Kingdom of Denmark, and in Estonia no formal approval is required for personal use. In the majority of the countries, forest owners have the freedom to cut down trees without any restrictions, others restrict the quantity an individual can harvest by him/herself (for example in Romania where owners can harvest less than 20m3 without a permit). Several countries in Eastern Europe only grant licenses to individuals with harvesting skills, and others such as Greece require the owner to contract a special firm for harvesting (Nichiforel et al., 2018). Ecological impacts of small-scale logging range from minimal to long-term. 3.3.4.3.2. Community Logging practice 3.3.4.3.2. Community Logging practice Community forest management involves the use and management of forests by communities. While community forestry often involves the management of large areas of forest relative to the average size of that managed by individual smallholders, the areas are still small relative to most industrial estates (100s of hectares compared with 1000s of hectares). Furthermore, the focus on multiple use management is strong in both community and smallholder forestry compared with the focus on timber production in industrial estates. Forest areas that are owned or managed by local communities have been increasing in the last decades and account for up to 15% of total forest area worldwide (513 million hectares) (Putraditama, Kim, & Baral, 2021). Collective forest tenure reforms in countries such as China (Yiwen, Kant, & Long, 2020) and Indonesia (Putraditama et al., 2021), although criticized in terms of effectiveness (Yiwen et al., 2020), are likely contributing to this upward trend in community forest area. The trend in moving away from industrial forestry towards landholder-based forest management and community forestry may be due to increased support for community forests as a form of sustainable development. From an ecological perspective, indigenous, low-intensity forest use has little negative impact on forest ecosystems (Gómez-Pompa, Whitmore, & Hadley, 1991). The effects of informal, more intensive timber harvest by the community in more forested landscapes in the Amazon and Central Africa, are limited to the easily accessible parts of the forests, where, after a while, the valuable species tend to disappear (Ferreira, Cunha, & Parolin, 2014). The environmental damage becomes stronger with the involvement of professional loggers, as they have the means for investments into infrastructure and heavy machinery. Although logging may be highly selective, the damage to the forest could be immense as it damages the remaining stand and changes its structure and tree composition in the long run (de Avila et al., 2017). However, the basic ecological functions of the forest remain as long as it is not converted for agricultural purposes. Independently of the type of ownership or management goals, community forest management has been supported across the globe by governments and donors as a way of combining socio-economic development with forest conservation. Box 3.15 Smallholder logging in Ucayali, Peruvian Amazon In Central Africa, when smallholder logging is driven by demand, chainsaw millers may penetrate deeper into the forest, and apply more effective tools such as portable saws in order to meet with a growing urban demand (Cerruti et al., 2017). Fallow-forestry allows the use of timber and other non-timber forest resources, while providing multiple contributions to people to regenerate soil fertility and conserve biodiversity (Pattanayak & Sills, 2001; Pyhälä, Brown, & Neil Adger, 2006). In such systems, smallholder farmers often conserve scarce timber species, such as Cedrela odorata, Swietenia macrophylla, and Dipteryx spp.), among others (Putzel, Padoch, & Ricse, 2013). Due to the individualized living schemes of small-scale farmers in the Amazon, there are not many social impacts of forest management. However, natural and planted forests are frequently affected by accidental fires caused during field preparation, which further reduces the attractiveness of forest investments (Hoch et al., 2012) and may lead to conflicts. Less frequent are wood robbery, and forest tenure conflicts in the remoter, less accessible forest parts of smallholder properties. Although not often discussed, smallholder logging often does not involve women in the operations, which may lead to some unequal distribution of benefits in the households undertaking logging, although women develop other activities in the farm and gardens (Colfer, 2005). Thousands of households manage forest fallows and trees as part of their customary livelihoods strategy that meets both subsistence and income needs (Pokorny & De Jong, 2015). Smallholder logging is only sustainable when it is done for subsistence or at low intensities. It constitutes a complementary activity that is shrinking over time due to the expansion of agriculture. Even with forest fallows and re-growing secondary forest, tree species composition and tree growth are affected from soil degradation caused by agricultural uses and fire. 245 3.3.4.3.2. Community Logging practice Transferring responsibilities from public (e.g., governments) or private (e.g., companies) entities to forest communities is believed to create conditions for better conservation and more sustainable use of forest ecosystems, as well as fostering social well-being and gender equity (Nandigama, 2020). There has been a high level of support for community forests managed under communal property rights, which suggests participatory engagement in common property resource management promotes environmental sustainability through improved livelihoods for the rural poor (Bluffstone et al., 2018; Okumu & Muchapondwa, 2020; Ostrom 2008, 2009) and decreases the costs of management (Gutiérrez-Zamora & Hernández Estrada, 2020; Nandigama, 2020; Shumsky, Hickey, Johns, Pelletier, & Galaty, 2014). Community forests also increase local resilience and enable better disaster preparedness for emergencies ranging from earthquakes to the COVID-19 pandemic (Gentle et al., 2020). Communities can have full, partial, or no formal ownership of the forests they manage. In the cases that communities hold ownership of the forest land, they often share forest management responsibilities, including its costs and benefits, with governments, non- 246 governmental organizations, etc., via different legal arrangements (Hyde, 2016). The mechanisms that transfer rights to use and management of forests from public or private land to communities greatly vary across the world. Legal arrangements range from the forestry regime of “baldios” in Portugal or the “montes comunales en mano commum” in Galiza/Spain (Carvalho Ribeiro, Sónia Maria, 1998; Skulska, Duarte, Rego, & Montiel- Molina, 2020), and the “van panchayats” in the Himalayas (Thakur et al., 2020). In Mediterranean European countries, the existence of common property institutions and community forests in particular dates to at least a thousand years (Cullotta et al., 2015; Skulska et al., 2020). Community forest management is also associated with use of forests in indigenous reserves or designated sustainable use areas including for example the sustainable use extractive reserves, some of which were created over the last decades, granting conditional local use rights on state lands for vast areas. In South America, often communities also manage land through forest concessions. Forest concessions are defined as a formal legal agreement signed with a concessionaire for the occupation and use of a territory. In these agreements, space units are demarcated for the use and management of ecosystems for specific uses and for a fixed time. 3.3.4.3.2. Community Logging practice There are at least 122 million hectares of tropical forests under concessions, equivalent to 14% of the world's public forests some of which are managed by forest communities. The industry is operated by small and medium forest enterprises which are largely left out of the forest statistics, planning and management, and yet it is growing rapidly in many tropical countries (Hoare, 2015). The small and medium forest enterprises are characterized by low level capital, informally trained workers and having potential for value addition (Osei- Tutu, Nketiah, Kyereh, Owusu-Ansah, & Faniyan, 2010). The industry contributes directly to the local economy in the form of improved livelihoods and cheap lumber for urban consumers. Small and medium forest enterprises are the main, additional or alternative income sources for a greater proportion of the local population as compared to the large-scale formal forest subsector in countries where the forestry sector is among the major income earner (Cerutti & Lescuyer, 2011; Osei-Tutu et al., 2010). This is because small and medium forest enterprises tend to accrue wealth locally, empower local entrepreneurship and seek local approval to operate (Osei-Tutu et al., 2010). Small-scale enterprises tend not to be adapted to landlocked, low population density, remote markets and high transportation, costs but can compete and replace forest concessions when public road infrastructures allow them easier access to the market (Karsenty, Drigo, Piketty, & Singer, 2008) (Box 3.15). Globally, about 15% of tropical forests are managed by communities (Arts & de Koning, 2017), many managed by indigenous peoples and local communities. As of 2020, indigenous peoples and local communities in Africa, South America and Asia, customarily managed at least 31% of land area corresponding to 571 M hectares (Khare, White, & Frechette, 2020). As of 2016, in Latin America nearly 33% of forests (232 million ha) were under some type of collective tenure regime owned by communities, most of which are of indigenous peoples, and another 8% of the area had been designated for their use. An important portion of these forests are used for meeting subsistence needs, but few of the communities undertake commercial logging operations, formally or informally. Traditional forest management for subsistence uses tends to be informed by traditional knowledge and customary 247 local regulations (Gibson, McKean, & Ostrom, 2000). In turn, community forestry for commercial purposes is informed by management plans that are based on scientific forestry with no obvious role for indigenous knowledge. 3.3.4.3.2. Community Logging practice While area in community forestry is small compared with industrial tenures, it makes important contributions to community development and diversifying the beneficiaries of forestry (Bullock & Hanna, 2007; McIlveen & Rhodes, 2016; Teitelbaum, 2016). 3.3.4.3.2. Community Logging practice Often, these plans are inspired by large-scale industrial timber-harvesting schemes. Since informal management schemes are considered by some to be ineffective or degrading, the management of forests by communities on the basis of the Reduced-Impact- Logging principles and formally authorized management plans has been widely promoted given assumptions that it would lead to sustainable outcomes in terms of biodiversity and local income. Accordingly, hundreds of initiatives across the tropics have promoted community forestry, in some cases also labelled as social forestry or collaborative forest management (Hajjar et al., 2021). The most developed cases of community forest management in Mesoamerica include Quintana Roo, Mexico, and Peten, Guatemala (see Supplementary material Box S3.1), as well as community forestry in the Amazon including Brazil, Bolivia, Peru. In Central Africa, community forestry has mainly developed in Cameroon, and to a lesser extent in Democratic Republic of the Congo. Especially in Latin America, the promotion of community forestry was accompanied by the formal recognition of tenure rights to indigenous peoples (RRI, 2015), which has been understood as a critical condition for achieving positive outcomes (Baynes, Herbohn, Smith, Fisher, & Bray, 2015). In developed countries, community forestry is less well established than in developing countries (Bullock & Hanna, 2007). Charnley and Poe (2007) reported only 2% community and indigenous ownership of forests in developed countries in comparison with the approximately 14% of community and indigenous owned forests in developing countries. Community forestry began to be implemented in the 1990s in Canada as a result of public controversies surrounding large-scale industrial forestry, and as of 2007 existed in Ontario, Quebec, and British Columbia (Box 3.17). In the Canadian context, forests remain state property but communities receive key management rights and responsibilities. In the United States of America community forestry initiatives have been supported through joint efforts across private, tribal, and public lands across the country. Despite widespread support for increased public participation in environmental decision-making in the United States of America, there has been resistance from the government and environmental groups to yielding actual control over land to local communities. Thus, in the United States of America collaborations between state and federal forest management agencies and local communities has been more common (Charnley & Poe, 2007). Box 3.17 Community forestry on public lands in Canada Community forestry has been a legally recognized form of forestry governance in Canada for over 50 years. Box 3.17 Community forestry on public lands in Canada Community forestry has been a legally recognized form of forestry governance in Canada for over 50 years. While area in community forestry is small compared with industrial tenures, it makes important contributions to community development and diversifying the beneficiaries of forestry (Bullock & Hanna, 2007; McIlveen & Rhodes, 2016; Teitelbaum, 2016). 248 Three provinces in Canada have institutionalized forms of community forestry on public land: British Columbia, Ontario, and Quebec. Since 1998, British Columbia has granted 25-year renewable licenses to more than 50 organizations and indigenous communities under the British Columbia Community Forest Agreement (Government of British Columbia, 2020). British Columbia also has a tenure specific to indigenous communities, the First Nations Woodland Licence (Government of British Columbia, 2020). Quebec was the second province to adopt a community forestry policy. Although implementation has been slow (Ministère des Ressources naturelles et de la Faune, 2011), a number of community forests have been created across the province (Bissonnette, Blouin, Bouthillier, & Teitelbaum, 2020; Teitelbaum, Beckley, & Nadeau, 2006). Many are located in proximity to small rural communities and are run by municipal or regional governments (Chiasson & Leclerc, 2013). A handful have also been allocated to indigenous communities and are largely run by the band council. The province of Ontario has a network of county and municipal forests, as well as forests owned and managed by Conservation Authorities (Teitelbaum & Bullock, 2012). Under this model, forestlands are owned outright by local government entities and have strong authority over management decisions. In contrast, the provinces of Quebec and British Columbia retain considerable control over management decisions such as allowable timber cut, wild species management and gathering. Community forestry entities in Quebec and Ontario also face substantial administrative burdens from a regulatory system designed for much larger operations (Ambus & Hoberg, 2011; R.L Trosper & Tindall, 2013). Timber harvesting is a main objective for many community forests and, in at least one case, generates significant employment in its region of British Columbia (McIlveen & Rhodes, 2016). However, there is considerable diversity in management values and priorities, with some strongly focused on protection of ecological functions and nature’s contributions to people. Some community forests have diversified their activities through development of recreation and/or alternative forest products. For example, one community forestry initiative in Quebec developed an innovative approach combining timber and wild blueberry production. Box 3.17 Community forestry on public lands in Canada Revenues have been sufficient to support research on optimal conditions for co-habitation of trees and blueberries (Fournier, 2013). The specific outcomes of community forestry initiatives largely depend on the biophysical conditions, tenure right situation, community characteristics, and the type of intervention. For the majority of cases, positive environmental and income-related outcomes are reported, but the need for formalization and the related bureaucratic and technical requirements negatively affect forest access and resource rights (Hajjar et al., 2021) and the attractiveness for the local resource users (Pokorny, 2013). Accordingly, the long-term success of community forestry initiatives largely relies on continuous external support, but only in some limited cases (Pokorny, Johnson, Medina, & Hoch, 2012). Traditional forest management for subsistence uses tends to be informed by traditional knowledge and customary local regulations (Gibson et al., 2000). In turn, community forestry for commercial purposes is informed by management plans that are based on scientific forestry with no obvious role for indigenous knowledge. Often, these plans are inspired by large-scale industrial timber-harvesting schemes. 249 In the Amazon, there are four general schemes of community timber harvesting: (1) traditional harvesting of forest products aimed to meet subsistence needs; (2) locally devised schemes to carry out commercial timber harvesting; (3) harvesting agreements between communities and loggers; and (4) formal community forestry on the basis of legally authorized management plans as described above (Sabogal, de Jong, Pokorny, & Louman, 2008). The species, volumes, areas, and management schemes of the forest operations vary strongly between and within these schemes and the context under which they occur. A key contextual factor is tenure. For example, some indigenous people and communities have been granted collective tenure, and others hold collective rights in extractive reserves, yet others have not been recognized with customary collective or individual tenure, which affects the community’s possibility to legally use timber. Informal logging operations tend to be highly selective of high-value species such as for example Swietenia macrophylla, Manilkara huberi, Mezilaurus itauba, Handroanthus serratifolia. While traditional logging practiced by communities works with motor-manual practices and concentrates on small areas up to 20 hectares with extraction volumes of around 50m³ in the entire area, formalized community forestry operations may cover extraction areas of up to 1,000 hectares and volumes extracted range from 5-20m³ per hectare. Box 3.18 Coomflona in Flona Tapajos, Para, Brazil Box 3.18 Coomflona in Flona Tapajos, Para, Brazil Tapajós National Forest is a government-owned land with community use designated as protected area with sustainable use of natural resources. Located in the state of Pará, in the Brazilian Amazon, Tapajós National Forest occupies an area of 527,319 ha of mostly dense tropical forest characterized by the dominance of large trees under a climatic regime of high temperatures and intense precipitation distributed throughout the year (Humphries, Andrade, & McGrath, 2015; IBAMA, 2004; Silva, de Carvalho, & Lopes, 1985). Over 24 forest-based communities are based in the area. Approximately 500 indigenous people and 5000 local people live within Tapajós National Forest. They have diversified livelihood strategies which include agriculture, non-timber forest products, timber, and fishing (Andrade, de Carvalho, ilva-Ribeiro, & Dantas, 2014; ICMBio, 2015). Tapajós National Forest residents founded a local timber cooperative, the Mixed Cooperative of the Tapajós National Forest (Coomflona) to manage tropical timber resources. The members include 150 forest residents from the 24 communities. With few exceptions, Coomflona hires external labor for work such as lawyer, forest engineer, and forestry machinery operators. The access to forest is collective, since every cooperative- member has the right to vote and make decisions over forest resource management. Tapajós National Forest is a government-owned land with community use designated as protected area with sustainable use of natural resources. Located in the state of Pará, in the Brazilian Amazon, Tapajós National Forest occupies an area of 527,319 ha of mostly dense tropical forest characterized by the dominance of large trees under a climatic regime of high temperatures and intense precipitation distributed throughout the year (Humphries, Andrade, & McGrath, 2015; IBAMA, 2004; Silva, de Carvalho, & Lopes, 1985). Over 24 forest-based communities are based in the area. Approximately 500 indigenous people and 5000 local people live within Tapajós National Forest. They have diversified livelihood strategies which include agriculture, non-timber forest products, timber, and fishing (Andrade, de Carvalho, ilva-Ribeiro, & Dantas, 2014; ICMBio, 2015). Tapajós National Forest residents founded a local timber cooperative, the Mixed Cooperative of the Tapajós National Forest (Coomflona) to manage tropical timber resources. The members include 150 forest residents from the 24 communities. With few exceptions, Coomflona hires external labor for work such as lawyer, forest engineer, and forestry machinery operators. The access to forest is collective, since every cooperative- member has the right to vote and make decisions over forest resource management. Box 3.17 Community forestry on public lands in Canada In accordance with Reduced-Impact-Logging principles, many in the Amazon region follow formally approved management plans drawing on timber inventories and respect defined cutting cycles (Sabogal et al., 2008). However, most frequently, timber on communal lands is harvested by local loggers on the basis of informal arrangements that pay the communities or the communitarian leader a lump sum for the right to harvest the forests. While these arrangements typically are unfair and often the logger doesn’t hold his promise, it provides communities the opportunity for an easy income (Medina, Pokorny, & Campbell, 2009) (Boxes 3.18 and 3.19). Box 3.18 Coomflona in Flona Tapajos, Para, Brazil Tapajós National Forest is a government-owned land with community use designated as protected area with sustainable use of natural resources. Located in the state of Pará, in the Brazilian Amazon, Tapajós National Forest occupies an area of 527,319 ha of mostly dense tropical forest characterized by the dominance of large trees under a climatic regime of high temperatures and intense precipitation distributed throughout the year (Humphries, Andrade, & McGrath, 2015; IBAMA, 2004; Silva, de Carvalho, & Lopes, 1985). Over 24 forest-based communities are based in the area. Approximately 500 indigenous people and 5000 local people live within Tapajós National Forest. They have diversified livelihood strategies which include agriculture, non-timber forest products, timber, and fishing (Andrade, de Carvalho, ilva-Ribeiro, & Dantas, 2014; ICMBio, 2015). Tapajós National Forest residents founded a local timber cooperative, the Mixed Cooperative of the Tapajós National Forest (Coomflona) to manage tropical timber resources. The members include 150 forest residents from the 24 communities. With few exceptions, Coomflona hires external labor for work such as lawyer, forest engineer, and forestry machinery operators. The access to forest is collective, since every cooperative- member has the right to vote and make decisions over forest resource management. 250 Decisions are made during general assemblies, held during the first three months of the year, and a cooperative executive committee operationalizes management decisions (Espada & Vasconcellos Sobrinho, 2019; Humphries, 2016). Coomflona has a permit to manage timber with non-onerous (zero-cost) concession from the federal government, and every year it has to submit an operational plan to get the approval from the government to execute timber-harvesting operations. Currently, the total timber harvest area covers 44,000 ha, and represents 8% of the total area of the Tapajós National Forest. Annually, Coomflona now manages an area of 1,500 ha, which has steadily increased since its first year of timber-harvesting operations in 2006. They manage for a cutting cycle of 30 years (Espada & Vasconcellos Sobrinho, 2019; Humphries, 2016). Coomflona implements reduced impact harvesting techniques, removing 3 to 4 whole trees per hectare. The main log extraction equipment in a skidder, and around of 30,000 meters cube of roundwood are harvest every year (Humphries et al., 2015). Coomflona, with the support of its partner organizations, achieved several notable accomplishments. First, the cooperative has secured financial resources for forestry operation costs, critical in community forestry. Box 3.18 Coomflona in Flona Tapajos, Para, Brazil Second, the cooperative created an innovative system of funds in which to allocate net profit from timber sales to benefit timber workers, their families, and communities, and beyond, local people that do not participate directly in the cooperative. Third, Coomflona invested in a portable sawmill and small-scale carpentry to verticalize timber production, aggregate value to timber products, expand market strategies, and engage additional community members in timber production. Fourth, Coomflona has established long-term and strong partnerships with diverse organizations. Fifth, Coomflona has become a model for other community-based groups aiming to manage timber resources in sustainable-use protected areas, as in the cases of extractive reserves. Sixth, Coomflona is running timber management with good practices considered in the forestry sector; the Forest Stewardship Council certification, for instance, certifies that Coomflona is maintaining forest health and ecosystem functions while provisioning both local social and economic benefits. Box 3.19 Ejido Petcacab-Quintana Roo, Mexico, drawn from (Wilshusen, 2005a, 2005b) Box 3.19 Ejido Petcacab-Quintana Roo, Mexico, drawn from (Wilshusen, 2005a, 2005b) Box 3.19 Ejido Petcacab-Quintana Roo, Mexico, drawn from (Wilshusen, 2005a, 2005b) Local communities own approximately 45% of Mexico's forests and have relative autonomy to manage them. Some of these communities have established community forest enterprises in order to generate benefits, such as jobs (Frey et al., 2019). In the Mexican state of Quintana Roo, tropical forest ecosystems dominate the landscape. Forest types vary by soil, topography and local climate: medium-stature forests (15 to 25 meters) are present on well- drained soils, while shorter forests occur on seasonally inundated wetlands depressions. Mahogany (Swietenia macrophylla) and Spanish cedar (Cedrela odorata) were historically the most important commercial tree species, but in recent decades lesser-known tropical species have come to constitute around 70% of the harvest (Ellis et al., 2015). As of 1992, the harvest was managed by four associations of forestry ejidos through community forest enterprises with a combined allowable cut of 10,580 m3 per year of mahogany and cedar from 393,481 ha of permanent forest areas (Flachsenberg & Galleti, 1999). Up until 1983, Local communities own approximately 45% of Mexico's forests and have relative autonomy to manage them. Some of these communities have established community forest enterprises in order to generate benefits, such as jobs (Frey et al., 2019). In the Mexican state of Quintana Roo, tropical forest ecosystems dominate the landscape. Forest types vary by soil, topography and local climate: medium-stature forests (15 to 25 meters) are present on well- drained soils, while shorter forests occur on seasonally inundated wetlands depressions. Mahogany (Swietenia macrophylla) and Spanish cedar (Cedrela odorata) were historically the most important commercial tree species, but in recent decades lesser-known tropical species have come to constitute around 70% of the harvest (Ellis et al., 2015). As of 1992, the harvest was managed by four associations of forestry ejidos through community forest enterprises with a combined allowable cut of 10,580 m3 per year of mahogany and cedar from 393,481 ha of permanent forest areas (Flachsenberg & Galleti, 1999). Up until 1983, 251 logging was carried out first by small private concessionaires and later by a parastatal company, with wildly fluctuating annual volumes between 10,000 and 50,000 m3. Box 3.19 Ejido Petcacab-Quintana Roo, Mexico, drawn from (Wilshusen, 2005a, 2005b) Beginning in 1984, community management produced a striking reduction and stabilization of harvests of mahogany and cedar going from 10,000 m3 annually, a 78% reduction from the last five years of the parastatal to around 5,000 m3 in 2018, and foresters consider this to be sustainable (Bray, 2020; Navarro-Martínez, Ellis, Hernández-Gómez, Romero-Montero, & Sánchez-Sánchez, 2018). Petcacab is an ejido, a common property land grant in Mexico’s agrarian system, inhabited by Mayan indigenous peoples. It is located in Central Quintana Roo, with an estimated population of 947 and 206 legal members of the ejido. The property regime is communal with a total land area of 46,000 hectares and permanent forest area of 32,500 hectares. Petcacab’s community forest enterprise was initially organized in the mid-1980s with external support from the Forest Pilot Plan, supported by the Mexican government and German foreign assistance. Petcacab initially organized its community forest enterprise as an entirely community-administered operation, supervised by community authorities. However, due to concerns about corruption, in 1996 Petcacab reorganized its community forest enterprise to be administered by what are termed “work groups” or coalitions of community members based on family clans and individual families. Access to communal lands by community groups, approved by the community assembly, was permitted by a 1992 reform to agrarian law. By 2000, Petcacab had 11 work groups who each received a proportional share of the annual authorized volume, and essentially managed themselves as small, separate community forest enterprises or microenterprises. All of the work groups operated under a single management program prepared by a professional forester and approved by the Mexican environmental agency. In the 2000s Petcacab had authorized harvest volumes of 1,499 m3 of mahogany, 2,545 m3 of tropical softwoods, 3, 927 m3 of tropical hardwoods and 10, 328 m3 of polewood, with a decline in the volume of mahogany in more recent years. Production is small-scale industrial, with the use of tractors and skidders for extraction and logwood is sold to a community sawmill or intermediaries. Harvests are regulated by Mexican forest and environmental laws and compliance is considered good. The work groups sell both logwood and sawnwood, after processing at a community sawmill. Most timber is sold domestically in Mexico. Benefits go to the individual work groups, with little or no reinvestment in the community or the community sawmill. Harvests are conducted according to the management programs with little or no input from indigenous knowledge. Box 3.19 Ejido Petcacab-Quintana Roo, Mexico, drawn from (Wilshusen, 2005a, 2005b) Harvests of mahogany have declined in recent years but are considered sustainable at current more reduced levels. Socially, the work groups have allowed for increased incomes at the work group and household level, but with a corresponding decline in community investments in public goods. The apportionment of the authorized volumes to individuals has led to a market in the shares of authorized volumes and increasing economic inequality with the ejido as some members purchase others shares. Economically the work groups appear to be profitable, and the work group arrangements appear to be sustainable. In protected areas an important timber management experience operated collectively by community-based enterprises takes place in the Mayan Biosphere Reserve, a protected area 252 of 2.1 million ha established in 1990s (Radachowsky, Ramos, McNab, Baur, & Kazakov, 2012). A total of 12-community concession contracts (for areas ranging from 7,000 ha to 85,000 ha for a total of 390,000 ha) were signed between 1994 and 2001 (Stoian, Rodas, Butler, Monterroso, & Hodgdon, 2018). All concession contracts required collective organization: three forms emerged i) limited liability companies or civil societies (Sociedades Civiles), ii) civil associations, and iii) cooperatives. Community concession contracts are legal agreements between the state and an organized group composed of members living in a given community. These 25-year concession contracts allowed concessionaire members rights to manage and extract timber and non-timber forest products recognizing also rights to implement nature- based tourism activities in protected areas. This system of community concessions in the Multiple Use Zone (MUZ) represents about 15% of the country’s total forest cover, including national parks (IARNA/URL/ILA, 2006). The area under forest concessions covers more than 480,000 hectares. To date, nine community concession contracts remain active (around 350,000 hectares) (Stoian et al., 2018). Compared to Latin America and South Asia, relatively little information on Africa was available. In Central Africa, the number of communities formally embracing community forest management has greatly increased over the last twenty years as all countries have included this management option in their forest legal frameworks. There are now more than a thousand of them, about 90% of which are in Cameroon. However, most of the community forestry operations validated by the authorities are either inactive, as in Cameroon (G. Lescuyer, Cerutti, & Tsanga, 2016), or oriented towards conservation, as in the Democratic Republic of Congo (Bauer, 2016). Box 3.19 Ejido Petcacab-Quintana Roo, Mexico, drawn from (Wilshusen, 2005a, 2005b) In total, only about 150 forestry communities are authorized in Cameroon, and about a hundred are created or in the process of being created in both Gabon and the Democratic Republic of Congo. There are no regional statistics on timber production from community forestry in Central Africa. Yet, based on case studies in each country, a maximum of 50,000m3 would be legally extracted from community forestry harvesting operations in the Congo Basin (Beauchamp & Ingram, 2011; Julve et al., 2013). Due to inadequate regulatory texts that are costly to apply for (Cuny, 2011), the vast majority of community forests that exploit timber do so illegally, destined for domestic markets that do not require timber of legal origin. There are no statistics on these illegal practices, but numerous reports from Cameroon (Nzoyem, Vabi, Kouokam, & Azanga, 2010) and Gabon (Ondo, Medik, Mijola, & Boussougou, 2020) indicate that informal timber-harvesting from community forestry operations far exceeds the volume legally extracted. Generally, South Asia’s forest dependent communities are the ones who are also the more disadvantaged in the region. Two categories of forest dependent people are identifiable. First, those who are traditionally the forest communities residing in and around forest areas for generations, such as tribal communities in India. Altogether, the population of this group is estimated to be 150 million in the region (World Bank, 2005). Second, people who depend on forests for a variety of products and nature’s contributions to people but do not directly reside inside or in the vicinity of the forest. There are around 400 million forest users in this category (Poffenberger, 2000). More recently, rapid rural and urban and even overseas migration of youths has led to change in the conventional patterns of forest dependence, with reduced use of forest products in livelihoods (Ojha et al., 2017). 253 In terms of policy shifts, South Asia has notable community forestry initiatives in terms of scale and demonstrated outcomes, although the actual form and operational modalities vary greatly across the countries and sub-national regions. Likewise, a variety of local regimes of community forestry are found: formally handed over state forests, jointly managed forests, ‘sacred groves’ with cultural values, community plantations, and other forms of collective land use for trees and pastures. The beliefs and rituals linked to sacred groves have helped to conserve biodiversity, although they are under threat due to changing values and perceptions (see section 3.3.5). Box 3.19 Ejido Petcacab-Quintana Roo, Mexico, drawn from (Wilshusen, 2005a, 2005b) Forest harvesting status and trends data for community forestry are not readily available for most countries in South Asia. Available evidence suggests all the countries and their sub- national authorities are struggling to optimize forest harvesting in a sustainable, efficient, and equitable way. A significant part of the forest landscape is under protected area management, and there have been participatory and co-management shifts in this regime too, especially since early 1990s. Studies show that such participatory shift in protected area management has resulted in more active use of resources, as found by a study in Bangladesh (K. Islam, Nath, Jashimuddin, & Rahman, 2019). In Bhutan, conservation rather than sustainable use mindsets dominate forest management policy and programs, and strategies and methodologies to promote sustainable harvesting are slow to develop (Phuntsho, 2011). Despite having nearly 70% of area under forest, Bhutan has kept harvesting level to a minimum, favoring import of forest products. The most significant wood-based import item is charcoal. In 2012, Bhutan imported charcoal worth 16.8 million United States dollars, comprising 1.4 percent of total imports and 60 percent of wood-based imports (MoF 2013, cited in World Bank (2019). The first national forest inventory published in 2017 and provides detailed data and information on Bhutan’s forests, showing the Bhutan is under harvesting its forest below the potential (World Bank, 2019). A bottom-up approach to forest management in Bhutan began after the 1979 royal decree that called for the involvement of local people in tree planting activities (Phuntsho, 2011). Bhutan’s community forestry policy emphasizes protection, conservation and sustainable use of forest resources in the country, together with contributions to poverty reduction and local democratization (Phuntsho, 2011). The level of harvesting in community forestry is no different than the national scenario. Following the adoption of a more decentralized and people- centered approach to forestry in the early 2000s, the number of community forestry management groups has increased rapidly since 2007. By 2018, there were 781 community forest management groups involving 32,402 rural households managing 92,165 hectares (3.0 percent) of forest land (MoAF, 2018; World Bank, 2019). Although the government is supportive to the implementation of the community forestry program, community forest management groups continue to face administrative hurdles with regard to timber marketing, leading to under-harvesting of forest stock (Samdrup, 2011). Box 3.19 Ejido Petcacab-Quintana Roo, Mexico, drawn from (Wilshusen, 2005a, 2005b) Community forestry in Nepal emerged in the mid-1970s and led to the devolution of management and user rights to forest user groups, largely as a shift in the approach to conserve the hill forests in the context of degradation and deforestation. During the last three decades Nepal’s community forestry programme has evolved in terms of coverage and institutional innovation, supported through appropriate changes in policies and legislation. Substantial international support has also helped to sustain community participation. Community forestry 254 has contributed to livelihoods of nearly one-third of the country’s total population, improved forest conditions and biodiversity, and above all, developed itself as a self-sustaining system involving a strong base of policy champions, service providers, and critical action researchers. By the end of 2019, over 22,000 community forestry user groups had been registered across the country, with rights granted to manage nearly two million hectares of forest areas (about a third of total forest areas of the country. Community forests are vital components of environmental resilience and nature’s contributions to people not only to the people close by but also to large populations downstream. Dissipating fears of desertification, community forestry has led to improvement in forest ecology, with 74% of the forest area managed by community forestry user groups reported as in “good” condition, compared to 19% in “degraded” condition (Kanel & Kandel, 2004). Community forestry user groups also compare favorably to government forests in terms of change in forest condition (Nagendra, Pareeth, Sharma, Schweik, & Adhikari, 2008). More recent empirical evidence confirms improved biodiversity outcomes from community forestry (Luintel, Bluffstone, & Scheller, 2018). Nepal has a strong legislation that allows communities to enjoy perpetual rights over designated community forest areas. Such perpetual and sustained rights of access to forests have been key for the success of community forestry in Nepal (Acharya, Adhikari, & Khanal, 2008). Though the land ownership remains with the government, the tenure of forest biomass is transferred to the community through a detailed approval process. Community forestry user groups retain 100 percent of revenues generated from their forest, but they have to allocate 25% of the income to forest development activities and 35% to programs that directly benefit the poorest households within the community forestry user group. Box 3.19 Ejido Petcacab-Quintana Roo, Mexico, drawn from (Wilshusen, 2005a, 2005b) Like Nepal and Bhutan, a conservative approach to forest harvesting dominates forest management practices across all regimes of public forests. harvesting dominates forest management practices across all regimes of public forests. India’s average annual yield of forest is estimated as 85.65 million m3, whereas the annual removal of only 5.85 million m3, which is 6.82% (FSI, 2019). Total growing stock is estimated to be 5915 million m3 and the growing stock of trees outside forest is 1642 m3 (FSI, 2019, p. 117). Total forest coverage between 2010 and 2020 increased in India by 0.38% (FAO, 2020a). Timber production from public forest meets only the 3.35% of the total demand, while trees from outside areas officially classified as forests provide 45% of the demand (Ghosh & Sinha, 2016). It is suggested that community forests managed by indigenous people and local communities are more likely to harvest in sustainable ways than those managed by the government (Sundar, 2017). Despite government efforts to raise domestic productivity, India’s overall timber production remains low. This is especially true for the tree species preferred by consumers such as teak, sheesham and pine (Norman & Canby, 2020). The International Union of Forest Research Organizations estimates that India was the third-largest importer of illegally logged timber in the world in 2016, after China and Vietnam (Kleinschmit et al., 2016). While its own forests are under harvested, India is emerging as a major importer of timber (Vanam, 2019). The demand for timber is growing from the current gross value added of 606 billion United States dollars in 2011 (FAO, 2014a). However, restrictive policy and regulatory barriers inhibit community forestry groups from harvesting and selling surplus timber from their forests even when supported by sustainable forest harvesting protocols (Shyamsundar, Ahlroth, Kristjanson, & Onder, 2020). India’s joint forest management is an arrangement for co- management between local communities and the Department of Forest. Typically, the joint forest management committee holds a joint account in the local public sector bank with the chairperson, vice-chairperson and the district forest officer or her nominee as joint signatories through which financial aid from donor and the government is channeled (Sundar, 2017). The district forest officer prepares forest management plans in consultation with the communities. Box 3.19 Ejido Petcacab-Quintana Roo, Mexico, drawn from (Wilshusen, 2005a, 2005b) The existing forest law provides communities with enough rights to choose their objectives of forest management and harvesting, but too often the actual practices of regulation and bureaucratic oversights hinder active management of forests beyond subsistence use. Nepal has achieved massive scale community forestry development in terms of enabling policy and institutional development, but the actual use of forest is less than 30% of the annual sustainable harvest level. Despite having 45% of the country’s area under forests, the contribution of the forest sector to local and national economy has remained much less than the potential in Nepal (Banjade, Paudel, Karki, Sunam, & Paudyal, 2011; Chhetri, Lund, & Nielsen, 2012; Luintel, Bluffstone, Scheller, & Adhikari, 2017; Thoms, 2008). As the national mood has recently shifted towards active forest management, several attempts have been made to develop and scale up silviculture innovations. These have stimulated debates in scientific forest management, though outcomes on the ground have remained limited. Community forestry in Sri Lanka has developed somewhat similarly to Nepal, but began in the 1990s. The community forestry project was initiated in Sri Lanka after a series of forest policy reforms and decentralization arrangements during the 1980s. Since 2003, the Department of Forest Conservation, a government department responsible for forestry in Sri Lanka, has been testing and trialing various approaches using the community forestry model (Ekanayake, Xie, Ahmad, Geldard, & Nissanka, 2020). Community-based forest management in Sri Lanka encompasses community-owned forests and agro-forests as well as government- owned forests managed by communities. Forests managed by communities produce timber and wood products in agroforestry systems, on agricultural lands and community lands including farmer woodlots and silvopastoral systems (De Zoysa, 2017). The home gardens, outside 255 natural and planted forests supply more than 70% of the timber and 80% of the fuel wood in Sri Lanka (De Zoysa, 2017). Recent studies have shown that impact of community forestry development has led to positive outcomes on livelihoods (Ekanayake et al., 2020). In India, large scale shifts from state control of forest to joint management with local communities has led to a large area of forest being managed under joint forest management. Joint forest management covers more than 22 million hectares which is about third of the forest land in India, engaging 25 million people through 104, in 729 committees across more than 100,000 villages (Sundar, 2017). 3.3.4.3.3. Industrial Logging practice In practice there are three major types of industrial logging: (i) most frequently, so-called private concessions grounded on an agreement between a private landholder and the logger or by the landowner himself, mostly on a short-term basis and sizes of some few hundred to thousand hectares; (ii) government granted concessions in public forests (~1.5 M hectares by 2019) (J. R. Ribeiro, Azevedo-Ramos, & Nascimento dos Santos, 2020) based on a set of technical, financial, and administrative requirements, that comprise several ten thousand hectares for an entire timber-harvesting cycle; (iii) the legal use of timber from authorized 256 forest conversion areas in private landholdings. Nearly all industrial logging is organized by sawmills to secure their supply. Large-scale industrial logging involves felling large numbers of trees in areas of more than 50,000 ha. The loggers have felling permits, use heavy machinery, have a processing plant and sell a number of wood products including logs, sawnwood, veneer, plywood, and wooden floors, almost exclusively for export (Cerutti & Lescuyer, 2011). In the last three to four decades, industrial logging has been the major source of globally traded wood products (FAO, 2009). For example, in Papua New Guinea, large-scale industrial logging companies export approximately 90% of the logs harvested in the country (PNGF, 2009). Large scale forestry operations occurring within managed forests and tree plantations were estimated to cover 26% of global forest area between 2001 and 2015 (mainly in America and Europe) (Curtis, Slay, Harris, Tyukavina, & Hansen, 2018). Within some tropical countries, a small number of large-scale companies who source much of their timber from small and medium sized enterprises dominate the export sector (Osei-Tutu et al., 2010). Allocation of forests or trees for large scale industrial logging in public and large-scale privately owned forests is predominantly done through the provision of forest concessions (Vilanova, Ramírez-Angulo, Ramírez, & Torres-Lezama, 2012), which is a common legal tool among forest policy decision-makers (Karsenty et al., 2008). Forest concessions have been carried out for hundreds of years in boreal, temperate and tropical public forests (Van Hensbergen, 2016). Forest concessions have been carried out in many of the Central African forests for over a century (since the colonial rule), and in South American countries for over three decades (Karsenty et al., 2008). 3.3.4.3.3. Industrial Logging practice More sustainable forest management is well planned so as to minimize damage on the remaining stand while effectively making use of costly heavy machinery. This includes infrastructure planning, spatial planning of harvesting operations, harvest planning based on an inventory of all commercially valuable trees, and skid-trail planning (Putz et al., 2012). In tropical and subtropical regions such harvesting is done by larger companies with the necessary human and financial resources and engaged in export activities often linked to Forest Stewardship Council (FSC) certification (Pokorny & Steinbrenner, 2005). Certification requires the demarcation of protected areas, and the timber-harvesting of a wider range of tree species, including the ones with lower commercial value, so as to reduce the pressure on the most valuable tree species (Putz et al., 2008). The engagement of the certifier has positive effects on the quality of the operation, the treatment of the workers and the local resource users living around the management unit. However, certified companies tend to work as enclaves in the forest landscape and prefer to work with non-local workers. They prioritize fast timber- harvesting and hesitate to invest in the long-term security of the management unit once the area has been logged. These practices place the long-term sustainability of these certified operations in question. Since the 1980s, variable retention forestry is being promoted as a sustainable forest management practice in temperate and boreal forests as opposed to clear-cutting (Fedrowitz et al., 2014; Franklin, Berg, Thornburgh, & Tappeiner, 1997; Harkema & Scott, 2002; Kuuluvainen & Grenfell, 2012). It is a system in which key structural components of the original stand are retained at the time of logging through selection cutting, gap cutting and modifications of clear cutting, and become part of a new stand that regrows after logging (Franklin et al., 1997; Gustafsson et al., 2010; Koivula et al., 2014; Koivula, Silvennoinen, Koivula, Tikkanen, & Tyrväinen, 2020; Puettmann, Messier, & Coates, 2009). Emerging research reveals that tree retention has the potential to reduce impacts of logging on forest biodiversity through creating favourable conditions that allow for complex and uneven forest structures similar to those of natural forests (Gustafsson et al., 2020; Moussaoui, Leduc, Fenton, Lafleur, & Bergeron, 2019; Opoku-Nyame, Leduc, & Fenton, 2021). The practice is being adopted with rather modest retention levels ranging from 30 to 40% (Beese, Deal, Dunsworth, Mitchell, & Philpott, 2019; Scott, Neyland, & Baker, 2019). 3.3.4.3.3. Industrial Logging practice Within Latin America, Southeast Asia and West & Central Africa, forest concessions cover about 123 million ha accounting for approximately 14% of the publicly owned forests (Van Hensbergen, 2016). Conventional logging is highly selective, sometimes concentrating on only one or two species. Selecting and felling trees often occurs without a complete inventory or thorough spatial planning. In large-scale conventional logging there may be issues with incorrect identification, poor labor conditions, insufficient training in best practices, and overly high felling rates. These conditions can lead to immense damage and economic losses (Piponiot et al., 2019). Since the 1950s, clear-cutting involves the use of heavy timber machinery (Boucher, Auger, Noël, Grondin, & Arseneault, 2017; Maleki, Nguema Allogo, & Lafleur, 2020; Mohr, Coppus, Iroumé, Huber, & Bronstert, 2013), which may lead to changes in tree composition and oversimplification of stand structure and species diversity (Boucher, Arseneault, Sirois, & Blais, 2009; Boucher et al., 2017; Gustafsson, Kouki, & Sverdrup-Thygeson, 2010). These activities can have negative effects on wild plant and animal populations (Berg et al., 1994; Gärdenfors, 2010; Hyvärinen, Juslén, Kemppainen, Uddström, & Liukko, 2019; Kålås, Viken, Henriksen, & Skjelseth, 2010). Log skidding, done with heavy machinery, can also be damaging if done during improper weather conditions or during the wrong season when soils are especially vulnerable. Industrial logging is done with and without legally authorized management plans. A correct tracing of the logs to their point of origin varies widely so loggers may use management plans to justify harvesting adjunct areas technically not under the plan. The vast majority of sawmills in the tropics work with this kind of timber-harvesting. They have small teams of 257 mostly non-local seasonal workers and use tractors for both the construction of access roads, secondary roads, and landings as well as for the skidding (Pokorny & Steinbrenner, 2005). Larger companies may also use skidders and stackers for loading. Sometimes machinery is owned by the sawmill, sometimes services are subcontracted. Transport distances from the forest to the sawmill may reach up to nearly 100 km (Pokorny & Steinbrenner, 2005). However, if the distance becomes too large, the saw lines are dismantled and rebuilt closer to the forest. Alternative “sustainable forest management” schemes are meant to ameliorate several of the concerns raised regarding species identification, spatial planning, proper use of equipment, and proper application of management plans. 3.3.4.3.3. Industrial Logging practice Though retaining small amounts of trees or patches is better than traditional clearfelling (Gustafsson et al., 2020; Koivula & Vanha-Majamaa, 2020), only retaining a minor proportion of the volume of harvestable timber (often 1-10%) makes it practically impossible to avoid edge effects and random demographic effects on the forest stands. Maintaining more of the mature forest characteristics in production forests would require lower harvest intensities in some areas than 258 is currently typical. Therefore, this low level of retention is still considered by some scholars as clear-felling (Fedrowitz et al., 2014). Prevailing retention practices have been reported to lack ecological credibility in safeguarding biodiversity and there are calls for their further development (Kuuluvainen, Lindberg, Vanha-Majamaa, Keto-Tokoi, & Punttila, 2019). Other studies have reported that it is not necessarily the level of retention of living trees, but rather, the microclimatic continuity, and maintenance and active increase of legacies such as existing coarse woody debris, very old trees, and tree species mixtures that significantly contribute to the conservation of forest species (Koivula & Vanha-Majamaa, 2020; Siitonen, 2001). Diversification of silvicultural harvesting techniques is recommended to enhance specific structural or compositional elements and the diversity of species in forest stands. Either clear-cutting, partial cutting or selective cutting can be carried out to match variations in stand conditions and effects of natural disturbances, biophysical site characteristics and succession processes (Bergeron, Gauthier, Kafka, Lefort, & Lesieur, 2001; Harvey & Bergeron, 1989; Maleki et al., 2020). Clear-cutting tends to allow cycling of early successional species into a single species dominant stand, while partial cutting and extended rotations can enable maintenance of a mixed species stand or stands that have some characteristics of older forests (Ruel, Fortin, & Pothier, 2013). Post-logging forest restoration greatly relies on post logging seedling generation. The ability of a species to be sustained through rotations depends on the growth and reproduction of surviving adults, juveniles and seedling regeneration (smith et al., 1997). 3.3.4.3.3. Industrial Logging practice However, many of the high-value timber species are nonpioneer light demanders whose seedlings occur at low densities in the forest understory due to limited shade tolerance (Grogan, Landis, Ashton, & Galvão, 2005; Gullison & Hubbell, 1992; Hall, Medjibe, Berlyn, & Ashton, 2003; Jones, 1956; Lamprecht, 1989; Medjibe & Hall, 2002; M Schulze, Vidal, Grogan, Zweede, & Zarin, 2005), such as wind-dispersed mahoganies and related genera in the family Meliaceae (Swietenia, Cedrela, Chukrasia, Entandrophragma, Khaya, Toona), Amburana, Cedrelinga, Couratari, Dinizia, Hymenolobium, and Tabebuia. These usually have limited post-logging regeneration (Dickinson & Whigham, 1999; Grogan, Galvão, Simões, & VerÍssimo, 2003; Gullison, Panfil, Strouse, & Hubbell, 1996; Schulze, 2003, p. 2003; Veríssimo, Barreto, Tarifa, & Uhl, 1995) and thus require adjustment in logging and silvicultural practices to promote their regeneration. To ensure sustained yield timber production from such timber species across the tropics, there are a number of silvicultural practices that should be taken into consideration (Grogan & Galvão, 2006). Economically, timber-harvesting is most profitable for the traders, particularly if engaged in export markets. Conventional, particularly illegal, timber-harvesting, is also profitable for the owner of the sawmill, but also provides urgently required income opportunities for local people, not so much in the forest operations, but in the sawmills (Pokorny, 2013). The benefits of large-scale industrial logging to the local economy are usually limited (Gray, 1999) to some low-paid work, but loss of non-timber forest products which many local people often rely on for subsistence or livelihood diversification can have serious negative impacts (Adams, 2009). Benefits to the national economy are restricted, because while value is added to the timber when it is sawn and made into products, this typically takes place elsewhere (Adams, 2009). The main products obtained are round logs which are directly 259 exported with very little in-country downstream processing. In instances where companies obtain concessions from private or community forests, these give royalties to the owners which depend on the tree species harvested. Nevertheless, large concessions seem to be a suitable tenure model in low-density areas where central or local governments are not capable of creating or maintaining adequate infrastructure to support regional economic issues and where only large-scale companies have the potential to do so (Karsenty et al., 2008). Social impacts are felt due to large amounts of migrant labor associated with industrial logging. 3.3.4.3.3. Industrial Logging practice A larger proportion of the workers in large-scale industrial timber-harvesting operations are permanent, but are brought from other regions and seldom become settled in a region. Concessionaires, especially including certified companies, have to effectively protect their management unit against informal harvest and encroachment. Accordingly, local resource users living in and around concessions suffer from restricted access to forest management areas. In Central Africa, the social impact of industrial timber-harvesting remains a contested issue. Taxation systems and services due to workers and the local populations are clear on paper, but there is limited transparency or availability of information about how much of the due amounts or promised services are actually paid into the State coffers or delivered locally. And while there seems to be a bit more clarity on the amounts of money that are collected and redistributed to local councils and villages, e.g., in Cameroon (Cerutti, Lescuyer, Assembe- Mvondo, & Tacconi, 2010) and the Democratic Republic of Congo (Tsanga, Cerutti, Bolika, Tibaldeschi, & Inkonkoy, 2020) much of the burden of redistributing benefits to local populations remains within the concessionaires themselves, which are not always willing or capable of playing that role (Cerruti et al., 2017). Ecological, economic and social sustainability can perhaps be achieved through continuous-cover forest management (e.g., Fedrowitz et al., 2014; Franklin et al., 1997; Kuuluvainen & Grenfell, 2012). This regime applies logging methods other than clear cutting and thus varies the amount and spatial distribution of retained trees, and the size of harvested openings. The logging methods include selection cutting, gap cutting and modifications of clear cutting, all characterized by maintaining a significant proportion of trees throughout the logging cycle (e.g., Koivula et al., 2014; Puettmann et al., 2009). Experimental evidence suggests that even modest retention of living trees in harvested blocks is beneficial for biodiversity (Koivula & Vanha-Majamaa, 2020). Also, based on landscape preference research, retention methods may be preferred over clear cutting by citizens who use forests for aesthetic pleasure, recreation, hunting, or harvesting (see 3.3.4.4). Clear cutting decreases the aesthetic and recreational values of forests (e.g., Arnberger et al., 2018; Karjalainen, 2006; Tyrväinen, Silvennoinen, & Hallikainen, 2017), whereas logging methods with a high amount of retained trees, such as selection cutting, are considered socially more acceptable (Putz et al., 2008; Ribe, 1989). Box 3.20 Industrial logging in the Amazon In 1998, the Brazilian Amazon generated 10.8 million cubic meters of native wood. Twenty years later, only 57% of this volume was produced (~ 6.2 million m3 (Lentini, Sobral, & Vieira, 2020). This was due to increasing competition with cheap supplies of forest products from tree plantations and contractions in the domestic market, as well as replacement with other materials such as plastics, steel and aluminum. An estimated 95% of the sawmills in the region are small family enterprises with very limited managerial capacity. Despite operations based on legally approved management plans, it is unclear whether this logging is sustainable. The extraction (cutting and skid trails) is performed mostly (61%) by third parties, while the rest (39%) is extracted by the processing industries themselves (D. Pereira et al., 2010). The Amazon has more than 300 species of trees considered commercially valuable (Martini, Rosa, & Uhl, 1994). However, for decades the very same 15 to 20 species of commercial interest were harvested. Some of the most strained and consequently most pressured species are: Hymenaea courbaril, Handroanthus sp, Apuleia leiocarpa, Goupia glabra Aubl., Manilkara alata, Himenolobium petreum , Couratari sp., Dinizia excelsa (Lentini et al., 2020). Manifold attempts to broadening this range have not been too successful. Only very few large companies have the interest and capacity to comply with the Forest Stewardship Council certification standard. It is estimated that less than a quarter of the timber produced in the Amazon is exported, as most of the timber is consumed in the big cities at the coast. Depending on the forest type and the market situation, between 10 to 25 m³ per hectare is harvested. Increments of commercial timber are low around 0.5 to 1.5 m³ per year and hectare, which mathematically result in harvesting cycles of around 25 to 35 years (Pereira et al., 2010). The regulations established in the Brazilian Amazon assume that a minimum harvest cycle of 25 to 30 years would guarantee the long-term sustainability of forest management. The legal requirements for industrial timber harvest include clarified tenure arrangements for the forest management unit, the formulation of a sustainable forest management plan, and annual operational plans. The volumes and products of harvested wood have to be reported in a document of forest origin (DFO) designed to accompany legally harvested wood at all stages of the transport and production chain (Waldhoff & Vidal, 2015). 3.3.4.3.3. Industrial Logging practice Citizens prefer forests with diverse tree ages, species, and sizes (Silvennoinen, Alho, Kolehmainen, & Pukkala, 2001; Silvennoinen, Pukkala, & Tahvanainen, 2002; Tyrväinen et al., 2017) with not too densely spaced trees (Ribe, 1989; Silvennoinen, 2017). Industrial logging is quite extensive in the tropics (Box 3.20). It takes place legally on public and private lands, and illegally on public forests designated for conservation. Logging also occurs on indigenous people and local communities’ lands and territories. Forest concessions have been widely used to allow companies to undertake large-scale timber- 260 harvesting operations, yet these areas have been shrinking over time, particularly in the Amazon (e.g., Bolivia, Peru) and Southeast Asia (e.g., Malaysia and Indonesia). There are significant areas of public lands granted as concessions on all continents. In 2009, small properties accounted for 28% of production, medium-sized properties extracted 41% of wood and large properties supplied 31% of roundwood (Pereira, Santos, Vedoveto, Guimarães, & Veríssimo, 2010). Only 29% of production in 2009 came from areas owned or leased by the timber industries. The remainder (71%) originated in third party areas. Box 3.20 Industrial logging in the Amazon The regulations foresee two categories of forest management: 1) Low-intensity forest management, normally by local communities, with a maximum harvest volume of up to 10 m3 per ha, a minimum harvest cycle of 10 years, and restrictions on the use of heavy machinery; 2) Complete forest management, which allows a maximum harvest volume of 261 30 m3 per hectare and year, minimum harvest cycles of 25 to 35 years, and without machinery restrictions (Pereira et al., 2010). Throughout the tropics, forestry regulations commonly grant rights to industrial, large- scale, export-oriented timber-harvesting concessions. These concessions require management plans, which are presumed to maintain forest cover and biodiversity. All countries in Central Africa follow the concessionary model, with the Ministries of Forests granting rights and responsibilities to the concessionaire (i.e., a private entity is given permission to manage a public property) either through public auctions or directly. The duration of the contract varies. In the Central African Republic, the concession is granted for the entire lifespan of the company, in all other countries there exist legal temporal limitations to the contractual agreement, which is 15 years in Cameroon, Republic of Congo and Equatorial Guinea, 25 years in the Democratic Republic of Congo, and 30 years in Gabon (Cerutti, Nasi, & Center for International Forestry Research (CIFOR), Kenya and Indonesia, 2020). Timber-harvesting concessions comprise a total area of 50 million ha in the Congo Basin, of which about half had approved management plans by 2020 (Cerutti et al., 2020). Management plans are generally based on a rotation period of about 30 years, with annual allowable cuts authorized for timber- harvesting each year by the forest administration. Logging in the boreal and temperate forests is mainly industrial in scale (Safford & Vallejo, 2019). Approximately 90% of the forest in Fennoscandia, and perhaps 40% and 60% of Canadian and Russian forests, respectively are subject to industrial tree harvest (Gauthier, Bernier, Kuuluvainen, Shvidenko, & Schepaschenko, 2015). Prior to the 20th century, selective cutting was the dominant logging practice in the temperate and boreal forests. An intensive era of clear-cutting targeting mainly conifer trees began in the 20th century due to economic factors (Dupuis, Danneyrolles, Laflamme, Boucher, & Arseneault, 2020; Koivula & Vanha-Majamaa, 2020; Lundmark, Josefsson, & Östlund, 2013; Storaunet, Rolstad, Gjerde, & Gundersen, 2005). Box 3.20 Industrial logging in the Amazon Clear-cutting continues to be the dominant logging practice (Curtis et al., 2018; Kålås et al., 2010; Siitonen, 2001), although trials for partial cutting practices, such as retention silviculture have been established to test their operational and biological feasibility (Bose, Harvey, Brais, Beaudet, & Leduc, 2014). In clear-cutting, mature trees are usually completely removed, followed by regeneration through site preparation, sowing or planting, tending of the emerging cohort of even-aged trees, and often a relatively short logging rotation (Koivula et al., 2020; Safford & Vallejo, 2019). An underlying rationale of clear-cutting is economic because it is seen as highly efficient and leading to sustained yields of timber (Koivula et al., 2020). The concept of sustained yield has been criticized for only concentrating on the maintenance of timber stocks over time, while other forest resources that are protected with site-specific practices are not explicitly considered in the management plans, consequently leading to their decline (Berg et al., 1994; Cyr, Gauthier, Bergeron, & Carcaillet, 2009; Luckert & Williamson, 2005). Tree retention is an emerging alternative to clear cut harvesting, practiced on several continents including North and South America, Oceania, and Europe (Gustafsson et al., 2020). Emerging research reveals that tree retention has the potential to reduce impacts of logging on forest biodiversity through creating favourable conditions that allow for complex and uneven forest structures similar to those of natural forests (Gustafsson et al., 2020; Moussaoui et al., 262 2019; Opoku-Nyame et al., 2021). Though even leaving small amounts of trees or patches is better than traditional clear-felling (Gustafsson et al., 2020), tree retention comprising a minor proportion of the volume of harvestable timber (often 1-10%), which makes it practically impossible to avoid edge effects and random demographic effects on the forest stands. Maintaining more of the mature forest characteristics in production forests would require lower harvest intensities in some areas than is currently typical. Determining exact levels that are required to secure long-term viable populations of different species, as well as the most cost- efficient implementation of these conservation measures, remains a major challenge for future research (Gustafsson et al., 2010). There is a continued increase in the amount of wood removals globally through industrial logging practices (Figure 3.56). In 2019, global wood removals were estimated at 3.97 billion m3, of which 2.02 billion m3 was industrial roundwood and 1.95 billion m3 fuel wood. Box 3.20 Industrial logging in the Amazon The year 2018 had the highest level of production and trade values for global wood removals and all major wood-based products since 1947 (data from the Food and Agriculture Organization of the United Nations, http://www.fao.org/faostat/en/#data). The demand for and the consumption of wood products is escalating in line with growing populations and incomes, a trend expected to continue in the coming decades (FAO, 2010b). North American and European countries have the highest global wood yields (Chaudhary, Carrasco, & Kastner, 2017), which is partly attributed to clear-felling regimes prevalent in temperate Europe/North American countries with high yields compared with low-yield selective logging in the tropics (Chaudhary, Burivalova, Koh, & Hellweg, 2016). There are also large-scale imports of timber products by a limited number of countries especially China and the United States of America. The globalization of trade has enabled such countries to reduce local forest exploitation and achieve forest transitions from net deforestation to net reforestation (Kastner, Erb, & Nonhebel, 2011; Meyfroidt, Rudel, & Lambin, 2010; Mills Busa, 2013). Figure 3.56 Global wood removals 1990-2019. Data from FAO database (FAO, 2021b) under license CC BY-NC- SA 3.0 IGO. See data management report for the figure at https://doi.org/10.5281/zenodo.6453131. Figure 3.56 Global wood removals 1990-2019. Data from FAO database (FAO, 2021b) under license CC BY-NC- SA 3.0 IGO. See data management report for the figure at https://doi.org/10.5281/zenodo.6453131. 263 Some of the logging practices in species-rich tropical forests have been reported to resemble mining operations at the species level (Gómez Pompa, 1989; N. Johnson & Cabarle, 1993; Moad & Whitmore, 1994; M Schulze et al., 2005), where a single, or group, or wider community of high value timber species are targeted for extraction. In the past, major target species included the big leaf Mahogany (Swietenia macrophylla), Brazilwood or Pau-brasil (Caesalpinia echinata), Brazil-nuts (Bertholletia excelsa), rosewood (Dalbergia nigra and Aniba rosaeodora) and others (Martini et al., 1994; Mark Schulze, Grogan, Landis, & Vidal, 2008; Veríssimo et al., 1995). Due to these practices, some species were reported endangered and added to Appendix II of Convention on International Trade in Endangered Species of Wild Fauna and Flora. With scarcity and restrictions in extraction and trade of the Convention on International Trade in Endangered Species of Wild Fauna and Flora listed species, new species are targeted. 3.3.4.4. Uses Like with the other practices reviewed in section 3.3, available knowledge on logging for a variety of uses was reviewed. In the case of logging, the relevant uses include decorative and aesthetic, energy, and shelter and construction. While it is certainly the case that many wood and tree products are used for ceremonial and cultural expression, food and feed, and medicine and hygiene, based on the definition of logging used in this assessment, these other uses (and the associated tree products) are discussed in the section on gathering (3.3.2). Box 3.20 Industrial logging in the Amazon This practice has led to severe and dense reductions of adult populations or old growth timber stocks, often at large spatial scales (Uhl, Veríssimo, Mattos, Brandino, & Vieira, 1991; Veríssimo, Barreto, Mattos, Tarifa, & Uhl, 1992; Veríssimo et al., 1995). Land occupation and timber extraction through conventional industrial logging has generated a culture of timber mining in many forest landscapes in the tropics, which has proved to be very persistent among some local stakeholders making an income from industrial timber extraction, which translates into low investments in operations or forest recovery. These cultural aspects of timber extraction in the tropics have been little studied, as well as shifts in social perceptions over time. 3.3.4.4.1. Decorative and aesthetic Harvesting timber for wood carvings is mainly a destructive process. The entire tree is felled at the trunk between 5 and 50 cm from the ground using a metal axe or chain saw and machetes, and the artists cut different lengths of timber from the fallen tree for their carvings (A. D. Griffiths, Philips, & Godjuwa, 2003; Koenig, Altman, & Griffiths, 2011; Purata, Brosi, & Chibnik, 2004). In other instances, only the prime section of the stem is removed, leaving the rest of wood in the forest. (B Belcher et al., 2002). Tree sizes are selected based on the size and nature of the sculpture to be made. This can depend on the cultural subject matter (Koenig et al., 2011). The average diameter of trees harvested for small sculptures such as birds would be smaller than those harvested to make canoes. However, the average diameter of trees harvested 264 has significant implications on the sustainability of the tree species. Cutting down smaller sized trees before they produce and disperse seeds could affect the population of the tree species. Wood for woodcarvings continues to be mainly harvested from the wild (Ellery, Cunningham, & Choge, 2005; Griffiths et al., 2003; Purata et al., 2004). These include forests on public land, communal and private forests (Ellery et al., 2005; A. D. Griffiths et al., 2003; Koenig et al., 2011; Matose, 2006). However, there are no certain global statistics of volumes of wood extracted for wood carvings as this is primarily an informal activity (Ellery et al., 2005), however that does not mean it is small in scale and scope. In 2002, woodcarving in Kenya were estimated to consume 50,000 trees per year (0.7% of the total round wood market share in Kenya). But although the amount of wood extracted for the purpose seems low, the wood carving practice relies on a selected number of species with desired qualities such as close grain, tensile strength and resistance to cracking or insect attack. In addition, a small range of different timbers are often favored as a result of social, cultural and historical factors (Cunningham et al., 2005), which end up being over exploited. This has led to over exploitation of the particular wild species, especially those with other purposes, of which some are listed among the endangered species (Cunningham et al., 2005; Ellery et al., 2005). 3.3.4.4.1. Decorative and aesthetic From carving small household items, to carving the interior and exterior of houses and temples, ritual objects and decorative pieces, fashioning idols for various articles of furniture and for ceremonial objects (Saville, 1925), carving traditions have mainly been associated with culture, technology and change (Cunningham et al., 2005). The practice was mainly associated with particular communities stretching back many generations, carving particular types of pieces that were mainly associated with long standing cultural significance. For example, in the tropics, subtropics, pre-industrial societies of Europe and some northern temperate regions, woodcarvings were and for some, are still the major sources of social and cultural materials (Cunningham et al., 2005). Whereas the practices have been socially and culturally sustainable among some woodcarving communities such as the Aboriginal wood carvers of Australia (Koenig, Altman, Griffiths, & Kohen, 2007), some communities such as those in the Mexican state of Oaxaca are engaged in carving novel creations without longstanding cultural significance (Purata et al., 2004). The wood carving industry has grown tremendously over the years, extending beyond the local and national to the international markets (Altman, 2005; Ellery et al., 2005) which has increased demand of the wood carvings. The carvings are sold in a number of arenas including family workshops, markets and craft shops, either within the villages or other cities and countries (Purata et al., 2004). An activity that was once predominantly a men’s activity (Cunningham et al., 2005) has progressively increased number of women and youths involved, becoming a family activity (Koenig et al., 2007; Purata et al., 2004). The women involved are mainly spouses and children of prominent wood carvers (Koenig et al., 2007). However, these are mainly involved in the less labor-intensive activities such as sanding, polishing and painting (Matose, 2006; Purata et al., 2004). Other than the aesthetic values, these products have earned households, communities and national economies income. Wood carving is a major source of income through facilitating purchase of livelihood needs (Purata et al., 2004) especially among communities in dry environments that suffer from lack of agricultural opportunities (Matose, 2006). However, it is not possible to obtain exact numbers of people involved (Ellery et al., 2005) and the value of 265 the industry as a whole is hard to determine (Griffiths et al., 2003) due to its dynamic nature. 3.3.4.4.1. Decorative and aesthetic The wood carving industry in Kenya generates about 20 million United States dollars per year in export revenue (Choge, 2002; Obunga, 1995), employing about 40% of thef national formal timber industry (Ellery et al., 2005). Around the Victoria falls in Zimbabwe, the industry provides a source of livelihood to nearly a thousand households in a dry part of the country with households getting around 14 to 60 United States dollars a month The timber trade and woodcarving are closely linked, particularly in Asia, where timber is intricately carved to make buildings, doors or furniture. Trade in carvings is not new. It is bigger than ever before; however, it has spread internationally, rather than regionally, and has focused on a far smaller resource base (Cunningham et al., 2005). Since many of the species used for wood carvings are endangered/threatened, their use and trade are restricted by both national regulations (for example sandalwood is restricted by the Kingdom of Tonga sandalwood regulations 2016, Tamil Nadu sandalwood possession rules, 1970, and the sandalwood (limitation of removal of sandalwood) order 1996 in Western Australia) and the Convention on International Trade in Endangered Species of Wild Fauna and Flora (Groves & Rutherford, 2015). Nevertheless, there are some initiatives to ensure sustainability of these species by both community and corporations. For example, some species have been in cultivation through plantation establishment and agroforestry practices; In Bali woodcarving has been put on a sound basis through shifts to a fast-growing species like Paraserianthus falcataria. In India there is the roadside, village-level and plantation production of Dalbergia sissoo. In coastal Kenya there are plantations of the neem trees. There is recommendation and adoption of community/corporate tree plantations for sandalwood (A. N. A. Kumar, Joshi, & Ram, 2012) in different parts of India with appropriate incentives and adequate protective measures. Australia has been raising large sandalwood plantations, and may be able to meet the global demands, with the world’s largest plantation of S. album established in the Kimberly, Western Australia. 3.3.4.4.2. Energy Energy security is one of the requirements for a good quality of life, and this includes availability and access to clean, reliable, affordable and sustainable energy without compromising health (UN, 2015). Yet globally, 1.1 billion (14%) people do not have access to electricity and 2.4 billion (approximately one-third of the global population) people rely on unclean ‘traditional biomass’ for energy (including charcoal, coal, crop waste, dung, kerosene and wood), with the associated health implications from household air pollution (IEA, 2017) (Figure 3.57a). Wood energy contributes 75-90% of sub-Saharan Africa’s household energy mix (Hoffmann, Brüntrup, & Dewes, 2016; World Bank, 2011). An estimated 880 million people globally log firewood or produce charcoal (FAO & UNEP, 2020). Reliance on wood biomass for cooking is highest in developing Asian countries and sub-Saharan Africa (IEA, 2017). One third of the world’s population (2.4 billion people) use fuel wood for cooking - which provides more nutrients than raw food - and other food preservation processes (e.g., smoking, drying), and one in ten people use fuel wood for boiling and sterilizing water (FAO & UNEP, 2020). 266 Most wild biomass energy is derived from wood, with implications for social and natural systems (Arnold et al., 2006; Bailis et al., 2005; Holdren et al., 2000; Miah et al., 2009; Munalula & Meincken, 2009; Smith & others, 2006). Logging for energy accounts for 50% of all wood consumed globally, and accounts for 90% of logged timber in Africa (FAO & UNEP, 2020). According to the Food and Agriculture Organization of the United Nations’ statistics (http://www.fao.org/faostat/en/#data/FO), global fuel wood removals have increased over time with approximately 2 billion m3 produced in 2019. There is great variation in fuel wood production and use in the different regions. Production is highest in Asia and Africa at approximately 713 million m3 and 706 million m3 respectively (Figure 3.57a). Whereas production levels are increasing in Africa (from 445 million m3 in 1990), the opposite is happening in Asia whose production has decreased from 897 million m3 in 1990. Latin America and the Caribbean have a fairly high level of production (268 million m3). Oceania has the lowest production of approximately 10 million m3 in 2019. Although absolute fuel wood consumption is increasing, especially in sub-Saharan Africa, per capita consumption is decreasing across all regions (Figure 3.57b). 3.3.4.4.2. Energy All regions reported minimal trade in fuel wood, implying that fuel wood are mainly consumed locally and in domestic markets. However, wood-based energy industry has the potential to grow in a number of countries. This has motivated the investment in biomass-based energy generation, and research and development of new energy products such as biodiesel (Asikainen et al., 2010). Although alternative energy sources reduce demand for fuel wood, in some areas fuel wood use persists due to habits, taste and custom (FAO, Schure, Ingram, & Yoo, 2017). Figure 3.57 Total a) and per capita b) fuel wood consumption trends by region. Source: (FAO, 2017b) © FAO, 2017. Figure 3.57 Total a) and per capita b) fuel wood consumption trends by region. Source: (FAO, 2017b) © FAO, 2017. 267 In several industrialized countries, wood energy provides nearly 25% of total energy supply, and the leading renewable energy source in Europe accounting for about 45% of primary energy from renewable sources (Francisco X. Aguilar, FAO, & UNECE, 2018). With the requirement of European Union states to have 27% of their energy generated from renewable energy by 2030 (European Commission, 2014), Europe’s wood consumption for energy generation is expected to grow and reach 752 million m3 in 2030 (Mantau et al., 2010). Logging for energy in North and Central America has been growing to meet increasing export demand for wood pellets. In Europe and North America, wood energy utilization is commonly integrated in forest management practices and the wood products industry. Wood energy feedstocks can be considered a co-product of forest management as part of silvicultural treatments inclusive of thinning, final integrated harvests and salvage logging, as well as a by-product of the forest industry during the production of sawn goods (Asikainen et al., 2010). Most of the wood used for energy comes indirectly through the forest industry as a co-product (58%) and a little over of a third of the wood mobilized for energy comes directly from forests (36%). Data from the Joint Wood Energy Enquiry for 2013 shows that the forest-based industry was the largest consumer of wood energy (44%), followed by the residential (36%) and combined heat and power (17%) sectors (F. X. Aguilar, Glavonjić, Hartkamp, Mabee, & Skog, 2015). 3.3.4.4.2. Energy Use of wood for energy creates job opportunities not only along the supply-chain of woody biomass feedstocks, but also through investments in technology development and energy conversion and final consumption (Francisco X. Aguilar et al., 2018). The FAO and the United Nations Environment Program (2020) estimated that over 40 million people are involved in commercial fuel wood activities to supply urban centers. Furthermore, fuel wood production generated an estimated 33 billion United States dollars in 2011 global revenue (FAO & UNEP, 2020). The number of jobs and net earnings is influenced by production method and organization of energy systems. For instance, the utilization of 390,000 dry tons of woody biomass estimated to feed a 100 megawatt power facility in the southern United States of America has been estimated to support 585 direct and 481 indirect jobs through the recovery of logging co-products, while direct and indirect employment associated with operation of the power plant were 281 and 115, respectively (Perez-Verdin, Grebner, Munn, Sun, & Grado, 2008). Global fuel wood demand peaked in the mid-1990s (Arnold, Köhlin, Persson, & Shepherd, 2003), instigating a declaration of a ‘fuelwood crisis’. However, the projected fuel wood supply-demand models predicting fuel wood stock collapse were an overestimation due to limited data and an incomplete understanding of social, economic and ecological interactions around wood energy (Dewees, 2020). Nevertheless, the available amounts of fuel wood may not be sufficient to meet local energy needs (Fabian, Volkmer, & Wiedemann, 2011; Swinkels, 2014). Household energy consumption is usually higher than fuel wood reported in official statistics, which mainly refer to wood from forests sources while leaving out other forms of wood biomass that contribute to household energy production. These include (for example in Europe) all by-products (sawmill by-products, other industrial wood residues and black liquor), solid wood fuels and post-consumer wood (Mantau et al., 2010). Although fuel wood demand can be met at a global, national or even regional scale, when comparing supply-demand balances, localized wood fuel scarcity persists (Arnold et al., 268 2003; FAO et al., 2017; Masera, Bailis, Drigo, Ghilardi, & Ruiz-Mercado, 2015). Fuel wood- scarcity ‘hotspots’ occur in areas where fuel wood is crucial for subsistence use and household- level well-being (Figure 3.58a) (Arnold et al., 2006; Sampson et al., 2005). 3.3.4.4.2. Energy In these areas, fuel wood users have few to no alternatives for cooking and heating, posing localized fuel wood driven challenges as most fuel wood (particularly firewood) is produced, harvested and consumed at a local level (Sampson et al., 2005). In addition, regions where logging rates exceed growth rates are likely to cause degradation or deforestation (Robert Bailis, Drigo, Ghilardi, & Masera, 2015; Masera et al., 2015). In 2009, 27-34% of fuel wood logging exceeded growth rates, predominantly in hotspots in South Asia and East Africa, affecting over 250 million rural people reliant on wood energy (Figure 3.58b) (Masera et al., 2015). The FAO estimate that one third of fuel wood logging was unsustainable and a major cause of forest degradation (FAO et al., 2017; FAO & UNEP, 2020). However, the link between fuel wood logging and deforestation or forest degradation is challenging to quantify and varies temporally and geographically (Rob Bailis, Wang, Drigo, Ghilardi, & Masera, 2017). Demand depends inter alia on household level preferences and economic context, vegetation species composition and physiognomy, the availability and cost of alternative energy sources (Rob Bailis et al., 2017). Supply may vary with land use, productivity (and associated edaphic and climatic determinants), and accessibility of wood (Rob Bailis et al., 2017). To further complicate quantification of fuel wood extraction, logging locations are not always from forests but are derived from many types of land cover (e.g., farms, roadside commons, home gardens), and may be a primary (logging specifically for fuel wood) or secondary activity (e.g., wood cleared from farms) (Rob Bailis et al., 2017). Thus, fuel wood logging is often not the sole cause of forest degradation, but unsustainable fuel wood logging in Africa, particularly charcoal logging in open access systems with uncertain or unclear forest tenure, can be the primary driver of forest degradation (FAO et al., 2017). The FAO found that fuel wood sustainability is strongly related to forest management rights and access, especially through permitting and/or taxation systems developed with local participation (FAO et al., 2017). However, beyond these areas of localized shortages, sustainably logged fuel wood has the potential to be a viable, renewable, energy source that provides income (FAO et al., 2017) and may be the preferred fuel source for cultural and economic reasons (P. 3.3.4.4.2. Energy Firewood trade can occur in conjunction with farmland clearing, with fuel wood being sold to wealthy urban clients through formal channels (Chidumayo & Gumbo, 2013; FAO et al., 2017; Gandar, 1994). Firewood is normally logged on foot, limiting the logging radius to 1-3km, although this distance can be higher in arid regions with low tree cover (Cardoso, Ladio, & Lozada, 2013). Increasing firewood demand, locally and in urban areas has resulted in logged wood being collected and transported vehicle or horseback (Cardoso et al., 2013; Matsika, Erasmus, & Twine, 2012), and may be by ‘outsiders’ (W. Twine, Saphugu, & Moshe, 2003). Localized shortages have also resulted in increased harvest time, changes in collected species or size classes, or harvest of live wood during deadwood shortages, often in violation of local traditional knowledge (Findlay & Twine, 2018). Higher income is associated with a reduction in both firewood and charcoal use, although there is substantial variation between countries (Arnold et al., 2003). The expectation that provisions of cleaner, more efficient energies and stoves would result in traditional energy users transitioning up the ‘energy ladder’ have largely not occurred, with households ‘stacking’ fuel, i.e., using multiple devices and fuels (Arnold et al., 2006; Hiemstra-Van der Horst & Hovorka, 2008; Masera et al., 2015; van der Kroon, Brouwer, & Van Beukering, 2013). Fuel wood use at household level is inelastic for a variety of reasons, including cultural and household taste preferences, high capital cost of appliances and energy, poor infrastructure and reflects dynamic, complex decision making at a household level (Arnold et al., 2006; Hiemstra- Van der Horst & Hovorka, 2008; IEA, 2017; Masera et al., 2015; van der Kroon et al., 2013). There are indications that energy stacking, and the availability of a diversity of energy resources represents the adaptive capacity of communities, favors the conservation of local species and contributes to broader social-ecological resilience (Cardoso et al., 2013). Energy stacking is a complex phenomenon, illustrated by a case in Patagonia, Argentina. A local village reliant on costly firewood, received subsidized liquefied petroleum gas (Betina Cardoso & González, 2019). This drastically reduced the amount of fuel wood collected in the region and reduced household air pollution, but not only did households continue to use wood burning stoves, their gas consumption to heat poorly insulated houses was extremely high (650 kilowatt-hour/m2) incurring substantial operational and environmental costs (M. Betina Cardoso & González, 2019). 3.3.4.4.2. Energy Munro, van der Horst, & Healy, 2017), provided air quality (indoor and outdoor) and climate change emissions are mitigated (Rob Bailis et al., 2017). 269 Figure 3.58 a) Global population reliant on traditional biomass, including fuel wood and animal waste, and b) fuel wood supply/demand balance with circles on major deficit “hotspots”. Sources: a) based on data from (IEA, 2020) © OECD/IEA; b) (Masera et al., 2015) See data management report for the figure at https://doi.org/10.5281/zenodo.6453135. Figure 3.58 a) Global population reliant on traditional biomass, including fuel wood and animal waste, and b) fuel wood supply/demand balance with circles on major deficit “hotspots”. Sources: a) based on data from (IEA, 2020) © OECD/IEA; b) (Masera et al., 2015) See data management report for the figure at https://doi.org/10.5281/zenodo.6453135. Figure 3.58 a) Global population reliant on traditional biomass, including fuel wood and animal waste, and b) fuel wood supply/demand balance with circles on major deficit “hotspots”. Sources: a) based on data from (IEA, 2020) © OECD/IEA; b) (Masera et al., 2015) See data management report for the figure at https://doi.org/10.5281/zenodo.6453135. Figure 3.58 a) Global population reliant on traditional biomass, including fuel wood and animal waste, and b) fuel wood supply/demand balance with circles on major deficit “hotspots”. Sources: a) based on data from (IEA, 2020) © OECD/IEA; b) (Masera et al., 2015) See data management report for the figure at https://doi.org/10.5281/zenodo.6453135. 270 In low-income countries, fuel wood use occurs predominantly at the household scale for lighting, cooking and heating, but can support local and village-level industry. Commercial involvement with fuel wood, both firewood and charcoal, provides supplemental or an occasional income source (Arnold et al., 2006), or an activity to fall back on as a ‘safety net’. For example, firewood trading (and household subsistence use) increased in South Africa during economic shocks, such as loss of urban employment or breadwinner death as a result of HIV/AIDS (Human Immunodeficiency Virus) (Shackleton & Shackleton, 2004) or in response to the covid-19 pandemic, such as the switch from liquefied petroleum gas to fuel wood in Kenya and Malawi (Shupler et al., 2020; Zalengera et al., 2020). Eastern Afghanistan’s forests have been an important energy resource during conflict-related crises in the region, although the forest wood stocks have been severely depleted (UNDP, 2014). 3.3.4.4.2. Energy The study’s recommendations were two-fold: insulate the houses and receive a return on investment in liquefied petroleum gas savings in 2.2 years, and consider subsiding a household preference-determined mix of cheaper fire wood and gas to reduce subsidization costs (M. Betina Cardoso & González, 2019). This example clearly demonstrates 271 the complexities in altering relative energy mixes and the potential trade-offs to social, economic and the environmental conditions. Although firewood use is slowly decreasing, charcoal demand in urban areas is growing, doubling over 25 years to about 207 million m3 wood for charcoal per annum in 2000 (Arnold et al., 2003). In tropical South America charcoal use varies across the region with Brazilian use mainly for manufacturing and Central American for the food industry and limited domestic use (Chidumayo & Gumbo, 2013). In sub-Saharan Africa charcoal is mainly used for household cooking, particularly in urban areas (Chidumayo & Gumbo, 2013). Rural-urban charcoal trade is increasing as wealthy, urban firewood users ‘transition’ to charcoal (Arnold et al., 2006). For example, 81% of energy use in Mozambique is fuel wood, with charcoal the predominant use in urban areas with the capital city, Maputo, garnering the highest prices for charcoal (Cuvilas, Jirjis, & Lucas, 2010). It is estimated that 91-99% of charcoal production is illegal (Cuvilas et al., 2010). The high value and demand of charcoal in urban areas further incentivizes increased production (Cuvilas et al., 2010). Charcoal production from plantations is increasing in the global tropics and charcoal is still predominantly derived from wild species in natural forests, and frequently related to deforestation or forest degradation (Chidumayo & Gumbo, 2013). Charcoal logging alone have resulted in the loss of 3 million hectares of forest cover in 2009 (Chidumayo & Gumbo, 2013). In Southern Africa charcoal production is valued at about 2-3% of gross domestic product (Malimbwi et al., 2010) and forms a significant income source with households able to earn 1000 to 10,000 United States dollars per annum although studies suggest this income is not sustained over the long-term and does not provide improvements in human well-being (Baumert et al., 2016; Smith et al., 2019). Like firewood, the broad extent of charcoal logging and impacts remain unknown due to the informal and dynamic use of the resource. Wood for firewood and charcoal are usually cut from main branches or the main stem, leaving the stump rooted in the ground. 3.3.4.4.2. Energy Many tree and shrub species logged for energy regenerate vegetatively, sprouting from the cut or damaged trunk, although the rate of coppice growth varies across species (Neke, Owen-Smith, & Witkowski, 2006), environmental context, post-logged land-use (Chidumayo & Gumbo, 2013), and the type of logging (Shackleton, 2000). Resprouting is a major source of regeneration in dry tropical forests and woodlands (Chidumayo, 2013; Tredennick & Hanan, 2015) and temperate forest regions, forming part of rotational logging management. A review of charcoal production reported 9-12 years logging rotations for Mali, Niger and Burkina Faso, 10-15 years for Mexico, 20-30 years in Zambia, and a wide 8–23-year range in Tanzania (Chidumayo & Gumbo, 2013). Underestimated coppice regeneration post-firewood and charcoal logging is one of the reasons that the ‘fuelwood crisis’ in which biomass stocks were predicted to collapse, has not occurred (Arnold et al., 2003; Mograbi et al., 2019; Twine & Holdo, 2016). Despite the significant productivity of woodlands and forests, fuel wood logging can alter floristic composition and vegetation structure (Mograbi et al., 2015; Tredennick & Hanan, 2015). Depending on the fuel wood logging intensity, these ecosystem changes can alter the amount or type of nature’s contributions to people derived from the forests (Chidumayo & Gumbo, 2013). For example, in Mozambique, charcoal production led to a reduction in firewood and construction material resources, with other natural resources such as wild food, medicinal plants and grazing mostly unaffected, although these trade-offs were mediated by village position and woodland resource 272 characteristics (Woollen et al., 2016). Variable ecosystem regeneration potential and context- specificity of environmental impacts and trade-offs in fuel wood logging are challenging to incorporate into large scale policy and management plans because of the social-ecological complexity and non-linear responses occurring across spatial and temporal scales. Gender is one of the predominant features of traditional energy harvest, use and management of wood energy, including their (lack of) involvement in trade of fuel wood. While much of the research and interventions on inequality in forest resource use and management, many of the same challenges and barriers are faced by other vulnerable groups, such as minority ethnic groups, migrants, indigenous peoples, youths, landless people and other socially-differentiated groups such as lower castes (Chaudhary, McGregor, Houston, & Chettri, 2018; Kristjanson et al., 2019). 3.3.4.4.2. Energy Gender gaps exist in almost aspects of natural resource use and management, including: disparities in participation; leadership; resource, land access, and tenure; forest use; division of labor and workloads; skills; access to technologies and inputs; access to information; access to forest services; access to benefits; access to credit; access to markets; policy engagement; and forest laws and regulations (Kristjanson et al., 2019). With respect to the use of wood fuel for energy, women bear the majority of the responsibility for logging and using wood fuel (Clancy, Ummar, Shakya, & Kelkar, 2007; IEA, 2017; Murphy, Berazneva, & Lee, 2018). Households spend 1.4 hours a day harvesting fuel - a significant amount of time for women and children that could be used on other livelihood activities and education (IEA, 2017). The physical burden of headloads is not insignificant with a bundle weighing between 25-50 kg (IEA, 2017). Lack of access to clean cooking methods also has implications for household health (IEA, 2017), with women and children the most vulnerable to household air pollution which is a major cause of death and illness in low-income countries (Masera et al., 2015). An innovative approach to track how women are benefitting from interventions in forest resource use, trade and management is the W+ certification standard (WOCAN, 2020). The standard was created to measure women’s empowerment and to accelerate investment to address gender inequality in access to resources and capital, specifically targeting improvements in: time, income and assets, health, leadership, education and knowledge, and food security (WOCAN, 2020). The standard provides certification for economic development and environment projects that improve socio-economic conditions for women. Benefits accrue to women through involvement in certified projects as well as from direct payments to women from the sale of W+ certification credits (WOCAN, 2020). Successful application of the W+ programme has demonstrated that interventions that save time and improve wood fuel efficiency are especially beneficial to women (Kristjanson et al., 2019). W+ certification involving biogas digester projects in Nepal and Indonesia have resulted in tangible time and energy savings for women with improvements in income, assets and leadership capacity (Kristjanson et al., 2019). Uptake of more fuel-efficient stoves has the potential for environmental benefits too. A case study in China documents a successful social media campaign to improve fuel-efficient stove uptake (DeWan, Green, Li, & Hayden, 2013). 3.3.4.4.2. Energy After two years, 43% of households had incorporated the stoves into their use, saving 40.1% on gathering time, and in the process saw a 23.7% reduction in newly felled trees in areas crucial to the conservation of the Sichuan Golden Snub-nosed Monkey (DeWan et al., 2013). 273 Whilst gender inequalities are certainly a rights-based issue (S. Chaudhary et al., 2018; Clancy et al., 2007; Rights and Resources Initiative, 2014), investment in targeted programmes for women are opportunities for the sustainable management of forests and poverty relief (Kristjanson et al., 2019). Ingram et al. (2016) document cases where male and female headed households harvest the same amount of wood, yet male households earned over three times more. In a Kenya study, women earn less than men in trading wood, and woodlots were mainly managed by men (Murphy et al., 2018). Yet women’s expenditures and increased roles in household expenditure decisions are associated with improvements in household nutrition, health and education (Ingram et al., 2016). Women’s income is a major determinant of household fuel choice and use (van der Kroon et al., 2013). Thus, gender responsive interventions in training and enabling women to access markets and boost income can serve as leverage points for improving community well-being (de Groot, Mohlakoana, Knox, & Bressers, 2017; Ingram et al., 2016). Similarly, opportunities for improved ecosystem health as empowering women’s leadership and technical capacity building have been found to improve sustainable management of forests (Mwangi & Mai, 2011; Mwangi, Meinzen-Dick, & Sun, 2011). However, if fuel wood demand declines significantly, there are many women reliant on fuel wood sale income that will have reduced earnings in the event of a lack of alternate opportunities (IEA, 2017). 3.3.4.4.3. Material and construction To have an idea of the amount of timber converted into wood for different purposes (sawn wood, energy, industrial round wood and paper and paper board), the assessment utilizes statistics on the production and trade of forest products over 245 countries and territories (FAO Stat, 2018). However, this does not include products from illegal timber trade.  Industrial round wood  Industrial round wood Industrial round wood is all roundwood used for any purpose other than energy. It comprises pulpwood, sawlogs and veneer logs. Global industrial roundwood removals have increased from 1.7 billion m3 in 1990 to 2.0 billion m3 in 2019 (Figure 3.59). Industrial round wood is all roundwood used for any purpose other than energy. It comprises pulpwood, sawlogs and veneer logs. Global industrial roundwood removals have increased from 1.7 billion m3 in 1990 to 2.0 billion m3 in 2019 (Figure 3.59). 274 274 Figure 3.59 Global trends in industrial roundwood use. Data from FAO database (FAO, 2021b) under license CC BY-NC- SA 3.0 IGO. See data management report for the figure at https://doi.org/10.5281/zenodo.6453131. Figure 3.59 Global trends in industrial roundwood use. Data from FAO database (FAO, 2021b) under license CC BY-NC- SA 3.0 IGO. See data management report for the figure at https://doi.org/10.5281/zenodo.6453131. The increase in production is across all the regions except North America. Europe and North America had significant decreases in production in 1995 and 2010 respectively, while Asia had its greatest increase in production in 2010. There is a slight increase in trade of industrial roundwood. In 2019, approximately 144 million m3 and 138 million m3 were imported and exported respectively, while 83 million m3 and 83 million m3 were imported and exported respectively in 1990. Asia is a net importer, importing about 30 million cubic meters higher in 2019 than in 1990. Other regions are net exporters. Europe is the main exporter followed by Oceania. Africa and the Latin America and the Caribbean import and export very minimal quantities of industrial round wood (data from the Food and Agriculture Organization of the United Nations; http://www.fao.org/faostat/en/#data).  Sawnwood Sawnwood encompasses planks, sleepers (cross-ties), beams, joists, boards, rafters, scantlings, laths, boxboards and “lumber”. There was an increase in sawnwood production from 463 million m3 in 1990 to 488 million m3 in 2019, with the largest increase in Asia (Figure 3.60). There are significant decreases in production between the two points in time happening in 2000 in Asia, 1995 in Europe and 2010 in North America. There is an increase in trade of sawnwood with 149 million m3 and 156 million m3 imported and exported respectively in 2019 as compared to 84 million m3 and 78 million m3 imported and exported respectively in 1990. Asia and Africa are net importer of sawnwood while the rest of the regions are net exporters. Asia is the major importer, importing about 47 million m3 more in 2019 than in 1990, 275 while Europe is the major exporter, exporting 71 million m3 more in 2019 than it exported in 1990 (data from the FAO; http://www.fao.org/faostat/en/#data). while Europe is the major exporter, exporting 71 million m3 more in 2019 than it exported in 1990 (data from the FAO; http://www.fao.org/faostat/en/#data). while Europe is the major exporter, exporting 71 million m3 more in 2019 than it exported in 1990 (data from the FAO; http://www.fao.org/faostat/en/#data). Figure 3.60 Global trends in sawnwood. Data from FAO database (FAO, 2021b) under license CC BY-NC- SA 3.0 IGO. See data management report for the figure at https://doi.org/10.5281/zenodo.6453131. Figure 3.60 Global trends in sawnwood. Data from FAO database (FAO, 2021b) under license CC BY-NC- SA 3.0 IGO. See data management report for the figure at https://doi.org/10.5281/zenodo.6453131. Figure 3.60 Global trends in sawnwood. Data from FAO database (FAO, 2021b) under license CC BY-NC- SA 3.0 IGO. See data management report for the figure at https://doi.org/10.5281/zenodo.6453131.  Wood based panels The wood-based panels’ product category consists of plywood (including blockboard and laminated veneer lumber), particle board, oriented strand board and fibreboard. In 2019, approximately 358 million m3 of wood-based panels were produced globally (Figure 3.61). This is an increase of 234 million m3 from a volume of 124 million m3 reported in 1990. The major producers of wood-based panels are Asia, Europe and North America, with Asia reporting the most tremendous increase of production from 25 million m3 in 1990 to 196 million m3 in 2019. Trade in wood-based products has also increased between 1990 to 2019 from approximately 28 million m3 of imports and exports in 1990 to 88 million m3 of imports and exports in 2019. Europe is the major trader of the product, followed by Asia and North America (data from the FAO; http://www.fao.org/faostat/en/#data). 276 Figure 3.61 Global trends in wood based panel production. Data from FAO database (FAO, 2021b) under license CC BY-NC- SA 3.0 IGO. See data management report for the figure at https://doi.org/10.5281/zenodo.6453131. Figure 3.61 Global trends in wood based panel production. Data from FAO database (FAO, 2021b) under license CC BY-NC- SA 3.0 IGO. See data management report for the figure at https://doi.org/10.5281/zenodo.6453131.  Paper and paper board  Paper and paper board The paper and paperboard product group comprises graphic papers (newsprint, printing and writing paper) and other paper and paperboard. There is an increase in global production of paper and paper boards (Figure 3.62). In 2019, approximately 404 million tons were produced, an increase of 165 million m3 from production volumes of 1990. The major producers and traders of paper and paper boards are Asia, followed by Europe and North America. Production levels of North America have fallen by approximately 11 million tons between 1990 and 2019, while those of Asia and Europe have increased by 138 million tons and 25 million tons respectively within the same time intervals. Trade in paper and paper boards has also increased with 110 million tons and 113 million tons imported and exported respectively. Asia is a net importer while Europe and North America are net exporters (data from the FAO; http://www.fao.org/faostat/en/#data). 277 Figure 3.62 Global trends in paper and paperboard production. Data from FAO database (FAO, 2021b) under license CC BY-NC- SA 3.0 IGO. See data management report for the figure at https://doi.org/10.5281/zenodo.6453131. Figure 3.62 Global trends in paper and paperboard production. Data from FAO database (FAO, 2021b) under license CC BY-NC- SA 3.0 IGO. See data management report for the figure at https://doi.org/10.5281/zenodo.6453131. 3.3.4.5.1. Covid-19 pandemic The COVID-19 pandemic has led to disruptions in international trade and supply chains of timber and its products globally. Many developing countries are heavily dependent on international trade of these products and the pandemic is having a significant effect on production and consumption patterns. For example, recent developments in the timber markets have increased the dependency on Chinese demand. With the pandemic triggered decline of exported round timber to China, stockpiles of export products are being built up in some places. This is further exacerbated by limited demand in typically strong markets such as Austria and Germany, while export markets in France, Italy and Spain are essentially at a standstill. Together these factors have resulted in a decrease in export incomes in developing countries (FAO, 2020c). As a result, the least developed timber-producing countries, in particular, may suffer directly from plummeting export volumes of roundwood and other wood products (FAO, 2020c). Nevertheless, in the post-COVID-19 environment, the trade and consumption of legal and sustainable wood products may be promoted through sustainable forest management for wood production, and can play a crucial role in economic recovery, especially considering efforts to promote a circular bioeconomy and climate change mitigation (FAO, 2020b). For indigenous people and local communities, negative effects of the COVID-19 pandemic on vulnerable communities, including women have been observed. Although, there 278 has been steady progress made to date to empower women by supporting their participation in legal and sustainable fuelwood and charcoal production, the COVID-19 crisis is expected to put increasing pressure on forest resources through illegal charcoal production. Situations where livelihoods are put under significant pressure often tend to result in a shift towards activities with quick economic gains at the sacrifice of legal activities. In some countries, restrictions on travel and movements may affect the transportation and trade of fuelwood (particularly charcoal) from production sites to market centers (mostly urban areas). This may affect reliable access to energy for cooking in urban areas (FAO, 2020b). The COVID-19 pandemic also set the progress of universal access to electricity and clean cooking back, with the number of people without electricity access forecast to rise by 2% in 2021 (IEA, 2021). The economic shock of the pandemic also resulted in a return to fuel wood, with many people unable to pay for modern, clean fuels (IEA, 2021). 3.3.5.1. Introduction: Significance of non-extractive practices 3.3.5.1. Introduction: Significance of non-extractive practices Non-extractive practices are widespread across the globe, occur in all ecoregions, and are essential to maintaining inter alia human relaxation, spiritual and cultural identity, connection to nature, belonging, sense of place, physical and psychological health, and inspiration. The contributions of wild species to people from non-extractive practices are often intangible and resist commodity-based valuation (with the exception of recreational tourism). Yet many of the non-extractive contributions from nature are core to the human experience and contribute to the well-being of people (Millennium Ecosystem Assessment, 2005; Russell et al., 2013). Knowing and experiencing nature is the foundation of cultural expression and identity; is inherent in the concept of biocultural diversity; forms the backdrop for social connections, religious experience and beauty; as well as contributing substantially to gross domestic product and local livelihoods (Russell et al., 2013). Although extractive practices are often the focus in the debate on what constitutes sustainable use of wild species (e.g., Abensperg-Traun, 2009; Di Minin et al., 2019; Link & Watson, 2019; Nijman, 2010; Zeller & Pauly, 2019), non-extractive practices may also have sustainability implications, both for wild species and for human well-being. Although, non- extractive practices, by its very definition, are viewed as having less of a direct impact on wild species and ecosystems than extractive practices, there are many documented detrimental impacts and sustainability concerns in this practice. This is particularly well-documented for the use of wild species for tourism and recreation (see Section 3.3.5.2.3.). However, many of the adverse impacts may be avoided or mitigated through context-based understanding and collaborative engagement with all stakeholders. Non-extractive benefits from wild species and nature are similar conceptually to the definitions of cultural nature’s contributions to people (Costanza et al., 1997) and non-material benefits (Millennium Ecosystem Assessment, 2005). In this assessment, non-extractive practices are defined based on the observation of wild species in a way that does not involve the harvest or removal of any part of the organism. The observation can imply some interaction 279 with the wild species, such as the activities of wild species tourism and whale watching or no interaction with the wild species, such as photography (see Chapter 1). Just as in extractive practices, the social contextual heterogeneity in the contributions from wild species to human well-being through their non-extractive use has implications for equitable environmental decision making (Martín-López et al., 2012). 3.3.5.1. Introduction: Significance of non-extractive practices The contributions from wild species to human well-being are perceived and valued differently, which influences the type and extent of use (Pascual et al., 2017; Satz et al., 2013). This also means that there may be conflict between different users of wild species (Pascual et al., 2017). For example, one study documented interpersonal conflicts both within and between two recreational user groups in Hawaii, scuba divers and snorkelers, that held different nature-oriented values, those who valued nature intrinsically and held protectionist beliefs versus an anthropocentric-utilitarian value (Philips, Szuster, & Needham, 2019). Similarly, local residents near North American ski resorts placed high emphasis on recreational access and came into conflict with city residents who preferred that the area remain pristine wilderness, unaffected by tourism activities (Saremba & Gill, 1991). One proposed solution to avoid these types of conflicts is to spatially or temporally partition regions that can cater for different stakeholder’s values (e.g., demarcated fishing and diving zones). There can also be a disconnect between the importance placed on non-extractive practices of nature at a local level, where they are used on a daily basis, and the level to which they are incorporated into regional, national and global decisions on ecosystem management, which are made from a more distanced level (Brondizio, Ostrom, & Young, 2009; S. Chaudhary, McGregor, Houston, & Chettri, 2019). Thus, governance systems play a large role in which non-extractive contributions from nature are delivered to people, by identifying which stakeholder group’s expectations and values are recognized (Gladkikh, Gould, & Coleman, 2019; Martín-López et al., 2012; Pert et al., 2015). 3.3.5.2. Uses 3.3.5.2. Uses Regarding non-extractive practices, the following uses are well-documented in the literature and available data sources: ceremony and cultural expression (section 3.3.5.2.1), medicine and hygiene (section 3.3.5.2.2.), recreation (section 3.3.5.2.3.), education and learning (section 3.3.5.2.4). The documentation of the non-extractive practices of nature, especially the use by indigenous peoples and local communities, often does not include species described at a species level, but frequently as part of a functional group (e.g., trees in urban green spaces; worship of sacred groves). For many indigenous and local communities their worldview and experiences are intimately connected with nature (Klain, Satterfield, & Chan, 2014; Pert et al., 2015). Indigenous and local knowledge is premised on the interdependence of what scientific knowledge may identify as distinct components of nature and culture, such that worship of sacred groves is a holistic practice that includes the species in the grove (e.g., forest plant and animal species community), the ecosystem processes (e.g., primary production, pollination), the landscape features (e.g., rocks, rivers), and the particular cultural practices and language of the human community. However, in order to keep the scope of this section pragmatic and practical, literature that deals with quantifiable and measurable use of wild species up to the 280 taxa level (e.g., trees) has been included in this review on non-extractive practices and literature on landscapes and landscape components (e.g., sacred pools, sacred mountains) was excluded. taxa level (e.g., trees) has been included in this review on non-extractive practices and literature on landscapes and landscape components (e.g., sacred pools, sacred mountains) was excluded. The following uses are not relevant to this practice and/or were not documented: decorative and aesthetic, energy, food and feed, materials and shelter. Although aesthetic The following uses are not relevant to this practice and/or were not documented: decorative and aesthetic, energy, food and feed, materials and shelter. Although aesthetic beauty and inspiration of nature are a form of non-extractive practice, this was excluded to maintain the scope of the assessment to quantifiable and measurable impacts of sustainable use. Keyword searches and methods for each review are detailed in each subsection. 3.3.5.2.1. Ceremony and cultural expression Ceremony and cultural expression refer to the use of wild species in spiritual observances and practices, valued for their role in maintaining cultural identity (Chapter 1). In the context of non-extractive practices, the use of wild species can be through worship of religious or culturally important species. In urban areas, similarly urban green spaces have become important analogues for worship and ceremonial rituals (Ngulani & Shackleton, 2019). Thus, wild species can underpin cultural and religious identity by supporting spiritual, intellectual and emotional features and contribute to literature, lifestyles, value systems, traditions and beliefs, and ways of living together. The use of wild species for ceremony and cultural expression supports social cohesion, belonging and identity (Satz et al., 2013). Wild species form part of history and cultural narratives (Pascual et al., 2017; Satz et al., 2013). Thus, unsustainable use of wild species central to cultural and ceremonial engagement can harm social relations (Millennium Ecosystem Assessment, 2005). Inversely, restoration of degraded forests and landscapes provides an opportunity for cultural and indigenous value revival (Constant & Taylor, 2020). The text below is based on a literature review (Web of Science) using the following strings of terms ("non-extractive use" OR "cultural ecosystem service" OR "non-material contribution" OR “non-consumptive use”) AND (spiritual OR ceremon* OR religion* OR ritual), generating 51 hits (see the data management report for Chapter 3 systematic literature review at https://doi.org/10.5281/zenodo.6452651). Articles that were recommended by citation databases were also considered, as well as harvested from personal libraries and recommendations from experts. The scope of this section is limited to the non-extractive practices of wild species for ceremonial and cultural expression where the impact of the use can be measured or assessed. After reviewing the title and abstract, 36 papers were selected for a full-text read. Relevance was determined by mention of either the status (current), trend (historical), or impacts of use of wild species (or taxa) in at least one dimension of sustainability (social, economic, or ecological). After a full text read of these papers, eight were deemed relevant and assessed for the literature review. These data form the basis of the text below. Of the reviewed articles, half (4 out of 8) covered the importance of trees for ceremonial use, particularly sacred groves in Africa. Research on sacred groves was mostly anthropological and social data of long-term (>10 years) trends on a regional (<100 km) scale. 3.3.5.2.1. Ceremony and cultural expression Of the reviewed articles, half (4 out of 8) covered the importance of trees for ceremonial use, particularly sacred groves in Africa. Research on sacred groves was mostly anthropological and social data of long-term (>10 years) trends on a regional (<100 km) scale. Sacred natural sites, such as sacred groves and burial sites, are an important feature across the world that can play a central role in biodiversity and resource conservation. These sites exhibit large diversity in their form and function, showing strong local context of both ecology and culture (Fournier, 2011; Juhé-Beaulaton & Salpeteur, 2017). Sacred groves are Sacred natural sites, such as sacred groves and burial sites, are an important feature across the world that can play a central role in biodiversity and resource conservation. These sites exhibit large diversity in their form and function, showing strong local context of both ecology and culture (Fournier, 2011; Juhé-Beaulaton & Salpeteur, 2017). Sacred groves are 281 places of spiritual and cultural importance, protected by the authority of tribal taboos and spiritual “caretakers”. In general, restrictions forbid cutting down or harvesting any part of the trees, including dead wood, to burn or harm the fauna and flora, or to remove soil or stones (Fournier, 2011). Depending on the tribal custom, picking herbaceous plants and grazing livestock near the sacred trees may be permitted (Fournier, 2011). Taboos also vary depending on the type of sacred grove. For example, the Tandroy clan in Madagascar allows the use of fire in honey groves - kept for medicinal, food, and spiritual purposes – but has more stringent taboos in burial forests (von Heland & Folke, 2014). The current status of sacred groves, or indeed of any wild species used for ceremonial and cultural purposes, is not well documented in the literature. But the limited data that do exist are mixed, with some evidence that taboos and traditional beliefs have protected sacred groves. Sacred groves can an important role in community-based conservation of biodiversity, acting as refugia for species. For example, India possesses relict populations of certain threatened tree species (Actinodaphne lawsonii, Hopea ponga, Madhuca neriifoli, and Syzygium zeylanicum, Myristica fatua and Gymnacranthera canarica) in numerous riparian groves. Sacred groves in the Karnataka state also shelter a high diversity of macrofungi, 49 out of 163 species are unique to sacred groves (Bhagwat & Rutte, 2006). 3.3.5.2.1. Ceremony and cultural expression Similarly, in central Tanzania, greater woody plant species richness was found in sacred groves than in a state-managed forest reserve (Mgumia & Oba, 2003). Despite droughts and pressure to use resources inside sacred forests, the ancestral forests in Ambonaivo have been preserved whereas elsewhere in Madagascar, sacred groves have been cut down for charcoal production (von Heland & Folke, 2014). Similarly, sacred groves in Morocco have been effectively conserved as a result of their sacred status (Frosch & Deil, 2011). Despite their significance, the protection offered to wooded shrines may be limited in extent and may only be for a certain period of time (Fournier, 2011). In Burkino Faso, the clearing of wooded shrines has also been blamed on “foreigners” fleeing worsening climatic conditions in the Sahel, who are (either intentionally or not) ignorant of local traditions (A. Fournier, 2011). In Benin most sacred groves have been neglected or cut down, but a few have been restored and are being managed for nature’s contributions to people (Juhé-Beaulaton & Salpeteur, 2017). A vegetation assessment of wooded shrines in West Africa found more groves were cut down than restored and although they were less used for extractive purposes than similar secular forests, they were still being used for extractive purposes (Fournier, 2011). Sacred groves are also not necessarily ecologically ‘pristine’ by conservation standards. Whilst the preference by locals is for sacred groves to “have trees”, preferably dense vegetation, the species of tree is considered unimportant and wooded shrines range from almost natural to highly modified (Fournier, 2011). The literature suggests the future of sacred groves is strongly dependent on how spiritual and religious practices adapt to changing socio-political conditions. The degrading social contract with nature and the erosion of ancestral and natural connections threatens the sustainability of sacred groves. Cultural trends show taboos around sacred groves are eroding as the elder “spiritual caretakers” who play an active role in supervising use of the groves, are not replaced (Fournier, 2011; von Heland & Folke, 2014). There are also changes to “social- ancestor contracts” which are being modernized, and more of the local people have converted to global religions (Juhé-Beaulaton & Salpeteur, 2017; von Heland & Folke, 2014). 3.3.5.2.1. Ceremony and cultural expression The 282 increasing assimilation of local peoples’ moral order into one more closely aligned with modern, Western, democratic ideals governed by the nation-state has eroded the traditional and ancestral social-ecological system central to their identity, as well as the associated protection afforded to their land and the species it contains (Findlay & Twine, 2018; von Heland & Folke, 2014). As local protection erodes for sacred sites, there is an opportunity for more formal protection, such as the promising case developing in Estonia where Estonian indigenous peoples and local communities (Maausk) and the government are planning to confer legal protection to approximately 550 sacred groves (Kaasik, 2012). There are also opportunities to conserve sacred groves for purposes other than cultural worship provided the other uses are compatible and respectful of the sacred status. This has occurred in West Africa where sacred groves are also used for heritage and cultural tourism (Juhé-Beaulaton & Salpeteur, 2017). The literature review on the use of wild species for ceremonial and cultural expression also described the use of urban green spaces for religious worship. However, increasing urbanization threatens the loss of green spaces used for worship, especially as these sacred sites are not associated with formal religious structures or buildings (Jackson & Ormsby, 2017). Use of urban green space for ceremonial purposes has been documented in Zimbabwe (Ngulani & Shackleton, 2019), Accra (Okyerefo & Fiaveh, 2017) and India (Gopal, von der Lippe, & Kowarik, 2019), but it is an underreported form of use of either formal or informal urban green spaces and has not received adequate research or policy attention (Jackson & Ormsby, 2017; Ngulani & Shackleton, 2019). No information on the use of urban green spaces for worship described whether this was an increasing phenomenon, or the sustainability of this use. Overall, the use of wild species for ceremonial and cultural purposes is likely widespread but poorly documented. There are little to no data on the status and trends, or sustainability of this use. However, the literature on sacred groves do suggest that cultural erosion is driving a decreasing trend of ceremonial use, and thus also an erosion of traditional protection that the use afforded these species. 3.3.5.2.2. Medicine and hygiene This section relates to the non-extractive practices of wild species for human health, both psychological and physical. The scope of this section is limited to the non-extractive practice of wild species for restorative and/or preventative effects and the impact this use has in terms of a measurable effect on the species. The text below is based on a literature review (Google Scholar) including the following search terms: ("non-extractive use" OR "cultural ecosystem service" OR "non-material contribution" OR “non-consumptive use”) AND (sustainab* OR "forest therapy" OR "human well-being" OR "human health" OR stress OR happiness OR dose- response) generating over 1 million hits (see the data management report for Chapter 3 systematic literature review at https://doi.org/10.5281/zenodo.6452651). Articles that were recommended by citation databases were also considered, as well as collected from personal libraries and recommendations by experts After a title and abstract read, 24 papers were selected for a full-text read. After a full text read of these papers, 13 were deemed relevant and assessed for the literature review. Relevance was determined by mention of either the status (current), trend (historical) or impacts of use of wild species (or taxa) in at least one dimension of sustainability (social, economic, or ecological). These data form the basis of the text below. 283 Relevant material from the review covered mostly the use of trees (10 papers) for health purposes, with a few mentions of terrestrial mammals and birds (4 papers). 46% of the studies on this topic were global overviews, with regional studies mostly representing Asia-Pacific and Europe Central. Relevant research was overwhelmingly short-term studies (<1 year), but spanned a variety of spatial scales: global, national, regional and local. There was an absence of information in the literature on trends in the non-extractive practices of wild species for health and hygiene. From the review, only one paper tracked trends in use for health over time. This was a paper that documented current and past trends in the use of forests for forest therapy in Korea (Shin et al., 2017). Similarly, no information was found reporting on the sustainability of health-based use of wild species on species or ecosystems. Although undocumented, negative impacts on wild species used for medicine and health likely include the effects of trampling during nature visits (see section 3.3.5.2.3. Recreational). It is possible that the health benefits obtained from wild species motivate people to support and protect their natural spaces. 3.3.5.2.2. Medicine and hygiene Research on environmental education supports that the more people learn from (and in) nature, the more likely they are to develop pro-nature behavior (M. Richardson, Cormack, McRobert, & Underhill, 2016). But this has not yet been documented for the non-extractive practices of wild species for medicine and hygiene use. It is not a given that the benefits provided by wild species always confer protection. For instance, street tree vandalism is a significant driver of urban tree mortality (e.g., Richardson & Shackleton, 2014) despite the numerous benefits provided by urban greening. The literature on this topic extensively deals with the beneficial impacts of nature, especially forests, on individual human well-being. A significant research gap exists on the impacts of health-based use of wild species on human community health (Nesbitt, Hotte, Barron, Cowan, & Sheppard, 2017). The rest of this section will describe the evidence and examples of the impacts of health use of wild species on human individual’s well-being. Rapid urbanization and industrialization have been related to the rise in chronic mental and physical health problems, mostly associated with stress (Ashworth, 2017), costing millions in healthcare-related expenses and lost work days (Moore, Gould, & Keary, 2003). Thus, preventive measures, including nature-based remedies, to deal with the modern-day health crisis are economically prudent, and are supported formally by some governments, such as the legislation passed by the Korean government for the use of forests for health (Kotte, Li, & Shin, 2019; Shin et al., 2017), or shinrin-yoku (“forest bathing”) by the Japanese Forestry Agency (Rajoo, Karam, & Abdullah, 2020). There are also documented case studies of forest therapy, and the increasing demand for cost-effective preventive medicine and stress management using forest therapy, in Southeast Asia and Northern Europe (Kotte et al., 2019; Lee et al., 2019). Shin et al., (2017) documented a significant increase in the use of Korean forests for recreational visitors, primarily for forest therapy and the health benefits of spending time in “healing forests”. This rise in health-based forest use has been facilitated by Korean government forests restoration programs, legislating certain forests specifically as “healing forests”, and substantial investment in forest therapy research (Shin et al., 2017). Although other studies mention that the demand for and use of nature for restoration and health has increased (Kotte et al., 2019; Lee et al., 2019; Rajoo et al., 2020), the quantitative change in this use has not been documented (Figure 3.63). 3.3.5.2.2. Medicine and hygiene 284 Figure 3.63 The graph (a) and photos (b) show the recovery of forest stocks in Young-il Gyeongsangnam-do, Korea from 1970-2013. Concomitantly, the number of “recreational visitors” to “Healing Forests” have increased over time as the number of recreational climbers have declined (a) with a mean increase of 117% in healing forest visits over 3 years (c). Source: (Shin et al., 2017) under license CC BY-SA 4.0. Figure 3.63 The graph (a) and photos (b) show the recovery of forest stocks in Young-il Gyeongsangnam-do, Korea from 1970-2013. Concomitantly, the number of “recreational visitors” to “Healing Forests” have increased over time as the number of recreational climbers have declined (a) with a mean increase of 117% in healing forest visits over 3 years (c). Source: (Shin et al., 2017) under license CC BY-SA 4.0. Figure 3.63 The graph (a) and photos (b) show the recovery of forest stocks in Young-il Gyeongsangnam-do, Korea from 1970-2013. Concomitantly, the number of “recreational visitors” to “Healing Forests” have increased over time as the number of recreational climbers have declined (a) with a mean increase of 117% in healing forest visits over 3 years (c). Source: (Shin et al., 2017) under license CC BY-SA 4.0. Figure 3.63 The graph (a) and photos (b) show the recovery of forest stocks in Young-il Gyeongsangnam-do, Korea from 1970-2013. Concomitantly, the number of “recreational visitors” to “Healing Forests” have increased over time as the number of recreational climbers have declined (a) with a mean increase of 117% in healing forest visits over 3 years (c). Source: (Shin et al., 2017) under license CC BY-SA 4.0. Reviews on the effects of forest therapy on human health found that most research reported positive effects (Frumkin et al., 2017; Kotte et al., 2019; Rajoo et al., 2020; Wolf et al., 2020). The benefits of natural settings for restorative effects, such as stress relief, decreased cognitive fatigue, and happiness (see Chapter 1), have been documented in both urban and non- urban settings. Natural settings have been associated with, amongst others, better cognitive functioning, fewer symptoms of depression and lower antidepressant use, reduced stress and 285 psychiatric disorders, reduced diabetes, and improved immune function (see review in Frumkin et al., 2017). Exposure to nature has also been shown to have a positive effect on infant birth weights, reductions in childhood obesity, and improved blood pressure (Aerts, Honnay, & Van Nieuwenhuyse, 2018; Frumkin et al., 2017). 3.3.5.2.2. Medicine and hygiene Exceptions include the negative effects of plant pollen and volatile organic compounds from trees (Wolf et al., 2020). Findings on the benefits wild species and ecosystems provide for mental and physical health have motivated for technology to provide this form of health benefit through virtual reality, and although exposure to nature through photographs or video does improve stress levels and reduce cognitive fatigue, the real experiences in nature significantly outperform virtual experiences for restorative benefits (Calogiuri et al., 2018). The studies mentioned above used a variety of self-reported measures to assess human well-being, with little research being done on clinical outcomes (Aerts et al., 2018). The research was also mostly based on a limited set of variables to describe nature. The majority of studies were based on proximity to nature (e.g., Xiao et al., 2019), or the number or cover of trees (Wolf et al., 2020). There were fewer studies on the diversity of wild species for human well-being (Methorst et al., 2021) and none was identified on specific wild species, rather focusing on functional groups such as trees or birds. The limited research on the effects of ecological quality (e.g., species richness) of trees suggests lower correlation with life satisfaction than overall abundance and denser tree cover, suggesting people were less affected by species diversity and more by the mere presence of trees (Marselle et al., 2020; Methorst et al., 2021), although the state of knowledge in this field is still mixed (Aerts et al., 2018). Certainly, people have expressed preference for particular species, especially those that were aesthetically pleasing or reminded them of their childhood home (C.M. Shackleton & Mograbi, 2020). Dose-response effects of wild species on human health have been demonstrated with trees and with birds. People living within 100m of higher street tree density had lower antidepressant prescriptions (Marselle et al., 2020). This effect was even more pronounced for individuals with low socio-economic status (Marselle et al., 2020). A study exploring self- reported life satisfaction across Europe in relation to several taxonomic groups and socio- economic indicators found that bird species richness was highly correlated with life satisfaction, comparable with that of net household income (Figure 3.64) (Methorst et al., 2021). Methorst et al., (2021) hypothesize that the direct multisensory experience of birds and/or the supporting landscape properties that support bird diversity benefit human life satisfaction. 3.3.5.2.2. Medicine and hygiene Another study found that vegetation cover and afternoon bird abundance was positively associated with lower depression, anxiety and stress (Cox et al., 2017). Cox et al., (2017) modelled neighborhood vegetation cover thresholds at which population prevalence of mental health issues were significantly lower: more than 20% for depression and stress, and more than 30% for anxiety. A dose-response model suggested that visits to nature of 30 minutes or more a week could reduce population prevalence of depression by 7% and high blood pressure by 9% (Shanahan et al., 2016). A significant reduction, especially considering that depression alone in Australia, where this study was conducted, was estimated at 12.6 billion Asutralian dollars per year (Shanahan et al., 2016). A study from the United Kingdom of Great Britain and Northern Ireland found that individuals spending at least 120 minutes a week in nature reported better health and well-being relative to people spending no time outdoors; 286 positive associations peaked at 200-300 minutes a week (White et al., 2019). These “Green Prescriptions” highlight the importance of species presence and diversity to human well-being, a cost-effective means of supporting a healthy population. Figure 3.64 Estimated life-satisfaction increase correlates to bird species richness and income across Europe. Estimates are based on the coefficients for log-transformed mean bird species richness and log-transformed net household income, both corrected for socio- and macro-economic factors. Source: (Methorst et al., 2021) under license CC BY-NC-ND 4.0. Figure 3.64 Estimated life-satisfaction increase correlates to bird species richness and income across Europe. Estimates are based on the coefficients for log-transformed mean bird species richness and log-transformed net household income, both corrected for socio- and macro-economic factors. Source: (Methorst et al., 2021) under license CC BY-NC-ND 4.0. There are significant socio-economic disparities in urban green space access, both as a result of restricted access (e.g., private space) and as a consequence of socio-economic class differentiation in urban planning (Venter, Shackleton, Van Staden, Selomane, & Masterson, 2020; J. Wu, He, Chen, Lin, & Wang, 2020). Gentrification, while making cities more attractive to wealthy residents and attracting investment, has environmental justice implications, especially on urban green space access by lower class or income communities (Kronenberg et al., 2020). 3.3.5.2.2. Medicine and hygiene Public space is also increasingly being ‘corporatized’, where public space maintenance is sponsored by private interests, and the urban green space is redesigned and highly controlled to meet the needs of the ‘owner’ rather than the general public (S. Schmidt, 2004). Research in North American cities on parks, urban forests and tree canopy cover found race, ethnicity and income disparities in tree distribution with non-white communities living in areas of lower tree density and lower quantity and quality of parks (Heynen, Perkins, & Roy, 2006; Rigolon, Browning, & Jennings, 2018). The disproportionate access to and distribution of urban green spaces creates inequitable health benefits derived from 287 exposure to nature, with lower income and minority communities in cities more likely to live in “riskscapes” – environments that increase the vulnerability of these communities to pollutants and hazards (Jennings, Johnson Gaither, & Gragg, 2012). “Green prescriptions” such as “forest bathing” are increasing as they improve human health, but there are also win-win opportunities for people and ecosystems through “reciprocal restoration”. Pilot initiatives with urban youth working in habitat restoration programs have shown greater anti-inflammatory capacity, cardiovascular fitness, resistance to endoparasites, resistance to infectious diseases, reduced sensitivity to allergens, reduced frequency of nervous and musculoskeletal disorders and a wide range of positive effects on mental health (Nabhan, Orlando, Smith Monti, & Aronson, 2020). Concurrently, habitats are restored including vegetation cover and soil microbial content (Nabhan et al., 2020). The hypothesized mechanisms for the documented improvements in mental and physical health include the Microbiome Rewilding Hypothesis where restoring soil microbial diversity enhances human gut microbiome health and boosts immune functioning, and the Psycho-Evolutionary Restoration Hypothesis where humans exposed to forested systems exposes them to phytoncides that may reduce depression and lower cortisol levels (Nabhan et al., 2020). In a critical review of the effects of environmental diversity on human health, Sandifer et al., (2015) found the only unambiguous causal relationship was the maintenance of a healthy immune system and reduction of inflammatory diseases through exposure to environmental microbial diversity. There is also a limit in research on the sustained, long-term effects of nature-based therapies (Rajoo et al., 2020). However, despite the limited information about the causal nature underlying the benefits of nature and biodiversity to human health, protecting and restoring a diversity of natural habitats seems crucial for maintaining human health in a developing world (Sandifer et al., 2015). 3.3.5.2.2. Medicine and hygiene Indeed, the improvement of human health is a powerful tool to leverage support from multiple stakeholders to enhance social-ecological health in a variety of ways. 3.3.5.2.3. Recreation Wildlife watching is an activity that involves the watching of wild species (animals and plants). Watching wild species is essentially an observational activity, although in some cases it can involve interactions with the animals being watched, such as touching or feeding them (UNEP/CMS, 2006). These recreational activities include nature-based tourism, hiking and nature walks, photography and cinematography, game watching and safaris, and snorkeling and scuba diving. The use of wild species for recreation is primarily for enjoyment but may also provide relaxation, restoration, physical exercise (see section 3.3.5.2.2. Medicine and hygiene), and educational experiences (see section 3.3.5.2.4. Education and learning). The scope of this section is limited to the non-extractive practices of wild species for recreation where the impact of the use can be measured or assessed. The text below is based on a literature review (Web of Science) using the following strings of terms ("non-extractive use" OR “non-consumptive” OR "cultural ecosystem service" OR "non-material contribution" OR “touris” OR “community based tourism” OR “ecotourism” OR “eco- tourism” OR “sustainable tourism” OR “recreational” OR “nature-based tourism” OR “wildlife watching” OR “wildlife viewing”) AND (sustainab OR trend), generating 16117 hits. 288 Articles that were recommended by citation databases were also considered, as well as collected from personal libraries and recommendations from experts. After a title and abstract read, 82 papers were selected for a full text read. After a full text read of these papers, 27 were deemed relevant and assessed for the literature review. Relevance was determined by mention of the status (current), trend (historical) or impacts of use of a wild species (or taxa) in at least one dimension of sustainability (social, economic, or ecological). These data form the basis of the text below. The literature covered fairly equally (4-6 papers each): vegetation (trees and shrubs); terrestrial mammals; birds (terrestrial and marine); marine mammals; fish, rays and sharks; and arthropods (marine and terrestrial). The temporal scale of the research articles was 10 short term (<1 year), 1 medium term (1-10 years), and 9 long term (>10 years) studies. The review included articles from every IPBES region. Most of the information in the text below relates to wildlife watching tourism, as 74% of relevant articles focused on tourism specifically. Wildlife watching does occur around people’s homes (Wilkinson, Waitt, & Gibbs, 2014; Zarazúa-Carbajal et al., 2020), but this is less well documented than wildlife watching tourism. 3.3.5.2.3. Recreation Wildlife watching tourism overlaps with various types of tourism, such as tours focused on seeing a specific kind of wild taxa (Table 3.19) and tourism where wildlife watching is an added advantage but not the main focus of the activity (e.g., adventure and sports tourism) (UNEP/CMS, 2006). Similarly, a specific type of nature-based tourism is eco-tourism, where the tourism activity aims to contribute to the conservation of natural and cultural heritage through the involvement of indigenous peoples and local communities (UNEP/CMS, 2006). Eco-tourism has relatively low numbers of tourism and is suited to small groups and independent tourists (UNEP/CMS, 2006). Table 3.19 Examples of species- and taxa-based wildlife watching across the globe. Source: (UNEP/CMS, 2006) under license CC-BY. Species being watched Tourism Activity Location example Butterflies Butterfly watching Monarch butterflies in Mexico, United States of America and Canada Glow worms Glow worm watching Springbrook National Park, Australia Crabs Red crab migration Christmas Island, Indian Ocean Corals and fish Snorkel/scuba diving Bunaken, Indonesia; Sian Ka’an, Mexico; Soufriere Marine Management Area, St. Lucia; Bonaire, Caribbean; Red Sea, Egypt Sharks Snorkel with whale sharks Seychelles; Ningaloo Reef, Australia Sharks Underwater watching/feeding of sharks Dyer Island, South Africa Stingrays Feeding and close interaction with stingrays Cayman Islands; Maldives; Australia Komodo dragons Watching Komodo dragons Komodo Island, Indonesia Snakes Watching pythons Bharatpur, India Crocodiles Watching crocodiles Black River, Jamaica; Kakadu Park, Australia Table 3.19 Examples of species- and taxa-based wildlife watching across the globe. Source: (UNEP/CMS, 2006) under license CC-BY. 3.19 Examples of species- and taxa-based wildlife watching across the globe. Source: /CMS, 2006) under license CC-BY. 3.3.5.2.3. Recreation 289 Turtles Watching turtles Projeto TAMAR-IBAMA, Brazil; Akumal, Yucatán Pennisula, Mexico; Cape Verde; Maputaland, South Africa; Sri Lanka; Indonesia Birds Independent or organized visits to reserves for bird- watching Bempton Cliffs, United Kingdom; Keoladeo, India; Pantanal, Brazil Albatrosses Independent or coach tours to see breeding colonies Taiaroa Head, New Zealand Cranes Watching cranes Müritz National Park, Germany; Platte River, United States of America Penguins Watching penguins and penguin colonies Antarctica; Peninsula Valdés, Argentina; Phillip Island, Australia Large African mammals Vehicle safaris to see large concentrations of mammals Serengeti National Park, Tanzania; Masai Mara, Kenya Tigers Tiger watching from hides or elephant back Chitwan National Park & Bardia National Park, Nepal Gorillas Mountain trek and camping to observe habituated gorillas Bwindi National Park, Uganda; Virunga National Park, Democratic Republic of Congo; Volcanoes National Park, Rwanda Orangutans Watching orangutans Sepilok Orangutan Centre & Danum Valley, Sabah Semenggok Wildlife Centre, Sarawak, Borneo Polar bears Watching polar bears Churchill, Canada Bats Watching bats Texas, United States of America Dolphins Watching dolphins Red Sea, Egypt; Mon Repos, Australia Whales Watching whales Peninsula Valdés, Argentina; Kaikoura, New Zealand; El Vizcaino, Baja California, Mexico; New England, United States of America; Plettenberg Bay, South Africa; Canary Islands  Social aspects  Social aspects Enjoyment of nature for tourism and recreation is recognized as the most prominent cultural nature’s contributions to people (Balmford et al., 2015). Over the last half a century the demand for nature-based tourism experiences has been on the rise, with the ever-increasing breadth and depth of its global penetration, integrating more and more natural areas into commercial processes (Balmford et al., 2009, 2015; Elmahdy, Haukeland, & Fredman, 2017; Hall, Harrison, Weaver, & Wall, 2013; Mowforth & Munt, 2015; D. Scott & Gössling, 2015). For example, according to rough estimations, world terrestrial protected nature areas currently receive approximately 8 billion visits per year, of which 80% are in Europe and North America (Balmford et al., 2015). In general, nature-based tourism and recreation are affected by the following global drivers of change, i.e., megatrends: social trends (population growth, urbanization, changes in household composition, aging population, health and well-being, changing work patterns, gender equality, values and lifestyle); technological (transportation, high-tech equipment, information and communication technologies); economic trends 290 (economic growth; sharing economy; fuel costs); environmental (climate change; land use and landscape change); political (political turbulence; changes in border regulations; health risks; geopolitics) (Elmahdy et al., 2017). 3.3.5.2.3. Recreation The complexities of these drivers are discussed in Chapter 4 of this assessment. For the purposes of Chapter 3, it is important to point out that a combination of these interconnected global trends is and will be significantly affecting demand for nature-based tourist experiences and the way people engage with nature. There is concern that the aforementioned global trends contribute to increasing disconnectedness of large masses of populations from natural phenomena and processes in their daily life, which generates interest to experience nature as a leisure activity in an organized, often commercialized setting (Buckley, 2000; Buckley, Gretzel, Scott, Weaver, & Becken, 2015; Curtin, 2005; Dwyer, 2003; Elmahdy et al., 2017). It has been observed that nature-based tourism and recreation are increasingly characterized by the importance of experiences, achievement, adventure and well-planned activities rather than simple leisure and social interaction. Many studies indicate that tourism and recreation in nature are becoming more specialized, diversified, motorized, sportified and adventurized (Öhman, Öhman, & Sandell, 2016; Sandell, Arnegård, & Backman, 2011). In this context nature is transformed into a setting, a scenic backdrop for tourist experiences (Fossgard & Fredman, 2019; Margaryan, 2017). This also affects tourists’ expectations regarding the availability of tourism-related services in nature. There is a growing demand for ‘wild’, ‘unspoiled’, ‘pristine’ nature in combination with high levels of comfort, accessibility and high-quality experiences (Elmahdy et al., 2017; Fredman, Wall-Reinius, & Grundén, 2012). These pristine landscapes are advertised for tourism in brochures with backgrounds of teeming game, but absent of the human communities that live alongside wild species (Montgomery, Borona, Kasozi, Mudumba, & Ogada, 2020). This marketing perpetuates that indigenous peoples and local communities are separate from the social-ecological system, constitute a threat to wild species conservation, and drives the alienation and displacement of indigenous peoples and local communities, often with indigenous peoples and local communities on the boundaries of conservation areas receiving few benefits from tourism activities taking place (Montgomery et al., 2020; Saarinen, Moswete, Atlhopheng, & Hambira, 2020). Indigenous peoples and local communities also suffer from the negative aspects of tourism, for example disease and predation adjacent to protected areas (Swemmer, Mmethi, & Twine, 2017), or tourist-related disturbance of their activities (e.g., snow mobile recreation in the vicinity of Saami reindeer herders in Lapland (Kluwe & Krumpe, 2003), or rock climbers disturbing Native American rituals on Devils Tower/Mato Tipila in Wyoming (Taylor and Geffen 2004). 3.3.5.2.3. Recreation There may also be a conflict in values between recreational and non-recreational users, especially around expected behavior in sacred areas or around traditional hunting practices (Zeppel, 2010). Tourists can also cause degradation of culturally important sites through or illegal removal of cultural heritage items (INTOSAI WGEA, 2013). Tourism may change local identities and values, through commercialization of local culture and standardization to meet tourists' expectations (INTOSAI WGEA, 2013). As the demand for wild species-related experiences is on the rise, wild species-related content on social media and wild species documentaries have become more popular than ever. For example, the British Broadcasting Corporation (BBC) Planet Earth I and II have been among the most watched documentaries worldwide (Jackson, 2016). The growth of media 291 attention and circulation of wild species-related content in the social media further stimulates demand to experience wild species in real life, as well as photograph and share ‘selfies’ with wild species. Between 2014 and 2017 there has been a documented increase of nearly 300% in the quantity of wild species selfies shared on the Instagram platform (World Animal Protection, 2017). Of these, over 40% could be classified as inappropriate wild species selfies – featuring handling, hugging, touching, feeding or other potentially detrimental interactions between humans and wild species (World Animal Protection, 2017). Tourism marketing and social media sharing influences the demand for extremely close interactions with wild species (Dou & Day, 2020). However, research has shown the dichotomy of tourists’ desires for intimate encounters with wild species and recognition of the detrimental effects on animal welfare as a result of these interactions (Dou & Day, 2020). Environmental education and increased awareness of wildlife watching sustainability can and does play a role in changing tourist behavior, such as that demonstrated in dolphin-watching tours where tourists were willing to trade-off close interactions for the purposes of dolphin welfare (Dou & Day, 2020). Research on Mozambiquan tourists showed low awareness of cetacean-based regulations, but the tourists were supportive of well-regulated activities, therefore educated tourists could increase operator compliance with regulations (Rocha et al., 2020). In their review on wildlife watching sustainability, Dou and Day (2020) caution that environmental awareness does not occur automatically from increased exposure to wild species, but rather from focused environmental education with targeted, actionable messages on biodiversity conservation. 3.3.5.2.3. Recreation Wildlife watching has emerged as a widespread and lucrative tourist activity and its popularity is growing rapidly (de Lima & Green, 2017; Dybsand, 2020; Hassan & Sharma, 2017; Karanth et al., 2017; Mowforth & Munt, 2015; World Animal Protection, 2017). International tourism has grown year after year for the last decade, driven in part by nature- based tourism (including extractive tourism activities) (UNWTO, 2019). Between 1990 and 2000, average annual international tourism growth was 4.4%, but wild species-rich countries like Madagascar, Brazil, Cuba and South Africa all experienced averages between 10-20% annually and Vietnam and Laos between 24-36% (UNEP/CMS, 2006). Regional share of wildlife watching tourism relative to overall tourism varies globally, from 36.3% in Africa to 1.6% in Europe (WTTC, 2019a). Domestic tourism is estimated to be ten times the scale, but the numbers are uncertain. Similarly, the proportion of non-extractive nature-based tourism in recreation and leisure tourism is difficult to unpack as increasingly tourism trends have seen a blend in various types of tourism, such as family holidays that involve urban, adventure tours and wildlife watching (UNEP/CMS, 2006), or visits that combine trophy hunting and wildlife watching. But comparisons of protected area visitation rates mirror overall tourism rates in low-income countries (Balmford et al., 2009). A global study estimates that protected areas receive 8 billion visits per annum, generating 600 billion United States dollars (Balmford et al., 2015). Revenue generated from tourism in protected areas far exceeds the cost of managing these areas (Balmford et al., 2015; WTTC, 2019a). Surveyed governments and tour operators overwhelming rank nature, national parks and wild species as their largest assets for tourism, a practice that is labor intensive and employs local communities, especially in remote areas where developing regions do not have many other employment options (UNWTO, 2015). Nature-based tourism has been increasing 292 over the last decade as a result of increased demand (increased knowledge of wild species from media and the internet) and shrinking supply (reduced habitats and wild species scarcity) (The World Bank, 2018). This is apparent in visitation data for the iconic nature-based tourism destination, the Galapagos Islands, which has recorded an increasing trend in visitors from 1989 (<50,000 visitors) to 2019 (about 271,000 visitors) (Díaz-Sánchez & Obaco, 2020). Recreational use of wild species also generates significant revenue, particularly through nature-based tourism. 3.3.5.2.3. Recreation Wildlife watching contributed 120.1 billion United States dollars in 2018 (343.6 billion United States dollars with multiplier effects) to global gross domestic product, five times the estimated value of the illegal wild species trade (WTTC, 2019a). Wildlife watching also sustained 21.8 million jobs (WTTC, 2019a). The global monetary potential value of whale watching was estimated at over 2.5 billion United States dollars in 2009 and supporting 19,000 jobs (Cisneros-Montemayor et al., 2010). Shark and ray watching generated over 314 million United States dollars per annum, directly supporting 10,000 jobs and is expected to double by 2033, generating over 780 million United States dollars globally (Cisneros-Montemayor, Barnes-Mauthe, Al-Abdulrazzak, Navarro-Holm, & Sumaila, 2013). In contrast the value of shark fisheries was estimated at 630 million United States dollars and has been on the decline over the last decade (Cisneros-Montemayor et al., 2013). The expected revenue from entrance tickets to the Galapagos Islands in 2020 was about 18 million United States dollars, although significant losses have been predicted as a result of the COVID-19 pandemic (Díaz-Sánchez & Obaco, 2020). This revenue is mainly allocated to Galapagos Island conservation programs (Díaz-Sánchez & Obaco, 2020). International tourism arrivals in Africa, in large part for wild species tourism (including extractive recreational tourism), in 2013 were 56 million people, generating 34.2 billion United States dollars, and 134 million tourists are expected in 2030 (World Tourism Organization, 2014). During 2000 in East Africa alone, 1 billion United States dollars was generated from foreign tourist arrivals (UNEP/CMS, 2006). In the United States of America, wildlife watching engaged 86 million people in the vicinity of their homes, and 81.1 million people travelled away from home to view wild species, generating 75.9 billion United States dollars (United States of America Department of the Interior, United States of America Fish and Wildlife Service, United States of America Department of Commerce, & United States of America Census Bureau, 2018). Recreation represents over 75% of the value of the United States of America national forests, higher than the value of timber extracted (Groom et al., 2006). Although tourism revenue is significant at the national level, for economic benefits to alleviate poverty, the World Tourism Organization (UNWTO) found local level employment, infrastructure benefits, supply of goods and services and support by the tourism enterprises, as well as other pro-poor approaches were important (UNEP/CMS, 2006). 3.3.5.2.3. Recreation If local communities and suppliers are able to meet the standard needed to cater to international tourists, considerable benefits can accrue to the local economies (Twining-Ward, Li, Bhammar, & Wright, 2018; UNEP/CMS, 2006). However, if supplies and expertise are sourced on imports, then 50% or more of the tourism revenue “leaks” from the local and national economies (UNEP/CMS, 2006). Wild species which have the biggest importance for the tourism and recreation practices are those which attract interest from the widest spectrum of tourists, i.e., the ‘flagship’ species – most often the megafauna, ‘charismatic’ mammals and birds, the ‘cute and cuddly’, 293 dangerous predators, threatened species, and species that are believed to display intelligence (Aguilera-Alcalá, Morales-Reyes, Martín-López, Moleón, & Sánchez-Zapata, 2020; Carr & Broom, 2018; Devillers & Beudels-Jamar, 2008; Higginbottom, 2004; Newsome, Moore, & Dowling, 2012). The growing awareness of biodiversity loss has created a demand to see places and wild species that might disappear, including “endangered experiences”, highly exclusive tourism packages offering unique opportunities (WTTC, 2019b). For example, in Eurasia and Africa, national parks that hold large mammals have much higher visitation rates than those which do not (Devillers & Beudels-Jamar, 2008). Difference in preferences for wild species has its roots in a range of evolutionary as well as cultural predispositions (Jacobs, 2009). While some countries have a long history of wildlife watching tourism (e.g., in the East and South Africa), recent rapid growth of this business has been observed in many new destinations, for example in Southeast Asia and the Amazon (Karanth et al., 2017; World Animal Protection, 2017). Overall, natural areas of high value for wildlife watching tourism tend to be characterized by (i) abundance of large animals, (ii) presence of charismatic species, and (iii) high biodiversity (Higginbottom, 2004; Newsome et al., 2012). It is expected that presence of tourism in such areas will only be increasing, so special attention needs to be paid to aspects of sustainability in these processes. Wildlife watching activities and tourism accrue considerable funds for conservation projects, as well as raising public awareness of the need for conservation. A case in point is Projeto Tamar which, through working with local communities and fishers, successfully promoted turtle conservation along the Brazilian coastline, improving turtle hatching success through protecting hatchery sites and establishing alternative employment and income opportunities based on tourism and turtle protection (UNEP/CMS, 2006). 3.3.5.2.3. Recreation Similarly, a public- private partnership in a heavily poached region resulted in increased revenue for local communities and provided alternative revenue, to such a degree that wild species are again abundant in Majete Wildlife Reserve, Malawi (Twining-Ward et al., 2018). Conservation of one of the last remaining nesting sites of Little Penguins (Eudyptula minor) on Australia’s Phillip Island Nature Park, on Bunurong Aboriginal Land, is funded by an inclusive, collaborative business plan for tourism (UNEP/CMS, 2006). The business plan is revised every five years with the community and stakeholders and the Bunurong community representatives are involved in education programs and project supervision of a high-quality, high-volume tourist enterprise (UNEP/CMS, 2006).  Ecological aspects g p Wildlife watching can have unintended consequences for wild species in three ways: changes to species behavior, changes to physiology, or damage to habitats (UNEP/CMS, 2006). Behavioral changes to wild species include changes to feeding or resting time, expending energy to try and move away from the disturbance, altering interactions between different species (UNEP/CMS, 2006), aggressive behavior, increased stress, or alternatively a reduction in fear towards humans, and dependency on non-natural and supplemental food sources especially at feeding sites (Dou & Day, 2020), or preventing optimal spatial distribution relative to resources (Blanc, Guillemain, Mouronval, Desmonts, & Fritz, 2006). Short-term changes in animal behavior as a result of human-wild species interactions in tourism contexts are easier to detect and well-studied, but long-term changes are under researched (Dou 294 & Day, 2020). Similarly, tourism effects on wild species individuals are more detectable and better documented than the repercussions of these individual effects at the population level (Blanc et al., 2006) The evasive nature of wild species together with tourists’ expectations for a close contact with wild species creates a strong incentive for tourist destination managers to minimize sighting uncertainty and decrease the watching distance through invasive practices ranging from baiting, attracting, and habituating, to capturing animals (Dybsand, 2020; Knight, 2009; Margaryan & Wall-Reinius, 2017), and driving off-road (Nortje, 2014). Commercial wildlife watching activities rely on wild species being made viewable, which is often achieved through highly unsustainable and unethical practices (Dybsand, 2020; Knight, 2009; World Animal Protection, 2017). For example, high tourist volumes in the Serengeti have created serious disturbance for wild species and the large area of the park makes it challenging to enforce responsible game watching behavior (UNEP/CMS, 2006). In one case, the cubs of a cheetah were scared away by 15 vehicles and assumed to be predated on by lions as they were never seen again (UNEP/CMS, 2006). Snorkeling and diving may also disturb the aquatic habitat and influence species behavior (Teresa, Romero, Casatti, & Sabino, 2011). The practice of fish feeding during diving may affect fish communities (Ilarri, Souza, Medeiros, Grempel, & Rosa, 2008). A long-term, intensive study of the detrimental impacts on wild species from even well-managed, low level, commercial watching and controlled feeding of bottle-nosed dolphins at Monkey Mia, Western Australia documented long-term dolphin responses to human-wild species interaction.  Ecological aspects Over decades of monitoring, dolphin abundance (immigration and mortality) and fecundity declined at the tourism sites but not the control sites (Higham & Bejder, 2008). Highly responsive management interventions were implemented based on the research recommendations (Higham & Bejder, 2008) and impacts were reduced after regulations limiting the duration of feeding events (Foroughirad & Mann, 2013). However, findings of this nature are of great concern for the unknown long-term sustainability at other, especially high-intensity and/or low management tourism sites, for cetaceans and other wild species (Dou & Day, 2020; Higham & Bejder, 2008). A similar activity has been conducted in the Negro River, in the Brazilian Amazon, directed to feeding the freshwater pink (or red) dolphin (Inia geoffrensis), but the potential effects of this activity on dolphin’s behavior are not well known, but potentially increase dolphin aggression and may be harmful to both the dolphins and tourists (Pinto de Sá Alves, Andriolo, Orams, & de Freitas Azevedo, 2013). White sharks (Carcharodon carcharias) watching activities elicited curiosity and aggressive behaviors associated with feeding, leading the authors to advise against intentional feeding to avoid human-shark-cage associated incidents and the conditioning of sharks to boats (Becerril‐García, Hoyos‐Padilla, Micarelli, Galván‐Magaña, & Sperone, 2019). Even relatively innocuous recreational activities can have an impact on animal behavior. Research using camera traps to assess the prevalence of human recreational activities in association with terrestrial mammal occurrence in a Canadian protected area showed avoidance of mountain biking by moose (Alces spp.) and grizzly bears (Ursus arctos), although all recorded mammal species avoided humans on trails, especially mountain bikes and motorized vehicles (Naidoo & Burton, 2020). Even “silent activities” such as windsurfing may 295 have impacts as they enable off-path access to otherwise “sanctuary” areas (Blanc et al., 2006). But the presence of tourists and vehicles can be reduced through spatial or temporal zonation to provide sanctuary for wild species. The adverse impacts of high volumes of tourists and vehicles on wild species is managed in the Serengeti through strict park zonation, where certain areas are designated “No-Go” zones where no wildlife watching is allowed, “Intensive” and “Low Use” zones have designated tourism activities and “Wilderness” zones where no vehicles are allowed and low numbers of tourists do walking tours (UNEP/CMS, 2006).  Ecological aspects Habituation (stress response decreases with repeated exposure to humans) or sensitization (stress response increases with repeated exposure to humans) varies across and within species, and with the type of stressor, the type of tourism, spatiotemporal aspects, life history traits and intraspecific characteristics (Geffroy, Samia, Bessa, & Blumstein, 2015). For example, African penguins (Spheniscus demersus) and Magellanic penguins (Sphenicus magellanicus) habituate to humans but yellow-eyed penguins (Megadyptes antipodes) sensitize to humans (Geffroy et al., 2015). Thus, the impacts of repeated exposure to humans are extremely context-dependent and need to be assessed locally, as well as monitored over the long-term. This has important repercussions for wild species, as behavioral changes as a result of tourist-exposure may compromise their susceptibility to poaching or their risk of predation by other animals (Geffroy et al., 2015). y y Wild species’ physiology may be affected by tourism activities even though their behavioral patterns have not altered (Dou & Day, 2020). Yellow-eyed penguins (Megadyptes antipodes) at unregulated tourism sites showed significantly higher stress-induced corticosterone concentrations, with lower breeding success and lower fledgling weights than penguins visited for monitoring purposes only (Ellenberg, Setiawan, Cree, Houston, & Seddon, 2007). The presence of roads and traffic can also increase animal stress levels (Lunde, Bech, Fyumagwa, Jackson, & Røskaft, 2016). A well-studied intensive tourism site at the Grand Cayman Islands where stingrays (Hypanus americanus) are visited and fed by recreational scuba divers since 1986 have shown haematological changes, increased parasite loads, high injury rates and open wounds from boat collisions, and major behavioral changes from being normally solitary to forming schools of 12-15 individuals, as well as switching to diurnal feeding at the dive sites (UNEP/CMS, 2006). Most of the stingrays’ food now comes from divers and the reduced dietary diversity has compromised their disease resistance and immune response (UNEP/CMS, 2006). However, these kinds of examples of poor tolerance of tourist activities by species are species, habitat, tour operator and regulator specific. For example, a comparison between the effects of provisioning and viewing on the Cayman stingrays, which has been shown as detrimental, against the highly self-regulated and limited number of shark- feeding tour operators in Fiji suggests no effects on bull shark (Carcharhinus leucas) fitness and health (Healy, Hill, Barnett, & Chin, 2020).  Ecological aspects The trend in using wild species as photo props for “selfies” as photographic souvenirs has driven an increase in captive and handling of wild species, like slow lorises (Nycticebus spp.) in Asia, which have their teeth clipped to reduce the risk of injury to tourists, and results in early death (Osterberg & Nekaris, 2015). A study of three-toed sloths (Bradypus variegatus) in Brazil and Peru found each sloth was held by on average five tourists, often by the claws and had their limbs stretched and manipulated (Carder et al., 2018). Wild species handled for 296 long durations have been shown to display increased behavioral and physiological stress responses, leading to injury, stress and death (Baird et al., 2016). Tourists and other recreational users of nature, especially in high volumes, can damage the environment and species habitats. Trampling vegetation and the creation of informal trails both damages the environment and reduces the appeal and restorative impact on human health and well-being of these areas to other recreational users (Taff, Benfield, Miller, D’Antonio, & Schwartz, 2019). There is evidence that scuba diving, even without fish feeding, may cause unintentional damage to aquatic organisms, such as corals and algae, which may be hit by divers (Di Franco, Milazzo, Baiata, Tomasello, & Chemello, 2009). The sunscreen from divers and swimmers has been associated with bleaching of coral reefs (Danovaro et al., 2008; Downs et al., 2014) and Hawaii has banned the use of sunscreens containing oxybenzone or octinoxate from the 1st of January 2021 with similar bans predicted to follow in other coral-reef containing countries (Raffa, Pergolizzi Jr, Taylor Jr, Kitzen, & Group, 2019). Even a single vehicle driving on sandy beaches has been estimated to crush up to 0.75% of the intertidal population (Schlacher, Thompson, & Price, 2007) and beach camping zones show a 20.2% reduction in dune vegetation (Thompson & Schlacher, 2008). A review of winter recreational activities in Alpine areas found ski resorts and associated infrastructure have negative impacts across all studied taxa, independent of geographic region or ski modification (Sato, Wood, & Lindenmayer, 2013). This is concerning as the area affected in Europe by ski-runs is large and increasing, currently spanning about 4000 km across Italy, Switzerland and Austria, although there is a suggestion that environmentally-friendly ski-run design could mitigate many of these impacts (Rolando, Caprio, Rinaldi, & Ellena, 2006).  Considerations for sustainable recreational use  Considerations for sustainable recreational use Based on the current trends one can expect further growth in demand for wildlife watching experiences and, consequently, an increasing number of wild species integrated into tourism operations. Particularly vulnerable in this perspective are the megafauna and ‘charismatic’ wild species, which, however, also receive the most media attention and conservation support (Carr & Broom, 2018). Megafauna are the best studied taxa of animals, whereas there is a lack of research on the impacts of tourism on the lesser fauna, e.g., ground-dwelling mammals, small reptiles, insects, etc. (Wolf, Croft, & Green, 2019). The interlinkages between tourism, representations of wild species on social media, conservation and sustainability have acquired great importance and need further research and policy attention. Likewise, the role of environmental education in changing tourist attitudes and behavior requires further research attention (Dou & Day, 2020). Specific attention needs to be paid to the emergence of the so-called tourist-driven destinations, which appear spontaneously based on a spike in media popularity and uncontrolled tourist demand, rather than coordinated marketing efforts of the local tourism actors. In addition, the expansion of tourism into remote, ‘pristine’ areas needs to be managed and monitored to avoid detrimental impacts to sensitive and vulnerable species (UNEP/CMS, 2006). As tourists prefer areas that are deemed ‘pristine’ (i.e., more ecologically and aesthetically sound), there are opportunities to increase recreational tourism by restoring ecosystems. For example, work in RAMSAR (the Convention of wetlands of international importance) listed wetlands in India suggest that annual recreational visits could increase by 13% if the water quality could be improved to maintain wild species and fisheries diversity and abundance (Sinclair, Ghermandi, Moses, & Joseph, 2019). Researchers have also highlighted the need for studies that integrate the ecological and social aspects of human-wild species interactions to inform the sustainable development of the tourism industry, local communities and wild species conservation (Dou & Day, 2020). Financial resources and operational experience are sorely needed at the human-wild species interaction interface, with many wild species populations attractive to tourists in countries least able to afford the research, management and monitoring needed in these sites (Dou & Day, 2020). Finding ways to mobilize the power of new communication technologies and channel them towards sustainable tourism practices will be crucial in achieving more sustainable wildlife watching operations.  Ecological aspects Tourism facilities (e.g., lodges, ablutions) and impacts from waste, as well as high water usage are concerns in the nature-based tourism industry (UNEP/CMS, 2006). Despite initiatives to foster sustainable travel behaviors (e.g., carbon offsetting for unavoidable travel emissions) and attempts to improve the eco-efficiencies of tourism industries, tourism carbon emissions have increased at 3.3% annually (Sun, Lin, & Higham, 2020), driven by increased travel frequency, long-haul flights and shorter stays per trip (Sun et al., 2020). Altering resource availability to wild species to increase watching potential can have unintended consequences on the surrounding ecosystem. The provision of artificial water points in Kruger National Park, South Africa, although intended to maintain herbivore numbers during droughts expanded the range of water-dependent species (e.g., zebra and wildebeest), and in association their predators (e.g., lions) to the detriment of less common species (e.g., roan antelope) (Harrington et al., 1999). The widespread availability of surface water has also been implicated in the reduction of vegetation structure by homogenizing elephant impacts across the landscape (Gaylard, Owen-Smith, & Redfern, 2003). These unintended effects to facilitate watching wild species demonstrate the complexity of tourism impacts on populations and ecosystems. As these impacts are species and context specific, there is much to be discovered about the potential of tourism impacts. Even under the best code of conduct, there might still be detrimental, often cryptic, effects on animal reproduction and long-term survival (Carr & Broom, 2018; Szott, Pretorius, Ganswindt, & Koyama, 2020; Tyagi et al., 2019). A review of tourist impacts on wild species cautions that although the literature overwhelmingly reports on negative impacts, the findings are strongly 297 dependent on the methods used and many findings (especially behavioral responses) could be interpreted as short-term coping strategies that do not necessarily have long-term repercussions (Bateman & Fleming, 2017). Considerable variation exists between and within species and locations, in tourism operator methods and regulations. Therefore, more serious and coordinated global multi-stakeholder efforts to regulate this practice, involving tourism businesses, local communities, science, governmental and non-governmental organizations, are needed.  Considerations for sustainable recreational use Sustainable nature-based tourism needs to make a positive impact to the natural and social setting that tourism takes place in, and should generate benefit for the host communities and indigenous peoples and local communities in a manner that does not compromise the future human well-being needs of indigenous peoples and local communities or the ecosystems (UNEP/CMS, 2006). Well-managed wildlife watching is a significant boon to community 298 development and revenue, as well as an important source of funding for wild species conservation (UNEP/CMS, 2006). However, tourism is only sustainable where the habitats and species being used recreationally are sufficiently resilient to the impacts related to the use, where tourism and the associated development is kept within manageable limits, where the tourism experience attracts a long-term and viable tourism economy, and where local communities and the local economy benefit from the activity (UNEP/CMS, 2006). The direct benefits range from increased income and employment through education and access to many new facilities, up to perception of pride and recognition. Although the direct economic benefits are most important to local residents (Akis, Peristianis, & Warner, 1996), the indirect benefits such as improved public infrastructure, education, public safety and healthcare facilities may reach even wider groups of people (e.g., Afenyo & Amuquandoh, 2014) and gain the support for tourism among those who do not benefit directly from the activity. Addressing the above points to plan and manage sustainable nature-based tourism requires stakeholder engagement in a process that helps identify diverse interests, provides expertise, and facilitates local commitment to managing tourism ventures and impacts (UNEP/CMS, 2006). An exemplary case study of stakeholder engagement in wild species tourism is that of Bunaken national marine park, Indonesia. Bunaken national marine park pioneered a co- management approach that is being modelled by other protected areas (UNEP/CMS, 2006). Bunaken national marine park is a popular dive site for international tourists, as well as the home of 30,000 people whose livelihoods depend on fishing. Park management is overseen by a multistakeholder advisory board, including governmental, non-governmental organizations, representations of the villages within the park, park authorities, Tourism and Fisheries Departments, the local universities and the private tourism sector (UNEP/CMS, 2006). Local community elders advised on the location of the marine sanctuaries and no-take zones, the local community is involved in reef restoration efforts, and advised where to place marine sanctuaries which replenish both diving and fishing sites.  Considerations for sustainable recreational use Proceeds from park fees are managed by the multistakeholder board and are used for conservation and development programs, village development schemes, plastic and waste disposal, environmental education of villagers, rehabilitation and restoration projects, and law enforcement and patrols for destructive fishing and tourism practices (UNEP/CMS, 2006). Stakeholder needs have evolved as the social and environmental landscape has changed, and management has recognized the need to be adaptive in this regard. In Bunaken, the growing popularity for tourism is placing additional stress on the reefs and the large numbers of dive operators are not all members of the stakeholder association. They are considering a mandatory license system rather than voluntary compliance to manage the number of divers, dive operators and boats (UNEP/CMS, 2006). In another example, an unexpected repercussion of a successful public-private partnership in a heavily poached reserve has resulted in a tourism and revenue boom in Malawi’s Majete wildlife reserve, but the now abundant wild species are affecting local communities' resources, increasing human-wildlife conflict (Twining-Ward et al., 2018). Stakeholders differing needs and perspectives need to be negotiated, as power imbalances between stakeholders can undercut effective collective management actions (Meza-Arce et al., 2020). Recreational use may also be at odds with the extractive natural resource use needs of the local communities. This highlights the need to manage both physical and cultural conflicts between recreational users and indigenous peoples and local 299 communities, through temporal or spatial zoning as well as by addressing the disparate cultural and social values of the respective stakeholders sensitively (Zeppel, 2010). Significant opportunities exist for tourism revenue to support indigenous peoples and local communities that are already involved in conservation practices through local and traditional practices. The Entim e Naimina Enkiyio (Forest of the Lost Child) is one of few ungazetted forests in Kenya supporting abundant wild species, including threatened and highly endemic species (Tebtebba Foundation, 2010). This site is estimated to have tourism potential of up to 40,000 United States dollars per annum, notwithstanding the other benefits supporting conservation would ensure, such as catchment protection, wild algae, fungi and plants, grazing, and spiritual and cultural value (Tebtebba Foundation, 2010). The communities conserving biodiversity, as well as managing the natural resources for their subsistence, should be supported and strengthened where appropriate. However, the benefits of tourism should not be overstated and require careful consideration of what is realistic (UNEP/CMS, 2006).  Considerations for sustainable recreational use For example, in a survey of World Bank Global Environnement Facility projects, most projects had positioned tourism as key to sustainable resource management and wild species conservation, but only 8% of the projects analyzed the tourism-derived income potential (UNEP/CMS, 2006). A key finding of this World Bank survey was that although tourism did generate revenue, it could not be solely relied on and was not even the most important source of funding (UNEP/CMS, 2006). An economic model of the impacts of increased tourism revenue in the Philippines demonstrated that although economic benefits are accrued to all local households in the short- term, over the long-term increased demand for natural resources driven by the tourism industry eroded local household incomes, particularly for households directly involved in the natural resources economy (Gilliland, Sanchirico, & Taylor, 2020). Similarly, tourism in Latin America was associated with increased agricultural expansion and deforestation to service tourist consumption (Gunter & Ceddia, 2020). Providing indigenous peoples and local communities with title deeds and land rights seemed to mitigate this effect, although the research authors caution this effect should not be overstated (Gunter & Ceddia, 2020). The Monarch Butterfly Forest Project is often lauded as a win-win-win success for tourism-livelihoods-conservation. Established in Mexico at forest locations where monarch butterflies (Danaus plexippus) congregate in winter, the project promoted recreation centers, established butterfly visitor centers and implemented tourism management at butterfly sanctuaries (UNEP/CMS, 2006). The project focuses on livelihood solutions for a region characterized by high unemployment, and has provided tourism training for local people, is involved in reforestation of areas critical to the butterfly habitat, and spans to managing logging impacts in Canada and the United States of America which threaten monarch summer habitat (UNEP/CMS, 2006). Without detracting from the immense strides the Monarch Butterfly Project has made in livelihoods and conservation, in some areas there is evidence of local residents returning to extractive activities as the project failed to yield the expected employment opportunities (Barkin, 2003). Although the rate of logging within the core areas of the Monarch Butterfly Biosphere Reserve have declined, logging is still present (Flores- Martínez et al., 2019; Vidal, López‐García, & Rendón‐Salinas, 2014), particularly small-scale logging (López-García & Navarro-Cerrillo, 2020).  Considerations for sustainable recreational use The Monarch Biosphere Reserve zonation policies restricting community use of natural resources and the subsequent compensation for 300 lost legal logging permits through payment for ecosystem services has had unintended consequences through provoking social conflict, often armed, in some areas (Gonzalez-Duarte, 2021). Indeed, the local communities who were ancestral inhabitants of what is now core biosphere areas do not share the biosphere reserve paradigm of a binary use/non-use landscape, but instead view the relationship with the forest ecosystem as a continuum of co-inhabitation and Gonzalez-Duarte (2021) suggests the enforced split in ancestral ecological practices has supported a fractured social compact, fostered illicit extractive activities, undermined community forest management, encouraged organized crime and has created a disregard for core areas where monarch butterflies do not overwinter. For an overview of the challenges facing Mexico’s biosphere reserves, see Sada (2019)). These challenges are by no means unique to the Monarch Butterfly Biosphere Reserve and occur in many of the global biospheres reserves where integration of core conservation areas is not adequately incorporated into the multi-use, social-ecological system that the core reserves and local communities exist within (Coetzer, Witkowski, & Erasmus, 2014). Often local livelihoods are believed to be in conflicting relation with conservation and therefore they are highly restricted by the rules and regulation that impede local economic development (Stone, 2015; West, Igoe, & Brockington, 2006). Cases of prohibition of traditional activities that involve unsustainable use of natural resources in favor of conservation were reported in Tanzania (Charnley, 2005), Bangladesh (Islam, Rahman, Iftekhar, & Rakkibu, 2013), Botswana (Sebele, 2010), and Nicaragua (de los Angeles Somarriba-Chang & Gunnarsdotter, 2012). The high dependence on natural resource for self-subsistence (Belsky, 2009; Moswete, Thapa, & Lacey, 2009; Prachvuthy, 2006; Rozemeijer, 2000; Wunder, 1999) often give communities no choice but to engage in illegal activities. For example, in a case study in Central Amazonian Rainforest, Brazil, some of the families were reported to risk starving because fishing became very difficult and the large-scale agriculture was prohibited in the conservation area (Lima & d’Hauteserre, 2011). It should be highlighted that nature-based tourism as a complementary activity that substitutes completely, or partially, unsustainable use of natural resources requires a fundamental re-organization of a community’s economic and social structure, which might trigger ideological opposition of those communities that have been relying on those activities for generations (Schweinsberg, Darcy, & Wearing, 2018).  Considerations for sustainable recreational use Local communities who participate in nature-based tourism and receive tangible benefits tend to become cautious in their use of natural resources and, therefore, more likely to support tourism and conservation (Lindberg, 2001). However, the employment in tourism must be high enough in terms of demand to maintain the workforce, and the financial benefits must be higher than gains from unsustainable activities (Kiss, 2004; Mbaiwa & Stronza, 2010). In destinations where community-based tourism is already in place, but it does not provide enough employment, the unsustainable use of resources is a common practice. The limited economic opportunities reduce or disable any incentives for conservation (Simmons, 1994). Immediately after the incentives for tourism development or benefits from it decrease, local residents go back to previous livelihood-supporting extractive activities (Wilkinson & Pratiwi, 1995). Direct employment is one of the most common limitations of many community- based tourism initiatives as often a small-scale project is not able to employ many people and still remain profitable. A study by Zapata et al. (2011) on 34 community-based tourism projects 301 in Nicaragua reported that they were able to generate an average of 6.8 permanent jobs and 12.2 part-time positions. However, it should be stressed that what is considered low or high employment is highly situational as it depends on the size of the community and their direct needs. When resource consumption is prohibited within the protected area, the high dependence on resource extractive activities may also have adverse effect on resources surrounding the area, as demands intensify due to a shrinking resource base (Durbin & Ralambo, 1994; Parry & Campbell, 1992). This might also have a negative effect on tourism itself that is based on supply of pristine landscape, biodiversity of animal and plant species. For example, in Wolong Nature Reserve, China, activities such as logging and clearing for fuelwood, agriculture, gathering of herbal medicinal plants, and ranching have significantly degraded and fragmented giant panda habitat that is the main tourism attraction offered by the local community-based tourism initiative (He et al., 2008). Nature-based tourism, and the associated reliance on tourism-derived funds for conservation, is also sensitive to economic shocks. For example, as a result of the COVID-19 pandemic, the predicted loss in park entrance fees to the Galapagos Islands is expected to cost between 35-55% of total revenue, monies mainly allocated for conservation (Díaz-Sánchez & Obaco, 2020).  Considerations for sustainable recreational use Continued conservation in the Galapagos will require alternate sources of funding or loans (Díaz-Sánchez & Obaco, 2020). The potential for detrimental effects of human-wild species interactions needs to be closely managed, requiring local community empowerment, supportive and cooperation from tour operators and enterprises, and buy-in from tourists (Dou & Day, 2020). The management of the recreational use of wild species needs complementary enforcement and voluntary compliance measures, especially in the tourism context, managing human-wild species interactions is in effect managing people (Dou & Day, 2020). Instilling and supporting a sense of pride and custodianship of wild species amongst tour operators can facilitate responsible tourism. In summary, for sustainable recreational use of wild species there needs to be: In summary, for sustainable recreational use of wild species there needs to be: 1. low impact on the wild species being used 2. long-term monitoring of wild species populations and habitats 3. long-term improvement in the livelihoods of local communities 4. awareness and support for conservation from all stakeholders 5. adaptive management and limits on “acceptable change” for wild species tourism, conservation and local communities, including the ability to limit further development 6. supportive regulatory frameworks from local and national government (UNEP/CMS, 2006) As every tourism initiative is different, there is no single set of suitable conditions that enable both conservation and local livelihoods to flourish (Beeton, 2008; Faulkner & Tideswell, 1997; Okazaki, 2008; Reimer & Walter, 2013). However, a number of factors emerged from a global analysis of over 100 community-based tourism case studies in natural areas (Yanes, Zielinski, Diaz Cano, & Kim, 2019; Zielinski, Kim, Botero, & Yanes, 2020). Aspects that are critical for a success are: Aspects that are critical for a success are: 1. the availability of financial resources 2. skills and technical expertise 302 3. political influence 3. political influence 4. local control over land and resources 5. community cohesion 6. involvement in local planning and management The external support provided by non-governmental organizations and governmental organization is crucial for ensuring the abovementioned conditions (Beeton, 2008; Okazaki, 2008)(Beeton, 2006; Okazaki, 2008). The external factors enabling community-based tourism are the political will and decentralization of power and control to the community. The main barriers for successful community-based tourism development are: The main barriers for successful community-based tourism development are 1. the lack of skills and expertise in areas required for tourism 2.  Considerations for sustainable recreational use lack of noticeable improvement of quality of life in the community (health, education, economy) 3. lack of independence in the decision-making process 4. lack of participative decision making 5. lack of community control over land and resources 6. low level of control over tourism activities in the area 7. internal conflict within community 8. high dependence on resource consumptive activities 9. lack of significant employment in tourism, among others. 3.3.5.2.4. Education and learning 3.3.5.2.4. Education and learning This section deals with the non-extractive practices of wild species for the production of knowledge (Chapter 1). The scope of this section is limited to the non-extractive practices of wild species for learning and education where the impact of this use has a measurable effect on the species. The text below is based on a literature review (Google Scholar) using the following keywords: wildlife, nature, environmental, education, and learning generating 119 000 hits. Articles that were recommended by citation databases were also considered, as well as collected from personal libraries and recommendations from experts. After a title and abstract read, 18 papers were selected for a full text read. After a full text read of these papers, 12 were deemed relevant and assessed for the literature review. Relevance was determined by mention of either the status (current), trend (historical) or impacts of use of a wild species (or taxa) in at least one dimension of sustainability (social, economic, or ecological) (see the data management report for Chapter 3 systematic literature review at https://doi.org/10.5281/zenodo.6452651). These data form the basis of the text below. Although the use of wild species and ecosystems for scientific research and environmental education, amongst other purposes, is certainly widespread, there is no indication whether this has increased over time or on the current status of use. Relevant articles represented all IPBES regions and most ecosystem types. The literature mostly addressed the use of ‘nature’ for education and learning, rather than a species/taxa specific approach, but where taxa were mentioned, they were either mammals (terrestrial and marine) or birds. There was little to no information in the literature about the sustainability or the effects of use on wild species or ecosystems. The exception was one article which mentioned concern over the routine use of outdoor teaching sites and their management plan to rotate use of environmentally 303 sensitive areas as needed (Ernst & Stanek, 2006). Although undocumented, the non-extractive practices of wild species are likely to experience similar impacts to recreational watching of wild species such as stress-related responses from wild species and habitat damage through trampling (see section 3.3.5.2.3. Ecological aspects of recreational use). There are two main methods of using wild species for learning and education. 3.3.5.2.4. Education and learning The first is via scientific research and the second through environmental education, mostly to school children and tourists, although a significant amount of informal, experiential learning and knowledge transfer occurs through other practices and uses of wild species, such as birdwatching (recreational use of wild species) (Zvonar & Weidensaul, 2015) and urban foraging (gathering) ( Poe, LeCompte, McLain, & Hurley, 2014). Scientific use of wild species is generated through measuring faunal and floral diversity, and population structure and ecological processes. A review of “intellectual ecosystem services” generated by South African National Parks showed a bias towards research on animals, particularly mammals (Smit, Roux, Swemmer, Boshoff, & Novellie, 2017). Similarly, the journals that published research from protected areas were mostly mammal dominated, with little to no focus on social science, environmental governance or social-ecological studies (Smit et al., 2017). Wild species use in education in Europe was dominated by threatened and charismatic species, such as wolves (Canis lupus), brown bears (Ursus arctos) and Imperial Eagles (Aquila adalberti) (Aguilera- Alcalá et al., 2020). These cases highlight the paucity of research conducted on less “popular” taxa, such as fungi and invertebrates, forbs and shrubs. Notwithstanding, the public’s interest in charismatic species has been harnessed effectively for scientific research, such as in the analysis of data such as camera traps (e.g., https://www.zooniverse.org/) or in data collection such as atlas projects (e.g., http://sabap2.birdmap.africa/). These citizen science projects both solve significant big-data processing and collecting challenges facing scientists, as well as providing enjoyment and ecological education to interested citizens. The second major use of wild species for education and learning is environmental education. Here environmental education is defined as a process that allows individuals to explore environmental issues, engage in problem solving, and take action to improve the environment. As a result, individuals develop a deeper understanding of environmental issues and have the skills to make informed and responsible decisions (EPA 2018: https://www.epa.gov/education/what-environmental-education Accessed on 9 January 2021). Most of this literature focuses on education and on nature rather than wild species per se. Most children today have more access to environmental knowledge through nature documentaries and films than all previous generations (Hudson, 2001). Ironically, such media- educated children in developed countries may fervently campaign for saving polar bears, cheetahs and whales, while they have almost no contact with wild animals or plants common in their own country (Hudson, 2001). 3.3.5.2.4. Education and learning There was consensus in the research on environmental education, especially for school children, that educational programs that use the environment for learning supported improved attitudes toward the environment and a desire to look after the environment. An education program specifically on wild Bornean orangutans (Pongo pygmaeus wurmbii) led to 13.6-40.4% increase in student knowledge and more positive attitudes towards conservation (Freund et al., 2020). In a study on primary and secondary school children in an environmental education program, 41% of students indicated their feelings about the environment had changed as a result of the nature-based excursion through 304 a combination of observation and instruction (R. Ballantyne & Packer, 2002). Responses include: “I had a better understanding of the impact of people on the forests.” (15-year-old) and “Don’t feed the native wildlife.” (15-year-old). The benefits of using wild species for learning and education are considerable. In terms of ecological benefits, scientific research on wild species is applied by wild species managers to improve sustainable conservation (Smit et al., 2017). Learning in (and from) nature engenders pro-nature behaviors (Richardson et al., 2016) such as fostering a sense of responsibility and stewardship, and changing attitudes and behavior via increased ecological knowledge (Kwan, Cheung, Law, Cheung, & Shin, 2017). This knowledge can ripple outwards from the primary recipients and be transmitted to parents and neighbors (Vaughan, Gack, Solorazano, & Ray, 2003). Educational courses and formal training on wild species and nature can build constituencies with neighboring communities, indigenous peoples and local communities and other stakeholders, as well as capacity building for future wild species research and management (Smit et al., 2017). Imparting environmental knowledge to tourists and students also provides employment, especially important when this is in local communities involved in these practices (Ternes, Gerhardinger, & Schiavetti, 2016; UNEP/CMS, 2006). The aspects of engaging with wild species that contribute significantly to conservation education in the public include: watching wild species in their natural habitat, opportunities for close encounters with wild species, opportunities to observe natural wild species behavior, engaging the public emotionally, connection with the public’s prior knowledge and experiences, convincing communication, and establishing a link between everyday actions and changes to these actions people can make to foster conservation outcomes, and providing incentives and activities to support behavior change (Ballantyne, Packer, Hughes, & Dierking, 2007). 3.3.5.2.4. Education and learning Beyond generating knowledge and awareness, there is concern on whether knowledge translates into action, and the longevity of pro-environmental awareness and behavior changes. In terms of longevity of pro-environmental awareness and attitudes, there is limited longitudinal research on this aspect. One study on the influence of a six-week bird feeding and monitoring program on school grounds showed that a year later, several schools had continued the program themselves, suggesting that such interventions have the potential to be maintained in the longer term (White, Eberstein, & Scott, 2018). Another example illustrates the benefits of a close engagement with a wild species over the longer term. Here secondary school students reared captive-born juvenile threatened Asian horseshoe crabs (Tachypleus tridentatus) for 14 months, which were then released into the wild (Kwan et al., 2017). Rearing involved training students to collect data, test water conditions, and provided opportunities to improve on the protocols through experimentation (Kwan et al., 2017). The students were also tasked with presentations on the importance of horseshoe crabs and after the horseshoe crabs were released, tagged individuals could be tracked by students to monitor their movements and growth (Kwan et al., 2017). The extended period of rearing and engagement with the horseshoe crabs engendered a strong emotional attachment and fostered a sense of responsibility, resulting in more pro-environmental attitudes and behavior (Kwan et al., 2017). Both Ernst and Stank (2006) and Freund et al., (2020) highlight self-efficacy as crucial to pro-environmental behavior. Self-efficacy engenders the belief that one can personally make a difference and empowerment is key to translating environmental education into pro- 305 environmental action. This is related to Hudson (2001) cautioning that environmental educators should avoid the “psychology of despair.” The overwhelming documenting of declines in the health of the natural world and species populations can create a sense of hopelessness for the future and negate the belief that an individual can make a difference. A drawback of environmental education is the limited reach of the programs. Although some ripple effect in increased awareness in the community (Vaughan et al., 2003), in communities reliant on natural resources and living in vulnerable ecosystems, it is the children who do not attend school who are more likely to be involved in illegal and unsustainable wild species activities in the future (Breuer & Mavinga, 2010). Furthermore, education alone cannot be solely responsible for changes in behavior. 3.3.5.2.4. Education and learning Environmental education programs need to be complemented by projects that alleviate poverty and develop alternative livelihood opportunities (Breuer & Mavinga, 2010). Conservationists and local governments should also provide information on the importance of ecological functions of wild species that cause problems in human-wildlife conflict, whilst mitigating the drawbacks of close contact with ‘problematic’ species (Hosaka, Sugimoto, & Numata, 2017). 3.4.1. Introduction Chapter 3 focuses on the status and trends of the use of wild species through its three interacting systems: the wild species themselves, the human practices by which they are obtained from nature, and the uses for which they are intended. Because it is impossible to include all wild species in the assessment, we have focused on those species which are more intensively utilized, those whose sustainable use is of particular concern, and those whose use exemplifies sustainable use in meaningful ways which are informative for overall consideration of the sustainable use of wild species discourse. Throughout the chapter we have followed the practices and uses typology outlined in chapter 1, with adaptations in each section in accordance to the standards in the various literatures and sectors reviewed. However, these use categories (and sometimes practice categories) are not exclusive. In this section we make an effort to consider the interactions among the uses and practices. While the specific practices and uses of particular wild species have been studied in greater detail, the interactions and influences among species and the related consequences for sustainable use of wild species has been much less examined. These interactions between, within and among wild species-related practices and uses, and their cross-influences relate to the notion of trade-offs and synergies. To avoid developing a compartmentalized and regimented understanding of sustainable use of wild species, the attempt in this section is to use the notions of trade-offs and synergies as analytical perspectives to understand how the practices and uses of wild species are connected in multiple ways, how they interact with each other and, in the process, how they engage with and cross-influence each other both negatively and positively. According to the IPBES Glossary (IPBES core glossary, 2021), a trade-off is a situation where an improvement in the status of one aspect of the environment or of human well-being is necessarily associated with a decline in or loss of a different aspect. Trade-offs characterize most complex systems and are important to consider when making decisions that aim to improve environmental and/or socio-economic outcomes. Synergies arise when the enhancement of one desirable outcome leads to enhancement of another. Trade-offs are distinct from synergies as the latter are also referred to as “win-win” scenarios. 3.3.5.3. Emerging issues Tourism is one of the practices most affected by the COVID-19 pandemic (Spenceley et al., 2021; UNCTAD, 2021). As a result, the COVID-19 pandemic has exposed the vulnerability of nature-based revenue streams to global economic shocks, and the reliance of communities and conservation funds on international tourism (Peter Lindsey et al., 2020; Rondeau, Perry, & Grimard, 2020; Spenceley et al., 2021). Loss of conservation funds has been severe as a result of decreased tourism revenue. For example, the predicted loss in park entrance fees to the Galapagos Islands is expected to cost between 35-55% of total revenue, monies mainly allocated for conservation (Díaz-Sánchez & Obaco, 2020). Continued conservation may require alternate sources of funding or loans (Díaz-Sánchez & Obaco, 2020; McCleery, Fletcher, Kruger, Govender, & Ferreira, 2020). Early evidence from the COVID-19 pandemic impacts suggests that communities reliant on nature-based tourism turned to extractive activities to meet their local livelihood needs (Spenceley et al., 2021), compounded by the return of migrant workers to rural areas and the associated increase in demand of local resources (Rondeau et al., 2020). There are preliminary indications of increase poaching and a surge in illegal logging during the pandemic, possibly as a result of decreased conservation authority presence and no wildlife watching tourists (Rondeau et al., 2020; Spenceley et al., 2021). In addition to the lack of tourism revenue, it is unknown what the impacts of COVID- 19 transmission from tourists on wild species will be (A. Gibbons, 2020) or from tourists to local communities (Hakim, 2020). But these early findings still need to be corroborated with more data as the effects of the pandemic on wild species, conservation funds, and local livelihoods becomes more understood. Another emerging issue is the non-extractive use of wild species through novel finance mechanisms, such as Rhino Impact Bonds (www.rhinoimpact.com), Lion Carbon (www.lionlandscapes.org/lioncarbon), The Lion’s Share Fund (www.thelionssharefund.com), or the Luc Hoffmann Institute’s Innovation Challenge “Beyond Tourism in Africa” (https://luchoffmanninstitute.org/beyond-tourism-in-africa-innovation-challenge) which seek 306 to support wild species conservation and sustainable livelihoods in the absence of recreational hunting or wildlife tourism. However, there is currently insufficient information on the use, trends or impacts of these finance mechanisms on wild species or wildlife economies. to support wild species conservation and sustainable livelihoods in the absence of recreational hunting or wildlife tourism. 3.3.5.3. Emerging issues However, there is currently insufficient information on the use, trends or impacts of these finance mechanisms on wild species or wildlife economies. 3.4.2. Conceptualizing trade-offs and synergies Based on the ecosystem services literature, a two-fold understanding of trade-offs and synergy is proposed: First, trade-offs or synergies only occur if the considered practice and use interact with each other (Bennett et al., 2009; García-Llorente et al., 2015). Second, trade-offs and synergies require assessment of supply, demand and use together and not separately (Geijzendorffer, Martín-López, & Roche, 2015). Following Turkelboom et al. (2016) a trade- off is a situation where one use or practice directly decreases the benefits supplied by another. A synergy is a situation where one use or practice directly increases the benefits supplied by another use or practice. Both synergies and trade-offs have spatial and temporal dimensions (see section 3.2). Trade-offs may depict an array of phenomena including conflicts, contestations, negative correlations, incompatibilities, rivalry and excludability in relation to sustainable use. The inverse of these phenomena signifies synergy. Both trade-offs and synergy are closely associated with benefits and well-being components, value dimensions, and management strategies (Iniesta-Arandia, García-Llorente, Aguilera, Montes, & Martín-López, 2014; Martín-López, Gómez-Baggethun, García-Llorente, & Montes, 2014; McShane et al., 2011). Trade-offs and synergies reflect a host of interactions, connections, relationships and linkages within, between and among practices and uses. If so, achieving the goal of sustainable use of wild species depends on the level of understanding of the key trade-offs and possible areas of synergy within and across practice areas. 3.4.1. Introduction While it is important to aim for a “win-win” synergy, this cannot be done without appropriate responses to the “win-lose” situations presented by existing and potential trade- offs between and among the practices and uses of wild species. Biophysical, economic and social factors all make it unlikely that multiple needs will be met simultaneously without deliberate efforts; so while there is still much interest in developing win-win outcomes there is little understanding of what is required for them to be achieved (Howe, Suich, Vira, & Mace, 2014; Tallis, Kareiva, Marvier, & Chang, 2008). 307 While win-wins may be attractive, they are not inevitable and may be unlikely in practice in the absence of carefully designed interventions (Bennett, Peterson, & Gordon, 2009). Howe et al., (2014, p. 263) suggest that “taking account of why trade-offs occur is more likely to create win-win situations than planning for a win-win from the outset. Consequently, taking a trade-off as opposed to a win-win approach, by having an awareness of and accounting for factors that predict a trade-off and the reasons why trade-offs are often the outcome, it may be possible to create the synergies we seek to achieve.”. Without attention to trade-offs, one is left with the notion that sustainability of wild species hinges separately on the individual practices and/or uses, which is both ecologically and socially unrealistic. 3.4.3.1. Trade-offs and synergies at intra-practice and intra-use level The lack or presence of a range of scientific and indigenous and local knowledge-based methods and their effective combinations for assessing the sustainability of wild species are linked to possible trade-offs and synergies. A diverse range of methods to analyze the status and trends of sustainable use of wild species under each practice category has been discussed in section 3.3. They include both scientific methods (e.g., stock assessment, biomass estimation) and the use of a variety of indigenous and local knowledge. However, there is a predominance of scientific methods for assessment of wild species even though use of indigenous and local knowledge is quite widespread. In fishing practices scientific assessments are publicly available for roughly half of the global fish catch while there is considerable effort to better understand the status of the remaining half of the stocks. This shows how science and technology are focused on only portions of wild species and not all that are important for human use. This may trigger undesirable trade-offs between assessed and non-assessed species. Addressing this may be tricky but not impossible. For example, the state of world fisheries and aquaculture by the FAO makes scientific assessment of status of 500 fish stocks worldwide, while the remaining almost half of the world’s stocks are covered through the expertise provided by expert knowledge to fill in the gap (Melnychuk et al., 2017). In some cases, this might mean that small stocks, especially the unassessed ones, are in a disadvantageous position of below target levels compared to large stocks which are often covered under scientific assessment (Costello et al., 2012). In order to ensure that partial nature of scientific information does not lead to ineffective decisions it may be combined with other types of knowledge, including indigenous knowledge, and use pluralistic and interdisciplinary forms of assessment of trade-offs. Further discussion on this appears in Chapter 5 and other sections of this assessment focusing on indigenous and local knowledge. Trade-offs between multiple uses under a specific practice may reallocate science, technology, investment and innovations in favor of new or emerging uses over the already established and traditional uses of wild species. This may dramatically alter any existing synergies between use categories and significantly impact the sustainability trajectories associated with individual use types of wild species. Section 3.3. 3.4.3. A framework to analyze trade-offs and synergies in the sustainable use of wild species The main purpose behind exploring trade-offs and synergies is to understand their implication for sustainable use of wild species, key trends and status. It is evident from section 3.3 that the assessment considered a large number of wild species, five broad categories of practices and sub-practices, and more than nine different types of uses. A simple three-pronged approach is used to consider the various trade-offs and synergies across these practices and uses of wild species by focusing on (i) trade-offs and synergies at intra-practice and intra-use level; (ii) trade-offs and synergies between practices and uses; and (iii) trade-offs and synergies involving the social, economic and environmental aspects of sustainable use. 308 3.4.3.1. Trade-offs and synergies at intra-practice and intra-use level 3.4.3.1. Trade-offs and synergies at intra-practice and intra-use level Small-scale fisheries communities have remained economically and politically marginalized, are highly vulnerable to change (including climate change), and until recently, remained largely invisible in policy debates in most countries and internationally (Berkes, 2015; FAO, 2015). These factors, together with increasing vulnerability due to climate, environmental, economic and policy drivers have created a global crisis in small-scale fisheries (Muzuka, et al., 2011; Paukert et al., 2017; Satumanatpan & Pollnac, 2017). (ii) Small-scale and large-scale / industrial fisheries: Data in Figure 3.65 below clearly shows that the annual catch in small-scale fisheries is higher than the large-scale fisheries both in the marine and inland fisheries practices. In the inland fisheries, small-scale fisheries have 14 times more catch than inland large-scale fisheries. Despite this lead in catch size and the significant contributions small-scale fisheries make to nutrition and food security, poverty alleviation and livelihoods, and local and national economies, especially in developing countries (Béné, Macfadyen, & Allison, 2007; Berkes, 2015; Lilian Ibengwe & Fatma Sobo, 2016), the policy attention this practice has received remains marginal. Small-scale fisheries communities have remained economically and politically marginalized, are highly vulnerable to change (including climate change), and until recently, remained largely invisible in policy debates in most countries and internationally (Berkes, 2015; FAO, 2015). These factors, together with increasing vulnerability due to climate, environmental, economic and policy drivers have created a global crisis in small-scale fisheries (Muzuka, et al., 2011; Paukert et al., 2017; Satumanatpan & Pollnac, 2017). In contrast, the large-scale fisheries practice has received significant policy attention across the national and international boundaries. A major example of this attention pertains to the extent of global subsidies to the tune of 35 billion United States dollars to the large-scale fisheries practice (Sumaila et al., 2019; Sumaila, Lam, Le Manach, Swartz, & Pauly, 2016). These discrepancies between the small- and large-scale fisheries signify intense levels of trade- offs between the two use types. Possible synergies can be built between these two practices within fishing practices if the small-scale fisheries can be recognized as a use type of wild species that is simply ‘Too Big To Ignore’ (Chuenpagdee, 2019; Chuenpagdee et al., 2019). Afterall, small-scale fisheries support over 90 percent of the 120 million people engaged in capture fisheries globally, about half of them are women, and it contributes approximately 45% of the global fish catch destined for direct human consumption (World Bank, 2012). 3.4.3.1. Trade-offs and synergies at intra-practice and intra-use level Even though it is not included in the scope of this assessment, reference to aquaculture is imperative because of the significant trade-offs it has with capture fisheries. (i) Capture fishery and aquaculture: the FAO estimates the total volume of capture fisheries as about 90 million metric tons which constitutes the largest wild food consumed by humans as well as one of the most established / traditional uses under fishing as a practice. Aquaculture as a new use category has gained momentum since the mid-1980’s but already in significant competition with capture fisheries. Figure 3.31 provides estimates of the global fish production as about 170 million metric tons with close to 50 percent coming from aquaculture. This is a consistently increasing trend in the last three decades whereby aquaculture is all set to takeover capture fisheries in the next decade or so. Not only that but aquaculture is reportedly encroaching into the dominant use of capture fisheries for the purpose of food for humans, i.e., out of the 90 million metric tons of fish obtained from capture sources over recent decades, about 60 million metric tons goes to direct human consumption and most of the rest is diverted as feed for aquaculture and livestock. Such trends might threaten to further marginalize the capture fisheries practice which is already experiencing a sharp decline in its biologically sustainable levels from 90 percent in 1974 to 65.8 percent in 2015 and stands at more than 34.2% stocks being overfished (FAO, 2020d). Even though it is not included in the scope of this assessment, reference to aquaculture is imperative because of the significant trade-offs it has with capture fisheries. (ii) Small-scale and large-scale / industrial fisheries: Data in Figure 3.65 below clearly shows that the annual catch in small-scale fisheries is higher than the large-scale fisheries both in the marine and inland fisheries practices. In the inland fisheries, small-scale fisheries have 14 times more catch than inland large-scale fisheries. Despite this lead in catch size and the significant contributions small-scale fisheries make to nutrition and food security, poverty alleviation and livelihoods, and local and national economies, especially in developing countries (Béné, Macfadyen, & Allison, 2007; Berkes, 2015; Lilian Ibengwe & Fatma Sobo, 2016), the policy attention this practice has received remains marginal. 3.4.3.1. Trade-offs and synergies at intra-practice and intra-use level offers adequate understanding that while some uses under a practice type are well-established and traditionally recognized, others may be new or emerging in nature. Despite the potential for synergy between these multiple use categories there seem to be inherent competition and overlapping contestations amongst them, ultimately affecting the levels of their sustainable use. In the process of competing with one another, some of the uses have become more prominent than others and thereby known to drive science, technology, investment and innovations away from existing use areas to the new uses that have the potential to negatively affect sustainable use of wild species as a whole. Several examples of these intense trade-offs ensue from the practices outlined in section 3.3. Fishing offers examples of two sets of mostly conflicting rather than complementary use categories that seem to pose significant challenges to the question of sustainability around this practice: (i) the overlapping interactions between capture fishery and aquaculture; (ii) the tussle between the invisibility of small-scale fisheries and the high visibility of large-scale / industrial fisheries. 309 (i) Capture fishery and aquaculture: the FAO estimates the total volume of capture fisheries as about 90 million metric tons which constitutes the largest wild food consumed by humans as well as one of the most established / traditional uses under fishing as a practice. Aquaculture as a new use category has gained momentum since the mid-1980’s but already in significant competition with capture fisheries. Figure 3.31 provides estimates of the global fish production as about 170 million metric tons with close to 50 percent coming from aquaculture. This is a consistently increasing trend in the last three decades whereby aquaculture is all set to takeover capture fisheries in the next decade or so. Not only that but aquaculture is reportedly encroaching into the dominant use of capture fisheries for the purpose of food for humans, i.e., out of the 90 million metric tons of fish obtained from capture sources over recent decades, about 60 million metric tons goes to direct human consumption and most of the rest is diverted as feed for aquaculture and livestock. Such trends might threaten to further marginalize the capture fisheries practice which is already experiencing a sharp decline in its biologically sustainable levels from 90 percent in 1974 to 65.8 percent in 2015 and stands at more than 34.2% stocks being overfished (FAO, 2020d). 3.4.3.1. Trade-offs and synergies at intra-practice and intra-use level Better synergies between these two use types under the fishing practice have the potential to 310 contribute to both goals of ecological conservation and global human development, all of which can potentially lead to sustainable outcomes. Figure 3.65 Total annual catch in small-scale and large-scale fisheries around the world. Abbreviations: MT: MillionTons, LSF: Large-Scale Fisheries, SSF: Small-Scale Fisheries. Source: (Toobigtoignore.Net - Big Numbers Project Report by World Bank/FAO/World Fish, 2010) under license CC-BY. Figure 3.65 Total annual catch in small-scale and large-scale fisheries around the world. Abbreviations: MT: MillionTons, LSF: Large-Scale Fisheries, SSF: Small-Scale Fisheries. Source: (Toobigtoignore.Net - Big Numbers Project Report by World Bank/FAO/World Fish, 2010) under license CC-BY. Fisheries bycatch is a growing trend and an example of how increased use of technology and the mechanization of vessel and gear types result in trade-offs. A related area is unreported volumes of fish discarded at sea. According to Figure 3.65, the global catch of fish reported by individual countries include only estimates of landing and do not include non-retained catch that are discarded at sea. Globally, estimated discards accounts for about 10% of total annual catches and most discards are generated by industrial (i.e., large‐scale) fisheries (Dirk Zeller et al., 2018). Compared to this, landing estimates for small- scale fisheries are widely regarded as an underestimation. 3.4.3.2. Trade-offs and synergies between practices and uses Trade-offs and synergies are inherently linked to fishing, gathering, terrestrial animal harvesting, logging, and non-extractive practices being treated exclusively or in isolation from each other. Several sections in 3.3 acknowledge the interconnections between practices. However, it was necessary to treat them in a somewhat stand-alone way for clarity in the systematic literature reviews and in reporting. This treatment exposes problems around synergies and trade-offs. Artificially created or not, the disconnections between major practice 311 categories are not healthy for sustainable use of wild species. For example, fishing is not all about fish and fishers alone. Aquatic systems that host fish habitats are integrally connected to terrestrial ecosystems as they mutually benefit or impact each other. Or when people harvest fruits from wild trees (gathering), they may also harvest the entire tree for firewood (logging). People who are primary users, engaged in one of these practices, tend to move between multiple practices and uses either as a seasonal livelihood routine or under pressure from multilevel drivers when their primary engagement in a specific practice is disrupted. First, several groups of wild species users are known to move between fishing, gathering and harvesting across a range of ecosystems which is influenced by their livelihood, cultural and occupational needs and complementary seasonality among the wild species, i.e., occurrence and availability. Second, even those who depend on one specific use or practice category as their primary source of food, subsistence or livelihoods are seen entering into other practices and uses of wild species due to unforeseen pressures. This includes increasing instances of how coastal inhabitants, primarily reliant on fishing, are forced to engage in the harvesting, gathering and use of wild species under non-fishing practice categories when they are faced with loss of fish and related income due to multilevel pressures. Natural disasters (i.e., cyclones, floods, tsunami, earthquakes, etc.) are known to push people from one practice and use category to others through temporary, semi-permanent and permanent displacement. It is understood that better synergies will facilitate this cross-practice mobility of users, especially in times of crisis and positively contribute to the sustainable use of wild species. It may also help resolve negative trade-offs between the practices or, at the minimum, bring them up for timely attention. 3.4.3.2. Trade-offs and synergies between practices and uses As discussed in the preceding paragraph, if trade-offs between practices are related to their separation from each other, the need to consider synergies between practices at global, regional, national and local policy and program levels cannot be underestimated. Trade-offs between uses within a practice may be related to differential policy attention each of the uses has received. For example, the use that generates the most revenue may move up in the hierarchy and receive most policy and related attention, and this may take place at the cost of other uses. It is evident that in the tussle between capture fisheries and aquaculture the later receives significantly higher attention compared to the former (section 3.3.1). Similarly, tourism tends to grab significant policy and program attention as compared to medicinal, ceremonial and cultural uses (section 3.3.5). The spaces and places where practices occur influence the nature of trade-offs and synergies. Section 3.3 offers numerous examples of this. Fishing practices are specific to marine, coastal, inland sectors as dominant fisheries within which multiple uses are operationalized by the users and the possibility of trade-offs and synergies are within and between these spaces. Similarly, gathering, harvesting, logging and non-extractive practices take place within multiple resource sectors that tend to interact and influence each other. Each of these resource sectors have their own social, economic, cultural, political and ecological characteristics which shape the nature of the practice and uses. Trade-offs and synergy result from how the practices and uses across these multiple sectors interact and influence each other. It is important to recognize that trade-offs and synergies may not only be considered to be existing between or within practices and uses but also the scale at which they operate has significant role. The tussle between the small-scale and large-scale fisheries is more about scale 312 than anything else (as discussed above). There can be multiple interpretations of how scale is linked with trade-offs and synergies in other practices and uses. Related to scale, understanding trade-offs and synergies between and within geographical contexts within which practices occur is important. The literature review on small-scale fisheries in section 3.3 provides detailed account of geographical context specificities by characterizing small-scale fisheries within Europe, Arica, Asia, Latin America, North America, and the Pacific. 3.4.3.2. Trade-offs and synergies between practices and uses It is important to note that the key characteristics and the major drivers influencing trade-offs and synergies in each of these geographical regions of the world may significantly vary. Section 3.2 offers a systematic analysis of the role of indicators in understanding sustainable use of wild species. Examination of the sustainable indicators has a lot to offer in terms of clarifying trade-offs and synergies. In fact, many sustainable use indicators are indicators of trade-offs and synergies. Tools such as monitoring in many indigenous peoples and local communities focus on interlinked social and ecological elements and can inform the development of local and global indicators that recognize these linkages. The acknowledgement of the value of including the knowledge of indigenous peoples and local communities contribute importantly to monitoring and assessment of the species and ecosystems used by these communities. Trade-offs and synergies between knowledge systems guiding the practices and uses is a whole new area to explore. While the politics and power dynamics between knowledge systems (Van Assche, Beunen, Duineveld, & Gruezmacher, 2017) may be inherently connected to trade-offs, systems of knowledge coproduction (Norström et al., 2020) signify synergies between sustainable use practices. In this context, indigenous knowledge is increasingly challenging to pass on because the environments in which indigenous and local communities live are threatened (3.3.5). Indigenous peoples and local communities report a loss of nature that supports their local livelihoods and well-being, in part as a result of natural resource extraction by both outsiders and locals (Ichii, Molnár, Obura, Purvis, & Willis, 2019). Increasing efforts to synthesize indigenous and local knowledge have shown that the natural indicators indigenous peoples and local communities use are reasonably compatible with scientific knowledge and show their deep connection with nature, albeit it at a very local scale (Ichii et al., 2019). Indigenous and local knowledge is increasingly being used to generate more accurate data on species trends, non-iconic species data and geospatially relevant data using technology (e.g., Cybertracker: (Ansell & Koenig, 2011; Liebenberg et al., 2017), participatory mapping using Google Earth (Peters-Guarin & McCall, 2012) and Open Data Kit (ODK): (Jeffers, Humber, Nohasiarivelo, Botosoamananto, & Anderson, 2019)). However, it is important to note that the goal in working with indigenous peoples and local communities is to honor their knowledge in its own right, not only when it is compatible with scientific knowledge or supportive thereof (Barron, Sthultz, Hurley, & Pringle, 2015). 3.4.3.3. Trade-offs and synergies involving the social, economic, environmental and policy aspects of sustainable use Sustainability is multidimensional but the essence of it can be captured by considering the social, economic and environmental aspects as inclusive categories. The questions about trade- offs and synergies are integrally linked to the three pillars of sustainability, i.e., economic viability, environmental protection and social equity (Purvis, Mao, & Robinson, 2019). Policy is also recognized as a supporting element of sustainability. In other words, economic, social, environmental and policy aspects of the sustainable uses of wild species help link practices and uses with key sustainability parameters. While negative trade-offs among and between these parameters threaten the viability of sustainable use, synergies among them provide pathways for sustainable use. In many contemporary societies, terrestrial animal harvesting has multiple functions and sustainability hinges on the synergies and trade-offs between social, ecological and economic dimensions of this specific practice. Human populations engage in animal harvesting (such as, hunting and trapping) to meet a range of nutritional, economic, medicinal, cultural and recreational needs and the level of synergy between these needs may have implications for the level of extraction of the resource, therefore its sustainability. International and national policy instruments and guidelines, along with civil society actions have supported processes to resolve negative trade-offs and potentially build synergies between practices and uses. There is strong evidence presented in section 3.3 to support this conclusion. The impact of fishing on marine ecosystems other than the target species and their habitats is well established. Several international instruments (such as agreements, policies, protocols, treaties) have been developed to help respond to these challenges and provide guidance for action. Prominent among those are United Nations Convention on the Law of the Sea, created in 1982, which established the 200-mile exclusive economic zone and the concept of maximum sustainable yield as an international measure for sustainable fisheries management. Given the inadequacies associated with the United Nations Convention on the Law of the Sea regarding fish stocks that range across multiple exclusive economic zones or in the high seas, the United Nations Fish Stocks Agreement, 2001 was brought into effect to offer international protocols for managing the overlapping stocks. Further, the Food and Agriculture Organization of the United Nations has put in place a range of international policy guidelines to promote sustainable use of aquatic ecosystems and facilitate the conservation of biodiversity of ecosystems by minimizing trade-offs in forms of competition, contestations and unsustainable practices. 3.4.3.2. Trade-offs and synergies between practices and uses Changing gender roles and dynamics can lead to the disruption of existing synergies and the creation of new trade-offs. One case that shows the complexity of trying to develop sustainable use practices based on gender assignments of particular practices and uses is the gathering practices of orchids in Tanzania. The majority of gatherers of wild edible orchids are female, orphans also commonly engage in this practice, and there are slightly more boys than girls among orphans affected by HIV/AIDS (Human Immunodeficiency Virus) in villages in 313 the southern highlands of Tanzania (Challe & Price, 2009; Challe, Struik, & Price, 2018). Children gather less species than adults and generally learn about the use and identification of wild species from their mothers and to a lesser extent also from their fathers (Cruz García, 2006; Łuczaj & Nieroda, 2011). When children's parents die before they share their knowledge, orphans teach each other and or learn from middlemen as a result of “trial and error”. This can lead to the gathering of too many non-marketable orchid tubers, which may in turn negatively affect the sustainability of the practice (Challe et al., 2018). 3.4.3.3. Trade-offs and synergies involving the social, economic, environmental and policy aspects of sustainable use These include: the 1995 Code of Conduct for Responsible Fisheries (FAO, 1995b); Voluntary International Plan of Action on reducing the incidental capture of 314 seabirds in longline fisheries (FAO, 1999a); International Plan of Action on the Conservation and Management of Sharks (FAO, 1999c); International Guidelines on Reducing Marine Turtle Fishing Mortality (Eric Gilman & Bianchi, 2010); International Guidelines on Managing Fisheries Bycatch (FAO, 2011); Small-Scale Fisheries Guidelines (FAO, 2015). y While these international policy measures have produced favorable results, there are gaps that still exist, such as the issue of the sustainability of non-target species relative to target fish stocks is still unclear. This indicates that species that are not covered by a treaty or international policy may be subject to overexploitation and, therefore, unsustainable or in the process of being so. In order to address this, further responses have come through the legally binding United Nations resolution 61.105 (2005) which provided for responsible fishing in vulnerable marine ecosystems and of non-target species. Additionally, the International Agreement on Port State Measures (FAO, 2016a) aims to prevent, deter and eliminate illegal, unreported and unregulated (IUU) fishing by preventing vessels engaged in illegal, unreported and unregulated fishing from using ports and landing their catches. These measures suggest that they are geared towards addressing factors (e.g., illegal, unreported and unregulated) that can trigger trade-offs and create barriers for possible synergies. Apart from the international responses to critical trade-offs, major efforts have also come from national governments and non-governmental organizations. For example, the formation of the Marine Stewardship Council (1997) to improve fisheries sustainability along with the initiation of several environmental non-governmental organizations for marine conservation, expansion of the science and management efforts by national and regional governments including the Common Fisheries Policy in the European Union are important landmarks. The above discussion suggests that the history of sustainable use of capture fisheries is closely tied with a number of critical international, national, regional policy guidelines, and non-governmental organizations and civil society action focusing on fisheries and their ecosystem conservation. These policy instruments and agreements provide a strong foundation for possible actions and responses to trade-offs and processes through which synergies for sustainable use can be achieved. 3.4.4. Selected case studies of trade-offs and synergies in sustainable use The following cases studies help explore the question ‘whether non-extractive uses can become an alternative to extractive uses? 3.4.4.1. Whaling and whale-watching Whale watching is commonly seen as the global success story of a non-extractive use replacing an extractive use, and in the process encouraging sustainable use, generating economic revenue and contributing to conservation. The growth in whale watching is a result of bans on whale hunting, the decline in whale-derived products, and environmental campaigns by non- governmental organizations to support whale watching as a sustainable alternative to whale hunting (Neves, 2010). Many whale populations are recovering after the global commercial moratorium was enacted on whaling in 1985, although determining the status for some 315 populations has remained challenging (IWC, 2020a). In addition, some countries continue to hunt whales under objection or reservation to the moritorium, or because they are not members of the International Whaling Commission (see section 3.3.1.4.5 above for additional discussion of this point). Whale watching has undoubtedly become a lucrative industry, particularly for tour operators in developing regions who often enjoy direct income streams considerably greater than existing levels of regional gross domestic product per capita (Mustika, Birtles, Welters, & Marsh, 2012) and so, by extension, for local communities that benefit from the tourism activities. Whale watching tourism has brought additional revenue to the Maoris in Kaikoura, New Zealand (Curtin, 2003), and the inhabitants of both Lajes in the Azores (L. Silva, 2015) and Baja, Mexico (Schwoerer, Knowler, & Garcia-Martinez, 2016), through direct expenditure on tours but also through the accompanying expenditure on transport, accommodation and hospitality. It has also brought positive attitudinal effects amongst whale-watching tourists and local populations. Mintzer et al. (2015) note that the creation of a sustainable development reserve and the presence of dolphin researchers have had positive effects on the attitudes and behaviors of an indigenous fishing community on the Amazon towards botos, which have in the past been killed for both bait and superstition. Wilson and Tisdell (2003) report that 78% of whale- watching tourists visiting Hervey Bay, Australia, find the experience convinces them of the need for a worldwide ban on whaling, 80% of the need for greater protection of whales in Australia, and 73% to be more likely to report whales that are stranded, injured or mistreated: biocentric effects that are supported by the findings of Gowreesunkar and Rycha (2015). However, whale watching is not without negative impacts on whales and marine ecosystems. 3.4.4.2. Recreational trophy hunting and wildlife watching tourism Trophy, sport or recreational hunting has attracted increasing negative attention, particularly since the widely publicized killing of “Cecil the Lion” in Zimbabwe in 2015. Trophy hunting has long been banned in some source countries (e.g., in Kenya since the 1970s) and efforts have been made to restrict it by banning imports of hunting trophies, at least from certain species, in consumer countries (e.g., France banned the imports of lion trophies in 2015, while the Netherlands and Australia banned imports from a wide range of species in 2016 (Ares, 2019). Trophy hunting can have negative impacts on wild species populations, particularly if offtake is too high or where infanticidal population dynamics exist (e.g. Loveridge, Searle, Murindagomo, & Macdonald, 2007; Milner, Nilsen, & Andreassen, 2007; Wielgus, Morrison, Cooley, & Maletzke, 2013) but can also positively impact conservation and local livelihoods, particularly by generating revenue from habitat and species conservation (Naidoo, Weaver, et al., 2016; Snyman et al., 2021). Debates have played out in the scientific literature and beyond as to the ecological, social and economic costs and benefits of hunting, but one key element of arguments against hunting has been that such extractive practices are repugnant because of ethical issues concerning certain types of harvesting of wild species. It has consequently been suggested that one solution would be to replace such practices with non-extractive uses, and in particular, with wildlife watching (e.g., photographic tourism). This argument assumes, in the first place, that wildlife watching is indeed a non- extractive use of wild species. Some commentators would argue against this on the basis of its negative ecological impacts on some species and ecosystems. For example, Ballantyne and Pickering (2013) identify tourism as a problem for 46% of threatened vascular plant species in Europe alone, while it has also been documented as limiting cheetah reproduction (Broekhuis, 2018). In addition, wildlife watching can have wide ecological impacts, including water use and carbon emissions (Gössling et al., 2012; Spenceley, 2005). A key argument for the conservation benefits of recreational hunting is similar to that made for photographic tourism, i.e., income is generated and this plays a role in in i) directly financing conservation agencies including national parks authorities (e.g., Brink, Smith, Skinner, & Leader-Williams, 2016; Lindsey et al., 2020), and ii) providing an incentive for habitat and biodiversity conservation beyond state-managed protected areas by communities and private landowners. 3.4.4.1. Whaling and whale-watching The International Whaling Commission has released a Whale Watching Handbook addressing these concerns and supporting sustainable whale watching (IWC, 2020b). In some areas, whale watching and whaling co-exist. Whale watching is the more economically lucrative and globally accepted activity. Although whaling depends on government subsidies, some indications are that public support for whaling in whaling countries like Japan and Iceland is growing as a perceived cultural and nationalistic right (Andersson, Gothall, & Wende, 2014; Cunningham, Huijbens, & Wearing, 2012). Yet, in both Japan and Iceland, whale watching tourism is booming (Cunningham et al., 2012), but there has been concern that continued whaling alongside tourism will negatively impact tourism industries (Bertulli, Leeney, Barreau, & Matassa, 2016; Cunningham et al., 2012; Hoyt & Hvenegaard, 2002; Kuo, Chen, & McAleer, 2012; Orams, 2001; Parsons & Draheim, 2009; Parsons & Rawles, 2003), the extremes of which could result in tourism boycotts such as happened in St. Vincent and the Grenadines (Hoyt & Hvenegaard, 2002). The whaling-whale watching nexus is complex and the discourse at the intersection of these activities needs further research (Cunningham et al., 2012). There are contradictory tensions involved in whaling-whale watching that need unpacking. Tourists who eat whale meat are also pro-conservation and support the ban on whale hunting (Burns, Lilja Öqvist, Angerbjörn, & Granquist, 2018). Ironically, the market for whale meat is strongest for tourists (Bertulli et al., 2016; Rasmussen, 2014). Iceland seems to have retained tourists who are tolerant of whaling (especially for subsistence) and who support local and cultural expression, but at the cost alienating tourists who cannot reconcile with whaling for commercial, scientific or indigenous reasons (Andersson et al., 2014). Although the number of whale watching 316 tourists has continued to grow in Iceland since 2002 when whaling resumed, the relative contribution of whale watching tourism to other tourist activities has declined (Andersson et al., 2014). Overall, whaling seems likely to face increased global resistance and unlikely to generate substantial economic incentives, whilst whale watching has global support and generates substantial revenue. It would be prudent for whaling countries to assess the implications of the negative impacts of whaling on their national “image” – their biggest tourism asset (Hoyt & Hvenegaard, 2002) – and conduct a thorough compatibility analysis. Conversely, highly visible national policy for cetacean conservation can attract tourists (Parsons & Draheim, 2009). 3.4.4.2. Recreational trophy hunting and wildlife watching tourism Opponents of hunting suggest that wild species are worth far more for wildlife watching tourism than for hunting. For example, a report by the David Sheldrick Wildlife Trust (2014) estimated that a single elephant may be worth 1.6 million United States 317 dollars over its lifetime through income from photographic tourism. A wider review by Lindsey et al., (2007) highlighted that photographic tourism undoubtedly generates greater gross revenues than trophy hunting at a continental scale across Africa. Importantly though, they note that even if smaller, “hunting revenues are significant because they enable wild species production to be a viable land use across a wider range of land uses than would be possible relying on revenues from photographic nature-based tourism alone.” Unlike wildlife watching tourists (generally, obviously exceptions may apply) hunters are often prepared to hunt in areas lacking attractive scenery, and require less infrastructure, therefore minimizing habitat degradation (Di Minin et al., 2016). Because wildlife watching tourism is not viable in all the places where hunting happens, the suggestion that one type of use can simply be replaced with another is thus naïve. For example, Lindsey et al. (2006) argue that not all land suitable for trophy hunting would be suitable for wildlife watching tourism, and that low visitor numbers would be unlikely to make it economically viable. Similarly, in Botswana, a ban on trophy hunting implemented in 2014 meant that communities were forced to shift their income earning opportunities from hunting to wildlife watching tourism (Mbaiwa, 2018). Photographic tour operators apparently had little interest in developing lodges in the concessions that lacked high tourism potential (Winterbach, Whitesell, & Somers, 2015). Consequently, there was a reduction of local benefits such as cash income, employment opportunities, scholarships and funeral insurance. This lack of local economic benefits had negative effects on conservation including negative attitudes by rural residents towards wild species conservation and an increase in poaching (Mbaiwa, 2018). Very few studies have directly compared the benefits of trophy hunting and wildlife watching tourism to the same people, in the same location. One that has is an analysis of communal conservancies in Namibia (Naidoo, Weaver, et al., 2016). The study looked at financial and in‐kind benefit streams from wildlife watching tourism and hunting on 77 Namibian communal conservancies from 1998 to 2013. 3.4.4.2. Recreational trophy hunting and wildlife watching tourism It found that although total benefits from hunting and tourism increased at roughly the same rate, conservancies typically started generating benefits from hunting within three years of formation compared to after six years for photographic tourism. Regarding the types of benefits, the majority (64%) of benefits from trophy hunting were in the form of cash for income for conservancy management, while 32% of benefits were meat for the community at large. In contrast, 58% of the benefits from wildlife watching tourism were in the form of jobs, with 30% used for conservancy management. A simulated ban on trophy hunting significantly reduced the number of conservancies that could cover their operating costs, whereas eliminating income from wildlife watching tourism was still negative but a less marked effect. The study concluded that maintaining both trophy hunting and wildlife watching tourism was likely to produce the greatest incentives for conservation while only focusing on one would reduce the competitiveness of wild species as a land‐use option and harm the viability of community‐based conservation efforts in Namibia, and possibly elsewhere. Other comparisons that have been made between hunting and wildlife watching relate to the broader environmental impacts of the two activities. Di Minin et al., (2016) argue because there are fewer trophy hunters compared to wildlife watchers and because it can generate more revenue from a smaller number of visitors, trophy hunting can have a smaller footprint than wildlife watching tourism in terms of carbon emissions and infrastructure development. One 318 case study where an analysis has been conducted between numbers of hunting tourists compared to photographic tourists is Timbavati Private Nature Reserve in South Africa (Timbavati Private Nature Reserve News, 2020). The annual operating budget of the reserve is currently 1.26 million United States dollars which is generated primarily through wildlife watching tourism and hunting. In 2016 an analysis by the reserve’s management team found that the conservation levies paid by the approximately 24,000 wildlife watching tourists who visited the reserve that year amounted to less than a third of the income earned from the 46 hunters who visited over the same period (Conservation Frontlines, 2020). The reserve has subsequently increased the fees charged to wildlife watching tourists to increase revenue without having to increase the number of bed-nights, and hence the human footprint. 3.4.4.2. Recreational trophy hunting and wildlife watching tourism Similarly in Tanzania, Estes (2015) suggests that trophy hunting and wildlife watching bring in similar amounts to the Tanzanian economy but the ratio of tourists who come to see the wild species and hunters who come to shoot it is many hundreds to one with one hunting tourist paying at least 10 times as much as every wildlife watcher. 3.4.4.3. Elasmobranch tourism opportunity and shark fishing Just as whale watching has contributed to the decline of whaling, there is opportunity for shark and ray watching tourism to mitigate shark fishing effects by providing additional income sources. In a review on elasmobranch tourism, Healy et al. (2020) demonstrate that the tourism value of individual sharks exceeds the fisheries value, contributing revenue to developing countries. In Palau, shark tourism contributed over 18 million United States dollars, 8% of the 2012 gross domestic product and the tourism value of sicklefin lemon sharks (Negaprion acutidens) in French Polynesia exceeds the payment received by fishers (Healy et al., 2020). Diving, snorkeling, feeding and cage diving currently occur in 42 countries focusing on 49 target species, predominantly in tropical and subtropical Africa, Oceania, Asia and the Caribbean, but also in temperate seas such as Canada, England, Scotland, Japan and New Zealand (Healy et al., 2020). There may be unintended social-ecological feedbacks between different uses (e.g., tourism, fishing) and wild species. An interesting example is the decline in white sharks (Carcharodon carcharias) in South Africa as part of the greater social ecological system. The decline has been of ecological concern, but also impacts on the white shark tourism industry. Killer whale (Orcinus orca) presence was initially attributed to the decline, as killer whales have been shown to displace white sharks (Jorgensen et al., 2019). However, continued white shark decline outside of the season niche overlap with killer whales has prompted speculation that demersal long line fishery of smaller shark species, a white shark resource, mostly exported to Australia for human consumption (Braccini, Blay, Harry, & Newman, 2020). This speculation, in turn, resulted in boycott calls against the Australian ‘fish and chip’ sales to protect South African white sharks, which has negatively affected an overall legitimate and sustainable Australian industry (Braccini et al., 2020).  Sea horse tours and extractive sea horse harvesting  Sea horse tours and extractive sea horse harvesting An interesting local example of non-extractive use replacing extractive use is the case of sea horse (Hippocampus reidii) tours by self-organized and self-governed ‘jangadeiros’ in a 319 Brazilian village (Ternes et al., 2016). Here the local communities impart their comprehensive local ecological knowledge of sea horses to tourists. They take tourists out by raft boat and dive sea horse specimens out to hold in glass jars for viewing by the tourists before releasing them back into their habitat (Ternes et al., 2016). The community involved in these tours no longer harvest sea horses for medicinal or ornamental purposes as they derive economic benefits from them in situ, unlike other villages in the region (Ternes et al., 2016). The authors of this case study suggest that by careful guiding this non-extractive approach could be expanded to other villages to benefit sea horse conservation and local livelihoods (Ternes et al., 2016). These case studies show that while non-extractive uses can improve the conservation status of wild species and improve livelihoods in a sustainable fashion, it is unlikely that complete extractive use will be halted. As always, careful consideration of the context and the implications of such a shift need to guide interventions. Furthermore, the eradication of extractive activities is not necessarily desirable, especially where the extractive use fosters cultural practices that result in conservation. Yet, where extractive indigenous peoples and local communities’ use occurs in conjunction with non-extractive use there is potential for conflict as a result of the opposing value systems between the user groups (see section 3.3.5.2.3). And there are nuances between different forms of extractive or non-extractive use. For example, illegal poaching has been shown to negatively impact wildlife tourism (Naidoo, Fisher, Manica, & Balmford, 2016) whereas hunting concession impacts on wildlife tourism can be avoided with careful management. Choices around sustainable use of wild species will not always be between extractive and non-extractive use. Novel financial mechanisms such as Lion Carbon (2020) or ‘rhino bonds’ (Aglionby, 2019) may provide important alternatives for some areas, but these currently are only nascent initiatives. It is important to recognize that this is not just about the degree of benefits but their distribution, as benefits from one type of use may be distributed very differently compared to another. 3.4.5. Key attributes necessary to respond to trade-offs and strengthen synergies in sustainable use In the use of wild species, there are synergies and trade-offs among the policies, practices and technologies used to address individually the issues of loss of biodiversity (wild species), land degradation, water pollution and climate change. Economic, ecological and social dimensions play pivotal roles in setting the context for use of wild species; the ways wild species are used differ under different economic conditions, law enforcement regimes, culture and traditional meanings and perception of users. Evidence supports that there are risks associated with the harvesting of wild populations under challenging conditions, and these are often highlighted in low-income countries (Leao, Lobo, & Scotson, 2017) although they can occur in developed countries as well. Therefore, the issues are strongly interconnected and cannot be addressed in isolation (Watson, 2005). Better understanding of the underlying mechanisms and motivations for trade-offs and synergies can be beneficial for planning and managing sustainable use through (i) predicting and anticipating where and when trade-offs might take place; (ii) reducing undesirable trade- offs and related conflicts; (iii) enhancing desirable synergies; (iv) promoting honest dialogue, creativity, and learning between concerned user / stakeholder groups; (v) creating more effective, efficient and credible management and governance decisions; and (vi) obtaining more equitable and fair outcomes by taking into account distributive impacts of trade-offs (Turkelboom et al., 2016). Key lessons on trade-offs and synergies pertaining to sustainable use of wild species include, but are not limited to:  Trade-offs and synergies reflect a host of interactions, connections, relationships and linkages within, between and among practices and uses. Without consideration of these interactions and their effects, sustainable use cannot be adequately assessed.  While trade-offs and synergies between uses within a practice is somewhat well understood, the exact nature of trade-offs and synergies between practices, for example the interactions among gathering and fishing, are not very well studied. This knowledge gap involving the lack of inter-practice trade-offs and synergies has the potential to adversely impact sustainable use of wild species.  Bifurcation of existing uses and the emergence of new uses within a practice area (e.g., capture vs. aquaculture within fishing practices; ceremony and cultural expression vs. recreation (tourism) within non-extractive practices) have led to a reconfiguration of intra-practice trade-offs and synergies.  Sea horse tours and extractive sea horse harvesting So, it may be tempting to conclude that because a non- extractive form of wild species use (e.g., shark watching) has the potential to generate more revenue and jobs than an extractive form (e.g., shark fishing) the former is more sustainable. The same applies in cases where extractive appears to be “better” than non-extractive use. However, sustainability is about more than just economics: the benefits and costs of different activities may accrue to very different stakeholder groups, which is likely to affect the degree to which each option is viewed as socially sustainable. Ultimately, wider non-economic aspects of sustainability of different uses (e.g., likely long-term impacts on the wild species population, interactions of that use with other conservation threats, resource demands of the users, perceived social acceptability etc.) should also be considered when examining trade-offs between different options. Furthermore, the likelihood of unsustainable activity should also be factored in to provide a reliable comparison, particularly the likelihood of land conversion to non-wildlife-based land uses under different scenarios. In all cases, understanding who benefits, and how, from the use of wild species is critical to designing effective policies and programmes that encourage the sustainability of that use and incentivize conservation over other land and resource use options. 320 3.4.5.1. Levels and scales at which trade-offs and synergies occur Trade-offs and synergies are scale-bound. IPBES Glossary defines scale as the spatial, temporal, quantitative and analytical dimensions used to measure and study any phenomenon, i.e., trade-off and synergy in this case (IPBES core glossary, 2021). The need for considering multiple scales (and levels) at which trade-offs and synergies around sustainable uses take place bears significance (Carpenter & Brock, 2006; Mayer, Pawlowski, & Cabezas, 2006). Empirical insights have been recorded from observations of modifications and reorganizations of system dynamics at the level of the ecosystem (Stephen R Carpenter & Kinne, 2003; Scheffer & van Nes, 2004). However, choices about scale of observation are not easily matched with strategies for intervention. For example, specific components of a wild species use regime can cross thresholds (understood as synergy) and lead to varying outcomes at substantially different temporal and spatial scales associated with the influences resulting from trade-offs. There is also an issue that boundaries that delineate units of scale (e.g., ecozones, de jure/formal administrative boundaries) do not always correspond to the reality of the ecosystems or human use which are instead (at best) 'soft' (as opposed to 'hard') boundaries (Norris, 2014; Veldhuis et al., 2019). Systematic treatment of trade-offs and synergies relevant to sustainable use of wild species will require scale-sensitive perspectives, and reflection on appropriate scales of understanding and intervention (Scheffer, Westley, & Brock, 2003). g y A related aspect of scale is to focus on the units of analysis for measuring trade-offs and synergies. Studies on ecosystem changes and shifts in use and management regimes tend to have mostly emphasized a single resource (or practice) type (Biggs, Carpenter, & Brock, 2009; S. R. Carpenter & Brock, 2006; Scheffer et al., 2003), including marine systems (Beaugrand, 2004; Mantua, 2004; Steele, 2004), lakes and lagoons (Gal & Anderson, 2010; Scheffer & van Nes, 2004), freshwater systems (Carpenter and Kinne, 2003), forests (Ludwig, Jones, & Holling, 1978), woodlands (Dublin, Sinclair, & McGlade, 1990), dry lands (Foley et al. 2003), rangelands (Skaggs et al., 2011), and agroecosystems (Gordon, Peterson, & Bennett, 2008), all of which act as sources of wild species. However, using individual resource systems (or practices) to define boundaries of sustainable use inevitably neglects the full range of human expectations from, and interactions with, the larger social, ecological and environmental system necessary to achieve sustainability (Nayak & Armitage, 2018). 3.4.5. Key attributes necessary to respond to trade-offs and strengthen synergies in sustainable use These changes drive technology, science, investment, policy focus, innovation away from existing use areas to the new uses that have the potential to negatively impact sustainable use of wild species as a whole.  Trade-offs and synergies between and among fishing, gathering, terrestrial animal harvesting, logging, and non-extractive practices are inherently linked but often treated exclusively or in isolation from each other. This exclusivity is reflected in the dominant culture of practice-specific policies leading to significant compartmentalization. Consideration of trade-offs and synergies between these practices and their use categories across global, regional, national and local policy and program levels could enhance sustainable use of wild species. 321  A combination of indigenous and local knowledge and scientific knowledge is effective to better understand and respond to the trade-offs and synergies relating to status and trends in sustainable use. Knowledge co-production processes based in ongoing collaborations are useful in this respect.  A combination of indigenous and local knowledge and scientific knowledge is effective to better understand and respond to the trade-offs and synergies relating to status and trends in sustainable use. Knowledge co-production processes based in ongoing collaborations are useful in this respect. Due to uncertainty and the plurality of values and information on wild species, addressing trade-offs requires inclusive adaptive co-governance that is sensitive to power dynamics, principles of justice and equity. 3.4.5.2. Equity and justice considerations in responding to trade-offs and negotiating synergies How can it be ensured that the outcomes of trade-offs and synergies associated with sustainable use of wild species are distributed equitably? The procedural and distributive aspects of trade- offs and synergies offer multiple pathways to sustainability, depending on the culture and the ecosystem. If inequity and injustice reign, there are few and often no sustainable pathways. Greater attention to equity and social justice considerations (i.e., winners and losers in the context of sustainable use) is needed to better understand the process and outcomes of trade- offs and synergies. Recognizing issues around sustainable use, trade-offs and synergies through the prism of social and environmental justice facilitates the identification of key motivations of users and main ingredients influencing these processes. Section 3.3 presents material that points towards equity and justice as both cause and effect of trade-offs and synergies. For example, an equity and social justice perspective helps clarify if outcomes from critical trade- offs disproportionately impact a multitude of users, e.g., poor, disempowered and other marginalized communities including women through a process of uneven distribution of benefits and impacts (see Walker & Bulkeley, 2006). Literature from multiple disciplines suggests that changes and shifts in ecosystem processes, structures, functions and services associated with sustainable use may redistribute benefits among stakeholders (Selkoe et al., 2015), and such redistribution may lack sensitivity to equity and justice issues. It is important to recognize that these shifts and redistribution processes are inherently linked to unresolved trade-offs and the absence of synergies among practices and uses of wild species. In economically and socially stratified social-ecological systems that host wild species, the outcomes of trade-offs and synergies pertaining to the diverse use regimes are often beneficial to some while adversely affecting others (Nayak, Armitage, & Andrachuk, 2016). For example, case studies by Nayak and Berkes (2010), Armitage and Marschke (2013), and others provide evidence that changes in the management practices in coastal and inland fisheries of Bay of Bengal and South China Sea (e.g., outcomes of the trade-offs from the introduction of aquaculture within a predominantly capture fishery system) have benefitted higher caste or wealthier aquaculture owners respectively but have proven negative for customary users. This trend is also evident in section 3.3. 3.4.5.1. Levels and scales at which trade-offs and synergies occur Incidentally, critical trade-offs and opportunities for building synergies may be missed if a narrow focus on the scale of sustainable use is adopted. Therefore, it is important to conceive what is an appropriate social-ecological unit within which to best capture trade-offs and synergies and why this is critical for observing trends and reporting status of wild species. Units of analysis of trade-offs and synergies in sustainable use may have both a physical (e.g., coastal line, bottom, rivers, 322 and vegetation, landscape) and a normative (e.g., culture, rituals, law, institutions, social interactions) dimension to their boundaries. Recognizing and understanding both these dimensions are useful from scale-sensitive perspectives. and vegetation, landscape) and a normative (e.g., culture, rituals, law, institutions, social interactions) dimension to their boundaries. Recognizing and understanding both these dimensions are useful from scale-sensitive perspectives. 3.4.5.3. Power dynamics and politics of use The appearance and disappearance of trade-offs and synergies, and the ways in which they are responded to and negotiated upon are not politically neutral. Social relations of power expressed through institutions, the position of different users in the society, and the language adopted to characterize trends in the use of wild species are crucial to understanding trade-offs and synergies. There is tremendous scope to comprehensively articulate the implications of power for sustainable use when it is under pressure from negative trade-offs, especially within a rapidly changing social-ecological context of the wild species (see similar arguments in Boonstra, 2016; Crépin, Biggs, Polasky, Troell, & de Zeeuw, 2012; Kull et al., 2018, 2018; Nayak et al., 2016). Important questions to further examine trade-off and synergy issues in sustainable use include: (i) What can be gained by assessing who wins and who loses in the context of changes in sustainable use of wild species and its emerging trends under the influence of multiple trade-offs? (ii) Is it possible to better assess the chances that a wild species use regime may be deliberately steered by some towards or away from other users? Such questions help to understand that sustainable use can benefit some and adversely impact others (see Armitage, Marschke, & van Tuyen, 2011; Ho, Ross, & Coutts, 2015). Divergent views on how a wild species use regime should be managed, who should benefit and who gets to decide on the essential features of the use system, and what needs to be done, are crucial questions with important consequences for how to respond to trade-offs and manage possible synergies. This is a highly context-specific issue, and no silver bullet exists. “What Works” in one context may be completely different in another. Further, this will require careful assessment of the dynamics associated with what Lebel et al. (2005) have termed as the “politics of scale” with attention to “politics of position” and “politics of place”, and this construct can be well placed in the analysis of trade-offs and synergies around sustainable use of wild species. Reid et al. (2006) adds to this view by highlighting the importance of user perspectives in problem formulation and analysis, and user knowledge to deal with governance and management issues. 3.4.5.2. Equity and justice considerations in responding to trade-offs and negotiating synergies These experiences clarify that trade- offs around sustainable use can create new opportunities and upward social and economic mobility for some users (and in this case those that were already upwardly mobile) but simultaneously exclude others (often those already marginalized). Such discrepancies in the nature and level of impacts are related to power and authority, structural advantage and institutional and political favor. Consequently, equity and social justice conditions influence how sustainable use related trade-offs, synergies and the outcomes thereof are ‘framed’ by certain groups as significant or not, and to what extent that framing can be used to design strategies to respond. 323 An additional consideration pertains to a multi/inter species justice dimension within the trade-offs and synergy discussions. This underscores the question whether sustainable development can really be accomplished without taking animals' own interests into account (Visseren-Hamakers, 2020). It is important to consider trade-offs and synergies between human and non-human justice leading to further explorations about the types of relationships humans can cultivate with animals so as to produce just outcomes. In doing so, neglecting the spiritual and cultural can also result in the lack of attention to the ways in which dominant Western cultural and spiritual forms sustain narrow conceptions of justice (Celermajer et al., 2021; Santiago-Ávila & Lynn, 2020). 3.4.5.4. Governing trade-offs and synergies for sustainable use What can be done when the outcomes of multiple, cross-cutting trade-offs between uses and practices become untenable for achieving sustainability of wild species, and when possible, synergies between and among use regimes and practices are not readily available? What approach could be useful when unresolved trade-offs have the potential to become stubborn and act as wicked problems, and configuring innovative synergies becomes a challenge? The question of adopting a governance approach to address these situations becomes important. Kooiman et al. (2005, p. 7) define governance as “the whole of interactions taken to solve societal problems and to create societal opportunities; including the formulation and application of principles guiding those interactions and care for institutions that enable and control them." According to this view, governance is qualitatively different from the related task of management in directing societal and environmental processes. It adds dimensions that are absent in a hands-on management approach. ‘Interactive governance’ emphasizes solving societal problems and creating societal opportunities through interactions among actors (Kooiman, J., Bavinck, M., Chuenpagdee, R., Mahon, R., & Pullin, R., 2008). The emphasis on ‘interactions’ constitutes the main innovation that fits appropriately with the need for responding to the trade-off and synergy related questions outlined at the beginning of this sub- section. IPBES Glossary (2021) adds rules, norms and actions as crucial elements of governance that can help structure, sustain, and regulate trade-offs and synergies. These multiple elements of governance help ensure dynamic problem-solving abilities based on values, principles, institutions and practices. Debates around sustainable use may trigger the need for biologically informed management and use targets that require an adaptive governance response (Selkoe et al., 2015). Here, governance refers to the “interrelated and increasingly integrated system of formal and informal rules, rule-making systems, and actor-networks at all levels of human society (from local to global) that are set up to steer societies toward preventing, mitigating, and adapting to global and local environmental change” (Biermann et al., 2009). Social and ecological processes, such as use regimes of wild species, influence and are influenced by governance arrangements in which social outcomes remain contingent upon ecological dynamics and vice- versa (Dale et al., 2000; Waltner-Toews & Kay, 2005). These interacting influences are very visible, for example, in section 3.3.5 regarding the dynamics in non-extractive use and governance, social, and ecological dimension of recreational tourism. 3.4.5.3. Power dynamics and politics of use Users’ own views of their situation reflect a rather different narrative and reality and failure to account for these diverse perspectives that emerge at different scales and from different users and actors can potentially restrict ability to deal with trade-offs and achieve sustainable use of wild species (Andrachuk & Armitage, 2015; Barron, Hartman, & Hagemann, 2020; Narayan, D., et al., 2001; Narayan, D., R. Patel, K. Schafft, A. Rademacher and S. Koch-Schulte., 2001; Nayak & Berkes, 2010). Berkes (2002) highlights numerous examples where higher scale perspectives and practices exert an influence 324 over or dominate lower scale realities, including through centralized decision-making, limited acceptance of alternative systems of knowledge in formal decision-making, nationalization of resources, influence of national and international markets, and top-down development policies and projects. These issues have significant connection with questions about trade-offs and synergy between and across practices and uses of wild species. 3.4.5.4. Governing trade-offs and synergies for sustainable use As explored in section 3.3.4 on logging, responses of social agents (users) in a given system to ecological change (wild species) have a direct bearing on outcomes (quality of life) (Following Lade, Tavoni, Levin, & Schlüter, 2013). In this respect, aggregated informal responses or coping strategies of local users to the shortage of wild species are important drivers of natural resource depletions, but often overlooked in the policy development of the natural resource management 325 (Ehara et al., 2018). These complex dynamics are visible across sections 3.3.3 and 3.3.5, for example, in relation to the interplay between the harvesting of wild meat for subsistence and protection of livestock, and the establishment of national parks in low-income countries throughout Africa to generate revenue. Both ecological variables (e.g., biodiversity, biogeochemical cycling, hydrological processes) as well as social variables influencing sustainable use, including human agency, social relations of power, institutions and rules that influence human behavior need to be assessed. As well, humans (users and other agents) both produce unsustainable use regimes and simultaneously adapt to them. Here the focus of governance will be on navigating or adapting, but in other cases the focus will be on steering towards more fundamental social transformation to avoid unsustainable use regimes under the influence of undesirable trade-offs and ensure stronger synergies between uses and practices (see Chapter 5 and 6). 3.5. Knowledge gaps However, the status of half of the world’s fisheries, largely from Southeast Asia, is not scientifically assessed (Costello et al., 2012). We know less about inland fisheries than marine fisheries. Marine mammals are especially susceptible to exploitation due to low reproductive rates and the many other threats they face, including noise pollution and climate change (Perrin, 2009). With regards to insects, fungi and microbes, insufficient taxonomic information makes it difficult to assess the sustainability of their use, and more generally knowledge on their roles in the supply of nature’s contributions to people is limited (Kassas, 2002). For example, it is believed that more than 90% of species remain unknown to science out of 148,000 species of fungi that have been scientifically identified (Antonelli et al., 2020). Sustainability of wild algae, fungi and plants harvesting is challenged by many factors and comprises interlinked dimensions such as socio-cultural, economic and political (Ghimire, 2008). Similarly, the sustainable management of medicinal trees requires knowledge on how different species respond to different harvesting techniques (Delvaux, Sinsin, Darchambeau, & Van Damme, 2009). As discussed in section 3.3.3.3.3 invertebrates provide an important source of nutrition in some areas, but data are missing on the sustainability or unsustainability of the gathering of edible insects. Overexploitation probably only concerns some species, but insects and fungi (sections 3.3.2.1; 3.3.3.2.3) on the whole are vulnerable due to the destruction of their habitats, to pesticides and other pollution, and to climate change (Arnold van Huis et al., 2013) Another limitation of indicators of sustainable use is related to spatial scales. Not all populations, taxa, systems and regions are equally or adequately represented in the scientific literature, meaning that while it is possible to assess the available knowledge, it is not actually possible to assess the sustainability of use. At the global level, there is a lack of pertinent data for many species of whales and seals, and the polar bear (Ursus maritimus) in the Arctic (Tierney et al., 2014) and for many small-scale fisheries in tropical developing countries, such as in Africa, Asia and South America (see small-scale fisheries section). There is a lack of data on how many species in each vertebrate class are used and how much is harvested. 3.5. Knowledge gaps There is an increasing tendency today to shift the focus away from sustainable use of wild species; whereas the emphasis is to view biodiversity conservation and sustainable use through the lens of ecosystem functioning and its capacity to produce ecosystem goods and services (Heywood, 2017). Therefore, it is very challenging to compile knowledge gaps on sustainable use of wild species as there is lack of consistency among worldwide databases to quantify the harvesting and use of wild species by people in different countries across the world. This happens because different countries and organizations have different accounting methodologies, making the merging of different datasets a huge challenge. Major knowledge gaps in the sustainable use of wild species are summarized here. (i) Across all practices, and especially in global fishing, existing data and reporting do not differentiate adequately between wild and non-wild species. Explained most explicitly in sections 3.2 (global overview), 3.3.1 (fishing), global indicators and data reported by the Food and Agriculture Organization of the United Nations and other agencies do not separate out wild and aquaculture, wild and farmed, wild and plantation, or wild and domesticated species when calculating global or regional off-takes. This makes it almost impossible to accurately assess and report on status and trends in sustainable use of wild species. There is vast legacy of available data on species taxonomy, conservation or economic value related to trade and markets rather than specifically on use as defined in the assessment. In addition, most of the datasets available lack detailed information on practices and uses of utilized and non-utilized species that challenges to make comparative account of population trends. (ii) Knowledge gap in status of taxonomic groups and their uses at different levels and scales. Information is available on the conservation status of vertebrates, particularly with regard to mammals and birds, to a lesser extent with amphibians and fish including demersal fish; however knowledge on conservation status and use is severely lacking for invertebrates (insects), fungi and microbial species (Coleman et al., 2019; Naranjo‐Ortiz & Gabaldón, 2019; Willis, 2018), and in some taxa, especially invertebrates and fungi, there are still thousands of 326 species yet to be described and being named. The knowledge gap also includes widely used and internationally traded species, for example porcini mushrooms (Boletus spp.). Marine species are especially susceptible to exploitation. 3.5. Knowledge gaps For example, data on harvested Arctic species are biased towards marine mammal and marine fish populations, and this could mask declines in some seabird colonies that are over-harvested (Tierney et al., 2014). Relatedly, many of the conservation models, protocols, procedures, monitoring and assessments are based on experience of animals, notably mammals and birds, and do not necessarily apply to plants, invertebrates or fungi (Heywood, 2017). (iii) Life histories and stocks of marine fish species not well understood. In most fisheries, there exist large gaps in understanding of life histories for many marine fish species, information on total cumulative anthropogenic levels of fishery removals from an individual population, knowledge of the conservation status of individual populations, and deficits in monitoring, including in data collection protocols, observer coverage rates, and sufficient time- series to detect the response in absolute population abundance of long-lived species to this anthropogenic mortality source (Gilman et al., 2014, 2020; Lewison, Crowder, Read, & Freeman, 2004b; Musick, 1999). Status of fish stocks of both large- and small-scale fishing is little understood for those countries and regions where fishing management intensity is low. 327 Further, there is data of status and trends individual fish stocks for IPBES regions such as Europe (e.g., https://www.eumofa.eu/) and North America, whereas data for other IPBES regions are missing. (iv) Knowledge gap in direct and collateral sources of fishing mortality on associated and dependent species. While there is increasing understanding of the status of stocks of principal market species of marine capture fisheries, albeit still incomplete especially in low-income countries, there remains a very large gap in knowledge of the effects of direct and collateral sources of fishing mortality on associated and dependent species including fecund species. For example, rare-event bycatch of species such as toothed whales and some pelagic sharks are unmonitored in most fisheries, there is a lack of knowledge of which populations are captured in individual fisheries, and as a result of these data quality constraints, extremely limited understanding of the sustainability of the ‘use’ of these wild species. For instance, 47 of 68 fisheries that catch marine resources managed by regional fisheries management organizations have no observer coverage (Gilman et al., 2014) for the vast majority of the ca. 4.6 million fishing vessels globally, information on non-retained catch is non-existent, and information on retained catch only is available in some cases. 3.5. Knowledge gaps While a target stock of a relatively productive species may be determined to be sustainable when assessed against various standards, the sustainability of the fishery and when assessed against impacts on incidentally captured species is very often unknown. Stock assessments which do not incorporate recreational fishing do not provide accurate assessments of global uptake and fish mortality. (v) Data gaps on sustainable use of wild species and their monitoring regarding small- scale fisheries, inland fisheries, marine and freshwater fisheries, and reef fisheries. One of the major challenges or data gaps to properly assess sustainable use of wild species, especially regarding small-scale fisheries and inland fisheries in tropical developing countries consists in the lack of long temporal series of data on resource use. Most of the small-scale fisheries worldwide show a chronic lack of monitoring data on time series of landings, fishing effort, biology of exploited species, among other relevant fisheries indicators (Welcomme, 2011). Similarly, there is no reliable information on value or number and diversity of sustainability of marine and freshwater ornamental fishery, and many species of reef fishes lack biological and ecological information. This indicates that conservation status of almost half of the species is still unknown (SOTWP, 2016). (v) Data gaps on sustainable use of wild species and their monitoring regarding small- scale fisheries, inland fisheries, marine and freshwater fisheries, and reef fisheries. One of the major challenges or data gaps to properly assess sustainable use of wild species, especially regarding small-scale fisheries and inland fisheries in tropical developing countries consists in the lack of long temporal series of data on resource use. Most of the small-scale fisheries worldwide show a chronic lack of monitoring data on time series of landings, fishing effort, biology of exploited species, among other relevant fisheries indicators (Welcomme, 2011). Similarly, there is no reliable information on value or number and diversity of sustainability of marine and freshwater ornamental fishery, and many species of reef fishes lack biological and ecological information. This indicates that conservation status of almost half of the species is still unknown (SOTWP, 2016). This lack of data precludes a proper assessment of the sustainability of most small-scale fisheries and inland fisheries. Furthermore, those indicators based on stock dynamics or population parameters, which have been widely applied in industrial fisheries, may not be suitable to complex, multispecies small-scale fisheries, or data needed to calculate these indicators cannot be gathered on a cost-effective and timely manner to inform policy intervention in many small-scale fisheries and inland fisheries. However, these limitations have been successfully addressed, in the context of small-scale fisheries, by studies adopting a scientific approach to record and analyze local or indigenous knowledge held by small-scale fishers on resource use over broad temporal scales (see section 3.3.1.3.2). 328 (vi) Research gap in gathering. Estimates on the number wild plant species that are used across different regions are unclear, despite documentation from (SOTWP, 2020) and (FOC, 2020). Also, there are limited information on wild species used as food, and these come mainly from ethnological or ecological inventories. As a global phenomenon, urban gathering that promotes positive cultural, ecological, economic and health outcomes research has received little scholarly attention and due emphasis has not been equally given in all regions of the globe. For example, 70% of the studies are from the Americas, Europe and Central Asia, 20% are from Africa, and the remaining are from Asia and the Pacific based on literature search retrieved for this assessment. (v) Data gaps on sustainable use of wild species and their monitoring regarding small- scale fisheries, inland fisheries, marine and freshwater fisheries, and reef fisheries. One of the major challenges or data gaps to properly assess sustainable use of wild species, especially regarding small-scale fisheries and inland fisheries in tropical developing countries consists in the lack of long temporal series of data on resource use. Most of the small-scale fisheries worldwide show a chronic lack of monitoring data on time series of landings, fishing effort, biology of exploited species, among other relevant fisheries indicators (Welcomme, 2011). Similarly, there is no reliable information on value or number and diversity of sustainability of marine and freshwater ornamental fishery, and many species of reef fishes lack biological and ecological information. This indicates that conservation status of almost half of the species is still unknown (SOTWP, 2016). Recently, an emerging gap has been in high demand of collection of recently described new species or rare species when their type localities were published in particular by specialized collectors. For this reason, an increasing number of scientists warn against publishing type localities (Lindenmayer & Scheele, 2017b); and the sustainability of this form of consumer-driven use is unclear. (vii) No data for global sale of cut flowers from wild and cultivated conditions. Cut flower or foliage of bromeliads, or ornamental plants like aloe and orchids share global market and these plant species are either gathered from cultivated or wild sources. But no data was available at the time of this assessment on the share of global market sales from wild vs cultivated plants. (viii) Gaps in ex-situ conservation of wild plant species. Botanic gardens gather live plant species from wild for conservation purpose, however, those botanical collections have focused mainly in the temperate parts of the world. For example, the PlantSearch database hosted by Botanic Gardens Conservation International indicates that 107,340 accepted species grow in botanic garden collections, representing 31% of vascular plant species. However, 93% of these species are held in temperate parts of the world. As a result, a temperate species has a 60% chance of being cultivated within the botanic garden network, whereas a tropical species has only a 25% chance. Similarly, the diversity of crop wild relatives is poorly represented in gene banks. For example, there are over 78,000 accessions representing about 688 species of crop wild relatives in gene banks, and over 70% of taxa are recommended as high priority for gathering so as to improve their representation in gene banks. However, gaps in gathering occur in the Mediterranean and the near East, Western and Southern Europe, Southeast and East Asia, and South America (Figure 3.45). (viii) Identification gaps in taxonomic groups of terrestrial animal harvesting. Some groups of terrestrial animals harvested mainly for trade lack proper identification. For example, more than 50% of all traded individuals of reptiles had no species-specific identification, and this makes implementation of species-based regulations ineffective. Further, scientific studies suggest that consumption of Didelphis marsupialis, a species of undeniable cultural significance for local communities in Latin America, but carrying a reservoir of parasites that cause severe diseases, should be the subject of further study. (ix) Insufficient information on recreation from green hunting. Green hunting that takes place with the help of tranquilizer dart guns is cheaper and less harmful compared to traditional (ix) Insufficient information on recreation from green hunting. Green hunting that takes place with the help of tranquilizer dart guns is cheaper and less harmful compared to traditional 329 hunting (section 3.3.3.4.2. However, green hunting is as of yet not a significant recreational activity. There exists insufficient information on the status, trends and/or impact of the activity with regards to its potential impact on sustainable use of terrestrial animal harvesting from wild. (x) Gap of trade of exotic pet animal species under the Convention on International Trade in Endangered Species of Wild Fauna and Flora list. Many wild animal species have been unsustainably traded to supply the international pet markets for natural breeding purpose, including rare and endemic species that are most threatened. Even with existing international regulations, the majority of species in exotic pet trade are not protected under the Convention on International Trade in Endangered Species of Wild Fauna and Flora, therefore, leaving international trade mostly unregulated and unmonitored of threatened species (Janssen & Shepherd, 2018) (section 3.3.3). (xii) Inadequate available information on wild species informal and formal trade, and consumption. Wild species are traded in informal and formal markets. Much of this trade goes unrecorded and is difficult to monitor. A complex and nuanced temporal association between the illegal and legal wild species trades exist (Tittensor et al., 2020). The gap is so great that in many cases the phrase, “we do not know what we do not know” applies. Cases where it is known that data are lacking include tropical fish for the aquarium trade, freshwater turtles and tortoises for terraria, recreational fishing (including catch and release) and spearfishing, amphibians, and reptiles (Alves, Rosa, et al., 2013; Castello, McGrath, & Beck, 2011; Costello et al., 2012; Schlaepfer, Hoover, & Dodd, 2005). Similarly, insects, especially butterflies and beetles, are harvested and traded all over the world, but there are few data about this exploitation and trade (Alan L. Yen, 2009). Further, the consumer-driven harvest of live specimens may have benefits for local peoples’ economically, however, sustainability of use is unclear. Wild meat harvest and trade are often excluded from official statistics (Pangau-Adam et al., 2012). Overall, there is much less information available on wild meat harvest in the Asian tropics, especially outside Borneo (Swamy & Pinedo-Vasquez, 2014). (ix) Insufficient information on recreation from green hunting. Green hunting that takes place with the help of tranquilizer dart guns is cheaper and less harmful compared to traditional A conspicuous knowledge gap concerning the causes of lion mortality has been identified, and this requires knowledge of both the existing population size and its dynamics over time and space (fecundity and mortality) (Macdonald et al., 2017). In addition, where markets in such species are monitored, often it is not clear whether sources are wild or domesticated. Existing data are available mainly for timber species traded in the global market (FAO, 2018a), but timber from illegal logging activities used within producing countries as well as across the transboundary are not available (Chaudhary et al., 2016). (xii) Knowledge gap in logging. Timbers are supplied to the markets; however, it is unclear to estimate which come from legal or illegal sources as well as differentiate timber from wild vs plantation sources. (xiii) Knowledge gap in non-extractive practice and uses. Assessment of knowledge gap in non-extractive practice and use is challenging as the non-extractive use of nature often does (xiii) Knowledge gap in non-extractive practice and uses. Assessment of knowledge gap in non-extractive practice and use is challenging as the non-extractive use of nature often does 330 not include species described at a species level, but frequently they appear as part of a functional group (e.g., trees in urban green spaces) or in terms of multifunctional landscapes (e.g., worship of sacred groves). Further research is especially needed to clarify the benefits of living in nature and focus on ecosystem elements. For example, in commercial wildlife watching, an increasing number of wild species such as megafauna and ‘charismatic’ wild species are integrated into tourism operations. Megafauna are well studied taxa of animals, whereas there is a lack of research on the impacts of tourism on the lesser fauna, e.g., ground- dwelling mammals, small reptiles, insects, etc. (Wolf et al., 2019). The literature on the non-extractive use of wild species for medicine and hygiene shows many positive benefits on human individuals, but there is an absence of research on the effects of wild species on human community health (Nesbitt et al., 2017). There is almost no information on the global or regional trends in the non-extractive use of wild species for human health. No research has looked at the sustained, long-term effects of nature-based therapies (Rajoo et al., 2020). (xiv) Gaps in inter-practice trade-offs and synergy. It is well known that different practices interact themselves and are connected with each other; however, the knowledge gap involves the lack of inter-practice trade-offs and synergies, such as between and among fishing, gathering, terrestrial animal harvesting, logging, and non-extractive practices across global, regional, national and local policy and program. (xv) Lack of critical linkages between nature’s contributions to people and quality of life and benefit gaps. There has been broad uptake of the critical linkages between nature’s contributions to people and quality of life. However, knowledge gap exists on the status of species and nature’s contribution to people linked to specific ecosystem functions, and interrelationships between gender equality, nature and nature’s contribution to people (IPBES, 2019). Therefore, enhanced attention is needed to develop specific variables and indicators to understand the multiple intricate ways in which peoples’ well-being / quality of life and nature’s contributions to people influence each other in a two-way feedback-oriented process (Chaplin-Kramer et al., 2019; Diaz, Demissew, Joly, Lonsdale, & Larigauderie, 2015; IPBES, 2019). (xiii) Knowledge gap in non-extractive practice and uses. Assessment of knowledge gap in non-extractive practice and use is challenging as the non-extractive use of nature often does It is also important to ascertain that such a connection draws on integration of indigenous and local knowledge and their effective participation pays judicious attention to scientific knowledge and strengthens linkage between nature and nature’s contribution to people ((Diaz et al., 2015). There are important methodological limitations to many of the studies exploring nature- based therapy or the presence of wild species on human health. Furthermore, the majority of the studies are correlative and the involvement of medical professionals is encouraged, as well as an increased diversity of study participants (Rajoo et al., 2020; Sandifer et al., 2015). The causal mechanisms that underlie the benefits people receive from health-based use of wild species is underexplored (Sandifer et al., 2015). Currently, there is limited evidence for environmental microbial exposure boosting human immune system response and no causal evidence for the phytoncides hypothesis was identified. Another important aspect to consider is that there is a poor understanding of how biodiversity affects people’s well-being and health through cultural pathways, and how that is 331 being affected by changes in the status and trends in sustainable use. A better understanding by linking biodiversity change with human culture values, well-being, and health might be profoundly important for biodiversity conservation and public health (N. E. Clark et al., 2014). It is believed that diversity of positive values is important for countering negative values and support conservation action when needed. (xvi) Inadequate economic valuation of wild species. A considerable body of valuation studies focuses on the economics of nature’s contributions to people at the global scale (e.g., carbon stocks and flows) delivered to people outside the countries where natural ecosystems and wild species occur (IPBES, 2019). However, the societal values of the gathering and use of wild species in local markets have not been properly addressed. Wild species are an integral component of ecosystems, and the value they provide in terms of services should be a standard part of ecosystem assessments (Puri, Yadav, & Joshi, 2019). Though, it needs to be recognized that there is no distinct division between wild (unmanaged) biodiversity and human managed biodiversity (Tisdell, 2015). A comprehensive assessment of the contributions (current or potential) of wild species in protected areas (terrestrial and marine), such as watching of animals, recreational tourism, recreational fishing, trophy hunting, among others to promote social and economic sustainability, besides ecological sustainability is lacking. 3.6.1. Challenges Major challenges related to status of and trends in sustainable use of wild species have been discussed. (xiii) Knowledge gap in non-extractive practice and uses. Assessment of knowledge gap in non-extractive practice and use is challenging as the non-extractive use of nature often does Yet there continues to be a need for methods, models, and approaches for integrative work (Barron et al., 2020; R. Hill et al., 2017). This approach seeks to examine the extent, range, and nature of the published literature (i.e., peer-reviewed and grey) that integrates and/or includes indigenous, local, and science-based knowledge in sustainable use of wild species research, monitoring, or management (Alexander, Provencher, Henri, Taylor, & Cooke, 2019). There is no solid mechanism developed for knowledge transfer from indigenous communities to scientific communities and vice versa, and as discussed in section 3.3.2, in many cases attempts to do so have led to issues of intellectual property and biopiracy (Barron et al., 2015; Berkes & Berkes, 2009; S. Devkota, 2006). Wild species are being used as a rich source of medicine because they produce a host of bioactive molecules, most of which probably evolved as chemical defenses against predation or infection. Wild plant species are chosen for pharmaceutical studies through different methods, one of the methods include ethnobotanical approach, i.e., indigenous uses of plant species based on indigenous and local knowledge that can offer strong clues to the biological activities of those plants (P. Cox & Balick, 1994). There are well-established drugs that were developed after scientists began to analyze the chemical constituents of plants used by indigenous peoples and local communities for medicinal or other biological effects. (xiii) Knowledge gap in non-extractive practice and uses. Assessment of knowledge gap in non-extractive practice and use is challenging as the non-extractive use of nature often does Further, several species of frogs in Africa, including endemic species (Conraua sp, Trichobatrachus sp. and Astylosternus sp.) are mainly harvested from wild used for local consumption and local trading; however, assessments of the value chains are poor, especially Central Africa regions (See section 3.3.3.3.3). (xvii) Knowledge gap on global scale of sustainable use of wild species among indigenous people and local communities. The importance of wild species that contribute to livelihood strategies, in particular for indigenous people and local communities is well recognized. However, little information exist in the available global indicator sets to comprehensively quantify the spatial and temporal scales of sustainable use of wild species occurring specifically in indigenous people and local communities across the globe; and the United Nations are aware of this gap. (xviii) Knowledge gap on quality assurance, safety and efficacy to assess traditional medicine. Wild species have been used in traditional medicinal practice for millennia. To control quality and to ensure safety and efficacy in production of traditional medicines is difficult. The World Health Organization has produced a series of technical documents in this field, including publications on good agricultural and collection practices and good manufacturing practices, along with other technical support, to assist with standardization and creation of high-quality products (WHO, 2013). The World Health Organization urges member states to cooperate with each other and to share knowledge while working to strengthen communication between conventional and traditional practitioners. Evaluation of quality, safety and efficacy based on research is needed to improve approaches to assessment of traditional medicines, a situation made difficult to remedy in light of historically inadequate public and private funding to address this growing concern (WHO, 2013). 332 (xix) Insufficient bridging of indigenous and science-based knowledge. The incorporation of multiple types of knowledge (e.g., science, indigenous knowledge, traditional ecological knowledge) is especially critical for the sustainable use of wild species, which can strengthen the evidence-base for policy advice, decision making, and environmental management (R. Hill et al., 2017). While the benefits of incorporating multiple types of knowledge in environmental research and management are many, successfully doing so has remained a challenge. In response there have been a number of recent reviews that have sought to better understand the bridging of indigenous, local, and science-based knowledge (Barron et al., 2015; Berkes, 2010; Berkes & Berkes, 2009). 3.6.1.4. Gathering A wide range of organisms are gathered worldwide for meeting a variety of needs (i.e., income, livelihoods, subsistence, social safety, identity), which are also traded in both domestic and international markets. A major challenge is that gathering practices are selective and based on specific organisms/group of organisms (e.g., ornamental fish and coral, orchids, cacti, bromeliads, succulent, palms and bamboos, medicinal plants and other wild algae, fungi and plants, wild biomass energy, edible insects and small terrestrial invertebrates, etc.). As organisms become more popular to harvest because of changing commodity chains or popular fads or trends, overharvesting can occur. The mix of temporal and spatial drivers and their direct effects on wild species are difficult to quantify since these same changes in demand lead to innovations in domestication and synthesizing similar materials. 3.6.1.3. Fishing Among fishing, small-scale fisheries are strongly connected with activities by local communities for their own consumption; and employ over 90 percent of fisheries workforce. Despite their importance, small-scale fisheries around the world are facing major challenges due to large-scale fisheries and increased global development activities as well as climate change. There is lack of data to evaluate the sustainability of small-scale fisheries on catches and measures of exploited stocks (i.e., size, proportion of juveniles caught, among others), especially over broader spatial or temporal scales. These challenges, in many cases, have placed the livelihoods, economy, food security, values and identity, and the viability of small- scale fisheries communities at risk. 3.6.1.2. Informal trade of wild species Informal trade of wild species in small quantities that do not enter the national trade or export statistics takes place through informal markets in most developing countries. Informal trade mainly includes subsistence small-scale coastal and freshwater fisheries, terrestrial animal harvesting, gathering of wild foodstuffs, medicinal plants, mushroom, and berry picking (FAO, 2019b). A challenge in such informal and largely unreported trade is that its ecological, economic and social impacts and importance to society remain invisible to decision-makers, hence unlikely to mainstream into policy-making. More research would be desirable to assess informal trade of wild species. Informal trade of wild species in small quantities that do not enter the national trade or export statistics takes place through informal markets in most developing countries. Informal trade mainly includes subsistence small-scale coastal and freshwater fisheries, terrestrial animal harvesting, gathering of wild foodstuffs, medicinal plants, mushroom, and berry picking (FAO, 2019b). A challenge in such informal and largely unreported trade is that its ecological, economic and social impacts and importance to society remain invisible to decision-makers, hence unlikely to mainstream into policy-making. More research would be desirable to assess informal trade of wild species. 3.6.1.1. Global scale and scope Fundamental challenges evaluating the role of sustainable use in biodiversity conservation and sustainable economic development pertain to the lack of guiding principles derived from analysis of spatial and temporal applications (Rands et al., 2010; Tierney et al., 2014). The scale and scope of these challenges involving sustainable use across regions and countries is immensely diverse and context specific. Studies that integrate and harmonize information from various sources and programs, where sustainable use has and has not been achieved, are needed to evaluate in order to better understand the likelihood of benefits and costs for both nature and people. However, these datasets, published from a variety of sources, are not sufficient in terms 333 of quantity or quality or both for an assessment of sustainability of harvest. Data need to be integrated and harmonized to evaluate status and trends in global use of many species. 3.6.1.6. Logging Harvesting of timber for wood carvings is a challenge because it involves destructive processes, which is not carefully monitored and remain somewhat hidden. Most commercial carving enterprises are based in homes or small production units (Cifor, 2002). In the past, wood carvings were mainly carried out to attain cultural materials, often as symbols of particular cultures or regions. Tree retention has the potential to reduce impact of logging on forest biodiversity, though determining exact levels that are required to secure long-term viable populations of different species in a natural forest in most cost-efficient conservation measures remains a major challenge for future research (Gustafsson et al., 2010). Another challenge is that harvesting has long been affected by changing tools and technology i.e., availability of axes, adzes and chisels made of iron, for example, increased both the speed with which wood could be carved and the range of species used. This includes endangered/threatened, for example sandalwood, whose use and trade are restricted by both national and international regulations. New policy instruments are emerging in some countries, such as in the United States of America, Australia and many European countries to prohibit the sale of illegally harvested wood and wood products. These regulations require operators to provide proof of certification of the identity of the species traded and the origin of their products. However, there is a mismatch between the legislated requirements and the capacity of importers to comply fully because existing methods for documenting species identity (wood anatomy and chemistry) and origin (mostly paper-based documentation, tagging) are insufficient, ambiguous and easily falsifiable (FAO, 2014c). While extensive literatures on the using of DNA analysis for forensic investigations in animal species exist, there is unfortunately a serious lack of information on wild plant species (Iyengar, 2014). 3.6.1.5. Terrestrial animal harvesting Throughout history human populations have been engaged in hunting and trapping to meet a range of nutritional, economic, medicinal, cultural and recreational needs. A major challenge is that overhunting, which is taking place at varying degrees of hunting pressure, often results 334 in faunal biomass collapses, mainly through declines of large-bodied species with low intrinsic rates of population increase, especially in Oceania, Africa and Asia. Trophy hunting is currently the subject of intense debate. However, some trophy hunting can produce simultaneous benefits of economic gains, and sustainable wild species exploitation and biodiversity conservation, even though well managed trophy hunting is rarely documented (Coad et al., 2019), and unsustainable hunting is common. Hunting becomes unsustainable when it causes species abundance on a trajectory of ongoing declines. 3.6.1.7. Non-extractive uses In the context of sustainable utilization of nature for economic and other benefits, nature tourism has created a growing demand for ‘watching wild species’, ‘un-spoilt habitat’, and ‘pristine nature’ in combination with high levels of comfort, accessibility and high-quality experiences. The ‘flagship’ species – most often the megafauna, ‘charismatic’ mammals and birds, the ‘cute and cuddly’, dangerous predators and species that are believed to display intelligence, play an important for tourism and recreation practices. The tourists’ preference of visiting a pristine natural habitat contributes to new challenges and creates pressure on the In the context of sustainable utilization of nature for economic and other benefits, nature tourism has created a growing demand for ‘watching wild species’, ‘un-spoilt habitat’, and ‘pristine nature’ in combination with high levels of comfort, accessibility and high-quality experiences. The ‘flagship’ species – most often the megafauna, ‘charismatic’ mammals and birds, the ‘cute and cuddly’, dangerous predators and species that are believed to display intelligence, play an important for tourism and recreation practices. The tourists’ preference of visiting a pristine natural habitat contributes to new challenges and creates pressure on the 335 ecosystems in general and wild species in particular. Consequently, special attention needs to be paid to the aspects of sustainability in these processes. ecosystems in general and wild species in particular. Consequently, special attention needs to be paid to the aspects of sustainability in these processes. 3.6.2. Research priorities An attempt has been made to identify common research priorities for status of and trends in sustainable use of wild species. This analysis is based on the assessment of: (i) key knowledge gaps to achieve global sustainability goals (Mastrángelo et al., 2019); (ii) knowledge gaps and challenges mentioned in this assessment; and (iii) selection of pertinent research questions that would substantially advance the goals of biodiversity conservation and sustainable development (Coleman et al., 2019). 3.6.2.1 Practices and uses In the practices and uses sectors, key prioritized areas include sustainable practices in fishing, gathering, terrestrial animal harvesting and logging as well as assessment of combined impact of wild species harvesting, fishing and hunting practices leading to habitat and global biodiversity loss. For example, gaps in fishing comprise: (i) amount of freshwater wild species harvested, consumed locally, and traded nationally and internationally; (ii) assessment of conservation status and sustainable small-scale fishery, and economically-important fish for food, live fish trade; and (iii) impact of international trade on fisheries and marine biodiversity, globally and regionally. Similarly, an emerging major challenge for future in logging remains to determine exact levels that are required to secure long-term viable populations of different species, as well as most cost-efficient implementation of these conservation measures (Gustafsson et al. 2010). It is estimated that Reduced Impact Logging provides guidelines to reduce environmental impact of logging, but it is unclear what intensity can sometimes result perverse effects; and more research is needed to clarify this type of practice. 3.6.2.2. Nature’s contributions to people & human well-being Some prioritized areas of research include: (i) evaluation of contributions of sustainable use of wild species including urban gathering to nature’s contributions to people that play key roles in regional and national scales; (ii) analysis of maximum benefits of nature tourism while minimizing adverse impacts on terrestrial, aquatic and marine ecosystems; (iv) assessment of effective livelihood support programs that meaningfully support nature conservation among marginalized communities and indigenous peoples and local communities; and (v) identification of key factors underlying win-win outcomes for sustainable use of wild species and poverty alleviation. 3.6.2.4. Social norms that affect uses and practices 3.6.2.4. Social norms that affect uses and practices There is growing interest in how socio-ecological dynamics relate to obtaining interdisciplinary and reliable data in research priorities, such as: (i) social science methods and approaches to obtaining reliable data on scale and patterns of uses of wild species; and (ii) evaluation of social norms (at local, regional and national scales) that affect use and practices including gathering and harvesting, fishing, logging, and hunting & poaching pressure. 3.6.2.3. Documenting under-researched taxa The emphasis should be on taxonomic assessment of under-researched taxa (e.g., invertebrates, insects, fungi, species that are pollinators or pest regulators, species or habitats with cultural value, rare or endemic species that are often viewed as the most important targets for 336 biodiversity conservation) being overlooked due to gaps in data, as well as inadequate enforcement of laws and principles that are particularly missing in the countries action plans. 3.6.2.4. Social norms that affect uses and practices There is growing interest in how socio-ecological dynamics relate to obtaining interdisciplinary and reliable data in research priorities, such as: (i) social science methods and approaches to obtaining reliable data on scale and patterns of uses of wild species; and (ii) evaluation of social norms (at local, regional and national scales) that affect use and practices including gathering and harvesting, fishing, logging, and hunting & poaching pressure. 3.6.2.5. Integrating indigenous local knowledge Indigenous and local knowledge research is increasingly being used to generate more accurate data on species trends, non-iconic species data and geospatially relevant data using technology. Participatory monitoring of use of wild species in close collaboration with local resource users can provide large amounts of reliable and much needed data to inform policy and management approaches in data-poor social-ecological systems. Biocultural approaches are being adopted by governments to policy that recognize both indigenous people and local communities’ territorial management practices and customary governance, thus countering the drivers of unsustainable resource use and offering alternative conceptualizations of the interrelations between people and nature (Brondízio et al., 2021). biodiversity conservation) being overlooked due to gaps in data, as well as inadequate enforcement of laws and principles that are particularly missing in the countries action plans. References Abel, D. C., & Grubbs, R. D. (2020). Shark Biology and Conservation: Essentials for Educators, Students, and Enthusiasts. Johns Hopkins University Press. Abensperg-Traun, M. (2009). CITES, sustainable use of wild species and incentive-driven conservation in developing countries, with an emphasis on southern Africa. Biological Conservation, 142(5), 948–963. https://doi.org/10.1016/j.biocon.2008.12.034 Abernethy, K., Maisels, F., & White, L. J. T. (2016). Environmental Issues in Central Africa. Annual Review of Environment and Resources, 41(1), 1–33. https://doi.org/10.1146/annurev-environ-110615-085415 Aburto, J., & Stotz, W. (2013). Learning about TURFs and natural variability: Failure of surf clam management in Chile. Ocean and Coastal Management, 71, 88–98. https://doi.org/10.1016/j.ocecoaman.2012.10.013 Acebes, J. M. V., Barr, Y., Pereda, J. M. R., & Santos, M. D. (2016). Characteristics of a previously undescribed fishery and habitat for Manta alfredi in the Philippines. Marine Biodiversity Records, 9(1). https://doi.org/10.1186/s41200-016-0098-2 Acharya, K. P., Adhikari, J., & Khanal, D. (2008). Forest Tenure Regimes and Their Impact on Livelihoods in Nepal. Journal of Forest and Livelihood, 14. Adams, C., Murrieta, R., Neves, W. A., & Harris, M. (Eds.). (2009). Amazon peasant societies in a changing environment: Political ecology, invisibility and modernity in the rainforest. New York: Springer. Adams, W. M. (2009). Green development: Environment and sustainability in a developing world (3rd ed). London ; New York: Routledge. Adamson, J. (2012). Whale as cosmos: Multi-species ethnography and contemporary indigenous cosmopolitics. Revista Canaria de Estudios Ingleses, 64, 29–45. Aerts, R., Honnay, O., & Van Nieuwenhuyse, A. (2018). Biodiversity and human health: Mechanisms and evidence of the positive health effects of diversity in nature and green spaces. British Medical Bulletin, 127(1), 5–22. https://doi.org/10.1093/bmb/ldy021 Afenyo, E. A., & Amuquandoh, F. E. (2014). Who Benefits from Community-based Ecotourism Development? Insights from Tafi Atome, Ghana. Tourism Planning & Development, 11(2), 179–190. https://doi.org/10.1080/21568316.2013.864994 Agius Darmanin, S., & Vella, A. (2019). First Central Mediterranean Scientific Field Study on Recreational Fishing Targeting the Ecosystem Approach to Sustainability. Frontiers in Marine Science, 6, 390. https://doi.org/10.3389/fmars.2019.00390 Aglionby, J. (2019, July 16). “Rhino bond” breaks new ground in conservation finance. Financial Times. Retrieved from https://www.ft.com/content/2f8bf9e6-a790-11e9- 984c-fac8325aaa04 Agnew, D. J., Pearce, J., Pramod, G., Peatman, T., Watson, R., Beddington, J. R., & Pitcher, T. J. (2009). Estimating the Worldwide Extent of Illegal Fishing. PLoS ONE, 4(2), e4570. https://doi.org/10.1371/journal.pone.0004570 Aguilar, F. X., Glavonjić, B., Hartkamp, R., Mabee, W., & Skog, K. (2015). Chapter 9: Wood Energy Market, 2014-2015. In: United Nations Forest Products Annual Market Review. 3.6.2.5. Integrating indigenous local knowledge Indigenous and local knowledge research is increasingly being used to generate more accurate data on species trends, non-iconic species data and geospatially relevant data using technology. Participatory monitoring of use of wild species in close collaboration with local resource users can provide large amounts of reliable and much needed data to inform policy and management approaches in data-poor social-ecological systems. Biocultural approaches are being adopted by governments to policy that recognize both indigenous people and local communities’ territorial management practices and customary governance, thus countering the drivers of unsustainable resource use and offering alternative conceptualizations of the interrelations between people and nature (Brondízio et al., 2021). Indigenous and local knowledge research is increasingly being used to generate more accurate data on species trends, non-iconic species data and geospatially relevant data using technology. Participatory monitoring of use of wild species in close collaboration with local resource users can provide large amounts of reliable and much needed data to inform policy and management approaches in data-poor social-ecological systems. Biocultural approaches are being adopted by governments to policy that recognize both indigenous people and local communities’ territorial management practices and customary governance, thus countering the drivers of unsustainable resource use and offering alternative conceptualizations of the interrelations between people and nature (Brondízio et al., 2021). 337 337 References In United Nations Forest Products Annual Market Review (pp. 91–104). Retrieved from http:// www.unece.org/fileadmin/DAM/timber/publications/2015- FPAMR-E.pdf 338 Aguilar, Francisco X., FAO, & UNECE (Eds.). (2018). Wood energy in the ECE Region: Data, trends and outlook in Europe, the Commonwealth of Independent States and North America. New York: United Nations. Retrieved from https://unece.org/DAM/timber/publications/SP-42-Interactive.pdf Aguilera-Alcalá, N., Morales-Reyes, Z., Martín-López, B., Moleón, M., & Sánchez-Zapata, J. A. (2020). Role of scavengers in providing non-material contributions to people. Ecological Indicators, 117, 106643. https://doi.org/10.1016/j.ecolind.2020.106643 Ahmad, K., Ahmad, M., & Weckerle, C. (2013). ETHNOBOTANICAL STUDIES OF THE EASTERN PLAINS OF TAKHT-E-SULAIMAN HILLS. PAKISTAN JOURNAL OF BOTANY, 45(1), 197–205. Ahmed, N., Rahman, S., Bunting, S. W., & Brugere, C. (2013). Socio-economic and ecological challenges of small-scale fishing and strategies for its sustainable management: A case study of the Old Brahmaputra River, Bangladesh. Singapore Journal of Tropical Geography, 34(1), 86–102. https://doi.org/10.1111/sjtg.12015 Ainsworth, C. H. (2011). Quantifying species abundance trends in the Northern Gulf of California using local ecological knowledge. Marine and Coastal Fisheries, 3(1), 190– 218. https://doi.org/10.1080/19425120.2010.549047 Ainsworth, C. H., Pitcher, T. J., & Rotinsulu, C. (2008). Evidence of fishery depletions and shifting cognitive baselines in Eastern Indonesia. Biological Conservation, 141(3), 848–859. https://doi.org/10.1016/j.biocon.2008.01.006 Akis, S., Peristianis, N., & Warner, J. (1996). Residents’ attitudes to tourism development: The case of Cyprus. Tourism Management, 17(7), 481–494. https://doi.org/10.1016/S0261- 5177(96)00066-0 Al-Abdulrazzak, D., Zeller, D., Belhabib, D., Tesfamichael, D., & Pauly, D. (2015). Total marine fisheries catches in the Persian/Arabian Gulf from 1950 to 2010. Regional Studies in Marine Science, 2, 28–34. Albrecht, M. A., & McCarthy, B. C. (2006). Comparative Analysis of Goldenseal (Hydrastis canadensis L.) Population Re-growth Following Human Harvest: Implications for Conservation. The American Midland Naturalist, 156(2), 229–236. https://doi.org/10.1674/0003-0031(2006)156[229:CAOGHC]2.0.CO;2 Alder, J., Campbell, B., Karpouzi, V., Kaschner, K., & Pauly, D. (2008). Forage Fish: From Ecosystems to Markets. Annual Review of Environment and Resources, 33(1), 153– 166. https://doi.org/10.1146/annurev.environ.33.020807.143204 Alexander, S. M., Provencher, J. F., Henri, D. A., Taylor, J. J., & Cooke, S. J. (2019). Bridging Indigenous and science-based knowledge in coastal-marine research, monitoring, and management in Canada: A systematic map protocol. Environmental Evidence, 8(1), 15. https://doi.org/10.1186/s13750-019-0159-1 Aliyu, B., Agnew, B., & Douglas, S. (2010). Croton megalocarpus (Musine) seeds as a potential source of bio-diesel. Biomass and Bioenergy, 34(10), 1495–1499. https://doi.org/10.1016/j.biombioe.2010.04.026 Allan, J. D., Abell, R., Hogan, Z., Revenga, C., Taylor, B. W., Welcomme, R. L., & Winemiller, K. (2005). Overfishing of Inland Waters. BioScience, 55(12), 1041–1051. https://doi.org/10.1641/0006-3568(2005)055[1041:OOIW]2.0.CO;2 339 Allen, C. R. References B., Brent, L. J. N., Motsentwa, T., Weiss, M. N., & Croft, D. P. (2020). Importance of old bulls: Leaders and followers in collective movements of all-male groups in African savannah elephants (Loxodonta africana). Scientific Reports, 10(1), 13996. https://doi.org/10.1038/s41598-020-70682-y Allwood, A., Vueti, E., Leblanc, L., & Bull, R. (2002). Eradication of introduced Bactrocera species (Diptera: Tephritidae) in Nauru using male annihilation and protein bait application techniques. Almeida, C., Vaz, S., Cabral, H., & Ziegler, F. (2014). Environmental assessment of sardine (Sardina pilchardus) purse seine fishery in Portugal with LCA methodology including biological impact categories. International Journal of Life Cycle Assessment, 19(2), 297–306. https://doi.org/10.1007/s11367-013-0646-5 Alonso-Fernández, A., Otero, J., Bañón, R., Campelos, J. M., Quintero, F., Ribó, J., … Molares, J. (2019). Inferring abundance trends of key species from a highly developed small-scale fishery off NE Atlantic. Fisheries Research, 209, 101–116. https://doi.org/10.1016/j.fishres.2018.09.011 Altherr, S., & Lameter, K. (2020). The Rush for the Rare: Reptiles and Amphibians in the European Pet Trade. Animals, 10(11). (WOS:000592859100001). https://doi.org/10.3390/ani10112085 Altherr, Sandra, Goyenechea, A., & Schubert, D. J. (2011). Canapés to extinction. The international trade in frogs’ legs and its ecological impact. Munich/Washington D.C.: Pro Wildlife/Defenders of Wildlife/ Animal Welfare Institute. Retrieved from https://www.prowildlife.de/wp-content/uploads/2016/02/Frogs- Legs_report_finalA4_web.pdf Altman, J. C. (2005). Brokering Aboriginal art: A critical perspective on marketing, institutions, and the state. Geelong, Vic: Deakin University. Álvarez, P., Espejel, I., Bocco, G., Cariño, M., & Seingier, G. (2018). Environmental history of Mexican North Pacific fishing communities. Ocean and Coastal Management, 165, 203–214. https://doi.org/10.1016/j.ocecoaman.2018.08.029 Alves, R. (2012). Relationship between fauna and people and the role of ethnozoology in animal conservation. Ethnobiology and Conservation, 1, 1–69. https://doi.org/10.15451/ec2012-8-1.2-1-69 Alves, R. R. N., Gonçalves, M. B. R., & Vieira, W. L. S. (2012). Caça, uso e conservação de vertebrados no semiárido Brasileiro. Tropical Conservation Science, 5(3), 394–416. https://doi.org/10.1177/194008291200500312 Alves, R. R. N., & Rosa, I. L. (2010). Trade of Animals Used in Brazilian Traditional Medicine: Trends and Implications for Conservation. Human Ecology, 38(5), 691–704. Alves, R. R. N., Rosa, I. L., Albuquerque, U. P., & Cunningham, A. B. (2013). Medicine from the Wild: An Overview of the Use and Trade of Animal Products in Traditional Medicines. In R. R. N. Alves & I. L. Rosa (Eds.), Animals in Traditional Folk Medicine (pp. 25–42). Berlin, Heidelberg: Springer Berlin Heidelberg. https://doi.org/10.1007/978-3-642-29026-8_3 Alves, R. R. N., & van Vliet, N. (2018). Chapter 10—Wild Fauna on the Menu. In R. R. Nóbrega Alves & U. P. References Albuquerque (Eds.), Ethnozoology (pp. 167–194). Academic Press. https://doi.org/10.1016/B978-0-12-809913-1.00010-7 340 Alves, R. R. N., Vieira, W. L. S., Santana, G. G., Vieira, K. S., & Montenegro, P. F. G. P. (2013). Herpetofauna Used in Traditional Folk Medicine: Conservation Implications. In R. R. N. Alves & I. L. Rosa (Eds.), Animals in Traditional Folk Medicine: Implications for Conservation (pp. 109–133). Berlin, Heidelberg: Springer. https://doi.org/10.1007/978-3-642-29026-8_7 Amato-Lourenco, L. F., Ranieri, G. R., de Oliveira Souza, V. C., Junior, F. B., Saldiva, P. H. N., & Mauad, T. (2020). Edible weeds: Are urban environments fit for foraging? Science of The Total Environment, 698, 133967. https://doi.org/10.1016/j.scitotenv.2019.133967 Ambrose, W. G., Clough, L. M., Johnson, J. C., Greenacre, M., Griffith, D. C., Carroll, M. L., & Whiting, A. (2014). Interpreting environmental change in coastal Alaska using traditional and scientific ecological knowledge. Frontiers in Marine Science, 1(SEP). https://doi.org/10.3389/fmars.2014.00040 Ambus, L., & Hoberg, G. (2011). The Evolution of Devolution: A Critical Analysis of the Community Forest Agreement in British Columbia. Society & Natural Resources, 24(9), 933–950. https://doi.org/10.1080/08941920.2010.520078 Amissah, J. N., Spiller, M., Oppong, A., Osei-Safo, D., Owusu-Darko, R., Debener, T., … Addae-Mensah, I. (2016). Genetic diversity and cryptolepine concentration of Cryptolepis sanguinolenta (Lindl). Schlt. From selected regions of Ghana. Journal of Applied Research on Medicinal and Aromatic Plants, 3(1), 34–41. https://doi.org/10.1016/j.jarmap.2015.12.005 Amoroso, R. O., Pitcher, C. R., Rijnsdorp, A. D., McConnaughey, R. A., Parma, A. M., Suuronen, P., … Jennings, S. (2018). Bottom trawl fishing footprints on the world’s continental shelves. Proceedings of the National Academy of Sciences, 115(43), E10275–E10282. https://doi.org/10.1073/pnas.1802379115 Amoroso, R., Parma, A., Pitcher, C., McConnaughey, R., & Jennings, S. (2018). Comment on “Tracking the global footprint of fisheries.” Science, 361(6404), eaat6713. Amos, A. M., & Claussen, J. D. (2009). Certification as a conservation tool in the marine aquarium trade: Challenges to effectiveness. Turnstone Consulting and Starling Resources Report, 51. Anderson, M. G., & Padding, P. I. (2015). The North American approach to waterfowl management: Synergy of hunting and habitat conservation. International Journal of Environmental Studies, 72(5), 810–829. https://doi.org/10.1080/00207233.2015.1019296 Anderson, S.C., Flemming, J. M., Watson, R., & Lotze, H. K. (2011). Rapid global expansion of invertebrate fisheries: Trends, drivers, and ecosystem effects. PLoS ONE, 6(3). Scopus. https://doi.org/10.1371/journal.pone.0014735 Anderson, Sean C, Flemming, J. M., Watson, R., & Lotze, H. K. (2011). Serial exploitation of global sea cucumber fisheries. Fish and Fisheries, 12(3), 317–339. https://doi.org/10.1111/j.1467-2979.2010.00397.x Andersson, T. D., Gothall, S. E., & Wende, B. D. (2014). References Iceland and the resumption of whaling: An empirical study of the attitudes of international tourists and whale-watch tour operators. In J. E. S. Higham & R. Williams (Eds.), Whale-watching: Sustainable 341 tourism and ecological management (pp. 95–109). New York: Cambridge University Press. Andrachuk, M., & Armitage, D. (2015). Understanding social-ecological change and transformation through community perceptions of system identity. Ecology and Society, 20(4), art26. https://doi.org/10.5751/ES-07759-200426 Andrade, D. F., de Carvalho, F. M., ilva-Ribeiro, R. B., & Dantas, J. B. (2014). Manejo Florestal Comunitário Como Estratégia de Gestão e Melhoria Da Qualidade de Vida Da População Tradicional Da Floresta Nacional Do Tapajós. In Simpósio Nacional de Áreas Protegidas, ed (pp. 249–256). Viçosa: Universidade Federal de Viçosa. Andrade-Erazo, V., & Galeano, G. (2015). La palma amarga (Sabal mauritiiformis, Arecaceae) en sistemas productivos del caribe colombiano: Estudios de caso en Piojó, Atlántico. Acta Biológica Colombiana, 21(1). https://doi.org/10.15446/abc.v21n1.47280 Angelsen, A., Jagger, P., Babigumira, R., Belcher, B., Hogarth, N. J., Bauch, S., … Wunder, S. (2014). Environmental income and rural livelihoods: A global-comparative analysis. World Development, 64, S12–S28. Anna, Z. (2017). Indonesian shrimp resource accounting for sustainable stock management. Biodiversitas, 18(1), 248–256. https://doi.org/10.13057/biodiv/d180132 Ansell, S., & Koenig, J. (2011). CyberTracker: An integral management tool used by rangers in the Djelk Indigenous Protected Area, central Arnhem Land, Australia. Ecological Management and Restoration, 12(1), 13–25. https://doi.org/10.1111/j.1442- 8903.2011.00575.x Antonelli, A., Fry, C., Smith, R. J., Simmonds, M. S. J., Kersey, P. J., Pritchard, H. W., … Zhang, B. G. (2020). State of the World’s Plants and Fungi 2020. Royal Botanic Gardens, Kew. https://doi.org/10.34885/172 Antonova, A. S. (2016). The rhetoric of “responsible fishing”: Notions of human rights and sustainability in the European Union’s bilateral fishing agreements with developing states. Marine Policy, 70, 77–84. https://doi.org/10.1016/j.marpol.2016.04.008 Antunes, A. P., Fewster, R. M., Venticinque, E. M., Peres, C. A., Levi, T., Rohe, F., & Shepard, G. H. (2016). Empty forest or empty rivers? A century of commercial hunting in Amazonia. Science Advances, 2(10), e1600936. https://doi.org/10.1126/sciadv.1600936 Antunes, C., Cobo, F., & Araújo, M. J. (2015). Iberian inland fisheries. In J. F. Craig (Ed.), Freshwater Fisheries Ecology (pp. 268–282). Oxford, UK: Wiley-Blackwell. https://doi.org/10.1002/9781118394380.ch23 Aqorau, T. (2009). Recent Developments in Pacific Tuna Fisheries: The Palau Arrangement and the Vessel Day Scheme. The International Journal of Marine and Coastal Law, 24(3), 557–581. https://doi.org/10.1163/157180809X455647 Araujo Catelani, P., Petry, A. C., Mayer Pelicice, F., & Azevedo Matias Silvano, R. (2021). References Fishers’ knowledge on the ecology, impacts and benefits of the non-native peacock bass Cichla kelberi in a coastal river in southeastern Brazil. Ethnobiology and Conservation. https://doi.org/10.15451/ec2020-09-10.04-1-16 Araújo, J. G. de, Santos, M. A. S. dos, Rebello, F. K., Prang, G., Almeida, M. C. de, & Isaac, V. J. (2020). Economic analysis of the threats posed to the harvesting of ornamental 342 fish by the operation of the Belo Monte hydroelectric dam in northern Brazil. Fisheries Research, 225, 105483. https://doi.org/10.1016/j.fishres.2019.105483 fish by the operation of the Belo Monte hydroelectric dam in northern Brazil. Fisheries Research, 225, 105483. https://doi.org/10.1016/j.fishres.2019.105483 Ares, E. (2019). Trophy Hunting (Briefing Paper No. 7908). London, U.K.: House of Commons Library. Retrieved from House of Commons Library website: https://researchbriefings.files.parliament.uk/documents/CBP-7908/CBP-7908.pdf Arets, E. J. M. M., Van der Meer, P. J., Verwer, C. C., Hengeveld, G. M., Tolkamp, G. W., Nabuurs, G. J., & Van Oorschot, M. (2011). Global wood production: Assessment of industrial round wood supply from forest management systems in different global regions (No. 1808). Alterra, Wageningen-UR. (No. 1808). Alterra: Wageningen-UR. Arias-arévalo, P., Gómez-baggethun, E., Martín-lópez, B., & Pérez-rincón, M. (2018). Widening the Evaluative Space for Ecosystem Services: A Taxonomy of Plural Values and Valuation Methods. (May). https://doi.org/10.3197/096327118X15144698637513 Arlinghaus, R., Tillner, R., & Bork, M. (2015). Explaining participation rates in recreational fishing across industrialised countries. Fisheries Management and Ecology, 22(1), 45– 55. https://doi.org/10.1111/fme.12075 Arlinghaus, Robert, & Cooke, S. J. (2009). Recreational Fisheries: Socioeconomic Importance, Conservation Issues and Management Challenges. In B. Dickson, J. Hutton, & W. M. Adams (Eds.), Recreational Hunting, Conservation and Rural Livelihoods (pp. 39–58). Oxford, UK: Wiley-Blackwell. https://doi.org/10.1002/9781444303179.ch3 Arlinghaus, Robert, Cooke, S. J., Lyman, J., Policansky, D., Schwab, A., Suski, C., … Thorstad, E. B. (2007). Understanding the Complexity of Catch-and-Release in Recreational Fishing: An Integrative Synthesis of Global Knowledge from Historical, Ethical, Social, and Biological Perspectives. Reviews in Fisheries Science, 15(1–2), 75– 167. https://doi.org/10.1080/10641260601149432 Armesto, J. J., Smith-Ramirez, C., & Rozzi, R. (1999). ABARE and Jaakko Pöyry (1999) Global Outlook for Plantations. Canberra, ACT: Australian Bureau of Agricultural and Resource Economics. Retrieved from http://www.fao.org/forestry/42688- 0a52e579757b86dd833ee20ba6e567078.pdf Armitage, D., & Marschke, M. (2013). Assessing the future of small-scale fishery systems in coastal Vietnam and the implications for policy. Environmental Science & Policy, 27, 184–194. https://doi.org/10.1016/j.envsci.2012.12.015 Armitage, D., Marschke, M., & van Tuyen, T. (2011). Early-stage transformation of coastal marine governance in Vietnam? Marine Policy, 35(5), 703–711. https://doi.org/10.1016/j.marpol.2011.02.011 Arnberger, A., Ebenberger, M., Schneider, I. Arnett, E. B., & Southwick, R. (2015). Economic and social benefits of hunting in North America. International Journal of Environmental Studies, 72(5), 734–745. https://doi.org/10.1080/00207233.2015.1033944 References E., Cottrell, S., Schlueter, A. C., von Ruschkowski, E., … Gobster, P. H. (2018). Visitor Preferences for Visual Changes in Bark Beetle-Impacted Forest Recreation Settings in the United States and Germany. Environmental Management, 61(2), 209–223. https://doi.org/10.1007/s00267-017- 0975-4 Arnett, E. B., & Southwick, R. (2015). Economic and social benefits of hunting in North America. International Journal of Environmental Studies, 72(5), 734–745. https://doi.org/10.1080/00207233.2015.1033944 343 Arnold, J. E. M., Köhlin, G., & Persson, R. (2006). Woodfuels, livelihoods, and policy interventions: Changing Perspectives. World Development, 34(3), 596–611. https://doi.org/10.1016/j.worlddev.2005.08.008 Arnold, J. E. M., Köhlin, G., Persson, R., & Shepherd, G. (2003). Fuelwood Revisited: What Has Changed in the Last Decade? (CIFOR Occasional Paper, 39). Retrieved from http://hdl.handle.net/10535/4692 Arora, D. (2008a). California Porcini: Three New Taxa, Observations on Their Harvest, and the Tragedy of No Commons. Economic Botany, 62(3), 356–375. https://doi.org/10.1007/s12231-008-9050-7 Arora, D. (2008b). The Houses That Matsutake Built. Economic Botany, 62(3), 278–290. https://doi.org/10.1007/s12231-008-9048-1 Arrington, A. (2021). Urban foraging of five non-native plants in NYC: Balancing ecosystem services and invasive species management. Urban Forestry & Urban Greening, 58, 126896. https://doi.org/10.1016/j.ufug.2020.126896 Arroyo-Quiroz, I., García-Barrios, R., Argueta-Villamar, A., Smith, R. J., & Salcido, R. P. G. (2017). Local Perspectives on Conflicts with Wildlife and Their Management in the Sierra Gorda Biosphere Reserve, Mexico. Journal of Ethnobiology, 37(4), 719–742. https://doi.org/10.2993/0278-0771-37.4.719 Artaud, H. (2016). Spelling out Sensations: Reflections on the ways in which the Natural Environment can infiltrate Meaning-Making. The Senses and Society, 11(3), 262–274. https://doi.org/10.1080/17458927.2016.1195109 Artaud, H. (2020). Piéger la rencontre. Billebaude, 16-L'art du leurre, 48–56. Artelle, K. A., Reynolds, J. D., Treves, A., Walsh, J. C., Paquet, P. C., & Darimont, C. T. (2018). Hallmarks of science missing from North American wildlife management. Science Advances, 4(3), eaao0167. https://doi.org/10.1126/sciadv.aao0167 Arts, B., & de Koning, J. (2017). Community Forest Management: An Assessment and Explanation of its Performance Through QCA. World Development, 96, 315–325. https://doi.org/10.1016/j.worlddev.2017.03.014 Arzel, P. (1987). Les Goémoniers. Douarnenez (France): Le Chasse-marée. Ashwell, D., & Walston, N. (2008). An overview of the use and trade of plants and animals in traditional medicine systems in Cambodia (p. 112) [TRAFFIC Southeast Asia, Greater Mekong Programme, Ha Noi, Viet Nam]. Retrieved from http://www.trafficj.org/publication/08_medical_plants_Cambodia.pdf Ashworth, M. (2017). How does stress affect us? Retrieved January 6, 2021, from PsychCentral website: https://psychcentral.com/lib/how-does-stress-affect-us#1 Asikainen, A., Anttila, P., Heinimö, J., Smith, T., Stupak, I., & Quirino, W. F. (2010). Forests and bioenergy production (Vol. 25, pp. 183-200). IUFRO. In IUFRO World Series Vol. 25. References Forests and Society-Responding to Global Drivers of Change (pp. 183–200). Vienna: International Union of Forest Research Organizations (IUFRO). Askerlund, P., & Almers, E. (2016). Forest gardens – new opportunities for urban children to understand and develop relationships with other organisms. Urban Forestry & Urban Greening, 20, 187–197. https://doi.org/10.1016/j.ufug.2016.08.007 344 Asner, G. P., Rudel, T. K., Aide, T. M., Defries, R., & Emerson, R. (2009). A Contemporary Assessment of Change in Humid Tropical Forests. Conservation Biology, 23(6), 1386– 1395. https://doi.org/10.1111/j.1523-1739.2009.01333.x Astuti, R. (1995). "The Vezo are not a kind of people”: Identity, difference, and “ethnicity” among a fishing people of western Madagascar. American Ethnologist, 22(3), 464–482. Aswani, S., & Hamilton, R. J. (2004). Integrating indigenous ecological knowledge and customary sea tenure with marine and social science for conservation of bumphead parrotfish (Bolbometopon muricatum) in the Roviana Lagoon, Solomon Islands. Environmental Conservation, 31(1), 69–83. https://doi.org/10.1017/S037689290400116X Auliya, M., Altherr, S., Ariano-Sanchez, D., Baard, E. H., Brown, C., Brown, R. M., … Ziegler, T. (2016). Trade in live reptiles, its impact on wild populations, and the role of the European market. Biological Conservation, 204, 103–119. https://doi.org/10.1016/j.biocon.2016.05.017 Ault, J. S., Bohnsack, J. A., Smith, S. G., & Luo, J. (2005). Towards sustainable multispecies fisheries in the Florida, USA, coral reef ecosystem. Bulletin of Marine Science, 76(2), 595–622. Ayunda, N., Sapota, M. R., & Pawelec, A. (2018). The impact of small-scale fisheries activities toward fisheries sustainability in Indonesia. In T. Zielinski, I. Sagan, & W. Surosz (Eds.), Interdisciplinary Approaches for Sustainable Development Goals: Economic Growth, Social Inclusion and Environmental Protection. Springer International Publishing. https://doi.org/10.1007/978-3-319-71788-3_11 Azzurro, E., Sbragaglia, V., Cerri, J., Bariche, M., Bolognini, L., Ben Souissi, J., … Moschella, P. (2019). Climate change, biological invasions, and the shifting distribution of Mediterranean fishes: A large-scale survey based on local ecological knowledge. Global Change Biology, 25(8), 2779–2792. https://doi.org/10.1111/gcb.14670 Azzurro, Ernesto, & Cerri, J. (2021). Participatory mapping of invasive species: A demonstration in a coastal lagoon. Marine Policy, 126, 104412. https://doi.org/10.1016/j.marpol.2021.104412 Azzurro, Ernesto, Moschella, P., & Maynou, F. (2011). Tracking Signals of Change in Mediterranean Fish Diversity Based on Local Ecological Knowledge. PLoS ONE, 6(9), e24885. https://doi.org/10.1371/journal.pone.0024885 Baeta, M., Breton, F., Ubach, R., & Ariza, E. (2018). A socio-ecological approach to the declining Catalan clam fisheries. Ocean and Coastal Management, 154, 143–154. https://doi.org/10.1016/j.ocecoaman.2018.01.012 Bahuchet, S., & de Garine, I. (1990). The art of trapping in the rain forest. In S. Bahuchet, C. M. Hladik, I. References de Garine, & Centre national de la recherche scientifique (France) (Eds.), Food and Nutrition in African Rain Forests (pp. 24–25). Paris, France: UNESCO / MAB. Baigún, C., Minotti, P., & Oldani, N. (2013). Assessment of sábalo (Prochilodus lineatus) fisheries in the lower Paraná river basin (Argentina) based on hydrological, biological, and fishery indicators. Neotropical Ichthyology, 11(1), 199–210. https://doi.org/10.1590/S1679-62252013000100023 345 Bailis, Rob, Wang, Y., Drigo, R., Ghilardi, A., & Masera, O. (2017). Getting the numbers right: Revisiting woodfuel sustainability in the developing world. Environmental Research Letters, 12(11), 115002. https://doi.org/10.1088/1748-9326/aa83ed Bailis, Robert, Drigo, R., Ghilardi, A., & Masera, O. (2015). The carbon footprint of traditional woodfuels. Nature Climate Change, 5(3), 266–272. https://doi.org/10.1038/nclimate2491 Bailis, Robert, Ezzati, M., & Kammen, D. M. (2005). Mortality and Greenhouse Gas Impacts of Biomass and Petroleum Energy Futures in Africa. Science, 308(5718), 98–103. https://doi.org/10.1126/science.1106881 Baird, B. A., Kuhar, C. W., Lukas, K. E., Amendolagine, L. A., Fuller, G. A., Nemet, J., … Schook, M. W. (2016). Program animal welfare: Using behavioral and physiological measures to assess the well-being of animals used for education programs in zoos. Applied Animal Behaviour Science, 176, 150–162. https://doi.org/10.1016/j.applanim.2015.12.004 Baird, I. G., & Flaherty, M. S. (2005). Mekong River Fish Conservation Zones in southern Laos: Assessing effectiveness using local ecological knowledge. Environmental Management, 36(3), 439–454. https://doi.org/10.1007/s00267-005-3093-7 Baker, W. J., & Dransfield, J. (2016). Beyond Genera Palmarum: Progress and prospects in palm systematics. Botanical Journal of the Linnean Society, 182(2), 207–233. https://doi.org/10.1111/boj.12401 Baker-Médard, M., & Faber, J. (2020). Fins and (Mis)fortunes: Managing shark populations for sustainability and food sovereignty. Marine Policy, 113. Scopus. https://doi.org/10.1016/j.marpol.2019.103805 Bakes, M. J., & Nichols, P. D. (1995). Lipid, fatty acid and squalene composition of liver oil from six species of deep-sea sharks collected in southern australian waters. Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology, 110(1), 267–275. https://doi.org/10.1016/0305-0491(94)00083-7 Bakun, A., Babcock, E. A., Lluch-Cota, S. E., Santora, C., & Salvadeo, C. J. (2010). Issues of ecosystem-based management of forage fisheries in “open” non-stationary ecosystems: The example of the sardine fishery in the Gulf of California. Reviews in Fish Biology and Fisheries, 20(1), 9–29. https://doi.org/10.1007/s11160-009-9118-1 Baldus, R. D., Damm, G. R., & Wollscheid, K.-U. (Eds.). (2008). Best practices in sustainable hunting a guide to best practices from around the world. Budakeszi: CIC. Retrieved from http://webdoc.sub.gwdg.de/ebook/serien/yo/CIC/01.pdf Balehegn, M., Balehey, S., Fu, C., & Liang, W. (2019). References Indigenous weather and climate forecasting knowledge among Afar pastoralists of north eastern Ethiopia: Role in adaptation to weather and climate variability. Pastoralism, 9(1), 8. https://doi.org/10.1186/s13570-019-0143-y Ballantyne, M., & Pickering, C. M. (2013). Tourism and recreation: A common threat to IUCN red-listed vascular plants in Europe. Biodiversity and Conservation, 22(13–14), 3027– 3044. https://doi.org/10.1007/s10531-013-0569-2 Ballantyne, R., & Packer, J. (2002). Nature-based Excursions: School Students’ Perceptions of Learning in Natural Environments. International Research in Geographical and 346 11(3), 218–236. Education, Ballantyne, R., Packer, J., Hughes, K., & Dierking, L. (2007). Conservation learning in wildlife tourism settings: Lessons from research in zoos and aquariums. Environmental Education Research, 13(3), 367–383. https://doi.org/10.1080/13504620701430604 Balmford, A., Beresford, J., Green, J., Naidoo, R., Walpole, M., & Manica, A. (2009). A global perspective on trends in nature-based tourism. PLoS Biol, 7(6), e1000144. https://doi.org/10.1371/journal.pbio.1000144 Balmford, A., Green, J. M., Anderson, M., Beresford, J., Huang, C., Naidoo, R., … Manica, A. (2015). Walk on the wild side: Estimating the global magnitude of visits to protected areas. PLoS Biol, 13(2), e1002074. https://doi.org/10.1371/journal.pbio.1002074 Ban, N. C., Eckert, L., McGreer, M., & Frid, A. (2017). Indigenous knowledge as data for modern fishery management: A case study of Dungeness crab in Pacific Canada. Ecosystem Health and Sustainability, 3(8), 1379887. https://doi.org/10.1080/20964129.2017.1379887 Banjade, M., Paudel, N. S., Karki, R., Sunam, R., & Paudyal, B. (2011). Putting timber into the hot seat: Discourse, policy and contestations over timber in Nepal. Discussion Paper Series 11: 2. Forest Action,. 16 p. (p. 16) [Discussion Paper Series 11: 2. Forest Action]. Kathmandu, Nepal. Bank Indonesia. (2020). Table V.13. Value of Non-Oil and Gas Export by Commodity. Jakarta, Indonesia: Bank Indonesia. Barange, M., Bernal, M., Cercole, M. C., Cubillos, L. A., Daskalov, G. M., Cunningham, C. L., … others. (2009). Current trends in the assessment and management of stocks. In Climate change and small pelagic fish (pp. 191–255). Cambridge University Press. Barber, C. V., & Talbott, K. (2003). The Chainsaw and the Gun: The Role of the Military in Deforesting Indonesia. Journal of Sustainable Forestry, 16(3–4), 131–160. https://doi.org/10.1300/J091v16n03_07 Barbosa-Filho, M. L. V., de Souza, G. B. G., Lopes, S. D. F., Siciliano, S., Hauser Davis, R. A., & Mourão, J. D. S. (2020). Evidence of shifting baseline and Fisher judgment on lane snapper (Lutjanus synagris) management in a Brazilian marine protected area. Ocean and Coastal Management, 183. https://doi.org/10.1016/j.ocecoaman.2019.105025 Barbosa-Filho, M. L. V., Hauser-Davis, R. A., Siciliano, S., Dias, T. L. P., Alves, R. References R. N., & Costa-Neto, E. M. (2019). Historical shark meat consumption and trade trends in a global richness hotspot. Ethnobiology Letters, 10(1), 97–103. https://doi.org/10.14237/ebl.10.1.2019.1560 Barboza, R. R. D., Lopes, S. F., Souto, W. M. S., Fernandes-Ferreira, H., & Alves, R. R. N. (2016). The role of game mammals as bushmeat In the Caatinga, northeast Brazil. Ecology and Society, 21(2), art2. https://doi.org/10.5751/ES-08358-210202 Barceló, C. M., Butí, E., Gras, A., Orriols, M., & Vallès, J. (2019). Ethnobotany in a “Masterpiece of the Oral and Intangible Heritage of Humanity”: Plants in “la Patum” Festivity (Berga, Catalonia, Iberian Peninsula)1. Economic Botany, 1–8. https://doi.org/10.1007/s12231-019-09474-z 347 Barkin, D. (2003). Alleviating Poverty Through Ecotourism: Promises and Reality in the Monarch Butterfly Reserve of Mexico. Environment, Development and Sustainability, 5(3/4), 371–382. https://doi.org/10.1023/A:1025725012903 Barnes-Mauthe, M., Oleson, K. L. L., & Zafindrasilivonona, B. (2013). The total economic value of small-scale fisheries with a characterization of post-landing trends: An application in Madagascar with global relevance. Fisheries Research, 147, 175–185. Scopus. https://doi.org/10.1016/j.fishres.2013.05.011 Barnett, R. (2000). Food for thought: The utilization of wild meat in eastern and southern Africa. Retrieved from https://portals.iucn.org/library/node/7963 Barney, J. N., & DiTomaso, J. M. (2010). Bioclimatic predictions of habitat suitability for the biofuel switchgrass in North America under current and future climate scenarios. Biomass and Bioenergy, 34(1), 124–133. https://doi.org/10.1016/j.biombioe.2009.10.009 Barron, E. S. (2010). Situated knowledge and fungal conservation: Morel mushroom management in the mid-Atlantic region of the United States. Rutgers The State University of New Jersey-New Brunswick. Barron, E. S. (2011). The emergence and coalescence of fungal conservation social networks in Europe and the U.S.A. Fungal Ecology, 4(2), 124–133. https://doi.org/10.1016/j.funeco.2010.09.009 Barron, E. S., Hartman, L., & Hagemann, F. (2020). From place to emplacement: The scalar politics of sustainability. Local Environment, 25(6), 447–462. https://doi.org/10.1080/13549839.2020.1768518 Barron, E. S., Sthultz, C., Hurley, D., & Pringle, A. (2015). Names matter: Interdisciplinary research on taxonomy and nomenclature for ecosystem management. Progress in Physical Geography, 39(5), 640–660. Barros, F. B., & de Aguiar Azevedo, P. (2014). Common opossum (Didelphis marsupialisLinnaeus, 1758): Food and medicine for people in the Amazon. Journal of Ethnobiology and Ethnomedicine, 10(1), 65. https://doi.org/10.1186/1746-4269-10-65 Barthem, R. B., Goulding, M., Leite, R. G., Cañas, C., Forsberg, B., Venticinque, E., … Mercado, A. (2017). Goliath catfish spawning in the far western Amazon confirmed by the distribution of mature adults, drifting larvae and migrating juveniles. Scientific Reports, 7(1), 41784. https://doi.org/10.1038/srep41784 Bartholomew, A., & Bohnsack, J. A. (2005). References A Review of Catch-and-Release Angling Mortality with Implications for No-take Reserves. Reviews in Fish Biology and Fisheries, 15(1–2), 129–154. https://doi.org/10.1007/s11160-005-2175-1 Bartley, D., De Graaf, G., Valbo‐Jørgensen, J., & Marmulla, G. (2015). Inland capture fisheries: Status and data issues. Fisheries Management and Ecology, 22(1), 71–77. Bastari, A., Beccacece, J., Ferretti, F., Micheli, F., & Cerrano, C. (2017). Local ecological knowledge indicates temporal trends of benthic invertebrates species of the Adriatic Sea. Frontiers in Marine Science, 4(MAY). Scopus. https://doi.org/10.3389/fmars.2017.00157 Bataille-Benguigui, M.-C. (1981). Bataille-Benguigui M-C. (1981) « La capture au requin du nœud coulant aux îles Tonga: Persistance et changements dans l’observation des 348 interdits », Journal de la Société des océanistes, n°72-73, tome 37, 1981. La pêche traditionnelle en Océanie. Pp. 239-250. Journal de La Société Des Océanistes, 37(72– 73), 239–250. https://doi.org/10.3406/jso.1981.3064 Bateman, P. W., & Fleming, P. A. (2017). Are negative effects of tourist activities on wildlife over-reported? A review of assessment methods and empirical results. Biological Conservation, 211, 10–19. https://doi.org/10.1016/j.biocon.2017.05.003 Battaglia, P., Andaloro, F., Consoli, P., Pedà, C., Raicevich, S., Spagnolo, M., & Romeo, T. (2017). Baseline data to characterize and manage the small-scale fishery (SSF) of an oncoming Marine Protected Area (Cape Milazzo, Italy) in the western Mediterranean Sea. Ocean and Coastal Management, 148, 231–244. https://doi.org/10.1016/j.ocecoaman.2017.08.014 Bauer, T. (2016). Cartographie Des Acteurs de La Foresterie Communautaire En RDC – Un Aperçu Des Intervenants, de La Vision et Les Défis Dans Sa Mise En Œuvre. Kinshasa, DRC: GIZ - Programme Biodiversité et Forêts. Retrieved from GIZ - Programme Biodiversité et Forêts website: https://www.researchgate.net/profile/Tina_Bauer2/publication/334729658_Cartograp hie_des_acteurs_de_la_Foresterie_Communautaire_en_RDC_- _un_apercu_des_intervenants_de_la_vision_et_les_defis_dans_sa_mise_en_oeuvre/li nks/5d3d3c16a6fdcc370a660f69/Cartographie-des-acteurs-de-la-Foresterie- Communautaire-en-RDC-un-apercu-des-intervenants-de-la-vision-et-les-defis-dans- sa-mise-en-oeuvre.pdf Baumert, S., Luz, A. C., Fisher, J., Vollmer, F., Ryan, C. M., Patenaude, G., … Macqueen, D. (2016). Charcoal supply chains from Mabalane to Maputo: Who benefits? Energy for Sustainable Development, 33, 129–138. https://doi.org/10.1016/j.esd.2016.06.003 Bavinck, M., Jentoft, S., & Scholtens, J. (2018). Fisheries as social struggle: A reinvigorated social science research agenda. Marine Policy, 94, 46–52. https://doi.org/10.1016/j.marpol.2018.04.026 Baynes, J., Herbohn, J., Smith, C., Fisher, R., & Bray, D. (2015). Key factors which influence the success of community forestry in developing countries. Global Environmental Change, 35, 226–238. https://doi.org/10.1016/j.gloenvcha.2015.09.011 Beauchamp, E., & Ingram, V. (2011). Impacts of community forests on livelihoods in Cameroon: Lessons from two case studies. International Forestry Review, 13(4), 389– 403. https://doi.org/10.1505/146554811798811371 Beaudreau, A. H., & Levin, P. S. (2014). Advancing the use of local ecological knowledge for assessing data-poor species in coastal ecosystems. Ecological Applications, 24(2), 244– 256. Scopus. References https://doi.org/10.1890/13-0817.1 Beaugrand, G. (2004). The North Sea regime shift: Evidence, causes, mechanisms and consequences. Progress in Oceanography, 60(2–4), 245–262. https://doi.org/10.1016/j.pocean.2004.02.018 Becerril‐García, E. E., Hoyos‐Padilla, E. M., Micarelli, P., Galván‐Magaña, F., & Sperone, E. (2019). The surface behaviour of white sharks during ecotourism: A baseline for monitoring this threatened species around Guadalupe Island, Mexico. Aquatic Conservation: Marine and Freshwater Ecosystems, 29(5), 773–782. https://doi.org/10.1002/aqc.3057 349 Becker, K., & Makkar, H. P. S. (2008). Jatropha curcas: A potential source for tomorrow’ s oil and biodiesel. Lipid Technology, 20(5), 104–107. https://doi.org/10.1002/lite.200800023 Beese, W. J., Deal, J., Dunsworth, B. G., Mitchell, S. J., & Philpott, T. J. (2019). Two decades of variable retention in British Columbia: A review of its implementation and effectiveness for biodiversity conservation. Ecological Processes, 8(1), 33. https://doi.org/10.1186/s13717-019-0181-9 Beeton, S. (2008). Community development through tourism. Collingwood, Victoria: Landlinks Press. Begossi, A., Salivonchyk, S., Lopes, P. F. M., & Silvano, R. A. M. (2016). Fishers’ knowledge on the coast of Brazil. Journal of Ethnobiology and Ethnomedicine, 12(1). https://doi.org/10.1186/s13002-016-0091-1 Begossi, A., Salivonchyk, S. V., Araujo, L. G., Andreoli, T. B., Clauzet, M., Martinelli, C. M., … Silvano, R. A. M. (2011). Ethnobiology of snappers (Lutjanidae): Target species and suggestions for management. Journal of Ethnobiology and Ethnomedicine, 7. https://doi.org/10.1186/1746-4269-7-11 Begossi, A., Salyvonchyk, S., Glamuzina, B., De Souza, S. P., Lopes, P. F. M., Priolli, R. H. G., … Silvano, R. A. M. (2019). Fishers and groupers (Epinephelus marginatus and E. morio) in the coast of Brazil: Integrating information for conservation. Journal of Ethnobiology and Ethnomedicine, 15(1). https://doi.org/10.1186/s13002-019-0331-2 Begossi, Alpina, Salivonchyk, S., & Silvano, R. (2016). Collaborative Research on dusky grouper (Epinephelus marginatus): Catches from the small-scale fishery of Copacabana Beach, Rio de Janeiro, Brazil. J Coast Zone Manag, 19(428), 21–23. Belcher, B, Braedt, O., Campbell, B., Cunningham, A., Choge, S., De Jong, W., … Standa- Gunda, W. (2002). Planning for woodcarving in the 21st century. Center for International Forestry Research (CIFOR). https://doi.org/10.17528/cifor/001164 Belhabib, D., Campredon, P., Lazar, N., Sumaila, U. R., Baye, B. C., Kane, E. A., & Pauly, D. (2016). Best for pleasure, not for business: Evaluating recreational marine fisheries in West Africa using unconventional sources of data. Palgrave Communications, 2. https://doi.org/10.1057/palcomms.2015.50 Belhabib, D., Sumaila, U. R., & Pauly, D. (2015). Feeding the poor: Contribution of West African fisheries toemployment and food security. Ocean and Coastal Management, 111, 72–81. https://doi.org/10.1016/j.ocecoaman.2015.04.010 Belhabib, Dyhia, Greer, K., & Pauly, D. (2018). References Trends in industrial and artisanal catch per effort in West African fisheries. Conservation Letters, 11(1), e12360. Bell, J. D., Allain, V., Allison, E. H., Andréfouët, S., Andrew, N. L., Batty, M. J., … Williams, P. (2015). Diversifying the use of tuna to improve food security and public health in Pacific Island countries and territories. Marine Policy, 51, 584–591. https://doi.org/10.1016/j.marpol.2014.10.005 Bell, J. D., & Secretariat of the Pacific Community (Eds.). (2011). Vulnerability of tropical Pacific fisheries and aquaculture to climate change: Summary for Pacific island countries and territories. Noumea, New Caledonia: Secretariat of the Pacific Community. 350 Belsky, J. M. (2009). Misrepresenting Communities: The Politics of Community-Based Rural Ecotourism in Gales Point Manatee, Belize. Rural Sociology, 64(4), 641–666. https://doi.org/10.1111/j.1549-0831.1999.tb00382.x Belton, B., Hossain, M. A. R., & Thilsted, S. H. (2018). Labour, Identity and Wellbeing in Bangladesh’s Dried Fish Value Chains. In D. S. Johnson, T. G. Acott, N. Stacey, & J. Urquhart (Eds.), Social Wellbeing and the Values of Small-scale Fisheries (pp. 217– 241). Cham: Springer International Publishing. https://doi.org/10.1007/978-3-319- 60750-4_10 Belton, B., & Thilsted, S. H. (2014). Fisheries in transition: Food and nutrition security implications for the global South. Global Food Security, 3(1), 59–66. https://doi.org/10.1016/j.gfs.2013.10.001 Bender, M. G., Floeter, S. R., & Hanazaki, N. (2013). Do traditional fishers recognise reef fish species declines? Shifting environmental baselines in Eastern Brazil. Fisheries Management and Ecology, 20(1), 58–67. https://doi.org/10.1111/fme.12006 Bender, M. G., Machado, G. R., De Azevedo Silva, P. J., Floeter, S. R., Monteiro-Netto, C., Luiz, O. J., & Ferreira, C. E. L. (2014). Local ecological knowledge and scientific data reveal overexploitation by multigear artisanal fisheries in the Southwestern Atlantic. PLoS ONE, 9(10). https://doi.org/10.1371/journal.pone.0110332 Béné, C., Macfadyen, G., & Allison, E. H. (2007). Increasing the contribution of small-scale fisheries to poverty alleviation and food security. Rome: Food and Agriculture Organization of the United Nations. Bengtsson, L. P., & Whitaker, J. H. (1988). Farm structures in tropical climates. A textbook for structural engineering and design. FAO,. Retrieved from FAO, website: https://www.fao.org/3/s1250e/S1250E00.htm Benítez, G. (2011). Animals used for medicinal and magico-religious purposes in western Granada Province, Andalusia (Spain). Journal of Ethnopharmacology, 137(3), 1113– 1123. https://doi.org/10.1016/j.jep.2011.07.036 Benjaminsen, T. A., & Svarstad, H. (2021). Discourses and Narratives on Environment and Development: The Example of Bioprospecting. In T. A. Benjaminsen & H. Svarstad (Eds.), Political Ecology: A Critical Engagement with Global Environmental Issues (pp. 59–87). Cham: Springer International Publishing. References https://doi.org/10.1007/978-3- 030-56036-2_3 Benkendorff, K., Rudd, D., Nongmaithem, B., Liu, L., Young, F., Edwards, V., … Abbott, C. (2015). Are the Traditional Medical Uses of Muricidae Molluscs Substantiated by Their Pharmacological Properties and Bioactive Compounds? Marine Drugs, 13(8), 5237– 5275. https://doi.org/10.3390/md13085237 Bennett, A., Patil, P., Kleisner, K., Rader, D., Virdin, J., & Basurto, X. (2018). Contribution of Fisheries to Food and Nutrition Security: Current Knowledge, Policy, and Research [NI Report 18-02]. Durham, NC: Duke University. Retrieved from https://nicholasinstitute.duke.edu/sites/default/files/publications/contribution_of_fishe ries_to_food_and_nutrition_security_0.pdf Bennett, E. M., Peterson, G. D., & Gordon, L. J. (2009). Understanding relationships among multiple ecosystem services: Relationships among multiple ecosystem services. 351 Ecology Letters, 12(12), 1394–1404. https://doi.org/10.1111/j.1461- 0248.2009.01387.x https://doi.org/10.1111/j.1461- Ecology Letters, 0248.2009.01387.x Bennett, E.L., & Rao, M. (2002). Wild meat consumption in Asian tropical forest countries: Is this a glimpse of the future for Africa? In S. Mainka & M. Trivedi (Eds.), Links between Biodiversity, Conservation, Livelihoods and Food Security: The Sustainable Use of Wild Species for Meat (pp. 9–44). Gland, Switzerland and Cambridge, UK: IUCN. Retrieved from https://www.iucn.org/content/links-between-biodiversity- conservation-livelihoods-and-food-security-sustainable-use-wild-species-meat Bennett, Elizabeth L., Blencowe, E., Brandon, K., Brown, D., Burn, R. W., Cowlishaw, G., … Wilkie, D. S. (2007). Hunting for Consensus: Reconciling Bushmeat Harvest, Conservation, and Development Policy in West and Central Africa. Conservation Biology, 21(3), 884–887. https://doi.org/10.1111/j.1523-1739.2006.00595.x Bennett, N. J., Finkbeiner, E. M., Ban, N. C., Belhabib, D., Jupiter, S. D., Kittinger, J. N., … Christie, P. (2020). The COVID-19 Pandemic, Small-Scale Fisheries and Coastal Fishing Communities. Coastal Management, 48(4), 336–347. https://doi.org/10.1080/08920753.2020.1766937 Bennett, R. (2003). Factors underlying the inclination to donate to particular types of charity. International Journal of Nonprofit and Voluntary Sector Marketing, 8(1), 12–29. https://doi.org/10.1002/nvsm.198 Berg, A., Ehnstrom, B., Gustafsson, L., Hallingback, T., Jonsell, M., & Weslien, J. (1994). Threatened Plant, Animal, and Fungus Species in Swedish Forests: Distribution and Habitat Associations. Conservation Biology, 8(3), 718–731. https://doi.org/10.1046/j.1523-1739.1994.08030718.x Berger, D. N. (2019). The Indigenous World 2019 (Berger, David Nathaniel). Copenhagen, Denmark: The International Work Group for Indigenous Affairs (IWGIA). Retrieved from https://www.iwgia.org/en/documents-and-publications/documents/publications- pdfs/english-publications/4-the-indigenous-world-2019/file.html Bergeron, Y., Gauthier, S., Kafka, V., Lefort, P., & Lesieur, D. (2001). Natural fire frequency for the eastern Canadian boreal forest: Consequences for sustainable forestry. Canadian Journal of Forest Research, 31(3), 384–391. https://doi.org/10.1139/x00-178 Bergstrom, R. D. (2008). The geographic and economic importance of hunting in Southwestern Montana, USA (Montana State University). Montana State University. Retrieved from https://scholarworks.montana.edu/xmlui/bitstream/handle/1/912/BergstromR0508.pdf ?sequence=1&isAllowed=y Berkes, F. (2002). Cross-scale institutional linkages: Perspectives from the bottom up. References In Drama of the Commons Ostrom E, Dietz T, Dolsak N, Stern PC, Stonich S, and Weber EU (pp. 293–322). National Academy Press. Berkes, F. (2010). Devolution of environment and resources governance: Trends and future. Environmental Conservation, 37(4), 489–500. https://doi.org/10.1017/S037689291000072X Berkes, F. (2015). Coasts for people: Interdisciplinary approaches to coastal and marine resource management (1 Edition). New York: Routledge, Taylor & Francis Group. 352 Berkes, F., & Berkes, M. K. (2009). Ecological complexity, fuzzy logic, and holism in indigenous knowledge. Futures, 41(1), 6–12. https://doi.org/10.1016/j.futures.2008.07.003 Bernal, R., Torres, C., García, N., Isaza, C., Navarro, J., Vallejo, M. I., … Balslev, H. (2011). Palm Management in South America. The Botanical Review, 77(4), 607–646. https://doi.org/10.1007/s12229-011-9088-6 Bertulli, C. G., Leeney, R. H., Barreau, T., & Matassa, D. S. (2016). Can whale-watching and whaling co-exist? Tourist perceptions in Iceland. Journal of the Marine Biological Association of the United Kingdom, 96(4), 969–977. https://doi.org/10.1017/S002531541400006X Bertwell, T. D., Kainer, K. A., Cropper Jr, W. P., Staudhammer, C. L., & de Oliveira Wadt, L. H. (2018). Are Brazil nut populations threatened by fruit harvest? Biotropica, 50(1), 50–59. https://doi.org/10.1111/btp.12505 BGCI. (2021). State of the worlds Trees. Retrieved from https://www.bgci.org/wp/wp- content/uploads/2021/08/FINAL-GTAReportMedRes-1.pdf Bhagawati, K., Sen, A., & Shukla, K. (2017). Seasonal Calendar and Gender Disaggregated Daily Activities of Indigenous Galo Farmers of Eastern Himalayan Region of India. Current Agriculture Research Journal, 5, 325–330. https://doi.org/10.12944/CARJ.5.3.10 Bhagwat, S. A., & Rutte, C. (2006). Sacred groves: Potential for biodiversity management Journal Item. Frontiers in Ecology and the Environment, 4(10), 519–524. https://doi.org/10.1890/1540-9295(2006)4[519:SGPFBM]2.0.CO;2 Bharucha, Z., & Pretty, J. (2010). The roles and values of wild foods in agricultural systems. Philosophical Transactions of the Royal Society B: Biological Sciences, 365(1554), 2913–2926. https://doi.org/10.1098/rstb.2010.0123 Bicknell, J. E., Struebig, M. J., & Davies, Z. G. (2015). Reconciling timber extraction with biodiversity conservation in tropical forests using reduced-impact logging. Journal of Applied Ecology, 52(2), 379–388. Bicknell, J. E., Struebig, M. J., Edwards, D. P., & Davies, Z. G. (2014). Improved timber harvest techniques maintain biodiversity in tropical forests. Current Biology, 24(23), R1119–R1120. https://doi.org/10.1016/j.cub.2014.10.067 Biermann F, Betsill MM, Gupta J, Kanie N, Lebel L, Liverman D, Schroeder H, Siebenhüner B. 6. (2009). Earth system governance: People, places and the planet. Science and Implementation Plan of the Earth System Governance Project. Earth System Governance Project Report No. 1. IHDP Report No. 20. International Human Dimensions Programme on Global Environmental Change. Biggs, R., Carpenter, S. R., & Brock, W. A. (2009). References Turning back from the brink: Detecting an impending regime shift in time to avert it. Proceedings of the National Academy of Sciences, 106(3), 826–831. https://doi.org/10.1073/pnas.0811729106 Bilchitz, D. (2016). Animal Interests and South African Law: The Elephant in the Room? In D. Cao & S. White (Eds.), Animal Law and Welfare—International Perspectives (pp. 131–155). Cham: Springer International Publishing. https://doi.org/10.1007/978-3- 319-26818-7_7 353 Bioeconomy.fi. (2017). The Finnish Bioeconomy Strategy. Edita Publishing. Retrieved from https://biotalous.fi/wp- content/uploads/2014/08/The_Finnish_Bioeconomy_Strategy_110620141.pdf Biondo, M. V. (2017). Quantifying the trade in marine ornamental fishes into Switzerland and an estimation of imports from the European Union. Global Ecology and Conservation, 11, 95–105. https://doi.org/10.1016/j.gecco.2017.05.006 Biondo, M. V. (2018). Importation of marine ornamental fishes to Switzerland. Global Ecology and Conservation, 15, e00418. https://doi.org/10.1016/j.gecco.2018.e00418 Biondo, M. V., & Burki, R. P. (2019). Monitoring the trade in marine ornamental fishes through the European Trade Control and Expert System TRACES: Challenges and possibilities. Marine Policy, 108, 103620. https://doi.org/10.1016/j.marpol.2019.103620 Biondo, M. V., & Burki, R. P. (2020). A Systematic Review of the Ornamental Fish Trade with Emphasis on Coral Reef Fishes—An Impossible Task. Animals, 10(11), 2014. https://doi.org/10.3390/ani10112014 Biondo, M. V., & Calado, R. (2021). The European Union Is Still Unable to Find Nemo and Dory-Time for a Reliable Traceability System for the Marine Aquarium Trade. Animals, 11(6), 1668. https://doi.org/10.3390/ani11061668 BirdLife International. (2008). State of the world’s birds: Indicators for our changing world. Birdlife International. Bissonnette, J.-F., Blouin, D., Bouthillier, L., & Teitelbaum, S. (2020). Vers des forêts de proximité en terres publiques ? Le « bricolage » institutionnel comme vecteur d’innovation en foresterie communautaire au Québec, Canada. Revue Gouvernance, 17(2), 52. https://doi.org/10.7202/1073111ar Bjerke, T., Østdahl, T., & Kleiven, J. (2003). Attitudes and activities related to urban wildlife: Pet owners and non-owners. Anthrozoos: A Multidisciplinary Journal of The Interactions of People & Animals, 16, 252–262. https://doi.org/10.2752/089279303786992125 Bjørn-Yoshimoto, W. E., Ramiro, I. B. L., Yandell, M., McIntosh, J. M., Olivera, B. M., Ellgaard, L., & Safavi-Hemami, H. (2020). Curses or Cures: A Review of the Numerous Benefits Versus the Biosecurity Concerns of Conotoxin Research. Biomedicines, 8(8), 235. https://doi.org/10.3390/biomedicines8080235 Blanc, R., Guillemain, M., Mouronval, J.-B., Desmonts, D., & Fritz, H. (2006). Effects of non- consumptive leisure disturbance to wildlife. Revue d’écologie. Blank, D., & Li, Y. (2021). Sustainable use of wildlife resources in Central Asia. Regional Sustainability, 2(2), 144–155. https://doi.org/10.1016/j.regsus.2021.05.001 Bluffstone, R. A., Somanathan, E., Jha, P., Luintel, H., Bista, R., Toman, M., … Adhikari, B. (2018). References Does Collective Action Sequester Carbon? Evidence from the Nepal Community Forestry Program. World Development, 101, 133–141. https://doi.org/10.1016/j.worlddev.2017.07.030 Blythe, J. L., Murray, G., & Flaherty, M. S. (2013). Historical perspectives and recent trends in the coastal Mozambican fishery. Ecology and Society, 18(4). https://doi.org/10.5751/ES-05759-180465 Boa, E. R. (2004). Wild edible fungi: A global overview of their use and importance to people. Rome: Food and Agriculture Organization of the United Nations. 354 Boakye, M. K., Pietersen, D. W., Kotzé, A., Dalton, D. L., & Jansen, R. (2014). Ethnomedicinal use of African pangolins by traditional medical practitioners in Sierra Leone. Journal of Ethnobiology and Ethnomedicine, 10(1), 76. https://doi.org/10.1186/1746-4269-10- 76 Bodmer, R. E., & Lozano, E. P. (2001). Rural Development and Sustainable Wildlife Use in Peru. Conservation Biology, 15(4), 1163–1170. https://doi.org/10.1046/j.1523- 1739.2001.0150041163.x Bonfil, R., Ricaño-Soriano, M., Mendoza-Vargas, O. U., Méndez-Loeza, I., Pérez-Jiménez, J. C., Bolaño-Martínez, N., & Palacios-Barreto, P. (2018). Tapping into local ecological knowledge to assess the former importance and current status of sawfishes in Mexico. Endangered Species Research, 36, 213–228. https://doi.org/10.3354/esr00899 Boom, K., Ben-Ami, D., Croft, D., Cushing, N., Ramp, D., & Boronyak, L. (2012). “Pest” and Resource: A Legal History of Australia’s Kangaroos. Animal Studies Journal, 1(1), 17– 40. Boonstra, W. J. (2016). Conceptualizing power to study social-ecological interactions. Ecology and Society, 21(1), art21. https://doi.org/10.5751/ES-07966-210121 Booth, H., Squires, D., & Milner-Gulland, E. (2019). The neglected complexities of shark fisheries, and priorities for holistic risk-based management. Ocean & Coastal Management, 182, 104994. Booth, V. R. (2010). Contribution of wildlife to national economies (No. 8). Budakeszi: FAO and CIC. Retrieved from FAO and CIC website: https://www1.sun.ac.za/awei/sites/default/files/Technical_series_8.pdf Borelli, T., Hunter, D., Powell, B., Ulian, T., Mattana, E., Termote, C., … Engels, J. (2020). Born to Eat Wild: An Integrated Conservation Approach to Secure Wild Food Plants for Food Security and Nutrition. Plants, 9(10), 1299. https://doi.org/10.3390/plants9101299 Borowitzka, M. A. (2018). Microalgae in Medicine and Human Health. In Microalgae in Health and Disease Prevention (pp. 195–210). Elsevier. https://doi.org/10.1016/B978- 0-12-811405-6.00009-8 Bose, A. K., Harvey, B. D., Brais, S., Beaudet, M., & Leduc, A. (2014). Constraints to partial cutting in the boreal forest of Canada in the context of natural disturbance-based management: A review. Forestry, 87(1), 11–28. https://doi.org/10.1093/forestry/cpt047 Boucher, Y., Arseneault, D., Sirois, L., & Blais, L. (2009). Logging pattern and landscape changes over the last century at the boreal and deciduous forest transition in Eastern Canada. Landscape Ecology, 24(2), 171–184. References https://doi.org/10.1007/s10980-008- 9294-8 Boucher, Y., Auger, I., Noël, J., Grondin, P., & Arseneault, D. (2017). Fire is a stronger driver of forest composition than logging in the boreal forest of eastern Canada. Journal of Vegetation Science, 28(1), 57–68. https://doi.org/10.1111/jvs.12466 Boudreau, S. A., & Worm, B. (2010). Top-down control of lobster in the Gulf of Maine: Insights from local ecological knowledge and research surveys. Marine Ecology Progress Series, 403, 181–191. Scopus. https://doi.org/10.3354/meps08473 355 Boughedir, W., Rifi, M., Shakman, E., Maynou, F., Ghanem, R., Ben Souissi, J., & Azzurro, E. (2015). Tracking the invasion of Hemiramphus far and Saurida undosquamis along the southern Mediterranean coasts: A Local Ecological Knowledge study. Mediterranean Marine Science, 16(3), 628. https://doi.org/10.12681/mms.1179 Bowles, S., & Choi, J.-K. (2013). Coevolution of farming and private property during the early Holocene. Proceedings of the National Academy of Sciences, 110(22), 8830–8835. https://doi.org/10.1073/pnas.1212149110 Boyd, C. E., D’Abramo, L. R., Glencross, B. D., Huyben, D. C., Juarez, L. M., Lockwood, G. S., … Valenti, W. C. (2020). Achieving sustainable aquaculture: Historical and current perspectives and future needs and challenges. Journal of the World Aquaculture Society, 51(3), 578–633. https://doi.org/10.1111/jwas.12714 Bozzeda, F., Marín, S. L., & Nahuelhual, L. (2019). An uncertainty-based decision support tool to evaluate the southern king crab (Lithodes santolla) fishery in a scarce information context. Progress in Oceanography, 174, 64–71. https://doi.org/10.1016/j.pocean.2018.10.013 BPS. (2020). Profil Industri Mikro Dan Kecil 2019. Jakarta, Indonesia: Badan Pusat Statistik. Retrieved from Badan Pusat Statistik website: https://www.bps.go.id/publication/2020/11/16/db2fdf158825afb80a113b6a/profil- industri-mikro-dan-kecil-2019.html Braccini, M., Blay, N., Harry, A., & Newman, S. J. (2020). Would ending shark meat consumption in Australia contribute to the conservation of white sharks in South Africa? Marine Policy, 120, 104144. https://doi.org/10.1016/j.marpol.2020.104144 Braden, K. (2014). Illegal recreational hunting in Russia: The role of social norms and elite violators. Eurasian Geography and Economics, 55(5), 457–490. https://doi.org/10.1080/15387216.2015.1020320 Braga, H. O., Azeiteiro, U. M., Oliveira, H. M. F., & Pardal, M. A. (2017). Evaluating fishermen’s conservation attitudes and local ecological knowledge of the European sardine (Sardina pilchardus), Peniche, Portugal. Journal of Ethnobiology and Ethnomedicine, 13(1). Scopus. https://doi.org/10.1186/s13002-017-0154-y Braga, H. O., Pardal, M. Â., & Azeiteiro, U. M. (2018). Incorporation of Local Ecological Knowledge (LEK) into Biodiversity Management and Climate Change Variability Scenarios for Threatened Fish Species and Fishing Communities—Communication Patterns Among BioResources Users as a Prerequisite for Co-management: A Case Study of Berlenga MNR, Portugal and Resex-Mar of Arraial do Cabo, RJ, Brazil. In W. Leal Filho, E. Manolas, A. Azul, U. References Azeiteiro, & H. McGhie (Eds.), Handbook of Climate Change Communication (pp. 237–262). Cham: Springer. https://doi.org/10.1007/978-3-319-70066-3_16 Braga, H. O., Pereira, M. J., Morgado, F., Soares, A. M. V. M., & Azeiteiro, U. M. (2019). Ethnozoological knowledge of traditional fishing villages about the anadromous sea lamprey (Petromyzon marinus) in the Minho river, Portugal. Journal of Ethnobiology and Ethnomedicine, 15(1). https://doi.org/10.1186/s13002-019-0345-9 Bragagnolo, C., Gama, G. M., Vieira, F. A. S., Campos-Silva, J. V., Bernard, E., Malhado, A. C. M., … Ladle, R. J. (2019). Hunting in Brazil: What are the options? Perspectives in Ecology and Conservation, 17(2), 71–79. https://doi.org/10.1016/j.pecon.2019.03.001 356 Bragina, E. V., Ives, A. R., Pidgeon, A. M., Balčiauskas, L., Csányi, S., Khoyetskyy, P., … others. (2018). Wildlife population changes across Eastern Europe after the collapse of socialism. Frontiers in Ecology and the Environment, 16(2), 77–81. Brainerd, S. (2007). European Charter on Hunting and Biodiversity. https://doi.org/10.13140/RG.2.2.22386.38086 Branch, T. A., Lobo, A. S., & Purcell, S. W. (2013). Opportunistic exploitation: An overlooked pathway to extinction. Trends in Ecology & Evolution, 28(7), 409–413. https://doi.org/10.1016/j.tree.2013.03.003 Brashares, J., Prugh, L., Stoner, C. J., & Epps, C. (2013). Ecological and conservation implications of mesopredator release. In J. Terborgh & J. Estes (Eds.), Trophic Cascades: Predators, Prey, and the Changing Dynamics of Nature (pp. 221–240). Island Press. Bray, D. B. (2020). Mexico’s community forest enterprises: Success on the commons and the seeds of a good anthropocene. Tucson: The University of Arizona Press. Brendler, T., Brinckmann, J. A., & Schippmann, U. (2018). Sustainable supply, a foundation for natural product development: The case of Indian frankincense (Boswellia serrata Roxb. Ex Colebr.). Journal of Ethnopharmacology, 225(DITSL GmbH, Bonn 2011), 279–286. https://doi.org/10.1016/j.jep.2018.07.017 Breuer, T., & Mavinga, F. B. (2010). Education for the conservation of great apes and other wildlife in northern Congo-the importance of nature clubs. American Journal of Primatology, 72(5), 454–461. https://doi.org/10.1002/ajp.20774 Brinckmann, J. A., Luo, W., Xu, Q., He, X., Wu, J., & Cunningham, A. B. (2018). Sustainable harvest, people and pandas: Assessing a decade of managed wild harvest and trade in Schisandra sphenanthera. Journal of Ethnopharmacology, 224, 522–534. https://doi.org/10.1016/j.jep.2018.05.042 Brink, H., Smith, R. J., Skinner, K., & Leader-Williams, N. (2016). Sustainability and Long Term-Tenure: Lion Trophy Hunting in Tanzania. PLoS ONE, 11(9). https://doi.org/10.1371/journal.pone.0162610 Brito, L. A., & O’Hagan, D. T. (2014). Designing and building the next generation of improved vaccine adjuvants. Journal of Controlled Release, 190, 563–579. https://doi.org/10.1016/j.jconrel.2014.06.027 Brochet, A.-L., Van Den Bossche, W., Jbour, S., Ndang’Ang’A, P. References K., Jones, V. R., Abdou, W. A. L. I., … Butchart, S. H. M. (2016). Preliminary assessment of the scope and scale of illegal killing and taking of birds in the Mediterranean. Bird Conservation International, 26(1), 1–28. https://doi.org/10.1017/S0959270915000416 Brock, J. R., Dönmez, A. A., Beilstein, M. A., & Olsen, K. M. (2018). Phylogenetics of Camelina Crantz. (Brassicaceae) and insights on the origin of gold-of-pleasure (Camelina sativa). Molecular Phylogenetics and Evolution, 127, 834–842. https://doi.org/10.1016/j.ympev.2018.06.031 Broekhuis, F. (2018). Natural and anthropogenic drivers of cub recruitment in a large carnivore. Ecology and Evolution, 8(13), 6748–6755. https://doi.org/10.1002/ece3.4180 Broeren, M. L. M., Dellaert, S. N. C., Cok, B., Patel, M. K., Worrell, E., & Shen, L. (2017). Life cycle assessment of sisal fibre – Exploring how local practices can influence 357 environmental performance. Journal of Cleaner Production, 149, 818–827. https://doi.org/10.1016/j.jclepro.2017.02.073 Brokamp, G., Valderrama, N., Mittelbach, M., Grandez R., C. A., Barfod, A. S., & Weigend, M. (2011). Trade in Palm Products in North-Western South America. The Botanical Review, 77(4), 571–606. https://doi.org/10.1007/s12229-011-9087-7 Brondízio, E. S., Aumeeruddy-Thomas, Y., Bates, P., Carino, J., Fernández-Llamazares, Á., Ferrari, M. F., … Shrestha, U. B. (2021). Locally Based, Regionally Manifested, and Globally Relevant: Indigenous and Local Knowledge, Values, and Practices for Nature. Annual Review of Environment and Resources, 46(1), 481–509. https://doi.org/10.1146/annurev-environ-012220-012127 Brondizio, E. S., Ostrom, E., & Young, O. R. (2009). Connectivity and the governance of multilevel social-ecological systems: The role of social capital. Annual Review of Environment and Resources, 34. https://doi.org/10.1146/annurev.environ.020708.100707 and Brønstad, A., Newcomer, C. E., Decelle, T., Everitt, J. I., Guillen, J., & Laber, K. (2016). Current concepts of Harm-Benefit Analysis of Animal Experiments—Report from the AALAS-FELASA Working Group on Harm-Benefit Analysis—Part 1. Laboratory Animals, 50(1 Suppl), 1–20. https://doi.org/10.1177/0023677216642398 Brooks, J. S., & Tshering, D. (2010). A respected central government and other obstacles to community-based management of the matsutake mushroom in Bhutan. Environmental Conservation, 37(3), 336–346. https://doi.org/10.1017/S0376892910000573 Brooks, S. E., Allison, E. H., Gill, J. A., & Reynolds, J. D. (2010). Snake prices and crocodile appetites: Aquatic wildlife supply and demand on Tonle Sap Lake, Cambodia. Biological Conservation, 143(9), 2127–2135. https://doi.org/10.1016/j.biocon.2010.05.023 Brotz, L., Schiariti, A., López-Martínez, J., Álvarez-Tello, J., Peggy Hsieh, Y.-H., Jones, R. P., … Mianzan, H. (2017). Jellyfish fisheries in the Americas: Origin, state of the art, and perspectives on new fishing grounds. Reviews in Fish Biology and Fisheries, 27(1). https://doi.org/10.1007/s11160-016-9445-y Brown, C. (2000). The global outlook for future wood supply from forest plantations. Working paper GFPOS/WP/03 prepared for the 1999 Global Forest Products Outlook Study. Rome, Italy: Forestry Policy and Planning Division, FAO. Retrieved from Forestry Policy and Planning Division, FAO website: http://www.fao.org/3/x8423e/x8423e00.htm Brown, R. A., Rosenberg, N. J., Hays, C. J., Easterling, W. E., & Mearns, L. O. (2000). Potential production and environmental effects of switchgrass and traditional crops under current and greenhouse-altered climate in the central United States: A simulation study. Agriculture, Ecosystems & Environment, 78(1), 31–47. https://doi.org/10.1016/S0167-8809(99)00115-2 Brownscombe, J. W., Bower, S. D., Bowden, W., Nowell, L., Midwood, J. D., Johnson, N., & Cooke, S. J. (2014). Canadian Recreational Fisheries: 35 Years of Social, Biological, and Economic Dynamics from a National Survey. Fisheries, 39(6), 251–260. https://doi.org/10.1080/03632415.2014.915811 358 Buckley, R. (2000). Neat trends: Current issues in nature, eco-and adventure tourism. International Journal of Tourism Research, 2(6), 437–444. https://doi.org/10.1002/1522-1970(200011/12)2:63.3.CO;2-R Buckley, R., Gretzel, U., Scott, D., Weaver, D., & Becken, S. (2015). Tourism megatrends. Tourism Recreation Research, 40(1), 59–70. https://doi.org/10.1080/02508281.2015.1005942 Budidarsono, S., Susanti, A., & Zoomers, A. (2013). Oil Palm Plantations in Indonesia: The Implications for Migration, Settlement/Resettlement and Local Economic Development. In Z. Fang (Ed.), Biofuels—Economy, Environment and Sustainability. InTech. https://doi.org/10.5772/53586 Budiman, I., Fujiwara, T., Sato, N., & Pamungkas, D. (2020). Another Law in Indonesia: Customary Land Tenure System Coexisting with State Order in Mutis Forest. Jurnal Manajemen Hutan Tropika (Journal of Tropical Forest Management), 26(3), 244–253. https://doi.org/10.7226/jtfm.26.3.244 Bulengela, G., Onyango, P., Brehm, J., Staehr, P. A., & Sweke, E. (2019). and “Bring fishermen at the center”: The value of local knowledge for understanding fisheries resources and climate-related changes in Lake Tanganyika. Environment, Development and Sustainability. Scopus. https://doi.org/10.1007/s10668-019-00443-z Bull, G. Q., Bazett, M., Schwab, O., Nilsson, S., White, A., & Maginnis, S. (2006). Industrial forest plantation subsidies: Impacts and implications. Forest Policy and Economics, 9(1), 13–31. https://doi.org/10.1016/j.forpol.2005.01.004 Bullock, R., & Hanna, K. (2007). Community Forestry: Mitigating or Creating Conflict in British Columbia? Society & Natural Resources, 21(1), 77–85. https://doi.org/10.1080/08941920701561007 Bunce, M., Rodwell, L. D., Gibb, R., & Mee, L. (2008). Shifting baselines in fishers’ perceptions of island reef fishery degradation. Ocean and Coastal Management, 51(4), 285–302. https://doi.org/10.1016/j.ocecoaman.2007.09.006 Bundy, A., Chuenpagdee, R., Cooley, S. R., Defeo, O., Glaeser, B., Guillotreau, P., … Perry, R. I. (2016). A decision support tool for response to global change in marine systems: The IMBER-ADApT Framework. Fish and Fisheries, 17(4), 1183–1193. https://doi.org/10.1111/faf.12110 Bunge, A., Diemont, S. A. W., Bunge, J. A., & Harris, S. (2019). Urban foraging for food security and sovereignty: Quantifying edible forest yield in Syracuse, New York using four common fruit- and nut-producing street tree species. Journal of Urban Ecology, 5(1). https://doi.org/10.1093/jue/juy028 Büntgen, U., Egli, S., Camarero, J. J., Fischer, E. M., Stobbe, U., Kauserud, H., … Stenseth, N. C. (2012). Drought-induced decline in Mediterranean truffle harvest. Nature Climate Change, 2(12), 827–829. https://doi.org/10.1038/nclimate1733 Burkhart, E. P., & Jacobson, M. G. (2009). Transitioning from wild collection to forest cultivation of indigenous medicinal forest plants in eastern North America is constrained by lack of profitability. Agroforestry Systems, 76(2), 437–453. https://doi.org/10.1007/s10457-008-9173-y Burkhart, E. P., Jacobson, M. G., & Finley, J. (2012). A case study of stakeholder perspective and experience with wild American ginseng (Panax quinquefolius) conservation efforts 359 in Pennsylvania, U.S.A.: Limitations to a CITES driven, top-down regulatory approach. Biodiversity and Conservation, 21(14), 3657–3679. https://doi.org/10.1007/s10531- 012-0389-9 Burns, G. L., Lilja Öqvist, E., Angerbjörn, A., & Granquist, S. (2018). When the wildlife you watch becomes the food you eat: Exploring moral and ethical dilemmas when consumptive and non-consumptive tourism merge. In Routledge Research in the Ethics of Tourism Series. Animals, food, and tourism. New York: Routledge. Buschmann, A. H., Camus, C., Infante, J., Neori, A., Israel, Á., Hernández-González, M. C., … Critchley, A. T. (2017). Seaweed production: Overview of the global state of exploitation, farming and emerging research activity. European Journal of Phycology, 52(4), 391–406. https://doi.org/10.1080/09670262.2017.1365175 Bush, E. R., Short, R. E., Milner-Gulland, E. and J., Lennox, K., Samoilys, M., & Hill, N. (2017). Mosquito Net Use in an Artisanal East African Fishery. Conservation Letters, 10(4), 450–458. https://doi.org/10.1111/conl.12286 Busilacchi, S., Russ, G. R., Williams, A. J., Begg, G. A., & Sutton, S. G. (2013). Quantifying changes in the subsistence reef fishery of indigenous communities in Torres Strait, Australia. Fisheries Research, 137, 50–58. https://doi.org/10.1016/j.fishres.2012.08.017 But, P. P.-H., Cheng, L., Chan, P. K., Lau, D. T.-W., & But, J. W.-H. (2002). Nostoc flagelliforme and faked items retailed in Hong Kong. Journal of Applied Phycology, 14(2), 143–145. https://doi.org/10.1023/A:1019518329032 Butchart, S. H. M. (2008). Red List Indices to measure the sustainability of species use and impacts of invasive alien species. Bird Conservation International, 18(S1), S245–S262. https://doi.org/10.1017/S095927090800035X Butler, J. R. A., Tawake, A., Skewes, T., Tawake, L., & McGrath, V. (2012). Integrating traditional ecological knowledge and fisheries management in the torres strait, Australia:The catalytic role of turtles and dugong as cultural keystone species. Ecology and Society, 17(4). https://doi.org/10.5751/ES-05165-170434 Bye, R. A. J. (1981). Quelites. Ethnoecology of edible greens. Past, present and future. Journal of Ethnobiology, 1(1), 109–123. Byers, A. C., Byers, E., Shrestha, M., Thapa, D., & Sharma, B. (2020). Impacts of Yartsa Gunbu Harvesting on Alpine Ecosystems in the Barun Valley, Makalu-Barun National Park, Nepal. Himalaya, 39(2), 44–59. Cai, J., & Leung, P. (2017). Short-term projection of global fish demand and supply gaps. Rome: Food and Agriculture Organization of the United Nations. Caldwell, J. (2017). World trade in crocodilian skins 2013-2015 (p. 32) [UNEP-WCMC technical report]. Cambridge: UN Environment. Retrieved from UN Environment website: https://www.unep- wcmc.org/system/dataset_file_fields/files/000/000/479/original/World_Trade_in_Cro codilian_Skins_2013-2015.pdf?1507799294 Calogiuri, G., Litleskare, S., Fagerheim, K. A., Rydgren, T. L., Brambilla, E., & Thurston, M. (2018). Experiencing nature through immersive virtual environments: Environmental perceptions, physical engagement, and affective responses during a simulated nature walk. Frontiers in Psychology, 8, 2321. https://doi.org/10.3389/fpsyg.2017.02321 360 Campos-Silva, J. V., & Peres, C. A. (2016). Community-based management induces rapid recovery of a high-value tropical freshwater fishery. Scientific Reports, 6(1), 34745. https://doi.org/10.1038/srep34745 Cannon, P. F., Hywel-Jones, N. L., Maczey, N., Norbu, L., Tshitila, Samdup, T., & Lhendup, P. (2009). Steps towards sustainable harvest of Ophiocordyceps sinensis in Bhutan. Biodiversity and Conservation, 18(9), 2263–2281. https://doi.org/10.1007/s10531-009- 9587-5 Cardeñosa, D., Quinlan, J., Shea, K. H., & Chapman, D. D. (2018). Multiplex real-time PCR assay to detect illegal trade of CITES-listed shark species. Scientific Reports, 8(1), 16313. https://doi.org/10.1038/s41598-018-34663-6 Carder, G., Plese, T., Machado, F., Paterson, S., Matthews, N., McAnea, L., & D’Cruze, N. and (2018). The Impact of ‘Selfie’ Tourism on the Behaviour and Welfare of Brown- Throated Three-Toed Sloths. Animals, 8(11), 216. https://doi.org/10.3390/ani8110216 Cardon, D. (2007). Natural dyes: Sources, tradition, technology and science. London: Archetype. Cardon, D. (2010). Cardon D. 2010. Natural Dyes, Our Global Heritage of Colors. . University of Nebraska, Lincoln. In Symposium Proceedings Textile Society of America (Textile Society of America, pp. 1–10). Lincoln: University of Nebraska. Retrieved from https://digitalcommons.unl.edu/cgi/viewcontent.cgi?article=1011&context=tsaconf Cardoso, M. Betina, & González, A. D. (2019). Residential energy transition and thermal efficiency in an arid environment of northeast Patagonia, Argentina. Energy for Sustainable Development, 50, 82–90. https://doi.org/10.1016/j.esd.2019.03.007 Cardoso, M.B., Ladio, A. H., & Lozada, M. (2013). Fuelwood consumption patterns and resilience in two rural communities of the northwest Patagonian steppe, Argentina. Journal of Arid Environments, 98, 146–152. https://doi.org/10.1016/j.jaridenv.2012.09.013 Carle, J., & Homgren, P. (2008). Wood from planted forests. Forest Products Journal, 58(12), 6. Carle, J., & Homgren, P. (2008). Wood from planted forests. Forest Products Journal, 58(12), 6 Carothers, C., Sformo, T. L., Cotton, S., George, J. C., & Westley, P. A. H. (2019). Pacific salmon in the rapidly changing arctic: Exploring local knowledge and emerging fisheries in Utqiaġvik and Nuiqsut, Alaska. Arctic, 72(3), 273–288. https://doi.org/10.14430/arctic68876 Carpenter, A., Dublin, H., Lau, M., Syed, G., McKay, J., & Moore, R. (2007). Over-harvesting. In C. Gascon, J. Collins, R. Moore, D. Church, & J. McKay (Eds.), Amphibian Conservation Action Plan: Proceedings IUCN/SSC Amphibian Conservation Summit. Retrieved from https://www.researchgate.net/publication/265550329_AI_Carpenter_H_Dublin_M_L au_G_Syed_J_E_McKay_RD_Moore_2007_Chapter_5_Over- harvesting_IN_Amphibian_Conservation_Action_Plan_Proceedings_IUCNSSC_Am phibian_Conservation_Summit_ed%27s_Gascon_C_et_al_IUCNSSC Carpenter, A. I., Andreone, F., Moore, R. D., & Griffiths, R. A. (2014). A review of the international trade in amphibians: The types, levels and dynamics of trade in CITES- listed species. Oryx, 48(4), 565–574. https://doi.org/10.1017/S0030605312001627 361 Carpenter, S. R., & Brock, W. A. (2006). Rising variance: A leading indicator of ecological transition: Variance and ecological transition. Ecology Letters, 9(3), 311–318. https://doi.org/10.1111/j.1461-0248.2005.00877.x Carpenter, Stephen R, & Kinne, O. (2003). Regime shifts in lake ecosystems. Carr, N., & Broom, D. M. (2018). Tourism and animal welfare. CABI. Carrà, G., Monaco, C., & Peri, I. (2017). Local management plans for sustainability of small- scale fisheries: A case study. Quality - Access to Success, 18, 116–121. Scopus. Retrieved from Scopus. Carroll, M. S., Blatner, K. A., & Cohn, P. J. (2003). Somewhere Between: Social Embeddedness and the Spectrum of Wild Edible Huckleberry Harvest and Use. Rural Sociology, 68(3), 319–342. https://doi.org/10.1111/j.1549-0831.2003.tb00140.x Carter, N., & Linnell, J. (2016). and Co-Adaptation Is Key to Coexisting with Large Carnivores. Trends in Ecology and Evolution, 31. https://doi.org/10.1016/j.tree.2016.05.006 Carvalho, A. N., Vasconcelos, P., Piló, D., Pereira, F., & Gaspar, M. B. (2017). Socio- economic, operational and technical characterisation of the harvesting of gooseneck barnacle (Pollicipes pollicipes) in SW Portugal: Insights towards fishery co- management. Marine Policy, 78, 34–44. https://doi.org/10.1016/j.marpol.2017.01.008 Carvalho Ribeiro, S. M., Soares Filho, B., Leles Costa, W., Bachi, L., Ribeiro de Oliveira, A., Bilotta, P., … Cioce Sampaio, C. (2018). Can multifunctional livelihoods including recreational ecosystem services (RES) and non timber forest products (NTFP) maintain biodiverse forests in the Brazilian Amazon? Ecosystem Services, 31, 517–526. https://doi.org/10.1016/j.ecoser.2018.03.016 Carvalho Ribeiro, Sónia Maria. (1998). A participação dos compartes na gestão do baldio: Estudo de caso no Baldio da Ermida, concelho de Terras de Bouro, Parque Nacional da Peneda Gerês, Portugal. UTAD, Universidade de Trás os Montes e Alto Douro, licenciatura em Eng Florestal. Casas, A., & Barbera, G. (2002). Mesoamerican domestication and diffusion. In Cacti. Biology and uses (Nobel, Park S., pp. 143–162). Berkeley & Los Angeles: University of California Press,. Casas, A., Otero-Arnaiz, A., Pérez-Negrón, E., & Valiente-Banuet, A. (2007). In situ Management and Domestication of Plants in Mesoamerica. Annals of Botany, 100(5), 1101–1115. https://doi.org/10.1093/aob/mcm126 Case, M. A., Flinn, K. M., Jancaitis, J., Alley, A., & Paxton, A. (2007). Declining abundance of American ginseng (Panax quinquefolius L.) documented by herbarium specimens. Biological Conservation, 134(1), 22–30. https://doi.org/10.1016/j.biocon.2006.07.018 Cashion, T., Le Manach, F., Zeller, D., & Pauly, D. (2017). Most fish destined for fishmeal production are food-grade fish. Fish and Fisheries, 18(5), 837–844. https://doi.org/10.1111/faf.12209 Castañeda-Álvarez, N. P., Khoury, C. K., Achicanoy, H. A., Bernau, V., Dempewolf, H., Eastwood, R. J., … Toll, J. (2016). Global conservation priorities for crop wild relatives. 2(April), 1–6. https://doi.org/10.1038/nplants.2016.22 Castellanos-Galindo, G. A., Chong-Montenegro, C., Baos E, R. A., Zapata, L. A., Tompkins, P., Graham, R. T., & Craig, M. (2018). Using landing statistics and fishers’ traditional ecological knowledge to assess conservation threats to Pacific goliath grouper in 362 Colombia. Aquatic Conservation: Marine and Freshwater Ecosystems, 28(2), 305–314. https://doi.org/10.1002/aqc.2871 Castello, L., Viana, J. P., Watkins, G., Pinedo-Vasquez, M., & Luzadis, V. A. (2009). Lessons from integrating fishers of arapaima in small-scale fisheries management at the mamirauá reserve, amazon. Environmental Management, 43(2), 197–209. https://doi.org/10.1007/s00267-008-9220-5 Castello, Leandro, Arantes, C. C., Mcgrath, D. G., Stewart, D. J., & Sousa, F. S. D. (2015). Understanding fishing‐induced extinctions in the Amazon. and Aquatic Conservation: Marine and Freshwater Ecosystems, 25(5), 587–598. Castello, Leandro, McGrath, D. G., & Beck, P. S. A. (2011b). Resource sustainability in small- scale fisheries in the Lower Amazon floodplains. Fisheries Research, 110(2), 356–364. https://doi.org/10.1016/j.fishres.2011.05.002 Castilla, J. C., Espinosa, J., Yamashiro, C., Melo, O., & Gelcich, S. (2016). Telecoupling between Catch, Farming, and International Trade for the Gastropods Concholepas concholepas (Loco) and Haliotis spp. (Abalone). Journal of Shellfish Research, 35(2), 499–506. Scopus. https://doi.org/10.2983/035.035.0223 Catarino, M. F., Kahn, J. R., & Freitas, C. E. C. (2019). Stock assessment of prochilodus nigricans (Actinopterygii: Characiformes: Prochilodontidae) using two distinct algorithms, in the context of a small-scale amazonian fishery. Acta Ichthyologica et Piscatoria, 49(4), 373–380. https://doi.org/10.3750/AIEP/02623 Catarino, S., Duarte, M., Costa, E., Carrero, P., & Romeiras, M. M. (2019). Conservation and sustainable use of the medicinal Leguminosae plants from Angola. PeerJ, 7, e6736. https://doi.org/10.7717/peerj.6736 Cavieses Núñez, R. A., Ojeda Ruiz De La Penã, M. Á., Flores Irigollen, A., Rodríguez Rodríguez, M., & Jardim, E. (2018). Deep learning models for the prediction of small- scale fisheries catches: Finfish fishery in the region of “bahiá Magadalena-Almejas.” ICES Journal of Marine Science, 75(6), 2088–2096. https://doi.org/10.1093/icesjms/fsy065 Cavole, L. M., Arantes, C. C., & Castello, L. (2015). How illegal are tropical small-scale fisheries? An estimate for arapaima in the Amazon. Fisheries Research, 168, 1–5. Scopus. https://doi.org/10.1016/j.fishres.2015.03.012 Celermajer, D., Schlosberg, D., Rickards, L., Stewart-Harawira, M., Thaler, M., Tschakert, P., … Winter, C. (2021). Multispecies justice: Theories, challenges, and a research agenda for environmental politics. Environmental Politics, 30(1–2), 119–140. https://doi.org/10.1080/09644016.2020.1827608 Cerruti, P. O., Lescuyer, G., Tacconi, L., Eba’a Atyi, R., Essiane, E., Nasi, R., … Tsanga, R. (2017). Social impacts of the Forest Stewardship Council certification in the Congo basin. International Forestry Review, 19(4), 50–63. https://doi.org/10.1505/146554817822295920 Cerutti, P. O., & Lescuyer, G. (2011). The domestic market for small-scale chainsaw milling in Cameroon: Present situation, opportunities and challenges. Bogor, Indonesia.: CIFOR. 363 Cerutti, P. O., Lescuyer, G., Assembe-Mvondo, S., & Tacconi, L. (2010). The challenges of redistributing forest-related monetary benefits to local governments: A decade of logging area fees in Cameroon. International Forestry Review, 12(2), 130–138. Cerutti, P. O., Nasi, R., & Center for International Forestry Research (CIFOR), Kenya and Indonesia. (2020). Sustainable forest management (SFM) of tropical moist forests: The Congo Basin. Center for International Forestry Research (CIFOR), Kenya and Indonesia. https://doi.org/10.19103/AS.2020.0074.41 Chaber, A. L., Allebone-Webb, S., Lignereux, Y., Cunningham, A. A., & Rowcliffe, J. M. (2010). and The scale of illegal meat importation from Africa to Europe via Paris. https://doi.org/10.1111/j.1755-263X.2010.00121.x Challe, J. F., & Price, L. L. (2009). Endangered edible orchids and vulnerable gatherers in the context of HIV/AIDS in the Southern Highlands of Tanzania. Journal of Ethnobiology and Ethnomedicine, 5(1), 41. https://doi.org/10.1186/1746-4269-5-41 Challe, J. F. X., Struik, P. C., & Price, L. L. (2018). Perspectives of Children Orphaned by HIV/AIDS on Ecology and Gathering of Wild Orchids in Tanzania. Journal of Ethnobiology, 38(2), 223–243. https://doi.org/10.2993/0278-0771-38.2.223 Challender, D., & Cooney, R. (2016). Informing decisions on trophy hunting (p. 19) [Briefing Paper]. IUCN. Retrieved from IUCN website: http://the- eis.com/elibrary/sites/default/files/downloads/literature/iucn_informing%20decisions %20on%20trophy%20hunting%20v%201.pdf Chamberlain, James. L., Emery, M. R., & Patel-Weynand, T. (2018). Assessment of Nontimber Forest Products in the United States Under Changing Conditions. A report for the United Sates Department of Agriculture (p. 267). USDA Forest Service, Southern Research Station. Retrieved from USDA Forest Service, Southern Research Station website: https://doi.org/10.2737/SRS-GTR-232 Chao, S. (2012). Forest Peoples: Numbers across the World. United Kingdom: Forest Peoples Programme. Retrieved from https://www.forestpeoples.org/sites/fpp/files/publication/2012/05/forest-peoples- numbers-across-world-final_0.pdf Chaplin-Kramer, R., Sharp, R. P., Weil, C., Bennett, E. M., Pascual, U., Arkema, K. K., … Daily, G. C. (2019). Global modeling of nature’s contributions to people. Science, 366(6462), 255–258. https://doi.org/10.1126/science.aaw3372 Chapman, C. A., & Peres, C. A. (2001). Primate conservation in the new millennium: The role of scientists. Evolutionary Anthropology: Issues, News, and Reviews, 10(1), 16–33. https://doi.org/10.1002/1520-6505(2001)10:1<16::AID-EVAN1010>3.0.CO;2-O Charitonidou, M., Stara, K., Kougioumoutzis, K., & Halley, J. M. (2019). Implications of salep collection for the conservation of the Elder-flowered orchid (Dactylorhiza sambucina) in Epirus, Greece. Journal of Biological Research-Thessaloniki, 26(1). https://doi.org/ARTN 18 10.1186/s40709-019-0110-1 Charnley, S. (2005). From Nature Tourism to Ecotourism? The case of the Ngorongoro Conservation Area, Tanzania. Human Organization, 64(1). https://doi.org/00 18- 7259/05/010075-14$1.90/1 364 Charnley, S., McLain, R. J., & Poe, M. R. (2018). Natural resource access rights and wrongs: Nontimber forest products gathering in urban environments. Society & Natural Resources, 31(6), 734–750. https://doi.org/10.1080/08941920.2017.1413696 Charnley, S., & Poe, M. R. (2007). Community forestry in theory and practice: Where are we now? Annual Review of Anthropology, 36, 301–336. https://doi.org/10.1146/annurev.anthro.35.081705.123143 Chaudhary, A., Burivalova, Z., Koh, L. P., & Hellweg, S. (2016). Impact of Forest Management on Species Richness: Global Meta-Analysis and Economic Trade-Offs. Scientific Reports, 6(1), 23954. https://doi.org/10.1038/srep23954 Chaudhary, A., Carrasco, L. R., & Kastner, T. (2017). Linking national wood consumption with global biodiversity and ecosystem service losses. Science of The Total Environment, 586, 985–994. https://doi.org/10.1016/j.scitotenv.2017.02.078 Chaudhary, R. and P., Uprety, Y., & Rimal, S. K. (2016). Deforestation in Nepal: Causes, consequences and responses. Biological and Environmental Hazards, Risks, and Disasters, 335–372. Chaudhary, Ram P., Aase, T. H., Vetaas, O. R., & Subedi, B. P. (Eds.). (2007). Local effects of global changes in the Himalayas: Manang, Nepal. Kathmandu : Norway : University of Bergen: Tribhuvan University. Chaudhary, S., McGregor, A., Houston, D., & Chettri, N. (2018). Environmental justice and ecosystem services: A disaggregated analysis of community access to forest benefits in Nepal. Ecosystem Services, 29, 99–115. https://doi.org/10.1016/j.ecoser.2017.10.020 Chaudhary, S., McGregor, A., Houston, D., & Chettri, N. (2019). Spiritual enrichment or ecological protection?: A multi-scale analysis of cultural ecosystem services at the Mai Pokhari, a Ramsar site of Nepal. Ecosystem Services, 39, 100972. https://doi.org/10.1016/j.ecoser.2019.100972 Chaves, W. A., Valle, D., Tavares, A. S., Morcatty, T. Q., & Wilcove, D. S. (2021). Impacts of rural to urban migration, urbanization, and generational change on consumption of wild animals in the Amazon. Conservation Biology, 35(4), 1186–1197. https://doi.org/10.1111/cobi.13663 Chbel, Asmaa, Delgado, Aurelio Serrano, Soukri, Abdelaziz, & El Khalfi, Bouchra. (2021). Marine biomolecules: A promising approach in therapy and biotechnology. https://doi.org/10.5281/ZENODO.4384158 Chen, G., Sun, W., Wang, X., Kongkiatpaiboon, S., & Cai, X. (2019). Conserving threatened widespread species: A case study using a traditional medicinal plant in Asia. Biodiversity and Conservation, 28(1), 213–227. https://doi.org/10.1007/s10531-018- 1648-1 Cheng, J. J., & Timilsina, G. R. (2011). Status and barriers of advanced biofuel technologies: A review. Renewable Energy, 36(12), 3541–3549. https://doi.org/10.1016/j.renene.2011.04.031 Cheung, H., Mazerolle, L., Possingham, H. P., & Biggs, D. (2018). Medicinal Use and Legalized Trade of Rhinoceros Horn From the Perspective of Traditional Chinese Medicine Practitioners in Hong Kong. Tropical Conservation Science, 11, 1940082918787428. https://doi.org/10.1177/1940082918787428 365 Chhetri, B. B. K., Lund, J. F., & Nielsen, Ø. J. (2012). The public finance potential of community forestry in Nepal. Ecological Economics, 73, 113–121. https://doi.org/10.1016/j.ecolecon.2011.09.023 Chiasson, G., & Leclerc, É. (Eds.). (2013). La gouvernance locale des forêts publiques québécoises: Une avenue de développement des régions périphériques? Québec (Québec): Presses de l’Université du Québec. Chidumayo, E. N. (2013). Forest degradation and recovery in a miombo woodland landscape in Zambia: 22 years of observations on permanent sample plots. Forest Ecology and Management, 291, 154–161. https://doi.org/10.1016/j.foreco.2012.11.031 Chidumayo, E. N., & Gumbo, D. J. (2013). The environmental impacts of charcoal production in tropical ecosystems of the world: A synthesis. Energy for Sustainable Development, 17(2), 86–94. https://doi.org/10.1016/j.esd.2012.07.004 Child, B. (2019). and Sustainable governance of wildlife and community-based natural resource management: From economic principles to practical governance. Abingdon, Oxon ; New York, NY: Routledge/Taylor and Francis Group. Choge, S. K. (2002). Study of Economic aspects of the Wood Carving Industry in Kenya: Implications for policy development to make the industry more sustainable (Masters thesis). , University of Natal, South Africa. Choo, J., Zent, E. L., & Simpson, B. B. (2009). The Importance of Traditional Ecological Knowledge for Palm-weevil Cultivation in the Venezuelan Amazon. Journal of Ethnobiology, 29(1), 113–128. https://doi.org/10.2993/0278-0771-29.1.113 Christensen, D. L., & Gorchov, D. L. (2010). Population dynamics of goldenseal (Hydrastis canadensis) in the core of its historical range. Plant Ecology, 210(2), 195–211. https://doi.org/10.1007/s11258-010-9749-2 Christensen, M., Bhattarai, S., Devkota, S., & Larsen, H. O. (2008). Collection and Use of Wild Edible Fungi in Nepal. Economic Botany, 62(1), 12–23. https://doi.org/10.1007/s12231-007-9000-9 Christensen, V., Coll, M., Piroddi, C., Steenbeek, J., Buszowski, J., & Pauly, D. (2014). A century of fish biomass decline in the ocean. Marine Ecology Progress Series, 512, 155–166. Chuenpagdee, R. (Ed.). (2011). Too big to ignore: Global research network for the future of small-scale fisheries. In World small-scale fisheries: Contemporary visions (pp. 383– 394). Delft: Eburon Academic Publishers. Chuenpagdee, R. (2019). Too Big To Ignore – A Transdisciplinary Journey. In R. Chuenpagdee & S. Jentoft (Eds.), Transdisciplinarity for Small-Scale Fisheries Governance (pp. 15– 31). Cham: Springer International Publishing. https://doi.org/10.1007/978-3-319- 94938-3_2 Chuenpagdee, R., Rocklin, D., Bishop, D., Hynes, M., Greene, R., Lorenzi, M. R., & Devillers, R. (2019). The global information system on small-scale fisheries (ISSF): A crowdsourced knowledge platform. Marine Policy, 101, 158–166. https://doi.org/10.1016/j.marpol.2017.06.018 Chungu, D., Muimba-Kankolongo, A., Roux, J., & Malambo, F. M. (2007). Bark removal for medicinal use predisposes indigenous forest trees to wood degradation in Zambia. 366 157–163. Hemisphere Southern Hemisphere Forestry Journal, 69(3), 157–163. https://doi.org/10.2989/SHFJ.2007.69.3.4.354 Forestry Ciesla, W. M. (2002). Non-wood forest products from temperate broad-leaved trees. Rome: Food and Agriculture Organization of the United Nations. Ciesla, W. M. (2002). Non-wood forest products from temperate broad-leaved trees. Rome: Food and Agriculture Organization of the United Nations. Cifor. (2002). Planning for woodcarving in the 21st century. Center for International Forestry Research (CIFOR). https://doi.org/10.17528/cifor/001164 Cillari, T., Falautano, M., Castriota, L., Marino, V., Vivona, P., & Andaloro, F. (2012). The use of bottom longline on soft bottoms: An opportunity of development for fishing tourism along a coastal area of the Strait of Sicily (Mediterranean Sea). and Ocean & Coastal Management, 55, 20–26. https://doi.org/10.1016/j.ocecoaman.2011.10.007 Cinner, J. E., McClanahan, T. R., MacNeil, M. A., Graham, N. A. J., Daw, T. M., Mukminin, A., … Kuange, J. (2012). Comanagement of coral reef social-ecological systems. Proceedings of the National Academy of Sciences, 109(14), 5219–5222. https://doi.org/10.1073/pnas.1121215109 Cisneros-Montemayor, A. M., Barnes-Mauthe, M., Al-Abdulrazzak, D., Navarro-Holm, E., & Sumaila, U. R. (2013). Global economic value of shark ecotourism: Implications for conservation. Oryx, 47(3), 381–388. https://doi.org/10.1017/S0030605312001718 Cisneros-Montemayor, A. M., Pauly, D., Weatherdon, L. V., & Ota, Y. (2016). A global estimate of seafood consumption by coastal indigenous peoples. PLoS ONE, 11(12). https://doi.org/10.1371/journal.pone.0166681 Cisneros-Montemayor, A. M., Sumaila, U. R., Kaschner, K., & Pauly, D. (2010). The global potential for whale watching. Marine Policy, 34(6), 1273–1278. https://doi.org/10.1016/j.marpol.2010.05.005 Cissé, A. A., Blanchard, F., & Guyader, O. (2014). Sustainability of tropical small-scale fisheries: Integrated assessment in French Guiana. Marine Policy, 44, 397–405. https://doi.org/10.1016/j.marpol.2013.10.003 CITES. (2012). CITES Trade: Recent trends in international trade in Appendix II‐listed species (1996‐2010). UNEP‐WCMC. CITES. (2019). CITES CoP (No. 101). Retrieved from https://s3.us-west- 2.amazonaws.com/enb.iisd.org/archive/download/pdf/enb21101e.pdf?X-Amz- Content-Sha256=UNSIGNED-PAYLOAD&X-Amz-Algorithm=AWS4-HMAC- SHA256&X-Amz-Credential=AKIA6QW3YWTJ6YORWEEL%2F20210313%2Fus- west-2%2Fs3%2Faws4_request&X-Amz-Date=20210313T151120Z&X-Amz- SignedHeaders=host&X-Amz-Expires=60&X-Amz- Signature=b32ebe5b8fcb26d769869957949ebb47630b0298ded7d9efb7310f0e584a03 10 Clancy, J., Ummar, F., Shakya, I., & Kelkar, G. (2007). Appropriate gender-analysis tools for unpacking the gender-energy-poverty nexus. Gender & Development, 15(2), 241–257. https://doi.org/10.1080/13552070701391102 Clapham, P. J. (2015). Japan׳s whaling following the International Court of Justice ruling: Brave New World – Or business as usual? Marine Policy, 51, 238–241. https://doi.org/10.1016/j.marpol.2014.08.011 Clapham, P. J. (2015). Japan׳s whaling following the International Court of Justice ruling: Brave New World – Or business as usual? Marine Policy, 51, 238–241. https://doi.org/10.1016/j.marpol.2014.08.011 367 Clark, M. R., Althaus, F., Schlacher, T. A., Williams, A., Bowden, D. A., & Rowden, A. A. (2016). The impacts of deep-sea fisheries on benthic communities: A review. ICES Journal of Marine Science, 73(suppl_1), i51–i69. Clark, N. E., Lovell, R., Wheeler, B. W., Higgins, S. L., Depledge, M. H., & Norris, K. (2014). Biodiversity, cultural pathways, and human health: A framework. Trends in Ecology & Evolution, 29(4), 198–204. https://doi.org/10.1016/j.tree.2014.01.009 Clarke, S. C., McAllister, M. K., Milner-Gulland, E. J., Kirkwood, G., Michielsens, C. G., Agnew, D. J., … Shivji, M. S. (2006). Global estimates of shark catches using trade records from commercial markets. Ecology Letters, 9(10), 1115–1126. https://doi.org/10.1111/j.1461-0248.2006.00968.x Clavelle, T., Lester, S. E., Gentry, R., & Froehlich, H. E. (2019). Interactions and management for the future of marine aquaculture and capture fisheries. Fish and Fisheries, 20(2), 368–388. https://doi.org/10.1111/faf.12351 Clemann, N., Rowe, K. M. C., Rowe, K. and C., Raadik, T., Gomon, M., Menkhorst, P., … Melville, J. (2014). Value and impacts of collecting vertebrate voucher specimens, with guidelines for ethical collection. Memoirs of Museum Victoria, 72, 141–153. https://doi.org/10.24199/j.mmv.2014.72.09 Clement, C. R. (2006). Fruit trees and the transition to food production in Amazonia. In Studies in Historical Ecology. Time and Complexity in the Neotropical Lowlands (Balée W., Erickson C.L. (ed), pp. 165–185). New York: Columbia University Press. Retrieved from https://www.academia.edu/778447/Fruit_trees_and_the_transition_to_food_productio n_in_Amazonia https://www.academia.edu/778447/Fruit_trees_and_the_transition_to_food_productio n_in_Amazonia Coad, L., Fa, J., Abernethy, K., van Vliet, N., Santamaria, C., Wilkie, D., … Nasi, R. (2019). Towards a sustainable, participatory and inclusive wild meat sector. Bogor, Indonesia: CIFOR. https://doi.org/10.17528/cifor/007046 Coad, L., Schleicher, J., Milner‐Gulland, E. J., Marthews, T. R., Starkey, M., Manica, A., … Abernethy, K. A. (2013). Social and Ecological Change over a Decade in a Village Hunting System, Central Gabon. Conservation Biology, 27(2), 270–280. https://doi.org/10.1111/cobi.12012 Cochran, F. V., Brunsell, N. A., Cabalzar, A., van der Veld, P.-J., Azevedo, E., Azevedo, R. A., … Winegar, L. J. (2016). Indigenous ecological calendars define scales for climate change and sustainability assessments. Sustain Sci, 11(1), 69–89. https://doi.org/10.1007/s11625-015-0303-y Cochrane, K. L., Eggers, J., & Sauer, W. H. H. (2020). A diagnosis of the status and effectiveness of marine fisheries management in South Africa based on two representative case studies. Marine Policy, 112. Scopus. https://doi.org/10.1016/j.marpol.2019.103774 Codjia, J. E., & Yorou, N. S. (2014). Ethnicity and gender variability in the diversity, recognition and exploitation of Wild Useful Fungi in Pobè region (Benin, West Africa). Journal of Applied Biosciences, 78(1), 6729. https://doi.org/10.4314/jab.v78i1.14 Coetzer, K. L., Witkowski, E. T. F., & Erasmus, B. F. N. (2014). Reviewing Biosphere Reserves globally: Effective conservation action or bureaucratic label?: Reviewing 368 Biosphere Reserves globally. Biological Reviews, 89(1), 82–104. https://doi.org/10.1111/brv.12044 Cohen, F. P. A., Valenti, W. C., & Calado, R. (2013). Traceability Issues in the Trade of Marine Ornamental Species. Reviews in Fisheries Science, 21(2), 98–111. https://doi.org/10.1080/10641262.2012.760522 Cohen, P. J., & Alexander, T. J. (2013). Catch Rates, Composition and Fish Size from Reefs Managed with Periodically-Harvested Closures. PLoS ONE, 8(9). Scopus. https://doi.org/10.1371/journal.pone.0073383 Cohen, P. J., Cinner, J. E., & Foale, S. (2013). Fishing dynamics associated with periodically harvested marine closures. Global Environmental Change, 23(6), 1702–1713. Scopus. https://doi.org/10.1016/j.gloenvcha.2013.08.010 Cohen, P. J., & Foale, S. J. (2013a). Sustaining small-scale fisheries with periodically harvested marine reserves. Marine Policy, 37(1), 278–287. Scopus. https://doi.org/10.1016/j.marpol.2012.05.010 Cohen, P. J., & Foale, S. J. (2013b). Sustaining small-scale fisheries with periodically harvested marine reserves. and Marine Policy, 37(1), 278–287. Scopus. https://doi.org/10.1016/j.marpol.2012.05.010 Coleman, J. L., Ascher, J. S., Bickford, D., Buchori, D., Cabanban, A., Chisholm, R. A., … Carrasco, L. R. (2019). Top 100 research questions for biodiversity conservation in Southeast Asia. Biological Conservation, 234, 211–220. https://doi.org/10.1016/j.biocon.2019.03.028 Coleman, T. R., Carpenter, P. B., & Dunphy, W. G. (1996). The Xenopus Cdc6 protein is essential for the initiation of a single round of DNA replication in cell-free extracts. Cell, 87(1), 53–63. https://doi.org/10.1016/s0092-8674(00)81322-7 Colfer, C. J. P. (Ed.). (2005). The equitable forest: Diversity, community, and resource management. Washington, DC : Bogor, Indonesia: Resources for the Future ; Center for International Forestry Research. Coll, M., Carreras, M., Ciércoles, C., Cornax, M.-J., Gorelli, G., Morote, E., & Sáez, R. (2014). Assessing fishing and marine biodiversity changes using fishers’ perceptions: The Spanish Mediterranean and Gulf of Cadiz case study. PLoS ONE, 9(1). Scopus. https://doi.org/10.1371/journal.pone.0085670 Collar, N. J. (2000). Opinion. Collecting and conservation: Cause and effect. Bird Conservation International, 10(1), 1–15. https://doi.org/10.1017/s0959270900000010 Collar, N. J., Baral, H. S., Batbayar, N., Bhardwaj, S., Brahma, N., Burnside, R. J., … Kessler, A. E. (2017). Averting the extinction of bustards in Asia. Forktail, 33, 1–26. Collette, B. B., Carpenter, K. E., Polidoro, B. A., Juan-Jordá, M. J., Boustany, A., Die, D. J., … Yáñez, E. (2011). High Value and Long Life—Double Jeopardy for Tunas and Billfishes. Science, 333(6040), 291–292. https://doi.org/10.1126/science.1208730 Colloca, F., Scarcella, G., & Libralato, S. (2017). Recent trends and impacts of fisheries exploitation on Mediterranean stocks and ecosystems. Frontiers in Marine Science, 4(AUG). Scopus. https://doi.org/10.3389/fmars.2017.00244 Coltman, D. W., O’Donoghue, P., Jorgenson, J. T., Hogg, J. T., Strobeck, C., & Festa-Bianchet, M. (2003). Undesirable evolutionary consequences of trophy hunting. Nature, 426(6967), 655–658. https://doi.org/10.1038/nature02177 369 Comandini, O., & Rinaldi, A. C. (2020). Ethnomycology in Europe: The Past, the Present, and the Future. In J. Pérez-Moreno, A. Guerin-Laguette, R. Flores Arzú, & F.-Q. Yu (Eds.), Mushrooms, Humans and Nature in a Changing World (pp. 341–364). Cham: Springer International Publishing. https://doi.org/10.1007/978-3-030-37378-8_13 Conference Board of Canada. (2018). The Economic Footprint of Angling, Hunting, Trapping, and Sport Shooting in Canada. Conference Board of Canada. Connors, B. M., Cooper, A. B., Peterman, R. M., & Dulvy, N. K. (2014). The false classification of extinction risk in noisy environments. Proceedings of the Royal Society B: Biological Sciences, 281(1787), 20132935. https://doi.org/10.1098/rspb.2013.2935 Conrad, J. L., Greene, W. D., & Hiesl, P. (2018). A Review of Changes in US Logging Businesses 1980s–Present. and Journal of Forestry, 116(3), 291–303. https://doi.org/10.1093/jofore/fvx014 Conservation Frontlines. (2020, April 2). Staying in the Game—Financing the Timbavati Private Nature Reserve. Retrieved March 1, 2022, from Conservation Frontlines website: https://www.conservationfrontlines.org/2020/04/staying-in-the-game- financing-the-timbavati-private-nature-reserve/ Constant, N. L., & Taylor, P. J. (2020). Restoring the forest revives our culture: Ecosystem services and values for ecological restoration across the rural-urban nexus in South Africa. Forest Policy and Economics, 118, 102222. https://doi.org/10.1016/j.forpol.2020.102222 Constantino, P. de A. L. (2016). Deforestation and hunting effects on wildlife across Amazonian indigenous lands. Ecology and Society, 21(2), art3. https://doi.org/10.5751/ES-08323-210203 Cook, F. E. M., Leon, C. J., & Nesbitt, M. (2015). Potpourri as a Sustainable Plant Product: Identity, Origin, and Conservation Status1. Economic Botany, 69(4), 330–344. https://doi.org/10.1007/s12231-015-9325-8 Cooke, S. J., & Schramm, H. L. (2007). Catch-and-release science and its application to conservation and management of recreational fisheries. Fisheries Management and Ecology, 14(2), 73–79. https://doi.org/10.1111/j.1365-2400.2007.00527.x Cooke, S.J., & Murchie, K. J. (2015). Status of aboriginal, commercial and recreational inland fisheries in North America: Past, present and future. Fisheries Management and Ecology, 22(1), 1–13. Scopus. https://doi.org/10.1111/fme.12005 Cooke, Steven J., & Cowx, I. G. (2004). The Role of Recreational Fishing in Global Fish Crises. BioScience, 54(9), 857. https://doi.org/10.1641/0006- 3568(2004)054[0857:TRORFI]2.0.CO;2 Cooke, Steven J., & Cowx, I. G. (2006). Contrasting recreational and commercial fishing: Searching for common issues to promote unified conservation of fisheries resources and aquatic environments. Biological Conservation, 128(1), 93–108. https://doi.org/10.1016/j.biocon.2005.09.019 Cooke, Steven J, Twardek, W. M., Lennox, R. J., Zolderdo, A. J., Bower, S. D., Gutowsky, L. F. G., … Beard, D. (2018). The nexus of fun and nutrition: Recreational fishing is also about food. Fish and Fisheries, 19(2), 201–224. https://doi.org/10.1111/faf.12246 p g Cooke, Steven J., Twardek, W. M., Reid, A. J., Lennox, R. J., Danylchuk, S. C., Brownscombe, Cooke, Steven J., Twardek, W. M., Reid, A. J., Lennox, R. J., Danylchuk, S. C., Brownscombe, J. W., … Danylchuk, A. J. (2019). Searching for responsible and sustainable 370 recreational fisheries in the Anthropocene. Journal of Fish Biology, 94(6), 845–856. https://doi.org/10.1111/jfb.13935 Cooney, R. (2017). The baby and the bathwater: Trophy hunting, conservation and rural livelihoods. UNASYLVA-FAO. Retrieved from https://www.iucn.org/commissions/commission-environmental-economic-and-social- policy/our-work/specialist-group-sustainable-use-and-livelihoods-suli/resources-and- publications/baby-and-bathwater-trophy-hunting-conservation Cooney, Rosie, Roe, D., Dublin, H., & Booker, F. (2018). Wild life, Wild Livelihoods: Involving Communities in Sustainable Wildlife Management and Combatting the Illegal Wildlife Trade. United Nations Environment Programme, Nairobi, Kenya. Cooney, Rosie, Roe, D., Dublin, H., Phelps, J., Wilkie, D., Keane, A., … Biggs, D. (2017). and From Poachers to Protectors: Engaging Local Communities in Solutions to Illegal Wildlife Trade: Engage communities against illegal wildlife trade. Conservation Letters, 10(3), 367–374. https://doi.org/10.1111/conl.12294 Copeland, C., Baker, E., Koehn, J. D., Morris, S. G., & Cowx, I. G. (2017). Motivations of recreational fishers involved in fish habitat management. Fisheries Management and Ecology, 24(1), 82–92. https://doi.org/10.1111/fme.12204 Coppen, J. J. W. (2020a). Chapter 1.0 Overview of International Trade and Markets. The Network for Natural Gums and Resins in Africa (NGARA). Coppen, J. J. W. (2020b). Overview of International Trade and Markets. In Production and Marketing of Gum Resins 2. The Network for Natural Gums & Resins in Africa. Retrieved from https://ngara.org/wp-content/uploads/2020/06/Production-and- Marketing-of-Gum-Resins_2.pdf Cör, D., Knez, Ž., & Knez Hrnčič, M. (2018). Antitumour, Antimicrobial, Antioxidant and Antiacetylcholinesterase Effect of Ganoderma Lucidum Terpenoids and Polysaccharides: A Review. Molecules, 23(3), 649. https://doi.org/10.3390/molecules23030649 Cordell, H. K., Betz, C. J., Mou, S. H., & Gormanson, D. D. (2012). Outdoor Recreation in the Northern United States (No. NRS-GTR-100; p. NRS-GTR-100). Newtown Square, PA: U.S. Department of Agriculture, Forest Service, Northern Research Station. https://doi.org/10.2737/NRS-GTR-100 Cornicelli, L., Fulton, D. C., Grund, M. D., & Fieberg, J. (2011). Hunter perceptions and acceptance of alternative deer management regulations. Wildlife Society Bulletin, 35(3), 323–329. https://doi.org/10.1002/wsb.51 Corral, S., & Manrique de Lara, D. R. (2017). Participatory artisanal fisheries management in islands: Application to the Canary Islands (Spain). Marine Policy, 81, 45–52. https://doi.org/10.1016/j.marpol.2017.03.011 Cosentino, A. M., & Fisher, S. (2016). The Utilization of Aquatic Bushmeat from Small Cetaceans and Manatees in South America and West Africa. Frontiers in Marine Science, 3. https://doi.org/10.3389/fmars.2016.00163 Costa-Neto, E. M. (2005). Entomotherapy or the medicinal use of insects. Journal of Ethnobiology, 25(1), 93–114. https://doi.org/10.2993/0278- 0771(2005)25[93:EOTMUO]2.0.CO;2 371 Costanza, R., Arge, R., Groot, R. D., Farber, S., Hannon, B., Limburg, K., … Neill, R. V. O. (1997). The Value of the World’s Ecosystem Services and Natural Capital. Nature, 387(May), 253–260. http://dx.doi.org/10.1016/j.ijrobp.2010.07.1349 Costello, C., Ovando, D., Hilborn, R., Gaines, S. D., Deschenes, O., & Lester, S. E. (2012). Status and Solutions for the World’s Unassessed Fisheries. Science, 338(6106), 517– 520. https://doi.org/10.1126/science.1223389 Costello, Christopher, & Ovando, D. (2019). Status, Institutions, and Prospects for Global Capture Fisheries. Annual Review of Environment and Resources, 44(1), 177–200. https://doi.org/10.1146/annurev-environ-101718-033310 Costello, Christopher, Ovando, D., Clavelle, T., Strauss, C. K., Hilborn, R., Melnychuk, M. C., … Leland, A. (2016). Global fishery prospects under contrasting management regimes. Proceedings of the National Academy of Sciences, 113(18), 5125–5129. and https://doi.org/10.1073/pnas.1520420113 Courchamp, F., Caut, S., Bonnaud, E., Bourgeois, K., Angulo, E., & Watari, Y. (2011). Eradication of alien invasive species: Surprise effects and conservation successes. Cowlishaw, G., Mendelson, S., & Rowcliffe, J. M. (2004). The Bushmeat Commodity Chain: Patterns of trade and sustainability in a mature urban market in West Africa. 4. Cox, D. T. C., Shanahan, D. F., Hudson, H. L., Plummer, K. E., Siriwardena, G. M., Fuller, R. A., … Gaston, K. J. (2017). Doses of Neighborhood Nature: The Benefits for Mental Health of Living with Nature. BioScience, biw173. https://doi.org/10.1093/biosci/biw173 Cox, P., & Balick, M. (1994). The Ethnobotanical Approach to Drug Discovery. Scientific American, 270(6), 82–87. Craig, P., Green, A., & Tuilagi, F. (2008). Subsistence harvest of coral reef resources in the outer islands of American Samoa: Modern, historic and prehistoric catches. Fisheries Research, 89(3), 230–240. https://doi.org/10.1016/j.fishres.2007.08.018 Crépin, A.-S., Biggs, R., Polasky, S., Troell, M., & de Zeeuw, A. (2012). Regime shifts and management. Ecological Economics, 84, 15–22. https://doi.org/10.1016/j.ecolecon.2012.09.003 Cretois, B., Linnell, J., Grainger, M., Nilsen, E., & Rød, J. K. (2020). Hunters as citizen scientists: Contributions to biodiversity monitoring in Europe. Global Ecology and Conservation, 23, e01077. https://doi.org/10.1016/j.gecco.2020.e01077 Crona, B. I., Basurto, X., Squires, D., Gelcich, S., Daw, T. M., Khan, A., … Allison, E. H. (2016). Towards a typology of interactions between small-scale fisheries and global seafood trade. Marine Policy, 65, 1–10. https://doi.org/10.1016/j.marpol.2015.11.016 Cronkleton, P., & Larson, A. (2015). Formalization and Collective Appropriation of Space on Forest Frontiers: Comparing Communal and Individual Property Systems in the Peruvian and Ecuadoran Amazon. Society & Natural Resources, 28(5), 496–512. https://doi.org/10.1080/08941920.2015.1014609 Crosmary, W.-G., Loveridge, A. J., Ndaimani, H., Lebel, S., Booth, V., Côté, S. D., & Fritz, H. (2013). Trophy hunting in Africa: Long-term trends in antelope horn size. Animal Conservation, 16(6), 648–660. https://doi.org/10.1111/acv.12043 372 Cruz García, G. S. (2006). The mother – child nexus. Knowledge and valuation of wild food plants in Wayanad, Western Ghats, India. Journal of Ethnobiology and Ethnomedicine, 2(1), 39. https://doi.org/10.1186/1746-4269-2-39 Cruz, R. E. A., Kaplan, D. A., Santos, P. B., Ávila-da-Silva, A. O., Marques, E. E., & Isaac, V. J. (2020). Trends and environmental drivers of giant catfish catch in the lower Amazon River. Marine and Freshwater Research. https://doi.org/10.1071/MF20098 Cruz-Garcia, G. S., & Price, L. L. (2011). Ethnobotanical investigation of “wild” food plants used by rice farmers in Kalasin, Northeast Thailand. Journal of Ethnobiology and Ethnomedicine, 7(1), 33. https://doi.org/10.1186/1746-4269-7-33 Cruz-Trinidad, A., Aliño, P. and Renewable and Sustainable Energy Reviews, 14(7), 2139–2146. https://doi.org/10.1016/j.rser.2010.02.002 Cuvilas, C. A., Jirjis, R., & Lucas, C. (2010). Energy situation in Mozambique: A review. Renewable and Sustainable Energy Reviews, 14(7), 2139–2146. https://doi.org/10.1016/j.rser.2010.02.002 Cyr, D., Gauthier, S., Bergeron, Y., & Carcaillet, C. (2009). Forest management is driving the eastern North American boreal forest outside its natural range of variability. Frontiers in Ecology and the Environment, 7(10), 519–524. https://doi.org/10.1890/080088 d’Armengol, L., Prieto Castillo, M., Ruiz-Mallén, I., & Corbera, E. (2018). A systematic review of co-managed small-scale fisheries: Social diversity and adaptive management improve outcomes. Global Environmental Change, 52, 212–225. Scopus. https://doi.org/10.1016/j.gloenvcha.2018.07.009 da Silva Santos, S., de Lucena, R. F. P., de Lucena Soares, H. K., dos Santos Soares, V. M., Sales, N. S., & Mendonça, L. E. T. (2019). Use of mammals in a semi-arid region of Brazil: An approach to the use value and data analysis for conservation. Journal of Ethnobiology and Ethnomedicine, 15(1), 33. https://doi.org/10.1186/s13002-019-0313- 4 Dagorn, L., Holland, K. N., Restrepo, V., & Moreno, G. (2013). Is it good or bad to fish with FADs? What are the real impacts of the use of drifting FADs on pelagic marine ecosystems? Fish and Fisheries, 14(3), 391–415. https://doi.org/10.1111/j.1467- 2979.2012.00478.x Dai, Z. (1992). Review on the research of Nostoc flagelliforme. J. Ning Xia Univ, 13, 71–77. Dale, V. H., Brown, S., Haeuber, R. A., Hobbs, N. T., Huntly, N., Naiman, R. J., … Valone, T. J. (2000). Ecological Principles and Guidelines for Managing the Use of Land. Ecological Applications, 10(3), 639. https://doi.org/10.2307/2641032 Dale, Virginia H., Joyce, L. A., Mcnulty, S., Neilson, R. P., Ayres, M. P., Flannigan, M. D., … Michael Wotton, B. (2001). Climate Change and Forest Disturbances. BioScience, 51(9), 723. https://doi.org/10.1641/0006-3568(2001)051[0723:CCAFD]2.0.CO;2 Daly, B., & Morton, L. L. (2009). Empathic Differences in Adults as a Function of Childhood and Adult Pet Ownership and Pet Type. Anthrozoös, 22(4), 371–382. https://doi.org/10.2752/089279309X12538695316383 Damalas, D., Maravelias, C. D., Osio, G. C., Maynou, F., Sbrana, M., & Sartor, P. (2015). “Once upon a Time in the Mediterranean” long term trends of mediterranean fisheries resources based on fishers’ traditional ecological knowledge. PloS One, 10(3). Damasio, L. de M. A., Lopes, P. F. M., Guariento, R. D., & Carvalho, A. R. (2015). Matching Fishers’ Knowledge and Landing Data to Overcome Data Missing in Small-Scale Fisheries. PLOS ONE, 10(7), e0133122. https://doi.org/10.1371/journal.pone.0133122 Damm G.R. (2008). Recreational Trophy Hunting: “What do we know and what should we do? and M., Geronimo, R. C., & Cabral, R. B. (2014). Linking Food Security with Coral Reefs and Fisheries in the Coral Triangle. Coastal Management, 42(2), 160–182. https://doi.org/10.1080/08920753.2014.877761 CSIRO. (2014). Tiwi seasons and plants and animals calendars. Retrieved August 10, 2021, from https://www.csiro.au/en/research/natural-environment/land/about-the- calendars/tiwi Cuevas, E., Guzmán-Hernández, V., Uribe-Martínez, A., Raymundo-Sánchez, A., & Herrera- Pavon, R. (2018). Identification of potential sea turtle bycatch hotspots using a spatially explicit approach in the Yucatan Peninsula, Mexico. Chelonian Conservation and Biology, 17(1), 78–93. Cullotta, S., Bončina, A., Carvalho-Ribeiro, S. M., Chauvin, C., Farcy, C., Kurttila, M., & Maetzke, F. G. (2015). Forest planning across Europe: The spatial scale, tools, andinter- sectoral integration in land-use planning. Journal of Environmental Planning and Management, 58(8), 1384–1411. https://doi.org/10.1080/09640568.2014.927754 Cunningham, A. B., Campbell, B. M., Belcher, B. M., World Wildlife Fund, Unesco, & Royal Botanic Gardens, Kew (Eds.). (2005). Carving out a future: Forests, livelihoods and the international woodcarving trade. London ; Sterling, VA: Earthscan. Cunningham, Anthony Balfour, & Zondi, A. (1991). Use of animal parts for the commercial trade in traditional medicines. University of Natal, Institute of Natural Resources. Cunningham, P. A., Huijbens, E. H., & Wearing, S. L. (2012). From whaling to whale watching: Examining sustainability and cultural rhetoric. Journal of Sustainable Tourism, 20(1), 143–161. https://doi.org/10.1080/09669582.2011.632091 Cuny, P. (2011). Etat des lieux de la foresterie communautaire et communale au Cameron. Wageningen: Tropenbos International. Curtin, S. (2003). Whale-Watching in Kaikoura: Sustainable Destination Development? Journal of Ecotourism, 2(3), 173–195. https://doi.org/10.1080/14724040308668143 Curtin, S. (2005). Nature, Wild Animals and Tourism: An Experiential View. Journal of Ecotourism, 4(1), 1–15. https://doi.org/10.1080/14724040508668434 Curtin, S. (2005). Nature, Wild Animals and Tourism: An Experiential View. Journal of Ecotourism, 4(1), 1–15. https://doi.org/10.1080/14724040508668434 Curtis, P. G., Slay, C. M., Harris, N. L., Tyukavina, A., & Hansen, M. C. (2018). Classifying drivers of global forest loss. Science, 361(6407), 1108–1111. https://doi.org/10.1126/science.aau3445 Curtis, P. G., Slay, C. M., Harris, N. L., Tyukavina, A., & Hansen, M. C. (2018). Classifying drivers of global forest loss. Science, 361(6407), 1108–1111. https://doi.org/10.1126/science.aau3445 Cuthbert, R. (2010). Sustainability of hunting, population densities, intrinsic rates of increase and conservation of Papua New Guinean mammals: A quantitative review. Biological Conservation, 143, 1850–1859. https://doi.org/10.1016/j.biocon.2010.04.005 Cuthbert, R. (2010). Sustainability of hunting, population densities, intrinsic rates of increase and conservation of Papua New Guinean mammals: A quantitative review. Biological Conservation, 143, 1850–1859. https://doi.org/10.1016/j.biocon.2010.04.005 373 Cuvilas, C. A., Jirjis, R., & Lucas, C. (2010). Energy situation in Mozambique: A review. , P. (2008). The people of the sea: Environment, identity, and history in Oceania. Honolulu: University of Hawaii Press. and In Best Practices in Sustainable Hunting – A Guide to Best Practices from Around the World (pp. 5–11). Danovaro, R., Bongiorni, L., Corinaldesi, C., Giovannelli, D., Damiani, E., Astolfi, P., … Pusceddu, A. (2008). Sunscreens cause coral bleaching by promoting viral infections. Environmental Health Perspectives, 116(4), 441–447. https://doi.org/10.1289/ehp.10966 D’Arcy, P. (2008). The people of the sea: Environment, identity, and history in Oceania. Honolulu: University of Hawaii Press. 374 Das, D., & Afonso, P. (2017). Review of the diversity, ecology, and conservation of elasmobranchs in the Azores region, Mid-North Atlantic. Frontiers in Marine Science, 4(NOV). Scopus. https://doi.org/10.3389/fmars.2017.00354 Daskalov, G. (2003). Long-term changes in fish abundance and environmental indices in the Black Sea. Marine Ecology Progress Series, 255, 259–270. https://doi.org/10.3354/meps255259 Daskalov, G M, Demirel, N., Ulman, A., Georgieva, Y., & Zengin, M. (2020). Stock dynamics and predator-prey effects of bonito and bluefish as top predators in the Black Sea. ICES JMS, in print. Daskalov, G M, Prodanov, K., & Zengin, M. (2008). The Black Seas fisheries and ecosystem change: Discriminating between natural variability and human-related effects. Freshwater Biology, 54, 635–636. https://doi.org/10.1111/j.1365-2427.2008.02115.x Daskalov, Georgi M., Boicenco, L., Grishin, A. N., Lazar, L., Mihneva, V., Shlyakhov, V. A., & Zengin, M. (2017). Architecture of collapse: Regime shift and recovery in an hierarchically structured marine ecosystem. Global Change Biology, 23(4), 1486–1498. https://doi.org/10.1111/gcb.13508 David Sheldrick Wildlife Trust. (2014). Dead or Alive? Valuing an elephant. Surrey, U.K. Retrieved from https://issuu.com/davidsheldrickwildlifetrust/docs/dead_or_alive_final_lr Davidson, L. N. K., Krawchuk, M. A., & Dulvy, N. K. (2016). Why have global shark and ray landings declined: Improved management or overfishing? Fish and Fisheries, 17(2), 438–458. (WOS:000382495500008). https://doi.org/10.1111/faf.12119 Davies, T. D., & Baum, J. K. (2012). Extinction Risk and Overfishing: Reconciling Conservation and Fisheries Perspectives on the Status of Marine Fishes. Scientific Reports, 2(1), 561. https://doi.org/10.1038/srep00561 Davis JR & Garcia R. (1989). Malaria mosquito in Brazil. In Eradication of Exotic Pests (pp. 274–283). Daw, Tim M, Robinson, J., & Graham, N. A. (2011). Perceptions of trends in Seychelles artisanal trap fisheries: Comparing catch monitoring, underwater visual census and fishers’ knowledge. Environmental Conservation, 38(1), 75–88. Daw, T.M. (2008). Spatial distribution of effort by artisanal fishers: Exploring economic factors affecting the lobster fisheries of the Corn Islands, Nicaragua. Fisheries Research, 90(1–3), 17–25. Scopus. https://doi.org/10.1016/j.fishres.2007.09.027 de Albuquerque, U. P., de Lima Araújo, E., El-Deir, A. C. A., de Lima, A. L. A., Souto, A., Bezerra, B. M., … Severi, W. (2012). and Caatinga Revisited: Ecology and Conservation of an Important Seasonal Dry Forest. The Scientific World Journal, 2012. https://doi.org/10.1100/2012/205182 de Avila, A. L., Schwartz, G., Ruschel, A. R., Lopes, J. do C., Silva, J. N. M., Carvalho, J. O. P. de, … Bauhus, J. (2017). Recruitment, growth and recovery of commercial tree species over 30 years following logging and thinning in a tropical rain forest. Forest Ecology and Management, 385, 225–235. https://doi.org/10.1016/j.foreco.2016.11.039 De Borger, E., Tiano, J., Braeckman, U., Rijnsdorp, A. D., & Soetaert, K. (2021). Impact of bottom trawling on sediment biogeochemistry: A modelling approach. Biogeosciences, 18(8), 2539–2557. https://doi.org/10.5194/bg-18-2539-2021 375 De Figueiredo Silva, S. L., Camargo, M., & Estupiñán, R. A. (2012). Fishery management in a conservation area the case of the Oiapoque River in northern Brazil. Cybium, 36(1), 17–30. de Frutos, P. (2020). Changes in world patterns of wild edible mushrooms use measured through international trade flows. Forest Policy and Economics, 112, 102093. https://doi.org/10.1016/j.forpol.2020.102093 de Groot, J., Mohlakoana, N., Knox, A., & Bressers, H. (2017). Fuelling women’s empowerment? An exploration of the linkages between gender, entrepreneurship and access to energy in the informal food sector. Energy Research & Social Science, 28, 86–97. https://doi.org/10.1016/j.erss.2017.04.004 De la Cruz-González, F. J., Patiño-Valencia, J. L., Luna-Raya, M. C., & Cisneros-Montemayor, A. M. (2018). Self-empowerment and successful co-management in an artisanal fishing community: Santa Cruz de Miramar, Mexico. Ocean and Coastal Management, 154, 96–102. https://doi.org/10.1016/j.ocecoaman.2018.01.008 de la Torre, L., Valencia, R., Altamirano, C., & Ravnborg, H. M. (2011). Legal and Administrative Regulation of Palms and Other NTFPs in Colombia, Ecuador, Peru and Bolivia. The Botanical Review, 77(4), 327–369. https://doi.org/10.1007/s12229-011- 9066-z de Lima, I. B., & Green, R. J. (Eds.). (2017). Wildlife Tourism, Environmental Learning and Ethical Encounters. Springer. Retrieved from https://doi.org/10.1007/978-3-319- 55574-4 de los Angeles Somarriba-Chang, M., & Gunnarsdotter, Y. (2012). Local community participation in ecotourism and conservation issues in two nature reserves in Nicaragua. Journal of Sustainable Tourism, 20(8), 1025–1043. https://doi.org/10.1080/09669582.2012.681786 de Mello, N. G. R., Gulinck, H., Van den Broeck, P., & Parra, C. (2020). Social-ecological sustainability of non-timber forest products: A review and theoretical considerations for future research. Forest Policy and Economics, 112, 102109. https://doi.org/10.1016/j.forpol.2020.102109 de Souza Junior, O. G., Nunes, J. L. G., & Silvano, R. A. M. (2020). Biology, ecology and behavior of the acoupa weakfish Cynoscion acoupa (Lacepède, 1801) according to the local knowledge of fishermen in the northern coast of Brazil. Marine Policy. Scopus. https://doi.org/10.1016/j.marpol.2020.103870 De Zoysa, M. and (2017). Community-based forest management in Sri Lanka: Approaching a green economy and environment. The Sri Lanka Forester, 38, 1–23. DEA. (2015). Situation Analysis of Four Selected Sub-Sectors of the Biodiversity and Conservation Sector in South Africa, and Transformation Framework. Pretoria: South African Department of Environmental Affairs. Deb, A. K., Haque, C. E., & Thompson, S. (2015). ‘Man can’t give birth, woman can’t fish’: Gender dynamics in the small-scale fisheries of Bangladesh. Gender, Place & Culture, 22(3), 305–324. https://doi.org/10.1080/0966369X.2013.855626 Dee, L. E., Horii, S. S., & Thornhill, D. J. (2014). Conservation and management of ornamental coral reef wildlife: Successes, shortcomings, and future directions. Biological Conservation, 169, 225–237. https://doi.org/10.1016/j.biocon.2013.11.025 376 Defeo, O., Castrejón, M., Pérez-Castañeda, R., Castilla, J. C., Gutiérrez, N. L., Essington, T. E., & Folke, C. (2016). Co-management in Latin American small-scale shellfisheries: Assessment from long-term case studies. Fish and Fisheries, 17(1), 176–192. https://doi.org/10.1111/faf.12101 DeFilipps, R. A., Krupnick, G. A., & Krupnick, G. A. (2018). The medicinal plants of Myanmar. PhytoKeys, 102(102), 1–341. https://doi.org/10.3897/phytokeys.102.24380 Dehgan, B. (1984). Phylogenetic Significance of Interspecific Hybridization in Jatropha (Euphorbiaceae). Systematic Botany, 9(4), 467. https://doi.org/10.2307/2418796 Dejene, T., Oria-de-Rueda, J. A., & Martín-Pinto, P. (2017). Wild mushrooms in Ethiopia: A review and synthesis for future perspective. Forest Systems, 26(1), eR02. https://doi.org/10.5424/fs/2017261-10790 Dejouhanet, L., & de Bercegol, R. (2019). New Geographies of Collection: Crossed perspectives on modern ”gatherers”—Introduction. EchoGéo [Online], 47. https://doi.org/10.4000/echogeo.17468 Delaney, D. G., Teneva, L. T., Stamoulis, K. A., Giddens, J. L., Koike, H., Ogawa, T., … Kittinger, J. N. (2017). Patterns in artisanal coral reef fisheries revealed through local monitoring efforts. PeerJ, 2017(12). https://doi.org/10.7717/peerj.4089 Delgado, C. L., International Food Policy Research Institute, & WorldFish Center (Eds.). (2003). Fish to 2020: Supply and demand in changing global markets. Washington, D.C. : Penang, Malaysia: International Food Policy Research Institute ; WorldFish Center. Delvaux, C., Sinsin, B., Darchambeau, F., & Van Damme, P. (2009). Recovery from bark harvesting of 12 medicinal tree species in Benin, West Africa. Journal of Applied Ecology, 46(3), 703–712. https://doi.org/10.1111/j.1365-2664.2009.01639.x Demanget, M. (2010). Aux sources d’une communauté imaginée. Le tourisme chamanique à Huautla de Jimenez (Indiens mazatèques, Mexique). Ethnologies, 32(2), 199–232. https://doi.org/10.7202/1006310ar Denevan, W. M., & Padoch, C. (Eds.). (1987). Swidden-fallow agroforestry in the Peruvian Amazon. Bronx, N.Y., U.S.A: New York Botanical Garden. Denny, S., Latham-Green T., & Hazenberg R. (2021). Sustainable Driven Grouse Shooting? A summary of the evidence. University of Northampton. Dent, F., & Clarke, S. (2015). and State of the global market for shark products. FAO Fisheries and Aquaculture, Technical Paper no. 590. FAO. Rome. Dentinger, B. T. M., & Suz, L. M. (2014). What’s for dinner? Undescribed species of porcini in a commercial packet. PeerJ, 2, e570. https://doi.org/10.7717/peerj.570 d’Eon-Eggertson, F., Dulvy, N. K., & Peterman, R. M. (2015). Reliable Identification of Declining Populations in an Uncertain World: Identifying declines in an uncertain world. Conservation Letters, 8(2), 86–96. https://doi.org/10.1111/conl.12123 Deori, B. B., Deb, P., Singha, H., & Choudhury, M. R. (2017). Traditional honey harvesting by the Pnar community of South Assam, India. Our Nature, 14(1), 13–21. https://doi.org/10.3126/on.v14i1.16436 Deprez, P., Volkman, J., & Davenport, S. (1990). Squalene content and neutral lipis composition of Livers from Deep-sea sharks caught in Tasmanian waters. Marine and Freshwater Research, 41(3), 375. https://doi.org/10.1071/MF9900375 377 Dermawan, A. (2020). Wood Legality Verification Systems and Furniture Producers in Indonesia: Lessons from Jepara and Pasuruan. Bogor, Indonesia: CIFOR. Des, M., Rizki, & Fitri, M. (2019). Plants used in the traditional ceremony in kanagarian tiku. Journal of Physics: Conference Series, 1317(1), 012098. https://doi.org/10.1088/1742- 6596/1317/1/012098 Deutsch, S. (2017). The struggle of a marginalized community for ethnic renewal: The whale hunters of Neah Bay. Environmental Sociology, 3(3), 186–196. https://doi.org/10.1080/23251042.2017.1298183 Deutsche Welle. (2020). Foragers find a taste of nature amid London coronavirus lockdown \| DW \| 10.06.2020. Retrieved April 2, 2021, from DW.COM website: https://www.dw.com/en/foragers-find-a-taste-of-nature-amid-london-coronavirus- lockdown/a-53743633 Devillers, P., & Beudels-Jamar, R. C. (2008). The Role of Megafauna Restoration in Dryland, Natural and Cultural Heritage Conservation. In C. Lee & T. Schaaf (Eds.), The Future of Drylands (pp. 101–113). Springer. Devkota, S. (2006). Yarsagumba [Cordyceps sinensis (Berk.) Sacc.]; Traditional Utilization in Dolpa District, Western Nepal. Our Nature, 4(1), 48–52. https://doi.org/10.3126/on.v4i1.502 Devkota, S. (2008). Approach towards the harvesting of Cordyceps sinensis (Berk.) SACC. in pastures of Dolpa, Nepal. In Medicinal plants in Nepal: An Anthology of Contemporary Research (pp. 90–96). Kathmandu, Nepal: Ecological Society (ECOS). Devkota, S. (2009). The frequency and relationship of flowering plants on the distribution pattern of Ophiocordyceps sinensis (Yarchagunbu) in the highlands of Dolpa district, Nepal. Banko Janakari, 19(1), 29–36. https://doi.org/10.3126/banko.v19i1.2180 Devkota, Shiva, Chaudhary, R. P., Werth, S., & Scheidegger, C. (2017). Indigenous knowledge and use of lichens by the lichenophilic communities of the Nepal Himalaya. Journal of Ethnobiology and Ethnomedicine, 13(1), 15. https://doi.org/10.1186/s13002-017-0142- 2 DeWan, A., Green, K., Li, X., & Hayden, D. (2013). Dhyani, S. (2018). Impact of forest leaf litter harvesting to support traditional agriculture in Western Himalayas. 59(3), 473–488. Dey, V. (2016). The global trade in ornamental fish. Infofish International, 4(16), 23–29. and Using social marketing tools to increase fuel-efficient stove adoption for conservation of the golden snub-nosed monkey, Gansu Province, China. Conservation Evidence, 10(1), 32–36. Dewees, P. (2020). Whose problem is it anyway? Narratives and counter-narratives and their impact on woodfuel policy formulation. International Forestry Review, 22(1), 55–64. Dewhurst‐Richman, N., Jones, J., Northridge, S., Ahmed, B., Brook, S., Freeman, R., … Turvey, S. (2020). Fishing for the facts: River dolphin bycatch in a small‐scale freshwater fishery in Bangladesh. Animal Conservation, 23(2), 160–170. Dey, S., Choudhary, S. K., Dey, S., Deshpande, K., & Kelkar, N. (2019). Identifying potential causes of fish declines through local ecological knowledge of fishers in the Ganga River, eastern Bihar, India. Fisheries Management and Ecology. https://doi.org/10.1111/fme.12390 Dey, V. (2016). The global trade in ornamental fish. Infofish International, 4(16), 23–2 Dhyani, S. (2018). Impact of forest leaf litter harvesting to support traditional agriculture in Western Himalayas. 59(3), 473–488. 378 Dhyani, S., Maikhuri, R. K., & Dhyani, D. (2011). Energy budget of fodder harvesting pattern along the altitudinal gradient in Garhwal Himalaya, India. Biomass and Bioenergy, 35(5), 1823–1832. https://doi.org/10.1016/j.biombioe.2011.01.022 Di Franco, A., Milazzo, M., Baiata, P., Tomasello, A., & Chemello, R. (2009). Scuba diver behaviour and its effects on the biota of a Mediterranean marine protected area. Environmental Conservation, 36(01), 32. https://doi.org/10.1017/S0376892909005426 Di Minin, E., Brooks, T. M., Toivonen, T., Butchart, S. H. M., Heikinheimo, V., Watson, J. E. M., … Moilanen, A. (2019). Identifying global centers of unsustainable commercial harvesting of species. Science Advances, 5(4), eaau2879. https://doi.org/10.1126/sciadv.aau2879 Di Minin, E., Clements, H. S., Correia, R. A., Cortés-Capano, G., Fink, C., Haukka, A., … Bradshaw, C. J. A. (2021). Consequences of recreational hunting for biodiversity conservation and livelihoods. One Earth, 4(2), 238–253. https://doi.org/10.1016/j.oneear.2021.01.014 Di Minin, E., Leader-Williams, N., & Bradshaw, C. J. A. (2016). Banning Trophy Hunting Will Exacerbate Biodiversity Loss. Trends in Ecology & Evolution, 31(2), 99–102. https://doi.org/10.1016/j.tree.2015.12.006 Diao, Y., & Yang, Z. (2014). Evaluation of morphological variation and biomass growth of Nostoc commune under laboratory conditions. Journal of Environmental Biology, 35(3), 485. Dias, A. C. E., Cinti, A., Parma, A. M., & Seixas, C. S. (2020). Participatory monitoring of small-scale coastal fisheries in South America: Use of fishers’ knowledge and factors affecting participation. Reviews in Fish Biology and Fisheries, 30(2), 313–333. https://doi.org/10.1007/s11160-020-09602-2 Dias, D. A., Urban, S., & Roessner, U. (2012). A Historical Overview of Natural Products in Drug Discovery. Metabolites, 2(2), 303–336. https://doi.org/10.3390/metabo2020303 Díaz, B. G., Argollo, D. and M., Franco, M. C., Nucci, S. M., Siqueira, W. J., de Laat, D. M., & Colombo, C. A. (2017). High genetic diversity of Jatropha curcas assessed by ISSR. Genetics and Molecular Research, 16(2). https://doi.org/10.4238/gmr16029683 Diaz, S., Demissew, S., Joly, C., Lonsdale, W. M., & Larigauderie, A. (2015). A Rosetta Stone for nature’s benefits to people. PLoS Biology, 13(1), e1002040. https://doi.org/10.1371/journal.pbio.1002040 Díaz, S., Pascual, U., Stenseke M., Martín-López, B., Watson, R. T., Molnár, Z. H. R., … Shirayama, Y. (2018). Assessing nature’s contributions to people. Science, 359(6373), 270–272. https://doi.org/10.1126/science.aap8826 Díaz-Sánchez, J. P., & Obaco, M. (2020). The effects of Coronavirus (COVID-19) on expected tourism revenues for natural preservation. The case of the Galapagos Islands. Journal of Policy Research in Tourism, Leisure and Events, 1–5. https://doi.org/10.1080/19407963.2020.1813149 Dickinson, M. B., & Whigham, D. F. (1999). Regeneration of mahogany (Swietenia macrophylla) in the Yucatan. International Forestry Review, 1, 35–39. Dinesen, G. E., Neuenfeldt, S., Kokkalis, A., Lehmann, A., Egekvist, J., Kristensen, K., … Støttrup, J. G. (2019). Cod and climate: A systems approach for sustainable fisheries management of Atlantic cod (Gadus morhua) in coastal Danish waters. Journal of 379 Coastal Conservation, 23(5), 943–958. Scopus. https://doi.org/10.1007/s11852-019- 00711-0 Dirzo, R., Young, H. S., Galetti, M., Ceballos, G., Isaac, N. J. B., & Collen, B. (2014). Defaunation in the Anthropocene. Science, 345(6195), 401–406. https://doi.org/10.1126/science.1251817 Djagoun, C. A. M. S., Akpona, H. A., Mensah, Guy. A., Nuttman, C., & Sinsin, B. (2013). Wild Mammals Trade for Zootherapeutic and Mythic Purposes in Benin (West Africa): Capitalizing Species Involved, Provision Sources, and Implications for Conservation. In R. R. N. Alves & I. L. Rosa (Eds.), Animals in Traditional Folk Medicine (pp. 367– 381). Berlin, Heidelberg: Springer Berlin Heidelberg. https://doi.org/10.1007/978-3- 642-29026-8_17 Doe, P. (2017). Fish Drying and Smoking: Production and Quality (Routlege). Donegan, T. M. (2009). Type specimens, samples of live individuals and the Galapagos Pink Land Iguana. Zootaxa, 2201(1), 12–20. https://doi.org/10.11646/zootaxa.2201.1.3 Doria, C. R. C., Athayde, S., Lima, H. M. de, Carvajal-Vallejos, F. M., & Dutka-Gianelli, J. (2020). Challenges for the Governance of Small-Scale Fisheries on the Brazil-Bolivia Transboundary Region. Society & Natural Resources, 33(10), 1213–1231. https://doi.org/10.1080/08941920.2020.1771492 Dou, X., & Day, J. (2020). Human-wildlife interactions for tourism: A systematic review. Journal of Hospitality and Tourism Insights, 3(5), 529–547. https://doi.org/10.1108/JHTI-01-2020-0007 Dounias, E., & Aumeeruddy-Thomas, Y. (2017). Children’s ethnobiological knowledge: An introduction. AnthropoChildren. https://doi.org/10.25518/2034-8517.2799 Downs, C., Kramarsky-Winter, E., Fauth, J. E., Segal, R., Bronstein, O., Jeger, R., … others. (2014). and Toxicological effects of the sunscreen UV filter, benzophenone-2, on planulae and in vitro cells of the coral, Stylophora pistillata. Ecotoxicology, 23(2), 175–191. https://doi.org/10.1007/s10646-014-1211-0. Doyon, S. (2019). Les cueillettes commerciales au Québec: Capter la diversité socio- environnementale. EchoGéo [Online], 47. https://doi.org/:10.4000/echogeo.16873 Duarte, J. A., Hernández-Flores, A., Salas, S., & Seijo, J. C. (2018a). Is it sustainable fishing for Octopus maya Voss and Solis, 1966, during the breeding season using a bait-based fishing technique? Fisheries Research, 199, 119–126. https://doi.org/10.1016/j.fishres.2017.11.020 Duarte, J. A., Hernández-Flores, A., Salas, S., & Seijo, J. C. (2018b). Is it sustainable fishing for Octopus maya Voss and Solis, 1966, during the breeding season using a bait-based fishing technique? Fisheries Research, 199, 119–126. Scopus. https://doi.org/10.1016/j.fishres.2017.11.020 Dublin, H. T., Sinclair, A. R. E., & McGlade, J. (1990). Elephants and Fire as Causes of Multiple Stable States in the Serengeti-Mara Woodlands. The Journal of Animal Ecology, 59(3), 1147. https://doi.org/10.2307/5037 Ducos, L., Guillonneau, V., Le Manach, F., & Nouvian, C. (2015). La Belle et la Bête, du requin dans nos crèmes de beauté ! BLOOM NGO. Retrieved from http://www.bloomassociation.org/la-belle-et-la-bete-etude-exclusive-du-requin-dans- nos-cremes-de-beaute/ (accessed 23 feb 2021) 380 Duda, T. F., Bingham, J.-P., Livett, B. G., Kohn, A. J., Massilia, G. R., Schultz, J. R., … Sweedler, J. V. (2004). How much at risk are cone snails? Science (New York, N.Y.), 303(5660), 955–957; author reply 955-957. https://doi.org/10.1126/science.303.5660.955 Duhart, F. (2012). Contribution à l’anthropologie de la consommation de champignons à partir du cas du sud-ouest de la France (xvie-xxi. Revue d’ethnoécologie, (2). https://doi.org/10.4000/ethnoecologie.917 Dulvy, N. K., Fowler, S. L., Musick, J. A., Cavanagh, R. D., Kyne, P. M., Harrison, L. R., … White, W. T. (2014). Extinction risk and conservation of the world’s sharks and rays. ELife, 3, e00590. https://doi.org/10.7554/eLife.00590 Dulvy, N. K., Jennings, S., Goodwin, N. B., Grant, A., & Reynolds, J. D. (2005). Comparison of threat and exploitation status in North-East Atlantic marine populations: Do threat criteria raise false alarms? Journal of Applied Ecology, 42(5), 883–891. https://doi.org/10.1111/j.1365-2664.2005.01063.x Dulvy, N. K., Pacoureau, N., Rigby, C. L., Pollom, R. A., Jabado, R. W., Ebert, D. A., … others. (2021). Overfishing drives over one-third of all sharks and rays toward a global extinction crisis. Current Biology, 31(21), 4773–4787. https://doi.org/10.1016/j.cub.2021.08.062 Duncan, P. F., Brand, A. R., Strand, Ø., & Foucher, E. (2016). The European Scallop Fisheries for Pecten maximus, Aequipecten opercularis, Chlamys islandica, and Mimachlamys varia. Developments in Aquaculture and Fisheries Science, 40, 781–858. Scopus. and https://doi.org/10.1016/B978-0-444-62710-0.00019-5 Dupuis, S., Danneyrolles, V., Laflamme, J., Boucher, Y., & Arseneault, D. (2020). Forest Transformation Following European Settlement in the Saguenay-Lac-St-Jean Valley in Eastern Québec, Canada. Frontiers in Ecology and Evolution, 8, 257. https://doi.org/10.3389/fevo.2020.00257 Durbin, J. C., & Ralambo, J. A. (1994). The Role of Local People in the Successful Maintenance of Protected Areas in Madagascar. Environmental Conservation, 21(2), 115–120. Dutertre, S., Jin, A.-H., Vetter, I., Hamilton, B., Sunagar, K., Lavergne, V., … Lewis, R. J. (2014). Evolution of separate predation- and defence-evoked venoms in carnivorous cone snails. Nature Communications, 5(1), 3521. https://doi.org/10.1038/ncomms4521 Dwyer, L. (2003). Trends Underpinning Tourism to 2015: An Analysis of Key Drivers for Change. International Journal of Tourism Sciences, 3(2), 61–77. https://doi.org/10.1080/15980634.2003.11434550 Dybsand, H. N. H. (2020). In the absence of a main attraction–Perspectives from polar bear watching tourism participants. Tourism Management, 79, 104097. https://doi.org/10.1016/j.tourman.2020.104097 Dykstra, D. P., & Heinrich, R. (1996). FAO model code of forest harvesting practice. Rome : Lanham, MD: FAO. Eba’a Atyi, R., Lescuyer, G., Cerutti, P. O., Tsanga, R., Essiane Mendoula, E., & Collins, F. (2016). Domestic markets, cross-border trade and the role of the informal sector in Cote d’Ivoire, Cameroon and the Democratic Republic of Congo. (No. 4). Yaoundé, Cameroon: CIFOR report for ITTO. 381 Eckert, L. E., Ban, N. C., Frid, A., & McGreer, M. (2018). Diving back in time: Extending historical baselines for yelloweye rockfish with Indigenous knowledge. Aquatic Conservation: Marine and Freshwater Ecosystems, 28(1), 158–166. https://doi.org/10.1002/aqc.2834 Eduardo, L. N., Bertrand, A., Frédou, T., Lira, A. S., Lima, R. S., Ferreira, B. P., … Lucena- Frédou, F. (2020). Biodiversity, ecology, fisheries, and use and trade of Tetraodontiformes fishes reveal their socio-ecological significance along the tropical Brazilian continental shelf. Aquatic Conservation: Marine and Freshwater Ecosystems. Scopus. https://doi.org/10.1002/aqc.3278 Egli, S., Peter, M., Buser, C., Stahel, W., & Ayer, F. (2006). Mushroom picking does not impair future harvests – results of a long-term study in Switzerland. Biological Conservation, 129(2), 271–276. https://doi.org/10.1016/j.biocon.2005.10.042 Ehara, M., Hyakumura, K., Sato, R., Kurosawa, K., Araya, K., Sokh, H., & Kohsaka, R. (2018). Addressing Maladaptive Coping Strategies of Local Communities to Changes in Ecosystem Service Provisions Using the DPSIR Framework. Ecological Economics, 149, 226–238. https://doi.org/10.1016/j.ecolecon.2018.03.008 Ekanayake, E. M. B. P., Xie, Y., Ahmad, S., Geldard, R. P., & Nissanka, A. H. S. (2020). Community Forestry for livelihood Improvement: Evidence from the intermediate zone, Sri lanka. Journal of Sustainable Forestry, 1–17. https://doi.org/10.1080/10549811.2020.1794906 Eklund T. (2017). and Possibilities, Challenges and International Demand for Commercial Hunting Services in Finland—New economic growth from the Finnish bioeconomy (Master’s thesis, Management and Economy in the International Forest Sector, Tampere University of Applied Sciences). Management and Economy in the International Forest Sector, Tampere University of Applied Sciences. Retrieved from https://www.theseus.fi/bitstream/handle/10024/132914/Eklund_Tiina.pdf?sequence=1 &isAllowed=y El Bizri, H., Morcatty, T., Valsecchi, J., Mayor, P., Ribeiro, J., Vasconcelos Neto, C. F., … Fa, J. (2020). Urban wild meat consumption and trade in central Amazonia. Conservation Biology, 34, 438–448. https://doi.org/10.1111/cobi.13420 El Bizri, H. R., Morcatty, T. Q., Lima, J. J. S., & Valsecchi, J. (2015). The thrill of the chase: Uncovering illegal sport hunting in Brazil through YouTube™ posts. Ecology and Society, 20(3), art30. https://doi.org/10.5751/ES-07882-200330 El-Kamali, H. H. (2000). Folk medicinal use of some animal products in Central Sudan. Journal of Ethnopharmacology, 72(1–2), 279–282. https://doi.org/10.1016/S0378- 8741(00)00209-9 Elkinton, J. S., & Liebhold, A. M. (1990). Population Dynamics of Gypsy Moth in North America. Annual Review of Entomology, 35(1), 571–596. https://doi.org/10.1146/annurev.en.35.010190.003035 Ellenberg, U., Setiawan, A. N., Cree, A., Houston, D. M., & Seddon, P. J. (2007). Elevated hormonal stress response and reduced reproductive output in Yellow-eyed penguins exposed to unregulated tourism. General and Comparative Endocrinology, 152(1), 54– 63. https://doi.org/10.1016/j.ygcen.2007.02.022 382 Ellery, W., Cunningham, A., & Choge, S. K. (2005). Chasing the wooden rhino: The case of woodcarving in Kenya. In Brian Belcher, B. M. Campbell, & A. Cunningham (Eds.), Carving out a Future (p. 320). London: Routledge. Retrieved from http://197.248.75.118:8282/jspui/bitstream/123456789/481/1/CHASING%20THE%2 0WOODEN%20RHINO.pdf Ellis, E., Kainer, K., Sierra-Huelsz, J., Negreros-Castillo, P., Rodriguez-Ward, D., & DiGiano, M. (2015). Endurance and Adaptation of Community Forest Management in Quintana Roo, Mexico. Forests, 6(12), 4295–4327. https://doi.org/10.3390/f6114295 Elmahdy, Y. M., Haukeland, J. V., & Fredman, P. (2017). Tourism megatrends: A literature review focused on nature-based tourism. Retrieved from https://hdl.handle.net/11250/2648159 Elps, J., Carrasco, L. R., & Webb, E. L. (2014). A Framework for Assessing Supply-Side Wildlife Conservation. Conservation Biology, 28(1). https://doi.org/10.1111/cobi.12160 Elsey, R, Woodward, A., & Balaguera-Reina, S. (2019). Alligator mississippiensis. The IUCN Red List of Threatened Species. Elsey, Ruth, Woodward, A., & Sergio Balaguera-Reina, L. (2018). IUCN Red List of Threatened Species: Alligator mississippiensis. IUCN Red List of Threatened Species. Retrieved from https://www.iucnredlist.org/en Emery, M.R, Martin, S., & Dyke, A. (2006). Wild harvests from Scottish Woodlands: Social, cultural, and economic values of contemporary non-timber forest products (p. i-viii + 1-40) [Government report]. Edinburgh: Forestry Commission. Emery, M.R., & Pierce, A. R. (2005). and Interrupting the telos: Locating subsistence in contemporary US forests. Environment and Planning A, 37, 981–993. Emery, M. R. (1999). Social values of specialty forest products to rural communities. In: Josiah, Scott J., Ed. Proceedings of the North American Conference on Enterprise Development Through Agroforestry: Farming the Forest for Specialty Products. Minneapolis, MN. 25-32. Retrieved from https://www.fs.usda.gov/treesearch/pubs/18989 Emery, M. R. (2001). Who Knows?: Local Non-Timber Forest Product Knowledge and Stewardship Practices in N orthern M ichigan. Journal of Sustainable Forestry, 13(3– 4), 123–139. https://doi.org/10.1300/J091v13n03_11 Emery, M. R., & Barron, E. S. (2010). Using Local Ecological Knowledge to Assess Morel Decline in the U.S. Mid–Atlantic Region. Economic Botany, 64(3), 205–216. https://doi.org/10.1007/s12231-010-9127-y Emery, M. R., Pierce, A. R., & Schroeder, R. (2004). Criterion 6, indicator 47 area and percent of forest land used for subsistence purposes. In: Darr, David R., Coord. Data Report: A Supplement of the National Report on Sustainable Forests-2003. FS-766A. Washington, DC: U.S. Department of Agriculture. Retrieved from https://www.fs.usda.gov/treesearch/pubs/18428 Emery, M. R., Wrobel, A., Hansen, M. H., Dockry, M., Moser, W. K., Stark, K. J., & Gilbert, J. H. (2014). Using traditional ecological knowledge as a basis for targeted forest inventories: Paper birch (Betula papyrifera) in the US Great Lakes region. Journal of Forestry, 112(2), 207–214. 383 Enomoto, K., Ishikawa, S., Hori, M., Sitha, H., Song, S. L., Thuok, N., & Kurokura, H. (2011). Data mining and stock assessment of fisheries resources in Tonle Sap Lake, Cambodia. Fisheries Science, 77(5), 713–722. Scopus. https://doi.org/10.1007/s12562-011-0378- z Enrique, A.-C., Daniela, V.-E., & Fernando, R.-G. (2020). On birds of Santander-Bio Expeditions, quantifying the cost of collecting voucher specimens in Colombia. Acta Biológica Colombiana, 25. Epelboin, A. (2012). Le bon goût de la viande de primates. In L’animal cannibalisé. Festins d’Afrique (Cros M., Bondaz J., Michaud M. (ed), pp. 41–64). Paris: Editions des archives contemporaines. Epstein, Y. (2017). Killing Wolves to Save Them? Legal Responses to ‘Tolerance Hunting’ in the European Union and United States. Review of European, Comparative & International Environmental Law, 26(1), 19–29. https://doi.org/10.1111/reel.12188 Erickson, W. P., Johnson, G. D., & Young, D. P. J. (2005). A summary and comparison of bird mortality from anthropogenic causes with an emphasis on collisions. In: Ralph, C. John; Rich, Terrell D., Editors 2005. Bird Conservation Implementation and Integration in the Americas: Proceedings of the Third International Partners in Flight Conference. 2002 March 20-24; Asilomar, California, Volume 2 Gen. Tech. Rep. PSW- GTR-191. Albany, CA: U.S. Dept. and of Agriculture, Forest Service, Pacific Southwest Research Station: P. 1029-1042, 191. Retrieved from https://www.fs.usda.gov/treesearch/pubs/32103 Eriksson, H., & Clarke, S. (2015). Chinese market responses to overexploitation of sharks and sea cucumbers. Biological Conservation, 184, 163–173. Scopus. https://doi.org/10.1016/j.biocon.2015.01.018 Eriksson, H., Friedman, K., Amos, M., Bertram, I., Pakoa, K., Fisher, R., & Andrew, N. (2018). Geography limits island small-scale fishery production. Fish and Fisheries, 19(2), 308– 320. Scopus. https://doi.org/10.1111/faf.12255 Erisman, B., Mascarenas, I., Paredes, G., de Mitcheson, Y. S., Aburto-Oropeza, O., & Hastings, P. (2010). Seasonal, annual, and long-term trends in commercial fisheries for aggregating reef fishes in the Gulf of California, Mexico. Fisheries Research, 106(3), 279–288. Ernst, J. (Athman), & Stanek, D. (2006). The Prairie Science Class: A Model for Re-Visioning Environmental Education within the National Wildlife Refuge System. Human Dimensions of Wildlife, 11(4), 255–265. https://doi.org/10.1080/10871200600803010 Ertug, F. (2003). Gendering the tradition of gathering in central Anatolia (Turkey). In Women & Plants (pp. 183–196). London, U.K. & USA: Zed Books. Escalle, L., Brouwer, S., Phillips, J., Pilling, G., & PNA. (2017). Preliminary Analyses of PNA FAD Tracking Data from 2016 and 2017. WCPFC-SC13-2017/MI-WP-05. Western and Central Pacific Fisheries Commission. Escobal, J., & Aldana, U. (2003). Are Nontimber Forest Products the Antidote to Rainforest Degradation? Brazil Nut Extraction in Madre De Dios, Peru. World Development, 31(11), 1873–1887. https://doi.org/10.1016/j.worlddev.2003.08.001 384 Espada, A. L. V., & Vasconcellos Sobrinho, M. (2019). Logging Community-Based Forests in the Amazon: An Analysis of External Influences, Multi-Partner Governance, and Resilience. Forests, 10(6), 461. https://doi.org/10.3390/f10060461 Essington, T. E., Moriarty, P. E., Froehlich, H. E., Hodgson, E. E., Koehn, L. E., Oken, K. L., … Stawitz, C. C. (2015). Fishing amplifies forage fish population collapses. Proceedings of the National Academy of Sciences, 112(21), 6648–6652. https://doi.org/10.1073/pnas.1422020112 Estela, E. H. R., Ghermandi, R. L., & Margutti, L. (1995). Edible Weeds: A Scarcely Used Resource. Bulletin of the Ecological Society of America, 4. Estes, J. A., Terborgh, J., Brashares, J. S., Power, M. E., Berger, J., Bond, W. J., … Wardle, D. A. (2011). Trophic Downgrading of Planet Earth. Science, 333(6040), 301–306. https://doi.org/10.1126/science.1205106 Estes, R. D. (2015). Hunting Helps Conserve African Wildlife Habitat. Retrieved February 27, 2021, from African Indaba website: http://www.africanindaba.com/2015/09/hunting- helps-conserve-african-wildlife-habitat-september-2015-volume-13-4/ Estrada, A., Garber, P. A., & Chaudhary, A. (2019). Expanding global commodities trade and consumption place the world’s primates at risk of extinction. PeerJ, 7, e7068. https://doi.org/10.7717/peerj.7068 EUMOFA. (2019). Case study – Fishmeal and fish oil. Monthly Highlights (No. 4). and Retrieved from https://effop.org/wp-content/uploads/2019/06/EUMOFA-Monthly-Highlights- April-2019-Fishmeal-and-Fish-Oil.pdf European Commission. (2014). A policy framework for climate and energy in the period from 2020 to 2030. Brussels, Belgium.: European Commission. Retrieved from European Commission website: https://eur-lex.europa.eu/legal- content/EN/ALL/?uri=CELEX%3A52014DC0015 Evans, J. (2009). Planted forests: Uses, impacts, and sustainability. Wallingford Rome: CABI FAO. Evers, H., Pinnegar, J. K., & Taylor, M. I. (2019a). Where are they all from? – Sources and sustainability in the ornamental freshwater fish trade. Journal of Fish Biology, jfb.13930. https://doi.org/10.1111/jfb.13930 Evers, H., Pinnegar, J. K., & Taylor, M. I. (2019b). Where are they all from? – Sources and sustainability in the ornamental freshwater fish trade. Journal of Fish Biology, jfb.13930. https://doi.org/10.1111/jfb.13930 Fa, J. E., & Brown, D. (2009). Impacts of hunting on mammals in African tropical moist forests: A review and synthesis. Mammal Review, 39(4), 231–264. https://doi.org/10.1111/j.1365-2907.2009.00149.x Fa, J. E., & Peres, C. A. (2001). Game vertebrate extraction in African and Neotropical forests: An intercontinental comparison. 39. Fa, J. E., Peres, C. A., & Meeuwig, J. (2002). Bushmeat Exploitation in Tropical Forests: An Intercontinental Comparison. Conservation Biology, 16(1), 232–237. https://doi.org/10.1046/j.1523-1739.2002.00275.x Fa, J. E., Ryan, S. F., & Bell, D. J. (2005). Hunting vulnerability, ecological characteristics and harvest rates of bushmeat species in afrotropical forests. Biological Conservation, 121(2), 167–176. https://doi.org/10.1016/j.biocon.2004.04.016 385 Fabian, A., Volkmer, H., & Wiedemann, C. (2011). Microloans for Thermal Insulation: A Product Documentation Based on Experience in Tajik Gorno-Badakhshan. [Support for microfinance services in rural regions" and GTZ/DED/CIM project "Sustainable Management of Natural Resources in Gorno-Badakhshan]. GIZ. 2 April 2021. Retrieved from GIZ website: https://archnet.org/collections/378/publications/6830 Fabio, P., Silvia, C., Paolo, V., & Anelli Monti, M. (2016). Present and future status of artisanal fisheries in the Adriatic Sea (western Mediterranean Sea). Ocean and Coastal Management, 122, 49–56. Scopus. https://doi.org/10.1016/j.ocecoaman.2016.01.004 FairWild Foundation. (2010). FairWild Standard, Version 2.0. Weinfelden, Switzerland: FairWild Foundation. Fan, M.-F. (2019). Risk discourses and governance of high-level radioactive waste storage in Taiwan. Journal of Environmental Planning and Management, 62(2), 327–341. https://doi.org/10.1080/09640568.2017.1418303 FAO. (1967). World Symposium on Man-made Forests and their Industrial Importance. Unasylva, (21), 3–4. FAO. (1995a). Code of Conduct for Responsible Fisheries. Food and Agriculture Organization of the United Nations. FAO. (1995b). Code of Conduct for Responsible Fisheries. Rome: Food and Agriculture ORganization of the United Nations. FAO (Ed.). (1999a). International plan of action for reducing incidental catch of seabirds in longline fisheries. Rome: Food and Agriculture Organization of the United Nations. FAO. (1999b). and International Plan of Action for the Conservation and Management of Sharks. Food and Agriculture Organization of the United Nations. FAO (Ed.). (1999c). International plan of action for the conservation and management of sharks. Rome: Food and Agriculture Organization of the United Nations. FAO. (2009). Guidelines to Reduce Sea turtle Mortality in Fishing Operations. Food and Agriculture Organization of the United Nations. FAO. (2010a). Agreement on Port State Measures to Prevent, Deter and Eliminate Illegal, Unreported and Unregulated Fishing (p. 100). Rome: Food and Agriculture Organization of the United Nations. Retrieved from Food and Agriculture Organization of the United Nations website: https://www.fao.org/3/i1644t/i1644t.pdf FAO. (2010b). Global forest resources assessment 2010: Main report. Rome: Food and Agriculture Organization of the United Nations. FAO. (2010b). Global forest resources assessment 2010: Main report. Rome: Food and Agriculture Organization of the United Nations. FAO. (2010c). State of the worlds fisheries and aquaculture in 2010. (p. 197). Rome. https://doi.org/10.4060/ca9229en FAO. (2010c). State of the worlds fisheries and aquaculture in 2010. (p. 197). Rome. https://doi.org/10.4060/ca9229en FAO. (2011). International guidelines on bycatch management and reduction of discards. Food and Agriculture Organization of the United Nations. FAO. (2011). International guidelines on bycatch management and reduction of discards. Food and Agriculture Organization of the United Nations. FAO. (2012a). La situation mondiale des pêches et de l’aquaculture (SOFIA). Rome: Food and Agriculture Organization of the United Nations. Retrieved from Food and Agriculture Organization of the United Nations website: www.fao.org/3/a-i3720f.pdf FAO. (2012a). La situation mondiale des pêches et de l’aquaculture (SOFIA). Rome: Food and Agriculture Organization of the United Nations. Retrieved from Food and Agriculture Organization of the United Nations website: www.fao.org/3/a-i3720f.pdf FAO. (2012b). Recreational fisheries. Rome: Food and Agriculture Organization of the United Nations. 386 FAO. (2012c). The state of world fisheries and aquaculture 2012. Rome; London: Food and Agriculture Organization of the United Nations ; Eurospan [distributor. Retrieved from https://www.fao.org/3/i2727e/i2727e00.htm FAO. (2014a). State of the world’s forests 2014: Enhancing the socioeconomic benefits from forests. Rome: Food and Agricultural Organization of the United Nations. FAO. (2014b). State of the World‘s Forests Enhancing the socioeconomic benefits from forests. Retrieved from https://www.fao.org/3/i3710e/i3710e.pdf FAO. (2014c). The state of the world’s forest genetic resources. Rome: Commission on Genetic Resources for Food and Agriculture, Food and Agriculture Organization of the United Nations. FAO. (2014d). The State of World Fisheries and Aquaculture. Opportunities and Challenges. Food and Agriculture Organization of the United Nations. FAO (Ed.). (2015). and Voluntary guidelines for securing sustainable small-scale fisheries in the context of food security and poverty eradication. Rome. FAO. (2016a). Agreement on Port State Measures to Prevent, Deter and Eliminate Illegal, Unreported and Unregulated Fishing. Rome: Food and Agriculture Organization of the United Nations. FAO. (2016b). The State of World Fisheries and Aquaculture 2016. Contributing to food security and nutrition for all. FAO, Rome. F.A.O. (2017). FAOSTAT statistical database. Rome, Italy: FAOSTAT, FAO. FAO. (2017a). Guidelines on Assessing Biodiverse Foods in Dietary Intake Surveys. Retrieved from https://www.fao.org/3/i6717e/i6717e.pdf FAO. (2017b). Incentivising sustainable wood energy in sub-Saharan Africa—A way forward for policy makers. Rome: Food and Agriculture Organization of the United Nations. Retrieved from Food and Agriculture Organization of the United Nations website: http://www.fao.org/3/i6815e/i6815e.pdf FAO. (2018a). Forests pathways to sustainable development. Rome: Food and Agricultural Organization of the United Nations. FAO (Ed.). (2018c). The State of the world’s forests 2018—Forests pathways to sustainable development. Rome: Food and Agricultural Organization of the United Nations. FAO. (2018d). The State of World Fisheries and Aquaculture. Meeting the Sustainable Development Goals. Food and Agriculture Organization of the United Nations. FAO. (2019a). Global forest products facts and figures 2018. 20. FAO. (2019b). The state of the world’s biodiversity for food and agriculture (p. 572). Rome: Commission on Genetic Resources for Food and Agriculture. Retrieved from Commission on Genetic Resources for Food and Agriculture website: http://www.fao.org/3/CA3129EN/CA3129EN.pdf FAO. (2020a). Global Forest Resources Assessment (FRA) 2020: Main report. Rome: Food and Agricultural Organization of the United Nations. Retrieved from https://www.fao.org/documents/card/en/c/ca8753en/ FAO. (2020b). Impacts of COVID-19 on wood value chains and forest sector response. Rome: Food and Agricultural Organization of the United Nations. https://doi.org/10.4060/cb1987en 387 FAO. (2020c). The impacts of COVID-19 on the forest sector: How to respond? Rome: Food and Agricultural Organization of the United Nations. https://doi.org/10.4060/ca8844en FAO. (2020d). The State of World Fisheries and Aquaculture 2020: Sustainability in action. Rome: Food and Agricultural Organization of the United Nations. https://doi.org/10.4060/ca9229enAlso Available in:Chinese Spanish Arabic French Russian FAO. (2021a). Agreement on Port State Measures (PSMA) \| Food and Agriculture Organization of the United Nations. Retrieved April 2, 2021, from http://www.fao.org/port-state-measures/en/ FAO. (2021b). Forestry Production and Trade Database. License: CC BY-NC-SA 3.0 IGO. Extracted from: Http://www.fao.org/faostat/en/#data/FO. Data of Access: May 2021. FAO, Schure, J., Ingram, V., & Yoo, B. I. (2017). Sustainable woodfuel for food security: A smart choice: green, renewable and affordable. Rome: Food and Agriculture Organization of the United Nations. and FAO Stat. (2018). FAO statistics on forestry production and use. Retrieved from https://www.fao.org/faostat/en/#data/FO FAO, & UNEP. (2020). The State of the World’s Forests 2020: Forests, Biodiversity and People. Rome: Food and Agricultural Organization of the United Nations. https://doi.org/10.4060/ca8642en Farfán, B., Casas, A., Ibarra-Manríquez, G., & Pérez-Negrón, E. (2007). Mazahua Ethnobotany and Subsistence in the Monarch Butterfly Biosphere Reserve, Mexico. Economic Botany, 61(2), 173–191. https://doi.org/10.1663/0013- 0001(2007)61[173:MEASIT]2.0.CO;2 Farfán-Heredia, B., Casas, A., Moreno-Calles, A. I., García-Frapolli, E., & Castilleja, A. (2018). Ethnoecology of the interchange of wild and weedy plants and mushrooms in Phurépecha markets of Mexico: Economic motives of biotic resources management. Journal of Ethnobiology and Ethnomedicine, 14(1), 5. https://doi.org/10.1186/s13002- 018-0205-z Fargeot, C., Drouet-Hoguet, N., & Le Bel, S. (2017). The role of bushmeat in urban household consumption: Insights from Bangui, the capital city of the Central African Republic. Bois & Forets Des Tropiques, 332, 31–42. https://doi.org/10.19182/bft2017.332.a31331 Farrell, M., & Chabot, B. (2012). Assessing the growth potential and economic impact of the U.S. maple syrup industry. Journal of Agriculture, Food Systems, and Community Development, 11–27. https://doi.org/10.5304/jafscd.2012.022.009 Fashing, P. J. (2004). Mortality trends in the African cherry (Prunus africana) and the implications for colobus monkeys (Colobus guereza) in Kakamega Forest, Kenya. Biological Conservation, 120(4), 449–459. https://doi.org/10.1016/j.biocon.2004.03.018 Faulkner, B., & Tideswell, C. (1997). A framework for monitoring community impacts of tourism. Journal of Sustainable Tourism, 5(1), 3–28. Fearnside, P. M. (2004). Are climate change impacts already affecting tropical forest biomass? Global Environmental Change, 14(4), 299–302. https://doi.org/10.1016/j.gloenvcha.2004.02.001 388 Fédensieu, A. (1988). Cures saisonnières et plantes de cueillette en milieu cévenol. Savoirs, 1, 66–83. Fedrowitz, K., Koricheva, J., Baker, S. C., Lindenmayer, D. B., Palik, B., Rosenvald, R., … Gustafsson, L. (2014). REVIEW: Can retention forestry help conserve biodiversity? A meta‐analysis. Journal of Applied Ecology, 51(6), 1669–1679. https://doi.org/10.1111/1365-2664.12289 Feeney, K. (2017). Peyote as Commodity: An Examination of Market Actors and Access Mechanisms. Human Organization, 76(1), 59–72. https://doi.org/10.17730/0018- 7259.76.1.59 Feng, Y., Chen, X.-M., Zhao, M., He, Z., Sun, L., Wang, C.-Y., & Ding, W.-F. (2018). Edible insects in China: Utilization and prospects. Insect Science, 25(2), 184–198. https://doi.org/10.1111/1744-7917.12449 Fernandes, B. M. (2004). Espaços agrários de inclusão e exclusão social: Novas configurações do campo brasileiro. Agrária (São Paulo. Online), 0(1), 16. https://doi.org/10.11606/issn.1808-1150.v0i1p16-36 Fernandes, P. G., Ralph, G. M., Nieto, A., García Criado, M., Vasilakopoulos, P., Maravelias, C. D., … Carpenter, K. E. (2017). Coherent assessments of Europe’s marine fishes show regional divergence and megafauna loss. and Nature Ecology & Evolution, 1(7), 0170. https://doi.org/10.1038/s41559-017-0170 Fernández-Gil, A., Naves, J., Ordiz, A., Quevedo, M., Revilla, E., & Delibes, M. (2016). Conflict Misleads Large Carnivore Management and Conservation: Brown Bears and Wolves in Spain. PLOS ONE, 11(3), e0151541. https://doi.org/10.1371/journal.pone.0151541 Ferreira, L. V., Cunha, D. A., & Parolin, P. (2014). Effects of logging on Virola surinamensis in an Amazonian floodplain forest. Environment Conservation Journal, 15(3), 1–8. https://doi.org/10.36953/ECJ.2014.15301 Ferretti, F., Osio, G. C., Jenkins, C. J., Rosenberg, A. A., & Lotze, H. K. (2013). Long-term change in a meso-predator community in response to prolonged and heterogeneous human impact. Scientific Reports, 3(1), 1–11. https://doi.org/10.1038/srep01057 Ferse, S.C.A., Glaser, M., Neil, M., & Schwerdtner Máñez, K. (2014). To cope or to sustain? Eroding long-term sustainability in an Indonesian coral reef fishery. Regional Environmental Change, 14(6), 2053–2065. Scopus. https://doi.org/10.1007/s10113- 012-0342-1 Ferse, Sebastian C. A., Knittweis, L., Krause, G., Maddusila, A., & Glaser, M. (2012). Livelihoods of Ornamental Coral Fishermen in South Sulawesi/Indonesia: Implications for Management. Coastal Management, 40(5), 525–555. https://doi.org/10.1080/08920753.2012.694801 FFA. (2015). Economic Indicators Report. Pacific Islands Forum Fisheries Agency. Fialho, M. de S., Ludwig, G., & Valença-Montenegro, M. M. (2016). Legal International Trade in Live Neotropical Primates Originating from South America. 6. Fidalgo, O., & Prance, G. T. (1976). The Ethnomycology of the Sanama Indians. Mycologia, 68(1), 201–210. https://doi.org/10.1080/00275514.1976.12019902 Fields, A. T., Fischer, G. A., Shea, S. K. H., Zhang, H., Abercrombie, D. L., Feldheim, K. A., … Chapman, D. D. (2018). Species composition of the international shark fin trade 389 assessed through a retail-market survey in Hong Kong. Conservation Biology, 32(2), 376–389. Scopus. https://doi.org/10.1111/cobi.13043 Figus, E., Carothers, C., & Beaudreau, A. H. (2017). Using local ecological knowledge to inform fisheries assessment: Measuring agreement among Polish fishermen about the abundance and condition of Baltic cod (Gadus morhua). ICES Journal of Marine Science, 74(8), 2213–2222. https://doi.org/10.1093/icesjms/fsx061 Findlay, S., & Twine, W. (2018). Chiefs in a democracy: A case study of the ‘new’systems of regulating firewood harvesting in post-apartheid South Africa. Land, 7(1), 35. Finlayson, A. (1994). Fishing for truth: A sociological analysis of northern cod stock assessments from 1977-1990 (Vol. 52). St. Johns, Newfoundland, Canada: Institute of Social and Economic Research, Memorial University of Newfoundland. Fischer, A., Sandström, C., Delibes-Mateos, M., Arroyo, B., Tadie, D., Randall, D., … Majić, A. (2013). On the multifunctionality of hunting – an institutional analysis of eight cases from Europe and Africa. Journal of Environmental Planning and Management, 56(4), 531–552. and https://doi.org/10.1080/09640568.2012.689615 Fischer, C. (2010). Does Trade Help or Hinder the Conservation of Natural Resources? Review of Environmental Economics and Policy, 4(1), 103–121. (WOS:000274089000007). https://doi.org/10.1093/reep/rep023 Fischer, L. K., & Kowarik, I. (2020). Connecting people to biodiversity in cities of tomorrow: Is urban foraging a powerful tool? Ecological Indicators, 112, 106087. https://doi.org/10.1016/j.ecolind.2020.106087 Fisher, R., Maginnis, S., Jackson, W., Barrow, E., & Jeanrenaud, S. (Eds.). (2008). Linking conservation and poverty reduction: Landscapes, people and power. London ; Sterling, VA: Earthscan. Fisheries Agency of Japan. (2007). Report of the Joint Meeting of Tuna RFMOs. Fisheries Agency of Japan. Fitzgerald, S. P., Wilson, J. R., & Lenihan, H. S. (2018). Detecting a need for improved management in a data-limited crab fishery. Fisheries Research, 208, 133–144. Scopus. https://doi.org/10.1016/j.fishres.2018.07.012 Flachsenberg, H., & Galleti, H. A. (1999). El Manejo Forestal de La Selva En Quintana Roo, México. In In La Selva Maya: Conservación y Desarrollo. island press. Retrieved from http://repositorio.cenpat- conicet.gob.ar:8081/xmlui/bitstream/handle/123456789/469/laSelvaMaya.pdf?sequen ce=1 Flannery, T. F. (2000). The Pleistocene mammal fauna of Kelangurr Cave, central montane Irian Jaya, Indonesia. Records of the Western Australian Museum, 57, 341–350. Flecks, M., Weinsheimer, F., Böhme, W., Chenga, J., Lötters, S., & Rödder, D. (2012). Watching extinction happen: The dramatic population decline of the critically endangered Tanzanian Turquoise Dwarf Gecko, Lygodactylus williamsi. Salamandra, 48(1), 12–20. Flores-Martínez, J. J., Martínez-Pacheco, A., Rendón-Salinas, E., Rickards, J., Sarkar, S., & Sánchez-Cordero, V. (2019). Recent Forest Cover Loss in the Core Zones of the Monarch Butterfly Biosphere Reserve in Mexico. Frontiers in Environmental Science, 7, 167. https://doi.org/10.3389/fenvs.2019.00167 390 Flores-Palacios, A., Bustamante-Molina, A., Corona-López, A., & Valencia-Díaz, S. (2015). Seed number, germination and longevity in wild dry forest Tillandsia species of horticultural value. Scientia Horticulturae, 187, 72–79. https://doi.org/10.1016/j.scienta.2015.03.003 Flowers, N. (2014). Economia, Subsistência e Trabalho: Sistema em Mudança. In Antropologia e História Xavante em Perspectiva (Coimbra Jr. CEA, Welch JR. (ed), pp. 67-86.). Rio de Janeiro: Museu do Índio/FUNAI. Retrieved from https://acervo.socioambiental.org/acervo/livros/antropologia-e-historia-xavante-em- perspectiva Floyd, M. F., Nicholas, L., Lee, I., Lee, J.-H., & Scott, D. (2006). Social Stratification in Recreational Fishing Participation: Research and Policy Implications. Leisure Sciences, 28(4), 351–368. https://doi.org/10.1080/01490400600745860 Foale, S., Cohen, P., Januchowski‐Hartley, S., Wenger, A., & Macintyre, M. (2011). Tenure and taboos: Origins and implications for fisheries in the Pacific. Fish and Fisheries, 12(4), 357–369. https://doi.org/10.1111/j.1467-2979.2010.00395.x FOC. (2020). The Flora of China (FOC). Retrieved from http://www.iplant.cn/frps/jingji/2?page=2 Foote, L., & Wenzel, G. (2009). Polar bear conservation hunting in Canada: Economics, culture and unintended consequences. and In M. R. Milton & L. Foote (Eds.), Inuit, Polar Bears and Sustainable Use: Local, National and International Perspectives (pp. 13–24). University of Alberta Press. Forest Europe. (2020). State of Europe’s Forests 2020. Retrieved from www.foresteurope.org Foroughirad, V., & Mann, J. (2013). Long-term impacts of fish provisioning on the behavior and survival of wild bottlenose dolphins. Biological Conservation, 160, 242–249. https://doi.org/10.1016/j.biocon.2013.01.001 Forrest, R. E., & Walters, C. J. (2009). Estimating thresholds to optimal harvest rate for long- lived, low-fecundity sharks accounting for selectivity and density dependence in recruitment. Canadian Journal of Fisheries and Aquatic Sciences, 66(12), 2062–2080. Fortibuoni, T., Borme, D., Franceschini, G., Giovanardi, O., & Raicevich, S. (2016). Common, rare or extirpated? Shifting baselines for common angelshark, Squatina squatina (Elasmobranchii: Squatinidae), in the Northern Adriatic Sea (Mediterranean Sea). Hydrobiologia, 772(1), 247–259. https://doi.org/10.1007/s10750-016-2671-4 Fossgard, K., & Fredman, P. (2019). Dimensions in the nature-based tourism experiencescape: An explorative analysis. Journal of Outdoor Recreation and Tourism, 28, 100219. https://doi.org/10.1016/j.jort.2019.04.001 Fotiou, E. (2016). The Globalization of Ayahuasca Shamanism and the Erasure of Indigenous Shamanism. Anthropology of Consciousness, 27(2), 151–179. https://doi.org/10.1111/anoc.12056 Fournier, A. (2011). Consequences of wooded shrine rituals on vegetation conservation in West Africa: A case study from the Bwaba cultural area (West Burkina Faso). Biodiversity and Conservation, 20(9), 1895–1910. https://doi.org/10.1007/s10531-011-0065-5 Fournier, A. (2011). Consequences of wooded shrine rituals on vegetation conservation in West Africa: A case study from the Bwaba cultural area (West Burkina Faso). Biodiversity and Conservation, 20(9), 1895–1910. https://doi.org/10.1007/s10531-011-0065-5 Fournier, J. (2013). Facteurs de succès et de contraintes à la foresterie communautaire: Étude de cas et évaluation de deux initiatives (Master’s thesis, Université du Québec à Fournier, J. (2013). Facteurs de succès et de contraintes à la foresterie communautaire: Étude de cas et évaluation de deux initiatives (Master’s thesis, Université du Québec à 391 Montréal). Université du Québec à Montréal. Retrieved from https://core.ac.uk/download/pdf/18491099.pdf Fourt, M., Faget, D., Dailianis, T., Koutsoubas, D., & Pérez, T. (2020). Past and present of a Mediterranean small-scale fishery: The Greek sponge fishery—Its resilience and sustainability. Regional Environmental Change, 20(1). Scopus. https://doi.org/10.1007/s10113-020-01581-1 Franco, F. M. (2015). Calendars and Ecosystem Management: Some Observations. Hum Ecol, 43(2), 355–359. https://doi.org/10.1007/s10745-015-9740-6 Frangoudes, K., & Garineaud, C. (2015). Governability of kelp forest small-scale harvesting in Iroise sea, France. In Interactive governance for small-scale fisheries (Jentoft S., Chuenpagdee R. (ed), Vol. 13, pp. 101–115). Amsterdam: MARE Publication Series. Retrieved from https://vdoc.pub/documents/interactive-governance-for-small-scale- fisheries-global-reflections-7ev51kilp040 Frank, E. G., & Wilcove, D. S. (2019). and https://doi.org/10.1016/j.forpol.2019.05.009 harvest by community forest enterprises in Mexico. Forest Policy and Economics, 107, 101923. https://doi.org/10.1016/j.forpol.2019.05.009 Freyfogle, E. T., & Goble, D. (2019). Wildlife law: A primer (Second edition). Washington, DC: Island Press. Frezza, P. E., & Clem, S. E. (2015). Using local fishers’ knowledge to characterize historical trends in the Florida Bay bonefish population and fishery. Environmental Biology of Fishes, 98(11), 2187–2202. Scopus. https://doi.org/10.1007/s10641-015-0442-0 Friday, J., & Okano, D. (2006). Calophyllum inophyllum (kamani). Species Profiles for Pacific Island Agroforestry, 2(1), 1–17. Friedlander, A. M., Shackeroff, J. M., & Kittinger, J. N. (2013). Customary marine resource knowledge and use in contemporary Hawai’i. Pacific Science, 67(3), 441–460. https://doi.org/10.2984/67.3.10 Friedlander, A. M., Stamoulis, K. A., Kittinger, J. N., Drazen, J. C., & Tissot, B. N. (2014). Understanding the Scale of Marine Protection in Hawai’i: From Community-Based Management to the Remote Northwestern Hawaiian Islands (p. 203). https://doi.org/10.1016/B978-0-12-800214-8.00005-0 Friedman, K., Gabriel, S., Abe, O., Nuruddin, A. A., Ali, A., Hassan, R. B. R., … Ye, Y. (2018). Examining the impact of CITES listing of sharks and rays in Southeast Asian fisheries. Fish and Fisheries, 19(4), 662–676. https://doi.org/10.1111/faf.12281 Froehlich, H. E., Jacobsen, N. S., Essington, T. E., Clavelle, T., & Halpern, B. S. (2018). Avoiding the ecological limits of forage fish for fed aquaculture. Nature Sustainability, 1(6), 298–303. https://doi.org/10.1038/s41893-018-0077-1 Frosch, B., & Deil, U. (2011). Forest vegetation on sacred sites of the Tangier Peninsula (NW Morocco) – discussed in a SW-Mediterranean context. Phytocoenologia, 41(3), 153– 181. https://doi.org/10.1127/0340-269X/2011/0041-0503 Frumkin, H., Bratman, G. N., Breslow, S. J., Cochran, B., Kahn Jr, P. H., Lawler, J. J., … Wood, S. A. (2017). Nature Contact and Human Health: A Research Agenda. Environmental Health Perspectives, 125(7), 075001. https://doi.org/10.1289/EHP1663 Wood, S. A. (2017). Nature Contact and Human Health: A Research Agenda. Environmental Health Perspectives, 125(7), 075001. https://doi.org/10.1289/EHP1663 FSC. (2012). Strategic review on the future of forest plantations. Helsinki, Finland. FSC. (2012). Strategic review on the future of forest plantations. Helsinki, Finland. FSI. (2019). India State of Forest Report 2019. Deheradun, Forest Survey of India, Ministry of Environment, Forest and Climate Change. Fu, Y., Grumbine, R. E., Wilkes, A., Wang, Y., Xu, J.-C., & Yang, Y.-P. (2012). Climate change adaptation among Tibetan pastoralists: Challenges in enhancing local adaptation through policy support. Environ Manage, 50(4), 607–621. https://doi.org/10.1007/s00267-012-9918-2 Fu, Y., Yang, J., Cunningham, A. B., Towns, A. M., Zhang, Y., Yang, H., … Yang, X. (2018). and Long delays in banning trade in threatened species. Science, 363(6428), 686–688. https://doi.org/10.1126/science.aav4013 Frank, S., Ordiz, A., Gosselin, J., Hertel, A., Kindberg, J., Leclerc, M., … Swenson, J. (2017). Indirect effects of bear hunting: A review from Scandinavia. Ursus, 28, 150–164. https://doi.org/10.2192/URSU-D-16-00028.1 Franklin, J. F., Berg, D. E., Thornburgh, D. A., & Tappeiner, J. C. (1997). Alternative silvicultural approaches to timber harvest: Variable retention harvest systems. Pages 111–139 in K.A. Kohm & J.F. Franklin, editors. Creating a forestry for the 21st century. Island Press, Covelo , California. In Creating a forestry for the 21st century: The science of ecosystem management (pp. 111–139). Washington DC: Island Press. Fraser, W., New Zealand & Department of Conservation. (2000). Status and conservation role of recreational hunting on conservation land. Wellington, N.Z.: Dept. of Conservation. Fredman, P., Wall-Reinius, S., & Grundén, A. (2012). The Nature of Nature in Nature-based Tourism Scandinavian Journal of Hospitality and Tourism 12(4) 289 309 Fraser, W., New Zealand & Department of Conservation. (2000). Status and conservation role of recreational hunting on conservation land. Wellington, N.Z.: Dept. of Conservation. Fredman, P., Wall-Reinius, S., & Grundén, A. (2012). The Nature of Nature in Nature-based Tourism. Scandinavian Journal of Hospitality and Tourism, 12(4), 289–309. https://doi.org/10.1080/15022250.2012.752893 Freire, K. M. F., Belhabib, D., Espedido, J. C., Hood, L., Kleisner, K. M., Lam, V. W. L., … Pauly, D. (2020). Estimating Global Catches of Marine Recreational Fisheries. Frontiers in Marine Science, 7, 12. https://doi.org/10.3389/fmars.2020.00012 Freitas, C. T., Espírito-Santo, H. M. V., Campos-Silva, J. V., Peres, C. A., & Lopes, P. F. M. (2020). Resource co-management as a step towards gender equity in fisheries. Ecological Economics, 176, 106709. https://doi.org/10.1016/j.ecolecon.2020.106709 Freund, C. A., Achmad, M., Kanisius, P., Naruri, R., Tang, E., & Knott, C. D. (2020). Conserving orangutans one classroom at a time: Evaluating the effectiveness of a wildlife education program for school‐aged children in Indonesia. Animal Conservation, 23(1), 18–27. https://doi.org/10.1111/acv.12513 Frey, G. E., Chamberlain, J. L., & Prestemon, J. P. (2018). The potential for a backward- bending supply curve of non-timber forest products: An empirical case study of wild American ginseng production. Forest Policy and Economics, 97(World Dev. 29 2001), 97–109. https://doi.org/10.1016/j.forpol.2018.09.011 Frey, G. E., Cubbage, F. W., Holmes, T. P., Reyes-Retana, G., Davis, R. R., Megevand, C., … Chemor-Salas, D. N. (2019). Competitiveness, certification, and support of timber 392 harvest by community forest enterprises in Mexico. Forest Policy and Economics, 107, 101923. and A billion cups: The diversity, traditional uses, safety issues and potential of Chinese herbal teas. Journal of Ethnopharmacology, 222, 217–228. https://doi.org/10.1016/j.jep.2018.04.026 Fugler, C. M. (1985). A proposed management programme for the Indian bullfrog, Rana tigrina, in Bangladesh, comments pertaining to its intensive cultivation with observations on the status of the exploited chelonians. Retrieved from https://agris.fao.org/agris-search/search.do?recordID=XF8552409 393 Fui, F. S., Saikim, F. H., Kulip, J., & Seelan, J. S. S. (2018). Distribution and ethnomycological knowledge of wild edible mushrooms in Sabah (Northern Borneo), Malaysia. Journal of Tropical Biology & Conservation (JTBC), 203â – 222. Fukuda, Y., Webb, G., Edwards, G., Saalfeld, K., & Whitehead, P. (2020). Harvesting predators: Simulation of population recovery and controlled harvest of saltwater crocodiles Crocodylus porosus. Wildlife Research, 48(3), 252–263. https://doi.org/10.1071/WR20033 Fukuda, Y., Webb, G., Manolis, C., Delaney, R., Letnic, M., Lindner, G., & Whitehead, P. (2011). Recovery of saltwater crocodiles following unregulated hunting in tidal rivers of the Northern Territory, Australia. Journal of Wildlife Management, 75(6), 1253– 1266. https://doi.org/10.1002/jwmg.191 Fukushima, C. S., Mammola, S., & Cardoso, P. (2020). Global wildlife trade permeates the Tree of Life. Biological Conservation, 247, 108503. https://doi.org/10.1016/j.biocon.2020.108503 Fulanda, B., Ohtomi, J., Mueni, E., & Kimani, E. (2011). Fishery trends, resource-use and management system in the Ungwana Bay fishery Kenya. Ocean and Coastal Management, 54(5), 401–414. Scopus. https://doi.org/10.1016/j.ocecoaman.2010.12.010 Furst, P. T. (1972). Flesh of the Gods: The ritual Use of Hallucinogens. New York: Praeger Publishers. Retrieved from https://archive.org/details/fleshofgodsritua0000furs Fusté-Forné, F. (2019). Seasonality in food tourism: Wild foods in peripheral areas. Tourism Geographies, 1–21. https://doi.org/10.1080/14616688.2018.1558453 Gaffric, G. (2013). Do Waves Have Memories? Human and Ocean Issues in Taiwan Indigenous Writer Syaman Rapongan’s Writing. TRANS - Revue de Littérature Générale et Comparée, 16. https://doi.org/10.4000/trans.867 Gal, G., & Anderson, W. (2010). A novel approach to detecting a regime shift in a lake ecosystem: Detecting regime shifts. Methods in Ecology and Evolution, 1(1), 45–52. https://doi.org/10.1111/j.2041-210X.2009.00006.x Gallon, R. K., Robuchon, M., Leroy, B., Le Gall, L., Valero, M., & Feunteun, E. (2014). Twenty years of observed and predicted changes in subtidal red seaweed assemblages along a biogeographical transition zone: Inferring potential causes from environmental data. Journal of Biogeography, 41(12), 2293–2306. https://doi.org/10.1111/jbi.12380 Gamboa-Álvarez, M. Á., López-Rocha, J. A., Poot-López, G. R., Aguilar-Perera, A., & Villegas-Hernández, H. (2020). Rise and decline of the sea cucumber fishery in Campeche Bank, Mexico. Ocean and Coastal Management, 184. Scopus. https://doi.org/10.1016/j.ocecoaman.2019.105011 Gandar, M. (1994). Afforestation and woodland management in South Africa (No. 9). and Cape Town: Energy for Development Research Centre. Retrieved from Energy for Development Research Centre website: https://open.uct.ac.za/bitstream/handle/11427/22671/Gandar_1994.pdf?sequence=6 Ganseforth, S. (2021). Blue revitalization or dispossession? Reform of common resource management in Japanese small-scale fisheries. Geographical Journal. Scopus. https://doi.org/10.1111/geoj.12414 394 Gao, K. (1998). Chinese studies on the edible blue-green alga, Nostoc flagelliforme: A review. Journal of Applied Phycology, 10(1), 37–49. Garcez Costa Sousa, R., & de Carvalho Freitas, C. E. (2011). Seasonal catch distribution of tambaqui (Colossoma macropomum), Characidae in a central Amazon floodplain lake: Implications for sustainable fisheries management: Seasonal catch distribution of tambaqui. Journal of Applied Ichthyology, 27(1), 118–121. https://doi.org/10.1111/j.1439-0426.2010.01521.x Garcia, G. S. C. (2006). The mother—Child nexus. Knowledge and valuation of wild food plants in Wayanad, Western Ghats, India. Journal of Ethnobiology and Ethnomedicine, 2. https://doi.org/Artn 39 10.1186/1746-4269-2-39 García, N., Galeano, G., Bernal, R., & Balslev, H. (2013). Management of Astrocaryum standleyanum (Arecaceae) for Handicraft Production in Colombia. Ethnobotany Research and Applications, 11, 18. García, N., Galeano, G., Mesa, L., Castaño, N., Balslev, H., & Bernal, R. (2015). Management of the palm Astrocaryum chambira Burret (Arecaceae) in northwest Amazon. Acta Botanica Brasilica, 29(1), 45–57. https://doi.org/10.1590/0102-33062014abb3415 García, N., Torres, C., Bernal, R., Galeano, G., Valderrama, N., & Barrera, V. (2011). Management of the Spiny Palm Astrocaryum malybo in Colombia for the Production od Mats. Palms, 55(4), 10. Garcia-Barreda, S., Forcadell, R., Sánchez, S., Martín-Santafé, M., Marco, P., Camarero, J. J., & Reyna, S. (2018). Black Truffle Harvesting in Spanish Forests: Trends, Current Policies and Practices, and Implications on its Sustainability. Environmental Management, 61(4), 535–544. https://doi.org/10.1007/s00267-017-0973-6 García-Llorente, M., Iniesta-Arandia, I., Willaarts, B. A., Harrison, P. A., Berry, P., Bayo, M. del M., … Martín-López, B. (2015). Biophysical and sociocultural factors underlying spatial trade-offs of ecosystem services in semiarid watersheds. Ecology and Society, 20(3), art39. https://doi.org/10.5751/ES-07785-200339 Gärdenfors, U. (2010). Rödlistade arter i Sverige 2010 = The 2010 red list of Swedish species. Uppsala: ArtDatabanken-SLU i samarbete med Naturvårdsverket. Garekae, H., & Shackleton, C. M. (2020a). Foraging Wild Food in Urban Spaces: The Contribution of Wild Foods to Urban Dietary Diversity in South Africa. Sustainability, 12(2), 678. https://doi.org/10.3390/su12020678 Garekae, H., & Shackleton, C. M. (2020b). Urban foraging of wild plants in two medium-sized South African towns: People, perceptions and practices. Urban Forestry & Urban Greening, 49, 126581. https://doi.org/10.1016/j.ufug.2020.126581 Garibay-Orijel, R., Cifuentes, J., & Estrada-Torres, A. (2006). People using macro-fungal diversity in Oaxaca, Mexico. Fungal Diversity, 21, 46–67. Garineaud, C. (2015). and Pratiques manuelles ou mécanisées: La part de la main dans les perceptions sensorielles et dans les savoirs écologiques. Exemple des récoltants d’algues en Bretagne. ethnographiques.org, 31. Retrieved from https://www.ethnographiques.org/2015/Garineaud Garineaud, C. (2017). Récolter la mer: Des savoirs et des pratiques des collecteurs d’algues à la gestion durable des ressources côtières en Bretagne (Museum National d’Histoire Naturelle). Paris. 395 Garmendia, V., Subida, M. D., Aguilar, A., & Fernández, M. (2021). The use of fishers’ knowledge to assess benthic resource abundance across management regimes in Chilean artisanal fisheries. Marine Policy, 127, 104425. https://doi.org/10.1016/j.marpol.2021.104425 Garnett, S. T., Burgess, N. D., Fa, J. E., Fernández-Llamazares, Á., Molnár, Z., Robinson, C. J., … Leiper, I. (2018). A spatial overview of the global importance of Indigenous lands for conservation. Nature Sustainability, 1(7), 369–374. https://doi.org/10.1038/s41893- 018-0100-6 Gaspare, L., Bryceson, I., & Kulindwa, K. (2015). Complementarity of fishers’ traditional ecological knowledge and conventional science: Contributions to the management of groupers (Epinephelinae) fisheries around Mafia Island, Tanzania. Ocean and Coastal Management, 114, 88–101. Scopus. https://doi.org/10.1016/j.ocecoaman.2015.06.011 Gauthier, S., Bernier, P., Kuuluvainen, T., Shvidenko, A. Z., & Schepaschenko, D. G. (2015). Boreal forest health and global change. Science, 349(6250), 819–822. https://doi.org/10.1126/science.aaa9092 Gaylard, A., Owen-Smith, N., & Redfern, J. (2003). Surface water availability: Implications for heterogeneity and ecosystem processes. In J. T. Du Toit, K. H. Rogers, & H. C. Biggs (Eds.), The Kruger Experience: Ecology and Management of Savanna Heterogeneity. Washington DC, USA: Island Press. Retrieved from https://catalogue.solent.ac.uk/openurl/44SSU_INST/44SSU_INST:VU1?u.ignore_dat e_coverage=true&rft.mms_id=9997124104904796 Geffroy, B., Samia, D. S. M., Bessa, E., & Blumstein, D. T. (2015). How Nature-Based Tourism Might Increase Prey Vulnerability to Predators. Trends in Ecology & Evolution, 30(12), 755–765. https://doi.org/10.1016/j.tree.2015.09.010 Geijzendorffer, I. R., Martín-López, B., & Roche, P. K. (2015). Improving the identification of mismatches in ecosystem services assessments. Ecological Indicators, 52, 320–331. https://doi.org/10.1016/j.ecolind.2014.12.016 Gelcich, S., Cinner, J., Donlan, C. J., Tapia-Lewin, S., Godoy, N., & Castilla, J. C. (2017). Fishers’ perceptions on the Chilean coastal TURF system after two decades: Problems, benefits, and emerging needs. Bulletin of Marine Science, 93(1), 53–67. Scopus. https://doi.org/10.5343/bms.2015.1082 Gelcich, S., Hughes, T. P., Olsson, P., Folke, C., Defeo, O., Fernández, M., … Castilla, J. C. (2010). Navigating transformations in governance of Chilean marine coastal resources. Proceedings of the National Academy of Sciences of the United States of America, 107(39), 16794–16799. Scopus. https://doi.org/10.1073/pnas.1012021107 Gelinaud, G., Combreau, O., & Seddon, P. (1997). First breeding by captive-bred houbara bustards introduced in central Saudi Arabia. and Journal of Arid Environments, 35, 527– 534. https://doi.org/10.1006/jare.1996.0155 Geng, Yanfei, Hu, G., Ranjitkar, S., Shi, Y., Zhang, Y., & Wang, Y. (2017). The implications of ritual practices and ritual plant uses on nature conservation: A case study among the Naxi in Yunnan Province, Southwest China. Journal of Ethnobiology and Ethnomedicine, 13(1), 58. https://doi.org/10.1186/s13002-017-0186-3 Geng, YL, & Jiang, Z. (1991). Resource of Nostoc flagelliforme and its utilization in Ningxia. Chinese Wild Plant, 1, 37–38. 396 Genovesi, P. (2005). Eradications of Invasive Alien Species in Europe: A Review. Biological Invasions, 7, 127–133. https://doi.org/10.1007/s10530-004-9642-9 Genovesi, P., & Carnevali, L. (2011). Invasive alien species on European islands: Eradications and priorities for future work. Gentle, P., Maraseni, T. N., Paudel, D., Dahal, G. R., Kanel, T., & Pathak, B. (2020). Effectiveness of community forest user groups (CFUGs) in responding to the 2015 earthquakes and COVID-19 in Nepal. Research in Globalization, 2, 100025. https://doi.org/10.1016/j.resglo.2020.100025 Geraci, A., Amato, F., Di Noto, G., Bazan, G., & Schicchi, R. (2018). The wild taxa utilized as vegetables in Sicily (Italy): A traditional component of the Mediterranean diet. Journal of Ethnobiology and Ethnomedicine, 14(1), 14. https://doi.org/10.1186/s13002-018-0215-x Gerhardinger, L. C., Marenzi, R. C., Bertoncini, Á. A., Medeiros, R. P., & Hostim-Silva, M. (2006). Local ecological knowledge on the goliath grouper Epinephelus itajara (Teleostei: Serranidae) in southern Brazil. Neotropical Ichthyology, 4(4), 441–450. Scopus. Retrieved from Scopus. Gerstner, C. L., Ortega, H., Sanchez, H., & Graham, D. L. (2006). Effects of the freshwater aquarium trade on wild fish populations in differentially-fished areas of the Peruvian Amazon. Journal of Fish Biology, 68(3), 862–875. Scopus. https://doi.org/10.1111/j.0022-1112.2006.00978.x Ghasemi Fard, S., Wang, F., Sinclair, A. J., Elliott, G., & Turchini, G. M. (2019). How does high DHA fish oil affect health? A systematic review of evidence. Critical Reviews in Food Science and Nutrition, 59(11), 1684–1727. https://doi.org/10.1080/10408398.2018.1425978 Ghimire, S., Gimenez, O., Pradel, R., McKey, D., & Aumeeruddy‐Thomas, Y. (2008). Demographic variation and population viability in a threatened Himalayan medicinal and aromatic herb Nardostachys grandiflora: Matrix modelling of harvesting effects in two contrasting habitats. Journal of Applied Ecology, 45(1), 41–51. https://doi.org/10.1111/j.1365-2664.2007.01375.x Ghimire, S. K. (2008). Medicinal plants in the Nepal Himalaya: Current issues, sustainable harvesting, knowledge gaps and research priorities. 19. Ghimire, S., McKey, D., & Aumeeruddy-Thomas, Y. (2005). Conservation of Himalayan medicinal plants: Harvesting patterns and ecology of two threatened species, Nardostachys grandiflora DC. and Neopicrorhiza scrophulariiflora (Pennell) Hong. Biological Conservation, 124(4), 463–475. and https://doi.org/10.1016/j.biocon.2005.02.005 Ghorbani, A., Gravendeel, B., Naghibi, F., & de Boer, H. (2014). Wild orchid tuber collection in Iran: A wake-up call for conservation. Biodiversity and Conservation, 23(11), 2749– 2760. https://doi.org/10.1007/s10531-014-0746-y Ghosh, M., & Sinha, B. (2016). Impact of forest policies on timber production in India: A review: Mili Ghosh and Bhaskar Sinha / Natural Resources Forum. Natural Resources Forum, 40(1–2), 62–76. https://doi.org/10.1111/1477-8947.12094 GIAHS. (2020). Traditional Agricultural System in the Southern Espinhaço Range, Minas Gerais, Brazil. Retrieved April 2, 2022, from Globally Important Agricultural heritage 397 Systems website: https://www.fao.org/giahs/giahsaroundtheworld/designated- sites/latin-america-and-the-caribbean/semprevivas-minasgerais/en Systems website: https://www.fao.org/giahs/giahsaroundtheworld/designated- sites/latin-america-and-the-caribbean/semprevivas-minasgerais/en Gibbons, A. (2020). Ape researchers mobilize to save primates from coronavirus. Science, 368(6491), 566.1-566. https://doi.org/10.1126/science.368.6491.566-a Gibbons, J. W., Scott, D. E., Ryan, T. J., Buhlmann, K. A., Tuberville, T. D., Metts, B. S., … Winne, C. T. (2000). The Global Decline of Reptiles, Déjà Vu Amphibians: Reptile species are declining on a global scale. Six significant threats to reptile populations are habitat loss and degradation, introduced invasive species, environmental pollution, disease, unsustainable use, and global climate change. BioScience, 50(8), 653–666. https://doi.org/10.1641/0006-3568(2000)050[0653:TGDORD]2.0.CO;2 Gibson, C. C., McKean, M. A., & Ostrom, E. (Eds.). (2000). People and forests: Communities, institutions, and governance. Cambridge, Mass: MIT Press. Gibson, R. S., & Hotz, C. (2001). Dietary diversification/modification strategies to enhance micronutrient content and bioavailability of diets in developing countries. British Journal of Nutrition, 85(S2), S159–S166. https://doi.org/10.1079/BJN2001309 Giglio, V. J., & Bornatowski, H. (2016). Fishers’ ecological knowledge of smalleye hammerhead, Sphyrna tudes, in a tropical estuary. Neotropical Ichthyology, 14(2). Scopus. https://doi.org/10.1590/1982-0224-20150103 Giglio, V. J., Luiz, O. J., & Gerhardinger, L. C. (2015). Depletion of marine megafauna and shifting baselines among artisanal fishers in eastern Brazil. Animal Conservation, 18(4), 348–358. Scopus. https://doi.org/10.1111/acv.12178 Gill, D. J. C., Fa, J. E., Rowcliffe, J. M., & Kümpel, N. F. (2012). Drivers of Change in Hunter Offtake and Hunting Strategies in Sendje, Equatorial Guinea: Gill et al . Conservation Biology, 26(6), 1052–1060. https://doi.org/10.1111/j.1523-1739.2012.01876.x Gillett, R. (2009). Fisheries in the Economies of Pacific Island Countries and Territories. Asian Development Bank. Gilliland, T. E., Sanchirico, J. N., & Taylor, J. E. (2020). Market-driven bioeconomic general equilibrium impacts of tourism on resource-dependent local economies: A case from the western Philippines. Journal of Environmental Management, 271, 110968. https://doi.org/10.1016/j.jenvman.2020.110968 Gilman, E. L. (2011). Bycatch governance and best practice mitigation technology in global tuna fisheries. Marine Policy, 35(5), 590–609. https://doi.org/10.1016/j.marpol.2011.01.021 Gilman, E., Perez Roda, A., Huntington, T., Kennelly, S. and J., Suuronen, P., Chaloupka, M., & Medley, P. A. H. (2020). Benchmarking global fisheries discards. Scientific Reports, 10(1), 14017. https://doi.org/10.1038/s41598-020-71021-x Gilman, Eric, Allain, V., Collette, B. B., Hampton, J., & Lehodey, P. (2016). Effects of Ocean Warming on Pelagic Tunas, a Review. In D. Laffoley & J. M. Baxter (Eds.), Explaining Ocean Warming: Causes, scale, effects and consequences (pp. 255–272). IUCN, International Union for Conservation of Nature. https://doi.org/10.2305/IUCN.CH.2016.08.en Gilman, Eric, & Bianchi, G. (2010). Guidelines to Reduce Sea turtle Mortality in Fishing Operations. FAO Technical Guidelines for Responsible Fisheries. 398 Gilman, Eric, Clarke, S., Brothers, N., Alfaro-Shigueto, J., Mandelman, J., Mangel, J., … others. (2008). Shark interactions in pelagic longline fisheries. Marine Policy, 32(1), 1–18. Gilman, Eric, Passfield, K., & Nakamura, K. (2014). Performance of regional fisheries management organizations: Ecosystem-based governance of bycatch and discards. Fish and Fisheries, 15(2), 327–351. https://doi.org/10.1111/faf.12021 Gilman, Eric, Perez Roda, A., Huntington, T., Kennelly, S. J., Suuronen, P., Chaloupka, M., & Medley, P. A. H. (2020). Benchmarking global fisheries discards. Scientific Reports, 10(1), 14017. https://doi.org/10.1038/s41598-020-71021-x Giraud, N. (2020). Sustainable foraging of wild edible plants in Norway: A Biocultural Approach. Norwegian University of Life Sciences (NMBU) and Institut supérieur d’agriculture Rhône-Alpes (ISARA-Lyon). Givens, G. H., & Heide-Jørgensen, M. P. (2021). Abundance. In The Bowhead Whale (pp. 77– 86). Elsevier. Gladkikh, T. M., Gould, R. K., & Coleman, K. J. (2019). Cultural ecosystem services and the well-being of refugee communities. Ecosystem Services, 40, 101036. https://doi.org/10.1016/j.ecoser.2019.101036 Glaser, M., & Diele, K. (2004). Asymmetric outcomes: Assessing central aspects of the biological, economic and social sustainability of a mangrove crab fishery, Ucides cordatus (Ocypodidae), in North Brazil. Ecological Economics, 49(3), 361–373. https://doi.org/10.1016/j.ecolecon.2004.01.017 Global Tree Assessment. (2020). State of the World’s Trees Report. Retrieved from https://www.globaltreeassessment.org/progress/%20Rivers,%202020) Global Tree Assessment. (2021). State of the World’s Trees Report. Retrieved from https://www.bgci.org/wp/wp-content/uploads/2021/08/FINAL-GTAReportMedRes- 1.pdf Godoy, N., Gelcich, S., Vasquez, J. A., & Castilla, J. C. (2010). Spearfishing to depletion: Evidence from temperate reef fishes in Chile. Ecological Applications, 20(6), 1504– 1511. Scopus. https://doi.org/10.1890/09-1806.1 Goel, G., Makkar, H. P. S., Francis, G., & Becker, K. (2007). Phorbol Esters: Structure, Biological Activity, and Toxicity in Animals. International Journal of Toxicology, 26(4), 279–288. https://doi.org/10.1080/10915810701464641 Goettsch, B., Hilton-Taylor, C., Cruz-Piñón, G., Duffy, J. P., Frances, A., Hernández, H. M., … Gaston, K. J. (2015). High proportion of cactus species threatened with extinction. Nature Plants, 1(10), 15142. https://doi.org/10.1038/nplants.2015.142 Goetze, J., Langlois, T., Claudet, J., Januchowski-Hartley, F., & Jupiter, S. D. and (2016). Periodically harvested closures require full protection of vulnerable species and longer closure periods. Biological Conservation, 203, 67–74. Scopus. https://doi.org/10.1016/j.biocon.2016.08.038 Golden, A. S., Naisilsisili, W., Ligairi, I., & Drew, J. A. (2014). Combining natural history collections with fisher knowledge for community-based conservation in Fiji. PLoS ONE, 9(5). Scopus. https://doi.org/10.1371/journal.pone.0098036 399 Gomes, I., Erzini, K., & Mcclanahan, T. R. (2014). Trap modification opens new gates to achieve sustainable coral reef fisheries. Aquatic Conservation: Marine and Freshwater Ecosystems, 24(5), 680–695. Scopus. https://doi.org/10.1002/aqc.2389 Gómez Pompa, P. (1989). Colección de ejercicios de ingeniería rural (hidráulica). Cáceres: Universidad de Extremadura. Servicio de Publicaciones. Gómez-Pompa, A., Whitmore, T. C., & Hadley, M. (Eds.). (1991). Rain forest regeneration and management. Paris: Unesco. Gonwouo, L. N., & Rödel, M.-O. (2008). The importance of frogs to the livelihood of the Bakossi people around Mount Manengouba, Cameroon, with special consideration of the Hairy Frog, Trichobatrachus robustus. Salamandra, 44, 23–34. Gonzalez-Duarte, C. (2021). Butterflies, organized crime, and “sad trees”: A critique of the Monarch Butterfly Biosphere Reserve Program in a context of rural violence. World Development, 142, 105420. https://doi.org/10.1016/j.worlddev.2021.105420 González-Tejero, M. R., Martínez-Lirola, M. J., Casares-Porcel, M., & Molero-Mesa, J. (1995). Three lichens used in popular medicine in Eastern Andalucia (Spain). Economic Botany, 49(1), 96–98. https://doi.org/10.1007/BF02862281 Goode, M. J., Horrace, W. C., Sredl, M. J., & Howland, J. M. (2005). Habitat destruction by collectors associated with decreased abundance of rock-dwelling lizards. Biological Conservation, 125(1), 47–54. https://doi.org/10.1016/j.biocon.2005.03.010 Gopal, D., von der Lippe, M., & Kowarik, I. (2019). Sacred sites, biodiversity and urbanization in an Indian megacity. Urban Ecosystems, 22(1), 161–172. https://doi.org/10.1007/s11252-018-0804-4 Gordoa, A., Dedeu, A. L., & Boada, J. (2019). Recreational fishing in Spain: First national estimates of fisher population size, fishing activity and fisher social profile. Fisheries Research, 211, 1–12. https://doi.org/10.1016/j.fishres.2018.10.026 Gordon, L. J., Peterson, G. D., & Bennett, E. M. (2008). Agricultural modifications of hydrological flows create ecological surprises. Trends in Ecology & Evolution, 23(4), 211–219. https://doi.org/10.1016/j.tree.2007.11.011 Gortázar, C., Acevedo, P., Ruiz-Fons, F., & Vicente, J. (2006). Disease risks and overabundance of game species. European Journal of Wildlife Research, 52(2), 81–87. https://doi.org/10.1007/s10344-005-0022-2 Gorzula, S. (1996). The Trade in Dendrobatid Frogs from 1987 to 1993. Retrieved August 12, 2019, from ResearchGate website: https://www.researchgate.net/publication/284781050_The_Trade_in_Dendrobatid_Fr ogs_from_1987_to_1993 Gössling, S., Peeters, P., Hall, C. M., Ceron, J.-P., Dubois, G., Lehmann, L. V., & Scott, D. (2012). Tourism and water use: Supply, demand, and security. An international review. Tourism Management, 33(1), 1–15. and https://doi.org/10.1016/j.tourman.2011.03.015 Goulding, M., Venticinque, E., Ribeiro, M. L. de B., Barthem, R. B., Leite, R. G., Forsberg, B., … Cañas, C. (2019). Ecosystem-based management of Amazon fisheries and wetlands. Fish and Fisheries, 20(1), 138–158. https://doi.org/10.1111/faf.12328 Government of British Columbia. (2020). Community Forest Agreements. Issued and Invited Community Forests (as of April 24, 2020). Government of British Columbia. Retrieved from Government of British Columbia website: 400 https://www2.gov.bc.ca/assets/gov/farming-natural-resources-and- industry/forestry/timber-tenures/community-forest- agreements/issued_cfa_status_report_april-24-2020.pdf Gowreesunkar, G., & Rycha, I. (2015). A Study on the Impacts of Dolphin Watching as a Tourism Activity: Western Mauritius as Case Study. International Journal of Trade, Economics and Finance, 6(1), 67–72. https://doi.org/10.7763/IJTEF.2015.V6.445 Gowreesunkar, G., & Rycha, I. (2015). A Study on the Impacts of Dolphin Watching as a Tourism Activity: Western Mauritius as Case Study. International Journal of Trade, Economics and Finance, 6(1), 67–72. https://doi.org/10.7763/IJTEF.2015.V6.445 Grabbatin, B., Hurley, P. T., & Halfacre, A. (2011). “I Still Have the Old Tradition”: The co- production of sweetgrass basketry and coastal development. Geoforum, 42(6), 638– 649. https://doi.org/10.1016/j.geoforum.2011.06.007 Grabek-Lejko, D., Kasprzyk, I., Zagu\la, G., & Puchalski, C. (2017). The bioactive and mineral compounds in birch sap collected in different types of habitats. Baltic Forestry, 23(1). Grace, O. M., Lovett, J. C., Gore, C. J. N., Moat, J., Ondo, I., Pironon, S., … Wilkin, P. (2020). Plant Power: Opportunities and challenges for meeting sustainable energy needs from the plant and fungal kingdoms. PLANTS, PEOPLE, PLANET, 2(5), 446–462. https://doi.org/10.1002/ppp3.10147 Graham, C. H., Ferrier, S., Huettman, F., Moritz, C., & Peterson, A. T. (2004). New developments in museum-based informatics and applications in biodiversity analysis. Trends in Ecology & Evolution, 19(9), 497–503. https://doi.org/10.1016/j.tree.2004.07.006 Grant, M. C., Mallard J., Leigh, S., & Thompson, P. S. (2012). The costs and benefits of grouse moor management to biodiversity and aspects of the wider environment: A review. UK: Sandy. Grantham, H. S., Duncan, A., Evans, T. D., Jones, K. R., Beyer, H. L., Schuster, R., … Watson, J. E. M. (2020). Anthropogenic modification of forests means only 40% of remaining forests have high ecosystem integrity. Nature Communications, 11(1), 5978. https://doi.org/10.1038/s41467-020-19493-3 Grati, F., Aladžuz, A., Azzurro, E., Bolognini, L., Carbonara, P., Çobani, M., … Milone, N. (2018). Seasonal dynamics of small-scale fisheries in the Adriatic Sea. Mediterranean Marine Science, 19(1), 21–35. Scopus. https://doi.org/10.12681/mms.2153 Graves, P., Mosman, K., & Rogers, S. (2012). 2011 LEGISLATIVE REVIEW AND ADMINISTRATIVE REVIEW \| Animal Legal & Historical Center. Animal Law, 18, 361–426. Gray, J. (1999). and Regime de propriedade florestal e valoração de floresta públicas no Brasil. Programa Nacional de Florestas. Brasília, Brazil: Ministério do Meio Ambiente. Gray, T. N. E., Phommachak, A., Vannachomchan, K., & Guegan, F. (2017). Using local ecological knowledge to monitor threatened Mekong megafauna in Lao PDR. PLoS ONE, 12(8). Scopus. https://doi.org/10.1371/journal.pone.0183247 Green, E. (2003). International trade in marine aquarium species: Using the global marine aquarium database. Marine Ornamental Species: Collection, Culture & Conservation, 29–48. Greyling, M., McCay, M., & Douglas-Hamilton, I. (2004). Green hunting as an alternative to lethal hunting. Proceedings of the Symposium on Human-Elephant Relationships and Conflicts. 401 Griffiths, A. D., Philips, A., & Godjuwa, C. (2003). Harvest of Bombax ceiba for the Aboriginal arts industry, central Arnhem Land, Australia. Biological Conservation, 113(2), 295–305. Readcube. https://doi.org/10.1016/s0006-3207(02)00419-6 Griffiths, S. P., Pollock, K. H., Lyle, J. M., Pepperell, J. G., Tonks, M. L., & Sawynok, W. (2010). Following the chain to elusive anglers: Following the chain to elusive anglers. Fish and Fisheries, 11(2), 220–228. https://doi.org/10.1111/j.1467-2979.2010.00354.x Grimble, A. F. (1989). Writings on the Atoll Culture of the Gilbert Islands (Maude H.E. (ed)). Honolulu: University of Hawaii Press. Retrieved from https://core.ac.uk/download/pdf/211329399.pdf GRIN-WEP. (2020). GRIN-Global Species Data. Retrieved from https://npgsweb.ars- grin.gov/gringlobal/taxon/taxonomysearch Griscom, B., Ellis, P., & Putz, F. E. (2014). Carbon emissions performance of commercial logging in East Kalimantan, Indonesia. Global Change Biology, 20(3), 923–937. https://doi.org/10.1111/gcb.12386 Grogan, J., & Galvão, J. (2006). Factors Limiting Post-logging Seedling Regeneration by Big- leaf Mahogany ( Swietenia macrophylla ) in Southeastern Amazonia, Brazil, and Implications for Sustainable Management 1: Mahogany Seed Availability. Biotropica, 38(2), 219–228. https://doi.org/10.1111/j.1744-7429.2006.00121.x Grogan, J., Galvão, J., Simões, L., & VerÍssimo, A. (2003). Regeneration of Big-Leaf Mahogany in Closed and Logged Forests of Southeastern Pará, Brazil. In A. E. Lugo, J. C. Figueroa Colón, & M. Alayón (Eds.), Big-Leaf Mahogany (pp. 193–208). New York: Springer-Verlag. https://doi.org/10.1007/0-387-21778-9_10 Grogan, J., Landis, R. M., Ashton, M. S., & Galvão, J. (2005). Growth response by big-leaf mahogany (Swietenia macrophylla) advance seedling regeneration to overhead canopy release in southeast Pará, Brazil. Forest Ecology and Management, 204(2–3), 399–412. https://doi.org/10.1016/j.foreco.2004.09.013 Groom, M. J., Meffe, G. K., & Carroll, C. R. (2006). Principles of Conservation Biology (3rd Edition). Gross, L. (2008). No Place for Predators? PLOS Biology, 6(2), e40. https://doi.org/10.1371/journal.pbio.0060040 Groves, M., & Rutherford, C. (2015). CITES and timber guide: A guide to CITES-listed tree species. Richmond (GB): Kew publishing. Retrieved from https://jordbruksverket.se/download/18.7d044c501710eb9f893e4656/1585320243840 /CITES-and-Timber-a-guide-to-CITES-listed-tree-species.pdf Guan, J., Cerutti, P. and O., Masiero, M., Pettenella, D., Andrighetto, N., & Dawson, T. (2016). Quantifying Illegal Logging and Related Timber Trade. In IUFRO World Series: Vol. 35. Illegal logging and related timber trade: Dimensions, drivers, impacts and responses: A global scientific rapid response assessment report (pp. 37–59). International Union of Forest Research Organizations (IUFRO). Retrieved from http://www.iufro.org/science/gfep/illegal-timber-trade-rapid-response/report/ Guan, Z., Chen, X., Xu, Y., & Liu, Y. (2020). Are imports of illegal timber in China, India, Japan and South Korea considerable? Based on a historic trade balance analysis method. International Wood Products Journal, 11(4), 211–225. https://doi.org/10.1080/20426445.2020.1785604 402 Guariguata, M. R., Cronkleton, P., Duchelle, A. E., & Zuidema, P. A. (2017). Revisiting the ‘cornerstone of Amazonian conservation’: A socioecological assessment of Brazil nut exploitation. Biodiversity and Conservation, 26(9), 2007–2027. https://doi.org/10.1007/s10531-017-1355-3 Gucu, A. C. (1997). Role of fishing in the Black Sea ecosystem. In E. Özsoy & A. Mikaelyan (Eds.), Sensitivity to Change: Black Sea, Baltic Sea and North Sea. Dordrecht: Springer Netherlands. https://doi.org/10.1007/978-94-011-5758-2 Guebert-Bartholo, F. M., Barletta, M., Costa, M. F., Lucena, L. R., & Da Silva, C. P. (2011). Fishery and the use of space in a tropical semi-arid estuarine region of Northeast Brazil: Subsistence and overexploitation. Journal of Coastal Research, (SPEC. ISSUE 64), 398–402. Scopus. Retrieved from Scopus. Guedes, A. M. M., Antoniassi, R., & de Faria-Machado, A. F. (2017). Pequi: A Brazilian fruit with potential uses for the fat industry. OCL, 24(5), D507. https://doi.org/10.1051/ocl/2017040 Guidetti, P., & Claudet, J. (2010). Comanagement practices enhance fisheries in marine protected areas. Conservation Biology, 24(1), 312–318. Guissou, K. M. L., Lykke, A. M., Sankara, P., & Guinko, S. (2008). Declining Wild Mushroom Recognition and Usage in Burkina Faso. Economic Botany, 62(3), 530–539. https://doi.org/10.1007/s12231-008-9028-5 Gullison, R. E., & Hubbell, S. P. (1992). Regeneracion natural de la mara (Swietenia macrophylla) en el bosque Chimanes, Bol. Ecologia En Bolivia, 19, 43–56. Gullison, R. E., Panfil, S. N., Strouse, J. J., & Hubbell, S. P. (1996). Ecology and management of mahogany (Swietenia macrophylla King) in the Chimanes Forest, Beni, Bolivia. Botanical Journal of the Linnean Society, 122(1), 9–34. https://doi.org/10.1111/j.1095- 8339.1996.tb02060.x Gunnarsdotter, Y. (2007). 13 What happens in a Swedish rural community when the local moose hunt meets hunting tourism? In B. Lovelock, Tourism and the Consumption of Wildlife: Hunting, Shooting and Sport Fishing. Routledge. Gunter, U., & Ceddia, M. G. (2020). Can Indigenous and Community-Based Ecotourism Serve as a Catalyst for Land Sparing in Latin America? and Journal of Travel Research, 004728752094968. https://doi.org/10.1177/0047287520949687 Gustafsson, L., Hannerz, M., Koivula, M., Shorohova, E., Vanha-Majamaa, I., & Weslien, J. (2020). Research on retention forestry in Northern Europe. Ecological Processes, 9(1), 3. https://doi.org/10.1186/s13717-019-0208-2 Gustafsson, L., Kouki, J., & Sverdrup-Thygeson, A. (2010). Tree retention as a conservation measure in clear-cut forests of northern Europe: A review of ecological consequences. Scandinavian Journal of Forest Research, 25(4), 295–308. https://doi.org/10.1080/02827581.2010.497495 Gustavo Hallwass, Luís Henrique Tomazoni da Silva, Paula Nagl, Mariana Clauzet, & Alpina Begossi. (2020). Small-scale fisheries, livelihoods and food security of riverine people. In Fish and Fisheries in the Brazilian Amazon: People, Ecology and Conservation in Black and Clear Water Rivers. São Paulo, Brazil: Springer International Publishing. Gutiérrez-Zamora, V., & Hernández Estrada, M. (2020). Responsibilization and state territorialization: Governing socio-territorial conflicts in community forestry in 403 Mexico. Forest Policy and Economics, 116, 102188. https://doi.org/10.1016/j.forpol.2020.102188 Economics, 116, 102188. Policy Economics, Forest and Economics, 116, and Guyader, O., Berthou, P., Koutsikopoulos, C., Alban, F., Demanèche, S., Gaspar, M. B., … Maynou, F. (2013). Small scale fisheries in Europe: A comparative analysis based on a selection of case studies. Fisheries Research, 140, 1–13. Scopus. https://doi.org/10.1016/j.fishres.2012.11.008 Guzmán, G. (2008). Diversity and Use of Traditional Mexican Medicinal Fungi. A Review. International Journal of Medicinal Mushrooms, 10(3), 209–217. https://doi.org/10.1615/IntJMedMushr.v10.i3.20 Guzmán Maldonado, A., Macedo Lopes, P. F., Rodríguez Fernández, C. A., Lasso Alcala, C. A., & Sumalia, U. R. (2017). Transboundary fisheries management in the Amazon: Assessing current policies for the management of the ornamental silver arawana (Osteoglossum bicirrhosum). Marine Policy, 76, 192–199. https://doi.org/10.1016/j.marpol.2016.11.021 Hágsater, E., Soto-Arenas, M. A., Salazar-Chávez, G. A., Jiménez-Machorro, R., López-Rosas, M. A., & Dressler, R. L. (2015). Las orquídeas de México (pp. 101–103). Mexico City, Mexico: Instituto Chinoín. Retrieved from Instituto Chinoín website: https://www.biodiversitylibrary.org/part/267080 Hair, C., Foale, S., Kinch, J., Yaman, L., & Southgate, P. C. (2016). Beyond boom, bust and ban: The sandfish (Holothuria scabra) fishery in the Tigak Islands, Papua New Guinea. Regional Studies in Marine Science, 5, 69–79. Scopus. https://doi.org/10.1016/j.rsma.2016.02.001 Hajjar, R., Oldekop, J. A., Cronkleton, P., Newton, P., Russell, A. J. M., & Zhou, W. (2021). A global analysis of the social and environmental outcomes of community forests. Nature Sustainability, 4(3), 216–224. https://doi.org/10.1038/s41893-020-00633-y Hakim, L. (2020). COVID-19, tourism, and small islands in Indonesia: Protecting fragile communities in the global Coronavirus pandemic. Journal of Marine and Island Cultures, 9(1). https://doi.org/10.21463/jmic.2020.09.1.08 Hall, C. M., Harrison, D., Weaver, D., & Wall, G. (2013). and Vanishing peripheries: Does tourism consume places? Tourism Recreation Research, 38(1), 71–92. https://doi.org/10.1080/02508281.2013.11081730 Hall, J. S., Medjibe, V., Berlyn, G. P., & Ashton, P. M. S. (2003). Seedling growth of three co- occurring Entandrophragma species (Meliaceae) under simulated light environments: Implications for forest management in central Africa. Forest Ecology and Management, 179(1–3), 135–144. https://doi.org/10.1016/S0378-1127(02)00488-7 Hall, M. A., Alverson, D. L., & Metuzals, K. I. (2000). By-Catch: Problems and Solutions. Marine Pollution Bulletin, 41(1–6), 204–219. https://doi.org/10.1016/S0025- 326X(00)00111-9 Hallwass, G., Lopes, P. F., Juras, A. A., & Silvano, R. A. M. (2013). Fishers’ knowledge identifies environmental changes and fish abundance trends in impounded tropical rivers. Ecological Applications, 23(2), 392–407. Scopus. https://doi.org/10.1890/12- 0429.1 404 Hallwass, G., Schiavetti, A., & Silvano, R. A. M. (2019). Fishers’ knowledge indicates temporal changes in composition and abundance of fishing resources in Amazon protected areas. Animal Conservation, acv.12504. https://doi.org/10.1111/acv.12504 Hallwass, Gustavo, Lopes, P. F., Juras, A. A., & Silvano, R. A. M. (2011). Fishing Effort and Catch Composition of Urban Market and Rural Villages in Brazilian Amazon. Environmental Management, 47(2), 188–200. https://doi.org/10.1007/s00267-010- 9584-1 Hallwass, Gustavo, Lopes, P. F., Juras, A. A., & Silvano, R. A. M. (2013). Fishers’ knowledge identifies environmental changes and fish abundance trends in impounded tropical rivers. Ecological Applications, 23(2), 392–407. https://doi.org/10.1890/12-0429.1 Hallwass, Gustavo, & Silvano, R. A. (2016). Patterns of selectiveness in the Amazonian freshwater fisheries: Implications for management. Journal of Environmental Planning and Management, 59(9), 1537–1559. Halpern, B. S., Walbridge, S., Selkoe, K. A., Kappel, C. V., Micheli, F., D’Agrosa, C., … Watson, R. (2008). A Global Map of Human Impact on Marine Ecosystems. Science, 319(5865), 948–952. https://doi.org/10.1126/science.1149345 Hamilton, A. C. (2004). Medicinal plants, conservation and livelihoods. Biodiversity and Conservation, 13(8), 1477–1517. https://doi.org/10.1023/B:BIOC.0000021333.23413.42 Hamilton, Richard J., Hughes, A., Brown, C. J., Leve, T., & Kama, W. (2019). Community- based management fails to halt declines of bumphead parrotfish and humphead wrasse in Roviana Lagoon, Solomon Islands. Coral Reefs, 38(3), 455–465. https://doi.org/10.1007/s00338-019-01801-z Hamilton, R.J., Giningele, M., Aswani, S., & Ecochard, J. L. (2012). Fishing in the dark-local knowledge, night spearfishing and spawning aggregations in the Western Solomon Islands. Biological Conservation, 145(1), 246–257. https://doi.org/10.1016/j.biocon.2011.11.020 Hamunen, K., Kurttila, M., Miina, J., Peltola, R., & Tikkanen, J. (2019). Sustainability of Nordic non-timber forest product-related businesses—A case study on bilberry. Forest Policy and Economics, 109. https://doi.org/ARTN 102002 10.1016/j.forpol.2019.102002 Hapke, H. M. (2001). Gender, Work, and Household Survival in South Indian Fishing Communities: A Preliminary Analysis. The Professional Geographer, 53(3), 313–331. and https://doi.org/10.1111/0033-0124.00287 Hara, M., & Njaya, F. (2015). Between a rock and a hard place: The need for and challenges to implementation of Rights Based Fisheries Management in small-scale fisheries of southern Lake Malawi. Fisheries Research, 174, 10–18. Scopus. https://doi.org/10.1016/j.fishres.2015.08.005 Harfoot, M., Glaser, S. A. M., Tittensor, D. P., Britten, G. L., McLardy, C., Malsch, K., & Burgess, N. D. (2018). Unveiling the patterns and trends in 40 years of global trade in CITES-listed wildlife. Biological Conservation, 223, 47–57. https://doi.org/10.1016/j.biocon.2018.04.017 Harkema, J., & Scott, M. (2002). The Retention Systemt: Maintaining Forest Ecosystem Diversity. Ministry of Forests, Forest Practices Branch. British Columbia, 7. 405 Haro-Luna, M. X., Ruan-Soto, F., & Guzmán-Dávalos, L. (2019). Traditional knowledge, uses, and perceptions of mushrooms among the Wixaritari and mestizos of Villa Guerrero, Jalisco, Mexico. IMA Fungus, 10(1), 16. https://doi.org/10.1186/s43008-019-0014-6 Harrington, R., Owen-Smith, N., Viljoen, P. C., Biggs, H. C., Mason, D. R., & Funston, P. (1999). Establishing the causes of the roan antelope decline in the Kruger National Park, South Africa. Biological Conservation, 90(1), 69–78. https://doi.org/10.1016/S0006- 3207(98)00120-7 Harris, F. M. A., & Mohammed, S. (2003). Relying on nature: Wild foods in Northern Nigeria. Ambio, 32(1), 24–29. https://doi.org/Doi 10.1639/0044- 7447(2003)032[0024:Ronwfi]2.0.Co;2 Harrison, H. L., & Loring, P. A. (2016). Urban harvests: Food security and local fish and shellfish in Southcentral Alaska. Agriculture and Food Security, 5(1). Scopus. https://doi.org/10.1186/s40066-016-0065-5 Harrison, R. D., Sreekar, R., Brodie, J. F., Brook, S., Luskin, M., O’Kelly, H., … Velho, N. (2016). Impacts of hunting on tropical forests in Southeast Asia: Hunting in Tropical Forests. Conservation Biology, 30(5), 972–981. https://doi.org/10.1111/cobi.12785 Harry, A. V., Tobin, A. J., Simpfendorfer, C. A., Welch, D. J., Mapleston, A., White, J., … Stapley, J. (2011). Evaluating catch and mitigating risk in a multispecies, tropical, inshore shark fishery within the Great Barrier Reef World Heritage Area. Marine and Freshwater Research, 62(6), 710–721. Hart, G. M., Ticktin, T., Kelman, D., Wright, A. D., & Tabandera, N. (2014). Contemporary Gathering Practice and Antioxidant Benefit of Wild Seaweeds in Hawai’i. Economic Botany, 68(1), 30–43. https://doi.org/10.1007/s12231-014-9258-7 Hartung, T. (2010). Comparative analysis of the revised Directive 2010/6106/EU for the protection of laboratory animals with its predecessor 86/609/EEEEC – a t4 report. ALTEX - Alternatives to Animal Experimentation, 27(4), 285–303. https://doi.org/10.14573/altex.2010.4.285 Harvey, B. D., & Bergeron, Y. (1989). Site patterns of natural regeneration following clear- cutting in northwestern Quebec. Canadian Journal of Forest Research, 19(11), 1458– 1469. https://doi.org/10.1139/x89-222 Harwood, J. L. (1996). and Recent advances in the biosynthesis of plant fatty acids. Biochimica et Biophysica Acta (BBA) - Lipids and Lipid Metabolism, 1301(1–2), 7–56. https://doi.org/10.1016/0005-2760(95)00242-1 Harwood, J. L., & Guschina, I. A. (2009). The versatility of algae and their lipid metabolism. Biochimie, 91(6), 679–684. https://doi.org/10.1016/j.biochi.2008.11.004 Hasan, M., & Halwart, M. (2009). Fish as feed inputs for aquaculture: Practices, sustainability and implications. FAO, Roma (Italia). Hassan, A., & Sharma, A. (2017). Wildlife Tourism for Visitors’ Learning Experiences: Some Evidences on the Royal Bengal Tiger in Bangladesh and India. In I. B. de Lima & R. Green (Eds.), Wildlife Tourism, Environmental Learning and Ethical Encounters (pp. 155–168). Springer. Hawkes, K., O’Connell, J. F., & Blurton Jones, N. G. (2001). Hadza meat sharing. Evolution and Human Behavior, 22(2), 113–142. https://doi.org/10.1016/S1090-5138(00)00066- 0 0 406 He, G., Chen, X., Liu, W., Bearer, S., Zhou, S., Cheng, L. Y., … Liu, J. (2008). Distribution of Economic Benefits from Ecotourism: A Case Study of Wolong Nature Reserve for Giant Pandas in China. Environmental Management, 42(6), 1017–1025. https://doi.org/10.1007/s00267-008-9214-3 He, J. (2018). Harvest and trade of caterpillar mushroom (Ophiocordyceps sinensis) and the implications for sustainable use in the Tibet Region of Southwest China. Journal of Ethnopharmacology, 221, 86–90. https://doi.org/10.1016/j.jep.2018.04.022 Healy, T. J., Hill, N. J., Barnett, A., & Chin, A. (2020). A global review of elasmobranch tourism activities, management and risk. Marine Policy, 118, 103964. https://doi.org/10.1016/j.marpol.2020.103964 Heberling, J. M., Prather, L. A., & Tonsor, S. J. (2019). The Changing Uses of Herbarium Data in an Era of Global Change: An Overview Using Automated Content Analysis. BioScience, 69(10), 812–822. https://doi.org/10.1093/biosci/biz094 Heffelfinger, J., Geist, V., & Wishart, W. (2013). The role of hunting in North American wildlife conservation. International Journal of Environmental Studies, 70. https://doi.org/10.1080/00207233.2013.800383 Heim, R., & Wasson, R. G. (1958). Heim R, Wasson RG. 1958. Les champignons hallucinogènes du Mexique. Paris: Muséum National d’Histoire Naturelle. Retrieved from https://sciencepress.mnhn.fr/sites/default/files/articles/pdf/archives_du_museum_serie _7_tome_6_-_les_champignons_hallucinogenes_du_mexique_- _etudes_ethnologiques_taxinomiques_biologiques_physiologiques_et_chimiques_- _med.pdf Heim, R., & Wasson, R. G. (1958). Heim R, Wasson RG. 1958. Les champignons hallucinogènes du Mexique. Paris: Muséum National d’Histoire Naturelle. Retrieved from https://sciencepress.mnhn.fr/sites/default/files/articles/pdf/archives_du_museum_seri _etudes_ethnologiques_taxinomiques_biologiques_physiologiques_et_chimiques_- _med.pdf Heino, M., Díaz Pauli, B., & Dieckmann, U. (2015). Fisheries-Induced Evolution. Annual Review of Ecology, Evolution, and Systematics, 46(1), 461–480. https://doi.org/10.1146/annurev-ecolsys-112414-054339 Heinrich, S., Wittmann, T. A., Prowse, T. A. A., Ross, J. V., Delean, S., Shepherd, C. R., & Cassey, P. (2016). Where did all the pangolins go? International CITES trade in pangolin species. Global Ecology and Conservation, 8, 241–253. https://doi.org/10.1016/j.gecco.2016.09.007 Hemmelgarn, H. and L., & Munsell, J. F. (2021). Exploring ‘beyond‐food’ opportunities for biocultural conservation in urban forest gardens. Urban Agriculture & Regional Food Systems, 6(1). https://doi.org/10.1002/uar2.20009 Henen, B. T. (2016). Do scientific collecting and conservation conflict. Herpetol Conserv Biol, 11, 13–18. Heppell, S. S. (1998). Application of Life-History Theory and Population Model Analysis to Turtle Conservation. Copeia, 1998(2), 367. https://doi.org/10.2307/1447430 Herfaut, J., Levrel, H., Thébaud, O., & Véron, G. (2013). The nationwide assessment of marine recreational fishing: A French example. Ocean & Coastal Management, 78, 121–131. https://doi.org/10.1016/j.ocecoaman.2013.02.026 Herrmann, H. L., Babbitt, K. J., Baber, M. J., & Congalton, R. G. (2005). Effects of landscape characteristics on amphibian distribution in a forest-dominated landscape. Biological Conservation, 123(2), 139–149. https://doi.org/10.1016/j.biocon.2004.05.025 407 Heynen, N., Perkins, H. A., & Roy, P. (2006). The Political Ecology of Uneven Urban Green Space: The Impact of Political Economy on Race and Ethnicity in Producing Environmental Inequality in Milwaukee. Urban Affairs Review, 42(1), 3–25. https://doi.org/10.1177/1078087406290729 Heywood, V. H. (2017). Plant conservation in the Anthropocene – Challenges and future prospects. Plant Diversity, 39(6), 314–330. https://doi.org/10.1016/j.pld.2017.10.004 Hicks, C. C., Cohen, P. J., Graham, N. A. J., Nash, K. L., Allison, E. H., D’Lima, C., … MacNeil, M. A. (2019). Harnessing global fisheries to tackle micronutrient deficiencies. Nature, 574(7776), 95–98. https://doi.org/10.1038/s41586-019-1592-6 Hiddink, J. G., Jennings, S., Sciberras, M., Bolam, S. G., Cambiè, G., McConnaughey, R. A., … Rijnsdorp, A. D. (2019). Assessing bottom trawling impacts based on the longevity of benthic invertebrates. Journal of Applied Ecology, 56(5), 1075–1084. https://doi.org/10.1111/1365-2664.13278 Hiddink, J. G., Jennings, S., Sciberras, M., Szostek, C. L., Hughes, K. M., Ellis, N., … others. (2017). Global analysis of depletion and recovery of seabed biota after bottom trawling disturbance. Proceedings of the National Academy of Sciences, 114(31), 8301–8306. Hiemstra-Van der Horst, G., & Hovorka, A. J. (2008). Reassessing the “energy ladder”: Household energy use in Maun, Botswana. Energy Policy, 36(9), 3333–3344. Higginbottom, K. (Ed.). (2004). Wildlife tourism: Impacts, management and planning. Altona, Vic: Common Ground Publishing. Higham, J. E. S., & Bejder, L. (2008). Managing Wildlife-based Tourism: Edging Slowly Towards Sustainability? Current Issues in Tourism, 11(1), 75–83. https://doi.org/10.2167/cit345.0 Hilborn, R. (2019). Measuring fisheries performance using the “Goldilocks plot.” ICES Journal of Marine Science, 76(1), 45–49. https://doi.org/10.1093/icesjms/fsy138 Hilborn, R., Amoroso, R. O., Anderson, C. M., Baum, J. K., Branch, T. A., Costello, C., … Ye, Y. (2020). Effective fisheries management instrumental in improving fish stock status. and Proceedings of the National Academy of Sciences, 117(4), 2218–2224. https://doi.org/10.1073/pnas.1909726116 Hilborn, R., & Costello, C. (2018). The potential for blue growth in marine fish yield, profit and abundance of fish in the ocean. Marine Policy, 87, 350–355. https://doi.org/10.1016/j.marpol.2017.02.003 Hilborn, R., & Hilborn, U. (2019). Ocean Recovery: A sustainable future for global fisheries? Oxford University Press. Hilborn, R., Hively, D. J., Loke, N. B., Moor, C. L., Kurota, H., Kathena, J. N., … Melnychuk, M. C. (2021). Global status of groundfish stocks. Fish and Fisheries, 22(5), 911–928. https://doi.org/10.1111/faf.12560 Hilborn, R., & Ovando, D. (2014). Reflections on the success of traditional fisheries management. ICES Journal of Marine Science, 71(5), 1040–1046. https://doi.org/10.1093/icesjms/fsu034 Hilborn, R., & Walters, C. J. (1992). Stock and Recruitment. In R. Hilborn & C. J. Walters, Quantitative Fisheries Stock Assessment (pp. 241–296). Boston, MA: Springer US. https://doi.org/10.1007/978-1-4615-3598-0_7 408 Hill, A., Guralnick, R., Smith, A., Sallans, A., Gillespie, R., Denslow, M., … Fortson, L. (2012). The notes from nature tool for unlocking biodiversity records from museum records through citizen science. ZooKeys, 209, 219–233. https://doi.org/10.3897/zookeys.209.3472 Hill, R., Malmer, P., Tengo, M., Raymond, C. M., Spierenburg, M., Danielsen, F., … Folke, C. (2017). ScienceDirect Weaving knowledge systems in IPBES , CBD and beyond— Lessons learned for sustainability. 17–25. https://doi.org/10.1016/j.cosust.2016.12.005 Hiller, M. A., Jarvis, B. C., Lisa, H., Paulson, L. J., Pollard, E. H. B., & Stanley, S. A. (2004). Recent Trends in Illegal Logging and a Brief Discussion of Their Causes: A Case Study from Gunung Palung National Park, Indonesia. Journal of Sustainable Forestry, 19(1– 3), 181–212. https://doi.org/10.1300/J091v19n01_09 Hines, D. A., & Eckman, K. (1993). Indigenous multipurpose trees of Tanzania: Uses and economic benefits for people. Ottawa: Cultural Survival Canada. Hinsley, A., de Boer, H. J., Fay, M. F., Gale, S. W., Gardiner, L. M., Gunasekara, R. S., … Phelps, J. (2018). A review of the trade in orchids and its implications for conservation. Botanical Journal of the Linnean Society, 186(4), 435–455. https://doi.org/10.1093/botlinnean/box083 Hirschfeld, A., Attard, G., & Scott, L. (2019). An analysis of bag ﬁgures and the potential impact on the conservation of threatened species. British Birds, 14. Ho, N. T. T., Ross, H., & Coutts, J. (2015). Power sharing in fisheries co-management in Tam Giang Lagoon, Vietnam. Marine Policy, 53, 171–179. https://doi.org/10.1016/j.marpol.2014.12.006 Hoare, A. (2015). Tackling illegal logging and the related trade. What progress and where next. London: Chatham House. Hobbs, R. C., Reeves, R. R., Prewitt, J. and S., Desportes, G., Breton-Honeyman, K., Christensen, T., … Garde, E. (2019). Global review of the conservation status of monodontid stocks. Marine Fisheries Review, 81(3–4), 1–62. Hoch, L., Pokorny, B., & de Jong, W. (2012). Financial attractiveness of smallholder tree plantations in the Amazon: Bridging external expectations and local realities. Agroforestry Systems, 84(3), 361–375. https://doi.org/10.1007/s10457-012-9480-1 Hochkirch, A., Samways, M. J., Gerlach, J., Böhm, M., Williams, P., Cardoso, P., … Dijkstra, K.-D. B. (2021). A strategy for the next decade to address data deficiency in neglected biodiversity. Conservation Biology, 35(2), 502–509. https://doi.org/10.1111/cobi.13589 Hocking, D., & Babbitt, K. (2014). Amphibian contributions to ecosystem services. Herpetological Conservation and Biology, 9, 1–17. Hoeppe, G. (2007). Conversations on the beach. Fishermen’s knowledge, metaphor and environmental change in South India. New York: Berghahn Books. Hoffman, L. C., & Cawthorn, D.-M. (2012). What is the role and contribution of meat from wildlife in providing high quality protein for consumption? Animal Frontiers, 2(4), 40– 53. https://doi.org/10.2527/af.2012-0061 Hoffmann, H., Brüntrup, M., & Dewes, C. (2016). Wood energy in sub-Saharan Africa: How to make a shadow business sustainable. Briefing Paper. 409 Holdren, J. P., Smith, K. R., Kjellstrom, T., Streets, D., Wang, X., & Fischer, S. (2000). Energy, the Environment, and Health. In World Energy Assessment. Energy and the challenge of sustainability. New York: United Nations Development Programme. Retrieved from http://large.stanford.edu/courses/2017/ph240/fleming2/docs/wea-2000.pdf#page=74 Hollins, J., Thambithurai, D., Koeck, B., Crespel, A., Bailey, D. M., Cooke, S. J., … Killen, S. S. (2018). A physiological perspective on fisheries-induced evolution. Evolutionary Applications, 11(5), 561–576. https://doi.org/10.1111/eva.12597 Hope, A. G., Sandercock, B. K., & Malaney, J. L. (2018). Collection of Scientific Specimens: Benefits for Biodiversity Sciences and Limited Impacts on Communities of Small Mammals. BioScience, 68(1), 35–42. https://doi.org/10.1093/biosci/bix141 Hornborg, S., & Främberg, A. (2019). Carp (Cyprinidae) Fisheries in Swedish Lakes: A Combined Environmental Assessment Approach to Evaluate Data-limited Freshwater Fish Resources as Food. Environmental Management. Scopus. https://doi.org/10.1007/s00267-019-01241-z Hosaka, T., Sugimoto, K., & Numata, S. (2017). Effects of childhood experience with nature on tolerance of urban residents toward hornets and wild boars in Japan. PLOS ONE, 12(4), e0175243. https://doi.org/10.1371/journal.pone.0175243 Hosonuma, N., Herold, M., De Sy, V., De Fries, R. S., Brockhaus, M., Verchot, L., … Romijn, E. (2012). An assessment of deforestation and forest degradation drivers in developing countries. Environmental Research Letters, 7(4), 044009. https://doi.org/10.1088/1748-9326/7/4/044009 Hossain, M. A., Thompson, B. S., Chowdhury, G. W., Mohsanin, S., Fahad, Z. H., Koldewey, H. J., & Islam, M. A. (2015). and Sawfish exploitation and status in Bangladesh. Aquatic Conservation: Marine and Freshwater Ecosystems, 25(6), 781–799. Scopus. https://doi.org/10.1002/aqc.2466 Houdt, S., Brown, R. P., Wanger, T. C., Twine, W., Fynn, R., Uiseb, K., … Traill, L. W. (2021). Divergent views on trophy hunting in Africa, and what this may mean for research and policy. Conservation Letters. https://doi.org/10.1111/conl.12840 Howard, P. L. (Ed.). (2003). Women & plants: Gender relations in biodiversity management and conservation. New York : Eschborn, Germany: Zed Books ; Deutsche Gesellschaft für Technische Zusammenarbeit. Howe, C., Suich, H., Vira, B., & Mace, G. M. (2014). Creating win-wins from trade-offs? Ecosystem services for human well-being: A meta-analysis of ecosystem service trade- offs and synergies in the real world. Global Environmental Change, 28, 263–275. https://doi.org/10.1016/j.gloenvcha.2014.07.005 Hoyt, E., & Hvenegaard, G. T. (2002). A Review of Whale-Watching and Whaling with Applications for the Caribbean. Coastal Management, 30(4), 381–399. https://doi.org/10.1080/089207502900273 HSI/HSUS. (2016). Trophy Hunting by the Numbers: The United States’ Role in Global Trophy Hunting. (p. 19). Retrieved from https://www.hsi.org/wp- content/uploads/assets/pdfs/report_trophy_hunting_by_the.pdf Hua, K., Cobcroft, J. M., Cole, A., Condon, K., Jerry, D. R., Mangott, A., … Strugnell, J. M. (2019). The Future of Aquatic Protein: Implications for Protein Sources in Aquaculture Diets. One Earth, 1(3), 316–329. https://doi.org/10.1016/j.oneear.2019.10.018 410 Hua, R., Chen, Z., & Fu, W. (2017). An Overview of Wild Edible Fungi Resource Conservation and Its Utilization in Yunnan. Journal of Agricultural Science, 9(5), 158. https://doi.org/10.5539/jas.v9n5p158 Huber, F. K., Ineichen, R., Yang, Y., & Weckerle, C. S. (2010). Livelihood and Conservation Aspects of Non-wood Forest Product Collection in the Shaxi Valley, Southwest China. Economic Botany, 64(3), 189–204. https://doi.org/10.1007/s12231-010-9126-z Huchzermeyer. (2003a). Crocodiles—Biology, Husbandry and Diseases \| Lymphatic System \| Aorta. Retrieved August 7, 2019, from Scribd website: https://www.scribd.com/doc/97354553/Crocodiles-Biology-Husbandry-and-Diseases Huchzermeyer, M. (2003b). A legacy of control? The capital subsidy for housing, and informal settlement intervention in South Africa. International Journal of Urban and Regional Research, 27(3), 591–612. https://doi.org/10.1111/1468-2427.00468 Hudson, S. J. (2001). Challenges for Environmental Education: Issues and Ideas for the 21st Century. BioScience, 51(4), 283. https://doi.org/10.1641/0006- 3568(2001)051[0283:CFEEIA]2.0.CO;2 Hughen, K. A., Eglinton, T. I., Xu, L., & Makou, M. (2004). Abrupt Tropical Vegetation Response to Rapid Climate Changes. Science, 304(5679), 1955–1959. https://doi.org/10.1126/science.1092995 Human Society of United States. (2014). Celebrating Animals, Confronting Cruelty, Annual report 2014. Retrieved from https://www.humanesociety.org/sites/default/files/docs/2014-hsus-annual-report.pdf Human Society of United States. (2014). Celebrating Animals, Confronting Cruelty, Annual report 2014. Retrieved from https://www.humanesociety.org/sites/default/files/docs/2014-hsus-annual-report.pdf Humphries, S. (2016). and Financial Viability and Income Generation for a Community Forestry Cooperative in Brazil. San Francisco. Humphries, S. (2016). Financial Viability and Income Generation for a Community Forestry Cooperative in Brazil. San Francisco. Humphries, S., Andrade, D., & McGrath, D. (2015). COOMFLONA : A Successful Community-Based Forest Enterprise in Brazil. San Francisco, CA, USA. Huntington, H., Sakakibara, C., Noongwook, G., Kanayurak, N., Skhauge, V., Zdor, E., … Lyberth, B. (2021). Whale hunting in Indigenous Arctic cultures. In The Bowhead Whale (pp. 501–517). Elsevier. Hurley, P. T., Grabbatin, B., Goetcheus, C., & Halfacre, A. (2013). Gathering, Buying, and Growing Sweetgrass (Muhlenbergia sericea): Urbanization and Social Networking in the Sweetgrass Basket-Making Industry of Lowcountry South Carolina. In R. Voeks & J. Rashford (Eds.), African Ethnobotany in the Americas (pp. 153–173). New York, NY: Springer New York. https://doi.org/10.1007/978-1-4614-0836-9_6 Hutniczak, B., Delpeuch, C., & Leroy, A. (2019). Closing Gaps in National Regulations Against IUU Fishing (OECD Food, Agriculture and Fisheries Papers No. 120). https://doi.org/10.1787/9b86ba08-en Hyde, W. F. (2016). Whereabouts devolution and collective forest management? New Frontiers of Forest Economics: Forest Economics beyond the Perfectly Competitive Commodity Markets, 72, 85–91. https://doi.org/10.1016/j.forpol.2016.06.018 Hyvärinen, E., Juslén, A. K., Kemppainen, E., Uddström, A., & Liukko, U.-M. (2019). Suomen lajien uhanalaisuus 2019-Punainen kirja: The 2019 Red List of Finnish Species. Helsinki: Ympäristöministeriö & Suomen ympäristökeskus. 411 IARNA/URL/ILA. (2006). Perfil Ambiental de Guatemala: Tendencias y reflexiones sobre la gestion ambiental. Guatemala: Instituto de Agricultura, Recursos Naturales y Abiente, Universidad Rafael Landivar and Asociacion Instituto de Incidencia Ambiental. IBAMA. (2004). Floresta Nacional Do Tapajós: Plano de Manejo.” Vol. I-Inform. Brasília, DF, Brasil: Instituto Brasileiro de Meio Ambiente e dos Recursos Naturais Renováveis. Retrieved from Instituto Brasileiro de Meio Ambiente e dos Recursos Naturais Renováveis. website: http://www.icmbio.gov.br/portal/images/stories/imgs-unidades- coservacao/flona_tapajoss.pdf ICES. (2018). EU request on analysis of the IUCN process for the assessment of the conservation status of marine species in comparison to the process used by fisheries management bodies. Copenhagen, Denmark: International Council for the Exploration of the Seas (ICES). Ichii, K., Molnár, Z., Obura, D., Purvis, A., & Willis, K. (2019). Chapter 2.2 Status and Trends—Nature. Secretariat of the Intergovernmental Science-Policy Platform on Biodiversity and Ecosystem Services, Global Ass(May). ICMBio. (2015). Relatório: Levantamento de Famílias Da Floresta Nacional Do Tapajós. Santarém, PA, Brasil: Instituto Chico Mendes de Conservação da Biodiversidade. IEA. (2017). Energy Access Outlook 2017: From poverty to prosperity [World Energy Outlook Special Report]. Paris: International Energy Agency. and Retrieved from International Energy Agency website: https://www.iea.org/reports/energy-access-outlook-2017 IEA. (2020). SDG7: Data and Projections © OECD/IEA, Paris. Database: Https://www.iea.org/reports/sdg7-data-and-projections. Accessed: May 2021. IEA. (2021). World Energy Outlook 2021 (p. 386). Paris: International Energy Agency. IEA. (2020). SDG7: Data and Projections © OECD/IEA, Paris. Database: Https://www.iea.org/reports/sdg7-data-and-projections. Accessed: May 2021. IEA. (2021). World Energy Outlook 2021 (p. 386). Paris: International Energy Agency. Retrieved from International Energy Agency website: https://www.iea.org/reports/world-energy-outlook-2021 IFOAM/ITC. (2007). Overview of world production and marketing or organic wild collected products. Retrieved from https://www.intracen.org/uploadedFiles/intracenorg/Content/Exporters/Sectors/Fair_tr ade_and_environmental_exports/Biodiversity/Overview_World_Production_Marketin g_Organic_Wild_Collected_Products.pdf Retrieved Ikiriza, H., Engeu, O., Peter, E. L., Hedmon, O., Umba, C., Abubaker, M., & Abdalla, A. (2019). Dioscorea bulbifera, a highly threatened African medicinal plant, a review. Cogent Biology, 5(1). https://doi.org/10.1080/23312025.2019.1631561 Ilarri, M. D. I., Souza, A. T. de, Medeiros, P. R. de, Grempel, R. G., & Rosa, I. M. de L. (2008). Effects of tourist visitation and supplementary feeding on fish assemblage composition on a tropical reef in the Southwestern Atlantic. Neotropical Ichthyology, 6(4), 651–656. https://doi.org/10.1590/S1679-62252008000400014 Imathiu, S. (2020). Benefits and food safety concerns associated with consumption of edible insects. NFS Journal, 18, 1–11. https://doi.org/10.1016/j.nfs.2019.11.002 Ingram, D. J., Coad, L., Collen, B., Kümpel, N. F., Breuer, T., Fa, J. E., … Scharlemann, J. P. W. (2015). Indicators for wild animal offtake: Methods and case study for African mammals and birds. Ecology and Society, 20(3), art40. https://doi.org/10.5751/ES- 07823-200340 412 Ingram, V., Haverhals, M., Petersen, S., Elias, M., Sijapati Basnett, B., & Sola, P. (2016). Gender and Forest, Tree and Agroforestry Value Chains: Evidence from Literature. In C. J. P. Colfer, B. Sijapati Basnett, & M. Elias (Eds.), Gender and forests: Climate change, tenure, value chains and emerging issues. London ; New York: Routledge, Taylor & Francis Group. Iniesta-Arandia, I., García-Llorente, M., Aguilera, P. A., Montes, C., & Martín-López, B. (2014). Socio-cultural valuation of ecosystem services: Uncovering the links between values, drivers of change, and human well-being. Ecological Economics, 108, 36–48. https://doi.org/10.1016/j.ecolecon.2014.09.028 International Finance Corporation (IFC). (2018). Wild Harvest Value Chain Assessment Report—Armenia. World Bank Group’s Armenia Gender Project. Retrieved from https://documents1.worldbank.org/curated/pt/258201534170791650/pdf/129405-WP- PUBLIC-ReportWildHarvestSectorReviewJune.pdf International Whaling Commission. (2021). Total catches. Available at https://iwc.int/total- catches, Downloaded August 2021. INTOSAI WGEA. (2013). Impact of Tourism on Wildlife Conservation (p. 44). Retrieved from http://iced.cag.gov.in/wp-content/uploads/2014/02/2013_wgea_Wild-Life_view.pdf IPBES. (2018a). The IPBES regional assessment report on Biodiversity and Ecosystem Services for Asia and the Pacific. Bonn, Germany: Secretariat of the Intergovernmental Science-Policy Platform on Biodiversity and Ecosystem Services. Retrieved from https://doi.org/10.5281/zenodo.3237373 IPBES. (2018b). The IPBES regional assessment report on biodiversity and ecosystem services for Europe and Central Asia. (M. Rounsevell, M. Fischer, A. Torre-Marin Rando, & A. Mader, Eds.). Bonn, Germany. Retrieved from https://doi.org/10.5281/zenodo.3237428 IPBES. (2018c). The IPBES regional assessment report on Biodiversity and Ecosystem Services for the Americas (Vol. 1; J. Rice, C. S. Seixas, M. E. Zaccagini, M. Bedoya- Gaitán, & N. Valderram, Eds.). Bonn, Germany: Secretariat of the Intergovernmental Science-Policy Platform on Biodiversity and Ecosystem Services. https://doi.org/10.1016/B978-0-12-384719-5.00349-X IPBES. (2018d). Retrieved The IPBES regional assessment report on Biodiversity and Ecosystem Sevices for Africa (E. Archer, L. Dziba, K. J. Mulongoy, M. A. Maoela, & M. Walters, Eds.). Bonn, Germany: Secretariat of the Intergovernmental Science-Policy Platform on Biodiversity and Ecosystem Services. Retrieved from http://doi.org/10.5281/zenodo.3236178 IPBES. (2019). Summary for policymakers of the global assessment report on biodiversity and ecosystem services of the Intergovernmental Science-Policy Platform on Biodiversity and Ecosystem Services. Bonn, Germany: IPBES Secretariat. Retrieved from https://doi.org/10.5281/zenodo.3831673 IPBES core glossary. (2021). IPBES core glossary. Retrieved March 26, 2021, from IPBES website: http://www.ipbes.net/glossary Isaza, C., Galeano, G., & Bernal, R. (2014). Manejo actual del Asaí (Euterpe precatoria Mart.) para la producción de frutos en el sur de la Amazonia colombiana. Colombia Forestal, 17(1), 77. https://doi.org/10.14483/udistrital.jour.colomb.for.2014.1.a05 413 Islam, G. M. N., Noh, K. M., Sidique, S. F., & Noh, A. F. M. (2014). Economic impact of artificial reefs: A case study of small scale fishers in Terengganu, Peninsular Malaysia. Fisheries Research, 151, 122–129. Scopus. https://doi.org/10.1016/j.fishres.2013.10.018 Islam, K., Nath, T. K., Jashimuddin, M., & Rahman, Md. F. (2019). Forest dependency, co- management and improvement of peoples’ livelihood capital: Evidence from Chunati Wildlife Sanctuary, Bangladesh. Environmental Development, 32, 100456. https://doi.org/10.1016/j.envdev.2019.100456 Islam, Md. W., Rahman, Md. M., Iftekhar, Md. S., & Rakkibu, Md. G. (2013). Can community- based tourism facilitate conservation of the Bangladesh Sundarbans? Journal of Ecotourism, 12(2), 119–129. https://doi.org/10.1080/14724049.2013.820309 ISSF. (2016). Tuna Stock Status Update – 2016. International Seafood Sustainability Foundation. ISSF. (2020). Status of the World Fisheries for Tuna. International Seafood Sustainability Foundation. IUCN. (2004). Application of the IUCN Sustainable Use Policy to sustainable consumptive use of wildlife and recreational hunting in southern Africa. REC 3.093. Retrieved from http://www2.ecolex.org/server2neu.php/libcat/docs/LI/WCC_2004_REC_93_EN.pdf IUCN. (2014). Thunnus orientalis: Collette, B., Fox, W., Juan Jorda, M., Nelson, R., Pollard, D., Suzuki, N. & Teo, S.: The IUCN Red List of Threatened Species 2014: e.T170341A65166749 [Data set]. International Union for Conservation of Nature. https://doi.org/10.2305/IUCN.UK.2014-3.RLTS.T170341A65166749.en IUCN. (2016). Informing decisions on trophy hunting: A briefing paper for European Union decision-makers regarding potential plans for restriction of imports of hunting trophies. IUCN. Retrieved from IUCN website: https://www.iucn.org/sites/dev/files/iucn_sept_briefing_paper_- _informingdecisionstrophyhunting.pdf IUCN. (2020a). General use and trade classification scheme (Version 1.0). IUCN. IUCN. (2020b). The IUCN Red List Data. Retrieved September 28, 2020, from IUCN Red List of Threatened Species website: https://www.iucnredlist.org/en IUCN Standards and Petitions Committee. (2019). Guidelines for Using the IUCN Red List Categories and Criteria. Version 14. IUCN Standards and Petitions Committee. Retrieved Retrieved from http://www.iucnredlist.org/documents/RedListGuidelines.pdf IUFRO. (2017). IUFRO: Glossary of Wildlife Management Terms and Definitions / SilvaVoc Terminology Project / Special Programmes and Projects. Retrieved from https://www.iufro.org/science/special/silvavoc/wildlife-glossary/ Ivanoff, J. (1992). Équilibre paradoxal: Sédentarité et sacralité chez les nomades marins moken. Bulletin de l’École Française d’Extrême-Orient, 79(2), 103–130. https://doi.org/10.3406/befeo.1992.1874 Iverson, L., & Matthews, S. (2018). Appendix 2: Assessment of risk due to climate change: Sugar maple (Acer saccharum Marshall). Chamberlain J, Emery MR, Patel-Waynand T (Eds), 249–251. 414 Iwasaki-Goodman, M., & Nomoto, M. (2001). Revitalizing the relationship between Ainu and salmon: Salmon rituals in the present. Senri Ethnological Studies, 59, 27–46. https://doi.org/10.15021/00002785 IWC. (2020a). Population Status. Retrieved December 22, 2020, from International Whaling Commission website: https://iwc.int/status IWC. (2020b). Whale Watching Handbook. International Whaling Commission. Retrieved from International Whaling Commission website: https://wwhandbook.iwc.int/en/ IWC. (2021). Report Of The Scientific Committee SC68C (No. 19276). Cambrige, UK. https://archive.iwc.int/pages/view.php?ref=17766&search=%21collection29+&order_ by=title&offset=0&restypes=&starsearch=&archive=&per_page=240&default_sort_d irection=DESC&sort=DESC&context=Root&k=&curpos=&go=previous&. Retrieved from https://archive.iwc.int/pages/view.php?ref=17766&search=%21collection29+&order_ by=title&offset=0&restypes=&starsearch=&archive=&per_page=240&default_sort_d irection=DESC&sort=DESC&context=Root&k=&curpos=&go=previous& https://archive.iwc.int/pages/view.php?ref=17766&search=%21collection29+&order_ by=title&offset=0&restypes=&starsearch=&archive=&per_page=240&default_sort_d irection=DESC&sort=DESC&context=Root&k=&curpos=&go=previous& IWC. (2019a). Description of the aboriginal subsistence hunt in Chukotka, Russian Federation. Retrieved November 11, 2020, from International Whaling Commission website: https://iwc.int/russian-federation IWC. (2019b). Description of the USA aboriginal subsistence hunt: Makah Tribe. Retrieved November 11, 2020, from International Whaling Commission website: https://iwc.int/makah-tribe IWC. (n.d.). Management and utilization of large whales in Greenland. Retrieved November 11, 2020, from International Whaling Commission website: https://iwc.int/makah-tribe Iyengar, A. (2014). Forensic DNA analysis for animal protection and biodiversity conservation: A review. Journal for Nature Conservation, 22(3), 195–205. https://doi.org/10.1016/j.jnc.2013.12.001 Izquierdo-Peña, V., Lluch-Cota, S. E., Hernandez-Rivas, M. E., & Martínez-Rincón, R. O. (2019). Revisiting the Regime Problem hypothesis: 25 years later. Deep Sea Research Part II: Topical Studies in Oceanography, 159, 4–10. https://doi.org/10.1016/j.dsr2.2018.11.003 Jabado, R. W., Al Baharna, R. A., Al Ali, S. R., Al Suwaidi, K. O., Al Blooshi, A. Y., & Al Dhaheri, S. S. (2017). Is this the last stand of the Critically Endangered green sawfish Pristis zijsron in the Arabian Gulf? Endangered Species Research, 32(1), 265–275. Scopus. https://doi.org/10.3354/esr00805 Jackson, A., & Newton, R. W. (2016). Project to Model the Use of Fisheries by-products in the Production of Marine Ingredients with Special Reference to omega-3 Fatty Acids EPA and DHA. Institute of Aquaculture, University of Stirling & IFFO, the Marine Ingredients Organisation. Jackson, J. (2016, July 11). Planet Earth II most watched natural history show for 15 years. The Guardian. Retrieved from https://www.theguardian.com/tv-and- radio/2016/nov/07/planet-earth-ii-bbc1-most-watched-natural-history-show-for-15- years Jackson, J. B. C. (2001). Historical Overfishing and the Recent Collapse of Coastal Ecosystems. Science, 293(5530), 629–637. Retrieved https://doi.org/10.1126/science.1059199 415 Jackson, W., & Ormsby, A. (2017). Urban sacred natural sites–a call for research. Urban Ecosystems, 20(3), 675–681. https://doi.org/10.1007/s11252-016-0623-4 Jacobs, M. H. (2009). Why Do We Like or Dislike Animals? Human Dimensions of Wildlife, 14(1), 1–11. https://doi.org/10.1080/10871200802545765 Jacquet, J., Fox, H., Motta, H., Ngusaru, A., & Zeller, D. (2010). Few data but many fish: Marine small-scale fisheries catches for Mozambique and Tanzania. African Journal of Marine Science, 32(2), 197–206. Jahan, I., Ahsan, D., & Farque, M. H. (2017). Fishers’ local knowledge on impact of climate change and anthropogenic interferences on Hilsa fishery in South Asia: Evidence from Bangladesh. Environment, Development and Sustainability, 19(2), 461–478. Scopus. https://doi.org/10.1007/s10668-015-9740-0 Jahirul, M. I., Brown, J. R., Senadeera, W., Ashwath, N., Laing, C., Leski-Taylor, J., & Rasul, M. G. (2013). Optimisation of Bio-Oil Extraction Process from Beauty Leaf (Calophyllum Inophyllum) Oil Seed as a Second Generation Biodiesel Source. Procedia Engineering, 56, 619–624. https://doi.org/10.1016/j.proeng.2013.03.168 Jaiteh, V. F., Hordyk, A. R., Braccini, M., Warren, C., & Loneragan, N. R. (2017). Shark finning in eastern Indonesia: Assessing the sustainability of a data-poor fishery. ICES Journal of Marine Science, 74(1), 242–253. Scopus. https://doi.org/10.1093/icesjms/fsw170 Jamu, D., Banda, M., Njaya, F., & Hecky, R. E. (2011). Challenges to sustainable management of the lakes of Malawi. Journal of Great Lakes Research, 37(SUPPL. 1), 3–14. Scopus. https://doi.org/10.1016/j.jglr.2010.11.017 Janssen, J., & Shepherd, C. R. (2018). Challenges in documenting trade in non CITES-listed species: A case study on crocodile skinks (Tribolonotus spp.). Journal of Asia-Pacific Biodiversity, 11(4), 476–481. (WOS:000514194200002). https://doi.org/10.1016/j.japb.2018.09.003 Jeffers, V. F., Humber, F., Nohasiarivelo, T., Botosoamananto, R., & Anderson, L. G. (2019). Trialling the use of smartphones as a tool to address gaps in small-scale fisheries catch data in southwest Madagascar. Marine Policy, 99(May 2018), 267–274. https://doi.org/10.1016/j.marpol.2018.10.040 Jennings, S., & Cotter, A. J. R. (1999). Fishing effects in northeast Atlantic shelf seas: Patterns in ®shing effort, diversity and community structure. I. Introduction. Fisheries Research, 4. Jennings, V., Johnson Gaither, C., & Gragg, R. S. (2012). Promoting Environmental Justice Through Urban Green Space Access: A Synopsis. Environmental Justice, 5(1), 1–7. https://doi.org/10.1089/env.2011.0007 Jennings, V., Larson, L., & Yun, J. (2016). Advancing Sustainability through Urban Green Space: Cultural Ecosystem Services, Equity, and Social Determinants of Health. International Journal of Environmental Research and Public Health, 13(2), 196. https://doi.org/10.3390/ijerph13020196 Jensen, A., & Meilby, H. (2008). Does commercialization of a non-timber forest product reduce ecological impact? A case study of the Critically Endangered Aquilaria crassna in Lao PDR. Retrieved Oryx, 42(2), 214–221. https://doi.org/10.1017/S0030605308007825 416 Jensen, Anders, & Meilby, H. (2010). Returns from Harvesting a Commercial Non-timber Forest Product and Particular Characteristics of Harvesters and Their Strategies: Aquilaria crassna and Agarwood in Lao PDR1. Economic Botany, 64(1), 34–45. Readcube. https://doi.org/10.1007/s12231-010-9108-1 Jentoft, S., & Chuenpagdee, R. (2009). Fisheries and coastal governance as a wicked problem. Marine Policy, 33(4), 553–560. https://doi.org/10.1016/j.marpol.2008.12.002 Jentoft, S., & Chuenpagdee, R. (Eds.). (2015). Interactive Governance for Small-Scale Fisheries. Cham: Springer International Publishing. https://doi.org/10.1007/978-3-319- 17034-3 Jentoft, S., & Eide, A. (Eds.). (2011). Poverty Mosaics: Realities and Prospects in Small-Scale Fisheries. Dordrecht: Springer Netherlands. https://doi.org/10.1007/978-94-007-1582- 0 Jerozolimski, A., & Peres, C. A. (2003). Bringing home the biggest bacon: A cross-site analysis of the structure of hunter-kill profiles in Neotropical forests. Biological Conservation, 111(3), 415–425. https://doi.org/10.1016/S0006-3207(02)00310-5 Jetz, W., McGeoch, M. A., Guralnick, R., Ferrier, S., Beck, J., Costello, M. J., … Turak, E. (2019). Essential biodiversity variables for mapping and monitoring species populations. Nature Ecology & Evolution, 3(4), 539–551. https://doi.org/10.1038/s41559-019-0826-1 Ji, Y., Su, A., Ma, G., Tao, T., Fang, D., Zhao, L., & Hu, Q. (2020). Comparison of bioactive constituents and effects on gut microbiota by in vitro fermentation between Ophicordyceps sinensis and Cordyceps militaris. Journal of Functional Foods, 68, 103901. https://doi.org/10.1016/j.jff.2020.103901 Jiao, Y., Li, X., Liang, L., Takeuchi, K., Okuro, T., Zhang, D., & Sun, L. (2012). Indigenous ecological knowledge and natural resource management in the cultural landscape of China’s Hani Terraces. Ecol Res, 27(2), 247–263. https://doi.org/10.1007/s11284-011- 0895-3 Jimenez, É. A., Barboza, R. S. L., Amaral, M. T., & Lucena Frédou, F. (2019). Understanding changes to fish stock abundance and associated conflicts: Perceptions of small-scale fishers from the Amazon coast of Brazil. Ocean and Coastal Management, 182. Scopus. https://doi.org/10.1016/j.ocecoaman.2019.104954 Jiménez-Ruiz, A., Thomé-Ortiz, H., Espinoza-Ortega, A., & Vizcarra Bordi, I. (2017). Aprovechamiento recreativo de los hongos comestibles silvestres: Casos de micoturismo en el mundo con énfasis en México. Bosque (Valdivia), 38(3), 447–456. https://doi.org/10.4067/S0717-92002017000300002 Joanen, T., Merchant, M., Griffith, R., Linscombe, J., & Guidry, A. (2021). Evaluation of Effects of Harvest on Alligator Populations in Louisiana. The Journal of Wildlife Management, 85(4), 696–705. https://doi.org/10.1002/jwmg.22028 Johannes, Robert E, Freeman, M. M., & Hamilton, R. J. (2000). Ignore fishers’ knowledge and miss the boat. Fish and Fisheries, 1(3), 257–271. Johannes, Robert Earle. (1981). Words of the lagoon: Fishing and marine lore in the Palau district of Micronesia. Berkeley/Los Angeles/London: University of California Press. Retrieved from from 417 https://books.google.fr/books?id=TloVDfV7QLoC&pg=PR3&hl=fr&source=gbs_sel ected_pages&cad=3#v=onepage&q&f=false https://books.google.fr/books?id=TloVDfV7QLoC&pg=PR3&hl=fr&source=gbs_sel ected_pages&cad=3#v=onepage&q&f=false Johns, A. Retrieved D. (1992). Vertebrate Responses to Selective Logging: Implications for the Design of Logging Systems. Philosophical Transactions of the Royal Society of London., 335(1275), 7. Johnson, N., & Cabarle, B. (1993). Surviving the cut: Natural forest management in the humid tropics. Washington, D.C., USA: World Resources Institute. Johnson, S., DeCarlo, A., Satyal, P., Dosoky, N. S., Sorensen, A., & Setzer, W. N. (2019). Organic Certification is Not Enough: The Case of the Methoxydecane Frankincense. Plants, 8(4), 88. https://doi.org/10.3390/plants8040088 Jones, E. W. (1956). Ecological Studies on the Rain Forest of Southern Nigeria: IV (Continued). The Plateau Forest of the Okomu Forest Reserve. The Journal of Ecology, 44(1), 83. https://doi.org/10.2307/2257155 Jorgensen, S. J., Anderson, S., Ferretti, F., Tietz, J. R., Chapple, T., Kanive, P., … Block, B. A. (2019). Killer whales redistribute white shark foraging pressure on seals. Scientific Reports, 9(1), 6153. https://doi.org/10.1038/s41598-019-39356-2 Juan-Jordá, M. J., Mosqueira, I., Freire, J., & Dulvy, N. K. (2013). The Conservation and Management of Tunas and Their Relatives: Setting Life History Research Priorities. PLOS ONE, 8(8), e70405. https://doi.org/10.1371/journal.pone.0070405 Judd, W. S. (Ed.). (1999). Plant systematics: A phylogenetic approach. Sunderland, Mass: Sinauer Associates. Juhé-Beaulaton, D., & Salpeteur, M. (2017). “Sacred groves” in African contexts (Benin, Cameroon): Insights from history and anthropology. Routledge Research in Landscape and Environmental Design, Taylor & Francis Ltd. Julliand, C., Pinton, F., Garreta, R., & Lescure, J.-P. (2019). Normaliser le sauvage: L’expérience française des cueilleurs professionnels. EchoGéo, (47). https://doi.org/10.4000/echogeo.16987 Julve, C., Eckebil, T. P., Nadège, N. S., Tchantchouang, J.-C., Kerkohf, B., Beauquin, A., … Lescuyer, G. (2013). Forêts communautaires camerounaises et Plan d’action «Forest Law Enforcement, Governance and Trade»(FLEGT): Quel prix pour la légalité?." Bois et forêts des tropiques 317, no. 3 (2013): 71-80. Bois et Forêts Des Tropiques, 317(3), 71–80. Jürgensen, C., Kollert, W., & Lebedys, A. (2014). Assessment of industrial roundwood production from planted forests. FAO Planted Forests and Trees Working Paper FP/48/E. 40. Kaasik, A. (2012). Conserving sacred natural sites in Estonia. In J. M. Mallarach i Carrera, T. Papagiannēs, & R. Väisänen (Eds.), The diversity of sacred lands in Europe: Proceedings of the Third Workshop of the Delos Initiative, Inari/Aanaar, Finland, 1-3 July 2010. Gland, Switzerland: IUCN. Kaczynski, V. M., & Fluharty, D. L. (2002). European policies in West Africa: Who benefits from fisheries agreements? Marine Policy, 26(2), 75–93. https://doi.org/10.1016/S0308-597X(01)00039-2 Kahn, F. (1997). Les palmiers de l’Eldorado. Paris: ORSTOM. Kahn, F. (1997). Les palmiers de l’Eldorado. Paris: ORSTOM. Retrieved 418 Kahn, F., & Arana, C. (2008). Las palmeras en el marco de la investigación para el desarrollo en América del Sur. Revista Peruana de Biología, 15(supl.1), 5–6. Kaiser, M. J., Hormbrey, S., Booth, J. R., Hinz, H., & Hiddink, J. G. (2018). Recovery linked to life history of sessile epifauna following exclusion of towed mobile fishing gear. Journal of Applied Ecology, 55(3), 1060–1070. https://doi.org/10.1111/1365- 2664.13087 Kala, C. (2009). Aboriginal uses and management of ethnobotanical species in deciduous forests of Chhattisgarh state in India. Journal of Ethnobiology and Ethnomedicine, 5(1), 20. https://doi.org/10.1186/1746-4269-5-20 Kålås, J. A., Viken, Å., Henriksen, S., & Skjelseth, S. (2010). Norsk rødliste for arter 2010 = The 2010 Norwegian red list for species. Trondheim: Artsdatabanken. Kallio, M. H., Kanninen, M., & Krisnawati, H. (2012). Smallholder teak plantations in two villages in Central Java: Silvicultural activity and stand performance. Forests, Trees and Livelihoods, 21(3), 158–175. https://doi.org/10.1080/14728028.2012.734127 Kamini, & Raina, R. (2013). Review of Nardostachys grandiflora: An Important Endangered Medicinal and Aromatic Plant of Western Himalaya. Forest Products Journal, 63(1), 67–71. https://doi.org/10.13073/FPJ-D-12-00092 Kanagavel, A., Parvathy, S., Nameer, P. O., & Raghavan, R. (2016). Conservation implications of wildlife utilization by indigenous communities in the southern Western Ghats of India. Journal of Asia-Pacific Biodiversity, 9(3), 271–279. https://doi.org/10.1016/j.japb.2016.04.003 Kanel, K. R., & Kandel, B. R. (2004). Community Forestry in Nepal: Achievements and Challenges. Journal of Forest and Livelihood, 4(1). Retrieved from https://www.forestaction.org/app/webroot/vendor/tinymce/editor/plugins/filemanager/ files/8.%20CF_policy_Kanel%20and%20Kandel%20final_june%2029.pdf Kaoma, H., & Shackleton, C. M. (2015). The direct-use value of urban tree non-timber forest products to household income in poorer suburbs in South African towns. Forest Policy and Economics, 61, 104–112. https://doi.org/10.1016/j.forpol.2015.08.005 Karanth, K. K., Jain, S., & Mariyam, D. (2017). 14 Emerging Trends in Wildlife and Tiger Tourism in India. In J. S. Chen & N. K. Prebensen (Eds.), Nature tourism (p. 220). Routledge. Karanth, K. K., Nichols, J. D., Karanth, K. U., Hines, J. E., & Christensen, N. L. (2010). The shrinking ark: Patterns of large mammal extinctions in India. Proceedings of the Royal Society B: Biological Sciences, 277(1690), 1971–1979. https://doi.org/10.1098/rspb.2010.0171 Karjalainen, E. (2006). The visual preferences for forest regeneration and field afforestation— Four case studies in Finland. Dissertationes Forestales, 2006(31). https://doi.org/10.14214/df.31 Karsenty, A., Drigo, I. G., Piketty, M.-G., & Singer, B. (2008). Regulating industrial forest concessions in Central Africa and South America. Forest Ecology and Management, 256(7), 1498–1508. https://doi.org/10.1016/j.foreco.2008.07.001 Kassas, M. (2002). Biodiversity: Gaps in knowledge. 8. 419 Kasso, M., & Balakrishnan, M. (2013). Retrieved Ex Situ Conservation of Biodiversity with Particular Emphasis to Ethiopia. ISRN Biodiversity, 2013, e985037. https://doi.org/10.1155/2013/985037 Kastner, T., Erb, K.-H., & Nonhebel, S. (2011). International wood trade and forest change: A global analysis. Global Environmental Change, 21(3), 947–956. https://doi.org/10.1016/j.gloenvcha.2011.05.003 Katikiro, R. E. (2014). Perceptions on the shifting baseline among coastal fishers of Tanga, Northeast Tanzania. Ocean and Coastal Management, 91, 23–31. Scopus. https://doi.org/10.1016/j.ocecoaman.2014.01.009 Katz, E., López, C. L., Fleury, M., Miller, R. P., Payê, V., Dias, T., … Moreira, E. (2012). No greens in the forest? Note on the limited consumption of greens in the Amazon. Acta Societatis Botanicorum Poloniae, 81(4). https://doi.org/10.5586/asbp.2012.048 Katz, Esther, García, C., & Goloubinoff, M. (2002). Sumatra Benzoin (Styrax spp.). In Tapping the Green Market. Certification and Management of Non-Timber Forest Products (Guillen A, Laird S, Shanley P, Pierce A. (ed), pp. 182–190). United Kingdom.: Earthscan/WWF/UNESCO People and Plants/Kew Gardens. Retrieved from https://www.researchgate.net/publication/272743208_Tapping_the_Green_Market_C ertification_and_Management_of_Non-timber_Forest_Products Kawarazuka, N., & Béné, C. (2010). Linking small-scale fisheries and aquaculture to household nutritional security: An overview. Food Security, 2(4), 343–357. https://doi.org/10.1007/s12571-010-0079-y Kay, M. C., Lenihan, H. S., Guenther, C. M., Wilson, J. R., Miller, C. J., & Shrout, S. W. (2012). Collaborative assessment of California spiny lobster population and fishery responses to a marine reserve network. Ecological Applications, 22(1), 322–335. Scopus. https://doi.org/10.1890/11-0155.1 Kelleher, K., Westlund, L., Hoshino, E., Mills, D., Willmann, R., de Graaf, G., & Brummett, R. (2012). Hidden harvest: The global contribution of capture fisheries. Worldbank; WorldFish. Keppeler, Friedrich Wofgang, Hallwass, G., Santos, F., da Silva, L. H. T., & Silvano, R. A. M. (2020). What makes a good catch? Effects of variables from individual to regional scales on tropical small-scale fisheries. Fisheries Research, 229, 105571. https://doi.org/10.1016/j.fishres.2020.105571 Keppeler, F.W., Hallwass, G., & Silvano, R. A. M. (2017). Influence of protected areas on fish assemblages and fisheries in a large tropical river. ORYX, 51(2), 268–279. Scopus. https://doi.org/10.1017/S0030605316000247 Kerns, J. A., Allen, M. S., & Harris, J. E. (2012). Importance of Assessing Population-Level Impact of Catch-and-Release Mortality. Fisheries, 37(11), 502–503. https://doi.org/10.1080/03632415.2012.731878 Kersey, P. J., Collemare, J., Cockel, C., Das, D., Dulloo, E. M., Kelly, L. J., … Leitch, I. J. (2020). Selecting for useful properties of plants and fungi – Novel approaches, opportunities, and challenges. PLANTS, PEOPLE, PLANET, 2(5), 409–420. https://doi.org/10.1002/ppp3.10136 Keskar, A., Raghavan, R., Kumkar, P., Padhye, A., & Dahanukar, N. (2017). Retrieved Assessing the sustainability of subsistence fisheries of small indigenous fish species: Fishing 420 mortality and exploitation of hill stream loaches in India. Aquatic Living Resources, 30. Scopus. https://doi.org/10.1051/alr/2016036 mortality and exploitation of hill stream loaches in India. Aquatic Living Resources, 30. Scopus. https://doi.org/10.1051/alr/2016036 KEW. (2020). State of the Worlds Plant and Fungi. Royal Botanic Gardens. Khan, A. M. A., Gray, T. S., Mill, A. C., & Polunin, N. V. C. (2018). Impact of a fishing moratorium on a tuna pole-and-line fishery in eastern Indonesia. Marine Policy, 94, 143–149. Scopus. https://doi.org/10.1016/j.marpol.2018.05.014 Khare, A., White, A., & Frechette, A. (2020). Estimate of the area of land and territories of Indigenous Peoples, local communities, and Afro- descendants where their rights have not been recognized (p. 32) [Technical Report]. Rights and Resources Initiative (RRI). Retrieved from Rights and Resources Initiative (RRI) website: https://rightsandresources.org/wp-content/uploads/2020/09/Area-Study-Final-1.pdf Khasanah, M., Nurdin, N., Sadovy de Mitcheson, Y., & Jompa, J. (2020). Management of the Grouper Export Trade in Indonesia. Reviews in Fisheries Science and Aquaculture, 28(1), 1–15. Scopus. https://doi.org/10.1080/23308249.2018.1542420 Khoury, C. K., Amariles, D., Soto, J. S., Diaz, M. V., Sotelo, S., Sosa, C. C., … Jarvis, A. (2019). Comprehensiveness of conservation of useful wild plants: An operational indicator for biodiversity and sustainable development targets. Ecological Indicators, 98, 420–429. https://doi.org/10.1016/j.ecolind.2018.11.016 Kideghesho, J. R. (2009). The potentials of traditional African cultural practices in mitigating overexploitation of wildlife species and habitat loss: Experience of Tanzania. International Journal of Biodiversity Science & Management, 5(2), 83–94. https://doi.org/10.1080/17451590903065579 Kindscher, K., Martin, L. M., & Long, Q. (2019). The Sustainable Harvest of Wild Populations of Oshá (Ligusticum porteri) in Southern Colorado for the Herbal Products Trade. Economic Botany, 1–16. https://doi.org/10.1007/s12231-019-09456-1 Kininmonth, S., Crona, B., Bodin, Ö., Vaccaro, I., Chapman, L. J., & Chapman, C. A. (2017). Microeconomic relationships between and among fishers and traders influence the ability to respond to social-ecological changes in a small-scale fishery. Ecology and Society, 22(2). Scopus. https://doi.org/10.5751/ES-08833-220226 Kiss, A. (2004). Is community-based ecotourism a good use of biodiversity conservation funds? Trends in Ecology & Evolution, 19(5), 232–237. https://doi.org/10.1016/j.tree.2004.03.010 Kissinger, G., Herold, M., & De Sy, V. (2012). Drivers of deforestation and forest degradation: A synthesis report for REDD+ policymakers (p. 48). Lexeme Consulting. Retrieved from Lexeme Consulting website: https://www.forestcarbonpartnership.org/sites/fcp/files/DriversOfDeforestation.pdf_N _S.pdf Kittinger, J. N., Teneva, L. T., Koike, H., Stamoulis, K. A., Kittinger, D. S., Oleson, K. L. L., … Friedlander, A. M. (2015). Retrieved From reef to table: Social and ecological factors affecting coral reef fisheries, artisanal seafood supply chains, and seafood security. PLoS ONE, 10(8). Scopus. https://doi.org/10.1371/journal.pone.0123856 Klain, S. C., Satterfield, T. A., & Chan, K. M. (2014). What matters and why? Ecosystem services and their bundled qualities. Ecological Economics, 107, 310–320. https://doi.org/10.1016/j.ecolecon.2014.09.003 421 Kleinschmit, D., Mansourian, S., Wildburger, C., & Purret, A. (Eds.). (2016). Illegal logging and related timber trade: Dimensions, drivers, impacts and responses ; a global scientific rapid response assessment report. Vienna: IUFRO. Klemens, M. W., & Thorbjarnarson, J. B. (1995). Reptiles as a food resource. Biodiversity & Conservation, 4(3), 281–298. https://doi.org/10.1007/BF00055974 Kletter, C., & Kriechbaum, M. (2001). Tibetan medicinal plants. CRC Press. Kline, K. S., Bruch, R. M., & Binkowski, F. P. (2012). People of the sturgeon: Wisconsin’s love affair with an ancient fish. Wisconsin Historical Society. Klinger, D., & Naylor, R. (2012). Searching for Solutions in Aquaculture: Charting a Sustainable Course. Annual Review of Environment and Resources, 37(1), 247–276. https://doi.org/10.1146/annurev-environ-021111-161531 Kluwe, J., & Krumpe, E. E. (2003). Interpersonal and societal aspects of use conflicts. International Journal of Wilderness, 9(3), 28–33. Knell, R. J., & Martínez-Ruiz, C. (2017). Selective harvest focused on sexual signal traits can lead to extinction under directional environmental change. Proceedings of the Royal Society B: Biological Sciences, 284(1868), 20171788. https://doi.org/10.1098/rspb.2017.1788 Knight, J. (2009). Making wildlife viewable: Habituation and attraction. Society & Animals, 17(2), 167–184. https://doi.org/10.1163/156853009X418091 Knowler, D. (2005). Reassessing the costs of biological invasion: Mnemiopsis leidyi in the Black sea. Ecological Economics, 52(2), 187–199. https://doi.org/10.1016/j.ecolecon.2004.06.013 Koehn, F. E., & Carter, G. T. (2005). The evolving role of natural products in drug discovery. Nature Reviews Drug Discovery, 4(3), 206–220. https://doi.org/10.1038/nrd1657 Koenig, J., Altman, J. C., & Griffiths, A. D. (2011). Artists as Harvesters: Natural Resource Use by Indigenous Woodcarvers in Central Arnhem Land, Australia. Human Ecology, 39(4), 407–419. https://doi.org/10.1007/s10745-011-9413-z Koenig, J., Altman, J. C., Griffiths, A. D., & Kohen, A. (2007). 20 Years of Aboriginal Woodcarving in Arnhem land, Australia: Using art sales records to examine the Dynamics of Sculpture Production. Forests, Trees and Livelihoods, 17(1), 43–60. https://doi.org/10.1080/14728028.2007.9752580 Kohn, A. (2018). Conus Envenomation of Humans: In Fact and Fiction. Toxins, 11(1), 10. https://doi.org/10.3390/toxins11010010 Koivula, M., Kuuluvainen, T., Hallman, E., Kouki, J., Siitonen, J., & Valkonen, S. (2014). Forest management inspired by natural disturbance dynamics (DISTDYN) – a long- term research and development project in Finland. Scandinavian Journal of Forest Research, 29(6), 579–592. Retrieved https://doi.org/10.1080/02827581.2014.938110 Koivula, M., Silvennoinen, H., Koivula, H., Tikkanen, J., & Tyrväinen, L. (2020). Continuous- cover management and attractiveness of managed Scots pine forests. Canadian Journal of Forest Research, 50(8), 819–828. https://doi.org/10.1139/cjfr-2019-0431 Koivula, M., & Vanha-Majamaa, I. (2020). Experimental evidence on biodiversity impacts of variable retention forestry, prescribed burning, and deadwood manipulation in Fennoscandia. Ecological Processes, 9(1), 11. https://doi.org/10.1186/s13717-019- 0209-1 422 Kolding, J., Béné, C., & Bavinck, M. (2014). Small-scale fisheries: Importance, vulnerability and deficient knowledge. Governance of Marine Fisheries and Biodiversity Conservation, 317–331. Kolm, N., & Berglund, A. (2003). Wild populations of a reef fish suffer from the “nondestructive” aquarium trade fishery. Conservation Biology, 17(3), 910–914. Scopus. https://doi.org/10.1046/j.1523-1739.2003.01522.x Koning, A. A., Perales, K. M., Fluet-Chouinard, E., & McIntyre, P. B. (2020). A network of grassroots reserves protects tropical river fish diversity. Nature, 588(7839), 631–635. https://doi.org/10.1038/s41586-020-2944-y Konsam, S., Thongam, B., & Handique, A. K. (2016). Assessment of wild leafy vegetables traditionally consumed by the ethnic communities of Manipur, northeast India. Journal of Ethnobiology and Ethnomedicine, 12(1), 11. https://doi.org/10.1186/s13002-016- 0083-1 Kooiman, J., Bavinck, M., Chuenpagdee, R., Mahon, R., & Pullin, R. (2008). Interactive Governance and Governability: An Introduction. Kooiman, J., Bavinck, M., Jentoft, S., & Pullin, R. (Eds.). (2005). Fish for Life: Interactive Governance for Fisheries. Amsterdam: Amsterdam University Press. https://doi.org/10.5117/9789053566862 Kosaka, Y., Xayvongsa, L., Vilayphone, A., Chanthavong, H., Takeda, S., & Kato, M. (2013). Wild Edible Herbs in Paddy Fields and Their Sale in a Mixture in Houaphan Province, the Lao People’s Democratic Republic. Economic Botany, 67(4), 335-349. https://doi.org/10.1007/s12231-013-9251-6 Koster, J. (2008). The impact of hunting with dogs on wildlife harvests in the Bosawas Reserve, Nicaragua. Environmental Conservation, 35, 211–220. https://doi.org/10.1017/S0376892908005055 Kotte, D., Li, Q., & Shin, W. S. (2019). International Handbook of Forest Therapy. Newcastle- upon-Tyne: Cambridge Scholars Publisher. Retrieved from https://public.ebookcentral.proquest.com/choice/publicfullrecord.aspx?p=5962801 Krainovic, P., Almeida, D. de, Desconci, D., Veiga-Júnior, V. da, & Sampaio, P. de. (2017). Sequential Management of Commercial Rosewood (Aniba rosaeodora Ducke) Plantations in Central Amazonia: Seeking Sustainable Models for Essential Oil Production. Forests, 8(12), 438. https://doi.org/10.3390/f8120438 Kreziou, A., de Boer, H., & Gravendeel, B. (2016). Harvesting of salep orchids in north- western Greece continues to threaten natural populations. Oryx, 50(3), 393–396. https://doi.org/10.1017/S0030605315000265 Kristjanson, P., Bah, T., Kuriakose, A., Shakirova, M., Segura, G., Siegmann, K., & Granat, M. (2019). Taking action on gender gaps in forest landscapes [Working Paper]. PROFOR. Retrieved from PROFOR website: https://www.profor.info/sites/profor.info/files/Working%20Paper_Taking%20Action %20on%20Gender%20Gaps%20in%20Forest%20Landscapes.pdf Kroloff, E. K. N., Heinen, J. T., Braddock, K. Retrieved N., Rehage, J. S., & Santos, R. O. (2019). Understanding the decline of catch-and-release fishery with angler knowledge: A key informant approach applied to South Florida bonefish. Environmental Biology of Fishes, 102(2), 319–328. Scopus. https://doi.org/10.1007/s10641-018-0812-5 423 Kronen, M., Magron, F., McArdle, B., & Vunisea, A. (2010). Reef finfishing pressure risk model for Pacific Island countries and territories. Fisheries Research, 101(1–2), 1–10. Scopus. https://doi.org/10.1016/j.fishres.2009.08.011 Kronenberg, J., Haase, A., Łaszkiewicz, E., Antal, A., Baravikova, A., Biernacka, M., … Onose, D. A. (2020). Environmental justice in the context of urban green space availability, accessibility, and attractiveness in postsocialist cities. Cities, 106, 102862. https://doi.org/10.1016/j.cities.2020.102862 Kroodsma, D. A., Mayorga, J., Hochberg, T., Miller, N. A., Boerder, K., Ferretti, F., … Worm, B. (2018). Tracking the global footprint of fisheries. Science, 359(6378), 904–908. https://doi.org/10.1126/science.aao5646 Kuijper, D. P. J., Kleine, C., Churski, M., Hooft, P., Bubnicki, J., & Jedrzejewska, B. (2013). Landscape of fear in Europe: Wolves affect spatial patterns of ungulate browsing in Bialowieża Primeval Forest, Poland. Ecography, 36, 1263–1275. https://doi.org/10.1111/j.1600-0587.2013.00266.x Kull, C. A., Kueffer, C., Richardson, D. M., Vaz, A. S., Vicente, J. R., & Honrado, J. P. (2018). Using the “regime shift” concept in addressing social-ecological change: Social- ecological regime shifts. Geographical Research, 56(1), 26–41. https://doi.org/10.1111/1745-5871.12267 Kumar, A. N. A., Joshi, G., & Ram, H. Y. M. (2012). Sandalwood: History, uses, present status and the future. CURRENT SCIENCE, 103(12), 10. Kumar, R. S., Parthiban, K. T., Hemalatha, P., Kalaiselvi, T., & Rao, M. G. (2009). Investigation on Cross-Compatibility Barriers in the Biofuel Crop Jatropha curcas L. with Wild Jatropha Species. Crop Science, 49(5), 1667–1674. https://doi.org/10.2135/cropsci2008.10.0601 Kuniyal, C. P., & Sundriyal, R. C. (2013). Conservation salvage of Cordyceps sinensis collection in the Himalayan mountains is neglected. Ecosystem Services, 3, e40–e43. https://doi.org/10.1016/j.ecoser.2012.12.004 Kuo, H.-I., Chen, C.-C., & McAleer, M. (2012). Estimating the impact of whaling on global whale-watching. Tourism Management, 33(6), 1321–1328. https://doi.org/10.1016/j.tourman.2011.12.015 Kurlansky, M. (1997). Cod: A Biography of the Fish that Changed the World. New York: Walker and Co. Kusrini, M. D., & Alford, R. A. (2006). Indonesia’s exports of frogs’ legs. TRAFFIC Bulletin, 21, 13–24. Kuuluvainen, T., & Grenfell, R. (2012). Natural disturbance emulation in boreal forest ecosystem management—Theories, strategies, and a comparison with conventional even-aged management 1 This article is one of a selection of papers from the 7th International Conference on Disturbance Dynamics in Boreal Forests. Canadian Journal of Forest Research, 42(7), 1185–1203. Retrieved https://doi.org/10.1139/x2012-064 Kuuluvainen, T., Lindberg, H., Vanha-Majamaa, I., Keto-Tokoi, P., & Punttila, P. (2019). Low-level retention forestry, certification, and biodiversity: Case Finland. Ecological Processes, 8(1), 47. https://doi.org/10.1186/s13717-019-0198-0 p g Kwan, B. K. Y., Cheung, J. H. Y., Law, A. C. K., Cheung, S. G., & Shin, P. K. S. (2017). Conservation education program for threatened Asian horseshoe crabs: A step towards 424 reducing community apathy to environmental conservation. Journal for Nature Conservation, 35, 53–65. https://doi.org/10.1016/j.jnc.2016.12.002 reducing community apathy to environmental conservation. Journal for Nature Conservation, 35, 53–65. https://doi.org/10.1016/j.jnc.2016.12.002 Kyne, P. M., & Simpfendorfer, C. A. (2007). A collation and summarization of available data on deepwater Chondrichthyans: Biodiversity, life history and fisheries. IUCN SSC Shark Specialist Group for the Marine Conservation Biology Institute. Lade, S. J., Tavoni, A., Levin, S. A., & Schlüter, M. (2013). Regime shifts in a social-ecological system. Theoretical Ecology, 6(3), 359–372. https://doi.org/10.1007/s12080-013-0187- 3 Ladislau, D. S., Ribeiro, M. W. S., Castro, P. D. S., Aride, P. H. R., Paiva, A. J. V., Polese, M. F., … Oliveira, A. T. (2020). Ornamental fishing in the region of Barcelos, Amazonas: Socioeconomic description and scenario of activity in the view of “piabeiros.” Brazilian Journal of Biology, 80(3), 544–556. https://doi.org/10.1590/1519-6984.215806 Lam, V. W., & Pauly, D. (2019). Status of fisheries in 13 Asian large marine ecosystems. Deep Sea Research Part II: Topical Studies in Oceanography, 163, 57–64. Lam, V. Y. Y., & Sadovy De Mitcheson, Y. (2011a). The sharks of South East Asia— Unknown, unmonitored and unmanaged. Fish and Fisheries, 12(1), 51–74. Scopus. https://doi.org/10.1111/j.1467-2979.2010.00383.x Lam, V. Y. Y., & Sadovy De Mitcheson, Y. (2011b). The sharks of South East Asia— Unknown, unmonitored and unmanaged. Fish and Fisheries, 12(1), 51–74. Scopus. https://doi.org/10.1111/j.1467-2979.2010.00383.x Lamprecht, H. (1989). Silviculture in the Tropics: Tropical Forest Ecosystems and Their Tree Species-Possibilities and Methods for Their Long-Term Utilization. Eschborn: Federal Republic of Germany. Lamrani-Alaoui, M., & Hassikou, R. (2018). Rapid risk assessment to harvesting of wild medicinal and aromatic plant species in Morocco for conservation and sustainable management purposes. Biodiversity and Conservation, 27(10), 2729–2745. https://doi.org/10.1007/s10531-018-1565-3 Landor-Yamagata, J., Kowarik, I., & Fischer, L. (2018). Urban Foraging in Berlin: People, Plants and Practices within the Metropolitan Green Infrastructure. Sustainability, 10(6), 1873. https://doi.org/10.3390/su10061873 Lange, D. (2006). International trade in medicinal and aromatic plants: Actors, volumes and commodities. Frontis, 155–170. Lanker, U., Malik, A. R., Gupta, N. K., & Butola, J. S. (2010). Retrieved Natural regeneration status of the endangered medicinal plant, Taxus baccata Hook. F. syn. T. wallichiana, in northwest Himalaya. International Journal of Biodiversity Science, Ecosystem Services & Management, 6(1–2), 20–27. https://doi.org/10.1080/21513732.2010.527302 Larrère, R. (1982). Des cueillettes, des conflits, des contrôles. Etudes Rurales, 87–88(La chasse et la cueillette aujourd’hui), 191–208. Larrère, R., & La Soudière, M. de. (1985). Cueillir la montagne: Plantes, fleurs, champignons en Gévaudan, Auvergne, et Limousin. Lyon: Manufacture. Larsen, H. (2005). Impact of replanting on regeneration of the medicinal plantNardostachys grandiflora DC. (Valerianaceae). Economic Botany, 59(3), 213–220. https://doi.org/10.1663/0013-0001(2005)059[0213:IORORO]2.0.CO;2 425 Laugrand, F. (2015). L’ontologie sur la glace. Les Inuit de l’Arctique central canadien et leurs animaux. La Lettre Du Collège de France, 114, 986–988. https://doi.org/10.4000/annuaire-cdf.12036 Laugrand, F. (2015). L’ontologie sur la glace. Les Inuit de l’Arctique central canadien et leurs animaux. La Lettre Du Collège de France, 114, 986–988. https://doi.org/10.4000/annuaire-cdf.12036 Laurance, W. (2004). The perils of payoff: Corruption as a threat to global biodiversity. Trends in Ecology & Evolution, 19(8), 399–401. https://doi.org/10.1016/j.tree.2004.06.001 Lavides, M. N., Molina, E. P. V., De La Rosa, G. E., Mill, A. C., Rushton, S. P., Stead, S. M., & Polunin, N. V. C. (2016). Patterns of coral-reef finfish species disappearances inferred from fishers’ knowledge in global epicentre of marine shorefish diversity. PLoS ONE, 11(5). Scopus. https://doi.org/10.1371/journal.pone.0155752 Lavorgna, A., Rutherford, C., Vaglica, V., Smith, M. J., & Sajeva, M. (2018). CITES, wild plants, and opportunities for crime. European Journal on Criminal Policy and Research, 24(3), 269–288. https://doi.org/10.1007/s10610-017-9354-1 Lawin, I. F., Houetchegnon, T., Fandohan, A. B., Salako, V. K., Assogbadjo, A. E., & Ouinsavi, C. A. (2019). Knowledge and uses of Cola millenii K. Schum. (Malvaceae) in the Guinean and Sudano-Guinean zones of Benin. Bois Et Forets Des Tropiques, (339), 61–74. https://doi.org/10.19182/bft2019.339.a31716 Laws, B. (2010). Fifty plants that changed the course of history. David and Charles International, Ltd. UK. Le Fur, J., Guilavogui, A., & Teitelbaum, A. (2011). Contribution of local fishermen to improving knowledge of the marine ecosystem and resources in the Republic of Guinea, West Africa. Canadian Journal of Fisheries and Aquatic Sciences, 68(8), 1454–1469. Scopus. https://doi.org/10.1139/f2011-061 Le Manach, F., Gough, C., Harris, A., Humber, F., Harper, S., & Zeller, D. (2012). Unreported fishing, hungry people and political turmoil: The recipe for a food security crisis in Madagascar? Marine Policy, 36(1), 218–225. Scopus. https://doi.org/10.1016/j.marpol.2011.05.007 Le Manacha, F., Goughb, C., Humberb, F., Harperc, S., & Zellerc, D. (2011). Reconstruction of total marine fisheries catches for Madagascar. Retrieved Fisheries Centre Research Reports, 19(4), 21. Leal, M. C., Vaz, M. C. M., Puga, J., Rocha, R. J. M., Brown, C., Rosa, R., & Calado, R. (2016). Marine ornamental fish imports in the European Union: An economic perspective. Fish and Fisheries, 17(2), 459–468. https://doi.org/10.1111/faf.12120 Leao, T. C., Lobo, D., & Scotson, L. (2017). Economic and biological conditions influence the sustainability of Harvest of wild animals and plants in developing countries. Ecological Economics, 140, 14–21. Lebel, L., Garden, P., & Imamura, M. (2005). The Politics of Scale, Position, and Place in the Governance of Water Resources in the Mekong Region. Ecology and Society, 10(2), art18. https://doi.org/10.5751/ES-01543-100218 Leblan, V. (2017). Aux frontières du singe. Relations entre hommes et chimpanzés au Kakandé, (XIXe-XXIe siècle). Paris: Editions de l’EHESS. Leclerc, M., Frank, S. C., Zedrosser, A., Swenson, J. E., & Pelletier, F. (2017). Hunting promotes spatial reorganization and sexually selected infanticide. Scientific Reports, 7(1), 45222. https://doi.org/10.1038/srep45222 426 Lee, D. S. (2017). Inuit and narwhal. In Narwhal: Revealing and Arctic legend (pp. 105–120). Hanover, NH: IPI Press and Smithsonian Institution. Lee, H. J., Son, Y.-H., Kim, S., & Lee, D. K. (2019). Healing experiences of middle-aged women through an urban forest therapy program. Urban Forestry & Urban Greening, 38, 383–391. https://doi.org/10.1016/j.ufug.2019.01.017 Leeney, R. H. (2016). Fishers’ ecological knowledge of sawfishes in Lake Piso, Liberia. Aquatic Conservation: Marine and Freshwater Ecosystems, 26(2), 381–385. Scopus. https://doi.org/10.1002/aqc.2542 Leeney, R. H. (2017). Are sawfishes still present in Mozambique? A baseline ecological study. PeerJ, 2017(2). Scopus. https://doi.org/10.7717/peerj.2950 Leeney, R. H., & Poncelet, P. (2015). Using fishers’ ecological knowledge to assess the status and cultural importance of sawfish in Guinea-Bissau. Aquatic Conservation: Marine and Freshwater Ecosystems, 25(3), 411–430. Scopus. https://doi.org/10.1002/aqc.2419 Leif, J. (2010). Plant fact sheet for sweetgrass [Hierochloe odorata (L.) P. Beauv. Rose Lake Plant Materials Center, East Lansing, MI 48823: USDA-Natural Resources Conservation Service. Leite, M. C., & Gasalla, M. A. (2013). A method for assessing fishers’ ecological knowledge as a practical tool for ecosystem-based fisheries management: Seeking consensus in Southeastern Brazil. Fisheries Research, 145, 43–53. Leleu, K., Pelletier, D., Charbonnel, E., Letourneur, Y., Alban, F., Bachet, F., & Boudouresque, C. F. (2014). Métiers, effort and catches of a Mediterranean small-scale coastal fishery: The case of the CÔte Bleue Marine Park. Fisheries Research, 154, 93–101. Scopus. https://doi.org/10.1016/j.fishres.2014.02.006 Lentini, M., Sobral, L., & Vieira, R. (2020). Como o Mercado Dos Produtos Madeireiros Da Amazônia Evoluiu Nas Últimas Duas Décadas (1998-2018)? Retrieved Boletim TIMBERFlow, (02), 11. Leopold, A. (1933). Game management. New York; London: C. Scribner’s Sons. Léopold, M., David, G., Raubani, J., Kaltavara, J., Hood, L., & Zeller, D. (2017). An improved reconstruction of total marine fisheries catches for the New Hebrides and the Republic of Vanuatu, 1950-2014. Frontiers in Marine Science, 4(OCT). Scopus. https://doi.org/10.3389/fmars.2017.00306 Lescure, J. P., Thévenin, T., Garreta, R., & Morisson, B. (2015). Les plantes faisant l’objet de cueillettes commerciales sur le territoire métropolitain. Une liste commentée. Le Monde Des Plantes, 517, 19–39. Lescuyer, G., Cerutti, P. O., & Tsanga, R. (2016). Contributions of community and individual small-scale logging to sustainable timber management in Cameroon. International Forestry Review, 18(1), 40–51. https://doi.org/10.1505/146554816819683744 Lescuyer, Guillaume, & Cerutti, P. (2013). Politiques de Gestion Durable Des Forêts En Afrique Centrale: Prendre En Compte Le Secteur Informel. Perspective, 21(21), 4. Lescuyer, Guillaume, Cerutti, P. O., & Robiglio, V. (2013). Artisanal chainsaw milling to support decentralized management of timber in Central Africa? An analysis through the theory of access. Forest Policy and Economics, 32, 68–77. https://doi.org/10.1016/j.forpol.2013.02.010 427 Lescuyer, Guillaume, Tsanga, R., Mendoula, E. E., Ahanda, B. X. E., Ouedraogo, H. A., Fung, O., … Logo, P. B. (2017). National demand for sawnwood in Cameroon. 72. Lesniewska, F., & McDermott, C. L. (2014). FLEGT VPAs: Laying a pathway to sustainability via legality lessons from Ghana and Indonesia. Forest Policy and Economics, 48, 16– 23. https://doi.org/10.1016/j.forpol.2014.01.005 Lewin, W.-C., Arlinghaus, R., & Mehner, T. (2006). Documented and Potential Biological Impacts of Recreational Fishing: Insights for Management and Conservation. Reviews in Fisheries Science, 14(4), 305–367. https://doi.org/10.1080/10641260600886455 Lewis, D. (1994). We, the Navigators: The Ancient Art of Landfinding in the Pacific. Honolulu: University of Hawaii Press. Lewison, R., Crowder, L., Read, A., & Freeman, S. (2004a). Understanding impacts of fisheries bycatch on marine megafauna. Trends in Ecology & Evolution, 19(11), 598–604. https://doi.org/10.1016/j.tree.2004.09.004 Lewison, R., Crowder, L., Read, A., & Freeman, S. (2004b). Understanding impacts of fisheries bycatch on marine megafauna. Trends in Ecology & Evolution, 19(11), 598– 604. https://doi.org/10.1016/j.tree.2004.09.004 Li, S. (1596). Compendium of Materia Medica. Nanjin, China. Li, X., Liu, Q., Li, W., Li, Q., Qian, Z., Liu, X., & Dong, C. (2019). A breakthrough in the artificial cultivation of Chinese cordyceps on a large-scale and its impact on science, the economy, and industry. Critical Reviews in Biotechnology, 39(2), 181–191. https://doi.org/10.1080/07388551.2018.1531820 Liao, Y., Hsieh, H.-L., Xu, S., Zhong, Q., Lei, J., Liang, M., … Kwan, B. K. Y. Retrieved (2019). Wisdom of Crowds reveals decline of Asian horseshoe crabs in Beibu Gulf, China. ORYX, 53(2), 222–229. Scopus. https://doi.org/10.1017/S003060531700117X Liebenberg, L., Steventon, J., Brahman, !Nate, Benadie, K., Minye, J., Langwane, H. (Karoha), & Xhukwe, Q. (Uase). (2017). Smartphone Icon User Interface design for non-literate trackers and its implications for an inclusive citizen science. Biological Conservation, 208, 155–162. https://doi.org/10.1016/j.biocon.2016.04.033 Lilian Ibengwe & Fatma Sobo. (2016). The Value of Tanzania Fisheries and Aquaculture: Assessment of the Contribution of the Sector to Gross Domestic Product. Rome, Italy: East Lansing, Michigan, USA : Bethesda, Maryland, USA: Food and Agriculture Organization of the United Nations ; Michigan State University ; American Fisheries society. Lima, E. G., Begossi, A., Hallwass, G., & Silvano, R. A. (2016). Fishers’ knowledge indicates short-term temporal changes in the amount and composition of catches in the southwestern Atlantic. Marine Policy, 71, 111–120. Lima, I. B., & d’Hauteserre, A.-M. (2011). Community capitals and ecotourism for enhancing Amazonian forest livelihoods. Anatolia, 22(2), 184–203. https://doi.org/10.1080/13032917.2011.597933 Lindberg, K. (2001). Economic impacts. In D. B. Weaver (Ed.), The encyclopedia of ecotourism. Oxon, UK ; New York, NY: CABI Pub. Lindenmayer, D., & Scheele, B. (2017a). Do not publish. Science. (world). https://doi.org/10.1126/science.aan1362 428 Lindenmayer, D., & Scheele, B. (2017b). Do not publish. Science, 356(6340), 800–801. https://doi.org/10.1126/science.aan1362 Lindsey, P. (2011). An analysis of game meat production and wildlife-based land uses on freehold land in Namibia: Links with food security. Harare, Zimbabwe: TRAFFIC East/Southern Africa. Lindsey, P. A., Alexander, R., Frank, L. G., Mathieson, A., & Romanach, S. S. (2006). Cretois. Animal Conservation, 9(3), 283–291. https://doi.org/10.1111/j.1469- 1795.2006.00034.x Lindsey, P. A., Roulet, P. A., & Romañach, S. S. (2007). Economic and conservation significance of the trophy hunting industry in sub-Saharan Africa. Biological Conservation, 134(4), 455–469. https://doi.org/10.1016/j.biocon.2006.09.005 Lindsey, P., Alexander, R., Balme, G., Midlane, N., & Craig, J. (2012). Possible Relationships between the South African Captive-Bred Lion Hunting Industry and the Hunting and Conservation of Lions Elsewhere in Africa. South African Journal of Wildlife Research, 42(1), 11–22. https://doi.org/10.3957/056.042.0103 Lindsey, P., Balme, G. A., Booth, V. R., & Midlane, N. (2012). The Significance of African Lions for the Financial Viability of Trophy Hunting and the Maintenance of Wild Land. PLoS ONE, 7(1), e29332. https://doi.org/10.1371/journal.pone.0029332 Lindsey, Peter, Allan, J., Brehony, P., Dickman, A., Robson, A., Begg, C., … Tyrrell, P. (2020). Conserving Africa’s wildlife and wildlands through the COVID-19 crisis and beyond. Nature Ecology & Evolution, 4(10), 1300–1310. Retrieved https://doi.org/10.1038/s41559-020-1275-6 Lindsey, Peter, & Bento, C. (2012). Illegal hunting and the bushmeat trade in Central Mozambique. A case-study from Coutada 9, Manica Province. 84. Liner, E. A. (2005). The culinary herpetologist. Salt Lake City: Bibliomania. Link, J. S., & Watson, R. A. (2019). Global ecosystem overfishing: Clear delineation within real limits to production. Science Advances, 5(6), eaav0474. https://doi.org/10.1126/sciadv.aav0474 Linnell, J D C, & Cretois, B. (2018). The revival of wolves and other large predators and its impact on farmers and their livelihood in rural regions of Europe. 106. Linnell, J D C, Cretois, B., Nilsen, E. B., Rolandsen, C. M., Solberg, E. J., Veiberg, V., … Kaltenborn, B. (2020). The challenges and opportunities of coexisting with wild ungulates in the human-dominated landscapes of Europe’s Anthropocene. Biological Conservation, 244, 108500. https://doi.org/10.1016/j.biocon.2020.108500 Linnell, John D. C. (2015). Defining scales for managing biodiversity and natural resources in the face of conflicts. In J. C. Young, K. A. Wood, R. J. Gutiérrez, & S. M. Redpath (Eds.), Conflicts in Conservation: Navigating Towards Solutions (pp. 212–225). Cambridge: Cambridge University Press. https://doi.org/10.1017/CBO9781139084574.016 Lion Landscapes. (2020). Lion Carbon. Retrieved February 27, 2021, from https://www.lionlandscapes.org/lioncarbon Liu, D., Tian, Y., Ma, S., Li, J., Sun, P., Ye, Z., … Zhou, S. (2021). Long-Term Variability of Piscivorous Fish in China Seas Under Climate Change With Implication for Fisheries 429 Management. Frontiers in Marine Science, 8. Scopus. https://doi.org/10.3389/fmars.2021.581952 in Liu, Dongyang, Cheng, H., Bussmann, R. W., Guo, Z., Liu, B., & Long, C. (2018). An ethnobotanical survey of edible fungi in Chuxiong City, Yunnan, China. Journal of Ethnobiology and Ethnomedicine, 14(1), 42. https://doi.org/10.1186/s13002-018-0239- 2 Liu, H., Luo, Y. B., Heinen, J., Bhat, M., & Liu, Z. J. (2014). Eat your orchid and have it too: A potentially new conservation formula for Chinese epiphytic medicinal orchids. Biodiversity and Conservation, 23(5), 1215–1228. https://doi.org/10.1007/s10531-014- 0661-2 Liu, Hong, Gale, S. W., Cheuk, M. L., & Fischer, G. A. (2019). Conservation impacts of commercial cultivation of endangered and overharvested plants. Conservation Biology, 33(2), 288–299. https://doi.org/10.1111/cobi.13216 Liu, Hong, Liu, Z., Jin, X., Gao, J., Chen, Y., Liu, Q., & Zhang, D.-Y. (2020). Assessing conservation efforts against threats to wild orchids in China. Biological Conservation, 243, 108484. https://doi.org/10.1016/j.biocon.2020.108484 Liu, J., Yong, D. L., Choi, C.-Y., & Gibson, L. (2020). Transboundary Frontiers: An Emerging Priority for Biodiversity Conservation. Trends in Ecology & Evolution, 35(8), 679–690. https://doi.org/10.1016/j.tree.2020.03.004 Liu, U., Breman, E., Cossu, T. A., & Kenney, S. (2018). Retrieved The conservation value of germplasm stored at the Millennium Seed Bank, Royal Botanic Gardens, Kew, UK. Biodiversity and Conservation, 27(6), 1347–1386. https://doi.org/10.1007/s10531-018-1497-y Lloret, J, Biton-Porsmoguer, S., Carreño, A., Di Franco, A., Sahyoun, R., Melià, P., … Font, T. (2020). Recreational and small-scale fisheries may pose a threat to vulnerable species in coastal and offshore waters of the western Mediterranean. ICES Journal of Marine Science, 77(6), 2255–2264. https://doi.org/10.1093/icesjms/fsz071 Lloret, J., Cowx, I. G., Cabral, H., Castro, M., Font, T., Gonçalves, J. M. S., … Erzini, K. (2018). Small-scale coastal fisheries in European Seas are not what they were: Ecological, social and economic changes. Marine Policy, 98, 176–186. Scopus. https://doi.org/10.1016/j.marpol.2016.11.007 Lloret, Josep, Sabatés, A., Muñoz, M., Demestre, M., Solé, I., Font, T., … Gómez, S. (2015). How a multidisciplinary approach involving ethnoecology, biology and fisheries can help explain the spatio‐temporal changes in marine fish abundance resulting from climate change. Global Ecology and Biogeography, 24(4), 448–461. Løbach, T., Petersson, T., Haberkon, E., & Mannini, P. (2020). Regional fisheries management organizations and advisory bodies. Activities and developments, 2000–2017. [AO Fisheries and Aquaculture Technical Paper No. 651]. Rome: FAO. https://doi.org/10.4060/ca7843en Locatelli, B., Brockhaus, M., Buck, A., & Thompson, I. (2010). Forests and Adaptation to Climate Change: Challenges and Opportunities. In IUFRO World Series: Vol. v. 25. Forests and society: Responding to global drivers of change. Vienna: International Union of Forest Research Organizations. Lodge, M., Anderson, D., & Lobach, T. (2007). Recommended Best Practices for Regional Fisheries Management Organizations. Report of an Independent Panel to Develop a 430 Model for Improved Governance by Regional Fisheries Management Organizations. Chatham House. Lopes, P.F.M., Rosa, E. M., Salyvonchyk, S., Nora, V., & Begossi, A. (2013). Suggestions for fixing top-down coastal fisheries management through participatory approaches. Marine Policy, 40(1), 100–110. Scopus. https://doi.org/10.1016/j.marpol.2012.12.033 Lopes, P.F.M., Verba, J. T., Begossi, A., & Pennino, M. G. (2019). Predicting species distribution from fishers’ local ecological knowledge: A new alternative for data-poor management. Canadian Journal of Fisheries and Aquatic Sciences, 76(8), 1423–1431. Scopus. https://doi.org/10.1139/cjfas-2018-0148 Lopes, Priscila F. M., Silvano, R. A. M., Nora, V. A., & Begossi, A. (2013). Transboundary Socio-Ecological Effects of a Marine Protected Area in the Southwest Atlantic. AMBIO, 42(8), 963–974. https://doi.org/10.1007/s13280-013-0452-0 López, C. (2005). Amate, Mexican bark paper: Resourceful harvest strategies to meet market demands. In Case Studies of Non-Timber Forest Product System: Vol. 3. Forest Products, Livelihoods and Conservation. Case Studies of Non-Timber Forest Product Systems. Retrieved Volume 3 – Latin America (Alexiades M.N., Shanley P. (ed), pp. 365–390). Bogor (Indonesia).: CIFOR. Retrieved from hhttps://doi.org/10.17528/cifor/002281 López-Angarita, J., Tilley, A., Díaz, J. M., Hawkins, J. P., Cagua, E. F., & Roberts, C. M. (2018). Winners and Losers in Area-Based Management of a Small-Scale Fishery in the Colombian Pacific. Frontiers in Marine Science, 5, 23. https://doi.org/10.3389/fmars.2018.00023 López-García, J., & Navarro-Cerrillo, R. M. (2020). Disturbance and forest recovery in the Monarch Butterfly Biosphere Reserve, Mexico. Journal of Forestry Research, 31(5), 1551–1566. https://doi.org/10.1007/s11676-019-00964-3 Lopez-Toledo, L., Horn, C., & Endress, B. A. (2011). Distribution and population patterns of the threatened palm Brahea aculeata in a tropical dry forest in Sonora, Mexico. Forest Ecology and Management, 261(11), 1901–1910. https://doi.org/10.1016/j.foreco.2011.02.013 Lorenzen, K., Leber, K. M., & Blankenship, H. L. (2010). Responsible Approach to Marine Stock Enhancement: An Update. Reviews in Fisheries Science, 18(2), 189–210. https://doi.org/10.1080/10641262.2010.491564 Loring, P. A., Harrison, H. L., & Gerlach, S. C. (2014). Local Perceptions of the Sustainability of Alaska’s Highly Contested Cook Inlet Salmon Fisheries. Society and Natural Resources, 27(2), 185–199. Scopus. https://doi.org/10.1080/08941920.2013.819955 Lotze, H. K., Milewski, I., Fast, J., Kay, L., & Worm, B. (2019). Ecosystem-based management of seaweed harvesting. Botanica Marina, 62(5), 395–409. https://doi.org/10.1515/bot- 2019-0027 Loveridge, A. J., Searle, A. W., Murindagomo, F., & Macdonald, D. W. (2007). The impact of sport-hunting on the population dynamics of an African lion population in a protected area. Biological Conservation, 134(4), 548–558. https://doi.org/10.1016/j.biocon.2006.09.010 Loveridge, A. J., Valeix, M., Chapron, G., Davidson, Z., Mtare, G., & Macdonald, D. (2016). Conservation of large predator populations: Demographic and spatial responses of African lions to the intensity of trophy hunting. Biological Conservation, 204B. 431 Retrieved from https://ora.ox.ac.uk/objects/uuid:400fdc15-f5cd-4dfb-bd9a- cea1635dff0c Loveridge, Andrew J., Reynolds, J. C., & Milner-Gulland, E. J. (2006). Does sport hunting benefit conservation? Key Topics in Conservation Biology. https://doi.org/10.3758/s13423-016-1123-5 Lovrić, M., Da Re, R., Vidale, E., Prokofieva, I., Wong, J., Pettenella, D., … Mavsar, R. (2020). Non-wood forest products in Europe – A quantitative overview. Forest Policy and Economics, 116, 102175. https://doi.org/10.1016/j.forpol.2020.102175 Lozano-Montes, H. M., Pitcher, T. J., & Haggan, N. (2008). Shifting environmental and cognitive baselines in the upper Gulf of California. Frontiers in Ecology and the Environment, 6(2), 75–80. Scopus. https://doi.org/10.1890/070056 Lüchtrath, A., & Schraml, U. (2015). The missing lynx—Understanding hunters’ opposition to large carnivores. Wildlife Biology, 21(2), 110–119. https://doi.org/10.2981/wlb.00068 Luckert, M. (Marty), & Williamson, T. (2005). Should sustained yield be part of sustainable forest management? Retrieved Canadian Journal of Forest Research, 35(2), 356–364. https://doi.org/10.1139/x04-172 Łuczaj, Ł., & Dolina, K. (2015). A hundred years of change in wild vegetable use in southern Herzegovina. Journal of Ethnopharmacology, 166, 297–304. https://doi.org/10.1016/j.jep.2015.02.033 Łuczaj, Ł., & Nieroda, Z. (2011). Collecting and Learning to Identify Edible Fungi in Southeastern Poland: Age and Gender Differences. Ecology of Food and Nutrition, 50(4), 319–336. https://doi.org/10.1080/03670244.2011.586314 Łuczaj, Ł., Pieroni, A., Tardío, J., Pardo-de-Santayana, M., Sõukand, R., Svanberg, I., & Kalle, R. (2012). Wild food plant use in 21st century Europe: The disappearance of old traditions and the search for new cuisines involving wild edibles. Acta Societatis Botanicorum Poloniae, 81(4), 359–370. https://doi.org/10.5586/asbp.2012.031 Łuczaj, Ł., Wilde, M., & Townsend, L. (2021). The Ethnobiology of Contemporary British Foragers: Foods They Teach, Their Sources of Inspiration and Impact. Sustainability, 13(6), 3478. https://doi.org/10.3390/su13063478 Łuczaj, Ł., Zovko Končić, M., Miličević, T., Dolina, K., & Pandža, M. (2013). Wild vegetable mixes sold in the markets of Dalmatia (southern Croatia). Journal of Ethnobiology and Ethnomedicine, 9(1), 2. https://doi.org/10.1186/1746-4269-9-2 Łuczaj, Łukasz, Jug-Dujaković, M., Dolina, K., Jeričević, M., & Vitasović-Kosić, I. (2019). The ethnobotany and biogeography of wild vegetables in the Adriatic islands Jug- Dujaković, M., Dolina, K., Jeričević M., Vitasović-Kosić I. Journal of Ethnobiology and Ethnomedicine, 15(article n° 18), 1–17. https://doi.org/10.1186/s13002-019-0297- 0 Ludwig, D., Jones, D. D., & Holling, C. S. (1978). Qualitative Analysis of Insect Outbreak Systems: The Spruce Budworm and Forest. The Journal of Animal Ecology, 47(1), 315. https://doi.org/10.2307/3939 Luintel, H., Bluffstone, R. A., & Scheller, R. M. (2018). The effects of the Nepal community forestry program on biodiversity conservation and carbon storage. PLOS ONE, 13(6), e0199526. https://doi.org/10.1371/journal.pone.0199526 432 Luintel, H., Bluffstone, R. A., Scheller, R. M., & Adhikari, B. (2017). The Effect of the Nepal Community Forestry Program on Equity in Benefit Sharing. The Journal of Environment & Development, 26(3), 297–321. https://doi.org/10.1177/1070496517707305 Lunde, E. T., Bech, C., Fyumagwa, R. D., Jackson, C. R., & Røskaft, E. (2016). Assessing the effect of roads on impala ( Aepyceros melampus ) stress levels using faecal glucocorticoid metabolites. African Journal of Ecology, 54(4), 434–441. https://doi.org/10.1111/aje.12302 Lundmark, H., Josefsson, T., & Östlund, L. (2013). The history of clear-cutting in northern Sweden – Driving forces and myths in boreal silviculture. Forest Ecology and Management, 307, 112–122. https://doi.org/10.1016/j.foreco.2013.07.003 Luo, H., Tang, Q., Shang, Y., Liang, S., Yang, M., Robinson, N., & Liu, J. (2020). Can Chinese Medicine Be Used for Prevention of Corona Virus Disease 2019 (COVID-19)? Retrieved A Review of Historical Classics, Research Evidence and Current Prevention Programs. Chinese Journal of Integrative Medicine, 26(4), 243–250. https://doi.org/10.1007/s11655-020-3192-6 Luoma, D. L., Eberhart, J. L., Abbott, R., Moore, A., Amaranthus, M. P., & Pilz, D. (2006). Effects of mushroom harvest technique on subsequent American matsutake production. Forest Ecology and Management, 236(1), 65–75. https://doi.org/10.1016/j.foreco.2006.08.342 Lute, M. L., Carter, N. H., López-Bao, J. V., & Linnell, J. D. (2018). Conservation professionals agree on challenges to coexisting with large carnivores but not on solutions. 223-232. https://doi.org/10.1016/j.biocon.2017.12.035 Lyons, J. A., & Natusch, D. J. D. (2013). Effects of consumer preferences for rarity on the harvest of wild populations within a species. Ecological Economics, 93, 278–283. https://doi.org/10.1016/j.ecolecon.2013.06.004 Mac Monagail, M., Cornish, L., Morrison, L., Araujo, R., & Critchley, A. T. (2017). Sustainable harvesting of wild seaweed resources. European Journal of Phycology, 52(4), 371–390. https://doi.org/10.1080/09670262.2017.1365273 Macdonald, C., & Soll, J. (2020). Shark conservation risks associated with the use of shark liver oil in SARS-CoV-2 vaccine development [Preprint]. Ecology. https://doi.org/10.1101/2020.10.14.338053 Macdonald, D. W., & Willis, K. (2013). Elephants in the room: Tough choices for a maturing discipline. 467–494. Macdonald, David W., Loveridge, A. J., Dickman, A., Johnson, P. J., Jacobsen, K. S., & Du Preez, B. (2017). Lions, trophy hunting and beyond: Knowledge gaps and why they matter. Mammal Review, 47(4), 247–253. https://doi.org/10.1111/mam.12096 Macdonald, P., Angus, C. H., Cleasby, I. R., & Marshall, C. T. (2014). Fishers’ knowledge as an indicator of spatial and temporal trends in abundance of commercial fish species: Megrim (Lepidorhombus whiffiagonis) in the northern North Sea. Marine Policy, 45, 228–239. Scopus. https://doi.org/10.1016/j.marpol.2013.11.001 Mace, G. M., Collar, N. J., Gaston, K. J., Hilton-Taylor, C., Akçakaya, H. R., Leader-Williams, N., … Stuart, S. N. (2008). Quantification of extinction risk: IUCN’s system for 433 classifying threatened species. Conservation Biology, 22(6), 1424–1442. https://doi.org/10.1111/j.1523-1739.2008.01044.x Mace, G. M., & Hudson, E. J. (1999). Attitudes toward Sustainability and Extinction. Conservation Biology, 13(2), 242–246. https://doi.org/10.1046/j.1523- 1739.1999.013002242.x MacFadden, B. J. (2019). Broader Impacts of Science on Society. Cambridge: Cambridge University Press. https://doi.org/10.1017/9781108377577 Macía, M. J., Armesilla, P. J., Cámara-Leret, R., Paniagua-Zambrana, N., Villalba, S., Balslev, H., & Pardo-de-Santayana, M. (2011). Palm Uses in Northwestern South America: A Quantitative Review. The Botanical Review, 77(4), 462–570. https://doi.org/10.1007/s12229-011-9086-8 Mack, A., & West, P. (2005). Ten thousand tonnes of small animals: Wildlife consumption in Papua New Guinea, a vital resource in need of management (p. 23). Macleod, R., Sinding, M. H. S., Olsen, M. Retrieved T., Collins, M. J., & Rowland, S. J. (2020). DNA preserved in jetsam whale ambergris. Biology Letters, 16(2). https://doi.org/ARTN 20190819 10.1098/rsbl.2019.0819 MacMillan, D. C., & Leitch, K. (2008). Conservation with a Gun: Understanding Landowner Attitudes to Deer Hunting in the Scottish Highlands. Human Ecology, 36(4), 473–484. https://doi.org/10.1007/s10745-008-9170-9 MacNeil, M. A., Chapman, D. D., Heupel, M., Simpfendorfer, C. A., Heithaus, M., Meekan, M., … others. (2020). Global status and conservation potential of reef sharks. Nature, 583(7818), 801–806. https://doi.org/10.1038/s41586-020-2519-y Macusi, E. D., Laya-og, M. E., & Abreo, N. A. S. (2019). Wild lobster (Panulirus ornatus) fry fishery in Balete bay, Davao Oriental: Catch trends and implications to fisheries management. Ocean and Coastal Management, 168, 340–349. Scopus. https://doi.org/10.1016/j.ocecoaman.2018.11.010 Mahoney, J., & Rueschemeyer, D. (2003). COMPARATIVE HISTORICAL ANALYSIS: ACHIEVEMENTS AND AGENDAS. In D. Rueschemeyer & J. Mahoney (Eds.), Comparative Historical Analysis in the Social Sciences (pp. 3–38). Cambridge: Cambridge University Press. https://doi.org/10.1017/CBO9780511803963.002 Mahoney, P., & Geist, V. (2019). The North American Model of Wildlife Conservation. Johns Hopkins University Press Books. Retrieved from https://jhupbooks.press.jhu.edu/title/north-american-model-wildlife-conservation Maia, H. A., Morais, R. A., Siqueira, A. C., Hanazaki, N., Floeter, S. R., & Bender, M. G. (2018). Shifting baselines among traditional fishers in São Tomé and Príncipe islands, Gulf of Guinea. Ocean and Coastal Management, 154, 133–142. Scopus. https://doi.org/10.1016/j.ocecoaman.2018.01.006 Maikhuri, R., Rawat, L., Negi, V., Purohit, V., Rao, K., & Saxena, K. (2011). Managing natural resources through simple and appropriate technological interventions for sustainable mountain development. CURRENT SCIENCE, 100(7), 992–997. Maisels, F., Keming, E., Kemei, M., & Toh, C. (2001). The extirpation of large mammals and implications for montane forest conservation: The case of the Kilum-Ijim Forest, North- west Province, Cameroon. Oryx, 35(4), 322–331. https://doi.org/10.1046/j.1365- 3008.2001.00204.x 434 Majkowski, J. (2005). Tuna and tuna-like species. In Review of the State of World Marine Fishery Resources (FAO Fisheries Technical Paper 457). Food and Agriculture Organization of the United Nations. Majkowski, J. (2007). Global Fishery Resources of Tuna and Tuna-like Species (FAO Fisheries Technical Paper 483). Food and Agriculture Organization of the United Nations. Makino, M., Matsuda, H., & Sakurai, Y. (2009). Expanding fisheries co-management to ecosystem-based management: A case in the Shiretoko World Natural Heritage area, Japan. Marine Policy, 33(2), 207–214. Scopus. https://doi.org/10.1016/j.marpol.2008.05.013 Maleki, K., Nguema Allogo, F., & Lafleur, B. (2020). Natural Regeneration Following Partial and Clear-Cut Harvesting in Mature Aspen-Jack Pine Stands in Eastern Canada. Forests, 11(7), 741. https://doi.org/10.3390/f11070741 Malhi, Y., & Phillips, O. L. (2004). Retrieved Tropical forests and global atmospheric change: A synthesis. Philosophical Transactions of the Royal Society of London. Series B: Biological Sciences, 359(1443), 549–555. https://doi.org/10.1098/rstb.2003.1449 Malimbwi, R., Chidumayo, E., Zahabu, E., Kingazi, S., Misana, S., Luoga, E., & Nduwamungu, J. (2010). Woodfuel. In E. N. Chidumayo & D. J. Gumbo (Eds.), The dry forests and woodlands of Africa: Managing for products and services (pp. 155– 178). London, UK: Earthscan. Mantau, U., Saal, U., Prins, K., Steierer, F., Lindner, M., Verkerk, H., & Anttila, P. (2010). Real potential for changes in growth and use of EU forests. Hamburg: EUwood, [Methodology report]. Hamburg/Germany, June 2010. Retrieved from http://www.unece.org/fileadmin/DAM/timber/meetings/20110321/euwood_final_repo rt.pdf Mantua, N. (2004). Methods for detecting regime shifts in large marine ecosystems: A review with approaches applied to North Pacific data. Progress in Oceanography, 60(2–4), 165–182. https://doi.org/10.1016/j.pocean.2004.02.016 Marcos, C., Torres, I., López-Capel, A., & Pérez-Ruzafa, A. (2015). Long term evolution of fisheries in a coastal lagoon related to changes in lagoon ecology and human pressures. Reviews in Fish Biology and Fisheries, 25(4), 689–713. Scopus. https://doi.org/10.1007/s11160-015-9397-7 Marengo, M., Culioli, J.-M., Santoni, M.-C., Marchand, B., & Durieux, E. D. H. (2015). Comparative analysis of artisanal and recreational fisheries for Dentex dentex in a Marine Protected Area. Fisheries Management and Ecology, 22(3), 249–260. Scopus. https://doi.org/10.1111/fme.12110 Margaryan, L. (2017). Commercialization of nature through tourism (PhD Thesis, Mid Sweden University). Mid Sweden University. Retrieved from https://www.diva- portal.org/smash/record.jsf?pid=diva2%3A1147748&dswid=-2503 Margaryan, L., & Wall-Reinius, S. (2017). Commercializing the unpredictable: Perspectives from wildlife watching tourism entrepreneurs in Sweden. Human Dimensions of Wildlife, 22(5), 406–421. https://doi.org/10.1080/10871209.2017.1334842 Maron, D.F. (2019). This shy Caribbean lizard is now a coveted pet—And critically endangered. How did this happen? ICRF Reptiles & Amphibians, 26(2), 167–169. 435 Maroyi, A. (2013). Use of weeds as traditional vegetables in Shurugwi District, Zimbabwe. Journal of Ethnobiology and Ethnomedicine, 9(1), 60. https://doi.org/10.1186/1746- 4269-9-60 Marselle, M. R., Bowler, D. E., Watzema, J., Eichenberg, D., Kirsten, T., & Bonn, A. (2020). Urban street tree biodiversity and antidepressant prescriptions. Scientific Reports, 10(1), 22445. https://doi.org/10.1038/s41598-020-79924-5 Marsh, S. M. E., Hoffmann, M., Burgess, N. D., Brooks, T. M., Challender, D. W. S., Cremona, P. J., … Böhm, M. (2021). Prevalence of sustainable and unsustainable use of wild species inferred from the IUCN Red List of Threatened Species. Conservation Biology, cobi.13844. https://doi.org/10.1111/cobi.13844 Martin, P. A., Newton, A. C., Pfeifer, M., Khoo, M., & Bullock, J. M. (2015). Impacts of tropical selective logging on carbon storage and tree species richness: A meta-analysis. Retrieved Forest Ecology and Management, 356, 224–233. https://doi.org/10.1016/j.foreco.2015.07.010 Martin, R. O. (2018). The wild bird trade and African parrots: Past, present and future challenges. Ostrich, 89(2), 139–143. https://doi.org/10.2989/00306525.2017.1397787 Martin, R. O., Perrin, M. R., Boyes, R. S., Abebe, Y. D., Annorbah, N. D., Asamoah, A., … Wondafrash, M. (2014). Research and conservation of the larger parrots of Africa and Madagascar: A review of knowledge gaps and opportunities. Ostrich, 85(3), 205–233. https://doi.org/10.2989/00306525.2014.948943 Martínez Carrera, M., D. Morales, P. Pellicer González, E. León, H. Aguilar, A. Ramírez, P. Ortega, P. Largo, A. Bonilla, M. Gómez. (2002). Studies on the traditional management, and processing of matsutake mushrooms In Oaxaca, Mexico. Micología Aplicada International. Retrieved from https://www.redalyc.org/articulo.oa?id=68514203 Martinez-Balleste, A., & Mandujano, M. C. (2013). The Consequences of Harvesting on Regeneration of a Non-timber Wax Producing Species (Euphorbia antisyphilitica Zucc.) of the Chihuahuan Desert. Economic Botany, 67(2), 121–136. https://doi.org/10.1007/s12231-013-9229-4 Martinez-Balleste, A., Martorell, C., & Caballero, J. (2008). The effect of Maya traditional harvesting on the leaf production, and demographic parameters of Sabal palm in the Yucatan Peninsula, Mexico. Forest Ecology and Management, 256(6), 1320–1324. https://doi.org/10.1016/j.foreco.2008.06.029 Martínez-Candelas, I. A., Pérez-Jiménez, J. C., Espinoza-Tenorio, A., McClenachan, L., & Méndez-Loeza, I. (2020). Use of historical data to assess changes in the vulnerability of sharks. Fisheries Research, 226. Scopus. https://doi.org/10.1016/j.fishres.2020.105526 Martini, A. M. Z., Rosa, N. de A., & Uhl, C. (1994). An Attempt to Predict Which Amazonian Tree Species May be Threatened by Logging Activities. Environmental Conservation, 21(2), 152–162. https://doi.org/10.1017/S0376892900024589 Martín-López, B., Gómez-Baggethun, E., García-Llorente, M., & Montes, C. (2014). Trade- offs across value-domains in ecosystem services assessment. Ecological Indicators, 37, 220–228. https://doi.org/10.1016/j.ecolind.2013.03.003 436 Martín-López, B., Iniesta-Arandia, I., García-Llorente, M., Palomo, I., Casado-Arzuaga, I., Del Amo, D. G., … others. (2012). Uncovering ecosystem service bundles through social preferences. PLoS One, 7(6), e38970. https://doi.org/10.1371/journal.pone.0038970 Martins, A. P. B., Feitosa, L. M., Lessa, R. P., Almeida, Z. S., Heupel, M., Silva, W. M., … Nunes, J. L. S. (2018). Analysis of the supply chain and conservation status of sharks (Elasmobranchii: Superorder Selachimorpha) based on fisher knowledge. PLoS ONE, 13(3). Scopus. https://doi.org/10.1371/journal.pone.0193969 Martins, I. M., Medeiros, R. P., Di Domenico, M., & Hanazaki, N. (2018). What fishers’ local ecological knowledge can reveal about the changes in exploited fish catches. Fisheries Research, 198, 109–116. Scopus. https://doi.org/10.1016/j.fishres.2017.10.008 Masera, O. R., Bailis, R., Drigo, R., Ghilardi, A., & Ruiz-Mercado, I. (2015). Environmental burden of traditional bioenergy use. Annual Review of Environment and Resources, 40, 121–150. Retrieved https://doi.org/10.1146/annurev-environ-102014-021318 Masters, S., van Andel, T., de Boer, H. J., Heijungs, R., & Gravendeel, B. (2020). Patent analysis as a novel method for exploring commercial interest in wild harvested species. Biological Conservation, 243. https://doi.org/UNSP 108454 10.1016/j.biocon.2020.108454 Matias, D. M. S., Borgemeister, C., & von Wehrden, H. (2018). Ecological changes and local knowledge in a giant honey bee (Apis dorsata F.) hunting community in Palawan, Philippines. Ambio, 47(8), 924–934. https://doi.org/10.1007/s13280-018-1038-7 Matose, F. (2006). Access mapping and chains: The woodcraft curio market around Victoria Falls, Zimbabwe. In Survival of the Commons: Mounting Challenges and New Realities, the 11th Conference of the International Association for the Study of Common Property, 16. Bali, Indonesia. Matsika, R., Erasmus, B. F. N. N., & Twine, W. C. (2012). A tale of two villages: Assessing the dynamics of fuelwood supply in communal landscapes in South Africa. Environmental Conservation, 40(01), 71–83. https://doi.org/10.1017/S0376892912000264 Matsuda, H., Makino, M., & Sakurai, Y. (2009). Development of an adaptive marine ecosystem management and co-management plan at the Shiretoko World Natural Heritage Site. Biological Conservation, 142(9), 1937–1942. Scopus. https://doi.org/10.1016/j.biocon.2009.03.017 Mattson, N. S. (2006). Conservation and enhancement of fisheries: The case of the Lower Mekong Basin. International Journal of Ecology and Environmental Sciences, 32(1), 109–117. Scopus. Retrieved from Scopus. Mattsson, L. (2008). Jakten i Sverige: Ekonomiska värden och attityder jaktåret 2005/06. Adaptiv förvaltning av vilt och fisk, Sveriges lantbruksuniversitet. Mausel, D. L., Waupochick, A., & Pecore, M. (2017). Menominee Forestry: Past, Present, Future. Journal of Forestry, 115(5), 366–369. https://doi.org/10.5849/jof.16-046 Mavruk, S., Saygu, İ., Bengil, F., Alan, V., & Azzurro, E. (2018). Grouper fishery in the Northeastern Mediterranean: An assessment based on interviews on resource users. Marine Policy, 87, 141–148. Scopus. https://doi.org/10.1016/j.marpol.2017.10.018 437 Maxwell, S. L., Fuller, R. A., Brooks, T. M., & Watson, J. E. M. (2016). Biodiversity: The ravages of guns, nets and bulldozers. Nature News, 536(7615), 143. https://doi.org/10.1038/536143a Maya, E. M. A., & Gómez, B. (2016). Insects and other invertebrates in the Pjiekakjoo (Tlahuica) culture in Mexico State, Mexico. Journal of Insects as Food and Feed, 2(1), 43–52. https://doi.org/10.3920/jiff2015.0090 Mayer, A. L., Pawlowski, C. W., & Cabezas, H. (2006). Fisher Information and dynamic regime changes in ecological systems. Ecological Modelling, 195(1–2), 72–82. https://doi.org/10.1016/j.ecolmodel.2005.11.011 Mayfield, S., Mundy, C., Gorfine, H., Hart, A. M., & Worthington, D. (2012). Fifty years of sustained production from the Australian abalone fisheries. Reviews in Fisheries Science, 20(4), 220–250. Scopus. https://doi.org/10.1080/10641262.2012.725434 Maynou, F., Martínez-Baños, P., Demestre, M., & Franquesa, R. Retrieved (2014). Bio-economic analysis of the Mar Menor (Murcia, SE Spain) small-scale lagoon fishery. Journal of Applied Ichthyology, 30(5), 978–985. Scopus. https://doi.org/10.1111/jai.12460 Maynou, F., Morales-Nin, B., Cabanellas-Reboredo, M., Palmer, M., García, E., & Grau, A. M. (2013). Small-scale fishery in the Balearic Islands (W Mediterranean): A socio- economic approach. Fisheries Research, 139, 11–17. Scopus. https://doi.org/10.1016/j.fishres.2012.11.006 Maynou, Francesc, Sbrana, M., Sartor, P., Maravelias, C., Kavadas, S., Damalas, D., … Osio, G. (2011). Estimating trends of population decline in long-lived marine species in the Mediterranean Sea based on fishers’ perceptions. PLoS One, 6(7), e21818. Mazor, T., Pitcher, C. R., Rochester, W., Kaiser, M. J., Hiddink, J. G., Jennings, S., … Hilborn, R. (2021). Trawl fishing impacts on the status of seabed fauna in diverse regions of the globe. Fish and Fisheries, 22(1), 72–86. https://doi.org/10.1111/faf.12506 Mazumder, S. K., Das, S. K., Ghaffar, M. A., Rahman, M. H., Majumder, M. K., & Basak, L. R. (2016). Role of co-management in wetland productivity: A case study from Hail haor in Bangladesh. AACL Bioflux, 9(3), 466–482. Scopus. Retrieved from Scopus. Mbaiwa, J. E. (2018). Effects of the safari hunting tourism ban on rural livelihoods and wildlife conservation in Northern Botswana. South African Geographical Journal, 100(1), 41– 61. https://doi.org/10.1080/03736245.2017.1299639 Mbaiwa, J. E., & Stronza, A. L. (2010). The effects of tourism development on rural livelihoods in the Okavango Delta, Botswana. Journal of Sustainable Tourism, 18(5), 635–656. https://doi.org/10.1080/09669581003653500 Mbata, K. J., Chidumayo, E. N., & Lwatula, C. M. (2002). Traditional regulation of edible caterpillar exploitation in the Kopa area of Mpika district in northern Zambia. Journal of Insect Conservation, 6(2), 115–130. https://doi.org/Doi 10.1023/A:1020953030648 McCafferty, J. R., Ellender, B. R., Weyl, O. L. F., & Britz, P. J. (2012). The use of water resources for inland fisheries in South Africa. Water SA, 38(2), 327–344. Scopus. https://doi.org/10.4314/wsa.v38i2.18 Mccarthy, A., Hepburn, C., Scott, N., Schweikert, K., Turner, R., & Moller, H. (2014). Local people see and care most? Severe depletion of inshore fisheries and its consequences for Māori communities in New Zealand. Aquatic Conservation: Marine and Freshwater Ecosystems, 24(3), 369–390. Scopus. https://doi.org/10.1002/aqc.2378 438 McCarthy, J. F. (2002). Power and Interest on Sumatra’s Rainforest Frontier: Clientelist Coalitions, Illegal Logging and Conservation in the Alas Valley. Journal of Southeast Asian Studies, 33(1), 77–106. https://doi.org/10.1017/S0022463402000048 McCauley, D. J., Jablonicky, C., Allison, E. H., Golden, C. D., Joyce, F. H., Mayorga, J., & Kroodsma, D. (2018). Wealthy countries dominate industrial fishing. Science Advances, 4(8), eaau2161. Retrieved https://doi.org/10.1126/sciadv.aau2161 McCleery, R. A., Fletcher, R. J., Kruger, L. M., Govender, D., & Ferreira, S. M. (2020). Conservation needs a COVID-19 bailout. Science, 369(6503), 515–516. https://doi.org/10.1126/science.abd2854 Mcclenachan, L., & Kittinger, J. N. (2013). Multicentury trends and the sustainability of coral reef fisheries in Hawai’i and Florida. Fish and Fisheries, 14(3), 239–255. Scopus. https://doi.org/10.1111/j.1467-2979.2012.00465.x McConnaughey, R. A., Hiddink, J. G., Jennings, S., Pitcher, C. R., Kaiser, M. J., Suuronen, P., … Hilborn, R. (2020). Choosing best practices for managing impacts of trawl fishing on seabed habitats and biota. https://doi.org/10.1111/faf.12431 McEwan, A., Marchi, E., Spinelli, R., & Brink, M. (2020). Past, present and future of industrial plantation forestry and implication on future timber harvesting technology. Journal of Forestry Research, 31(2), 339–351. https://doi.org/10.1007/s11676-019-01019-3 McIlveen, K., & Rhodes, M. (2016). Community forestry in an age of crisis: Structural change, the mountain pine beetle, and the evolution of the Burns Lake community forest. In Community forestry: Lessons from policy and practice (pp. 179–209). Vancouver, BC: UBC Press. McLain, R. J., MacFarland, K., Brody, L., Hebert, J., Hurley, P., Poe, M., … Charnley, S. (2012). Gathering in the city: An annotated bibliography and review of the literature about human-plant interactions in urban ecosystems (No. PNW-GTR-849; p. PNW- GTR-849). Portland, OR: U.S. Department of Agriculture, Forest Service, Pacific Northwest Research Station. McLain, R. J., Poe, M. R., Urgenson, L. S., Blahna, D. J., & Buttolph, L. P. (2017). Urban non- timber forest products stewardship practices among foragers in Seattle, washington (USA). Urban Forestry & Urban Greening, 28, 36–42. https://doi.org/10.1016/j.ufug.2017.10.005 McLain, Rebecca J. (2008). Constructing a Wild Mushroom Panopticon: The Extension of Nation-State Control over the Forest Understory in Oregon, USA. Economic Botany, 62(3), 343–355. https://doi.org/10.1007/s12231-008-9025-8 McLain, Rebecca J, Hurley, P. T., Emery, M. R., & Poe, M. R. (2014). Gathering “wild” food in the city: Rethinking the role of foraging in urban ecosystem planning and management. Local Environment, 19(2), 220–240. https://doi.org/10.1080/13549839.2013.841659 McLaughlin, S. B., de la Torre Ugarte, D. G., Garten, C. T., Lynd, L. R., Sanderson, M. A., Tolbert, V. R., & Wolf, D. D. (2002). High-Value Renewable Energy from Prairie Grasses. Environmental Science & Technology, 36(10), 2122–2129. https://doi.org/10.1021/es010963d 439 Mclean, E. L., & Forrester, G. E. (2018). Comparing fishers’ and scientific estimates of size at maturity and maximum body size as indicators for overfishing. Ecological Applications, 28(3), 668–680. Scopus. https://doi.org/10.1002/eap.1675 McNicol, I. M., Ryan, C. M., & Mitchard, E. T. A. (2018). Retrieved Carbon losses from deforestation and widespread degradation offset by extensive growth in African woodlands. Nature Communications, 9(1), 3045. https://doi.org/10.1038/s41467-018-05386-z McRae, L., Freeman, R., Geldmann, J., Moss, G. B., Kjær-Hansen, L., & Burgess, N. D. (2022). A global indicator of utilized wildlife populations: Regional trends and the impact of management. One Earth, 5(4), 422–433. https://doi.org/10.1101/2020.11.02.365031 McShane, T. O., Hirsch, P. D., Trung, T. C., Songorwa, A. N., Kinzig, A., Monteferri, B., … O’Connor, S. (2011). Hard choices: Making trade-offs between biodiversity conservation and human well-being. Biological Conservation, 144(3), 966–972. https://doi.org/10.1016/j.biocon.2010.04.038 Medina, G., Pokorny, B., & Campbell, B. (2009). Loggers, Development Agents and the Exercise of Power in Amazonia. Development and Change, 40(4), 745–767. https://doi.org/10.1111/j.1467-7660.2009.01570.x Medjibe, V., & Hall, J. S. (2002). Seed dispersal and its implications for silviculture of African mahogany (Entandrophragma spp.) in undisturbed forest in the Central African Republic. Forest Ecology and Management, 170(1–3), 249–257. https://doi.org/10.1016/S0378-1127(01)00769-1 Meissa, B., Gascuel, D., & Rivot, E. (2013). Assessing stocks in data-poor African fisheries: A case study on the white grouper Epinephelus aeneus of Mauritania. African Journal of Marine Science, 35(2), 253–267. Scopus. https://doi.org/10.2989/1814232X.2013.798244 Mejia, E., Pacheco, P., Muzo, A., & Torres, B. (2015). Smallholders and timber extraction in the Ecuadorian Amazon: Amidst market opportunities and regulatory constraints. International Forestry Review, 17(1), 38–50. https://doi.org/10.1505/146554815814668954 Melnychuk, M. C., Peterson, E., Elliott, M., & Hilborn, R. (2017). Fisheries management impacts on target species status. Proceedings of the National Academy of Sciences, 114(1), 178–183. https://doi.org/10.1073/pnas.1609915114 Menchaca García, R. A., Lozano Rodríguez, M. Á., & Sánchez Morales, L. (2012). Strategies for the sustainable harvesting of Mexican orchids. Revista Mexicana de Ciencias Forestales, 3(13), 09–16. Mensah, J. T., & Elofsson, K. (2017). An Empirical Analysis of Hunting Lease Pricing and Value of Game in Sweden. Land Economics, 93(2), 292–308. Mera Ovando, L. M., Castro Lara, D., & Bye Boettler, R. A. (2011). Especies vegetales poco valoradas: Una alternativa para la seguridad alimentaria. Mesa, L., & Galeano, G. (2013). Palms uses in the Colombian Amazon. Caldasia, 35, 351– 369. Mesnildrey, L., Jacob, C., Frangoudes, K., Reunavot, M., & Lesueur, M. (2012). La filière des macro-algues en France. Rapport d’étude. Netalgae. (Vol. 9). Rennes: Les publications du Pôle halieutique Agrocampus Ouest. 440 Mesquita, E. M. C., Cruz, R. E. A., Hallwass, G., & Isaac, V. J. (2019). Fishery parameters and population dynamics of silver croaker on the Xingu river, Brazilian Amazon. Boletim Do Instituto de Pesca, 45(2), e.423. Retrieved https://doi.org/10.20950/1678-2305.2019.45.2.423 Methorst, J., Rehdanz, K., Mueller, T., Hansjürgens, B., Bonn, A., & Böhning-Gaese, K. (2021). The importance of species diversity for human well-being in Europe. Ecological Economics, 181, 106917. https://doi.org/10.1016/j.ecolecon.2020.106917 Meyfroidt, P., Rudel, T. K., & Lambin, E. F. (2010). Forest transitions, trade, and the global displacement of land use. Proceedings of the National Academy of Sciences, 107(49), 20917–20922. https://doi.org/10.1073/pnas.1014773107 Meza-Arce, M. I., Malpica-Cruz, L., Hoyos-Padilla, M. E., Mojica, F. J., Arredondo-García, M. C., Leyva, C., … Santana-Morales, O. (2020). Unraveling the white shark observation tourism at Guadalupe Island, Mexico: Actors, needs and sustainability. Marine Policy, 119, 104056. https://doi.org/10.1016/j.marpol.2020.104056 Mgana, H., Kraemer, B. M., O’Reilly, C. M., Staehr, P. A., Kimirei, I. A., Apse, C., … McIntyre, P. B. (2019). Adoption and consequences of new light-fishing technology (LEDs) on Lake Tanganyika, East Africa. PLoS ONE, 14(10). Scopus. https://doi.org/10.1371/journal.pone.0216580 Mgumia, F. H., & Oba, G. (2003). Potential role of sacred groves in biodiversity conservation in Tanzania. Environmental Conservation, 30(3), 259–265. https://doi.org/10.1017/S0376892903000250 Miah, M. D., Al Rashid, H., & Shin, M. Y. (2009). Wood fuel use in the traditional cooking stoves in the rural floodplain areas of Bangladesh: A socio-environmental perspective. Biomass and Bioenergy, 33(1), 70–78. Milbrandt, A., & Overend, R. (2011). Assessment of biomass resources in Afghanistan. No. National Renewable Energy Lab.(NREL), Golden, CO (United States), 2011. (Technical Report No. NREL/TP-6A20-49358.). National Renewable Energy Laboratory. Milenge Kamalebo, H., Nshimba Seya Wa Malale, H., Masumbuko Ndabaga, C., Degreef, J., & De Kesel, A. (2018). Uses and importance of wild fungi: Traditional knowledge from the Tshopo province in the Democratic Republic of the Congo. Journal of Ethnobiology and Ethnomedicine, 14(1), 13. https://doi.org/10.1186/s13002-017-0203-6 Militz, T. A., Kinch, J., Foale, S., & Southgate, P. C. (2016). Fish rejections in the marine aquarium trade: An initial case study raises concern for village-based fisheries. PLoS One, 11(3), e0151624. Millar, J., Robinson, W., Baumgartner, L., Homsombath, K., Chittavong, M., Phommavong, T., & Singhanouvong, D. (2019). Local perceptions of changes in the use and management of floodplain fisheries commons: The case of Pak Peung wetland in Lao PDR. Environment, Development and Sustainability, 21(4), 1835–1852. Scopus. https://doi.org/10.1007/s10668-018-0105-3 Millennium Ecosystem Assessment. (2005). Ecosystems and Human Well-Being: Synthesis. Washington DC: Island Press. Retrieved from http://www.who.int/entity/globalchange/ecosystems/ecosys.pdf 441 Miller, D. C., Mansourian, S., & Wildburger, C. (2020). Forests, Trees and the Eradication of Poverty: Potential and Limitations. A Global Assessment Report [A Global Assessment Report]. Vienna.: International Union of Forest Research Organizations (IUFRO). Retrieved Mills Busa, J. H. (2013). Deforestation beyond borders: Addressing the disparity between production and consumption of global resources: Deforestation beyond borders. Conservation Letters, 6(3), 192–199. https://doi.org/10.1111/j.1755- 263X.2012.00304.x Milner, J. M., Nilsen, E. B., & Andreassen, H. P. (2007). Demographic side effects of selective hunting in ungulates and carnivores. Conservation Biology: The Journal of the Society for Conservation Biology, 21(1), 36–47. https://doi.org/10.1111/j.1523- 1739.2006.00591.x Milner-Gulland, E. J., & Bennett, E. L. (2003). Wild meat: The bigger picture. Trends in Ecology & Evolution, 18(7), 351–357. https://doi.org/10.1016/S0169-5347(03)00123- X Mingaila, J., Čiuldienė, D., Viškelis, P., Bartkevičius, E., Vilimas, V., & Armolaitis, K. (2020). The Quantity and Biochemical Composition of Sap Collected from Silver Birch (Betula pendula Roth) Trees Growing in Different Soils. Forests, 11(4), 365. https://doi.org/10.3390/f11040365 Ministère des Ressources naturelles et de la Faune. (2011). Proposals for the selection , establishment and operation of local forests. Consultation paper. Quebec. Retrieved from https://mffp.gouv.qc.ca/forets/gestion/pdf/document-consultation-proximite- ang.pdf Ministry of Ecology and Environment of the People’s Republic of China, & Chinese Academy of Sciences. (2018). 中国生物多样性红色名录—大型真菌卷—China’s Red List of Biodiversity—MacroFungal Assessment Report. Retrieved from http://www.mee.gov.cn/xxgk2018/xxgk/xxgk01/201805/W020180926382629921552. pdf Minteer, B. A., Collins, J. P., Love, K. E., & Puschendorf, R. (2014). Avoiding (Re)extinction. Science, 344(6181), 260–261. https://doi.org/10.1126/science.1250953 Mintzer, V. J., Schmink, M., Lorenzen, K., Frazer, T. K., Martin, A. R., & da Silva, V. M. F. (2015). Attitudes and behaviors toward Amazon River dolphins (Inia geoffrensis) in a sustainable use protected area. Biodiversity and Conservation, 24(2), 247–269. https://doi.org/10.1007/s10531-014-0805-4 Mirera, D. O., Ochiewo, J., Munyi, F., & Muriuki, T. (2013). Heredity or traditional knowledge: Fishing tactics and dynamics of artisanal mangrove crab (Scylla serrata) fishery. Ocean and Coastal Management, 84, 119–129. Scopus. https://doi.org/10.1016/j.ocecoaman.2013.08.002 Misra, S., Maikhuri, R., Kala, C., Rao, K., & Saxena, K. (2008). Wild leafy vegetables: A study of their subsistence dietetic support to the inhabitants of Nanda Devi Biosphere Reserve, India. J Ethnobiology Ethnomedicine, 4(1), 15. https://doi.org/10.1186/1746- 4269-4-15 442 Misund, B., Oglend, A., & Pincinato, R. B. M. (2017). The rise of fish oil: From feed to human nutritional supplement. Aquaculture Economics & Management, 21(2), 185–210. https://doi.org/10.1080/13657305.2017.1284942 Mittelman, A. J., Lai, C. K., Byron, N., Michon, G., & Katz, E. (1997). Non-wood forest products outlook study for Asia and the Pacific: Towards 2010. Rome/Bangkok: FAO. Forest Policy and Planning Division / Regional Office for Asia and the Pacific. Retrieved from https://www.fao.org/publications/card/fr/c/e041130d-dd94-5502- 8b16-317e11afb26c Miyake, M., Guillotreau, P., & Sun, C. (2010). Recent Developments in the Tuna Industry. Retrieved Stocks, Fisheries, Management, Processing, Trade and Markets. Food and Agriculture Organization of the United Nations. Mkono, M. (2019). Neo-colonialism and greed: Africans’ views on trophy hunting in social media. Journal of Sustainable Tourism, 27(5), 689–704. https://doi.org/10.1080/09669582.2019.1604719 Mkuna, E., & Baiyegunhi, L. J. S. (2019a). Analysis of the technical efficiency of Nile perch (Lates niloticus) fishers in the Tanzanian portion of Lake Victoria: A stochastic frontier analysis. Lakes and Reservoirs: Research and Management, 24(3), 228–238. Scopus. https://doi.org/10.1111/lre.12274 Mkuna, E., & Baiyegunhi, L. J. S. (2019b). Determinants of Nile perch (Lates niloticus) overfishing and its intensity in Lake Victoria, Tanzania: A double-hurdle model approach. Hydrobiologia. Scopus. https://doi.org/10.1007/s10750-019-3932-9 Mlcek, J., Rop, O., Borkovcova, M., & Bednarova, M. (2014). A Comprehensive Look at the Possibilities of Edible Insects as Food in Europe – a Review. Polish Journal of Food and Nutrition Sciences, 64(3), 147–157. https://doi.org/10.2478/v10222-012-0099-8 Moad, A. S., & Whitmore, J. L. (1994). Tropical Forest Management in the Asia-Pacific Region. Journal of Sustainable Forestry, 1(4), 25–63. https://doi.org/10.1300/J091v01n04_02 MoAF. (2018). Statistics on Community Forests (CFs) as of December 2017. Thimpu, Bhutan: Ministry of Agriculture and Forests. Mograbi, P. J., Erasmus, B. F., Witkowski, E., Asner, G. P., Wessels, K. J., Mathieu, R., … Main, R. (2015). Biomass increases go under cover: Woody vegetation dynamics in South African rangelands. PLoS One, 10(5), e0127093. https://doi.org/10.1371/journal.pone.0127093 Mograbi, P. J., Witkowski, E. T., Erasmus, B. F., Asner, G. P., Fisher, J. T., Mathieu, R., & Wessels, K. J. (2019). Fuelwood extraction intensity drives compensatory regrowth in African savanna communal lands. Land Degradation & Development, 30(2), 190–201. https://doi.org/10.1002/ldr.3210 Mohanty, N. P., & Measey, J. (2019). The global pet trade in amphibians: Species traits, taxonomic bias, and future directions. Biodiversity and Conservation, 28(14), 3915– 3923. https://doi.org/10.1007/s10531-019-01857-x Mohapatra, R., Panda, S., Nair, M., Acharjyo, L., & Challender, D. (2015). A note on the illegal trade and use of pangolin body parts in India. TRAFFIC Bulletin, 27. Mohneke, M. (2011). (Un)sustainable use of frogs in West Africa and resulting consequences for the ecosystem. 443 Mohneke, M., Onadeko, A. B., & Rödel, M.-O. (2009). Exploitation of frogs – a review with a focus on West Africa. 10. Mohneke, M., Onadeko, A., Petersen, M., & Rödel, M.-O. (2010). Dried or fried: Amphibians in Local and Regional Food Markets in West Africa. Traffic, 22, 69–80. Mohr, C. H., Coppus, R., Iroumé, A., Huber, A., & Bronstert, A. (2013). Retrieved Runoff generation and soil erosion processes after clear cutting. Journal of Geophysical Research: Earth Surface, 118(2), 814–831. https://doi.org/10.1002/jgrf.20047 Mollee, E., Pouliot, M., & McDonald, M. A. (2017). Into the urban wild: Collection of wild urban plants for food and medicine in Kampala, Uganda. Land Use Policy, 63, 67–77. https://doi.org/10.1016/j.landusepol.2017.01.020 Moloney, P., & Turnbull, J. D. (2012). Estimates of harvest for deer, duck and quail in Victoria: Results from surveys of Victorian Game Licence holders in 2012. Mondragón Chaparro, D., & Ticktin, T. (2011). Demographic Effects of Harvesting Epiphytic Bromeliads and an Alternative Approach to Collection: Use and Conservation of Epiphytic Bromeliads. Conservation Biology, 25(4), 797–807. https://doi.org/10.1111/j.1523-1739.2011.01691.x Mondragón, D., Méndez-García, E. del, & Morillo, I. (2016). Prioritizing the Conservation of Epiphytic Bromeliads Using Ethnobotanical Information from a Traditional Mexican Market. Economic Botany, 70(1), 29–36. https://doi.org/10.1007/s12231-016-9332-4 Monteiro, F. T., Fávero, C., Costa Filho, A., Oliveira, M. N. S., Soldati, G. T., & Duque-Brasil, R. (2019). Sistema agrícola tradicional da Serra do Espinhaço Meridional, MG: transumância, biodiversidade e cultura nas paisagens manejadas pelos(as) apanhadores(as) de flores sempre-vivas. In Povos e comunidades tradicionais: Vol. 3. Sistemas agrícolas tradicionais no Brasil (Simoni Eidt J., Udry C. (ed)., pp. 93–140). Brasilia: Embrapa. Retrieved from https://www.embrapa.br/busca-de-publicacoes/- /publicacao/1109452/sistemas-agricolas-tradicionais-no-brasil Monteiro-Neto, C., Cunha, F. E. D. A., Nottingham, M. C., Araújo, M. E., Rosa, I. L., & Barros, G. M. L. (2003). Analysis of the marine ornamental fish trade at Ceará State, northeast Brazil. Biodiversity & Conservation, 12(6), 1287–1295. Montgomery, R. A., Borona, K., Kasozi, H., Mudumba, T., & Ogada, M. (2020). Positioning human heritage at the center of conservation practice. Conservation Biology, 34(5), 1122–1130. https://doi.org/10.1111/cobi.13483 Monticini, P. (2010). The ornamental fish trade: Production and commerce of ornamental fish: Technical-managerial and legislative aspects. Montoya, A., Hernández, N., Mapes, C., Kong, A., & Estrada–Torres, A. (2008). The Collection and Sale of Wild Mushrooms in a Community of Tlaxcala, Mexico. Economic Botany, 62(3), 413–424. https://doi.org/10.1007/s12231-008-9021-z Montufar, R., & Pintaud, J.-C. (2006). Variation in species composition, abundance and microhabitat preferences among western Amazonian terra firme palm communities. Botanical Journal of the Linnean Society, 151(1), 127–140. https://doi.org/10.1111/j.1095-8339.2006.00528.x Moore, M., Gould, P., & Keary, B. S. (2003). Global urbanization and impact on health. International Journal of Hygiene and Environmental Health, 206(4–5), 269–278. https://doi.org/10.1078/1438-4639-00223 444 Morales-Nin, B., Grau, A. M., Aguilar, J. S., Del Mar Gil, M., & Pastor, E. (2017). Balearic Islands boat seine fisheries: The transparent goby fishery an example of co- management. Retrieved ICES Journal of Marine Science, 74(7), 2053–2058. Scopus. https://doi.org/10.1093/icesjms/fsw227 Moreau, M.-A., & Coomes, O. T. (2007). Aquarium fish exploitation in western Amazonia: Conservation issues in Peru. Environmental Conservation, 34(1), 12–22. https://doi.org/10.1017/S0376892907003566 Morellet, N., Gaillard, J.-M., Hewison, A. J. M., Ballon, P., Boscardin, Y., Duncan, P., … Maillard, D. (2007). Indicators of ecological change: New tools for managing populations of large herbivores: Ecological indicators for large herbivore management. Journal of Applied Ecology, 44(3), 634–643. https://doi.org/10.1111/j.1365- 2664.2007.01307.x Moreno Fuentes, Á. (2014). Un recurso alimentario de los grupos originarios y mestizos de méxico: Los hongos silvestres. Anales de Antropología, 48(1), 241–272. https://doi.org/10.1016/S0185-1225(14)70496-5 Morton, O., Scheffers, B. R., Haugaasen, T., & Edwards, D. P. (2021). Impacts of wildlife trade on terrestrial biodiversity. Nature Ecology & Evolution. https://doi.org/10.1038/s41559-021-01399-y Moss, T., Voigt, F., & Becker, S. (2021). Digital urban nature: Probing a void in the smart city discourse. City, 25(3–4), 255–276. https://doi.org/10.1080/13604813.2021.1935513 Moswete, N., Thapa, B., & Lacey, G. (2009). Village-based tourism and community participation: A case study of the Matsheng villages in southwest Botswana. In J. Saarinen (Ed.), Sustainable tourism in Southern Africa: Local communities and natural resources in transition (pp. 189–209). Bristol, U.K.: Channel view publications. Moussaoui, L., Leduc, A., Fenton, N. J., Lafleur, B., & Bergeron, Y. (2019). Changes in forest structure along a chronosequence in the black spruce boreal forest: Identifying structures to be reproduced through silvicultural practices. Ecological Indicators, 97, 89–99. https://doi.org/10.1016/j.ecolind.2018.09.059 Mowforth, M., & Munt, I. (2015). Tourism and sustainability: Development, globalisation and new tourism in the third world. routledge. MSC. (2021). History of the MSC \| Marine Stewardship Council. Retrieved April 2, 2021, from https://www.msc.org/about-the-msc/our-history Muallil, R. N., Mamauag, S. S., Cababaro, J. T., Arceo, H. O., & Aliño, P. M. (2014). Catch trends in Philippine small-scale fisheries over the last five decades: The fishers perspectives. Marine Policy, 47, 110–117. Scopus. https://doi.org/10.1016/j.marpol.2014.02.008 Muallil, R. N., Mamauag, S. S., Cabral, R. B., Celeste-Dizon, E. O., & Aliño, P. M. (2014). Status, trends and challenges in the sustainability of small-scale fisheries in the Philippines: Insights from FISHDA (Fishing Industries’ Support in Handling Decisions Application) model. Marine Policy, 44, 212–221. Scopus. https://doi.org/10.1016/j.marpol.2013.08.026 Mugah, J. O., Chikamai, B. N., Mbiru, S. S., & Casadei, E. (1997). Conservation, management and utilization of plant gums, resins and essential oils. Proccedings of a regional 445 conference for Africa held in Nairobi, Kenya (6-10/10/97). Rome/Nairobi: FAO, Forestry Department, Forest Products Division/ KEFRI/ TWAS/ AIDGUM/ GTZ. Retrieved conference for Africa held in Nairobi, Kenya (6-10/10/97). Rome/Nairobi: FAO, Forestry Department, Forest Products Division/ KEFRI/ TWAS/ AIDGUM/ GTZ. Munadi, E. (2017). Furnitur, Produk Berdaya Saing Yang Butuh Perhatian. In Info Komoditi Furniture. Jakarta: Indonesia: Kementerian Perdagangan. Retrieved from http://bppp.kemendag.go.id/media_content/2017/10/Isi_BRIK_FURNITUR.pdf Munalula, F., & Meincken, M. (2009). An evaluation of South African fuelwood with regards to calorific value and environmental impact. Biomass and Bioenergy, 33(3), 415–420. Munn, I. A., Hussain, A., Spurlock, S., & Henderson, J. E. (2010). Economic Impact of Fishing, Hunting, and Wildlife-Associated Recreation Expenditures on the Southeast U.S. Regional Economy: An Input–Output Analysis. Human Dimensions of Wildlife, 15(6), 433–449. Readcube. https://doi.org/10.1080/10871209.2010.508193 Munro, G. R. (2000). The United Nations Fish Stocks Agreement of 1995: History and problems of implementation. Marine Resource Economics, 15(4), 265–280. Munro, P., van der Horst, G., & Healy, S. (2017). Energy justice for all? Rethinking sustainable development goal 7 through struggles over traditional energy practices in Sierra Leone. Energy Policy, 105, 635–641. Muoneke, M. I., & Childress, W. M. (1994). Hooking mortality: A review for recreational fisheries. Reviews in Fisheries Science, 2(2), 123–156. https://doi.org/10.1080/10641269409388555 Murphy, D. M. A., Berazneva, J., & Lee, D. R. (2018). Fuelwood source substitution, gender, and shadow prices in western Kenya. Environment and Development Economics, 23(6), 655–678. https://doi.org/10.1017/S1355770X1800027X Murray, G., Neis, B., Palmer, C. T., & Schneider, D. C. (2008). Mapping cod: Fisheries science, fish harvesters’ ecological knowledge and cod migrations in the Northern Gulf of St. Lawrence. Human Ecology, 36(4), 581–598. Scopus. https://doi.org/10.1007/s10745-008-9178-1 Murray, G., Neis, B., & Schneider, D. C. (2008). Lessons from a multi-scale historical reconstruction of newfoundland and labrador fisheries. Coastal Management, 36(1), 81–108. Scopus. https://doi.org/10.1080/08920750701682056 Musembi, P., Fulanda, B., Kairo, J., & Githaiga, M. (2019). Species composition, abundance and fishing methods of small-scale fisheries in the seagrass meadows of Gazi Bay, Kenya. Journal of the Indian Ocean Region, 15(2), 139–156. Scopus. https://doi.org/10.1080/19480881.2019.1603608 Musick, J. A. (Ed.). (1999). Life in the slow lane: Ecology and conservation of long-lived marine animals. Bethesda, Md: American Fisheries Society. Mustika, P. L. K., Birtles, A., Welters, R., & Marsh, H. (2012). The economic influence of community-based dolphin watching on a local economy in a developing country: Implications for conservation. Ecological Economics, 79, 11–20. https://doi.org/10.1016/j.ecolecon.2012.04.018 Mustin, K., Arroyo, B., Beja, P., Newey, S., Irivine, R. J., Kestler, J., & Redpath, S. M. (2018). Consequences of game bird management for non-game species in Europe. Journal of Applied Ecology, 55(5), 2285–2295. Retrieved https://doi.org/10.1111/1365-2664.13131 446 Mustin, K., Newey, S., Irvine, J., Arroyo, B., & Redpath, S. (2012). Biodiversity impacts of game bird hunting and associated management practices in Europe and North America. 72. Muzuka, N., Bwire, K.M., Shalli, M., Kyewalyanga, M., Jacob, G.M., Ibengwe, L. (2011). Enhancement of Adaptation Strategies of Coastal Communities Dependent on Coastal Panaeid Shrimps Fisheries to Impacts of Climate Change and Variability in Coast Region, Tanzania. Mwangi, E., & Mai, Y. H. (2011). Introduction to the Special Issue on Forests and Gender. International Forestry Review, 13(2), 119–122. https://doi.org/10.1505/146554811797406561 Mwangi, E., Meinzen-Dick, R., & Sun, Y. (2011). Gender and sustainable forest management in East Africa and Latin America. Ecology and Society, 16(1). https://doi.org/10.5751/ES-03873-160117 Mweetwa, T., Christianson, D., Becker, M., Creel, S., Rosenblatt, E., Merkle, J., … Simpamba, T. (2018). Quantifying lion (Panthera leo) demographic response following a three-year moratorium on trophy hunting. PLoS ONE, 13(5), e0197030–e0197030. Myers, R. A., & Worm, B. (2005). Extinction, survival or recovery of large predatory fishes. Philosophical Transactions of the Royal Society B: Biological Sciences, 360(1453), 13– 20. https://doi.org/10.1098/rstb.2004.1573 Nabhan, G. P., Orlando, L., Smith Monti, L., & Aronson, J. (2020). Hands-On Ecological Restoration as a Nature-Based Health Intervention: Reciprocal Restoration for People and Ecosystems. Ecopsychology, 12(3), 195–202. https://doi.org/10.1089/eco.2020.0003 NACSO. (2015). Scary wildlife count in North East Namibia. Retrieved from http://www.nacso.org.na/news/2015/10/scary-wildlife-count-in-north-east-namibia NACSO. (2019). KEEP NAMIBIA’S WILDLIFE ON THE LAND! Retrieved from http://www.nacso.org.na/sites/default/files/2019_Wildlife-on-the- land_rgb_F_201207s.pdf NACSO, & MET. (2018). The State of Community Conservation in Namibia—A review of communal conservancies, community forests and other CBNRM initiatives. Windhoek. Naegel, A. (2004). Plicopurpura pansa (Gould, 1853) from the Pacific coast of Mexico and Central America: A traditional source of Tyrian purple. Journal of Shellfish Research, 23, 211–214. Nagendra, H., Pareeth, S., Sharma, B., Schweik, C. M., & Adhikari, K. R. (2008). Forest fragmentation and regrowth in an institutional mosaic of community, government and private ownership in Nepal. Landscape Ecology, 23(1), 41–54. https://doi.org/10.1007/s10980-007-9162-y Naidoo, R., & Burton, A. C. (2020). Relative effects of recreational activities on a temperate terrestrial wildlife assemblage. Conservation Science and Practice, 2(10). https://doi.org/10.1111/csp2.271 Naidoo, R., Fisher, B., Manica, A., & Balmford, A. (2016). Estimating economic losses to tourism in Africa from the illegal killing of elephants. Nature Communications, 7(1), 13379. https://doi.org/10.1038/ncomms13379 447 Naidoo, R., Weaver, L. C., Diggle, R. W., Matongo, G., Stuart‐Hill, G., & Thouless, C. (2016). Complementary benefits of tourism and hunting to communal conservancies in Namibia. Conservation Biology, 30(3), 628–638. https://doi.org/10.1111/cobi.12643 Nair, M. Retrieved (2004). Gum tapping in Sterculia urens Roxb. Sterculiaceae Using Ethephon. US Forest Service Pacific Northwest Research Station General Technical Report PNW GTR, 604, 69–73. NAMMCO. (2018). Report of the NAMMCO Global Review of Monodontids. Nandigama, S. (2020). Performance of success and failure in grassroots conservation and development interventions: Gender dynamics in participatory forest management in India. Land Use Policy, 97, 103445. https://doi.org/10.1016/j.landusepol.2018.05.061 Naranjo‐Ortiz, M. A., & Gabaldón, T. (2019). Fungal evolution: Major ecological adaptations and evolutionary transitions. Biological Reviews, 94(4), 1443–1476. https://doi.org/10.1111/brv.12510 Narayan, D., et al. (2001). Voices of the poor: Crying out for change. Oxford University Narayan, D., et al. (2001). Voices of the poor: Crying out for change. Oxford University Press. Narayan, D., R. Patel, K. Schafft, A. Rademacher and S. Koch-Schulte. (2001). Voices of the Poor: Can Anyone Hear Us? New York: Oxford University Press. Narayan, D., R. Patel, K. Schafft, A. Rademacher and S. Koch-Schulte. (2001). Voices of the Poor: Can Anyone Hear Us? New York: Oxford University Press. Nasi, R., Brown, D., Wilkie, D., Bennett, E., Tutin, C., Van Tol, G., & Christophersen, T. (2008). Conservation and use of wildlife-based resources: The bushmeat crisis. Secretariat of the Convention on Biological Diversity, Montreal. And Center for International Forestry Research (CIFOR), Bogor. Technical Series, 50. Nasi, R., Taber, A., & Van Vliet, N. (2011). Empty forests, empty stomachs? Bushmeat and livelihoods in the Congo and Amazon Basins. International Forestry Review, 13(3), 355–368. https://doi.org/10.1505/146554811798293872 Natale, F., Hofherr, J., Fiore, G., & Virtanen, J. (2013). Interactions between aquaculture and fisheries. Marine Policy, 38, 205–213. https://doi.org/10.1016/j.marpol.2012.05.037 Natale, F., Hofherr, J., Fiore, G., & Virtanen, J. (2013). Interactions between aquaculture and fisheries. Marine Policy, 38, 205–213. https://doi.org/10.1016/j.marpol.2012.05.037 National Academies of Sciences, E. (2020). Biological Collections: Ensuring Critical Research and Education for the 21st Century. https://doi.org/10.17226/25592 National Academies of Sciences, E. (2020). Biological Collections: Ensuring Critical Research and Education for the 21st Century. https://doi.org/10.17226/25592 National Academies of Sciences, Engineering, and Medicine; Division on Earth and Life Studies; Institute for Laboratory Animal Research; Roundtable on Science and Welfare in Laboratory Animal Use. (2019). Care, Use, and Welfare of Marmosets as Animal Models for Gene Editing-Based Biomedical Research: Proceedings of a Workshop (L. Anestidou & A. F. Johnson, Eds.). Washington (DC): National Academies Press (US). Retrieved from http://www.ncbi.nlm.nih.gov/books/NBK544647/ Navarro, J. A., Galeano, G., & Bernal, R. (2011). Impact of Leaf Harvest on Populations of Lepidocaryum tenue, an Amazonian Understory Palm Used for Thatching. Retrieved Tropical Conservation Science, 4(1), 25–38. https://doi.org/10.1177/194008291100400104 Navarro-Martínez, A., Ellis, E. A., Hernández-Gómez, I., Romero-Montero, J. A., & Sánchez- Sánchez, O. (2018). Distribution and Abundance of Big-Leaf Mahogany ( Swietenia macrophylla ) on the Yucatan Peninsula, Mexico. Tropical Conservation Science, 11, 194008291876687. https://doi.org/10.1177/1940082918766875 Nayak, P. K., & Armitage, D. (2018). Social-ecological regime shifts (SERS) in coastal systems. Ocean & Coastal Management, 161, 84–95. https://doi.org/10.1016/j.ocecoaman.2018.04.020 448 Nayak, P. K., Armitage, D., & Andrachuk, M. (2016). Power and politics of social–ecological regime shifts in the Chilika lagoon, India and Tam Giang lagoon, Vietnam. Regional Environmental Change, 16(2), 325–339. https://doi.org/10.1007/s10113-015-0775-4 Nayak, P. K., & Berkes, F. (2010). Whose marginalisation? Politics around environmental injustices in India’s Chilika lagoon. Local Environment, 15(6), 553–567. Nayak, P. K., Dias, A. C. E., & Pradhan, S. K. (2021). Traditional Fishing Community and Sustainable Development. In Walter Leal Filho, A. M. Azul, L. Brandli, A. Lange Salvia, & T. Wall (Eds.), Life Below Water (pp. 1–18). Cham: Springer International Publishing. https://doi.org/10.1007/978-3-319-71064-8_88-1 Naylor, R. L., Hardy, R. W., Bureau, D. P., Chiu, A., Elliott, M., Farrell, A. P., … Nichols, P. D. (2009). Feeding aquaculture in an era of finite resources. Proceedings of the National Academy of Sciences, 106(36), 15103–15110. https://doi.org/10.1073/pnas.0905235106 Naylor, Rosamond L., Goldburg, R. J., Primavera, J. H., Kautsky, N., Beveridge, M. C. M., Clay, J., … Troell, M. (2000). Effect of aquaculture on world fish supplies. Nature, 405(6790), 1017. https://doi.org/10.1038/35016500 Nazih, H., & Bard, J.-M. (2018). Microalgae in human health: Interest as a functional food. Microalgae in Health and Disease Prevention, 211–226. Negrelle, B. R. R., & Anacleto, A. (2012). Bromeliads wild harvesting in State of Parana. Ciência Rural, Santa Maria, 42(6), 981–986. Neis, B. (1999). 3. Familial and Social Patriarchy in the Newfoundland Fishing Industry. In D. Newell & R. Ommer (Eds.), Fishing Places, Fishing People. Toronto: University of Toronto Press. https://doi.org/10.3138/9781442674936-005 Neiva, J., Coelho, R., & Erzini, K. (2006). Feeding habits of the velvet belly lanternshark Etmopterus spinax (Chondrichthyes: Etmopteridae) off the Algarve, southern Portugal. Journal of the Marine Biological Association of the United Kingdom, 86(4), 835–841. https://doi.org/10.1017/S0025315406013762 Neke, K. S., Owen-Smith, N., & Witkowski, E. T. (2006). Comparative resprouting response of Savanna woody plant species following harvesting: The value of persistence. Forest Ecology and Management, 232(1–3), 114–123. https://doi.org/10.1016/j.foreco.2006.05.051 Nekratova, N., & Shurupova, M. (2016). Natural Resources of Medicinal Plants: Estimation of Reserves on Example of Rhaponticum carthamoides. Key Engineering Materials, 683, 433–439. Retrieved https://doi.org/10.4028/www.scientific.net/kem.683.433 Nellemann, C., International Criminal Police Organization, & GRID--Arendal. (2012). Green carbon, black trade: Illegal logging, tax fraud and laundering in the worlds tropical forests : a rapid response assessment. Nesbitt, L., Hotte, N., Barron, S., Cowan, J., & Sheppard, S. R. J. (2017). The social and economic value of cultural ecosystem services provided by urban forests in North America: A review and suggestions for future research. Urban Forestry & Urban Greening, 25, 103–111. https://doi.org/10.1016/j.ufug.2017.05.005 Neto, N. A. L., Voeks, R. A., Dias, T. L., & Alves, R. R. (2012). Mollusks of Candomblé: Symbolic and ritualistic importance. Journal of Ethnobiology and Ethnomedicine, 8(article n° 10), 1–10. https://doi.org/10.1186/1746-4269-8-10 449 Neubauer, P., Jensen, O. P., Hutchings, J. A., & Baum, J. K. (2013). Resilience and Recovery of Overexploited Marine Populations. Science, 340(6130), 347–349. (WOS:000317657500054). https://doi.org/10.1126/science.1230441 Neves, K. (2010). Cashing in on Cetourism: A Critical Ecological Engagement with Dominant E-NGO Discourses on Whaling, Cetacean Conservation, and Whale Watching1. Antipode, 42(3), 719–741. https://doi.org/10.1111/j.1467-8330.2010.00770.x Newell, S. L., & Doubleday, N. C. (2020). Sharing country food: Connecting health, food security and cultural continuity in Chesterfield Inlet, Nunavut. Polar Research. https://doi.org/10.33265/polar.v39.3755 Newman, D. J., & Cragg, G. M. (2007). Natural Products as Sources of New Drugs over the Last 25 Years. Journal of Natural Products, 70, 461–477. Newsome, D., Moore, S. A., & Dowling, R. K. (2012). Natural area tourism: Ecology, impacts and management (Vol. 58). Channel view publications. Ngansop, T. M., Biye, E. H., Fongnzossie, F. E., Forbi, P. F., & Chimi, D. C. (2019). Using transect sampling to determine the distribution of some key non-timber forest products across habitat types near Boumba-Bek National Park, South-east Cameroon. BMC Ecology, 19(1), 3. https://doi.org/10.1186/s12898-019-0219-y Ngo, H. C., Nguyen, T. Q., Phan, T. Q., van Schingen, M., & Ziegler, T. (2019). A case study on trade in threatened Tiger Geckos (Goniurosaurus) in Vietnam including updated information on the abundance of the Endangered G-catbaensis. Nature Conservation- Bulgaria, (33), 1–19. (WOS:000462997700001). https://doi.org/10.3897/natureconservation.33.33590 Ngulani, T., & Shackleton, C. (2019). Use of public urban green spaces for spiritual services in Bulawayo, Zimbabwe. Urban Forestry & Urban Greening, 38, 97–104. https://doi.org/10.1016/j.ufug.2018.11.009 Ngwenya, M. P. (2001). Implications of the medicinal animal trade for nature conservation in KwaZulu-Natal. Unpublished Ezemvelo KZN Wildlife Report No. NA 124, (04), 76. Nichiforel, L., Keary, K., Deuffic, P., Weiss, G., Thorsen, B. J., Winkel, G., … Bouriaud, L. (2018). How private are Europe’s private forests? A comparative property rights analysis. Retrieved Land Use Policy, 76, 535–552. https://doi.org/10.1016/j.landusepol.2018.02.034 Nichols, J. D., Runge, M. C., Johnson, F. A., & Williams, B. K. (2007). Adaptive harvest management of North American waterfowl populations: A brief history and future prospects. Journal of Ornithology, 148(2), 343–349. https://doi.org/10.1007/s10336- 007-0256-8 Nichols, P., Rayner, M., & Stevens, J. D. (2001). A pilot investigation of Northern Australian shark liver oils: Characterization and value-adding. CSIRO Marine Research. FRDC Project 99/369. Retrieved from http://frdc.com.au/Archived- Reports/FRDC%20Projects/1999-369-DLD.pdf (accessed 15 Feb 2021) Niedermüller, S., Ainsworth, G., de Juan, S., Garcia, R., Ospina-Alvarez, A., & Pita, P. (2021). The shark and ray meat network: A deep dive into a global affair. World Wildlife Fund. Niedziałkowski, K., Sidorovich, A., Kireyeu, V., & Shkaruba, A. (2021). Stimuli and barriers to innovation in wildlife policy – long-term institutional analysis of wolf management 450 in Belarus. Innovation: The European Journal of Social Science Research, 0(0), 1–21. https://doi.org/10.1080/13511610.2021.1995336 in Belarus. Innovation: The European Journal of Social Science Research, 0(0), 1–21. https://doi.org/10.1080/13511610.2021.1995336 Nijman, V. (2010). An overview of international wildlife trade from Southeast Asia. Biodiversity and Conservation, 19(4), 1101–1114. https://doi.org/10.1007/s10531-009- 9758-4 Nin, S., Petrucci, W. A., Del Bubba, M., Ancillotti, C., & Giordani, E. (2017). Effects of environmental factors on seed germination and seedling establishment in bilberry (Vaccinium myrtillus L.). Scientia Horticulturae, 226, 241–249. https://doi.org/10.1016/j.scienta.2017.08.049 Nisticò, R. (2017). Aquatic-Derived Biomaterials for a Sustainable Future: A European Opportunity. Resources, 6(4), 65. https://doi.org/10.3390/resources6040065 Nordbø, I., Turdumambetov, B., & Gulcan, B. (2018). Local opinions on trophy hunting in Kyrgyzstan. https://doi.org/10.1080/09669582.2017.1319843 Norman, M., & Canby, K. (2020). India’s wooden furniture and wood handicrafts: Risk of trade in illegally harvested woods. Forest Trends. Retrieved from https://www.forest- trends.org/wp-content/uploads/2020/09/India_Report_FINAL.pdf Norris, T. B. (2014). Bridging the great divide: State, civil society, and ‘participatory’ conservation mapping in a resource extraction zone. Applied Geography, 54, 262–274. https://doi.org/10.1016/j.apgeog.2014.05.016 Norström, A. V., Cvitanovic, C., Löf, M. F., West, S., Wyborn, C., Balvanera, P., … Österblom, H. (2020). Principles for knowledge co-production in sustainability research. Nature Sustainability, 3(3), 182–190. https://doi.org/10.1038/s41893-019- 0448-2 Nortje, G. P. (2014). Studies on the impacts of off-road driving and the influence of tourists’ consciousness and attitudes on soil compactionand associated vegetation in the Makuleke Contractual Park, Kruger National Park (PhD Thesis, University of Pretoria). University of Pretoria, Pretoria. Retrieved from http://hdl.handle.net/2263/40223 Noss, A. J. (1998). The Impacts of Cable Snare Hunting on Wildlife Populations in the Forests of the Central African Republic. Conservation Biology, 12(2), 390–398. JSTOR. Retrieved from JSTOR. Novaro, A. Retrieved J., Redford, K. H., & Bodmer, R. E. (2000). Effect of Hunting in Source-Sink Systems in the Neotropics. Conservation Biology, 14(3), 713–721. https://doi.org/10.1046/j.1523-1739.2000.98452.x NRB. (2015). A study on Impact of Yarsagumba on Nepali Economy (p. 48). Nunes, M. U. S., Cardoso, O. R., Soeth, M., Silvano, R. A. M., & Fávaro, L. F. (2021). Fishers’ ecological knowledge on the reproduction of fish and shrimp in a subtropical coastal ecosystem. Hydrobiologia, 848(4), 929–942. https://doi.org/10.1007/s10750-020- 04503-8 Nunes, M. U. S., Hallwass, G., & Silvano, R. A. M. (2019). Fishers’ local ecological knowledge indicate migration patterns of tropical freshwater fish in an Amazonian river. Hydrobiologia, 833(1), 197–215. 451 Nyman, M. (2019). Food, meaning-making and ontological uncertainty: Exploring ‘urban foraging’ and productive landscapes in London. Geoforum, 99, 170–180. https://doi.org/10.1016/j.geoforum.2018.10.009 Nzoyem, N., Vabi, M., Kouokam, R., & Azanga, C. (2010). Forêts communautaires contre la pauvreté, la déforestation et la dégradation des forêts: En faire une réalité au Cameroun. 24–26. Obegi, B. N., Sarfo, I., Morara, G. N., Boera, P., Waithaka, E., & Mutie, A. (2020). Bio- economic modeling of fishing activities in Kenya: The case of Lake Naivasha Ramsar site. Journal of Bioeconomics. Scopus. https://doi.org/10.1007/s10818-019-09292-2 Obunga, R. (1995). Sustainable Development Of Wood Carving Industry In Kenya [Technical progress report]. Obura, D., Wells, S., Church, J., & Horrill, C. (2002). Monitoring of fish and fish catches by local fishermen in Kenya and Tanzania. Marine and Freshwater Research, 53(2), 215– 222. Ochoa, J. J., & Ladio, A. H. (2014). Ethnoecology of Oxalis adenophylla Gillies ex Hook. & Arn. Journal of Ethnopharmacology, 155(1), 533–542. https://doi.org/10.1016/j.jep.2014.05.058 O’Donnell, K. P., Molloy, P. P., & Vincent, A. C. J. (2012). Comparing Fisher Interviews, Logbooks, and Catch Landings Estimates of Extraction Rates in a Small-Scale Fishery. Coastal Management, 40(6), 594–611. Scopus. https://doi.org/10.1080/08920753.2012.727734 O’Donnell, K. P., Pajaro, M. G., & Vincent, A. C. J. (2010). How does the accuracy of fisher knowledge affect seahorse conservation status? Animal Conservation, 13(6), 526–533. Scopus. https://doi.org/10.1111/j.1469-1795.2010.00377.x Oguz, T., & Gilbert, D. (2007). Abrupt transitions of the top-down controlled Black Sea pelagic ecosystem during 1960–2000: Evidence for regime-shifts under strong fishery exploitation and nutrient enrichment modulated by climate-induced variations. Deep- Sea Research I. https://doi.org/10.1016/j.dsr.200609.010 Ohl-Schacherer, J., Shepard, G. H., Kaplan, H., Peres, C. A., Levi, T., & Yu, D. W. (2007). The sustainability of subsistence hunting by Matsigenka native communities in Manu National Park, Peru. Conservation Biology: The Journal of the Society for Conservation Biology, 21(5), 1174–1185. Retrieved https://doi.org/10.1111/j.1523-1739.2007.00759.x Öhman, J., Öhman, M., & Sandell, K. (2016). Outdoor recreation in exergames: A new step in the detachment from nature? Journal of Adventure Education and Outdoor Learning, 16(4), 285–302. https://doi.org/10.1080/14729679.2016.1147965 OIE, WHO, & UNEP. (2021). Reducing public health risks associated with the sale of live wild animals of mammalian species in traditional food markets. OIE, WHO, UNEP. Retrieved from OIE, WHO, UNEP website: https://cdn.who.int/media/docs/default- source/food-safety/ig--121-1-food-safety-and-covid-19-guidance-for-traditional-food- markets-2021-04-12-en.pdf?sfvrsn=921ec66d_1&download=true Ojha, H. R., Shrestha, K. K., Subedi, Y. R., Shah, R., Nuberg, I., Heyojoo, B., … McManus, P. (2017). Agricultural land underutilisation in the hills of Nepal: Investigating socio- environmental pathways of change. Journal of Rural Studies, 53, 156–172. https://doi.org/10.1016/j.jrurstud.2017.05.012 452 Okazaki, E. (2008). A Community-Based Tourism Model: Its Conception and Use. Journal of Sustainable Tourism, 16(5), 511–529. https://doi.org/10.1080/09669580802159594 Okemwa, G., Kaunda-Arara, B., Kimani, E., & Ogutu, B. (2016). Catch composition and sustainability of the marine aquarium fishery in Kenya Fisheries Research 183 19– Okazaki, E. (2008). A Community-Based Tourism Model: Its Conception and Use. Journal of Sustainable Tourism, 16(5), 511–529. https://doi.org/10.1080/09669580802159594 Okemwa, G., Kaunda-Arara, B., Kimani, E., & Ogutu, B. (2016). Catch composition and sustainability of the marine aquarium fishery in Kenya. Fisheries Research, 183, 19– 31. Okumu, B., & Muchapondwa, E. (2020). Welfare and forest cover impacts of incentive based conservation: Evidence from Kenyan community forest associations. World Development, 129, 104890. https://doi.org/10.1016/j.worlddev.2020.104890 Okyerefo, M. P. K., & Fiaveh, D. Y. (2017). Prayer and health-seeking beliefs in Ghana: Understanding the ‘religious space’of the urban forest. Health Sociology Review, 26(3), 308–320. https://doi.org/10.1080/14461242.2016.1257360 Oliveira, M. N. S. de, Cruz, S. M., Sousa, A. M. de, Moreira, F. da C., & Tanaka, M. K. (2014). Implications of the harvest time on Syngonanthus nitens (Bong.) Ruhland (Eriocaulaceae) management in the state of Minas Gerais. Brazilian Journal of Botany, 37(2), 95–103. https://doi.org/10.1007/s40415-014-0049-2 Olivera, B. M. (2006). Conus peptides: Biodiversity-based discovery and exogenomics. The Journal of Biological Chemistry, 281(42), 31173–31177. https://doi.org/10.1074/jbc.R600020200 Olivero, J., Fa, J. E., Farfán, M. A., Márquez, A. L., Vargas, J. M., Real, R., & Nasi, R. (2016). Protected African rainforest mammals and climate change. African Journal of Ecology, 54(3), 392–397. https://doi.org/10.1111/aje.12313 Olivier, K. (2001). The ornamental fish market. Olivier, K. (2001). The ornamental fish market. Olsen, C. S., & Larsen, H. O. (2003). Alpine medicinal plant trade and Himalayan 5BlackwellPublishingLtd mountain livelihood strategies. 12. Ommer, R. E. (2007). Coasts Under Stress: Restructuring and Social-Ecological Health. Montreal: McGill-Queen’s University Press. Öndes, F., Kaiser, M. Retrieved J., & Güçlüsoy, H. (2020). Human impacts on the endangered fan mussel, Pinna nobilis. Aquatic Conservation: Marine and Freshwater Ecosystems, 30(1), 31– 41. Scopus. https://doi.org/10.1002/aqc.3237 Ondo, R., Medik, A., Mijola, J., & Boussougou, A. C. (2020). Légalité et Traçabilité Des Bois Des Forêts Communautaires Du Gabon (Province de l’Ogooue Ivindo). Libreville, DRC: UE-FAO FLEGT. Retrieved from UE-FAO FLEGT. website: http://www.keva- in.org/wp-content/uploads/2020/06/L%C3%A9galit%C3%A9-et- tra%C3%A7abilit%C3%A9-des-bois-des-for%C3%AAts-communautaires-au- Gabon.pdf O’Neill, F. G., & Ivanović, A. (2016). The physical impact of towed demersal fishing gears on soft sediments. ICES Journal of Marine Science, 73(suppl_1), i5–i14. https://doi.org/10.1093/icesjms/fsv125 Opoku-Nyame, J., Leduc, A., & Fenton, N. J. (2021). Bryophyte Conservation in Managed Boreal Landscapes: Fourteen-Year Impacts of Partial Cuts on Epixylic Bryophytes. Frontiers in Forests and Global Change, 4, 674887. https://doi.org/10.3389/ffgc.2021.674887 453 Orams, M. B. (2001). From Whale Hunting to Whale Watching in Tonga: A Sustainable Future? Journal of Sustainable Tourism, 9(2), 128–146. https://doi.org/10.1080/09669580108667394 O’Regan, S. M. (2015). Harvesters’ perspectives on the management of British Columbia’s giant red sea cucumber fishery. Marine Policy, 51, 103–110. Scopus. https://doi.org/10.1016/j.marpol.2014.07.025 Organ, J. F., Decker, T. A., & Lama, T. M. (2016). The North American model and captive cervid facilities—What is the threat? Wildlife Society Bulletin, 40(1), 10–13. https://doi.org/10.1002/wsb.637 Ortez, P. (2021). Foraging in Tucson’s Parks: Interest, Barriers, and Opportunities. The University of Arizona. Ortuño Crespo, G., & Dunn, D. C. (2017). A review of the impacts of fisheries on open-ocean ecosystems. ICES Journal of Marine Science, 74(9), 2283–2297. https://doi.org/10.1093/icesjms/fsx084 Osarenkhoe, O. O., John, O. A., & Theophilus, D. A. (2014). Ethnomycological Conspectus of West African Mushrooms: An Awareness Document. Advances in Microbiology, 04(01), 39–54. https://doi.org/10.4236/aim.2014.41008 Osei-Tutu, P., Nketiah, K., Kyereh, B., Owusu-Ansah, M., & Faniyan, J. (2010). Hidden forestry revealed: Characteristics, constraints and opportunities for small and medium forest enterprises in Ghana. London: International Institute for Environment and Development. Osterberg, P., & Nekaris, K. A. I. (2015). The use of animals as photo props to attract tourists in Thailand: A case study of the slow loris Nycticebus spp. (TRAFFIC Bulletin No. 27; pp. 13–18). Retrieved from https://www.traffic.org/site/assets/files/3008/traffic_pub_bulletin_27_1.pdf#page=17 https://www.traffic.org/site/assets/files/3008/traffic_pub_bulletin_27_1.pdf#page=17 Ostrom, E. (2008). INSTITUTIONS AND THE ENVIRONMENT. Economic A 24–31. https://doi.org/10.1111/j.1468-0270.2008.00840.x Ostrom, E. (2009). A General Framework for Analyzing Sustainability of Social-Ecological Systems. Science, 325(5939), 419–422. https://doi.org/10.1126/science.1172133 Ottolenghi, F., Silvestri, C., Giordano, P., Lovatelli, A., New, M. B., & others. (2004). Capture- based aquaculture: The fattening of eels, groupers, tunas and yellowtails. FAO. Oyetayo, O. V. (2011). Retrieved Medicinal uses of mushrooms in Nigeria: Towards full and sustainable exploitation. African Journal of Traditional, Complementary, and Alternative Medicines: AJTCAM, 8(3), 267–274. https://doi.org/10.4314/ajtcam.v8i3.65289 Özturk, B. (Ed.). (1996). Proceedings of the First International Symposium on the Marine Mammals of the Black Sea. UNEP, Istanbul. P. McPhee, D., Leadbitter, D., & A. Skilleter, G. (2002). Swallowing the bait: Is recreational fishing in Australia ecologically sustainable? Pacific Conservation Biology, 8(1), 40. https://doi.org/10.1071/PC020040 Pace, M. L., Cole, J. J., Carpenter, S. R., & Kitchell, J. F. (1999). Trophic cascades revealed in diverse ecosystems. Trends in Ecology & Evolution, 14(12), 483–488. https://doi.org/10.1016/S0169-5347(99)01723-1 Pacheco, P. (2009). Smallholder Livelihoods, Wealth and Deforestation in the Eastern Amazon. Human Ecology, 37(1), 27–41. https://doi.org/10.1007/s10745-009-9220-y 454 Pacheco, P. (2012). Smallholders and communities in timber markets: Conditions shaping diverse forms of engagement in tropical Latin America. Conservation and Society, 10(2), 114. https://doi.org/10.4103/0972-4923.97484 Pacheco, P., Mejía, E., Cano, W., & de Jong, W. (2016). Smallholder Forestry in the Western Amazon: Outcomes from Forest Reforms and Emerging Policy Perspectives. Forests, 7(12), 193. https://doi.org/10.3390/f7090193 Packer, C., Kosmala, M., Cooley, H. S., Brink, H., Pintea, L., Garshelis, D., … Nowell, K. (2009). Sport Hunting, Predator Control and Conservation of Large Carnivores. PLOS ONE, 4(6), e5941. https://doi.org/10.1371/journal.pone.0005941 Pacoureau, N., Rigby, C. L., Kyne, P. M., Sherley, R. B., Winker, H., Carlson, J. K., … Dulvy, N. K. (2021). Half a century of global decline in oceanic sharks and rays. Nature, 589(7843), 567–571. https://doi.org/10.1038/s41586-020-03173-9 Padmanabhan, P., Correa-Betanzo, J., & Paliyath, P. (2016). Berries and Related Fruits. In B. Caballero, P. M. Finglas, & F. Toldrá (Eds.), Encyclopedia of food and health. Amsterdam ; Boston: Academic Press is an imprint of Elsevier. Padoch, C., & Pinedo-Vásquez, M. (2006). 10. Concurrent Activities and Invisible Technologies: An Example of Timber Management in Amazonia. In D. A. Posey & M. J. Balick (Eds.), Human Impacts on Amazonia (pp. 172–180). Columbia University Press. https://doi.org/10.7312/pose10588-013 Palacios-Abrantes, J., Herrera-Correal, J., Rodríguez, S., Brunkow, J., & Molina, R. (2018). Evaluating the bio-economic performance of a Callo de hacha (Atrina maura, Atrina tuberculosa & Pinna rugosa) fishery restoration plan in La Paz, Mexico. PLoS ONE, 13(12). Scopus. https://doi.org/10.1371/journal.pone.0209431 Palliwoda, J., Kowarik, I., & von der Lippe, M. (2017). Human-biodiversity interactions in urban parks: The species level matters. Landscape and Urban Planning, 157, 394–406. https://doi.org/10.1016/j.landurbplan.2016.09.003 Palmer, M., Tolosa, B., Grau, A. M., del Mar Gil, M., Obregón, C., & Morales-Nin, B. (2017). Retrieved Combining sale records of landings and fishers knowledge for predicting métiers in a small-scale, multi-gear, multispecies fishery. Fisheries Research, 195, 59–70. Palomares, M., & Pauly, D. (2019). On the creeping increase of vessels’ fishing power. Ecology and Society, 24(3). https://doi.org/10.5751/ES-11136-240331 Panatto, D., Haag, M., Lai, P. L., Tomczyk, S., Amicizia, D., & Lino, M. M. (2020). Enhanced Passive Safety Surveillance (EPSS) confirms an optimal safety profile of the use of MF59 ® ‐adjuvanted influenza vaccine in older adults: Results from three consecutive seasons. Influenza and Other Respiratory Viruses, 14(1), 61–66. https://doi.org/10.1111/irv.12685 Pangau-Adam, M., & Noske, R. (2010). Wildlife hunting and bird trade in northern Papua (Irian Jaya), Indonesia. Ethno-Ornithology. Global Studies in Indigenous Ornithology: Culture, Society and Conservation, 73–86. Pangau-Adam, M., Noske, R., & Muehlenberg, M. (2012). Wildmeat or Bushmeat? Subsistence Hunting and Commercial Harvesting in Papua (West New Guinea), Indonesia. Human Ecology, 40(4), 611–621. https://doi.org/10.1007/s10745-012-9492- 5 5 455 Paoletti, M. G., Buscardo, E., & Dufour, D. L. (2000). Edible Invertebrates Among Amazonian Indians: A Critical Review of Disappearing Knowledge. Environment, Development and Sustainability, 2(3/4), 195–225. https://doi.org/10.1023/a:1011461907591 Paoli, G. D., Peart, D. R., Leighton, M., & Samsoedin, I. (2001). An Ecological and Economic Assessment of the Nontimber Forest Product Gaharu Wood in Gunung Palung National Park, West Kalimantan, Indonesia. Conservation Biology, 15(6), 1721–1732. https://doi.org/10.1046/j.1523-1739.2001.98586.x Papworth, S. K., Rist, J., Coad, L., & Milner-Gulland, E. J. (2009). Evidence for shifting baseline syndrome in conservation. Conservation Letters. https://doi.org/10.1111/j.1755-263X.2009.00049.x Paradis, E. (2020). Modelling transition in land cover highlights forest losses and gains in Southeast Asia. Biodiversity and Conservation, 29(8), 2539–2551. https://doi.org/10.1007/s10531-020-01987-7 Pardo, S. A., Kindsvater, H. K., Reynolds, J. D., & Dulvy, N. K. (2016). Maximum intrinsic rate of population increase in sharks, rays, and chimaeras: The importance of survival to maturity. Canadian Journal of Fisheries and Aquatic Sciences, 73(8), 1159–1163. https://doi.org/10.1139/cjfas-2016-0069 Parkkila, K., Arlinghaus, R., Artell, J., Gentner, B., Haider, W., Aas, Ø., … Hickley, P. (2010). Methodologies for assessing socio-economic benefits of European inland recreational fisheries. EIFAAC Occasional Paper, (46), I. Parks, C. G., & Schmitt, C. L. (1997). Wild edible mushrooms in the Blue Mountains: Resource and issues. (No. PNW-GTR-393; p. PNW-GTR-393). Portland, OR: U.S. Department of Agriculture, Forest Service, Pacific Northwest Research Station. https://doi.org/10.2737/PNW-GTR-393 Parrish, J. D., Braun, D. P., & Unnasch, R. S. (2003). Are We Conserving What We Say We Are? Measuring Ecological Integrity within Protected Areas. BioScience, 53(9), 851. https://doi.org/10.1641/0006-3568(2003)053[0851:AWCWWS]2.0.CO;2 Parry, D., & Campbell, B. (1992). 26–27, Retrieved Attitudes of rural communities to animal wildlife and its utilization in Chobe Enclave and Mababe Depression, Botswana. Environmental Conservation, 19(3), 245–252. Parsons, E. C. M., & Draheim, M. (2009). A reason not to support whaling – a tourism impact case study from the Dominican Republic. Current Issues in Tourism, 12(4), 397–403. https://doi.org/10.1080/13683500902730460 Parsons, E. C. M., & Rawles, C. (2003). The Resumption of Whaling by Iceland and the Potential Negative Impact in the Icelandic Whale-watching Market. Current Issues in Tourism, 6(5), 444–448. https://doi.org/10.1080/13683500308667964 Pascual, U., Balvanera, P., Diaz, S., Pataki, G., Roth, E., Stenseke, M., … Yagi, N. (2017). Valuing nature’s contributions to people: The IPBES approach. Current Opinion in Environmental Sustainability, 26–27, 7–16. https://doi.org/10.1016/j.cosust.2016.12.006 Patarra, R. F., Iha, C., Pereira, L., & Neto, A. I. (2019). Concise review of the species Pterocladiella capillacea (SG Gmelin) Santelices & Hommersand. Journal of Applied Phycology. https://doi.org/10.1007/s10811-019-02009-y 456 Paton, A., Antonelli, A., Carine, M., Forzza, R. C., Davies, N., Demissew, S., … Dickie, J. (2020). Plant and fungal collections: Current status, future perspectives. PLANTS, PEOPLE, PLANET, 2(5), 499–514. https://doi.org/10.1002/ppp3.10141 Pattanayak, S. K., & Sills, E. O. (2001). Do Tropical Forests Provide Natural Insurance? The Microeconomics of Non-Timber Forest Product Collection in the Brazilian Amazon. Land Economics, 77(4), 595–612. https://doi.org/10.2307/3146943 Pattiselanno, F. (2006). The Wildlife Hunting in Papua. Biota, 11, 59–61. Paukert, C. P., Lynch, A. J., Beard, T. D., Chen, Y., Cooke, S. J., Cooperman, M. S., … Winfield, I. J. (2017). Designing a global assessment of climate change on inland fishes and fisheries: Knowns and needs. Reviews in Fish Biology and Fisheries, 27(2), 393– 409. https://doi.org/10.1007/s11160-017-9477-y Pauly, D. (1995). Anecdotes and the shifting baseline syndrome of fisheries. Trends in Ecology & Evolution, 10(10), 430. Pauly, D., Belhabib, D., Blomeyer, R., Cheung, W. W. W. L., Cisneros‐Montemayor, A. M., Copeland, D., … Zeller, D. (2014). China’s distant‐water fisheries in the 21st century. Fish and Fisheries, 15(3), 474–488. https://doi.org/10.1111/faf.12032 Pawson, M. G., Glenn, H., & Padda, G. (2008). The definition of marine recreational fishing in Europe. Marine Policy, 32(3), 339–350. https://doi.org/10.1016/j.marpol.2007.07.001 Paye, G. D. (2000). Cultural Uses of Plants: A Guide to Learning About Ethnobotany. New York Botanical Garden PressDept. Payne, C. L. R., Badolo, A., Cox, S., Sagnon, B., Dobermann, D., Milbank, C., … Balmford, A. (2020). The contribution of “chitoumou”, the edible caterpillar Cirina butyrospermi, to the food security of smallholder farmers in southwestern Burkina Faso. Food Security, 12(1), 221–234. Retrieved https://doi.org/10.1007/s12571-019-00994-z Payne, C. L. R., Badolo, A., Sagnon, B., Cox, S., Pearson, S., Sanon, A., … Balmford, A. (2020). Effects of defoliation by the edible caterpillar “chitoumou” (Cirina butyrospermi) on harvests of shea (Vitellaria paradoxa) and growth of maize (Zea mays). Agroforestry Systems, 94(1), 231–240. https://doi.org/10.1007/s10457-019- 00385-5 Pearce, T. R., Antonelli, A., Brearley, F. Q., Couch, C., Campostrini Forzza, R., Gonçalves, S. C., … Breman, E. (2020). International collaboration between collections‐based institutes for halting biodiversity loss and unlocking the useful properties of plants and fungi. PLANTS, PEOPLE, PLANET, 2(5), 515–534. https://doi.org/10.1002/ppp3.10149 Peintner, U., Schwarz, S., Mešić, A., Moreau, P.-A., Moreno, G., & Saviuc, P. (2013). Mycophilic or Mycophobic? Legislation and Guidelines on Wild Mushroom Commerce Reveal Different Consumption Behaviour in European Countries. PLoS ONE, 8(5), e63926. https://doi.org/10.1371/journal.pone.0063926 Penning, M., Reid, G., Koldewey, H., Dick, G., Andrews, B., Arai, K., … Tanner, K. (2009). Turning the tide: A global aquarium strategy for conservation and sustainability. World Association of Zoos and Aquariums, Berna, Suiza. Pereira, D., Santos, D., Vedoveto, M., Guimarães, J., & Veríssimo, A. (2010). Fatos Florestais da Amazônia. IMAZON-Instituto do Homem e Meio Ambiente da Amazônia. 457 Pereira, F., Vasconcelos, P., Moreno, A., & Gaspar, M. B. (2019). Catches of Sepia officinalis in the small-scale cuttlefish trap fishery off the Algarve coast (southern Portugal). Fisheries Research, 214, 117–125. Scopus. https://doi.org/10.1016/j.fishres.2019.01.022 Pereira, R., Zweede, J., Asner, G. P., & Keller, M. (2002). Forest canopy damage and recovery in reduced-impact and conventional selective logging in eastern Para, Brazil. Forest Ecology and Management, 168(1–3), 77–89. https://doi.org/10.1016/S0378- 1127(01)00732-0 Peres, C. A., Baider, C., Zuidema, P. A., Wadt, L. H. O., Kainer, K. A., Gomes-Silva, D. A. P., … Freckleton, R. P. (2003). Demographic Threats to the Sustainability of Brazil Nut Exploitation. Science, 302(5653), 2112–2114. https://doi.org/10.1126/science.1091698 Peres, C. A., & Nascimento, H. S. (2006). Impact of game hunting by the Kayapo´ of south- eastern Amazonia: Implications for wildlife conservation in tropical forest indigenous reserves. 28. Pérez Roda, M. A., Gilman, E., Huntington, T., Kennelly, S. J., Suuronen, P., Chaloupka, M., … Food and Agriculture Organization of the United Nations. (2019). A third assessment of global marine fisheries discards / by Maria Amparo Pérez Roda, Eric Gilman, Tim Huntington, Steven J. Kennelly, Petri Suuronen, Milani Chaloupka, and Paul A. H. Medley. Pérez-Moreno, J., Martínez-Reyes, M., Yescas-Pérez, A., Delgado-Alvarado, A., & Xoconostle-Cázares, B. (2008). Wild Mushroom Markets in Central Mexico and a Case Study at Ozumba. Retrieved Economic Botany, 62(3), 425–436. https://doi.org/10.1007/s12231- 008-9043-6 Perez-Verdin, G., Grebner, D. L., Munn, I. A., Sun, C., & Grado, S. C. (2008). Economic impacts of woody biomass utilization for bioenergy in Mississippi. FOREST PRODUCTS JOURNAL, 58(11), 10. Perrin, W. F. (Ed.). (2009). Encyclopedia of marine mammals (2. ed). Burlington,MA: Academic Press. Perron, F. E. (1981). Larval Growth and Metamorphosis of Conus (Castropoda: Toxoglossa) in Hawaii. Retrieved from http://scholarspace.manoa.hawaii.edu/handle/10125/536 Pert, P. L., Hill, R., Maclean, K., Dale, A., Rist, P., Schmider, J., … Tawake, L. (2015). Mapping cultural ecosystem services with rainforest aboriginal peoples: Integrating biocultural diversity, governance and social variation. Ecosystem Services, 13, 41–56. https://doi.org/10.1016/j.ecoser.2014.10.012 Pet Food Manufactures Association. (2014). Pet Population 2014. Retrieved April 15, 2022, from https://www.pfma.org.uk/pet-population-2014 Peters, H., O’Leary, B. C., Hawkins, J. P., & Roberts, C. M. (2016). The cone snails of Cape Verde: Marine endemism at a terrestrial scale. Global Ecology and Conservation, 7, 201–213. https://doi.org/10.1016/j.gecco.2016.06.006 Petersen, L., Reid, A. M., Moll, E. J., & Hockings, M. T. (2017). Perspectives of wild medicine harvesters from Cape Town, South Africa. South African Journal of Science, 113(9/10), 8–8. https://doi.org/10.17159/sajs.2017/20160260 Petersen, T. A., Brum, S. M., Rossoni, F., Silveira, G. F. V., & Castello, L. (2016). Recovery of Arapaima sp. populations by community-based management in floodplains of the 458 Purus River, Amazon: Recovery of arapaima sp. populations. Journal of Fish Biology, 89(1), 241–248. https://doi.org/10.1111/jfb.12968 Purus River, Amazon: Recovery of arapaima sp. populations. Journal of Fish Biology, 89(1), 241–248. https://doi.org/10.1111/jfb.12968 Peters-Guarin, G., & McCall, M. K. (2012). Participatory mapping and monitoring of forest carbon services using freeware: Cybertracker and Google Earth. In M. Skutsch (Ed.), Community Forest Monitoring for the Carbon Market: Opportunities Under REDD (pp. 94–106). London, U.K.: Earthscan. Retrieved from https://books.google.co.za/books?id=-Q5obfJhX5QC Peterson, M. N., & Nelson, M. (2017). Why the North American Model of Wildlife Conservation is Problematic for Modern Wildlife Management. https://doi.org/10.1080/10871209.2016.1234009 Peterson, N., & Rigsby, B. (2014). Customary marine tenure in Australia. Sydney: Sydney University Press. Retrieved from https://dx.doi.org/10.30722/sup.9781743323892 Petrere, M., Barthem, R. B., Córdoba, E. A., & Gómez, B. C. (2004). Review of the large catfish fisheries in the upper Amazon and the stock depletion of piraíba (Brachyplatystoma filamentosumLichtenstein). Reviews in Fish Biology and Fisheries, 14(4), 403–414. Petriello, M. A., & Stronza, A. L. (2020). Campesino hunting and conservation in Latin America. Conservation Biology, 34(2), 338–353. https://doi.org/10.1111/cobi.13396 Pezzuti, J. C. B., Lima, J. P., da Silva, D. F., & Begossi, A. (2010). Retrieved Uses and Taboos of Turtles and Tortoises Along Rio Negro, Amazon Basin. Journal of Ethnobiology, 30(1), 153– 168. https://doi.org/10.2993/0278-0771-30.1.153 Pfab, M. F., & Scholes, M. A. (2004). Is the collection of Aloe peglerae from the wild sustainable? An evaluation using stochastic population modelling. Biological Conservation, 118(5), 695–701. https://doi.org/10.1016/j.biocon.2003.10.018 Phelps, J., & Webb, E. L. (2015). “Invisible” wildlife trades: Southeast Asia’s undocumented illegal trade in wild ornamental plants. Biological Conservation, 186, 296–305. https://doi.org/10.1016/j.biocon.2015.03.030 Philips, L. P., Szuster, B. W., & Needham, M. D. (2019). Tourist value orientations and conflicts at a marine protected area in Hawaii. International Journal of Tourism Research, 21(6), 868–881. https://doi.org/10.1002/jtr.2311 Phuntsho, S. (2011). Forests, community forestry and their significance in Bhutan. In Community forestry in Bhutan: Putting people at the heart of poverty reduction (pp. 1– 3). Bhutan: Ugyen Wangchuck Institute for Conservation and Environment (UWICE). Picard, N., Gourlet-Fleury, S., & Forni, É. (2012). Estimating damage from selective logging and implications for tropical forest management. Canadian Journal of Forest Research, 42(3), 605–613. https://doi.org/10.1139/x2012-018 Pierce, A. R., & Emery, M. R. (2005). The use of forests in times of crisis: Ecological literacy as a safety net. Forests Trees and Livelihoods, 15(3), 249–252. https://doi.org/10.1080/14728028.2005.9752525 Pieroni, A. (2016). The changing ethnoecological cobweb of white truffle (Tuber magnatum Pico) gatherers in South Piedmont, NW Italy. Journal of Ethnobiology and Ethnomedicine, 12(1), 18. https://doi.org/10.1186/s13002-016-0088-9 459 Pieroni, A., Nebel, S., Santoro, F. R., & Heinrick, M. (2005). Food for Two Seasons: Culinary Uses of Non-Cultivated Local Vegetables and Mushrooms in a South Italian Village. International Journal of Food Sciences and Nutrition, 56, 245–272. Pikitch, E. K. (2015). Stop-loss order for forage fish fisheries. Proceedings of the National Academy of Sciences, 112(21), 6529–6530. https://doi.org/10.1073/pnas.1505403112 Pikitch, E. K., Rountos, K. J., Essington, T. E., Santora, C., Pauly, D., Watson, R., … Munch, S. B. (2014). The global contribution of forage fish to marine fisheries and ecosystems. Fish and Fisheries, 15(1), 43–64. https://doi.org/10.1111/faf.12004 Piña, C. I., Lucero, L. E., Simoncini, M. S., Peterson, G. B., & Tavella, M. (2017). Lipid profile of yacarés overo meat fed with diets enriched with flax seeds. Zootecnia Tropical, 34. Pinard, M. A., Adam, K. A., Cobbinah, J. R., Nutukor, E., Damnyang, L., Nyarko, C., … Abrebesse, O. M. (2006). Processing lumber with chainsaws: Relevance for households in the forest zone of Ghana. Retrieved In A Cut for the Poor, Proceedings of the International Conference on Managing Forests for Poverty Reduction: Capturing Opportunities in Forest Harvesting and Wood Processing for the Benefit of the Poor (pp. 3–6). Ho Chi Min City, Vietnam. Retrieved from http://www.fao.org/3/ag131e/ag131e19.htm Pinard, M. A., Putz, F. E., Tay, J., & Sullivan, T. (1995). Creating timber harvest guidelines for a reduced-impact logging project in Malaysia. Journal of Forestry, 93(10), 41–45. Pintaud, J.-C., Galeano, G., Balslev, H., Bernal, R., Ferreira, E., de Granville, J.-J., … Stauffer, F. W. (2008). Las palmeras de América del Sur: Diversidad, distribución e historia evolutiva The palms of South America: Diversity, distribution and evolutionary history. 24. Pinto de Sá Alves, L. C., Andriolo, A., Orams, M. B., & de Freitas Azevedo, A. (2013). Resource defence and dominance hierarchy in the boto (Inia geoffrensis) during a provisioning program. Acta Ethologica, 16(1), 9–19. https://doi.org/10.1007/s10211- 012-0132-2 Pinto, M. F., Mourão, J. S., & Alves, R. R. N. (2015). Use of ichthyofauna by artisanal fishermen at two protected areas along the coast of Northeast Brazil. Journal of Ethnobiology and Ethnomedicine, 11(1). Scopus. https://doi.org/10.1186/s13002-015- 0007-5 Piponiot, C., Rödig, E., Putz, F. E., Rutishauser, E., Sist, P., Ascarrunz, N., … Hérault, B. (2019). Can timber provision from Amazonian production forests be sustainable? Environmental Research Letters, 14(6), 064014. https://doi.org/10.1088/1748- 9326/ab195e Pires, S., & Moreto, W. (2016). The Illegal Wildlife Trade. In Oxford Handbooks Online (pp. 1–41). https://doi.org/10.1093/oxfordhb/9780199935383.013.161 Pita, P., Fernández-Márquez, D., Antelo, M., Macho, G., & Villasante, S. (2019). Socioecological changes in data-poor S-fisheries: A hidden shellfisheries crisis in Galicia (NW Spain). Marine Policy, 101, 208–224. Scopus. https://doi.org/10.1016/j.marpol.2018.09.018 Pitcher, C. R., Ellis, N., Jennings, S., Hiddink, J. G., Mazor, T., Kaiser, M. J., … Hilborn, R. (2017). Estimating the sustainability of towed fishing‐gear impacts on seabed habitats: A simple quantitative risk assessment method applicable to data‐limited fisheries. 460 Methods in Ecology and Evolution, 8(4), 472–480. https://doi.org/10.1111/2041- 210X.12705 Methods in Ecology and Evolution, 8(4), 472–480. https://doi.org/10.1111/2041- 210X.12705 Ploeg, A. (2007). The volume of the ornamental fish trade. International Transport of Live Fish in the Ornamental Aquatic Industry: OFI Educational Publication; Ornamental Fish International: Maarsen, The Netherlands, 7. Plotkin, M. J., Famolare, L., Conservation International, & Asociación Nacional para la Conservación de la Naturaleza (Eds.). (1992). Sustainable harvest and marketing of rain forest products. Washington, D.C: Island Press. PNGF. (2009). PAPUA NEW GUINEA FORESTRY OUTLOOK STUDY. Papua New Guinea Forest Authority. Retrieved Retrieved from Papua New Guinea Forest Authority website: http://www.fao.org/3/am614e/am614e00.pdf Poe, M. R., LeCompte, J., McLain, R., & Hurley, P. (2014). Urban foraging and the relational ecologies of belonging. Social & Cultural Geography, 15(8), 901–919. https://doi.org/10.1080/14649365.2014.908232 Poe, S., & Armijo, B. (2014). Lack of effect of herpetological collecting on the population structure of a community of Anolis (Squamata: Dactyloidae) in a disturbed habitat. Herpetology Notes, 7, 153–157. Poffenberger, M. (2000). Communities and forest management in South Asia. IUCN. Pokorny, B. (2013). Smallholders, forest management and rural development in the amazon. Place of publication not identified: ROUTLEDGE. Pokorny, B., & De Jong, W. (2015). Smallholders and forest landscape transitions: Locally devised development strategies of the tropical Americas. International Forestry Review, 17(1), 1–19. https://doi.org/10.1505/146554815814668981 Pokorny, B., Johnson, J., Medina, G., & Hoch, L. (2012). Market-based conservation of the Amazonian forests: Revisiting win–win expectations. Geoforum, 43(3), 387–401. https://doi.org/10.1016/j.geoforum.2010.08.002 Pokorny, B., & Steinbrenner, M. (2005). Collaborative Monitoring of Production and Costs of Timber Harvest Operations in the Brazilian Amazon. Ecology and Society, 10(1), art3. https://doi.org/10.5751/ES-01224-100103 Polovina, J., Abecassis, M., Howell, E., & Woodworth, P. (2009). Increases in the relative abundance of mid-trophic level fishes concurrent with declines in apex predators in the subtropical North Pacific, 1996-2006. Fisheries Bulletin, 107, 523–531. Pons, M., Branch, T. A., Melnychuk, M. C., Jensen, O. P., Brodziak, J., Fromentin, J. M., … Hilborn, R. (2017). Effects of biological, economic and management factors on tuna and billfish stock status. Fish and Fisheries, 18(1), 1–21. https://doi.org/10.1111/faf.12163 Popescu, V., Artelle, K., Pop, M. I., Manolache, S., & Rozylowicz, L. (2016). Assessing biological realism of wildlife population estimates in data‐poor systems. https://doi.org/10.1111/1365-2664.12660 Porro, R., Lopez-Feldman, A., W. Vela-Alvarado, J., QuiÑonez-Ruíz, L., P. Seijas-Cardenas, Z., Vásquez-Macedo, M., … Cardenas-Ruiz, J. (2014). Forest Use and Agriculture in Ucayali, Peruvian Amazon: Interactions Among Livelihood Strategies, Income and Environmental Outcomes. Tropics, 23(2), 47–62. https://doi.org/10.3759/tropics.23.47 461 Porszt, E. J., Peterman, R. M., Dulvy, N. K., Cooper, A. B., & Irvine, J. R. (2012). Reliability of Indicators of Decline in Abundance: Reliability of Indicators of Decline. Conservation Biology, 26(5), 894–904. https://doi.org/10.1111/j.1523- 1739.2012.01882.x Porter, L., & Lai, H. Y. (2017). Marine Mammals in Asian Societies; Trends in Consumption, Bait, and Traditional Use. Frontiers in Marine Science, 4. https://doi.org/10.3389/fmars.2017.00047 Post, J. R., Sullivan, M., Cox, S., Lester, N. P., Walters, C. J., Parkinson, E. A., … Shuter, B. J. (2002). Canada’s Recreational Fisheries: The Invisible Collapse? Fisheries, 27(1), 6–17. https://doi.org/10.1577/1548-8446(2002)027<0006:CRF>2.0.CO;2 Potts, W. Retrieved M., Childs, A.-R., Sauer, W. H. H., & Duarte, A. D. C. (2009). Characteristics and economic contribution of a developing recreational fishery in southern Angola. Fisheries Management and Ecology, 16(1), 14–20. https://doi.org/10.1111/j.1365- 2400.2008.00617.x Poudeyal, M., Meilby, H., Shrestha, B., & Ghimire, S. (2019). Harvest effects on density and biomass of Neopicrorhiza scrophulariiflora vary along environmental gradients in the Nepalese Himalayas. Ecol Evol, 9(13), 7726–7740. https://doi.org/10.1002/ece3.5355 Poudyal, B. H., Maraseni, T., & Cockfield, G. (2018). Evolutionary dynamics of selective logging in the tropics: A systematic review of impact studies and their effectiveness in sustainable forest management. Forest Ecology and Management, 430, 166–175. https://doi.org/10.1016/j.foreco.2018.08.006 Poudyal, N. C., Bowker, J. M., Green, G. T., & Tarrant, M. A. (2012). Supply of Private Acreage for Recreational Deer Hunting in Georgia. Human Dimensions of Wildlife, 17(2), 141–154. https://doi.org/10.1080/10871209.2011.604666 Pouil, S., Tlusty, M. F., Rhyne, A. L., & Metian, M. (2020). Aquaculture of marine ornamental fish: Overview of the production trends and the role of academia in research progress. Reviews in Aquaculture, 12(2), 1217–1230. https://doi.org/10.1111/raq.12381 Pouliot, M., Pyakurel, D., & Smith-Hall, C. (2018). High altitude organic gold: The production network for Ophiocordyceps sinensis from far-western Nepal. Journal of Ethnopharmacology, 218, 59–68. https://doi.org/10.1016/j.jep.2018.02.028 Pounds, J. A., Bustamante, M. R., Coloma, L. A., Consuegra, J. A., Fogden, M. P. L., Foster, P. N., … Young, B. E. (2006). Widespread amphibian extinctions from epidemic disease driven by global warming. Nature, 439(7073), 161–167. https://doi.org/10.1038/nature04246 Pounds, J. A., Fogden, M. P. L., & Campbell, J. H. (1999). Biological response to climate change on a tropical mountain. Nature, 398(6728), 611–615. https://doi.org/10.1038/19297 Pouta, E., Sievänen, T., & Neuvonen, M. (2006). Recreational Wild Berry Picking in Finland— Reflection of a Rural Lifestyle. Society & Natural Resources, 19(4), 285–304. https://doi.org/10.1080/08941920500519156 Powell, B., Thilsted, S. H., Ickowitz, A., Termote, C., Sunderland, T., & Herforth, A. (2015). Improving diets with wild and cultivated biodiversity from across the landscape. Food Security, 7(3), 535–554. https://doi.org/10.1007/s12571-015-0466-5 462 Prachvuthy, M. (2006). Tourism, Poverty, and Income Distribution: Chambok Community- based Ecotourism Development, Kirirom National Park, Kompong Speu Province, Cambodia. Journal of GMS Development Studies, 3, 25–40. Prato, G., Barrier, C., Francour, P., Cappanera, V., Markantonatou, V., Guidetti, P., … Gascuel, D. (2016). Assessing interacting impacts of artisanal and recreational fisheries in a small Marine Protected Area (Portofino, NW Mediterranean Sea). Ecosphere, 7(12). https://doi.org/10.1002/ecs2.1601 Prescott, J., Riwu, J., Prasetyo, A. P., & Stacey, N. (2017). Retrieved The money side of livelihoods: Economics of an unregulated small-scale Indonesian sea cucumber fishery in the Timor Sea. Marine Policy, 82, 197–205. Scopus. https://doi.org/10.1016/j.marpol.2017.03.033 Pritchard, P.C.H. (1996). The Galápagos Tortoises: Nomenclatural and Survival Status (Chelonian Research Monographs). Prober, S. M., O’Connor, M. H., & Walsh, F. J. (2011). Australian Aboriginal Peoples’ Seasonal Knowledge: A Potential Basis for Shared Understanding in Environmental Management. Ecology and Society, 16(2). Retrieved from https://www.jstor.org/stable/26268886 Prugh, L. R., Stoner, C. J., Epps, C. W., Bean, W. T., Ripple, W. J., Laliberte, A. S., & Brashares, J. S. (2009). The Rise of the Mesopredator. BioScience, 59(9), 779–791. https://doi.org/10.1525/bio.2009.59.9.9 Puettmann, K., Messier, C., & Coates, K. D. (2009). A Critique of Silviculture: Managing For Complexity. Washington D.C.: Island Press. Puillandre, N., Kantor, Y. I., Sysoev, A., Couloux, A., Meyer, C., Rawlings, T., … Bouchet, P. (2011). The dragon tamed? A molecular phylogeny of the Conoidea (Gastropoda). Journal of Molluscan Studies, 77(3), 259–272. https://doi.org/10.1093/mollus/eyr015 Puillandre, Nicolas, Stöcklin, R., Favreau, P., Bianchi, E., Perret, F., Rivasseau, A., … Bouchet, P. (2014). When everything converges: Integrative taxonomy with shell, DNA and venomic data reveals Conus conco, a new species of cone snails (Gastropoda: Conoidea). Molecular Phylogenetics and Evolution, 80(0), 186–192. https://doi.org/10.1016/j.ympev.2014.06.024 Pujiati, R. (2017). Produksi Furniture Indonesia. In Info Komoditi Furniture (pp. 7–36). Jakarta, Indonesia. Retrieved from http://bppp.kemendag.go.id/media_content/2017/10/Isi_BRIK_FURNITUR.pdf Pulido, M. T., & Caballero, J. (2006). The impact of shifting agriculture on the availability of non-timber forest products: The example of Sabal yapa in the Maya lowlands of Mexico. Forest Ecology and Management, 222(1–3), 399–409. https://doi.org/10.1016/j.foreco.2005.10.043 Purata, S. E., Brosi, B. J., & Chibnik, M. (2004). Alebrijes, wood carvings. In Riches of the forest: Fruits, remedies and handicrafts in Latin America (p. 5). Desa Putra, Indonesia: Center for International Forestry Research (CIFOR). Retrieved from http://www.cifor.org/library/1612/riches-of-the-forest-fruits-remedies-and- handicrafts-in-latin-america/ Puri, K., Yadav, V., & Joshi, R. (2019). Functional Role of Elephants in Maintaining Forest Ecosystem and Biodiversity: Lessons from Northwestern Elephant Range in India. 463 1–8. Ecology, Asian Journal of Environment Environment & Journal of Asian Journal of Environment & Ecology, 1 8. https://doi.org/10.9734/ajee/2019/v9i230091 https://doi.org/10.9734/ajee/2019/v9i230091 Purvis, B., Mao, Y., & Robinson, D. (2019). Three pillars of sustainability: In search of conceptual origins. Sustainability Science, 14(3), 681–695. https://doi.org/10.1007/s11625-018-0627-5 Purvis, B., Mao, Y., & Robinson, D. (2019). Three pillars of sustainability: In search of conceptual origins. Sustainability Science, 14(3), 681–695. https://doi.org/10.1007/s11625-018-0627-5 Pusceddu, A., Bianchelli, S., Martin, J., Puig, P., Palanques, A., Masque, P., & Danovaro, R. (2014). Retrieved Chronic and intensive bottom trawling impairs deep-sea biodiversity and ecosystem functioning. Proceedings of the National Academy of Sciences, 111(24), 8861–8866. https://doi.org/10.1073/pnas.1405454111 Putraditama, A., Kim, Y.-S., & Baral, H. (2021). Where to put community-based forestry?: Reconciling conservation and livelihood in Lampung, Indonesia. Trees, Forests and People, 4, 100062. https://doi.org/10.1016/j.tfp.2021.100062 Putz, F. (2018). SUSTAINABLE = GOOD, BETTER, OR RESPONSIBLE. JOURNAL OF TROPICAL FOREST SCIENCE, 30(5), 415–417. https://doi.org/10.26525/jtfs2018.30.5.415417 Putz, F. E., Dykstra, D. P., & Heinrich, R. (2000). Why Poor Logging Practices Persist in the Tropics. Conservation Biology, 14(4), 951–956. https://doi.org/10.1046/j.1523- 1739.2000.99137.x Putz, F. E., Sist, P., Fredericksen, T., & Dykstra, D. (2008). Reduced-impact logging: Challenges and opportunities. Forest Ecology and Management, 256(7), 1427–1433. https://doi.org/10.1016/j.foreco.2008.03.036 Putz, F. E., Zuidema, P. A., Synnott, T., Peña-Claros, M., Pinard, M. A., Sheil, D., … Zagt, R. (2012). Sustaining conservation values in selectively logged tropical forests: The attained and the attainable: Sustaining tropical forests with forestry. Conservation Letters, 5(4), 296–303. https://doi.org/10.1111/j.1755-263X.2012.00242.x Putzel, L., Padoch, C., & Ricse, A. (2013). Putting Back the Trees: Smallholder Silvicultural Enrichment of Post-Logged Concession Forest in Peruvian Amazonia. Small-Scale Forestry, 12(3), 421–436. https://doi.org/10.1007/s11842-012-9221-3 Pyhälä, A., Brown, K., & Neil Adger, W. (2006). Implications of Livelihood Dependence on Non-Timber Products in Peruvian Amazonia. Ecosystems, 9(8), 1328–1341. https://doi.org/10.1007/s10021-005-0154-y Queiroz, N., Humphries, N. E., Couto, A., Vedor, M., Da Costa, I., Sequeira, A. M., … others. (2019). Global spatial risk assessment of sharks under the footprint of fisheries. Nature, 572(7770), 461–466. https://doi.org/10.1038/s41586-019-1444-4 Quetglas, A., Merino, G., González, J., Ordines, F., Garau, A., Grau, A. M., … Massutí, E. (2017). Harvest strategies for an ecosystem approach to fisheries management in western Mediterranean demersal fisheries. Frontiers in Marine Science, 4(APR). Scopus. https://doi.org/10.3389/fmars.2017.00106 Quetglas, A., Merino, G., Ordines, F., Guijarro, B., Garau, A., Grau, A. M., … Massutí, E. (2016). Assessment and management of western Mediterranean small-scale fisheries. Ocean and Coastal Management, 133, 95–104. Scopus. https://doi.org/10.1016/j.ocecoaman.2016.09.013 R. Froese & D. Pauly. (2019). FishBase. World Wide Web electronic publication. 464 Radachowsky, J., Ramos, V. H., McNab, R., Baur, E. H., & Kazakov, N. (2012). Forest concessions in the Maya Biosphere Reserve, Guatemala: A decade later. Forest Ecology and Management, 268, 18–28. https://doi.org/10.1016/j.foreco.2011.08.043 Radomir, M., Mesud, A., & Žaklina, M. (2018). Conservation and trade of wild edible mushrooms of Serbia – history, state of the art and perspectives. Nature Conservation, 25, 31–53. https://doi.org/10.3897/natureconservation.25.21919 Raffa, R. B., Pergolizzi Jr, J. V., Taylor Jr, R., Kitzen, J. M., & Group, N. R. Retrieved (2019). Sunscreen bans: Coral reefs and skin cancer. Journal of Clinical Pharmacy and Therapeutics, 44(1), 134–139. https://doi.org/10.1111/jcpt.12778 Raghavan, R., Dahanukar, N., Tlusty, M. F., Rhyne, A. L., Krishna Kumar, K., Molur, S., & Rosser, A. M. (2013). Uncovering an obscure trade: Threatened freshwater fishes and the aquarium pet markets. Biological Conservation, 164, 158–169. https://doi.org/10.1016/j.biocon.2013.04.019 Rajoo, K. S., Karam, D. S., & Abdullah, M. Z. (2020). The physiological and psychosocial effects of forest therapy: A systematic review. Urban Forestry & Urban Greening, 54, 126744. https://doi.org/10.1016/j.ufug.2020.126744 RAM Legacy Stock Assessment Database. (2018). RAM Legacy Stock Assessment Database v4.44 [Data set]. Zenodo. https://doi.org/10.5281/ZENODO.2542919 Ramírez-Amaro, S., & Galván-Magaña, F. (2019). Effect of gillnet selectivity on elasmobranchs off the northwestern coast of Mexico. Ocean and Coastal Management, 172, 105–116. Scopus. https://doi.org/10.1016/j.ocecoaman.2019.02.001 Ramos-Elorduy, J. (2006). Threatened edible insects in Hidalgo, Mexico and some measures to preserve them. Journal of Ethnobiology and Ethnomedicine, 2, article n°51. https://doi.org/10.1186/1746-4269-2-51 Ramos-Elorduy, J., Pino-Moreno, J. M., & Martínez-Camacho, V. H. (2012). Could grasshoppers be a nutritive meal? Food and Nutrition Sciences, 3, 164–175. http://dx.doi.org/10.4236/fns.2012.32025 Rands, M. R. W., Adams, W. M., Bennun, L., Butchart, S. H. M., Clements, A., Coomes, D., … Vira, B. (2010). Biodiversity Conservation: Challenges Beyond 2010. Science, 329(5997), 1298–1303. https://doi.org/10.1126/science.1189138 Rangel-Landa, S., Casas, A., García-Frapolli, E., & Lira, R. (2017). Sociocultural and ecological factors influencing management of edible and non-edible plants: The case of Ixcatlán, Mexico. Journal of Ethnobiology and Ethnomedicine, 13(1), 59. https://doi.org/10.1186/s13002-017-0185-4 Rasethe, M. T., Semenya, S. S., & Maroyi, A. (2019). Medicinal Plants Traded in Informal Herbal Medicine Markets of the Limpopo Province, South Africa. Evidence-Based Complementary and Alternative Medicine. https://doi.org/Artn 2609532 10.1155/2019/2609532 Rashid, W., Shi, J., Rahim, I. ur, Dong, S., & Sultan, H. (2020). Issues and Opportunities Associated with Trophy Hunting and Tourism in Khunjerab National Park, Northern Pakistan. Animals : An Open Access Journal from MDPI, 10(4), 597. https://doi.org/10.3390/ani10040597 Rasmussen, M. (2014). The whaling versus whale-watching debate: The resumption of Icelandic whaling. In J. E. S. Higham & R. Williams (Eds.), Whale-watching: 465 Sustainable tourism and ecological management (pp. 81–94). New York: Cambridge University Press. Sustainable tourism and ecological management (pp. 81–94). New York: Cambridge University Press. Rassweiler, A., Lauer, M., Lester, S. E., Holbrook, S. J., Schmitt, R. J., Madi Moussa, R., … Claudet, J. (2020). Perceptions and responses of Pacific Island fishers to changing coral reefs. Ambio, 49(1), 130–143. Scopus. https://doi.org/10.1007/s13280-019-01154-5 Ravenel, R. M. (2004). Retrieved Community-Based Logging and De Facto Decentralization: Illegal Logging in the Gunung Palung Area of West Kalimantan, Indonesia. Journal of Sustainable Forestry, 19(1–3), 213–237. https://doi.org/10.1300/J091v19n01_10 Raya, M. L. R., & Berdugo, J. E. F. (2019). Decision Making in the Campeche Maya Octopus fishery in two fishing communities. Maritime Studies, 18(1), 91–101. Raybaud, V., Beaugrand, G., Goberville, E., Delebecq, G., Destombe, C., Valero, M., … Gevaert, F. (2013). Decline in kelp in West Europe and climate. 8(6): 1–10. PLoS ONE, 8(6), 1–10. https://doi.org/10.1371/journal.pone.0066044 Rebours, C., Friis Pedersen, S., Øvsthus, I., & Roleda, M. (2014). Seaweed-a resource for organic farming. Bioforsk Fokus, 9(2), 107. RECOFTC. (2020). Survey finds forest communities in Thailand face multiple hardships from COVID-19. Retrieved from https://www.recoftc.org/news/survey-finds-forest- communities-thailand-face-multiple- hardships-covid-19). Redmond. (2006). Bushmeat-Trade-Report-2006.pdf. Retrieved August 5, 2019, from Google Docs website: https://docs.google.com/file/d/0B9- 2g_Nw6ywWcXd3NVJfQ3VKMWs/edit?usp=embed_facebook 2g_Nw6ywWcXd3NVJfQ3VKMWs/edit?usp=embed_facebook Redzic, S., Barudanovic, S., & Pilipovic, S. (2010). Wild mushrooms and lichens used as human food for survival in war conditions; Podrinje-Zepa Region (Bosnia and Herzegovina, W. Balkan). Human Ecology Review, 175–187. Rehage, J. S., Santos, R. O., Kroloff, E. K. N., Heinen, J. T., Lai, Q., Black, B. D., … Adams, A. J. (2019). How has the quality of bonefishing changed over the past 40 years? Using local ecological knowledge to quantitatively inform population declines in the South Florida flats fishery. Environmental Biology of Fishes, 102(2), 285–298. Scopus. https://doi.org/10.1007/s10641-018-0831-2 Rehren, J., Wolff, M., & Jiddawi, N. (2018). Fisheries assessment of Chwaka Bay (Zanzibar) – following a holistic approach. Journal of Applied Ichthyology, 34(1), 117–128. Scopus. https://doi.org/10.1111/jai.13578 Reich, P. B., & Frelich, L. (2002). Temperate Deciduous Forests. In H. A. Mooney & J. G. Canadell (Eds.), The earth system: Biological and ecological dimensions of global environmental change. (Vol. 2, pp. 565–569). Chichester ; New York: Wiley. Retrieved from http://citeseerx.ist.psu.edu/viewdoc/download;jsessionid=C37252852ED31209F37AE 030E316BAA2?doi=10.1.1.657.2202&rep=rep1&type=pdf http://citeseerx.ist.psu.edu/viewdoc/download;jsessionid=C37252852ED31209F37AE 030E316BAA2?doi=10.1.1.657.2202&rep=rep1&type=pdf Reid, J., & Rout, M. (2018). Can sustainability auditing be indigenized? Agriculture and Human Values, 35(2), 283–294. https://doi.org/10.1007/s10460-017-9821-9 Reid, J., & Rout, M. (2020). Developing sustainability indicators – The need for radical transparency. Ecological Indicators, 110, 105941. https://doi.org/10.1016/j.ecolind.2019.105941 030E316BAA2?doi=10.1.1.657.2202&rep=rep1&type=pdf Reid, J., & Rout, M. (2018). Can sustainability auditing be indigenized? Agriculture and Human Values, 35(2), 283–294. https://doi.org/10.1007/s10460-017-9821-9 Reid, J., & Rout, M. (2020). Developing sustainability indicators – The need for radical transparency. Ecological Indicators, 110, 105941. https://doi.org/10.1016/j.ecolind.2019.105941 Reid, J., & Rout, M. (2018). Can sustainability auditing be indigenized? Agriculture and Human Values, 35(2), 283–294. https://doi.org/10.1007/s10460-017-9821-9 Reid, J., & Rout, M. (2020). Retrieved Developing sustainability indicators – The need for radical transparency. Ecological Indicators, 110, 105941. https://doi.org/10.1016/j.ecolind.2019.105941 466 Reid, W., Berkes, F., Wilbanks, T., & Capistrano, D. (2006). Bridging scales and knowledge systems: Linking global science and local knowledge in assessments. Millennium Ecosystem Assessment and Island Press, Washington DC. Reimer, J. K. (Kila), & Walter, P. (2013). How do you know it when you see it? Community- based ecotourism in the Cardamom Mountains of southwestern Cambodia. Tourism Management, 34, 122–132. https://doi.org/10.1016/j.tourman.2012.04.002 Reimoser, F., & Reimoser, S. (2016). Long-term trends of hunting bags and wildlife populations in Central Europe. 41, 29–43. Reinert, T. R., & Winter, K. A. (2002). Sustainability of harvested pacú (Colossoma macropomum) populations in the northeastern Bolivian Amazon. Conservation Biology, 16(5), 1344–1351. Remm, L., Runkla, M., & Lohmus, A. (2018). How Bilberry Pickers Use Estonian Forests: Implications for Sustaining a Non-Timber Value. Baltic Forestry, 24(2), 287–295. Remsen, J. V. (1995). The importance of continued collecting of bird specimens to ornithology and bird conservation. Bird Conservation International, 5(2–3), 146–180. https://doi.org/10.1017/S095927090000099X Rendón-Carmona, H., Martínez-Yrízar, A., Balvanera, P., & Pérez-Salicrup, D. (2009). Selective cutting of woody species in a Mexican tropical dry forest: Incompatibility between use and conservation. Forest Ecology and Management, 257(2), 567–579. https://doi.org/10.1016/j.foreco.2008.09.031 Reyes-Garcia, V., Menendez-Baceta, G., Aceituno-Mata, L., Acosta-Naranjo, R., Calvet-Mir, L., Dominguez, P., … Pardo-de-Santayana, M. (2015). From famine foods to delicatessen: Interpreting trends in the use of wild edible plants through cultural ecosystem services. Ecological Economics, 120, 303–311. https://doi.org/10.1016/j.ecolecon.2015.11.003 Reynolds, J. D., & Mace, G. M. (1999). Risk assessments of threatened species. Trends in Ecology & Evolution, 14(6), 215–217. https://doi.org/10.1016/S0169-5347(99)01629- 8 Rhodes, K. L., Tupper, M. H., & Wichilmel, C. B. (2008). Characterization and management of the commercial sector of the Pohnpei coral reef fishery, Micronesia. Coral Reefs, 27(2), 443–454. Scopus. https://doi.org/10.1007/s00338-007-0331-x Rhyne, A. L., Tlusty, M. F., Schofield, P. J., Kaufman, L., Morris, J. A., & Bruckner, A. W. (2012). Revealing the Appetite of the Marine Aquarium Fish Trade: The Volume and Biodiversity of Fish Imported into the United States. PLoS ONE, 7(5), e35808. https://doi.org/10.1371/journal.pone.0035808 Rhyne, A. L., Tlusty, M. F., Szczebak, J. T., & Holmberg, R. J. (2017). Expanding our understanding of the trade in marine aquarium animals. PeerJ, 5, e2949. https://doi.org/10.7717/peerj.2949 Ribe, R. G. (1989). The aesthetics of forestry: What has empirical preference research taught us? Environmental Management, 13(1), 55–74. https://doi.org/10.1007/BF01867587 Ribeiro, A. R., Damasio, L. M. A., & Silvano, R. A. M. (2021). Retrieved Fishers’ ecological knowledge to support conservation of reef fish (groupers) in the tropical Atlantic. Ocean & Coastal Management, 204, 105543. https://doi.org/10.1016/j.ocecoaman.2021.105543 467 Ribeiro, J. R., Azevedo-Ramos, C., & Nascimento dos Santos, R. B. (2020). Impact of forest concessions on local jobs in central amazon. Trees, Forests and People, 2, 100021. https://doi.org/10.1016/j.tfp.2020.100021 Ribeiro, N. S., Snook, L. K., Vaz, I. C. N. de C., & Alves, T. (2019). Gathering honey from wild and traditional hives in the Miombo woodlands of the Niassa National Reserve, Mozambique: What are the impacts on tree populations? Global Ecology and Conservation, 17(Parks 23 2 2017), e00552. https://doi.org/10.1016/j.gecco.2019.e00552 Ricard, D., Minto, C., Jensen, O. P., & Baum, J. K. (2012). Examining the knowledge base and status of commercially exploited marine species with the RAM Legacy Stock Assessment Database: The RAM Legacy Stock Assessment Database. Fish and Fisheries, 13(4), 380–398. https://doi.org/10.1111/j.1467-2979.2011.00435.x Rice, J. C., Shelton, P. A., Rivard, D., Chouinard, G. A., & Fréchet, A. (2003). Recovering Canadian Atlantic cod stocks: The shape of things to come? (No. CM 2003/U:06; p. 23). Copenhagen: International Council for Exploration of the Seas. Richards, S. J. & Suryadi, S. (2000). A Biodiversity Assessment of Yongsu – Cyclop Mountains and the Southern Mamberamo basin, Papua. Conservation International, Washington DC. Retrieved from https://www.nhbs.com/a-biodiversity-assessment-of-the-yongsu- cyclops-mountains-and-the-southern-mamberamo-basin-papua-indonesia-book Richardson, E., & Shackleton, C. M. (2014). The extent and perceptions of vandalism as a cause of street tree damage in small towns in the Eastern Cape, South Africa. Urban Forestry & Urban Greening, 13(3), 425–432. https://doi.org/10.1016/j.ufug.2014.04.003 Richardson, M., Cormack, A., McRobert, L., & Underhill, R. (2016). 30 days wild: Development and evaluation of a large-scale nature engagement campaign to improve well-being. PloS One, 11(2), e0149777. https://doi.org/10.1371/journal.pone.0149777 Richer, E. (2016, June 27). Chinese Furniture Exports Reach All-Time High in 2015. Retrieved March 31, 2021, from Forest Trends website: https://www.forest- trends.org/blog/chinese-furniture-exports-reach-all-time-high-in-2015/ Rife, A. N., Aburto-Oropeza, O., Hastings, P. A., Erisman, B., Ballantyne, F., Wielgus, J., … Gerber, L. (2013). Long-term effectiveness of a multi-use marine protected area on reef fish assemblages and fisheries landings. Journal of Environmental Management, 117, 276–283. Rights and Resources Initiative. (2014). What future for reform? Progress and slowdown in forest tenure reform since 2002. Washington DC. Retrieved from https://rightsandresources.org/en/publication/view/what-future-for-reform/ Rigolon, A., Browning, M., & Jennings, V. (2018). Inequities in the quality of urban park systems: An environmental justice investigation of cities in the United States. Landscape and Urban Planning, 178, 156–169. https://doi.org/10.1016/j.landurbplan.2018.05.026 Ripple, W. Retrieved J., Estes, J. A., Beschta, R. L., Wilmers, C. C., Ritchie, E. G., Hebblewhite, M., … Wirsing, A. J. (2014). Status and Ecological Effects of the World’s Largest Carnivores. Science, 343(6167), 1241484–1241484. https://doi.org/10.1126/science.1241484 468 Ripple, William J., Abernethy, K., Betts, M. G., Chapron, G., Dirzo, R., Galetti, M., … Young, H. (2016). Bushmeat hunting and extinction risk to the world’s mammals. Royal Society Open Science, 3(10), 160498. https://doi.org/10.1098/rsos.160498 Ripple, William J., Newsome, T. M., Wolf, C., Dirzo, R., Everatt, K. T., Galetti, M., … Valkenburgh, B. V. (2015). Collapse of the world’s largest herbivores. Science Advances, 1(4), e1400103. https://doi.org/10.1126/sciadv.1400103 Rivera, A., Gelcich, S., García-Flórez, L., & Acuña, J. L. (2016). Assessing the sustainability and adaptive capacity of the gooseneck barnacle co-management system in Asturias, N. Spain. Ambio, 45(2), 230–240. Scopus. https://doi.org/10.1007/s13280-015-0687-z Rivera, A., Gelcich, S., García-Flórez, L., & Acuña, J. L. (2017). Trends, drivers, and lessons from a long-term data series of the Asturian (northern Spain) gooseneck barnacle territorial use rights system. Bulletin of Marine Science, 93(1), 35–51. Scopus. https://doi.org/10.5343/bms.2015.1080 Robbins, P., Emery, M., & Rice, J. L. (2008). Gathering in Thoreau’s backyard: Nontimber forest product harvesting as practice. Area, 40(2), 265–277. https://doi.org/10.1111/j.1475-4762.2008.00794.x Roberts, D. L., & Solow, A. R. (2008). The effect of the Convention on International Trade in Endangered Species on scientific collections. Proceedings. Biological Sciences / The Royal Society, 275(1637), 987–989. https://doi.org/10.1098/rspb.2007.1683 Robertson, J. M. Y., & van Schaik, C. P. (2001). Causal factors underlying the dramatic decline of the Sumatran orang-utan. 13. Robiglio, V., Acevedo, M. R., & Simauchi, E. C. (2015). Diagnóstico de los productores familiares en la Amazonía Peruana. Lima, Perú.: ICRAF Oficina Regional para América Latina. Robinson, J., Cinner, J. E., & Graham, N. A. J. (2014). The influence of fisher knowledge on the susceptibility of reef fish aggregations to fishing. PLoS ONE, 9(3). Scopus. https://doi.org/10.1371/journal.pone.0091296 Robinson, J. G., & Bennett, E. L. (2004). Having your wildlife and eating it too: An analysis of hunting sustainability across tropical ecosystems. Animal Conservation, 7(4), 397– 408. https://doi.org/10.1017/S1367943004001532 Robinson, N. B., Krieger, K., Khan, F. M., Huffman, W., Chang, M., Naik, A., … Gaudino, M. (2019). The current state of animal models in research: A review. International Journal of Surgery (London, England), 72, 9–13. https://doi.org/10.1016/j.ijsu.2019.10.015 Robotham, H., Bustos, E., Ther-Rios, F., Avila, M., Robotham, M., Hidalgo, C., & Muñoz, J. (2019). Contribution to the study of sustainability of small-scale artisanal fisheries in Chile. Marine Policy, 106. Retrieved Scopus. https://doi.org/10.1016/j.marpol.2019.103514 Rocha, D., Drakeford, B., Marley, S. A., Potts, J., Hale, M., & Gullan, A. (2020). Moving towards a sustainable cetacean-based tourism industry – A case study from Mozambique. Marine Policy, 120, 104048. https://doi.org/10.1016/j.marpol.2020.104048 Rocha, L. A., Aleixo, A., Allen, G., Almeda, F., Baldwin, C. C., Barclay, M. V., … Witt, C. C. (2014). Specimen collection: An essential tool. Science, 344(6186), 814–815. https://doi.org/10.1126/science.344.6186.814 469 Roditi, K., & Vafidis, D. (2019). Net fisheries’métiers in the eastern Mediterranean: Insights for small-scale fishery management on Kalymnos Island. Water (Switzerland), 11(7). Scopus. https://doi.org/10.3390/w11071509 Rodríguez, C., Rollins-Smith, L., Ibáñez, R., Durant-Archibold, A. A., & Gutiérrez, M. (2017). Toxins and pharmacologically active compounds from species of the family Bufonidae (Amphibia, Anura). Journal of Ethnopharmacology, 198, 235–254. https://doi.org/10.1016/j.jep.2016.12.021 Roeger, J., Foale, S., & Sheaves, M. (2016). When “fishing down the food chain” results in improved food security: Evidence from a small pelagic fishery in Solomon Islands. Fisheries Research, 174, 250–259. Scopus. https://doi.org/10.1016/j.fishres.2015.10.016 Roeland, S., Moretti, M., Amorim, J. H., Branquinho, C., Fares, S., Morelli, F., … Calfapietra, C. (2019). Towards an integrative approach to evaluate the environmental ecosystem services provided by urban forest. Journal of Forestry Research, 30(6), 1981–1996. https://doi.org/10.1007/s11676-019-00916-x Rokaya, M. B., Münzbergová, Z., & Dostálek, T. (2017). Sustainable harvesting strategy of medicinal plant species in Nepal – results of a six-year study. Folia Geobotanica, 52(2), 239–252. https://doi.org/10.1007/s12224-017-9287-y Rolando, A., Caprio, E., Rinaldi, E., & Ellena, I. (2006). The impact of high-altitude ski-runs on alpine grassland bird communities: Ski-runs and alpine grassland bird communities. Journal of Applied Ecology, 44(1), 210–219. https://doi.org/10.1111/j.1365- 2664.2006.01253.x Rondeau, D., Perry, B., & Grimard, F. (2020). The Consequences of COVID-19 and Other Disasters for Wildlife and Biodiversity. Environmental and Resource Economics, 76(4), 945–961. https://doi.org/10.1007/s10640-020-00480-7 Rønsted, N., Zubov, D., Bruun-Lund, S., & Davis, A. P. (2013). Snowdrops falling slowly into place: An improved phylogeny for Galanthus (Amaryllidaceae). Molecular Phylogenetics and Evolution, 69(1), 205–217. https://doi.org/10.1016/j.ympev.2013.05.019 Root, T. L., Price, J. T., Hall, K. R., Schneider, S. H., Rosenzweig, C., & Pounds, J. A. (2003). Fingerprints of global warming on wild animals and plants. Nature, 421(6918), 57–60. https://doi.org/10.1038/nature01333 Rosa, I. L., Oliveira, T. P., Osório, F. M., Moraes, L. E., Castro, A. L., Barros, G. M., & Alves, R. R. (2011). Fisheries and trade of seahorses in Brazil: Historical perspective, current trends, and future directions. Biodiversity and Conservation, 20(9), 1951–1971. Rosenberg, A. A., Kleisner, K. M., Afflerbach, J., Anderson, S. Retrieved C., Dickey-Collas, M., Cooper, A. B., … Ye, Y. (2018). Applying a New Ensemble Approach to Estimating Stock Status of Marine Fisheries around the World: Estimating global fisheries status. Conservation Letters, 11(1), e12363. https://doi.org/10.1111/conl.12363 Rossant, J., & Mummery, C. (2012). NOBEL 2012 Physiology or medicine: Mature cells can be rejuvenated. Nature, 492(7427), 56. https://doi.org/10.1038/492056a Rounsevell, M. D., Harfoot, M., Harrison, P. A., Newbold, T., Gregory, R. D., & Mace, G. M. (2020). A biodiversity target based on species extinctions. Science, 368(6496), 1193– 1195. https://doi.org/10.1126/science.aba6592 470 ROUTES. (2020). Runway To Extinction—Wildlife Trafficking in the Air Transport Sector (p. 112). Retrieved from https://routespartnership.org/industry- resources/publications/routes_runwaytoextinction_fullreport.pdf/view from ROUTES. (2022). Wildlife Trafficking. Retrieved March 1, 2022, from ROUTES website: https://routespartnership.org/about-routes/background/background Roux, M.-J., Tallman, R. F., & Martin, Z. A. (2019). Small-scale fisheries in Canada’s Arctic: Combining science and fishers knowledge towards sustainable management. Marine Policy, 101, 177–186. Scopus. https://doi.org/10.1016/j.marpol.2018.01.016 Rowland, S. J., Sutton, P. A., & Knowles, T. D. J. (2019). The age of ambergris. Natural Product Research, 33(21), 3134–3142. https://doi.org/10.1080/14786419.2018.1523163 Royse, D. J. (2014). A global perspective on the high five: Agaricus, Pleurotus, Lentinula, Auricularia & Flammulina. Proceedings of the 8th International Conference on Mushroom Biology and Mushroom Products (ICMBMP8). Citeseer. Rozemeijer, N. (2000). Community-based tourism in Botswana: The SNV experience in 3 community tourism projects. In N. Rozemeijer, T. Gujadhur, C. Motshubi, E. van den Berg, & M. V. Flyman (Eds.), SNV/IUCN CBNRM Support Programme: Gaborone, Botswana (pp. 17–20). Retrieved from http://www.bibalex.org/Search4Dev/files/284060/116197.pdf Rozemeijer, N., & Aggrey, J. (2011). Securing legal domestic lumber supply through multi- stakeholder dialogue in Ghana. Wageningen: Tropenbos International. Retrieved from http://edepot.wur.nl/214614 RRI. (2015). Who Owns the World’s Land? A Global Baseline of Formally Recognized Indigenous and Community Land Rights. Washington DC: Rights and Resources Initiative. Retrieved from Rights and Resources Initiative website: https://rightsandresources.org/wp-content/uploads/GlobalBaseline_web.pdf Rubio-Cisneros, N. T., Aburto-Oropeza, O., & Ezcurra, E. (2016). Small-scale fisheries of lagoon estuarine complexes in northwest Mexico. Tropical Conservation Science, 9(1), 78–134. Scopus. https://doi.org/10.1177/194008291600900106 Rubio-Cisneros, N. T., Aburto-Oropeza, O., Jackson, J., & Ezcurra, E. (2017). Coastal exploitation throughout Marismas Nacionales Wetlands in Northwest Mexico. Tropical Conservation Science, 10. Scopus. https://doi.org/10.1177/1940082917697261 Ruddle, K., & Ishige, N. (2010). On the origins, diffusion and cultural context of fermented fish products in Southeast Asia. Globalization, Food and Social Identities in the Asia Pacific Region, 1–17. Ruel, J.-C., Fortin, D., & Pothier, D. (2013). Partial cutting in old-growth boreal stands: An integrated experiment. The Forestry Chronicle, 89(03), 360–369. https://doi.org/10.5558/tfc2013-066 Ruid, D. B., Paul, W. J., Roell, B. J., Wydeven, A. P., Willging, R. C., Jurewicz, R. L., & Lonsway, D. H. (2009). Wolf–Human Conflicts and Management in Minnesota, Wisconsin, and Michigan. In A. P. Wydeven, T. R. Van Deelen, & E. J. Heske (Eds.), Recovery of Gray Wolves in the Great Lakes Region of the United States (pp. 279–295). New York, NY: Springer New York. https://doi.org/10.1007/978-0-387-85952-1_18 471 Runstrom, A., Bruch, R. M., Reiter, D., & Cox, D. (2002). Lake sturgeon (Acipenser fulvescens) on the Menominee Indian Reservation: An effort toward co-management and population restoration. Journal of Applied Ichthyology, 18(4–6), 481–485. https://doi.org/10.1046/j.1439-0426.2002.00426.x Rupprecht, C. from D. D., Byrne, J. A., Garden, J. G., & Hero, J.-M. (2015). Informal urban green space: A trilingual systematic review of its role for biodiversity and trends in the literature. Urban Forestry & Urban Greening, 14(4), 883–908. https://doi.org/10.1016/j.ufug.2015.08.009 Russell, R., Guerry, A. D., Balvanera, P., Gould, R. K., Basurto, X., Chan, K. M., … Tam, J. (2013). Humans and nature: How knowing and experiencing nature affect well-being. Annual Review of Environment and Resources, 38, 473–502. https://doi.org/10.1146/annurev-environ-012312-110838 Russo, A., & Cirella, G. T. (2020). Edible Green Infrastructure for Urban Regeneration and Food Security: Case Studies from the Campania Region. Agriculture, 10(8), 358. https://doi.org/10.3390/agriculture10080358 Russo, A., Escobedo, F. J., Cirella, G. T., & Zerbe, S. (2017). Edible green infrastructure: An approach and review of provisioning ecosystem services and disservices in urban environments. Agriculture, Ecosystems & Environment, 242, 53–66. https://doi.org/10.1016/j.agee.2017.03.026 Russo, D., Ancillotto, L., Hughes, A. C., Galimberti, A., & Mori, E. (2017). Collection of voucher specimens for bat research: Conservation, ethical implications, reduction, and alternatives. Mammal Review, 47(4), 237–246. https://doi.org/10.1111/mam.12095 Russo, I.-R. M., Hoban, S., Bloomer, P., Kotzé, A., Segelbacher, G., Rushworth, I., … Bruford, M. W. (2019). ‘Intentional Genetic Manipulation’ as a conservation threat. Conservation Genetics Resources, 11(2), 237–247. https://doi.org/10.1007/s12686- 018-0983-6 Russow, L.-M., & Theran, P. (2003). Ethical issues concerning animal research outside the laboratory. ILAR Journal, 44(3), 187–190. https://doi.org/10.1093/ilar.44.3.187 Sá, R. M. M., da Silva, M. F., Sousa, F. M., & Minhós, T. (2012). The Trade and Ethnobiological Use of Chimpanzee Body Parts in Guinea-Bissau. TRAFFIC Bulletin, 24(1), 31–34. Saalfeld, K., Fukuda Y., Duldig T., & Fisher A. (2016). Management Program for the Saltwater Crocodile (Crocodylus porosus) in the Northern Territory of Australia, 2016- 2020. Northern Territory Department of Environment and Natural Resources, Darwin. Saarinen, J., Moswete, N., Atlhopheng, J. R., & Hambira, W. L. (2020). Changing socio- ecologies of Kalahari: Local perceptions towards environmental change and tourism in Kgalagadi, Botswana. Development Southern Africa, 37(5), 855–870. https://doi.org/10.1080/0376835X.2020.1809997 Saayman, M., van der Merwe, P., & Saayman, A. (2018). The economic impact of trophy hunting in the south African wildlife industry. Global Ecology and Conservation, 16, e00510. https://doi.org/10.1016/j.gecco.2018.e00510 Sabater, L. (2020). Gender, culture, and sustainability in the Mediterranean [IUCN: International Union for Conservation of Nature]. IUCN: International Union for Conservation of Nature. Retrieved from IUCN: International Union for Conservation 472 of Nature website: https://policycommons.net/artifacts/1368648/gender-culture-and- sustainability-in-the-mediterranean/1982816/ of Nature website: https://policycommons.net/artifacts/1368648/gender-culture-and- sustainability-in-the-mediterranean/1982816/ Sabogal, C., de Jong, W., Pokorny, B., & Louman, B. (Eds.). (2008). from Manejo forestal comunitario en América Latina experiencias, lecciones aprendidas y retos para el futuro. Belém, PA: CIFOR: CATIE. Sada, S. G. (2019). The Mexican biosphere reserves: Landscape and sustainability. In UNESCO Biosphere Reserves (pp. 47–48). Routledge. Sáenz-Arroyo, A., & Revollo-Fernández, D. (2016). Local ecological knowledge concurs with fishing statistics: An example from the abalone fishery in Baja California, Mexico. Marine Policy, 71, 217–221. Scopus. https://doi.org/10.1016/j.marpol.2016.06.006 Sáenz–Arroyo, A., Roberts, C. M., Torre, J., & Cariño‐Olvera, M. (2005). Using fishers’ anecdotes, naturalists’ observations and grey literature to reassess marine species at risk: The case of the Gulf grouper in the Gulf of California, Mexico. Fish and Fisheries, 6(2), 121–133. Safford, H. D., & Vallejo, V. R. (2019). Ecosystem management and ecological restoration in the Anthropocene: Integrating global change, soils, and disturbance in boreal and Mediterranean forests. In Developments in Soil Science (Vol. 36, pp. 259–308). Elsevier. https://doi.org/10.1016/B978-0-444-63998-1.00012-4 Sahley, C. T., Vargas, J. T., & Valdivia, J. S. (2007). Biological sustainability of live shearing of vicuna in Peru. Conserv Biol, 21(1), 98–105. https://doi.org/10.1111/j.1523- 1739.2006.00558.x Sainsbury, K. (2000). Design of operational management strategies for achieving fishery ecosystem objectives. ICES Journal of Marine Science, 57(3), 731–741. https://doi.org/10.1006/jmsc.2000.0737 Saito, H., & Mitsumata, G. (2008). Bidding Customs and Habitat Improvement for Matsutake (Tricholoma matsutake) in Japan. Economic Botany, 62(3), 257–268. https://doi.org/10.1007/s12231-008-9034-7 Sakai, S., Choy, Y. K., Kishimoto-Yamada, K., Takano, K. T., Ichikawa, M., Samejima, H., … Itioka, T. (2016). Social and ecological factors associated with the use of non-timber forest products by people in rural Borneo. Biological Conservation, 204(Plants 54 2009), 340–349. https://doi.org/10.1016/j.biocon.2016.10.022 Sakakibara, C. (2020). Whale Snow: Iñupiat, Climate Change, and Multispecies Resilience in Arctic Alaska. University of Arizona Press. Sala, E., Aburto-Oropeza, O., Reza, M., Paredes, G., & López-Lemus, L. G. (2004). Fishing Down Coastal Food Webs in the Gulf of California. Fisheries, 29(3), 19–25. https://doi.org/10.1577/1548-8446(2004)29[19:FDCFWI]2.0.CO;2 Saldaña-Ruiz, L. E., Sosa-Nishizaki, O., & Cartamil, D. (2017). Historical reconstruction of Gulf of California shark fishery landings and species composition, 1939–2014, in a data-poor fishery context. Fisheries Research, 195, 116–129. Scopus. https://doi.org/10.1016/j.fishres.2017.07.011 Salinas, E., Wallace, R., Painter, L., Lehm, Z., Loayza, O., Pabón, C., & Ramírez, A. (2017). The Environmental, Economic and Sociocultural Value of Indigenous Territorial Management in the Greater Madidi Landscape Executive Summary (p. 50). La Paz, Bolivia: CIPTA, CIPLA and WCS. 473 Salo, M., Sirén, A., & Kalliola, R. (2013). Changing the Law of the Jungle. In Diagnosing Wild Species Harvest (pp. 161–177). Elsevier. from https://doi.org/10.1016/B978-0-12-397204- 0.00009-7 Salvatori, V., Donfrancesco, V., Trouwborst, A., Boitani, L., Linnell, J. D. C., Alvares, F., … Ciucci, P. (2020). European agreements for nature conservation need to explicitly address wolf-dog hybridisation. Biological Conservation, 248, 108525. https://doi.org/10.1016/j.biocon.2020.108525 Samanta, C., Bhaumik, U., & Patra, B. (2016). Socio-economic status of the fish curers of the dry fish industry of the coastal areas of West Bengal, India. International Journal of Current Research and Academic Review, 4(5), 84–100. Samaranayaka, S., Perera, A. N. F., & Warnasuriya, N. (2013). Food Habits among Adolescents in Colombo , Sri Lanka. Samdrup, T. (2011). Improving the contribution of community forestry to poverty reduction in Bhutan. In Community forestry in Bhutan: Putting people at the heart of poverty reduction (pp. 5–16). Ugyen Wangchuck Institute for Conservation and Environment and Social Forestry Division,. Retrieved from http://www.uwice.gov.bt/admin_uwice/publications/publication_files/Reports/2011/U WICER-CFIB.pdf Samiti, R. S. (2020, May 15). Bajhang residents begin cultivation amidst ban on Yarsagumba collection. The Himalayan Times. Retrieved from https://thehimalayantimes.com/nepal/ bajhang-residents-begin-cultivation-amidst-ban- on-yarsagumba-harvest Sampaio, M. B., Schmidt, I. B., & Figueiredo, I. B. (2008). Harvesting Effects and Population Ecology of the Buriti Palm (Mauritia flexuosa L. f., Arecaceae) in the Jalapão Region, Central Brazil1. Economic Botany, 62(2), 171–181. https://doi.org/10.1007/s12231- 008-9017-8 Sampson, R. N., Bystriakova, N., Brown, S., Gonzalez, P., Irland, L. L., Kauppi, P., … Thompson, I. D. (2005). Timber, Fuel, and Fiber. In Current State and Trends. Millienium Ecosystem Assessment. Washington DC: Island Press. Retrieved from https://www.millenniumassessment.org/documents/document.278.aspx.pdf Samy-Kamal, M. (2015). Status of fisheries in Egypt: Reflections on past trends and management challenges. Reviews in Fish Biology and Fisheries, 25(4), 631–649. Samyshev, E. Z., & Rubinstein, I. G. (1988). Change in structure of plankton and benthos in the Black Sea by the anthropogenic factors. Proceedings of the III Vsecoyuznaya Konferentziya Po Morskoy Biologii, Sevastopol, Octobre 1988, Volume 2, Kiev., 137– 139. Sánchez-García, C., Urda, V., Lambarri, M., Prieto, I., Andueza, A., & Villanueva, L. F. (2021). Evaluation of the economics of sport hunting in Spain through regional surveys. International Journal of Environmental Studies, 78(3), 517–531. https://doi.org/10.1080/00207233.2020.1759305 Sánchez-Jiménez, A., Fujitani, M., MacMillan, D., Schlüter, A., & Wolff, M. (2019). Connecting a trophic model and local ecological knowledge to improve fisheries management: The case of gulf of Nicoya, Costa Rica. Frontiers in Marine Science, 6(MAR). Scopus. https://doi.org/10.3389/fmars.2019.00126 474 Sandell, K., Arnegård, J., & Backman, E. (Eds.). (2011). from Friluftssport och äventyrsidrott: Utmaningar för lärare, ledare och miljö i en föränderlig värld [Outdoor sport and adventure sport – Challenges for teachers, leaders and environments in a changing world]. Lund: Studentlitteratur. Sandifer, P. A., Sutton-Grier, A. E., & Ward, B. P. (2015). Exploring connections among nature, biodiversity, ecosystem services, and human health and well-being: Opportunities to enhance health and biodiversity conservation. Ecosystem Services, 12, 1–15. https://doi.org/10.1016/j.ecoser.2014.12.007 Santiago-Ávila, F. J., & Lynn, W. S. (2020). Bridging compassion and justice in conservation ethics. Biological Conservation, 248, 108648. https://doi.org/10.1016/j.biocon.2020.108648 SantoDomingo, A. F., Castro-Díaz, L., González-Uribe, C., Wayúu Community of Marbacella and El Horno, & Barí Community of Karikachaboquira. (2016). Ecosystem Research Experience with Two Indigenous Communities of Colombia: The Ecohealth Calendar as a Participatory and Innovative Methodological Tool. Ecohealth, 13(4), 687–697. https://doi.org/10.1007/s10393-016-1165-1 Santos, C. A. B., & Nóbrea Alves, R. R. (2016). Ethnoichthyology of the indigenous Truká people, Northeast Brazil. Journal of Ethnobiology and Ethnomedicine, 12(1). Scopus. https://doi.org/10.1186/s13002-015-0076-5 Santos, R. E., Pinto-Coelho, R. M., Fonseca, R., Simões, N. R., & Zanchi, F. B. (2018). The decline of fisheries on the Madeira River, Brazil: The high cost of the hydroelectric dams in the Amazon Basin. Fisheries Management and Ecology, 25(5), 380–391. https://doi.org/10.1111/fme.12305 Santos, R. O., Rehage, J. S., Kroloff, E. K. N., Heinen, J. E., & Adams, A. J. (2019). Combining data sources to elucidate spatial patterns in recreational catch and effort: Fisheries- dependent data and local ecological knowledge applied to the South Florida bonefish fishery. Environmental Biology of Fishes, 102(2), 299–317. Scopus. https://doi.org/10.1007/s10641-018-0828-x Santos Thykjaer, V., dos Santos Rodrigues, L., Haimovici, M., & Cardoso, L. G. (2019). Long- term changes in fishery resources of an estuary in southwestern Atlantic according to local ecological knowledge. Fisheries Management and Ecology. Scopus. https://doi.org/10.1111/fme.12398 Sanuma, O. I., Tokimoto, K., Sanuma, C., Autuori, J., Sanuma, L. R., Sanuma, M., … Apiamö, R. M. (2016). Cogumelos. Enciclopédia dos Alimentos Yanomami (Sanöma)/Ana amopö. Sanöma samakönö sama tökö nii pewö oa wi i tökö waheta. São Paulo/Boa Vista: Instituto Sociambiental/Hutukara Associação Yanomami. Retrieved from https://acervo.socioambiental.org/acervo/publicacoes-isa/enciclopedia-dos-alimentos- yanomami-sanoma-cogumelos Sanuma, Oscar Ipoko, Sanuma, C., Martins, M. S., Tokimoto, K., Instituto Socioambiental (Brazil), & Hutukara Associação Yanomami (Eds.). (2016). Sanöma samakönö sama tökö nii pewö oa wi ĩ tökö waheta. Ana amopö = Enciclopédia dos alimentos Yanomami (Sanöma). Cogumelos. Boa Vista, Roraima, Brasil : São Paulo, SP, Brasil: Hutukara Associação Yanomami ; Instituto Socioambiental. 475 Sardeshpande, M., & Shackleton, C. (2019). from Slow response of societies to new problems: Causes and costs. Ecosystems, 6(5), 493–502. Scheffers, B. R., Oliveira, B. F., Lamb, I., & Edwards, D. P. (2019). Global wildlife trade across the tree of life. Science, 366(6461), 71–76. https://doi.org/10.1126/science.aav5327 Schemmel, E., Friedlander, A. M., Andrade, P., Keakealani, K., Castro, L. M., Wiggins, C., … Kittinger, J. N. (2016). The codevelopment of coastal fisheries monitoring methods to support local management. Ecology and Society, 21(4). Scopus. https://doi.org/10.5751/ES-08818-210434 Schiller, L., Alava, J. J., Grove, J., Reck, G., & Pauly, D. (2015). The demise of Darwin’s fishes: Evidence of fishing down and illegal shark finning in the Galápagos Islands. Aquatic Conservation: Marine and Freshwater Ecosystems, 25(3), 431–446. Scopus. https://doi.org/10.1002/aqc.2458 Schlacher, T. A., Thompson, L., & Price, S. (2007). Vehicles versus conservation of invertebrates on sandy beaches: Mortalities inflicted by off-road vehicles on ghost crabs. Marine Ecology, 28(3), 354–367. https://doi.org/10.1111/j.1439- 0485.2007.00156.x Schlaepfer, M. A., Hoover, C., & Dodd, C. K. (2005b). Challenges in Evaluating the Impact of the Trade in Amphibians and Reptiles on Wild Populations. BioScience, 55(3), 256. https://doi.org/10.1641/0006-3568(2005)055[0256:CIETIO]2.0.CO;2 Schmidt, I. B., Figueiredo, I. B., & Scariot, A. (2007). Ethnobotany and effects of harvesting on the population ecology of Syngonanthus nitens (Bong.) Ruhland (Eriocaulaceae), a NTFP from Jalapao Region, Central Brazil. Economic Botany, 61(1), 73–85. https://doi.org/Doi 10.1663/0013-0001(2007)61[73:Eaeoho]2.0.Co;2 Schmidt, I. B., & Ticktin, T. (2012). When lessons from population models and local ecological knowledge coincide—Effects of flower stalk harvesting in the Brazilian savanna. Biological Conservation, 152, 187–195. https://doi.org/10.1016/j.biocon.2012.03.018 Schmidt, Isabel B., Mandle, L., Ticktin, T., & Gaoue, O. G. (2011). What do matrix population models reveal about the sustainability of non-timber forest product harvest?: Evaluating NTFP harvest sustainability. Journal of Applied Ecology, 48(4), 815–826. https://doi.org/10.1111/j.1365-2664.2011.01999.x Schmidt, J., Cruse-Sanders, J., Chamberlain, J. L., Ferreira, S., & Young, J. A. (2019). Explaining harvests of wild-harvested herbaceous plants: American ginseng as a case study. Biological Conservation, 231(Nature 452 2008), 139–149. https://doi.org/10.1016/j.biocon.2019.01.006 Schmidt, S. (2004). World wide plaza: The corporatization of urban public space. IEEE Technology and Society Magazine, 23(3), 17–18. Schmidt, S. (2004). World wide plaza: The corporatization of urban public space. IEEE Technology and Society Magazine, 23(3), 17–18. Schmink, M., & García, M. (2015). Under the canopy: Gender and forests in Amazonia. Center for International Forestry Research (CIFOR). https://doi.org/10.17528/cifor/005505 Schmitt, L., & Rempel, D. (2019). The Role of well-regulated Hunting Tourism in Namibia – in effective Conservation Management. In Universities, Entrepreneurship and Enterprise Development in Africa (Vol. from Wild Edible Fruits: A Systematic Review of an Under-Researched Multifunctional NTFP (Non-Timber Forest Product). Forests, 10(6), 467. https://doi.org/10.3390/f10060467 Sardeshpande, M., & Shackleton, C. (2020a). Fruits of the Veld: Ecological and Socioeconomic Patterns of Natural Resource Use across South Africa. Human Ecology, 48(6), 665–677. https://doi.org/10.1007/s10745-020-00185-x Sardeshpande, M., & Shackleton, C. (2020b). Urban foraging: Land management policy, perspectives, and potential. PLoS One, 15(4), e0230693. https://doi.org/10.1371/journal.pone.0230693 Saremba, J., & Gill, A. (1991). Value conflicts in mountain park settings. Annals of Tourism Research, 18(3), 455–472. https://doi.org/10.1016/0160-7383(91)90052-D Sartor, P., Carbonara, P., Cerasi, S., Lembo, G., Facchini, M. T., Lucchetti, A., … Spedicato, M. T. (2019). A selective and low impacting traditional fishery, sustaining the economy of small coastal villages in central Mediterranean: Keep or replace the small-scale driftnets? Fisheries Management and Ecology, 26(6), 661–673. Scopus. https://doi.org/10.1111/fme.12397 Sato, C. F., Wood, J. T., & Lindenmayer, D. B. (2013). The Effects of Winter Recreation on Alpine and Subalpine Fauna: A Systematic Review and Meta-Analysis. PLoS ONE, 8(5), e64282. https://doi.org/10.1371/journal.pone.0064282 Satumanatpan, S., & Pollnac, R. (2017). Factors influencing the well-being of small-scale fishers in the Gulf of Thailand. Ocean & Coastal Management, 142, 37–48. https://doi.org/10.1016/j.ocecoaman.2017.03.023 Satz, D., Gould, R. K., Chan, K. M., Guerry, A., Norton, B., Satterfield, T., … others. (2013). The challenges of incorporating cultural ecosystem services into environmental assessment. Ambio, 42(6), 675–684. https://doi.org/10.1007/s13280-013-0386-6 Savi, M. K., Noumonvi, R., Chadaré, F. J., Daïnou, K., Salako, V. K., Idohou, R., … Glèlè Kakaï, R. (2019). Synergy between traditional knowledge of use and tree population structure for sustainability of Cola nitida (Vent.) Schott. & Endl in Benin (West Africa). Environment, Development and Sustainability, 21(3), 1357–1368. https://doi.org/10.1007/s10668-018-0091-5 Saville, M. H. (1925). The Wood-Carver ́s Art in Ancient Mexico. New York, Museum of the American Inidan, Heye Foundation. Retrieved from https://archive.org/details/woodcarversartin00savi/page/n21/mode/2up Saylor, C. R., Alsharif, D. K. A., & Torres, H. (2017). The importance of traditional ecological knowledge in agroecological systems in Peru. International Journal of Biodiversity Science, Ecosystem Services & Management, 13(1), 150–161. https://doi.org/10.1080/21513732.2017.1285814 Schaal, B. (1993). Schaal B. 1993. Des remèdes et des corps, gérer ‘la force’: Aspects d’une approche ethnobotanique dans une vallée vosgienne. Écologie humaine, 11 (1): 23-45. Écologie humaine, 11(1), 23–45. Scheffer, M., & van Nes, E. H. (2004). Mechanisms for marine regime shifts: Can we use lakes as microcosms for oceans? Progress in Oceanography, 60(2–4), 303–319. https://doi.org/10.1016/j.pocean.2004.02.008 476 Scheffer, M., Westley, F., & Brock, W. (2003). from 7, pp. 98–117). German African University Partnership Platform for the Development of Entrepreneurs and Small/Medium Enterprises. Retrieved from https://ideas.repec.org/h/sau/ueedcc/07098-117.html 477 Schroeder, D. M., & Love, M. S. (2002). Recreational fishing and marine fish populations in California. California Cooperative Oceanic Fisheries Investigations Report, 182–190. Schroeder-Wildberg, E., & Carius, A. (2005). Illegal logging, conflict and the business sector in Indonesia. Berlin: InWEnt [u.a.]. Schuhbauer, A., & Sumaila, U. R. (2016). Economic viability and small-scale fisheries—A review. Ecological Economics, 124, 69–75. Scopus. https://doi.org/10.1016/j.ecolecon.2016.01.018 Schulp, C. J. E., Thuiller, W., & Verburg, P. H. (2014). Wild food in Europe: A synthesis of knowledge and data of terrestrial wild food as an ecosystem service. Ecological Economics, 105, 292–305. https://doi.org/10.1016/j.ecolecon.2014.06.018 Schultes, R. E., & Hofmann, A. (1979). Plants of the gods. London: McGraw & Hill,. Retrieved from https://archive.org/details/SchultesHofmannPlantsOfTheGodsHealingArts2001 Schultze, V., D’Agosto, V., Wack, A., Novicki, D., Zorn, J., & Hennig, R. (2008). Safety of MF59TM adjuvant. Vaccine, 26(26), 3209–3222. https://doi.org/10.1016/j.vaccine.2008.03.093 Schulze, M. D. (2003). Ecology and Behavior of Nine Timber Tree Species in Pará, Brazil: Links between Species Life History and Forest Management and Conservation. University Park: The Pennsylvania State University. Schulze, M, Vidal, E., Grogan, J., Zweede, J., & Zarin, D. (2005). Madeiras nobres em perigo: Práticas e leis atuais de manejo florestal não garantem exploração sustentável. Revista Ciência Hoje, 241(36), 66–69. Schulze, Mark, Grogan, J., Landis, R. M., & Vidal, E. (2008). How rare is too rare to harvest? Forest Ecology and Management, 256(7), 1443–1457. https://doi.org/10.1016/j.foreco.2008.02.051 Schunko, C., Grasser, S., & Vogl, C. R. (2015). Explaining the resurgent popularity of the wild: Motivations for wild plant gathering in the Biosphere Reserve Grosses Walsertal, Austria. Journal of Ethnobiology and Ethnomedicine, 11(1), 55. https://doi.org/10.1186/s13002-015-0032-4 Schunko, C., & Vogl, C. R. (2018). Is the Commercialization of Wild Plants by Organic Producers in Austria Neglected or Irrelevant? Sustainability, 10(11), 1–14. Schunko, C., Wild, A.-S., & Brandner, A. (2021). Exploring and limiting the ecological impacts of urban wild food foraging in Vienna, Austria. Urban Forestry & Urban Greening, 62, 127164. https://doi.org/10.1016/j.ufug.2021.127164 Schweinsberg, S., Darcy, S., & Wearing, S. L. (2018). Repertory grids and the measurement of levels of community support for rural ecotourism development. Journal of Ecotourism, 17(3), 239–251. https://doi.org/10.1080/14724049.2018.1502936 Schwoerer, T., Knowler, D., & Garcia-Martinez, S. (2016). The value of whale watching to local communities in Baja, Mexico: A case study using applied economic rent theory. Ecological Economics, 127, 90–101. https://doi.org/10.1016/j.ecolecon.2016.03.004 Sciberras, M., Hiddink, J. from G., Jennings, S., Szostek, C. L., Hughes, K. M., Kneafsey, B., … Kaiser, M. J. (2018). Response of benthic fauna to experimental bottom fishing: A global meta-analysis. Fish and Fisheries, 19(4), 698–715. https://doi.org/10.1111/faf.12283 478 Scott, D., & Gössling, S. (2015). What could the next 40 years hold for global tourism? Tourism Recreation Research, 40(3), 269–285. Scott, D., & Gössling, S. (2015). What could the next 40 years hold for global tourism? Tourism Recreation Research, 40(3), 269–285. https://doi.org/10.1080/02508281.2015.1075739 Scott, R. E., Neyland, M. G., & Baker, S. C. (2019). Variable retention in Tasmania, Australia: Trends over 16 years of monitoring and adaptive management. Ecological Processes, 8(1), 23. https://doi.org/10.1186/s13717-019-0174-8 Seafish. (2018). Fishmeal and fish oil facts and figures. Seafish. Retrieved from Seafish website: https://www.seafish.org/document/?id=1b08b6d5-75d9-4179-9094- 840195ceee4b Sears, R. R., Cronkleton, P., Polo Villanueva, F., Miranda Ruiz, M., & Pérez-Ojeda del Arco, M. (2018). Farm-forestry in the Peruvian Amazon and the feasibility of its regulation through forest policy reform. Forest Policy and Economics, 87, 49–58. https://doi.org/10.1016/j.forpol.2017.11.004 Sears, R. R., Pinedo-Vasquez, M., & Padoch, C. (2014). 26. From Fallow Timber to Urban Housing: Family Forestry and Tablilla Production in Peru. In The Social Lives of Forests: Past, Present, and Future of Woodland Resurgence (pp. 336–347). University of Chicago Press. https://doi.org/10.7208/chicago/9780226024134.001.0001 Sebele, L. S. (2010). Community-based tourism ventures, benefits and challenges: Khama Rhino Sanctuary Trust, Central District, Botswana. Tourism Management, 31(1), 136– 146. https://doi.org/10.1016/j.tourman.2009.01.005 Seddon, P., Knight, M., & Budd, K. (2009). Progress and Partnerships for Protected Areas in the Arabian Peninsula. 100. Seddon, P., & Launay, F. (2008). Arab Falconry: Changes, challenges and conservation opportunities of an ancient art. Seidu, I., Brobbey, L. K., Danquah, E., Oppong, S. K., van Beuningen, D., Seidu, M., & Dulvy, N. K. (2022). Fishing for survival: Importance of shark fisheries for the livelihoods of coastal communities in Western Ghana. Fisheries Research, 246, 106157. https://doi.org/10.1016/j.fishres.2021.106157 Seignobos, C. (2014). La chasse/pêche aux Batraciens: Aux origines de la vie des populations du bassin du lac tchad ? (L’exemple du diamaré, cameroun). Anthropozoologica, 49(2), 305–325. https://doi.org/10.5252/az2014n2a11 Sejersen, F. (2001). Hunting and Management of Beluga Whales (Delpinapterus leucas) in Greenland: Changing Strategies to Cope with New National and Local Interests. ARCTIC, 54(4), 431–443. https://doi.org/10.14430/arctic800 Selgrath, J. C., Gergel, S. E., & Vincent, A. C. J. (2018a). Incorporating spatial dynamics greatly increases estimates of long-term fishing effort: A participatory mapping approach. ICES Journal of Marine Science, 75(1), 210–220. Scopus. https://doi.org/10.1093/icesjms/fsx108 Selgrath, J. C., Gergel, S. from E., & Vincent, A. C. J. (2018b). Shifting gears: Diversification, intensification, and effort increases in small-scale fisheries (1950-2010). PLoS ONE, 13(3). Scopus. https://doi.org/10.1371/journal.pone.0190232 Selkoe, K. A., Blenckner, T., Caldwell, M. R., Crowder, L. B., Erickson, A. L., Essington, T. E., … Zedler, J. (2015). Principles for managing marine ecosystems prone to tipping 479 1–18. points. Ecosystem Health and Sustainability, 1(5), 1–18. https://doi.org/10.1890/EHS14-0024.1 1(5), Sustainability, and Sustainability, Health points. Ecosystem Health and Sustainabilit https://doi.org/10.1890/EHS14-0024.1 points. Ecosystem Health https://doi.org/10.1890/EHS14-0024.1 and Senkoro, A. M., Shackleton, C. M., Voeks, R. A., & Ribeiro, A. I. (2019). Uses, Knowledge, and Management of the Threatened Pepper-Bark Tree (Warburgia salutaris) in Southern Mozambique. Economic Botany, 73(3), 304–324. https://doi.org/10.1007/s12231-019-09468-x Senkoro, A. M., Shackleton, C. M., Voeks, R. A., & Ribeiro, A. I. (2019). Uses, Knowledge, and Management of the Threatened Pepper-Bark Tree (Warburgia salutaris) in Southern Mozambique. Economic Botany, 73(3), 304–324. https://doi.org/10.1007/s12231-019-09468-x Şereflişan, H., & Alkaya, A. (2016). The biology, economy, hunting and legislation of edible Frogs (Ranidae) Intended for Export in Turkey. Turkish Journal of Agriculture - Food Science and Technology, 4(7), 600–604. https://doi.org/10.24925/turjaf.v4i7.600- 604.654 Serra, A. B. (2020). Family Farming in the Amazon: A Dead End or the Way Ahead for Sustainable Development? A Case Study from the Trans-Amazon Highway in Brazil (Doctoral Thesis, University of Freiburg). University of Freiburg, Freiburg. Retrieved from https://d1wqtxts1xzle7.cloudfront.net/63141872/Thesis_Serra_20_02_202020200429- 90069-niu049.pdf?1588207416=&response-content- disposition=inline%3B+filename%3DFamily_Farming_in_the_Amazon_A_Dead_En d.pdf&Expires=1617126611&Signature=HB~sfM6ESgjCMwTxCeQGEHM~NV0Z8 yyL9uwwriAVfZc5XADWhGB1cKsta0VFD0fHR5OwdeJ6CySv- b7Q2BgOGsUEDRmXZxmsK013iZ2OwTHJGV5uKzmALybhgJU-r- qiupq3wRAj2vW9bDeK00jOBERjsq3l0ML5zZ7AiBDJ6PbZbvn4tQ1oANPj8cO4Sc qheZxpb18mX0a3Ot5BWfgRgr55mNTx0VEn24Szawd8aiLpWbmWuFkkoQyLiTKv 9QnwquuP0ZymqX7uf31AkGZmXReTNn0aHs0QDY5h5ennMX~QxivwZRKTlQY eREAya9EwsAbGW8AtFVYWLj5UQ12kIA__&Key-Pair- Id=APKAJLOHF5GGSLRBV4ZA Id=APKAJLOHF5GGSLRBV4ZA Seto, K., Belhabib, D., Mamie, J., Copeland, D., Vakily, J. M., Seilert, H., … Zylich, K. (2017). War, fish, and foreign fleets: The marine fisheries catches of Sierra Leone 1950–2015. Marine Policy, 83, 153–163. Shackleton, C., Hurley, P., Dahlberg, A., Emery, M., & Nagendra, H. (2017). Urban Foraging: A Ubiquitous Human Practice Overlooked by Urban Planners, Policy, and Research. Sustainability, 9(10), 1884. https://doi.org/10.3390/su9101884 Shackleton, C. M., Drescher, A., & Schlesinger, J. (2020). Urbanisation reshapes gendered engagement in land-based livelihood activities in mid-sized African towns. World Development, 130, 104946. https://doi.org/10.1016/j.worlddev.2020.104946 Shackleton, C., & Shackleton, S. (2004). The importance of non-timber forest products in rural livelihood security and as safety nets: A review of evidence from South Africa. South African Journal of Science, 100(11), 658–664. Shackleton, Charlie M. (2000). Stump size and the number of coppice shoots for selected savanna tree species. South African Journal of Botany, 66(2), 124–127. https://doi.org/10.1016/S0254-6299(15)31074-7 Shackleton, Charlie M., & de Vos, A. (2022). How many people globally actually use non- timber forest products? Forest Policy and Economics, 135, 102659. https://doi.org/10.1016/j.forpol.2021.102659 480 Shackleton, C.M., & Mograbi, P. J. (2020). Meeting a diversity of needs through a diversity of species: Urban residents’ favourite and disliked tree species across eleven towns in South Africa and Zimbabwe. Urban Forestry & Urban Greening, 48, 126507. https://doi.org/10.1016/j.ufug.2019.126507 Shackleton, S., Chinyimba, A., Hebinck, P., Shackleton, C., & Kaoma, H. (2015). Multiple benefits and values of trees in urban landscapes in two towns in northern South Africa. Landscape and Urban Planning, 136, 76–86. https://doi.org/10.1016/j.landurbplan.2014.12.004 Shanahan, D. F., Bush, R., Gaston, K. J., Lin, B. B., Dean, J., Barber, E., & Fuller, R. A. (2016). Health Benefits from Nature Experiences Depend on Dose. Scientific Reports, 6(1), 28551. https://doi.org/10.1038/srep28551 Shao, S.-C., Burgess, K. S., Cruse-Sanders, J. M., Liu, Q., Fan, X.-L., Huang, H., & Gao, J.- Y. (2017). Using In Situ Symbiotic Seed Germination to Restore Over-collected Medicinal Orchids in Southwest China. Frontiers in Plant Science, 8, 888. https://doi.org/10.3389/fpls.2017.00888 heikh, P. A., & Bermejo, L. F. (2019). International Trophy Hunting (No. R45615; p. 33 Sheikh, P. A., Bermejo, L. F., & Procita, K. (2019). Illegal logging: Background and issues. Congressional Research Service. Retrieved from Congressional Research Service. website: https://fas.org/sgp/crs/misc/IF11114.pdf Shephard, S., Josset, Q., Davidson, I., Kennedy, R., Magnusson, K., Gargan, P. G., … Poole, R. (2019). Combining empirical indicators and expert knowledge for surveillance of data-limited sea trout stocks. Ecological Indicators, 104, 96–106. Scopus. https://doi.org/10.1016/j.ecolind.2019.04.073 Shepherd, C. Id=APKAJLOHF5GGSLRBV4ZA J., & Jackson, A. J. (2013). Global fishmeal and fish-oil supply: Inputs, outputs and markets a: global production of fishmeal and fish-oil. Journal of Fish Biology, 83(4), 1046–1066. https://doi.org/10.1111/jfb.12224 Shepherd, J., & Bachis, E. (2014). CHANGING SUPPLY AND DEMAND FOR FISH OIL. Aquaculture Economics & Management, 18(4), 395–416. https://doi.org/10.1080/13657305.2014.959212 Sheppard, J. P., Chamberlain, J., Agúndez, D., Bhattacharya, P., Chirwa, P. W., Gontcharov, A., … Mutke, S. (2020). Sustainable Forest Management Beyond the Timber-Oriented Status Quo: Transitioning to Co-production of Timber and Non-wood Forest Products—a Global Perspective. Current Forestry Reports, 6(1), 26–40. https://doi.org/10.1007/s40725-019-00107-1 Sherley, R. B., Winker, H., Rigby, C. L., Kyne, P. M., Pollom, R., Pacoureau, N., … others. (2020). Estimating IUCN Red List population reduction: JARA—a decision-support tool applied to pelagic sharks. Conservation Letters, 13(2), e12688. https://doi.org/10.1111/conl.12688 Shin, W. S., Kim, J.-J., Lim, S. S., Yoo, R.-H., Jeong, M.-A., Lee, J., … others. (2017). Paradigm shift on forest utilization: Forest service for health promotion in the Republic of Korea. Net. J. Agric. Sci, 5, 53–57. Shiva, V. (2007). Bioprospecting as Sophisticated Biopiracy. Signs: Journal of Women in Culture and Society, 32(2), 307–313. https://doi.org/10.1086/508502 481 Shivambu, N., Shivambu, T. C., & Downs, C. T. (2020). Assessing the potential impacts of non-native small mammals in the South African pet trade. NeoBiota, 60, 1–18. https://doi.org/10.3897/neobiota.60.52871 Short Gianotti, A. G., & Hurley, P. T. (2016). Gathering plants and fungi along the urban-rural gradient: Uncovering differences in the attitudes and practices among urban, suburban, and rural landowners. Land Use Policy, 57, 555–563. https://doi.org/10.1016/j.landusepol.2016.06.019 Short, M. L., & Darimont, C. T. (2021). Global synthesis reveals that ecosystem degradation poses the primary threat to the world’s medicinal animals. Ecology and Society, 26(1), art21. https://doi.org/10.5751/ES-12174-260121 Shrestha, U. B., & Bawa, K. S. (2014). Economic contribution of Chinese caterpillar fungus to the livelihoods of mountain communities in Nepal. Biological Conservation, 177, 194– 202. https://doi.org/10.1016/j.biocon.2014.06.019 Shumsky, S., Hickey, G. M., Johns, T., Pelletier, B., & Galaty, J. (2014). Institutional factors affecting wild edible plant (WEP) harvest and consumption in semi-arid Kenya. Land Use Policy, 38, 48–69. https://doi.org/10.1016/j.landusepol.2013.10.014 Shupler, M., Mwitari, J., Gohole, A., Cuevas, R. A. de, Puzzolo, E., Čukić, I., … Pope, D. (2020). COVID-19 Lockdown in a Kenyan Informal Settlement: Impacts on Household Energy and Food Security [Preprint]. Public and Global Health. https://doi.org/10.1101/2020.05.27.20115113 Shyamsundar, P., Ahlroth, S., Kristjanson, P., & Onder, S. (2020). Supporting pathways to prosperity in forest landscapes – A PRIME framework. Silva, L. (2015). How ecotourism works at the community-level: The case of whale-watching in the Azores. Current Issues in Tourism, 18(3), 196–211. Id=APKAJLOHF5GGSLRBV4ZA World Development, 125, 104622. https://doi.org/10.1016/j.worlddev.2019.104622 SickKids. (2020). Ontario Poison Centre warns of risks of mushroom foraging, a COVID-19 pastime gaining popularity. Retrieved April 2, 2021, from SickKids website: https://www.sickkids.ca/en/news/archive/2020/ontario-poison-centre-warns-of-risks- of-mushroom-foraging-a-covid-19-pastime-gaining-popularity/ Siegmund-Schultze, M., Rischkowsky, B., da Veiga, J. B., & King, J. M. (2007). Cattle are cash generating assets for mixed smallholder farms in the Eastern Amazon. Agricultural Systems, 94(3), 738–749. https://doi.org/10.1016/j.agsy.2007.03.005 Šiftová, J. (2020). Foraging in Czechia: The nation’s precious hobby. Norsk Geografisk Tidsskrift - Norwegian Journal of Geography, 74(5), 310–320. https://doi.org/10.1080/00291951.2020.1851757 Siitonen, J. (2001). Forest Management, Coarse Woody Debris and Saproxylic Organisms: Fennoscandian Boreal Forests as an Example. 32. Sikes, R. S., & Paul, E. (2013). Fundamental Differences between Wildlife and Biomedical Research. ILAR Journal, 54(1), 5–13. https://doi.org/10.1093/ilar/ilt015 Silva, J. N. M., de Carvalho, J. O. P., & Lopes, J. do C. A. (1985). INVENTÁRIO FLORESTAL DE UMA ÁREA EXPERIMENTAL NA FLORESTA NACIONAL DO TAPAJÓS. Embrapa Amazônia Oriental-Artigo em periódico indexado (ALICE), (10), 69. Silva, L. (2015). How ecotourism works at the community-level: The case of whale-watching in the Azores. Current Issues in Tourism, 18(3), 196–211. 482 Silva, P., Cabral, H., Rangel, M., Pereira, J., & Pita, C. (2019a). Ready for co-management? Portuguese artisanal octopus fishers’ preferences for management and knowledge about the resource. Marine Policy, 101, 268–275. Scopus. https://doi.org/10.1016/j.marpol.2018.03.027 Silvano, R.A.M., & Begossi, A. (2012). Fishermen’s local ecological knowledge on southeastern Brazilian coastal fishes: Contributions to research, conservation, and management. Neotropical Ichthyology, 10(1), 133–147. Scopus. https://doi.org/10.1590/S1679-62252012000100013 Silvano, R.A.M., MacCord, P. F. L., Lima, R. V., & Begossi, A. (2006). When does this fish spawn? Fishermen’s local knowledge of migration and reproduction of Brazilian coastal fishes. Environmental Biology of Fishes, 76(2–4), 371–386. Scopus. https://doi.org/10.1007/s10641-006-9043-2 Silvano, Renato A. M., Hallwass, G., Lopes, P. F., Ribeiro, A. R., Lima, R. P., Hasenack, H., … Begossi, A. (2014). Co-management and Spatial Features Contribute to Secure Fish Abundance and Fishing Yields in Tropical Floodplain Lakes. Ecosystems, 17(2), 271– 285. https://doi.org/10.1007/s10021-013-9722-8 Silvano, Renato A.M. (Ed.). (2020). Fish and Fisheries in the Brazilian Amazon: People, Ecology and Conservation in Black and Clear Water Rivers. Cham: Springer International Publishing. https://doi.org/10.1007/978-3-030-49146-8 Silvano, Renato A.M., Nora, V., Andreoli, T. B., Lopes, P. F. M., & Begossi, A. (2017). The ‘ghost of past fishing’: Small-scale fisheries and conservation of threatened groupers in subtropical islands. Marine Policy, 75, 125–132. https://doi.org/10.1016/j.marpol.2016.10.002 Silvano, Renato Azevedo Matias, & Hallwass, G. (2020). Id=APKAJLOHF5GGSLRBV4ZA Participatory Research with Fishers to Improve Knowledge on Small-Scale Fisheries in Tropical Rivers. Sustainability, 12(11), 4487. https://doi.org/10.3390/su12114487 Silvennoinen, H. (2017). Metsämaiseman kauneus ja metsänhoidon vaikutus koettuun maisemaan metsikkötasolla. Dissertationes Forestales, 2017(242). https://doi.org/10.14214/df.242 Silvennoinen, H., Alho, J., Kolehmainen, O., & Pukkala, T. (2001). Prediction models of landscape preferences at the forest stand level. Landscape and Urban Planning, 56(1– 2), 11–20. https://doi.org/10.1016/S0169-2046(01)00163-3 Silvennoinen, H., Pukkala, T., & Tahvanainen, L. (2002). Effect of Cuttings on the Scenic Beauty of a Tree Stand. Scandinavian Journal of Forest Research, 17(3), 263–273. https://doi.org/10.1080/028275802753742936 Silverman, J., Suckow, M. A., & Murthy, S. (2000). The IACUC Handbook. Taylor & Francis. Šimat, V., Elabed, N., Kulawik, P., Ceylan, Z., Jamroz, E., Yazgan, H., … Özogul, F. (2020). Recent Advances in Marine-Based Nutraceuticals and Their Health Benefits. Marine Drugs, 18(12), 627. https://doi.org/10.3390/md18120627 Simberloff, D., Parker, I. M., & Windle, P. N. (2005). Introduced species policy, management, and future research needs. Frontiers in Ecology and the Environment, 3(1), 12–20. https://doi.org/10.1890/1540-9295(2005)003[0012:ISPMAF]2.0.CO;2 Simelane, T., & Kerley, G. (1998). Conservation implications of the use of vertebrates by Xhosa traditional healers in South Africa. South African Journal of Wildlife Research- 483 121–126. 28(4), Access, Delayed Open 24-Month Delayed Open Access, 28(4), 121–126. https://journals.co.za/doi/abs/10.10520/EJC117057 24-Month Delayed Open Acce https://journals.co.za/doi/abs/10.10520/EJC117057 https://journals.co.za/doi/abs/10.10520/EJC117057 Simmons, C. S., Walker, R., Aldrich, S., Arima, E., Pereira, R., Castro, E. M. R. de, … Antunes, A. (2019). Discipline and Develop: Destruction of the Brazil Nut Forest in the Lower Amazon Basin. Annals of the American Association of Geographers, 109(1), 242–265. https://doi.org/10.1080/24694452.2018.1489215 Simmons, D. G. (1994). Community participation in tourism planning. Tourism Management, 15(2), 98–108. https://doi.org/10.1016/0261-5177(94)90003-5 Simpfendorfer, C. A., & Dulvy, N. K. (2017). Bright spots of sustainable shark fishing. Current Biology, 27(3), R97–R98. https://doi.org/10.1016/j.cub.2016.12.017 Simpfendorfer, C. A., & Kyne, P. M. (2009). Limited potential to recover from overfishing raises concerns for deep-sea sharks, rays and chimaeras. Environmental Conservation, 36(2), 97–103. https://doi.org/10.1017/S0376892909990191 Sinclair, M., Ghermandi, A., Moses, S. A., & Joseph, S. (2019). Recreation and environmental quality of tropical wetlands: A social media based spatial analysis. Tourism Management, 71, 179–186. https://doi.org/10.1016/j.tourman.2018.10.018 Singh, B. P. (2020, June 22). Hundreds of collectors climb highlands despite ban in Yarsagumba harvest this season. Kathmandu Post. Retrieved from https://kathmandupost.com/sudurpaschim-province/2020/06/22/hundreds-of- collectors-climb-highlands-despite-ban-in-yarsagumba-harvest-this-season Sirotenko, M. D., Danilevskiy, M. M., & Shlyakhov, V. A. (1979). Dolphins. In K. S. Tkatcheva & Y. K. Benko (Eds.), Resources and Raw Materials in the Black Sea (pp. 242–246). Moskow: AztcherNIRO, Pishtchevaya promishlenist. Siry, J. Id=APKAJLOHF5GGSLRBV4ZA P., Cubbage, F. W., & Ahmed, M. R. (2005). Sustainable forest management: Global trends and opportunities. Forest Policy and Economics, 7(4), 551–561. https://doi.org/10.1016/j.forpol.2003.09.003 Sist, P. (2000). Reduced-impact logging in the tropics: Objectives, principles and impacts. The International Forestry Review, 3–10. Sist, P., & Ferreira, F. N. (2007). Sustainability of reduced-impact logging in the Eastern Amazon. Forest Ecology and Management, 243(2–3), 199–209. https://doi.org/10.1016/j.foreco.2007.02.014 Sist, P., Fimbel, R., Sheil, D., Nasi, R., & Chevallier, M.-H. (2003). Towards sustainable management of mixed dipterocarp forests of South-east Asia: Moving beyond minimum diameter cutting limits. Environmental Conservation, 30(4), 364–374. https://doi.org/10.1017/S0376892903000389 Sist, P., Nolan, T., Bertault, J.-G., & Dykstra, D. (1998). Harvesting intensity versus sustainability in Indonesia. Forest Ecology and Management, 108(3), 251–260. https://doi.org/10.1016/S0378-1127(98)00228-X Sitta, N., & Floriani, M. (2008). Nationalization and Globalization Trends in the Wild Mushroom Commerce of Italy with Emphasis on Porcini (Boletus edulis and Allied Species). Economic Botany, 62(3), 307–322. https://doi.org/10.1007/s12231-008- 9037-4 484 Skaggs, R., Edwards, Z., Bestelmeyer, B. T., Wright, J. B., Williamson, J., & Smith, P. (2011). Vegetation Maps at the Passage of the Taylor Grazing Act (1934): A Baseline to Evaluate Rangeland Change After a Regime Shift. 7. Skogen, K., Krange, O., & Figari, H. (2017). Wolf Conflicts. A Sociological Study. In Wolf Conflicts: A Sociological Study. https://doi.org/10.2307/j.ctvw04jgs Skogen, K., Mauz, I., & Krange, O. (2008). Cry Wolf!: Narratives of Wolf Recovery in France and Norway. Rural Sociology, 73(1), 105–133. https://doi.org/10.1526/003601108783575916 Skulska, I., Duarte, I., Rego, F. C., & Montiel-Molina, C. (2020). Relationships Between Wildfires, Management Modalities of Community Areas, and Ownership Types in Pine Forests of Mainland Portugal. Small-Scale Forestry, 19(2), 231–251. https://doi.org/10.1007/s11842-020-09445-6 Sloan, S., Meyfroidt, P., Rudel, T. K., Bongers, F., & Chazdon, R. (2019). The forest transformation: Planted tree cover and regional dynamics of tree gains and losses. Global Environmental Change, 59, 101988. https://doi.org/10.1016/j.gloenvcha.2019.101988 Small, C. J., Chamberlain, J. L., & Mathews, D. S. (2011). Recovery of Black Cohosh (Actaea racemosa L.) Following Experimental Harvests. The American Midland Naturalist, 166(2), 339–348. https://doi.org/10.1674/0003-0031-166.2.339 Smit, I. P. J., Roux, D. J., Swemmer, L. K., Boshoff, N., & Novellie, P. (2017). Protected areas as outdoor classrooms and global laboratories: Intellectual ecosystem services flowing to-and-from a National Park. Ecosystem Services, 28, 238–250. https://doi.org/10.1016/j.ecoser.2017.05.003 Smith, A. D. M., Brown, C. J., Bulman, C. M., Fulton, E. A., Johnson, P., Kaplan, I. C., … Tam, J. (2011). Impacts of Fishing Low-Trophic Level Species on Marine Ecosystems. Science, 333(6046), 1147–1150. Id=APKAJLOHF5GGSLRBV4ZA https://doi.org/10.1126/science.1209395 Smith, B., Wassersug, R., & Tyler, M. (2007). How frogs and humans interact: Influences beyond habitat destruction, epidemics and global warming. Applied Herpetology, 4(1), 1–18. https://doi.org/10.1163/157075407779766741 Smith, D. W., & Peterson, R. O. (2021). Intended and unintended consequences of wolf restoration to Yellowstone and Isle Royale National Parks. Conservation Science and Practice, 3(4). https://doi.org/10.1111/csp2.413 Smith, H. E., Ryan, C. M., Vollmer, F., Woollen, E., Keane, A., Fisher, J. A., … others. (2019). Impacts of land use intensification on human wellbeing: Evidence from rural Mozambique. Global Environmental Change, 59, 101976. https://doi.org/10.1016/j.gloenvcha.2019.101976 Smith, K. R. & others. (2006). Health impacts of household fuelwood use in developing countries. UNASYLVA-FAO-, 57(2), 41. Smith, L. E. D., Khoa, S. N., & Lorenzen, K. (2005). Livelihood functions of inland fisheries: Policy implications in developing countries. Water Policy, 7(4), 359–383. https://doi.org/10.2166/wp.2005.0023 Smith, N. S., & Zeller, D. (2016). Unreported catch and tourist demand on local fisheries of small island states: The case of The Bahamas, 1950-2010. Fishery Bulletin, 114(1). 485 Snyder, S. A., Butler, B. J., & Markowski-Lindsay, M. (2019). Small-Area Family Forest Ownerships in the USA. Small-Scale Forestry, 18(1), 127–147. https://doi.org/10.1007/s11842-018-9410-9 Snyman, S., Sumba, D., Vorhies, F., Gitari, E., Enders, C., Ahenkan, A., … Bengone, N. (2021). State of the Wildlife Economy in Africa. African Leadership University, School of Wildlife Conservation: Kigali, Rwanda. Retrieved from https://www.ogresearchconservation.org/state-of-the-wildlife-economy-in-africa-case- study Sodeinde, O., & Soewu, D. (1999). Pilot study of the traditional medicine trade in Nigeria. TRAFFIC BULLETIN-CAMBRIDGE-TRAFFIC INTERNATIONAL-, 18, 35–40. Soehartono, T., & Newton, A. C. (2001). Conservation and sustainable use of tropical trees in the genus Aquilaria II. The impact of gaharu harvesting in Indonesia. Biological Conservation, 97(1), 29–41. https://doi.org/10.1016/S0006-3207(00)00089-6 Soewu A Durojaye, & Sodeinde A Olufemi. (2015). Utilization of pangolins in Africa: Fuelling factors, diversity of uses and sustainability. International Journal of Biodiversity and Conservation, 7(1), 1–10. https://doi.org/10.5897/IJBC2014.0760 Somers, M., & Hayward, M. (2012). Fencing for conservation: Restriction of evolutionary potential or a riposte to threatening processes? New York, USA: Springer. https://doi.org/10.1007/978-1-4614-0902-1 Somesh, D., Rao, R., Murali, R., & Nagendra, H. (2021). Patterns of urban foraging in Bengaluru city. Urban Forestry & Urban Greening, 57, 126940. https://doi.org/10.1016/j.ufug.2020.126940 Sorrenti, S. & Food and Agriculture Organization of the United Nations. (2017). Non-wood forest products in international statistical systems. Rome: Food and Agriculture Organization of the United Nations. SOTWP. (2016). KEW, State of the World’s Plants. SOTWP. (2020). KEW, State of the World’s Plants. Id=APKAJLOHF5GGSLRBV4ZA Retrieved from Kew Science \| Kew Souchay, G., Besnard, A., Perrot, C., Jakob, C., & Ponce, F. (2018). Anthropic and natural factors drive variation of survival in the red-legged partridge in southern France. Wildlife Biology, 2018(1). https://doi.org/10.2981/wlb.00438 Sõukand, R., Quave, C. L., Pieroni, A., Pardo-de-Santayana, M., Tardío, J., Kalle, R., … Mustafa, B. (2013). Plants used for making recreational tea in Europe: A review based on specific research sites. Journal of Ethnobiology and Ethnomedicine, 9(1), 58. https://doi.org/10.1186/1746-4269-9-58 Soule, M. E. (1990). The Onslaught of Alien Species, and Other Challenges in the Coming Decades. Conservation Biology, 4(3), 233–239. Soule, M. E., Bolger, D. T., Alberts, A. C., Wrights, J., Sorice, M., & Hill, S. (1988). Reconstructed Dynamics of Rapid Extinctions of Chaparral-Requiring Birds in Urban Habitat Islands. Conservation Biology, 2(1), 75–92. https://doi.org/10.1111/j.1523- 1739.1988.tb00337.x Soulsbury, C. D., Gray, H. E., Smith, L. M., Braithwaite, V., Cotter, S. C., Elwood, R. W., … Collins, L. M. (2020). The welfare and ethics of research involving wild animals: A primer. Methods in Ecology and Evolution, 11(10), 1164–1181. https://doi.org/10.1111/2041-210X.13435 486 Spahr, D. L. (2009). Edible and medicinal mushrooms of New England and Eastern Canada: A photographic Guidebook to Finding and Using Key Species. Berkeley, CA USA: North Atlantic Books. Retrieved from https://www.google.com/search?q=turkey+tail+jewelry&rlz=1C5CHFA_enNO863N O864&oq=turkey+tail+jewelry&aqs=chrome..69i57.5967j0j7&sourceid=chrome&ie= UTF-8 SPC. (2015). Western and Central Pacific Fisheries Commission Tuna Fishery Yearbook 2014. [Oceanic Fisheries Programme, Secretariat of the Pacific Community, Noumea, New Caledonia.]. Retrieved from https://www.wcpfc.int/doc/wcpfc-tuna-fishery-yearbook- 2015 Spenceley, A. (2005). Nature-based Tourism and Environmental Sustainability in South Africa. Journal of Sustainable Tourism, 13(2), 136–170. https://doi.org/10.1080/09669580508668483 Spenceley, A., McCool, S., Newsome, D., Báez, A., Barborak, J. R., Blye, C.-J., … Zschiegner, A.-K. (2021). Tourism in protected and conserved areas amid the COVID-19 pandemic. PARKS, (27), 103–118. https://doi.org/10.2305/IUCN.CH.2021.PARKS-27-SIAS.en Stacey, N. E., Karam, J., Meekan, M. G., Pickering, S., & Ninef, J. (2012). Prospects for whale shark conservation in Eastern Indonesia through bajo traditional ecological knowledge and community-based monitoring. Conservation and Society, 10(1), 63–75. Scopus. https://doi.org/10.4103/0972-4923.92197 Stafford, C. A., Preziosi, R. F., & Sellers, W. I. (2017). A Cross-Site Analysis of Neotropical Bird Hunting Profiles. Tropical Conservation Science, 10, 1940082917736894. https://doi.org/10.1177/1940082917736894 Stanley, D., Voeks, R., & Short, L. (2012). Is Non-Timber Forest Product Harvest Sustainable in the Less Developed World? A Systematic Review of the Recent Economic and Ecological Literature. Ethnobiology and Conservation, 1. Retrieved from https://www.ethnobioconservation.com/index.php/ebc/article/view/19 bank. (2020). Whale hunts, pilot whales, and skinn (1951-2020). Statistics Faroe Islands. Stattersfield, A. J., Crosby, M. Id=APKAJLOHF5GGSLRBV4ZA J., Long, A. J., Wege, D. C., & Rayner, A. P. (1998). Endemic bird areas of the world: Priorities for biodiversity conservation. Retrieved from https://portals.iucn.org/library/node/25865 Steele, J. H. (2004). Regime shifts in the ocean: Reconciling observations and theory. Progress in Oceanography, 60(2–4), 135–141. https://doi.org/10.1016/j.pocean.2004.02.004 Stephenson, R. L., & Smedbol, R. K. (2019). Small Pelagic Species Fisheries. In Encyclopedia of Ocean Sciences (pp. 503–509). Elsevier. https://doi.org/10.1016/B978-0-12-409548- 9.11491-5 Stevens, C. H., Croft, D. P., Paull, G. C., & Tyler, C. R. (2017). Stress and welfare in ornamental fishes: What can be learned from aquaculture?: stress and welfare in ornamental fishes. Journal of Fish Biology, 91(2), 409–428. https://doi.org/10.1111/jfb.13377 Stevens, J. (2000). The effects of fishing on sharks, rays, and chimaeras (chondrichthyans), and the implications for marine ecosystems. ICES Journal of Marine Science, 57(3), 476– 494. https://doi.org/10.1006/jmsc.2000.0724 487 Stewart, K. (2009). Effects of bark harvest and other human activity on populations of the African cherry (Prunus africana) on Mount Oku, Cameroon. Forest Ecology and Management, 258(7), 1121–1128. https://doi.org/10.1016/j.foreco.2009.05.039 Stewart, K. M. (2003). The African cherry (Prunus africana): Can lessons be learned from an over-exploited medicinal tree? Journal of Ethnopharmacology, 89(1), 3–13. https://doi.org/10.1016/j.jep.2003.08.002 Stewart, K. R., Lewison, R. L., Dunn, D. C., Bjorkland, R. H., Kelez, S., Halpin, P. N., & Crowder, L. B. (2010). Characterizing Fishing Effort and Spatial Extent of Coastal Fisheries. PLoS ONE, 5(12), e14451. https://doi.org/10.1371/journal.pone.0014451 Stocks, A. P., Foster, S. J., Bat, N. K., Ha, N. M., & Vincent, A. C. J. (2019). Local Fishers’ Knowledge of Target and Incidental Seahorse Catch in Southern Vietnam. Human Ecology, 47(3), 397–408. Scopus. https://doi.org/10.1007/s10745-019-0073-8 Stocks, A. P., Foster, S. J., Bat, N. K., & Vincent, A. C. J. (2017). Catch as catch can: Targeted and indiscriminate small-scale fishing of seahorses in Vietnam. Fisheries Research, 196, 27–33. Scopus. https://doi.org/10.1016/j.fishres.2017.07.021 Stoian, D., Rodas, A., Butler, M., Monterroso, I., & Hodgdon, B. (2018). Forest concessions in Petén, Guatemala: A Systematic Analysis of the Socioeconomic Performance of Community Enterprises in the Maya Biosphere Reserve. CIFOR, 8. y p y p toker, S. W., & Krupnik, I. I. (1993). Subsistence whaling. The Bowhead Whale, 579–62 Stone, M. T. (2015). Community-based ecotourism: A collaborative partnerships perspective. Journal of Ecotourism, 14(2–3), 166–184. https://doi.org/10.1080/14724049.2015.1023309 Storaas, T., Gundersen, H., Henriksen, H., & Andreassen, H. P. (2001). The economic value of moose in Norway—A review. Alces, 37(1), 97–108. Storaunet, K., Rolstad, J., Gjerde, I., & Gundersen, V. (2005). Id=APKAJLOHF5GGSLRBV4ZA Historical logging, productivity, and structural characteristics of boreal coniferous forests in Norway. Silva Fennica, 39(3). https://doi.org/10.14214/sf.479 Strieder Philippsen, J., Minte-Vera, C. V., Okada, E. K., Carvalho, A. R., & Angelini, R. (2017). Fishers’ and scientific histories: An example of consensus from an inland fishery. Marine and Freshwater Research, 68(5), 980–992. Scopus. https://doi.org/10.1071/MF16053 Stryamets, N., Elbakidze, M., Ceuterick, M., Angelstam, P., & Axelsson, R. (2015). From economic survival to recreation: Contemporary uses of wild food and medicine in rural Sweden, Ukraine and NW Russia. Journal of Ethnobiology and Ethnomedicine, 11(1), 53. https://doi.org/10.1186/s13002-015-0036-0 Suarez, A. V., & Tsutsui, N. D. (2004). The Value of Museum Collections for Research and Society. BioScience, 54(1), 66. https://doi.org/10.1641/0006- 3568(2004)054[0066:tvomcf]2.0.co;2 Subedi, C. K., Chaudhary, R. P., Kunwar, R. M., Bussmann, W., & Oaniagua-Zambrana, N. Y. (2021). Jatropha curcas L. (Euphorbiaceae). In R. M. Kunwar, H. Sher, & R. W. Bussmann (Eds.), Ethnobotany of the Himalayas. Cham: Springer International Publishing. https://doi.org/10.1007/978-3-030-57408-6 488 Sujatha, M., & Prabakaran, A. J. (2003). New ornamental Jatropha hybrids through interspecific hybridization. Genetic Resources and Crop Evolution, 50(1), 75–82. https://doi.org/10.1023/A:1022961028064 Sumaila, U. R., Ebrahim, N., Schuhbauer, A., Skerritt, D., Li, Y., Kim, H. S., … Pauly, D. (2019). Updated estimates and analysis of global fisheries subsidies. Marine Policy, 109, 103695. https://doi.org/10.1016/j.marpol.2019.103695 Sumaila, U. R., Lam, V., Le Manach, F., Swartz, W., & Pauly, D. (2016). Global fisheries subsidies: An updated estimate. Marine Policy, 69, 189–193. https://doi.org/10.1016/j.marpol.2015.12.026 Sun, Y.-Y., Lin, P.-C., & Higham, J. (2020). Managing tourism emissions through optimizing the tourism demand mix: Concept and analysis. Tourism Management, 81, 104161. https://doi.org/10.1016/j.tourman.2020.104161 Sundar, B. (2017). Joint forest management in India – an assessment. International Forestry Review, 19(4), 495–511. https://doi.org/10.1505/1465548822272329 Susilowati, A., Rachmat, H., Elfiati, D., & Hasibuan, H. M. (2019). The composition and diversity of plant species in pasak bumi’s (Eurycoma longifolia) habitat in Batang Lubu Sutam forest, North Sumatra, Indonesia. Biodiversitas Journal of Biological Diversity, 20(2), 413–418. https://doi.org/10.13057/biodiv/d200215 Suydam, R., & George, J. (2021). Current indigenous whaling. In The Bowhead Whale (pp. 519–535). Elsevier. Svanberg, I., Sõukand, R., Luczaj, L., Kalle, R., Zyryanova, O., Dénes, A., … others. (2012). Uses of tree saps in northern and eastern parts of Europe. Acta Societatis Botanicorum Poloniae, 81(4). Svenning, J.-C., & Macı́a, M. J. (2002). Harvesting of Geonoma macrostachys Mart. leaves for thatch: An exploration of sustainability. Forest Ecology and Management, 167(1–3), 251–262. https://doi.org/10.1016/S0378-1127(01)00699-5 Svizzero, S. (2016). Foraging Wild Resources: Evolving Goals of an Ubiquitous Human Behavior. Id=APKAJLOHF5GGSLRBV4ZA Anthropology, 04(01). https://doi.org/10.4172/2332-0915.1000161 Swamy, V., & Pinedo-Vasquez, M. (2014). Bushmeat harvest in tropical forests. 32. Swemmer, L., Mmethi, H., & Twine, W. (2017). Tracing the cost/benefit pathway of protected areas: A case study of the Kruger National Park, South Africa. Ecosystem Services, 28, 162–172. https://doi.org/10.1016/j.ecoser.2017.09.002 Swenson, J., Schneider, M., Zedrosser, A., Söderberg, A., Franzén, R., & Kindberg, J. (2017). Challenges of managing a European brown bear population; lessons from Sweden, 1943–2013. Wlb.00251. https://doi.org/10.2981/wlb.00251 Swinkels, R. (2014). Assessment of household energy deprivation in Tajikistan: Policy options for socially responsible reform in the energy sector. Washington D.C: World Bank. Retrieved from World Bank website: http://documents1.worldbank.org/curated/en/944321468341064427/pdf/888370ESW0 whit0n0Energy0Deprivation.pdf Syampungani, S., Chirwa, P., Geldenhuys, C., Handavu, F., Chishaleshale, M., Rija, A., … Ribeiro, N. (2020). Managing Miombo: Ecological and Silvicultural Options for Sustainable Socio-Economic Benefits. https://doi.org/10.1007/978-3-030-50104-4_4 489 Synk, C. M., Kim, B. F., Davis, C. A., Harding, J., Rogers, V., Hurley, P. T., … Nachman, K. E. (2017). Gathering Baltimore’s bounty: Characterizing behaviors, motivations, and barriers of foragers in an urban ecosystem. Urban Forestry & Urban Greening, 28, 97– 102. https://doi.org/10.1016/j.ufug.2017.10.007 Szostek, C. L., Murray, L. G., Bell, E., & Kaiser, M. J. (2017). Filling the gap: Using fishers’ knowledge to map the extent and intensity of fishing activity. Marine Environmental Research, 129, 329–346. Scopus. https://doi.org/10.1016/j.marenvres.2017.06.012 Szott, I. D., Pretorius, Y., Ganswindt, A., & Koyama, N. F. (2020). Physiological stress response of African elephants to wildlife tourism in Madikwe Game Reserve, South Africa. Wildlife Research, 47(1), 34–43. https://doi.org/10.1071/WR19045 Szulecka, J., Pretzsch, J., & Secco, L. (2014). Paradigms in tropical forest plantations: A critical reflection on historical shifts in plantation approaches. International Forestry Review, 16(2), 128–143. https://doi.org/10.1505/146554814811724829 Tacconi, L., Cerutti, P. O., Leipold, S., Rodrigues, R. J., Savaresi, A., To, P., & Weng, X. (2016). Defining Illegal Forest Activities and Illegal Logging. In Illegal Logging and Related Timber Trade – Dimensions, Drivers, Impacts and Responses: A Global Scientific Rapid Response Assessment Report (pp. 23–35). International Union of Forest Research Organizations (IUFRO). Tacon, A. G. J., Hasan, M. R., & Metian, M. (2011). Demand and supply of feed ingredients for farmed fish and crustaceans. Rome: Food and Agriculture Organization of the United Nations. Tacon, A. G. J., & Metian, M. (2008a). Aquaculture Feed and Food Safety. Annals of the New York Academy of Sciences, 1140(1), 50–59. https://doi.org/10.1196/annals.1454.003 Tacon, A. G. J., & Metian, M. (2008b). Id=APKAJLOHF5GGSLRBV4ZA Global overview on the use of fish meal and fish oil in industrially compounded aquafeeds: Trends and future prospects. Aquaculture, 285(1– 4), 146–158. https://doi.org/10.1016/j.aquaculture.2008.08.015 Tacon, A. G. J., & Metian, M. (2009). Fishing for Aquaculture: Non-Food Use of Small Pelagic Forage Fish—A Global Perspective. Reviews in Fisheries Science, 17(3), 305–317. https://doi.org/10.1080/10641260802677074 Tacon, A. G. J., & Metian, M. (2013). Fish Matters: Importance of Aquatic Foods in Human Nutrition and Global Food Supply. Reviews in Fisheries Science, 21(1), 22–38. https://doi.org/10.1080/10641262.2012.753405 Tacon, A. G. J., & Metian, M. (2015). Feed Matters: Satisfying the Feed Demand of Aquaculture. Reviews in Fisheries Science & Aquaculture, 23(1), 1–10. https://doi.org/10.1080/23308249.2014.987209 Tadesse, W., Desalegn, G., & Alia, R. (2007). Natural gum and resin bearing species of Ethiopia and their potential applications. Investigación Agraria: Sistemas y Recursos Forestales, 16(3), 211. https://doi.org/10.5424/srf/2007163-01010 Tadesse, Wubalem, Dejene, T., Zeleke, G., & Desalegn, G. (2020). Underutilized Natural Gum and Resin Resources in Ethiopia for Future Directions and Commercial Utilization. World Journal of Agricultural Research, 8(2), 32–38. https://doi.org/10.12691/wjar-8- 2-2 490 Taff, Benfield, Miller, D’Antonio, & Schwartz. (2019). The Role of Tourism Impacts on Cultural Ecosystem Services. Environments, 6(4), 43. https://doi.org/10.3390/environments6040043 Tallis, H., Kareiva, P., Marvier, M., & Chang, A. (2008). An ecosystem services framework to support both practical conservation and economic development. Proceedings of the National Academy of Sciences, 105(28), 9457–9464. https://doi.org/10.1073/pnas.0705797105 Tapper, S., & Reynolds, J. (1996). The wild fur trade: Historical and ecological perspectives. In V. J. Taylor & N. Dunstone (Eds.), The Exploitation of Mammal Populations (pp. 28–44). Dordrecht: Springer Netherlands. https://doi.org/10.1007/978-94-009-1525- 1_3 Tareau, M.-A., Dejouhanet, L., Odonne, G., Palisse, M., & Ansoe, C. (2019). Wild medicinal plant collection in transitional societies: A case Analysis from French Guiana. EchoGéo, (47). https://doi.org/10.4000/echogeo.17260 Tavakoli, S., Luo, Y., Regenstein, J. M., Daneshvar, E., Bhatnagar, A., Tan, Y., & Hong, H. (2021). Sturgeon, Caviar, and Caviar Substitutes: From Production, Gastronomy, Nutrition, and Quality Change to Trade and Commercial Mimicry. Reviews in Fisheries Science & Aquaculture, 29(4), 753–768. https://doi.org/10.1080/23308249.2021.1873244 Taylor, N., & Signal, T. D. (2009). Pet, Pest, Profit: Isolating Differences in Attitudes towards the Treatment of Animals. Anthrozoös, 22(2), 129–135. https://doi.org/10.2752/175303709X434158 Taylor, W. A., Lindsey, P. A., Nicholson, S. K., Relton, C., & Davies-Mostert, H. T. (2020). Jobs, game meat and profits: The benefits of wildlife ranching on marginal lands in South Africa. Biological Conservation, 245, 108561. https://doi.org/10.1016/j.biocon.2020.108561 Tebtebba Foundation (Ed.). (2010). Sustaining & enhancing forests through traditional resource management. Id=APKAJLOHF5GGSLRBV4ZA Baguio City, Philippines: Tebtebba Foundation. Tefera, D. A., Zerihun, M. M., & Wolde-Meskel, Y. T. G. (2019). Catch distribution and size structure of Nile tilapia (Oreochromis niloticus) in Lake Tana, Ethiopia: Implications for fisheries management. African Journal of Aquatic Science, 44(3), 273–280. Scopus. https://doi.org/10.2989/16085914.2019.1637710 Teh, L. C. L., Teh, L. S. L., Abe, K., Ishimura, G., & Roman, R. (2020). Small-scale fisheries in developed countries: Looking beyond developing country narratives through Japan’s perspective. Marine Policy, 122. Scopus. https://doi.org/10.1016/j.marpol.2020.104274 Teichman, K. J., Cristescu, B., & Darimont, C. T. (2016). Hunting as a management tool? Cougar-human conflict is positively related to trophy hunting. BMC Ecology, 16(1), 44. https://doi.org/10.1186/s12898-016-0098-4 Teitelbaum, S. (Ed.). (2016). Community forestry in Canada: Lessons from policy and practice. Vancouver ; Toronto: UBC Press. Teitelbaum, S., Beckley, T., & Nadeau, S. (2006). A national portrait of community forestry on public land in Canada. The Forestry Chronicle, 82(3), 416–428. https://doi.org/10.5558/tfc82416-3 491 Teitelbaum, S., & Bullock, R. (2012). Are community forestry principles at work in Ontario’s County, Municipal, and Conservation Authority forests? The Forestry Chronicle, 88(06), 697–707. https://doi.org/10.5558/tfc2012-136 Teiwaki, R. (1988). Kiribati: Nation of water. Micronesian Politics, 1–37. Teresa, F. B., Romero, R. de M., Casatti, L., & Sabino, J. (2011). Fish as Indicators of Disturbance in Streams Used for Snorkeling Activities in a Tourist Region. Environmental Management, 47(5), 960–968. https://doi.org/10.1007/s00267-011- 9641-4 Ternes, M. L. F., Gerhardinger, L. C., & Schiavetti, A. (2016). Seahorses in focus: Local ecological knowledge of seahorse-watching operators in a tropical estuary. Journal of Ethnobiology and Ethnomedicine, 12(1), 52. https://doi.org/10.1186/s13002-016-0125- 8 Tesfamichael, D., Pitcher, T. J., & Pauly, D. (2014). Assessing Changes in Fisheries Using Fishers’ Knowledge to Generate Long Time Series of Catch Rates: A Case Study from the Red Sea. Ecology and Society, 19(1), art18. https://doi.org/10.5751/ES- 06151-190118 Tewfik, A., Babcock, E. A., Appeldoorn, R. S., & Gibson, J. (2019). Declining size of adults and juvenile harvest threatens sustainability of a tropical gastropod, Lobatus gigas, fishery. Aquatic Conservation: Marine and Freshwater Ecosystems, 29(10), 1587– 1607. Scopus. https://doi.org/10.1002/aqc.3147 Thakur, S., Negi, V. S., Pathak, R., Dhyani, R., Durgapal, K., & Rawal, R. S. (2020). Indicator based integrated vulnerability assessment of community forests in Indian west Himalaya. Forest Ecology and Management, 457, 117674. https://doi.org/10.1016/j.foreco.2019.117674 Thaman, B., Thaman, R. R., Balawa, A., & Veitayaki, J. (2017). The Recovery of a Tropical Marine Mollusk Fishery: A Transdisciplinary Community-Based Approach in Navakavu, Fiji. Journal of Ethnobiology, 37(3), 494–513. Scopus. https://doi.org/10.2993/0278-0771-37.3.494 Tharme, A. Id=APKAJLOHF5GGSLRBV4ZA P., Green, R. E., Baines, D., Bainbridge, I. P., & O’Brien, M. (2001). The effect of management for red grouse shooting on the population density of breeding birds on heather-dominated moorland: Grouse moor management and breeding birds. Journal of Applied Ecology, 38(2), 439–457. https://doi.org/10.1046/j.1365-2664.2001.00597.x The Economic Footprint of Angling, Hunting, Trapping and Sport Shooting in Canada. (2019). The Economic Footprint of Angling, Hunting, Trapping and Sport Shooting in Canada. Retrieved from https://bcwf.bc.ca/wp-content/uploads/2019/09/Economic-Footprint- Analysis-of-AHTS.pdf The New York Times. (2020). ‘I Have Never Seen So Many Toadstools.’ A Bumper Crop of Mushrooms in Ukraine. - The New York Times. Retrieved April 2, 2021, from https://www.nytimes.com/2020/11/29/world/europe/ukraine- mushrooms.html?action=click&module=News&pgtype=Homepage The World Bank. (2018). Growing Wildlife-Based Tourism Sustainably: A New Report and Q&A. Retrieved October 7, 2020, from https://www.worldbank.org/en/news/feature/2018/03/01/growing-wildlife-based- tourism-sustainably-a-new-report-and- 492 qahttps://www.worldbank.org/en/news/feature/2018/03/01/growing-wildlife-based- tourism-sustainably-a-new-report-and-qa qahttps://www.worldbank.org/en/news/feature/2018/03/01/growing-wildlife-based- tourism-sustainably-a-new-report-and-qa Theuerkauf, J., & Rouys, S. (2008). Habitat selection by ungulates in relation to predation risk by wolves and humans in the Białowieża Forest, Poland. Forest Ecology and Management, 256, 1325–1332. https://doi.org/10.1016/j.foreco.2008.06.030 Thilsted, S. H., James, D., Toppe, J., Subasinghe, R., & Karunasagar, I. (2014). Maximizing the contribution of fish to human nutrition. Thomas, E., Valdivia, J., Caicedo, C. A., Quaedvlieg, J., Wadt, L. H. O., & Corvera, R. (2017). NTFP harvesters as citizen scientists: Validating traditional and crowdsourced knowledge on seed production of Brazil nut trees in the Peruvian Amazon. PLoS ONE, 12(8), e0183743. https://doi.org/10.1371/journal.pone.0183743 Thomford, N., Senthebane, D., Rowe, A., Munro, D., Seele, P., Maroyi, A., & Dzobo, K. (2018). Natural Products for Drug Discovery in the 21st Century: Innovations for Novel Drug Discovery. International Journal of Molecular Sciences, 19(6), 1578. https://doi.org/10.3390/ijms19061578 Thompson, L. M. C., & Schlacher, T. A. (2008). Physical damage to coastal dunes and ecological impacts caused by vehicle tracks associated with beach camping on sandy shores: A case study from Fraser Island, Australia. Journal of Coastal Conservation, 12(2), 67–82. https://doi.org/10.1007/s11852-008-0032-9 Thoms, C. A. (2008). Community control of resources and the challenge of improving local livelihoods: A critical examination of community forestry in Nepal. Geoforum, 39(3), 1452–1465. https://doi.org/10.1016/j.geoforum.2008.01.006 Thornhill, D. J. (2012). Ecological impacts and practices of the coral reef wildlife trade. Defenders of Wildlife, 187. Thurstan, R. H., Buckley, S. M., Ortiz, J. C., & Pandolfi, J. M. (2016a). Setting the Record Straight: Assessing the Reliability of Retrospective Accounts of Change. Conservation Letters, 9(2), 98–105. Scopus. https://doi.org/10.1111/conl.12184 Thurstan, R. H., Buckley, S. M., Ortiz, J. C., & Pandolfi, J. M. (2016b). Id=APKAJLOHF5GGSLRBV4ZA Setting the Record Straight: Assessing the Reliability of Retrospective Accounts of Change. Conservation Letters, 9(2), 98–105. Scopus. https://doi.org/10.1111/conl.12184 Ticktin, T. (2004). The ecological implications of harvesting non-timber forest products: Ecological implications of non-timber harvesting. Journal of Applied Ecology, 41(1), 11–21. https://doi.org/10.1111/j.1365-2664.2004.00859.x Ticktin, Tamara, Mondragón, D., Lopez‐Toledo, L., Dutra‐Elliott, D., Aguirre‐León, E., & Hernández‐Apolinar, M. (2020). Synthesis of wild orchid trade and demography provides new insight on conservation strategies. Conservation Letters, 13(2). https://doi.org/10.1111/conl.12697 Tiencheu, B., & Womeni, H. M. (2017). Entomophagy: Insects as Food. In V. D. C. Shields (Ed.), Insect Physiology and Ecology. InTech. https://doi.org/10.5772/67384 Tierney, M., Almond, R., Stanwell-Smith, D., McRae, L., Zöckler, C., Collen, B., … de Bie, S. (2014). Use it or lose it: Measuring trends in wild species subject to substantial use. Oryx, 48(3), 420–429. https://doi.org/10.1017/S0030605313000653 p g Tilley, A., Hunnam, K. J., Mills, D. J., Steenbergen, D. J., Govan, H., Alonso-Poblacion, E., … Cohen, P. J. (2019). Evaluating the fit of co-management for small-scale fisheries 493 governance in timor-leste. Frontiers in Marine Science, 6(JUL). Scopus. https://doi.org/10.3389/fmars.2019.00392 Tilley, A., Wilkinson, S. P., Kolding, J., López-Angarita, J., Pereira, M., & Mills, D. J. (2019). Nearshore fish aggregating devices show positive outcomes for sustainable fisheries development in Timor-Leste. Frontiers in Marine Science, 6(JUL). Scopus. https://doi.org/10.3389/fmars.2019.00487 Timbavati Private Nature Reserve News. (2020). Staying in the Game – Financing the Timbavati Private Nature Reserve. Retrieved February 27, 2021, from Timbavatie Private Nature Reserve News website: https://timbavati.co.za/staying-in-the-game- financing-the-timbavati-private-nature-reserve/ Timoshyna, A., Ke, Z., Yang, Y., Liang, X., & Leaman, D. (2020). IN THE TIMES OF COVID- 19 AND THE ESSENTIAL JOURNEY TOWARDS SUSTAINABILITY. 15. Tisdell, C. A. (2015). The conservation and loss of wild biodiversity and natural ecosystems: Basic economic issues. In Sustaining Biodiversity and Ecosystem Functions (pp. 245– 274). Edward Elgar Publishing. Tisdell, C., & Svizzero, S. (2015). The persistence of hunting and gathering economies. Social Evolution & History, 14(2). Tittensor, D. P., Harfoot, M., McLardy, C., Britten, G. L., Kecse‐Nagy, K., Landry, B., … Malsch, K. (2020). Evaluating the relationships between the legal and illegal international wildlife trades. Conservation Letters, 13(5). https://doi.org/10.1111/conl.12724 Tiwary, A., Vilhar, U., Zhiyanski, M., Stojanovski, V., & Dinca, L. (2020). Management of nature-based goods and services provisioning from the urban common: A pan- European perspective. Urban Ecosystems, 23(3), 645–657. https://doi.org/10.1007/s11252-020-00951-1 Tocher, D. R. (2015). Omega-3 long-chain polyunsaturated fatty acids and aquaculture in perspective. Aquaculture, 449, 94–107. https://doi.org/10.1016/j.aquaculture.2015.01.010 Tocher, D. Id=APKAJLOHF5GGSLRBV4ZA R., Zheng, X., Schlechtriem, C., Hastings, N., Dick, J. R., & Teale, A. J. (2006). Highly unsaturated fatty acid synthesis in marine fish: Cloning, functional characterization, and nutritional regulation of fatty acyl Δ6 desaturase of Atlantic cod (Gadus morhua L.). Lipids, 41(11), 1003–1016. https://doi.org/10.1007/s11745-006- 5051-4 toobigtoignore.net—Big Numbers Project report by World Bank/FAO/World Fish. (2010). Retrieved November 22, 2021, from http://toobigtoignore.net/ Torres Romero, M. C., Galeano Garces, G. A., & Bernal, R. (2016). Cosecha y manejo de Copernicia tectorum (Kunth) Mart. Para uso artesanal en el Caribe colombiano. Colombia Forestal, 19(1), 5. https://doi.org/10.14483/udistrital.jour.colomb.for.2016.1.a01 5. Torres-Guevara, L. E., Lopez, M. C., & Schlüter, A. (2016). Understanding artisanal fishers’ behaviors: The case of Ciénaga Grande de Santa Marta, Colombia. Sustainability (Switzerland), 8(6). Scopus. https://doi.org/10.3390/su8060549 Tourenq, C., Combreau, O., Pole, S. B., Lawrence, M., Ageyev, V. S., Karpov, A. A., & Launay, F. (2004). Monitoring of Asian houbara bustard Chlamydotis macqueenii 494 populations in Kazakhstan reveals dramatic decline. Oryx, 38(1), 62–67. https://doi.org/10.1017/S0030605304000109 populations in Kazakhstan reveals dramatic decline. Oryx, 38(1), 62–67. https://doi.org/10.1017/S0030605304000109 Towns, A. M., & Shackleton, C. (2018). Traditional, Indigenous, or Leafy? A Definition, Typology, and Way Forward for African Vegetables. Economic Botany, 72(4), 461– 477. https://doi.org/10.1007/s12231-019-09448-1 TPL. (2020). The Plant List. Retrieved from http://www.theplantlist.org/ (acessed November 2020) TRAFFIC. (2008). What’s driving the wildlife trade? A review of expert opinion on economic and social drivers of the wildlife trade and trade control efforts in Cambodia, Indonesia, Lao PDR, and Vietnam (No. 46791; pp. 1–120). The World Bank. Retrieved from The World Bank website: http://documents.worldbank.org/curated/en/608621468139780146/Whats-driving-the- wildlife-trade-A-review-of-expert-opinion-on-economic-and-social-drivers-of-the- wildlife-trade-and-trade-control-efforts-in-Cambodia-Indonesia-Lao-PDR-and- Vietnam from TRAFIC. (2018). Wild at home TRAFIC. Retrieved from https://www.traffic.org/site/assets/files/9241/wild-at-home.pdf Tranquilli, S. (2014). Protected Areas in Tropical Africa: Assessing Threats and Conservation Activities. PLoS ONE. Tredennick, A. T., & Hanan, N. P. (2015). Effects of tree harvest on the stable-state dynamics of savanna and forest. The American Naturalist, 185(5), E153–E165. https://doi.org/10.1086/680475 Tregidgo, D. J., Barlow, J., Pompeu, P. S., de Almeida Rocha, M., & Parry, L. (2017). Rainforest metropolis casts 1,000-km defaunation shadow. Proceedings of the National Academy of Sciences, 114(32), 8655–8659. Trosper, R.L, & Tindall, D. B. (2013). Consultation and accommodation: Making losses visible. In Aboriginal Peoples and forest lands in Canada (pp. 313–325). Vancouver, BC: UBC Press. Trosper, Ronald L. (2012). Menominee implementation of the Chichilnisky criterion for sustainable forest management. Forest Policy and Economics, 25, 56–61. https://doi.org/10.1016/j.forpol.2012.08.002 Troudet, J., Vignes-Lebbe, R., Grandcolas, P., & Legendre, F. (2018). Id=APKAJLOHF5GGSLRBV4ZA The Increasing Disconnection of Primary Biodiversity Data from Specimens: How Does It Happen and How to Handle It? Systematic Biology, 67(6), 1110–1119. https://doi.org/10.1093/sysbio/syy044 Trujillo-González, A., & Militz, T. A. (2019). Taxonomically constrained reporting framework limits biodiversity data for aquarium fish imports to Australia. Wildlife Research, 46(4), 355. https://doi.org/10.1071/WR18135 Tsanga, R., Cerutti, P. O., Bolika, J. M., Tibaldeschi, P., & Inkonkoy, F. (2020). Independent monitoring of social clauses in the Democratic Republic of Congo. Report. Bogor, Indonesia: CIFOR. Tsing, A., Satsuka, S., & for the Matsutake Worlds Research Group. (2008). Diverging Understandings of Forest Management in Matsutake Science. Economic Botany, 62(3), 244–253. https://doi.org/10.1007/s12231-008-9035-6 495 Tuda, P. M., & Wolff, M. (2015). Evolving trends in the Kenyan artisanal reef fishery and its implications for fisheries management. Ocean and Coastal Management, 104, 36–44. Scopus. https://doi.org/10.1016/j.ocecoaman.2014.11.016 Tufts, B. L., Holden, J., & DeMille, M. (2015). Benefits arising from sustainable use of North America’s fishery resources: Economic and conservation impacts of recreational angling. International Journal of Environmental Studies, 72(5), 850–868. https://doi.org/10.1080/00207233.2015.1022987 Turkelboom, F., Thoonen, M., Jacobs, S., García-Llorente, M., Martín-López, B., & Berry, P. (2016). Ecosystem services trade-offs and synergies (draft). OpenNESS Ecosystem Services Reference Book. EC FP7 Grant Agreement, (308428). Turtiainen, M., Saastamoinen, O., Kangas, K., & Vaara, M. (2012). Picking of wild edible mushrooms in Finland in 1997–1999 and 2011. Silva Fennica, 46(4). https://doi.org/10.14214/sf.911 Turtiainen, M., Salo, K., & Saastamoinen, O. (2011). Variations of yield and utilisation of bilberries (Vaccinium myrtillus L.) and cowberries (V. vitis-idaea L.) in Finland. Silva Fennica, 45(2). https://doi.org/10.14214/sf.115 Turvey, S. T., Barrett, L. A., Yujiang, H., Lei, Z., Xinqiao, Z., Xianyan, W., … Ding, W. (2010). Rapidly shifting baselines in yangtze fishing communities and local memory of extinct species. Conservation Biology, 24(3), 778–787. Scopus. https://doi.org/10.1111/j.1523-1739.2009.01395.x Twine, W. C., & Holdo, R. M. (2016). Fuelwood sustainability revisited: Integrating size structure and resprouting into a spatially realistic fuelshed model. Journal of Applied Ecology, 53(6), 1766–1776. https://doi.org/10.1111/1365-2664.12713 Twine, W., Saphugu, V., & Moshe, D. (2003). Harvesting of communal resources by ‘outsiders’ in rural South Africa: A case of xenophobia or a real threat to sustainability? The International Journal of Sustainable Development & World Ecology, 10(3), 263– 274. Twining-Ward, L., Li, W., Bhammar, H., & Wright, E. (2018). Supporting sustainable livelihoods through wildlife tourism [Tourism for Development]. Washington DC, USA: The World Bank. Id=APKAJLOHF5GGSLRBV4ZA Retrieved from The World Bank website: https://econpapers.repec.org/scripts/redir.pf?u=https%3A%2F%2Fopenknowledge.wo rldbank.org%2Fbitstream%2Fhandle%2F10986%2F29417%2F123765-WP-P157432- PUBLIC.pdf%3Fsequence%3D6;h=repec:wbk:wboper:29417 Tyagi, A., Kumar, V., Kittur, S., Reddy, M., Naidenko, S., Ganswindt, A., & Umapathy, G. (2019). Physiological stress responses of tigers due to anthropogenic disturbance especially tourism in two central Indian tiger reserves. Conservation Physiology, 7(1), coz045. https://doi.org/10.1093/conphys/coz045 Tyrväinen, L., Silvennoinen, H., & Hallikainen, V. (2017). Effect of the season and forest management on the visual quality of the nature-based tourism environment: A case from Finnish Lapland. Scandinavian Journal of Forest Research, 32(4), 349–359. https://doi.org/10.1080/02827581.2016.1241892 Tzanatos, E., Castro, J., Forcada, A., Matić-Skoko, S., Gaspar, M., & Koutsikopoulos, C. (2013). A Métier-Sustainability-Index (MSI25) to evaluate fisheries components: 496 Assessment of cases from data-poor fisheries from southern Europe. ICES Journal of Marine Science, 70(1), 78–98. Scopus. https://doi.org/10.1093/icesjms/fss161 Assessment of cases from data-poor fisheries from southern Europe. ICES Journal of Marine Science, 70(1), 78–98. Scopus. https://doi.org/10.1093/icesjms/fss161 Uhl, C., Barreto, P., Vidal, E., Amaral, P., Barros, A. C., Souza, C., … Gerwing, J. (1997). Natural Resource Management in the Brazilian Amazon. BioScience, 47(3), 160–168. https://doi.org/10.2307/1313035 Uhl, C., Veríssimo, A., Mattos, M. M., Brandino, Z., & Vieira, I. C. G. (1991). Social, economic, and ecological consequences of selective logging in an Amazon frontier: The case of Tailândia. Forest Ecology and Management, 46(3–4), 243–273. Ulian, T., Diazgranados, M., Pironon, S., Padulosi, S., Liu, U., Davies, L., … Mattana, E. (2020). Unlocking plant resources to support food security and promote sustainable agriculture. PLANTS, PEOPLE, PLANET, 2(5), 421–445. https://doi.org/10.1002/ppp3.10145 Ulman, A., Bekisoglu, S., Zengin, M., Knudsen, S., Unal, V., Mathews, C., … Pauly, D. (2013). From bonito to anchovy: A reconstruction of Turkey’s marine fisheries catches (1950-2010). Mediterranean Marine Science, 14(2), 309–342. Ulman, A., Çiçek, B. A., Salihoglu, I., Petrou, A., Patsalidou, M., Pauly, D., & Zeller, D. (2015a). Unifying the catch data of a divided island: Cyprus’s marine fisheries catches, 1950-2010. Environment, Development and Sustainability, 17(4), 801–821. Scopus. https://doi.org/10.1007/s10668-014-9576-z Ulman, A., Çiçek, B. A., Salihoglu, I., Petrou, A., Patsalidou, M., Pauly, D., & Zeller, D. (2015b). Unifying the catch data of a divided island: Cyprus’s marine fisheries catches, 1950-2010. Environment, Development and Sustainability, 17(4), 801–821. Scopus. https://doi.org/10.1007/s10668-014-9576-z Ulman, A., & Pauly, D. (2016). Making history count: The shifting baselines of Turkish fisheries. Fisheries Research, 183, 74–79. Scopus. https://doi.org/10.1016/j.fishres.2016.05.013 UN. (2015). The Sustainable Development Agenda. Accessed from: Https://www.un.org/sustainabledevelopment/development-agenda/, [International]. Retrieved from Sustainable Development Goals website: The Sustainable Development Agenda. Accessed from: https://www.un.org/sustainabledevelopment/development- agenda/ Ünal, V., & Franquesa, R. (2010a). Id=APKAJLOHF5GGSLRBV4ZA A comparative study on socio-economic indicators and viability in small-scale fisheries of six districts along the Turkish coast: Technical note. Journal of Applied Ichthyology, 26(1), 26–34. Scopus. https://doi.org/10.1111/j.1439- 0426.2009.01346.x UNCTAD. (2017). 20 years of Biotrade. Connecting people, the planet and markets. Retrieved from https://www.greengrowthknowledge.org/sites/default/files/downloads/resource/20%2 0Years%20of%20BioTrade.pdf (acessed March 2021) UNCTAD. (2021). COVID-19 and Tourism: An Update—Assessing the economic consequences. United Nations Conference on Trade and Development. Retrieved from United Nations Conference on Trade and Development website: https://unctad.org/system/files/official-document/ditcinf2021d3_en_0.pdf 497 UNDP. (2014). United Nations Development Programme Country: Afghanistan PROJECT DOCUMENT “Establishing integrated models for protected areas and their co- management in Afghanistan. UNEP. (2021). The Species+ Website. Nairobi, Kenya. Compiled by UNEP-WCMC, Cambridge, UK. Available at: Www.speciesplus.net. [Accessed 25/02/2021]. Retrieved September 21, 2021, from https://speciesplus.net/ UNEP/CMS. (2006). Wildlife watching and tourism: A study on the benefits and risks of a fast growing tourism activity and its impacts on species (No. CMS/ScS14/Inf.8; p. 86). Bonn, Germany: UNEP / CMS Secretariat. Retrieved from UNEP / CMS Secretariat website: https://www.cms.int/sites/default/files/document/ScC14_Inf_08_Wildlife_Watching_ E_0.pdf https://www.cms.int/sites/default/files/document/ScC14_Inf_08_Wildlife_Watching_ E_0.pdf UNESCO. (2021). World Heritage Centre _ Interactive Map. Retrieved August 10, 2021, from https://whc.unesco.org/en/interactive-map/ UNGA. United Nations General Assembly Resolution 61/105. Sustainable fisheries, including through the 1995 Agreement for the Implementation of the Provisions of the United Nations Convention on the Law of the Sea of 10 December 1982 relating to the Conservation and Management of Straddling Fish Stocks and Highly Migratory Fish Stocks, and related instruments. , 61/105 § (2006). UNIDO/CFC. (2005). Product and Market Development of Sisal and Henequen (No. Project completion report Addendum A.3-Part One: Kenya). Retrieved from https://open.unido.org/api/documents/4788682/download/PRODUCT%20AND%20 MARKET%20DEVELOPMENT%20OF%20SISAL%20AND%20HENEQUEN.%20 VARIETY%20TRIALS%20IN%20ESTATES.%20PROJECT%20COMPLETION% 20REPORT-ADDENDUM%20A.3.%20PART%20ONE%20- %20KENYA.%20COMMON%20FUND%20FOR%20COMMODITIES%20PROJE CT%20CFC-FIGHF-07%20(23503.en) %20KENYA.%20COMMON%20FUND%20FOR%20COMMODITIES%20PROJE CT%20CFC-FIGHF-07%20(23503.en) United Nations. (2006a). Resolution Adopted by the General Assembly. 61/105. Sustainable Fisheries, including through the 1995 Agreement for the Implementation of the Provisions of the United Nations Convention on the Law of the Sea of 10 December 1982 relating to the Conservation and Management of straddling Fish Stocks and Highly Migratory Fish Stocks, and Related Instruments (United Nations General Assembly Doc. A/RES/61/105). United Nations. (2006b). Review Conference on the Agreement for the Implementation of the Provisions of the United Nations Convention on the Law of the Sea of 10 December 1982 relating to the Conservation and Management of Straddling Fish Stocks and Highly Migratory Fish Stocks, New York, 22 to 26 May 2006 (A/CONF.210/2006/15). United Nations. United Nations Department of Economic and Social Affairs. (2020). Resources on Data and Indicators \| United Nations For Indigenous Peoples. Retrieved April 15, 2020, from https://www.un.org/development/desa/indigenouspeoples/mandated-areas1/data-and- indicators/resources-on-data-and-indicators.html 498 UNODC. (2016). UNODC Annual Report. Retrieved from https://www.unodc.org/documents/AnnualReport2016/2016_UNODC_Annual_Repor t.pdf from Annual https://www.unodc.org/documents/AnnualReport2016/2016_UNODC_Annual_Repor t.pdf UNWTO. (2015). Towards Measuring the Economic Value of Wildlife Watching Tourism in Africa. UNWTO Madrid, Spain. UNWTO. (2019). International tourism highlights 2019. Madrid: World Tourism Organization. U.S. Department of the Interior. (2017). New 5-Year Report Shows 101.6 Million Americans Participated in Hunting, Fishing & Wildlife Activities. Retrieved April 15, 2020, from https://www.doi.gov/pressreleases/new-5-year-report-shows-1016-million-americans- participated-hunting-fishing-wildlife U.S. Department of the Interior. (2020, March 19). Sportsmen and Sportswomen Generate Nearly $1 Billion in Conservation Funding. Retrieved February 7, 2021, from Press Releases website: https://www.doi.gov/pressreleases/sportsmen-and-sportswomen- generate-nearly-1-billion-conservation-funding U.S. Department of the Interior, U.S. Fish and Wildlife Service, U.S. Department of Commerce, & U.S. Census Bureau. (2018). 2016 National Survey of Fishing, Hunting and Wildlife-Associated Recreation (No. FHW/16-NAT). Retrieved from https://www.fws.gov/wsfrprograms/subpages/nationalsurvey/nat_survey2016.pdf U.S. Fish and Wildlife Service. (2016). National Survey of Fishing, Hunting, and Wildlife- Associated Recreation. USDA, & NRCS. (2009). The PLANTS Database for gulfhairawn muhly (Muhlenbergia filipes). National Plant Data Center, Baton Rouge, LA. Retrieved from http://plants.usda.gov/ Uzoebo, C., Azeez, A., Akeredolu, O., Adetunji, A., Bolaji, O., & Abdulkadir, A. (2019). History of mushroom hunting and identification in Nigeria. Journal of Medicinal Plants Studies, 7(6), 89–91. Vallejo, M. I., Galeano, G., Bernal, R., & Zuidema, P. A. (2014). The fate of populations of Euterpe oleracea harvested for palm heart in Colombia. Forest Ecology and Management, 318, 274–284. https://doi.org/10.1016/j.foreco.2014.01.028 Vallejo, M. I., Valderrama, N., Bernal, R., Galeano, G., Arteaga, G., & Leal, C. (2011). Producción de palmito Euterpe oleracea Mart. (ARECACEAE) en la costa pacífica colombiana:estado actual y perspectivas. CT%20CFC-FIGHF-07%20(23503.en) Colombia Forestal, 14(2), 191. https://doi.org/10.14483/udistrital.jour.colomb.for.2011.2.a05 Van Assche, K., Beunen, R., Duineveld, M., & Gruezmacher, M. (2017). Power/knowledge and natural resource management: Foucaultian foundations in the analysis of adaptive governance. Journal of Environmental Policy & Planning, 19(3), 308–322. https://doi.org/10.1080/1523908X.2017.1338560 van der Knaap, M., & Ligtvoet, W. (2010). Is western consumption of nile perch from Lake Victoria sustainable? Aquatic Ecosystem Health and Management, 13(4), 429–436. Scopus. https://doi.org/10.1080/14634988.2010.526088 van der Kroon, B., Brouwer, R., & Van Beukering, P. J. (2013). The energy ladder: Theoretical myth or empirical truth? Results from a meta-analysis. Renewable and Sustainable Energy Reviews, 20, 504–513. 499 van Heezik, Y., & Ostrowski, S. (2001). Conservation breeding for reintroductions: Assessing survival in a captive flock of houbara bustards. Animal Conservation Forum, 4(3), 195– 201. https://doi.org/10.1017/S1367943001001238 Van Hensbergen, B. (2016). Forest Concessions—Past Present and Future. Forestry and Institutions Working Paper, 36. Rome, Italy: Food and Agriculture Organization of the United Nations. Retrieved from http://www.fao.org/forestry/45024- 0c63724580ace381a8f8104cf24a3cff3.pdf van Huis, A. (2020). Insects as food and feed, a new emerging agricultural sector: A review. Journal of Insects as Food and Feed, 6(1), 27–44. https://doi.org/10.3920/Jiff2019.0017 van Huis, A., & Oonincx, D. G. A. B. (2017). The environmental sustainability of insects as food and feed. A review. Agronomy for Sustainable Development, 37(5). https://doi.org/ARTN 43 10.1007/s13593-017-0452-8 van Huis, Arnold. (2018). Insects as Human Food. In Ethnozoology (pp. 195–213). Elsevier. https://doi.org/10.1016/B978-0-12-809913-1.00011-9 van Huis, Arnold, Itterbeeck, J. V., Klunder, H., Mertens, E., Halloran, A., Muir, G., & Vantomme, P. (2013). Edible insects: Future prospects for food and feed security (Vol. 171). Rome: Food and Agriculture Organization of the United Nations. Retrieved from https://www.fao.org/3/i3253e/i3253e.pdf Van, N. D. N., & Tap, N. (2008). An overview of the use of plants and animals in traditional medicine systems in Viet Nam [TRAFFIC Southeast Asia, Greater Mekong Programme, Ha Noi, Viet Nam]. Retrieved from http://www.trafficj.org/publication/08_medical_plants_Viet_Num.pdf van Putten, I. E., Frusher, S., Fulton, E. A., Hobday, A. J., Jennings, S. M., Metcalf, S., … Handling editor: Sarah Kraak. (2016). Empirical evidence for different cognitive effects in explaining the attribution of marine range shifts to climate change. ICES Journal of Marine Science, 73(5), 1306–1318. https://doi.org/10.1093/icesjms/fsv192 Van Schuylenbergh, P. (2009). Entre délinquance et résistance au Congo belge: L’interprétation coloniale du braconnage. ntre délinquance et résistance au Congo belge: l’interprétation coloniale du braconnage, 7, 25–48. Van Vliet, N., Milner-Gulland, E. J., Bousquet, F., Saqalli, M., & Nasi, R. (2010). CT%20CFC-FIGHF-07%20(23503.en) Effect of Small-Scale Heterogeneity of Prey and Hunter Distributions on the Sustainability of Bushmeat Hunting: Heterogeneity of Prey and Hunter Distributions. Conservation Biology, 24(5), 1327–1337. https://doi.org/10.1111/j.1523-1739.2010.01484.x van Vliet, N., Muhindo, J., Kambale Nyumu, J., Mushagalusa, O., & Nasi, R. (2018). Mammal Depletion Processes as Evidenced From Spatially Explicit and Temporal Local Ecological Knowledge. Tropical Conservation Science, 11, 194008291879949. https://doi.org/10.1177/1940082918799494 van Vliet, N., & Nasi, R. (2008). Hunting for Livelihood in Northeast Gabon: Patterns, Evolution, and Sustainability. Ecology and Society, 13(2), art33. https://doi.org/10.5751/ES-02560-130233 Vanam, B. (2019). Timber trade in India-challenges and policies. EPRA International Journal of Multidisciplinary Research (IJMR), 12(5), 119–122. 500 Vannuccini, S. (1999). Shark utilization, marketing and trade. FAO FISHERIES TECHNICAL PAPER. Retrieved from http://www.fao.org/3/x3690e/x3690e1d.htm (accessed 19 feb 2021). Vasconcellos, M., & Cochrane, K. (2005). Overview of world status of data-limited fisheries: Inferences from landings statistics. In Fisheries assessment and management in data- limited situations (Anchorage, AK: LowellWakefield Symposium, 21., pp. 1–20). V. F. Kruse Gallucci, et al. (Eds.). Vasconcelos, J., Sousa, R., Henriques, P., Amorim, A., Delgado, J., & Riera, R. (2020). Two sympatric, not externally discernible, and heavily exploited deepwater species with coastal migration during spawning season: Implications for sustainable stocks management of Aphanopus carbo and Aphanopus intermedius around madeira. Canadian Journal of Fisheries and Aquatic Sciences, 77(1), 124–131. Scopus. https://doi.org/10.1139/cjfas-2018-0423 Vaughan, C., Gack, J., Solorazano, H., & Ray, R. (2003). The Effect of Environmental Education on Schoolchildren, Their Parents, and Community Members: A Study of Intergenerational and Intercommunity Learning. The Journal of Environmental Education, 34(3), 12–21. https://doi.org/10.1080/00958960309603489 Veldhuis, M. P., Ritchie, M. E., Ogutu, J. O., Morrison, T. A., Beale, C. M., Estes, A. B., … Olff, H. (2019). Cross-boundary human impacts compromise the Serengeti-Mara ecosystem. Science, 363(6434), 1424–1428. https://doi.org/10.1126/science.aav0564 Venkatraman, P. D., Scott, K., & Liauw, C. (2020). Environmentally friendly and sustainable bark cloth for garment applications: Evaluation of fabric properties and apparel development. Sustainable Materials and Technologies, 23. https://doi.org/ARTN e00136 10.1016/j.susmat.2019.e00136 Venohr, M., Langhans, S. D., Peters, O., Hölker, F., Arlinghaus, R., Mitchell, L., & Wolter, C. (2018). The underestimated dynamics and impacts of water-based recreational activities on freshwater ecosystems. Environmental Reviews, 26(2), 199–213. https://doi.org/10.1139/er-2017-0024 Venter, Z. S., Shackleton, C. M., Van Staden, F., Selomane, O., & Masterson, V. A. (2020). Green Apartheid: Urban green infrastructure remains unequally distributed across income and race geographies in South Africa. Landscape and Urban Planning, 203, 103889. https://doi.org/10.1016/j.landurbplan.2020.103889 Venugopal, V. (2018). Nutrients and Nutraceuticals from Seafood. In J.-M. CT%20CFC-FIGHF-07%20(23503.en) Merillon & K. G. Ramawat (Eds.), Sweeteners (pp. 1–45). Cham: Springer International Publishing. https://doi.org/10.1007/978-3-319-54528-8_36-2 Vereinte Nationen. (2016). World wildlife crime report: Trafficking in protected species, 2016. New York: United Nations. Veríssimo, A., Barreto, P., Mattos, M., Tarifa, R., & Uhl, C. (1992). Logging impacts and prospects for sustainable forest management in an old Amazonian frontier: The case of Paragominas. Forest Ecology and Management, 55(1–4), 169–199. https://doi.org/10.1016/0378-1127(92)90099-U Veríssimo, A., Barreto, P., Tarifa, R., & Uhl, C. (1995). Extraction of a high-value natural resource in Amazonia: The case of mahogany. Forest Ecology and Management, 72(1), 39–60. https://doi.org/10.1016/0378-1127(94)03432-V 501 Verissimo, D., MacMillan, D. C., & Smith, R. J. (2011). Toward a systematic approach for identifying conservation flagships: Identifying conservation flagships. Conservation Letters, 4(1), 1–8. https://doi.org/10.1111/j.1755-263X.2010.00151.x Větrovský, T., Morais, D., Kohout, P., Lepinay, C., Algora, C., Awokunle Hollá, S., … Baldrian, P. (2020). GlobalFungi, a global database of fungal occurrences from high- throughput-sequencing metabarcoding studies. Scientific Data, 7(1), 228. https://doi.org/10.1038/s41597-020-0567-7 Vidal, O., López‐García, J., & Rendón‐Salinas, E. (2014). Trends in Deforestation and Forest Degradation after a Decade of Monitoring in the Monarch Butterfly Biosphere Reserve in Mexico. Conservation Biology, 28(1), 177–186. https://doi.org/10.1111/cobi.12138 Vilanova, E., Ramírez-Angulo, H., Ramírez, G., & Torres-Lezama, A. (2012). Compliance with sustainable forest management guidelines in three timber concessions in the Venezuelan Guayana: Analysis and implications. Forest Policy and Economics, 17, 3– 12. https://doi.org/10.1016/j.forpol.2011.11.001 Vincent, H., Wiersema, J., Kell, S., Fielder, H., Dobbie, S., Castañeda-Álvarez, N. P., … Maxted, N. (2013). A prioritized crop wild relative inventory to help underpin global food security. Biological Conservation, 167, 265–275. https://doi.org/10.1016/j.biocon.2013.08.011 Virgós, E., & Travaini, A. (2005). Relationship Between Small-game Hunting and Carnivore Diversity in Central Spain. Biodiversity & Conservation, 14(14), 3475. https://doi.org/10.1007/s10531-004-0823-8 Visseren-Hamakers, I. J. (2020). The 18th Sustainable Development Goal. Earth System Governance, 3, 100047. https://doi.org/10.1016/j.esg.2020.100047 Volker D. (2006). Studying Marine Mammal Cognition in the Wild: A Review of Four Decades of Playback Experiments. Aquatic Mammals, 32. https://doi.org/10.1578/AM.32.4.2006.461 von Heland, J., & Folke, C. (2014). A social contract with the ancestors—Culture and ecosystem services in southern Madagascar. Global Environmental Change, 24, 251– 264. https://doi.org/10.1016/j.gloenvcha.2013.11.003 von Hoffen, L. P., & Säumel, I. (2014). Orchards for edible cities: Cadmium and lead content in nuts, berries, pome and stone fruits harvested within the inner city neighbourhoods in Berlin, Germany. Ecotoxicology and Environmental Safety, 101, 233–239. https://doi.org/10.1016/j.ecoenv.2013.11.023 Wabnitz, C. (2003). From ocean to aquarium: The global trade in marine ornamental species. UNEP/Earthprint. Wadt, L. H. CT%20CFC-FIGHF-07%20(23503.en) O., Kainer, K. A., Staudhammer, C. L., & Serrano, R. O. P. (2008). Sustainable forest use in Brazilian extractive reserves: Natural regeneration of Brazil nut in exploited populations. Biological Conservation, 141(1), 332–346. https://doi.org/10.1016/j.biocon.2007.10.007 Wakild, E. (2020). Saving the Vicuña: The Political, Biophysical, and Cultural History of Wild Animal Conservation in Peru, 1964–2000. The American Historical Review, 125(1), 54–88. https://doi.org/10.1093/ahr/rhz939 502 Waldhoff, P., & Vidal, E. (2015). Community loggers attempting to legalize traditional timber harvesting in the Brazilian Amazon: An endless path. Forest Policy and Economics, 50, 311–318. https://doi.org/10.1016/j.forpol.2014.08.005 Walker, G., & Bulkeley, H. (2006). Geographies of environmental justice. Geoforum, 37(5), 655–659. https://doi.org/10.1016/j.geoforum.2005.12.002 Waller, D. M., & Reo, N. J. (2018). First stewards: Ecological outcomes of forest and wildlife stewardship by indigenous peoples of Wisconsin, USA. Ecology and Society. https://doi.org/10.5751/ES-09865-230145 Walls, R. H., & Dulvy, N. K. (2021). Tracking the rising extinction risk of sharks and rays in the Northeast Atlantic Ocean and Mediterranean Sea. Scientific Reports, 11(1), 1–15. https://doi.org/10.1038/s41598-021-94632-4 Walter, A., & Sam, C. (1999). Fruits d’Océanie. Paris: IRD Editions. Walters, G., Pathak Broome, N., Cracco, M., Dash, T., Dudley, N., Elías, S., … Van Vliet, N. (2021). COVID-19, Indigenous peoples, local communities and natural resource governance. PARKS, (27), 57–72. https://doi.org/10.2305/IUCN.CH.2021.PARKS-27- SIGW.en Waltner-Toews, D., & Kay, J. (2005). The Evolution of an Ecosystem Approach: The Diamond Schematic and an Adaptive Methodology for Ecosystem Sustainability and Health. Ecology and Society, 10(1), art38. https://doi.org/10.5751/ES-01214-100138 Wang, T., Feng, L., Mou, P., Wu, J., Smith, J. L. D., Xiao, W., … Ge, J. (2016). Amur tigers and leopards returning to China: Direct evidence and a landscape conservation plan. Landscape Ecology, 31(3), 491–503. https://doi.org/10.1007/s10980-015-0278-1 Wang, X.-L., & Yao, Y.-J. (2011). Host insect species of Ophiocordyceps sinensis: A review. ZooKeys, 127, 43–59. https://doi.org/10.3897/zookeys.127.802 Ward, P., & Myers, R. A. (2005). SHIFTS IN OPEN-OCEAN FISH COMMUNITIES COINCIDING WITH THE COMMENCEMENT OF COMMERCIAL FISHING. Ecology, 86(4), 835–847. https://doi.org/10.1890/03-0746 Wardojo, W., Suhariyanto, & Purnama, B. M. (2001). Law enforcement and forest protection in Indonesia: A retrospect andprospect. Bali, Indonesia. Retrieved from http://siteresources.worldbank.org/INTINDONESIA/FLEG/20171554/Law_Enforcem ent.pdf Warkentin, I. G., Bickford, D., Sodhi, N. S., & Bradshaw, C. J. A. (2009). Eating Frogs to Extinction. Conservation Biology, 23(4), 1056–1059. https://doi.org/10.1111/j.1523- 1739.2008.01165.x Warwick, C., & Steedman, C. (2021). Regulating pets using an objective positive list approach. Journal of Veterinary Behavior, 42, 53–63. https://doi.org/10.1016/j.jveb.2021.01.008 Watson, K. (2017). Alternative Economies of the Forest: Honey Production and Public Land Management in Northwest Florida. CT%20CFC-FIGHF-07%20(23503.en) Society & Natural Resources, 30(3), 331–346. https://doi.org/10.1080/08941920.2016.1209265 Watson, K., Christian, C. S., Emery, M. R., Hurley, P. T., McLain, R. J., & Wilmsen, C. (2018). Social dimensions of nontimber forest products. In: Chamberlain, James L.; Emery, Marla R.; Patel-Weynand, Toral, Eds. 2018. Assessment of Nontimber Forest Products in the United States under Changing Conditions. Gen. Tech. Rep. SRS–232. Asheville, 503 NC: U.S. Department of Agriculture, Forest Service, Southern Research Station, 2018, 102–117. NC: U.S. Department of Agriculture, Forest Service, Southern Research Station, 2018, 102–117. Watson, R. T. (2005). Turning science into policy: Challenges and experiences from the science–policy interface. Philosophical Transactions of the Royal Society B: Biological Sciences, 360(1454), 471–477. https://doi.org/10.1098/rstb.2004.1601 WCPFC. (2014). Conservation and Management Measure on Establishing a Harvest Strategy for Key Fisheries and Stocks in the Western and Central Pacific Ocean. Western and Central Pacific Fisheries Commission. Webb, G.J.W. (2014). Wildlife Conservation: In the Belly of the Beast. Charles Darwin University Press. Webb, Grahame J W. (2021). History of Crocodile Management in the Northern Territory of Australia: WMI. Webster, F. J., Cohen, P. J., Malimali, S., Tautai, M., Vidler, K., Mailau, S., … Fatongiatau, V. (2017). Detecting fisheries trends in a co-managed area in the Kingdom of Tonga. Fisheries Research, 186, 168–176. Scopus. https://doi.org/10.1016/j.fishres.2016.08.026 Wehi, P. M., & Wehi, W. L. (2010). Traditional Plant Harvesting in Contemporary Fragmented and Urban Landscapes. Conserv Biol, 24(2), 594–604. https://doi.org/10.1111/j.1523- 1739.2009.01376.x Weinbaum, K. Z., Brashares, J. S., Golden, C. D., & Getz, W. M. (2013). Searching for sustainability: Are assessments of wildlife harvests behind the times? https://doi.org/10.1111/ele.12008 Weiss, G., Ludvig, A., Asamer-Handler, M., Fischer, C. R., Vacik, H., & Zivojinovic, I. (2019). Rendering NWFPs innovative. In Bernhard Wolfslehner, I. Prokofieva, & R. Mavsar (Eds.), Non-wood forest products in Europe: Seeing the forest around the trees. Joensuu: European Forest Institute. Welcomme, R. L. (2011). An overview of global catch statistics for inland fish. ICES Journal of Marine Science, 68(8), 1751–1756. Wendiro, D., Wacoo, A. P., & Wise, G. (2019). Identifying indigenous practices for cultivation of wild saprophytic mushrooms: Responding to the need for sustainable utilization of natural resources. Journal of Ethnobiology and Ethnomedicine, 15(1), 64. https://doi.org/10.1186/s13002-019-0342-z Werling, B. P., Dickson, T. L., Isaacs, R., Gaines, H., Gratton, C., Gross, K. L., … Landis, D. A. (2014). Perennial grasslands enhance biodiversity and multiple ecosystem services in bioenergy landscapes. Proceedings of the National Academy of Sciences, 111(4), 1652–1657. CT%20CFC-FIGHF-07%20(23503.en) https://doi.org/10.1073/pnas.1309492111 West, P., Igoe, J., & Brockington, D. (2006). Parks and Peoples: The Social Impact of Protected Areas. Annual Review of Anthropology, 35(1), 251–277. https://doi.org/10.1146/annurev.anthro.35.081705.123308 West, T. A. P., Vidal, E., & Putz, F. E. (2014). Forest biomass recovery after conventional and reduced-impact logging in Amazonian Brazil. Forest Ecology and Management, 314, 59–63. https://doi.org/10.1016/j.foreco.2013.11.022 504 Wetzel, S., Duchesne, L. C., & Laporte, M. F. (2006). Decorative and Aesthetic Products. In Bioproducts from canada’s forests: New Partnerships in the Bioeconomy (pp. 147– 162). Springer. WFO. (2020). World Flora Online. Retrieved from http://www.worldfloraonline.org/(acessed Novemeber 2020) White, M. P., Alcock, I., Grellier, J., Wheeler, B. W., Hartig, T., Warber, S. L., … Fleming, L. E. (2019). Spending at least 120 minutes a week in nature is associated with good health and wellbeing. Scientific Reports, 9(1), 7730. https://doi.org/10.1038/s41598-019- 44097-3 White, R. L., Eberstein, K., & Scott, D. M. (2018). Birds in the playground: Evaluating the effectiveness of an urban environmental education project in enhancing school children’s awareness, knowledge and attitudes towards local wildlife. PLOS ONE, 13(3), e0193993. https://doi.org/10.1371/journal.pone.0193993 Whiting, M. J., Williams, V. L., & Hibbitts, T. J. (2013). Animals Traded for Traditional Medicine at the Faraday Market in South Africa: Species Diversity and Conservation Implications. In R. R. N. Alves & I. L. Rosa (Eds.), Animals in Traditional Folk Medicine: Implications for Conservation (pp. 421–473). Berlin, Heidelberg: Springer. https://doi.org/10.1007/978-3-642-29026-8_19 Whitman, K., Starfield, A. M., Quadling, H. S., & Packer, C. (2004). Sustainable trophy hunting of African lions. Nature, 428(6979), 175–178. https://doi.org/10.1038/nature02395 WHO (Ed.). (2013). WHO traditional medicine strategy. 2014-2023. Geneva: World Health Organization. Wiadnyana, N., Suharti, S., Ndobe, S., Triharyuni, S., Lilley, G., Risuana, S., … Moore, A. (2020). Population trends of Banggai cardinalfish in the Banggai Islands, Central Sulawesi, Indonesia. 420, 012033. IOP Publishing. Wielgus, R. B., Morrison, D. E., Cooley, H. S., & Maletzke, B. (2013). Effects of male trophy hunting on female carnivore population growth and persistence. Biological Conservation, 167, 69–75. https://doi.org/10.1016/j.biocon.2013.07.008 Wilkie, D. S., Bennett, E. L., Peres, C. A., & Cunningham, A. A. (2011). The empty forest revisited. Annals of the New York Academy of Sciences, 1223(1), 120–128. https://doi.org/10.1111/j.1749-6632.2010.05908.x Wilkinson, C., Waitt, G., & Gibbs, L. (2014). Understanding Place as ‘Home’ and ‘Away’ through Practices of Bird-watching. Australian Geographer, 45(2), 205–220. https://doi.org/10.1080/00049182.2014.899029 Wilkinson, P. F., & Pratiwi, W. (1995). Gender and tourism in an Indonesian village. Annals of Tourism Research, 22(2), 283–299. CT%20CFC-FIGHF-07%20(23503.en) https://doi.org/10.1016/0160-7383(94)00077-8 Williams, A., Althaus, F., Maguire, K., Green, M., Untiedt, C., Alderslade, P., … Schlacher, T. A. (2020). The Fate of Deep-Sea Coral Reefs on Seamounts in a Fishery-Seascape: What Are the Impacts, What Remains, and What Is Protected? Frontiers in Marine Science, 7, 567002. https://doi.org/10.3389/fmars.2020.567002 Williams, A., Schlacher, T. A., Rowden, A. A., Althaus, F., Clark, M. R., Bowden, D. A., … Kloser, R. J. (2010). Seamount megabenthic assemblages fail to recover from trawling 505 Ecology, 31, 183–199. 183–199. impacts: Trawling impacts. Marine Ecology, 31, 183–199. https://doi.org/10.1111/j.1439-0485.2010.00385.x impacts: Trawling impacts. Marine https://doi.org/10.1111/j.1439-0485.2010.00385.x Marine impacts. Williams, B. K., Johnson, F. A., & Wilkins, K. (1996). Uncertainty and the Adaptive Management of Waterfowl Harvests. The Journal of Wildlife Management, 60(2), 223– 232. https://doi.org/10.2307/3802220 Williams, B. K., Johnson, F. A., & Wilkins, K. (1996). Uncertainty and the Adaptive Management of Waterfowl Harvests. The Journal of Wildlife Management, 60(2), 223– 232. https://doi.org/10.2307/3802220 Williams, C., Walsh, A., Vaglica, V., Sirakaya, A., da Silva, M., Dalle, G., … Cowell, C. (2020). Conservation Policy: Helping or hindering science to unlock properties of plants and fungi. PLANTS, PEOPLE, PLANET, 2(5), 535–545. https://doi.org/10.1002/ppp3.10139 Williams, P. R. D., Inman, D., Aden, A., & Heath, G. A. (2009). Environmental and Sustainability Factors Associated With Next-Generation Biofuels in the U.S.: What Do We Really Know? Environmental Science & Technology, 43(13), 4763–4775. https://doi.org/10.1021/es900250d Williams, V. L., Victor, J. E., & Crouch, N. R. (2013). Red Listed medicinal plants of South Africa: Status, trends, and assessment challenges. South African Journal of Botany, 86, 23–35. https://doi.org/10.1016/j.sajb.2013.01.006 Williams, Vivienne L., Cunningham, A. B., Kemp, A. C., & Bruyns, R. K. (2014). Risks to Birds Traded for African Traditional Medicine: A Quantitative Assessment. PLOS ONE, 9(8), e105397. https://doi.org/10.1371/journal.pone.0105397 Williams, Vivienne Linda, & Whiting, M. J. (2016). A picture of health? Animal use and the Faraday traditional medicine market, South Africa. Journal of Ethnopharmacology, 179, 265–273. https://doi.org/10.1016/j.jep.2015.12.024 Williams, V.L., Loveridge, A. J., Newton, D. J., & Macdonald, D. W. (2017). A roaring trade? The legal trade in Panthera leo bones from Africa to East-Southeast Asia. PLOS ONE, 12(10), e0185996. https://doi.org/10.1371/journal.pone.0185996 Willis, K. J. (2018). State of the world’s fungi 2018. Report. (K. J. Willis, Ed.). Richmond, UK: Kew Publishing. Wilshusen, P. R. (2005a). Community adaptation or collective breakdown? The emergence of ‘work groups’ in two forestry ejidos in Quintana Roo, Mexico. In The community forests of Mexico: Managing for sustainable landscapes (pp. 151–179). CT%20CFC-FIGHF-07%20(23503.en) Austin: University of Texas Press. Wilshusen, P. R. (2005b). ITTO Country Case Study: Petcacab. Sociedad de Productores Forestales Ejidales de Quintana Roo (SPFEQR), Quintana Roo, México. Retrieved from https://rightsandresources.org/wp-content/exported-pdf/petcacabqr.pdf Wilson, C., & Tisdell, C. (2003). Conservation and economic benefits of wildlife-based marine tourism: Sea turtles and whales as case studies. Human Dimensions of Wildlife, 8(1), 49–58. Winker, K., Reed, J. M., Escalante, P., Askins, R. A., Cicero, C., Hough, G. E., & Bates, J. (2010). The Importance, Effects, and Ethics of Bird Collecting. The Auk, 127(3), 690– 695. https://doi.org/10.1525/auk.2010.09199 Winkler, D. (2008). Yartsa Gunbu (Cordyceps sinensis) and the Fungal Commodification of Tibet’s Rural Economy. Economic Botany, 62(3), 291–305. https://doi.org/10.1007/s12231-008-9038-3 506 Winterbach, C. W., Whitesell, C., & Somers, M. J. (2015). Wildlife Abundance and Diversity as Indicators of Tourism Potential in Northern Botswana. PLOS ONE, 10(8), e0135595. https://doi.org/10.1371/journal.pone.0135595 Wit, M., van Dam, J., Omar Cerutti, P., Lescuyer, G., & Mckeown, J. P. (2010). Chainsaw milling: Supplier to local markets—A synthesis. 16. WOCAN. (2020). About the W+ Standard. Women Organizing for Change in Agriculture and Natural Resource Management (WOCAN). Retrieved from https://www.wocan.org/what-we-do/wstandard Wolf, I. D., Croft, D. B., & Green, R. J. (2019). Nature Conservation and Nature-Based Tourism: A Paradox? Environments, 6(9), 104. https://doi.org/10.3390/environments6090104 Wolf, K. L., Lam, S. T., McKeen, J. K., Richardson, G. R. A., van den Bosch, M., & Bardekjian, A. C. (2020). Urban Trees and Human Health: A Scoping Review. International Journal of Environmental Research and Public Health, 17(12), 4371. https://doi.org/10.3390/ijerph17124371 Wolfslehner, B., Prokofieva, I., & Mavsar, R. (2019). Non-wood forest products in Europe: Seeing the forest around the trees. What Science Can Tell Us 10. European Forest Institute. Retrieved from European Forest Institute website: https://efi.int/sites/default/files/files/publication-bank/2019/efi_wsctu_10_2019.pdf Wong, S., & Liu, H. (2019). Wild-Orchid Trade in a Chinese E-Commerce Market. Economic Botany, 73(3), 357–374. https://doi.org/10.1007/s12231-019-09463-2 Woodhouse, E., McGowan, P., & Milner-Gulland, E. J. (2014). Fungal gold and firewood on the Tibetan plateau: Examining access to diverse ecosystem provisioning services within a rural community. Oryx, 48(1), 30–38. https://doi.org/10.1017/S0030605312001330 Woodward, E., Jackson, S., Finn, M., & McTaggart, P. M. (2012). Utilising Indigenous seasonal knowledge to understand aquatic resource use and inform water resource management in northern Australia. Ecological Management & Restoration, 13(1), 58– 64. https://doi.org/10.1111/j.1442-8903.2011.00622.x Woodward, E., & Marrfurra McTaggart, P. (2019). Co-developing Indigenous seasonal calendars to support ‘healthy Country, healthy people’ outcomes. Glob Health Promot, 26(3_suppl), 26–34. https://doi.org/10.1177/1757975919832241 Woollen, E., Ryan, C. World Bank. (2005). India: Unlocking Opportunities for Forest-Dependent People in India, Volume 2, Appendixes. Washington DC: World Bank. Retrieved from World Bank website: https://openknowledge.worldbank.org/handle/10986/8414 CT%20CFC-FIGHF-07%20(23503.en) M., Baumert, S., Vollmer, F., Grundy, I., Fisher, J., … Lisboa, S. N. (2016). Charcoal production in the Mopane woodlands of Mozambique: What are the trade-offs with other ecosystem services? Philosophical Transactions of the Royal Society B-Biological Sciences, 371, 1–14. https://doi.org/10.1098/rstb.2015.0315 World Animal Protection. (2017). A close up on cruelty: The harmful impact of wildlife selfies in the Amazon. Retrieved from https://www.worldanimalprotection.org/sites/default/files/int_files/amazon_selfies_re port.pd World Bank. (2005). India: Unlocking Opportunities for Forest-Dependent People in India, Volume 2, Appendixes. Washington DC: World Bank. Retrieved from World Bank website: https://openknowledge.worldbank.org/handle/10986/8414 507 World Bank. (2011). Wood-based biomass energy development for sub-Saharan Africa: Issues and approaches. World Bank. World Bank. (2012). The Hidden Harvest. The global contribution of capture fisheries. Retrieved from https://www.researchgate.net/publication/277664581_World_Bank_2012_The_Hidde n_Harvest_The_global_contribution_of_capture_fisheries World Bank. (2019). Bhutan Forest Note: Pathways for Sustainable Forest Management and Socio-equitable Economic Development. Washington, DC: World Bank. Retrieved from World Bank website: https://openknowledge.worldbank.org/handle/10986/32047 License: CC BY 3.0 IGO. World Customs Organization. (2019). INTERNATIONAL CONVENTION ON THE HARMONIZED COMMODITY DESCRIPTION AND CODING SYSTEM - AMENDMENTS TO THE NOMENCLATURE APPENDED AS AN ANNEX TO THE CONVENTION. Retrieved from http://www.wcoomd.org/- /media/wco/public/global/pdf/topics/nomenclature/instruments-and-tools/hs- nomenclature-2022/ng0262b1.pdf?db=web World Tourism Organization. (2014). Towards measuring the economic value of wildlife watching tourism in Africa—Briefing paper. Madrid: UNWTO. Worm, B., Barbier, E. B., Beaumont, N., Duffy, J. E., Folke, C., Halpern, B. S., … Watson, R. (2006). Impacts of Biodiversity Loss on Ocean Ecosystem Services. Science, 314(5800), 787–790. https://doi.org/10.1126/science.1132294 Worm, Boris, Davis, B., Kettemer, L., Ward-Paige, C. A., Chapman, D., Heithaus, M. R., … Gruber, S. H. (2013). Global catches, exploitation rates, and rebuilding options for sharks. Marine Policy, 40, 194–204. https://doi.org/10.1016/j.marpol.2012.12.034 Woziwoda, B., Dyderski, M. K., & Jagodziński, A. M. (2020). Forest land use discontinuity and northern red oak Quercus rubra introduction change biomass allocation and life strategy of lingonberry Vaccinium vitis-idaea [Preprint]. In Review. https://doi.org/10.21203/rs.3.rs-38732/v1 WTTC. (2019a). The economic impact of global wildlife tourism: Travel & tourism as an economic tool for the protection of wildlife. World Travel & Tourism Council. Retrieved from World Travel & Tourism Council website: https://wttc.org/Portals/0/Documents/Reports/2019/Sustainable%20Growth- Economic%20Impact%20of%20Global%20Wildlife%20Tourism- Aug%202019.pdf?ver=2021-02-25-182802-167 WTTC. (2019b). World, Transformed: Megatrends and their implications for travel and tourism. World Travel & Tourism Council. Retrieved from World Travel & Tourism Council website: https://tourismknowledgecenter.com/publication/world-transformed- megatrends-and-their-implications-for-travel-tourism Wu, F., Zhou, L.-W., Yang, Z.-L., Bau, T., Li, T.-H., & Dai, Y.-C. (2019). Resource diversity of Chinese macrofungi: Edible, medicinal and poisonous species. Fungal Diversity, 98(1), 1–76. CT%20CFC-FIGHF-07%20(23503.en) https://doi.org/10.1007/s13225-019-00432-7 Wu, F., Zhou, L.-W., Yang, Z.-L., Bau, T., Li, T.-H., & Dai, Y.-C. (2019). Resource diversity of Chinese macrofungi: Edible, medicinal and poisonous species. Fungal Diversity, 98(1), 1–76. https://doi.org/10.1007/s13225-019-00432-7 Wu, J., He, Q., Chen, Y., Lin, J., & Wang, S. (2020). Dismantling the fence for social justice? Evidence based on the inequity of urban green space accessibility in the central urban Wu, J., He, Q., Chen, Y., Lin, J., & Wang, S. (2020). Dismantling the fence for social justice? Evidence based on the inequity of urban green space accessibility in the central urban 508 area of Beijing. Environment and Planning B: Urban Analytics and City Science, 47(4), 626–644. https://doi.org/10.1177/2399808318793139 area of Beijing. Environment and Planning B: Urban Analytics and City Science, 47(4), 626–644. https://doi.org/10.1177/2399808318793139 Wujisguleng, W., & Khasbagen, K. (2010). An integrated assessment of wild vegetable resources in Inner Mongolian Autonomous Region, China. Journal of Ethnobiology and Ethnomedicine, 6(1), 34. https://doi.org/10.1186/1746-4269-6-34 Wunder, S. (1999). Promoting forest conservation through ecotourism income: A case study from the Ecuadorian Amazon region. CIFOR Occasional Paper, (21), 24. Wunder, S., Angelsen, A., & Belcher, B. (2014). Forests, Livelihoods, and Conservation: Broadening the Empirical Base. World Development, 64, S1–S11. https://doi.org/10.1016/j.worlddev.2014.03.007 WWF China. (2012). Standards for Giant Panda Friendly Products (Version March 2012). WWF China Chengdu Programme Office, Chengdu: WWF China. Xego, S., Kambizi, L., & Nchu, F. (2016). Threatened medicinal plants of South Africa: Case of the family hyacinthaceae. African Journal of Traditional, Complementary and Alternative Medicines, 13(3), 169–180. https://doi.org/10.4314/ajtcam.v13i3.20 Xiao, Y., Wang, D., & Fang, J. (2019). Exploring the disparities in park access through mobile phone data: Evidence from Shanghai, China. Landscape and Urban Planning, 181, 80– 91. https://doi.org/10.1016/j.landurbplan.2018.09.013 Yan, H. F., Kyne, P. M., Jabado, R. W., Leeney, R. H., Davidson, L. N., Derrick, D. H., … Dulvy, N. K. (2021a). Overfishing and habitat loss drive range contraction of iconic marine fishes to near extinction. Science Advances, 7(7), eabb6026. https://doi.org/10.1126/sciadv.abb6026 Yan, H. F., Kyne, P. M., Jabado, R. W., Leeney, R. H., Davidson, L. N. K., Derrick, D. H., … Dulvy, N. K. (2021b). Overfishing and habitat loss drive range contraction of iconic marine fishes to near extinction. Science Advances, 7(7), eabb6026. https://doi.org/10.1126/sciadv.abb6026 Yanes, A., Zielinski, S., Diaz Cano, M., & Kim, S. (2019). Community-Based Tourism in Developing Countries: A Framework for Policy Evaluation. Sustainability, 11(9), 2506. https://doi.org/10.3390/su11092506 Yang, D., & Pomeroy, R. (2017). CT%20CFC-FIGHF-07%20(23503.en) The impact of community-based fisheries management (CBFM) on equity and sustainability of small-scale coastal fisheries in the Philippines. Marine Policy, 86, 173–181. Scopus. https://doi.org/10.1016/j.marpol.2017.09.027 Yang, H., Ranjitkar, S., Zhai, D., Zhong, M., Goldberg, S. D., Salim, M. A., … Xu, J. (2019). Role of Traditional Ecological Knowledge and Seasonal Calendars in the Context of Climate Change: A Case Study from China. Sustainability, 11(12), 3243. https://doi.org/10.3390/su11123243 Yang, J. H., Liu, Y. J., Li, J. K., Huang, J. X., Zhang, W. Y., & Li, S. Y. (2013). Potential Species and Character of Wild Diesel Plant in Tianjin. Advanced Materials Research, 641–642, 578–582. https://doi.org/10.4028/www.scientific.net/AMR.641-642.578 Yen, A. L., & Ro, S. (2013). The sale of tarantulas in Cambodia for food or medicine: Is it sustainable? Journal of Threatened Taxa, 5(1), 3548–3551. https://doi.org/10.11609/jott.o3149.153 Yen, Alan L. (2009). Edible insects: Traditional knowledge or western phobia? Entomological Research, 39(5), 289–298. https://doi.org/10.1111/j.1748-5967.2009.00239.x 509 Yen, Alan L. (2015). Insects as food and feed in the Asia Pacific region: Current perspectives and future directions. Journal of Insects as Food and Feed, 1(1), 33–55. https://doi.org/10.3920/JIFF2014.0017 Yetman, D., Búrquez Montijo, A., Hultine, K., Sanderson, M. J., & Crosswhite, F. S. (2020). The saguaro cactus: A natural history. Tucson: The University of Arizona Press. Yijian, Y., Jiangchun, W., Wenying, Z., Lei, C., Dongmei, L., Junsheng, L., … 5 School of Food Science and Engineering, Yangzhou University, Yangzhou, Jiangsu 225127. (2020). Development of red list assessment of macrofungi in China. Biodiversity Science, 28(1), 4–10. https://doi.org/10.17520/biods.2019173 Yiwen, Z., Kant, S., & Long, H. (2020). Collective Action Dilemma after China’s Forest Tenure Reform: Operationalizing Forest Devolution in a Rapidly Changing Society. Land, 9(2), 58. https://doi.org/10.3390/land9020058 Yonariza, & Webb, E. L. (2007). Rural household participation in illegal timber felling in a protected area of West Sumatra, Indonesia. Environmental Conservation, 34(1), 73–82. https://doi.org/10.1017/S0376892907003542 Yorou, N. S., Koné, N., Guissou, M.-L., Guelly, A. K., Maba, D. L., Ekué, M. R. M., & Kesel, A. (2014). Biodiversity and Sustainable Use of Wild Edible Fungi in the Sudanian Centre of Endemism: A Plea for Valorisation. In Ectomycorrhizal Symbioses in Tropical and Neotropical Forests. CRC Press. Young, G. C. (2007). Texas safari: The game hunter’s guide to Texas. Houston, Tex. : John M. Hardy Pub. Retrieved from http://archive.org/details/texassafarigameh0000youn Yue, K., Ye, M., Lin, X., & Zhou, Z. (2013). The Artificial Cultivation of Medicinal Caterpillar Fungus, Ophiocordyceps sinensis (Ascomycetes): A Review. International Journal of Medicinal Mushrooms, 15(5), 425–434. CT%20CFC-FIGHF-07%20(23503.en) https://doi.org/10.1615/IntJMedMushr.v15.i5.10 Zaitsev, Y., & Mamaev, V. (1998). Marine Biological Diversity in the Black Sea: A Study of Change and Decline. Colonial Waterbirds, 21(1), 113. https://doi.org/10.2307/1521749 Zalengera, C., Chitedze, I., To, L. S., Chitawo, M., Mwale, V., & Maroyi, T. (2020). Impacts and Coping Mechanisms for the Covid-19 Pandemic in Malawi’s Energy Sector (pp. 1–12) [Workshop Report]. Energy and Economic Growth: Applied Research Programme. Retrieved from Energy and Economic Growth: Applied Research Programme website: pstorage-loughborough- 53465.s3.amazonaws.com/25189643/MzuzuUniWorkshopReport.pdf Zapata, M. J., Hall, C. M., Lindo, P., & Vanderschaeghe, M. (2011). Can community-based tourism contribute to development and poverty alleviation? Lessons from Nicaragua. Current Issues in Tourism, 14(8), 725–749. https://doi.org/10.1080/13683500.2011.559200 Zapelini, C., Bender, M. G., Giglio, V. J., & Schiavetti, A. (2019). Tracking interactions: Shifting baseline and fisheries networks in the largest Southwestern Atlantic reef system. Aquatic Conservation: Marine and Freshwater Ecosystems, 29(12), 2092– 2106. Scopus. https://doi.org/10.1002/aqc.3224 Zapelini, Cleverson, Giglio, V. J., Carvalho, R. C., Bender, M. G., & Gerhardinger, L. C. (2017). Assessing Fishing Experts’ Knowledge to Improve Conservation Strategies for 510 an Endangered Grouper in the Southwestern Atlantic. Journal of Ethnobiology, 37(3), 478–493. Zarazúa-Carbajal, M., Chávez-Gutiérrez, M., Romero-Bautista, Y., Rangel-Landa, S., Moreno- Calles, A. I., Ramos, L. F. A., … Casas, A. (2020). Use and management of wild fauna by people of the Tehuacán-Cuicatlán Valley and surrounding areas, Mexico. Journal of Ethnobiology and Ethnomedicine, 16(1), 4. https://doi.org/10.1186/s13002-020-0354- 8 Zeller, D., Harper, S., Zylich, K., & Pauly, D. (2015). Synthesis of underreported small-scale fisheries catch in Pacific island waters. Coral Reefs, 34(1), 25–39. https://doi.org/10.1007/s00338-014-1219-1 Zeller, D., Palomares, M. L. D., Tavakolie, A., Ang, M., Belhabib, D., Cheung, W. W. L., … Pauly, D. (2016). Still catching attention: Sea Around Us reconstructed global catch data, their spatial expression and public accessibility. Marine Policy, 70, 145–152. https://doi.org/10.1016/j.marpol.2016.04.046 Zeller, Dirk, Cashion, T., Palomares, M., & Pauly, D. (2018). Global marine fisheries discards: A synthesis of reconstructed data. Fish and Fisheries, 19(1), 30–39. https://doi.org/10.1111/faf.12233 Zeller, Dirk, & Pauly, D. (2019). Viewpoint: Back to the future for fisheries, where will we choose to go? Global Sustainability, 2, e11. https://doi.org/10.1017/sus.2019.8 Zent, E. L. (2008). Mushrooms for Life among the Jotï in the Venezuelan Guayana. Economic Botany, 62(3), 471–481. https://doi.org/10.1007/s12231-008-9039-2 Zent, E. L., Zent, S., & Iturriaga, T. (2004). Knowledge and Use of Fungi by a Mycophilic Society of the Venezuelan Amazon. Economic Botany, 58(2), 214–226. https://doi.org/10.1663/0013-0001(2004)058[0214:KAUOFB]2.0.CO;2 Zenteno, M., Zuidema, P. A., de Jong, W., & Boot, R. G. A. CT%20CFC-FIGHF-07%20(23503.en) (2013). Livelihood strategies and forest dependence: New insights from Bolivian forest communities. Forest Policy and Economics, 26, 12–21. https://doi.org/10.1016/j.forpol.2012.09.011 Zeppel, H. (2010). Managing cultural values in sustainable tourism: Conflicts in protected areas. Tourism and Hospitality Research, 10(2), 93–104. https://doi.org/10.1057/thr.2009.28 Zerner, C. (2003). Sounding the Makassar Strait: The poetics and politics of an Indonesian marine environment. In Culture and the question of rights. Forests, coasts, and seas in Southeast Asia (Zerner C. (ed), pp. 56–108). Durham & London: Duke University Press. Retrieved from https://doi.org/10.1515/9780822383819-005 Zielinski, S., Kim, S., Botero, C., & Yanes, A. (2020). Factors that facilitate and inhibit community-based tourism initiatives in developing countries. Current Issues in Tourism, 23(6), 723–739. https://doi.org/10.1080/13683500.2018.1543254 Zukowski, S., Curtis, A., & Watts, R. J. (2011). Using fisher local ecological knowledge to improve management: The Murray crayfish in Australia. Fisheries Research, 110(1), 120–127. Scopus. https://doi.org/10.1016/j.fishres.2011.03.020 Zvonar, A., & Weidensaul, A. (2015). Bird study in urban environmental education. In A. Russ (Ed.), Urban Environmental Education (pp. 95–99). Cornell university Civic Ecology Lab, NAAEE, EECapacity. Retrieved from https://www.researchgate.net/profile/Angelique- 511 Hjarding/publication/302877963_Urban_Planning_and_Environmental_Education/lin ks/5732474208ae9ace84047dd9/Urban-Planning-and-Environmental- Education.pdf#page=97 Артеага В. (2019, November 1). Старая песня про зубра. Старая Песня Про Зубра. 512 512
https://openalex.org/W1931981173	https://www.ntnu.no/ojs/index.php/etikk_i_praksis/article/download/1774/1771	Norwegian	null	Mangfold og felles utfordringer i anvendt etikk	Etikk i praksis	2,012	cc-by	1,271	Rune Nydal, Berge Solberg & Bjørn Myskja Da dette tidsskriftet ble etablert for seks år siden, var det ut fra tanken om at det finnes noen felles problemstillinger innen etikk og politikk som får en spesiell karakter i nor- diske land. Den velferdsmodellen vi har utviklet innenfor rammene av et deltakende demokrati, gir vilkår for debatten som vi ikke deler fullt ut med de fleste andre demokrati i verden. Mange av de artiklene vi har trykket i disse årene, har vist betydningen av denne nordiske konteksten. Det gjelder også noen av bidragene i dette nummeret. Dette nummeret presenterer fem bidrag hentet fra fem ulike temaområder som dis- kuterer spørsmål av typen: I hvilken grad kan europeiske land stilles til ansvar for urett begått mange tiår tilbake? Bør bransjeetiske retningslinjer få vekt i rettslige resonnemen- ter? Hvordan kan en dyktiggjøre sykepleiere til likebehandling når ens egne fordommer mot marginaliserte grupper først slår inn i en praksissituasjon? Hvilke konfliktfylte beslutningsfaktorer må en bedriftsleder være spesielt oppmerksom på, og hvordan kan en styrke en organisasjons kapasitet til å diskutere organisasjonens formål og utøvelse? Denne sammenstillingen av ulike problemområder demonstrerer det mangfoldet i anvendt etikk som vi ønsker å presentere i ett og samme tidsskrift. En fordel med et bredt anlagt tidsskrift innenfor anvendt etikk er at utfordringer som deles av ulike grener av fel- tet, kan bli tydeligere. Spørsmålene som behandles av forfatterne i dette nummeret, er spørsmål som gjen- kjennes som etiske, men som samtidig er utmyntet innenfor ett eller flere fagområder som setter de etiske utfordringene. Selv om de aktuelle etiske problemstillinger kan være svært presserende, er spørsmålene ofte blitt lite systematisk behandlet. Arbeid innenfor anvendt etikk innebærer ofte en type nybrottsarbeid – som vi ser eksempler på i dette nummeret – som det kan være vanskelig å finne et egnet tidsskrift for. Det er heller ikke opplagt hvem som best kan vurdere og fremdyrke denne type faglitteratur ettersom teo- ritilfanget for anvendt etikk både favner bredt og samtidig alltid krysser over flere fagfelt. Idet normative spørsmål reises, viser det seg ofte vanskelig å avgjøre hvilke fagtradisjoner argumentasjonen burde prøves ut mot. En av de funksjoner teorier har, er at de er retningsgivende, og de setter måter å se og vurdere fenomener på. Når normative spørsmål reises i en praktisk fagkontekst, reises ikke bare et spørsmål om hvordan og hvilke normative perspektiver som bør tilføres. Det reises også et spørsmål til det aktuelle fagperspektivets normative forutsetninger. Etikk i praksis. Nordic Journal of Applied Ethics (2012), 6 (1), s. 1–3. Mangfold og felles utfordringer i anvendt etikk Rune Nydal, Berge Solberg & Bjørn Myskja Artikler i dette nummeret Rettferdighet er sentralt i etisk og politisk teori, selv om det fortsatt er stor faglig uenighet om hva begrepet innebærer og under hvilke betingelser man kan fremme rettferdighets- krav. Den grunnleggende analysen av begrepet finner vi hos Aristoteles, som skiller mellom fordelingsrettferdighet og gjenopprettende rettferdighet. Den siste typen rettfer- dighet dreier seg om å gjenopprette balansen når urett er begått. Dette er utgangspunktet for Göran Collste sin artikkel, «Betydelsen av historisk rättvisa efter kolonialismen». Artikkelen diskuterer hvorvidt det er rimelig å reise rettferdighetsbaserte krav etter over- grep begått av kolonistater mot enkeltpersoner og grupper. Hvem kan reise slike krav, og hvem er ansvarlig for disse overgrepene? Collste diskuterer to historiske hendelser: Stor- britannias overgrep under Mau Mau-opprøret i Kenya og det tyske keiserrikets folkemord på Herero-folket i det nåværende Namibia. Ved å analysere disse eksemplene kartlegger Collste sentrale spørsmål og mulige svar på hvordan man kan håndtere rettferdighetskrav som følge av historisk urett. Børge Aadland undersøker betydningen av en juridisk forankring av profesjonsetiske kodekser i artikkelen «Den rettslige betydningen av yrkesetiske regler med utgangspunkt i lovfestede spesielle god skikk-regler». Aadland sammenligner fem bransjer der yrkese- tiske regler har fått en lovmessig forankring ut fra en hypotese om at slike regler vil regu- lere fem elementer: profesjonsansvar, integritet, objektivitet, taushetsplikt og kompetan- sekrav. En slik lovfesting av yrkesetiske regler gir anledning til større grad av realitetsbe- handling av brudd på yrkesetiske regler, argumenter Aadland. For selv om rettspraksis tyder på at de ulovfestede yrkesregler har hatt betydning for rettsvurderinger, har argu- mentasjonen i liten grad gitt anledning til innholdsvurdering utover argumentasjon om at forventninger til yrkesutøvere – ut fra en sedvane etablert i yrkeskodekser – må være bindende for domstolene. Gitt denne analysen ser lovregulering ut til å gi domstolen større handlingsrom slik at den kan foreta en selvstendig normativ vurdering av aktørers profesjonelle samfunnsansvar. Trine Myhrvold diskuterer hvordan man skal bevisstgjøre sykepleiestudenter om de helsemessige utfordringene som kan knyttes til grupper som lever i samfunnets periferi. I artikkelen «Marginalisation as a Possible Health Issue: an Exercise in Practice-Based Ethical Education» beskriver hun et pilotprosjekt der studentene møter representanter for slike marginaliserte grupper og reflekterer over de moralske og politiske plikter de – som helsearbeidere – har overfor dem. Dette skjer gjennom en dialogisk tilnærming som involverer frivillige organisasjoner og lavterskeltilbud rettet mot dem som faller utenfor de normale helse- og velferdstilbudene. Rune Nydal, Berge Solberg & Bjørn Myskja Norma- tive diskusjoner synes å måtte utmyntes i skjæringspunktet mellom ulike teoretiske per- spektiver, og behandles i spørsmålenes situerte kontekst. Dette utgjør en av de tilbakeven- dende teoretisk-metodiske utfordringer for anvendt etikk, noe som på ulikt vis blir demonstrert i artiklene i dette nummeret. 1 1 ETIKK I PRAKSIS NR. 1 2012 Artikler i dette nummeret Pilotprosjektet innebærer flere interessante grep som kan følges opp i større undersøkelser, slik som samarbeidet med institusjoner som vanligvis ikke inngår i sykepleieutdannelsen, og det direkte møtet med sosialt ekskluderte mennesker som en del av helseutdannelsen. Øyvind Kvalnes og Einar Øverenget diskuterer etisk navigasjon for ledere i sin artikkel «Ethical Navigation in Leadership Training». Utgangspunktet for Kvalnes og Øverengets artikkel er ledere som jevnlig møter etiske utfordringer og dilemmaer i sitt virke. Hvor- dan kan man treffe gode etiske beslutninger? Er dette i det hele tatt noe man kan bli bedre på gjennom trening og målrettet arbeid? Kvalnes og Øverenget har et bekreftende svar på 2 ETIKK I PRAKSIS NR. 1 2012 dette. «Navigasjonshjulet» er verktøyet de introduserer for lederen som vil trene på å møte etiske utfordringer i sin ledertilværelse på en god måte. Navigasjonshjulet er ment å ta høyde for alle de aspekter som er relevante for næringslivslederes etiske beslutninger. Fra helsevesenet kjenner vi «de fire prinsippers etikk», som har blitt et populært «naviga- sjonshjul» for helsevesenet i svært mange land. Kvalnes og Øverengets navigasjonshjul er spesialtilpasset næringslivslederens utfordringer. Deres erfaring etter mer enn ti års kon- takt med næringslivet er at etisk trening er mulig, og at navigasjonshjulet er et verktøy som styrker refleksiviteten til lederne og artikulerer og kultiverer deres etiske oppfatnin- ger. I artikkelen «Organising Ethics: The Case of the Norwegian Army» foreslår Ellen- Marie Forsberg, Are Eidhamar og Svein-Tore Kristiansen en modell for hvordan en kan styrke en organisasjons kapasitet til å håndtere etiske utfordringer i organisasjonen. Artikkelen er basert på en studie av Forsvaret, men forfatterne hevder modellen kan anvendes på enhver organisasjon. Ut fra denne modellen skal tiltak i organisasjonen bygge på en empirisk identifisering av svake eller mangelfulle diskusjonsarenaer i organi- sasjonen. Denne empiriske undersøkelsen er strukturert og opplyst av Richard Scotts organisasjonsteori. Ett av forfatternes poenger er at tradisjonelle leverandører av norma- tiv teori (som de identifiserer som filosofi og teologi) ikke alene gir tilfredsstillende red- skaper for analyse og håndtering av organisasjonsetiske utfordringer. Her tydeliggjøres en av de metodiske og teoretiske kjerneutfordringer i anvendt etikk. Idet teorier fra ulike fag- felt inndras i normative analyser, synliggjøres behovet for å klargjøre disse teoriers nor- mative forutsetninger. Mangfold og felles utfordringer i anvendt etikk Rune Nydal, Berge Solberg & Bjørn Myskja 3
https://openalex.org/W4292708055	https://www.frontiersin.org/articles/10.3389/fendo.2022.990668/pdf	English	null	Editorial: The role of genetic alterations in thyroid carcinoma	Frontiers in endocrinology	2,022	cc-by	1,375	TYPE Editorial PUBLISHED 23 August 2022 DOI 10.3389/fendo.2022.990668 TYPE Editorial PUBLISHED 23 August 2022 DOI 10.3389/fendo.2022.990668 TYPE Editorial PUBLISHED 23 August 2022 DOI 10.3389/fendo.2022.990668 Editorial on the Research Topic The role of genetic alterations in thyroid cancer Editorial on the Research Topic The role of genetic alterations in thyroid cancer Thyroid carcinomas are detected with increasing incidence rates in many countries. This is mainly contributed to more frequent diagnostic procedures around the world. © 2022 Feldkamp. This is an open- access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, Most of the tumors are papillary and follicular thyroid carcinomas, followed by non- follicular medullary thyroid carcinomas and anaplastic carcinomas. The clinical spectrum shows a wide range from sometimes almost benign disease in patients with papillary microcarcinomas, especially in the elderly and when detected incidentally, to the rare cases of anaplastic thyroid carcinomas and primary squamous cell carcinoma of the thyroid with an unfavorable outcome. For some types of thyroid carcinomas genetic causes are best known. 25% of medullary thyroid carcinomas (MTC) arise in the context of multiple endocrine neoplasia type 2 A and B. The knowledge of causative genetic alterations on cancer development has led to a strong improvement in the survival of patients with MEN-2 disease. A genotype-phenotype association showing different outcomes with speciﬁc mutations has led to individualized therapy strategies with prophylactic surgery in many cases, detected by family screening. Hansen et al. investigated the role of the Leu56Met variant in the RET gene by clinical follow up of a Danish RET Leu56Met cohort. Using predeﬁned criteria none of the patients exhibited evidence of MEN 2. With an allele frequency of 0.59% the Leu56Met variant suggests that it is a common variant in the population with no pathogenetic importance. Less frequent are genetic causes of non-medullary thyroid carcinomas (NMTC). About 3% of NFTC are estimated to be of hereditary origin. Most of these tumors arise in the context of syndromic diseases such as familiary adenomous polyposis coli and PTEN- Hamartoma-tumor-Syndrome such as Cowden-syndrome. Further entities include Carney-complex, Werner-Syndrome and DICER-1-syndrome. Several single genes are involved or suspected to be to be involved in the pathogenesis of NMTC. Many studies in recent years gave insight in the important role of genetic alterations in the development and growth progression in papillary thyroid carcinoma (PTC). Genetic alterations often involve the mitogen-activated protein kinase (MAPK). KEYWORDS thyroid carcinoma, differentiated, anaplastic, mutation, medullary thyroid cancer OPEN ACCESS Joachim Feldkamp* Academic Department of Endocrinology and Diabetes, General Internal Medicine, Infectiology, Medical School and University Medical Center East Westphalia-Lippe, Klinikum Bielefeld, Bielefeld University, Bielefeld, Germany Editorial on the Research Topic The role of genetic alterations in thyroid cancer BRAF as part of the RAF/MAPK pathway is an effector of cell proliferation. Many BRAF Frontiers in Endocrinology frontiersin.org 01 10.3389/fendo.2022.990668 Feldkamp Lenvatinib and sorafenib have been shown to improve disease- free survival in patients with radio-refractory thyroid carcinoma while cabozantinib has its role as second line therapy in these patients. Selpercatinib is a treatment option for patients with RET fusions in PTC and medullary thyroid carcinomas (3). In MTC, vandetanib and cabozantinib have shown their therapeutic potential already for a couple of years. The most aggressive tumors of the thyroid are anaplastic carcinomas. Understanding the underlying mechanisms of disease and concomitant factors is important for an adequate management of these patients. mutations have been described in papillary thyroid carcinomas. The BRAFV600E mutation is the most frequent mutation found in papillary thyroid carcinoma and associated with a poorer prognosis, lymph node involvement, extrathyroidal tumor expansion and distant metastases. Due to its high kinase activity secondary genetic alterations such as mutations in phosphoinositide 3-kinase-Akt serine/threonine kinase (PI3K- AKT) pathway may occur leading to more aggressive tumors and an unfavorable clinical outcome for the patients. Parvathareddy et al. could conﬁrm that BRAFV600E mutations are the most frequent mutations in papillary microcarcinomas. They could detect BRAFV600E mutations in 45.7% of their cases (84 of 184 patients in a middle east population). Beside BRAF V600E mutations telomerase reverse transcriptase (TERT) promoter mutations play a relevant role in the aggressiveness of tumor growth in papillary thyroid carcinoma. TERT promoter mutations were detected in the series of Parvathareddy in 8.7% cases and were signiﬁcantly associated with distant metastases and shorter metastasis-free survival in multivariate analysis. Jin et al. used the Surveillance, Epidemiology, and End Results Program (SEER) data base (2004-2015) to compare differences in characteristics between anaplastic thyroid carcinoma (ATC) and primary squamous cell carcinoma of the thyroid (PSCCTh) and establish prognostic models. For patients with PSCCTh (n=124) prognostic factors inﬂuencing the cancer-speciﬁc survival were age, radiotherapy, multiple primary tumors, and surgery. For ATC patients (n=1164) these factors were different comprising age, sex, radiotherapy, chemotherapy, surgery, multiple primary tumors, marital status, and distant metastasis. This may lead to the need of different clinical treatment and management in this group of patients. Gao et al. investigated if dysregulation of miRNA plays a role in PCT wit BRAF mutations. In PTC an upregulation of miR- 222-3p has already been demonstrated. Author contributions All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication. Editorial on the Research Topic The role of genetic alterations in thyroid cancer The actual study showed that miR-222-3p led to more aggressive clinical manifestations of PTC promoted via the snail transcription factor. Conclusively, the understanding of genetic alterations in thyroid carcinoma is growing fast, leading to new treatment options for our patients. With surgery and radioiodine therapy most of the patients with differentiated thyroid carcinoma have a favorable outcome. But in radioiodine refractory carcinomas overall survival and progression-free survival time is limited. Clinical management of patients can be improved if the response to radioactive iodine can be detected early. Liu et al. used expression proﬁles of mRNAs and miRNAs in addition to clinical data of PTC patients. They could apply a two-RNA model (IPCEF1 and hsa-mir-486-5p) associated with the prognosis of RAI-therapy. Together with their RNA-based risk score calculated on the Cox coefﬁcient of the individual RNA and clinical parameters (age at diagnosis and tumor stage) the authors were able to demonstrate high precision in predicting the RAI response of PTC patients. Conﬂict of interest The author declares that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. Rearranged- during transfection (RET) kinase as a protooncogene can also be involved in tumorigenesis of thyroid carcinoma. Besides its role in hereditary forms of medullary thyroid carcinoma, chromosomal RET rearrangements are found almost only in PTC (1). Ret/PTC1 and Ret/PTC 3 are the most common rearrangements. Both forms are found more frequently in younger patients and RET/ PTC3 rearrangement has been linked to external radiation. Publisher’s note All claims expressed in this article are solely those of the authors and do not necessarily represent those of their afﬁliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. The knowledge and understanding of the role of genetic alterations in thyroid carcinoma has led to advances in treatment options for patients with radio refractory thyroid carcinomas. Multikinase inhibitors target different pathways including antiangiogenic activity, RET and BRAFV600E and others (2). Frontiers in Endocrinology frontiersin.org 02 10.3389/fendo.2022.990668 Feldkamp 2. Tiedje V, Fagin JA. Therapeutic breakthroughs for metastatic thyroid cancer Nat Rev Endocrinol (2020) 16(2):77–8. doi: 10.1038/s41574-019-0307-2 3. Jaber T, Dadu R, Hu MI. Medullary thyroid carcinoma. Curr Opin Endocrinol Diabetes Obes (2021) 28(5):540–6. doi: 10.1097/MED.0000000000000662 10.3389/fendo.2022.990668 1. Li AY, McCusker MG, Russo A, Scilla KA, Gittens A, Arensmeyer K, et al. RET fusions in thyroid tumors. Cancer Treat Rev (2019) 81:101911. doi: 10.1016/ j.ctrv.2019.101911 References 1. Li AY, McCusker MG, Russo A, Scilla KA, Gittens A, Arensmeyer K, et al. RET fusions in thyroid tumors. Cancer Treat Rev (2019) 81:101911. doi: 10.1016/ j.ctrv.2019.101911 Frontiers in Endocrinology frontiersin.org 03
https://openalex.org/W3173656731	https://revista.redipe.org/index.php/1/article/download/1334/1249	English	null	Study of potential application of alternative power cycles in Colombia	Boletín Redipe	2,021	cc-by-sa	6,573	1 Departamento de Matemáticas y Estadística, Uni- versidad Francisco de Paula Santander, Norte de Santander, Cúcuta, Colombia, correo: mawencyvergel@ufps.edu.co Or- cid: https://orcid.org/0000-0001-8285-2968 2 Facultad de Ingenierías, Universidad Francisco de Paula Santander, Cúcuta, Colombia. Correo jhanpiero- rojas@ufps.edu.co ORCID iD: https://orcid.org/0000-0003- 2682-9880 3 Facultad de Ciencias Empresariales, Universidad Francisco de Paula Santander, Cúcuta, Colombia. Correo: sofiaorjuela@ufps.edu.co ORCID: https://orcid.org/0000- 0002-9742-8673 Sofia Orjuela Abril3 Universidad Francisco de Paula Santander, Cúcuta, Colombia Universidad Francisco de Paula Santander, Cúcuta, Colombia · 3 8 2 3 Facultad de Ciencias Empresariales, Universidad Francisco de Paula Santander, Cúcuta, Colombia. Correo: sofiaorjuela@ufps.edu.co ORCID: https://orcid.org/0000- 0002-9742-8673 2 Facultad de Ingenierías, Universidad Francisco de Paula Santander, Cúcuta, Colombia. Correo jhanpiero- rojas@ufps.edu.co ORCID iD: https://orcid.org/0000-0003- 2682-9880 1 Departamento de Matemáticas y Estadística, Uni- versidad Francisco de Paula Santander, Norte de Santander, Cúcuta, Colombia, correo: mawencyvergel@ufps.edu.co Or- cid: https://orcid.org/0000-0001-8285-2968 RECIBIDO EL 7 DE NOVIEMBRE DE 2020 - ACEPTADO EL 8 DE FEBRERO DE 2021 RECIBIDO EL 7 DE NOVIEMBRE DE 2020 - ACEPTADO EL 8 DE FEBRERO DE 2021 Study of potential application of alternative power cycles in colombia Estudio de la potencial aplicación de ciclos de potencia alternativos en Colombia Mawency Vergel Ortega1 Jhan Piero Rojas Suarez2 Sofia Orjuela Abril3 Universidad Francisco de Paula Santander, Cúcuta, Colombia Mawency Vergel Ortega1 Jhan Piero Rojas Suarez2 Sofia Orjuela Abril3 Universidad Francisco de Paula Santander, Cúcuta, Colombia Mawency Vergel Ortega1 Jhan Piero Rojas Suarez2 Mawency Vergel Ortega1 Jhan Piero Rojas Suarez2 Sofia Orjuela Abril3 PALABRAS CLAVE: the support of financing instruments and policies at the national level is a latent need in Colombia. Ciclos alternativos, Eficiencia energética, Política energética, Potencial, Energías renovables. Ciclos alternativos, Eficiencia energética, Política energética, Potencial, Energías renovables. KEYWORDS: Alternative Cycles, Efficiency Energy, Energy Policy, Potential, Renewable Energy. Alternative Cycles, Efficiency Energy, Energy Policy, Potential, Renewable Energy. INTRODUCTION The efficiency of energy use is a current necessity, and the world has this course due to various factors. One of them is the growing trend of energy demand. U.S Energy Information Administration published a projection of 50% growth in energy use between 2018 and 2050. Other factors are sustainability, energy security, distribution, among other factors derived from the use of non-renewable primary energy. However, the use of non-renewable primary energy has been fundamental for world economic development (Hook & Tang, 2013). In addition, it has brought negative effects that have been reflected in recent decades. Such as global warming due to the high emission of greenhouse gases (GHG), studies estimate an increase in GHG emissions from 25% to 90% between 2000 and 2030 (IPCC, 2008). ABSTRACT sectors of the economy where alternative power cycles like Goswami, Kalina, and ORC have great potential in both energy recovery and the use of renewable energy resources. In Colombia, the potential for using these technologies is wide in the industry since sectors such as Steel, cement, textile, among others, are made up. Where these cycles represent an opportunity for efficient energy use and, on the other hand, in the use of renewable energies such as geothermal and solar, these cycles have again proven to be viable, and due to the geographical location and characteristics of its soil, Colombia has a wide potential. All this shows the necessity for national policies that encourage efficient use through subsidies, investment, training, etc. For the application of alternative power cycles because their costs are so high and The energy cost and environmental problems such as greenhouse gas emissions have made efficient energy and renewable energy resources use a necessity for the care of the environment and for the economic and productive growth of the different sectors of a country’s economy. This has been achieved through policies that encourage the application of technologies that increase the processes efficiency and take advantage of the means available for generating power in different R E V I S T A B O L E T Í N R E D I P E 1 0 ( 6 ) : 3 8 2 - 3 9 2 - J U N I O 2 0 2 1 - I S S N 2 2 5 6 - 1 5 3 6 RESUMEN El costo de la energía y los problemas ambientales como las emisiones de gases de efecto invernadero han hecho que el uso eficiente de la energía y de los recursos energéticos renovables sea una necesidad para el cuidado del medio ambiente y para el crecimiento económico y productivo de los diferentes sectores de la economía de un país. Esto se ha logrado a través de políticas que incentiven la aplicación de tecnologías que aumenten la eficiencia de los procesos y aprovechen los medios disponibles para la generación de energía en los diferentes sectores de la economía donde los ciclos alternativos de potencia como Goswami, Kalina y ORC tienen un gran potencial tanto en la recuperación de energía como en el uso de recursos energéticos renovables. En Colombia, el potencial de uso de estas tecnologías es amplio en la industria ya que se conforman sectores como el Acero, el cemento, el textil, entre otros. Donde estos ciclos representan una oportunidad para el uso eficiente de la energía y, por otro lado, en el uso de energías renovables como la geotérmica y la solar, estos ciclos han demostrado nuevamente ser viables, y por la ubicación geográfica y características de su suelo, Colombia tiene un amplio potencial. Todo esto muestra la necesidad de políticas nacionales que incentiven el uso eficiente a través de subsidios, inversión, capacitación, etc. Para la aplicación de los ciclos alternativos de energía por sus altos costos y el apoyo de instrumentos y políticas de financiación a nivel nacional es una necesidad latente en Colombia. 3 8 3 Methods and new technologies are being implemented for the recovery of waste energy (Broberg & Johansson, 2014). With this, a great environmental and economic impact has been achieved. This is the reason why the application of power generation systems through alternative thermal cycles such as Kalina, Organic Rankine (ORC), and Goswami (Karimi M., Dutta, Kaushik, Bansal, & Haque, 2015) (CASATI, 2014) has been considered an important option for efficient energy use and this energy problem mitigation (Broberg Viklund & Johansson, 2014). In the energy recovery area, the industry potential is vast. (Hammond & Norman, 2014) estimated 52 PJ/yr in the industrial sector in the UK and found that the greatest potential is in low-temperature waste energy, where ORC is the most common. R.C. (McKenna & Norman, In the energy recovery area, the industry potential is vast. RESUMEN (Hammond & Norman, 2014) estimated 52 PJ/yr in the industrial sector in the UK and found that the greatest potential is in low-temperature waste energy, where ORC is the most common. R.C. (McKenna & Norman, R E V I S T A B O L E T Í N R E D I P E 1 0 ( 6 ) : 3 8 2 - 3 9 2 - J U N I O 2 0 2 1 - I S S N 2 2 5 6 - 1 5 3 6 - 3 9 2 - J U N I O 2 0 2 1 - I S S N 2 2 5 6 - 1 5 3 6 S T U D Y O F P O T E N T I A L A P P L I C A T I O N O F A L T E R N A T I V E P O W E R C Y C L E S I N C O L O M B I A Wolpert et al. (Wolpert & Riffat, 1996) used ORC for domestic applications taking advantage of solar energy in the UK and Mexico. Kalina and Leibowitz (Kalina & Leibowitz, Application of the Kalina cycle technology to geothermal power generation, 1989) presented a Kalina cycle application using geothermal energy. Lolos et al. (Lolos & Rogdakis, 2009)studied the Kalina application powered by solar collectors. Cheng et al. (Cheng, Sanjayan, & Amer, 2005) studied ORC use to produce at least 10MW with solar energy in Malaysia. 2010)estimated the energy recovery potential of 36 - 71 PJ/yr for the industry in the same country. In 2012 it was estimated 4GWe in the cement industry of waste energy with 49% of total potential corresponding to heat at low temperatures, 60% in Steel industry with a total potential estimated of 2.9 GWTh (Lu, 2015). (Broberg Viklund & Johansson, 2014) calculated 25GWh/year of electricity or 91GWh/year of district heating using excesses of thermal energy with the technologies proposed in Gävleborg, Sweden. (Bailey & Worrel, 2005)estimated the potential for energy recovery of 750MWe in the USA, 500 MWe in Germany, and 300 MWe in Europe. Considering the review of the art state, there is no doubt about the potential of using power cycles for efficient energy use, climate change, and, therefore, the economic benefits that this brings. RESUMEN Also, we can observe the necessity to create and implement public policies that promote efficient energy use in order to promote the development of systems of this type and thus mitigate the environmental energy problem that is projected for the following decades. Relatively demanding measures have already been taken in different countries through public policies. Encouraging and promoting laws, training, subsidies, among others. Unfortunately, even so, be aware, not all government states have progressed in the same way in terms of efficient energy use, as happens, for example, at the national level, even so, having a great energy potential. By another hand, Studies have (Haddad, Périlhon, Danlos, François, & Descombes, 2014) determined that the highest percentage of waste energy is in low-temperature ranges between 100 - 200°C. This is why in this area, these cycles have been quite studied and used. One of the most widely used cycles is the ORC, which has proven to be an efficient cycle for power generation with low-temperature sources (Yamada, Tominaga, & Yoshida, 2014). Lemmens y Lecompte (Lemmens & Lecompte, 2017) studied an application case of a Rankine organic cycle (ORC) in energy recovery in the Flanders region, Belgium. Kwak, Binns, and Kim (Kwak, Binns, & Kim, 2014) have investigated several scenarios for ORC implementation. Kalina proposed a thermodynamic cycle for energy recovery that has his name (Kalina A., Combined cycle and waste heat recovery power systems based on a novel thermodynamic energy cycle utilizing low-temperature, 1983). Paredes et al. (Paredes Sanchez, Restrepo Baena, Álvarez Rodríguez, Osorio Correa, & Restrepo, 2015) studied energy recovery in the Portland cement industry. 3 8 4 The energy policies that favor the implementation and development of the energy recovery power cycles in Colombia are implemented by the Ministry of Mines and Energy (MME). An entity whose main objective is to formulate and adopt timely policies, plans, programs, projects, and regulations for the mining and energy sector, in accordance with the guidelines of the National Government (Ministerio de minas y energía, s.f.). In Colombia, we have some laws and decrees that enhance the technologies application for alternative energy development. 3.1 KALINA CYCLE Alexander Kalina proposed a group of the cycle which use ammonia-water mixture like a working fluid (United Estate Patente nº 4346561, 1982) (Kalina A., Combined cycle and waste heat recovery power systems based on a novel thermodynamic energy cycle utilizing low-temperature for power generation, 1983a) (Kalina A., Combined-cycle system with novel bottoming cycle, 1984). This cycle was thought to improve efficiency and installation cost per kilowatt at that moment (Kalina A., Combined- cycle system with novel bottoming cycle, 1984). The main feature of this family of cycles is the working fluid, and this mixture allows work with different conditions for several reasons. One of them is because the composition of the mixture could be fixed (Ogriseck, 2009) that make this cycle more flexible, and in some case is necessary a separator that facilities a working range. Ammonia-water mixture has thermophysical properties like variable boiling temperature, which can thus be fitter to the falling temperature of heat source that results in a reduction entropy generation (Valdimarsson & Eliasson, 2003) and allow capture more of available potential work (Mlcak, 2002). Legal entities that directly make investments in control, conservation, and improvement of the environment will have the law to deduct 25% of the investments they have made in the respective taxable year from their income tax (article 255 ET). Exemption from taxes on the import of machinery and equipment for projects development or activities that are exporters of certificates of carbon emissions reduction and that contribute to reducing greenhouse gas emission and, therefore, to sustainable development (Article 428 lit i). 3 8 5 By law 1715 of 2014, incentives for investment in non-conventional energy source projects (FNCE) are established, such as accelerated depreciation of assets applied to the machinery, equipment, and civil works necessary for the pre-investment, investment, and operation of the generation with FNCE, that are acquired and/ or built, exclusively for that purpose, from the effective date of this law. For these purposes, the annual depreciation rate will be no more than twenty percent (20%) as the annual global rate (Article 14). Cycles with these characteristics have been developed for several operational conditions. We can find various Kalina Cycle System (KCS). The first one published by Kalina (Kalina A., Combined-cycle system with novel bottoming cycle, 1984) was designated as KCS 1. 3. RESULTS AND DISCUSSION In 2001, Law 697 was issued, in which rational and efficient energy use is promoted, the use of alternative energy is encouraged, and other provisions are issued. In order to progress, through this law, the rational energy use program (PROURE) was created by MME (Law 697), regulated by decree 3683 of 2003 and the intersectoral Commission for the rational and efficient use of the energy and unconventional energy sources (CIURE) (Decree 3683 of 2003). Decree was modified in 2008 by decree number 2688 (Decree 2688 of 2003). 2. METHOD In the energy use of renewable sources, these cycles have been widely used and studied. R E V I S T A B O L E T Í N R E D I P E 1 0 ( 6 ) : 3 8 2 - 3 9 2 - J U N I O 2 0 2 1 - I S S N 2 2 5 6 - 1 5 3 6 S T U D Y O F P O T E N T I A L A P P L I C A T I O N O F A L T E R N A T I V E P O W E R C Y C L E S I N C O L O M B I A 3.1 KALINA CYCLE KCS 6 was developed as an improved variant with an increase of 10% in efficiency (Kalina A., Novel power cycle for combined-cycle system and utility power plants(ESL-IE-86-06-39), 1986). We can find KCS 34 and KCS 34g, which are suitable for temperatures below 121°C (Mlcak, 2002). KCS 34g is suitable for smaller size plants, while KCS 34 is used for combined power production and downstream district heating applications (Mlcak, In addition, this law promotes the development of energies from Biomass, solar, geothermal energy, among others. In which the alternative cycles of power generation have an application (Law 1715 of 2014). R E V I S T A B O L E T Í N R E D I P E 1 0 ( 6 ) : 3 8 2 - 3 9 2 - J U N I O 2 0 2 1 - I S S N 2 2 5 6 - 1 5 3 6 - 3 9 2 - J U N I O 2 0 2 1 - I S S N 2 2 5 6 - 1 5 3 6 S T U D Y O F P O T E N T I A L A P P L I C A T I O N O F A L T E R N A T I V E P O W E R C Y C L E S I N C O L O M B I A Jobson, Smith, & Perry, 2016) (Crook, 1994). This cycle is a variation of the Rankine cycle, which uses an organic fluid as work fluid (Devotta & Holland, 1985) that allows operation with a low-temperature heat source. Moreover, ORC has some benefits like low maintenance costs and desirables operating pressures (Tchance, Lambrinos, Frangoudakis, & Papadakis, 2011). 2002). We can find several designs in literature for different conditions and applications, and the most common application is geothermal energy, but the Kalina cycle is not just for geothermal energy. KCS 1-2 is an option to heat recovery in a cement kiln (Mirolli, Cementing Kalina cycle effectiveness, The Kalina Cycle for cement klin waste-heat-recovery power plants, 2006) which has shown better results than organic Rankine cycle, although in energy recovery, this is better. Fluid selection is an important parameter in the design of ORC. 3.2 GOSWAMI CYCLE This cycle was created by Dr. Yogi Goswami in 1998. It is characterized by being a combination of the Rankine power cycle and an absorption power cycle (Karimi M., Dutta, Kaushik, Bansal, & Haque, 2015). Also, its working fluid is an ammonia and water mixture, which reduces the heat transfer irreversibilities, especially for low- temperature finite heat sources such as heat from solar collectors and geothermal heat (Feng, Goswami, & Sunil, 2000). 3.4 COMPARATIVE ANALYSIS WITH EUROPEAN UNION AND U.S.A. The European Union developed the Europe 2020 strategy, where the European Commission aims to 20% of reduction in GHG, an increase of 20% of renewable energy production, and an increase of 20% in energy efficiency compared to 1990. This objective was proposed for 2020 (EC. Europe 2020, s.f.). In 2014 the efficiency target was changed by 27% in 2030. In 2018 was fixed a 32.5% by 2030 in a reduction of energy consumption, which was an energy efficiency target. All these proposals focused on economic growth, a European sustainable and eco-friendly, and obtained good results with a decreasing ODEX index in Household, Transport, Services, and Industry (Ciucci), as we can see in Figure 1. 3 8 6 A series of benefits or advantages that can be obtained with this cycle can be the production of energy and cooling in the same cycle, efficient conversion of heat sources of moderate temperature, in addition, the possibility of improved resource utilization compared to separate power and cooling systems (Karimi M., Dutta, Kaushik, Bansal, & Haque, 2015) Comparative studies with the Rankine cycle showed a 10-20% improvement in thermal efficiency (Maloney & Robertson, 1953). Following this, another study carried out shows that the cycle proposed by Goswami preserves the advantages of the Kalina cycle but eliminates the restrictions that the Kalina cycle presents (Feng, Goswami, & Sunil, 2000). In 2012 European Union published an act that is mainly for efficient energy use (Directive 2012/27/EU of the European Parliament and of the Council of 25 October 2012 on energy efficiency, 2012). 3.1 KALINA CYCLE A good selection of work fluid has a greater influence on performance and output net power (Javanshir & Sarunac, 2017). And each application and operational condition determines the correct work fluid (Freeman, Hellgardt, & Markides, 2015) (Desai & Bandyopadhyay, 2016) (Drescher & Brüggemann, 2007). R E V I S T A B O L E T Í N R E D I P E 1 0 ( 6 ) : 3 8 2 - 3 9 2 - J U N I O 2 0 2 1 - I S S N 2 2 5 6 - 1 5 3 6 3.3 ORGANIC RANKINE CYCLE (ORC) They aim 25% - 50% of energy saving by 2020 -2030 in homes, buildings, and industries (Energy efficiency and renewable energy, s.f.). 3 8 7 In the United States, each state has a group of incentives by efficiency energy use and renewable energy. From household to industry. We can find subsidies, credits to investment, energy credits, and other ones (DSIRE, s.f.). Through the Energy Policy Act of 2005, the United States promotes the use of geothermal energy where Kalina, ORC, and Goswami could be used. With the Energy Policy Act of 1992, the United States promotes energy efficiency in all countries. Where ORC, Kalina, and Goswami could be used in heat recovery. This directive covered some technologies for energy recovery or cogeneration and include ORC, a combined cycle gas turbine with heat recovery, a gas turbine with heat recovery, and any other technology that meet the definition of cogeneration established by the act in Paragraph 30 in article 2. 3.3 ORGANIC RANKINE CYCLE (ORC) The Organic Rankine Cycle is the most common candidate for waste heat recovery and is considered a mature technology (Oluleye, R E V I S T A B O L E T Í N R E D I P E 1 0 ( 6 ) : 3 8 2 - 3 9 2 - J U N I O 2 0 2 1 - I S S N 2 2 5 6 - 1 5 3 S T U D Y O F P O T E N T I A L A P P L I C A T I O N O F A L T E R N A T I V E P O W E R C Y C L E S I N C O L O M B I A electric generation by cogeneration or heat waste recovery, as we can see in Figure 2. Figure 2: Electricity production by CHP in Germany (Umwelt bundesamt, s.f.). The U.S. Department of Energy Efficiency & Renewable Energy (EERE) has established some objectives to increase efficiency energy use. They aim 25% - 50% of energy saving by 2020 -2030 in homes, buildings, and industries (Energy efficiency and renewable energy, s.f.). Figure 1: ODEX index in Europe Union (European environment agency, s.f.). Figure 1: ODEX index in Europe Union (European environment agency, s.f.). Figure 1: ODEX index in Europe Union (European environment agency, s.f.). electric generation by cogeneration or heat waste recovery, as we can see in Figure 2. Figure 2: Electricity production by CHP in Germany (Umwelt bundesamt, s.f.). In this act (Directive 2012/27/EU of the European Parliament and of the Council of 25 October 2012 on energy efficiency, 2012), article 12 established a group of instruments and policies to promote the change, and these ones include fiscals incentives, access to finance, subsidies, workplace activities, an exemplary project. On the other hand, Article 14 promotes efficiency in heating and cooling. Moreover, paragraph 11 of this article promotes the use of waste heat for energy production with the objective of saving primary energy, and additionally, paragraphs 35 and 36 of the act talk about waste heat recovery. The U.S. Department of Energy Efficiency & Renewable Energy (EERE) has established some objectives to increase efficiency energy use. In the transport sector, studies have been carried out, applying power cycles for energy recovery. Through the Rankine cycle, ways have been found to optimize and take advantage of waste heat in vehicles (Macián, Serrano, Dolz, & Sánchez, 2013) in order to reduce energy losses that are reflected in economic losses for the country. Colombia loses approximately 3,000 million dollars per year in this sector due to the inefficiency of the predominant equipment and technologies (UPME, 2016). The Colombian industry is mainly made up of sectors such as manufacturing, textile, cement, steel, ceramics, etc., in which energy consumption is high due to the productive process of each sector. Each of these, where heat demand is high, are possible areas of application for alternative cycles as an option to make the Colombian industry more efficient and competent. 3 8 8 From another energy perspective, 73% of our energy consumption comes from fossil sources. We have crude oil reserves until 2024 and gas reserves until 2029. One of the main objectives is to increase the capacity to generate clean energy as stipulated in the country’s national development plan (Duque, 2018). Among these clean energy sources are geothermal energy and solar energy, where alternative power cycles have a great application for the use of energy resources (Tchanche, Pétrissans, & Papadakis, 2014) (Kalina & Leibowitz, Application of the Kalina cycle technology to geothermal power generation, 1989), being these a viable option for the energy improvement of the country. In the cement industry, production processes represent approximately 40% (Moya, Pardo Garcia, & Mercier, 2011) of the product cost, where 90% from 40% correspond to heat in the drying process (Kermeli, Worrell, & Masanet, 2008). 3.5 ENERGETIC POTENTIAL FOR ALTERNATIVE CYCLES POWER USE IN COLOMBIA This directive was a significant advance in the promotion of alternative cycles to heat recovery and efficient energy use. This produced developed in the application of these technologies. An example is Germany, where the federal government establishes goals of Observing the last Colombian energy balance, carried out by UPME in 2015, wasted energy costs of close to 4.7 billion dollars a year, it is clear that the theoretical potential of Colombia to improve energy efficiency is significant (UPME, R E V I S T A B O L E T Í N R E D I P E 1 0 ( 6 ) : 3 8 2 - 3 9 2 - J U N I O 2 0 2 1 - I S S N 2 2 5 6 - 1 5 3 6 S T U D Y O F P O T E N T I A L A P P L I C A T I O N O F A L T E R N A T I V E P O W E R C Y C L E S I N C O L O M B I A 2016). As can be seen in Figure 3, the greatest energy demand is destined for the transport sector and the energy sector, being here where the greatest losses are generated. use. These studies have determined to promise the use of these technologies, which represents an opportunity for energy use for alternative power cycles where this energy can be harnessed and thus increase efficiency significantly in the production process in Colombia. Figure 3: Distribution of final energy consumption in Colombia (UPME, 2016). The metal casting represents 10% (UPME-BRP, 2007)of the energy consumed in the Colombian industry, where 84.7% corresponds to thermal energy used in the process (UPME, Acciones y perspectivas en eficiencia energetica, 2014). In Colombia, a study was carried out analyzing 5 foundry businesses in the Valle del Cauca and Cauca. in which they propose the option for organic Rankine cycle application (Carabali, Forero, & Cadavid, 2018). Therefore, strengthening the efficiency of the production process with a focus on waste heat recovery is important to the improvement of the industry The Kalina and ORC cycle has been studied (Barbosa, Ponce , Ponce, & Ferreira, 2019) (Moreira & Arrieta, 2019)(Paredes Sanchez, Restrepo Baena, Álvarez Rodríguez, Osorio Correa, & Restrepo, 2015) for its use in the recovery of waste heat as an option for energy R E V I S T A B O L E T Í N R E D I P E 1 0 ( 6 ) : 3 8 2 - 3 9 2 - J U N I O 2 0 2 1 - I S S N 2 2 5 6 - 1 5 3 6 S T U D Y O F P O T E N T I A L A P P L I C A T I O N O F A L T E R N A T I V E P O W E R C Y C L E S I N C O L O M B I A The applicability potential of these alternative power cycles for growth in efficient energy use can be observed. What represents the different sectors of the country more international competitiveness. Colombia requires the use of technologies that propel it to be a more efficient and productive country, and these cycles represent a step to achieve it. However, it has not been enough to control energy losses and greenhouse gas emissions, a problem with a negative impact that could collapse in a couple of years. 5. BIBLIOGRAPHIC REFERENCES Bailey, O., & Worrel, E. (2005). Clean Energy Technologies A Preliminary Inventory of the Potential for Electricity Generation. Enerst Orlando Lawrence Berkeley National Laboratory. 4. CONCLUSIONS It can be concluded that the application of power cycles such as Kalina, ORC, and Goswami are viable for the mitigation of the energy and environmental problem that is being experienced in the world. In addition, these cycles are a good option to boost the economic development of a country in the most demanding energy sectors, such as industrial and transport. Broberg Viklund, S., & Johansson, M. (2014). Technologies for utilization of industrial excess heat: Potentials for energy recovery and CO2 emission reduction. Energy Conversion and Management, 369-379. Broberg, S., & Johansson, M. (2014). Technologies for utilization of industrial excess heat; Potentials for energy recovery and CO2 emisson reduction. Energy Conversion and Management, 369-379. Colombia is a country with great energy potential that has not yet been exploited in the use of alternatives cycles of power generation. Due to its geographical location, soil, and climates characteristics, it has a renewable natural resource and an industry with the capacity to be developed efficient energy use that makes it an attractive place for the use of alternative cycle as Kalina, ORC, and Goswami. 3 8 9 CASATI, E. I. (2014). New concepts for organic Rankine cycle power systems. Cheng, E., Sanjayan, V., & Amer , D. (2005). Solar thermal organic Rankine cycle as a renewable energy option. Jurnal Mekanikal, 68-77. Energy policies that promote efficient energy and the renewable energies use are so important and necessary for the development of a country because they potentiate and incentivize the investment of companies and investors in the development of expensive energy projects, benefiting the country, the investment entity, and all those who would enjoy the energy quality. Ciucci, M. (n.d.). Fact Sheets on the European Union. Retrieved from https://www.europarl. europa.eu/factsheets/en/sheet/69/energy- efficiency Crook, A. (1994). Profiting from low-grade heat. The Watt Committee on Energy Report. Desai, N., & Bandyopadhyay, S. (2016). Thermo- economic analysis and selection of working fluid for solar organic rankine cycle. Applied Thermal Engineering, 471-481. Globally, not all countries have advanced in the same way in terms of developing public energy policies. Some countries, including the European Union, have faced the energy issue with greater commitment. On the other hand, Colombia has relatively extensive environmental legislation. 4. CONCLUSIONS R E V I S T A B O L E T Í N R E D I P E 1 0 ( 6 ) : 3 8 2 - 3 9 2 - J U N I O 2 0 2 1 - I S S N 2 2 5 6 - 1 5 3 6 S T U D Y O F P O T E N T I A L A P P L I C A T I O N O F A L T E R N A T I V E P O W E R C Y C L E S I N C O L O M B I A articles/721-kalina-cycle-power-systems- in-waste-heat-recovery-applications Devotta, S., & Holland, F. (1985). Comparison of Theoretical Rankine Power Cycle Performance Data For 24 Working Fluids. Heat Recovery Systems, 503-510. Haddad, C., Périlhon, C., Danlos, A., François, M.-X., & Descombes, G. (2014). Some efficient solutions to recover low and medium waste heat: competitiveness of the thermoacoustic technology. Energy Procedia, 1056-1069. Directive 2012/27/EU of the European Parliament and of the Council of 25 October 2012 on energy efficiency. (2012). Retrieved from https://eur-lex.europa.eu/legal-content/EN/ TXT/?uri=celex%3A32012L0027 Hammond, G., & Norman, J. (2014). Heat recovery opportunities in UK industry. Applied Energy, 387-397. Drescher, U., & Brüggemann, D. (2007). Fluid selection for the Organic Rankine Cycle (ORC) in biomass power and heat plants. Applied Thermal Engineering, 223-228. Hook, M., & Tang, X. (2013). Depletion of fossil fuels and anthropogenic climate change. Energy Policy, 797-809. DSIRE. (n.d.). Retrieved from https://www. dsireusa.org/ INMIS energy. (n.d.). Retrieved from http://www. inmis-energy.com/5-0-heat-recovery/5- 2-industrial-applications/5-2-2-selected- references Duque, I. (2018). Plan nacional de desarrollo 2018-2022. EC. Europe 2020. (n.d.). Retrieved from http:// ec.europa.eu/europe2020/europe-2020-in- a-nutshell/targets/index_en.htm International energy agency. (2019). Retrieved from World Energy Outlook 2019: https:// www.iea.org/reports/world-energy- outlook-2019/energy-efficiency · 3 9 0 Energy efficiency and renewable energy. (n.d.). Retrieved from https://www.energy.gov/ eere/about-office-energy-efficiency-and- renewable-energy IPCC. (2008). Cambio climatico 2007. Ginebra, suiza. European environmet agency. (n.d.). Retrieved from https://www.eea.europa.eu/data- and-maps/indicators/progress-on-energy- efficiency-in-europe-3/assessment Javanshir, A., & Sarunac , N. (2017). Effect of Working Fluid on Performance of the ORC and Combined Brayton/ORC Cycle. ASME. Kalina, A. (1982). United Estate Patent No. 4346561. Feng, X., Goswami, Y., & Sunil, S. (2000). A combined power/cooling cycle. Energy , 233-246. Kalina, A. (1983). Combined cycle and waste heat recovery power systems based on a novel thermodynamic energy cycle utilizing low-temperatura. ASME. Freeman, J., Hellgardt, K., & Markides, C. (2015). 4. CONCLUSIONS An assessment of solar-powered organic Rankine cycle systems for combined heating and power in Uk domestic applications. Applied Energy, 605-620. Kalina, A. (1983a). Combined cycle and waste heat recovery power systems based on a novel thermodynamic energy cycle utilizing low-temperature for power generation. ASME, 1-5. Global Cement. (2012, August 6). Retrieved from https://www.globalcement.com/magazine/ R E V I S T A B O L E T Í N R E D I P E 1 0 ( 6 ) : 3 8 2 - 3 9 2 - J U N I O 2 0 2 1 - I S S N 2 2 5 6 - 1 5 3 6 S T U D Y O F P O T E N T I A L A P P L I C A T I O N O F A L T E R N A T I V E P O W E R C Y C L E S I N C O L O M B I A Kalina, A. (1984). Combined-cycle system with novel bottoming cycle. ASME. Analysis of Waste HEat Potential in Chinese Industry and Policy Options for Waste Heat to Power Generation. Ernest Orlando Lawrence Berkeley National Laboratory. Kalina, A. (1986). Novel power cycle for combined-cycle system and utility power plants(ESL-IE-86-06-39). Proceedings from the eighth annual industrial energy technology confenrece, Houston, TX. Macián, V., Serrano, J., Dolz, V., & Sánchez, J. (2013). Methodology to design a bottoming Rankine cycle, as a waste energy recovering system in vehicles. recovering system in vehicles. Study in a HDD engine. Applied Energy, 758-771. Kalina, A., & Leibowitz, H. (1989). Application of the Kalina cycle technology to geothermal power generation. Getehermal Resource Council Trans, 605-611. Maloney, J., & Robertson, R. (1953). Thermodinamic study of ammonia-water heat power cycles. Oak Ridge National Lab. Karimi, M., Dutta, A., Kaushik, A., Bansal, H., & Haque, S. (2015). A Review of Organic Rankine, Kalina and Goswami Cycle . International Journal of Engineering Technology, Management and Applied Sciences, 90-105. McKenna, R., & Norman, J. (2010). Spatial Modelling of industrial heat loads and recovery potentials in the UK. Energy Policy, 5878-5891. Ministerio de minas y energía. (n.d.). Retrieved from https://www.minenergia.gov.co/ mision-y-vision Karimi, M., Dutta, A., Kaushik, A., Bansal, H., & Haque, S. (2015a). A Review of Organic Rankine, Kalina and Goswami Cycle. IJETMAS, 90-105. · 3 9 1 Mirolli, M. (2006). T A B O L E T Í N R E D I P E 1 0 ( 6 ) : 3 8 2 - 3 9 2 - J U N I O 2 0 2 1 - I S S N 2 2 5 6 - 1 5 3 6 4. CONCLUSIONS Cementing Kalina cycle effectiveness, The Kalina Cycle for cement klin waste-heat-recovery power plants. IEEE Industry Applications Magazine. Kwak, D.-H., Binns, M., & Kim, J.-K. (2014). Integrated design and optimization of technologies for utilizing low grade heat in process industries. Applied Energy, 307- 322. Mirolli, M. (n.d.). The Kalina cycle for cement kiln waste heat recovery power plants. Lajorla, J. (1995). Electricity from industrial waste heat using high-speed organic Rankine cycle(ORC). International journal of production economics, 227-235. Mlcak, H. (2002). Kaline cycle concept for low temperature geothermal. Geothermal Resources Council transaction. Ogriseck, S. (2009). Integration of Kaline cycle combined heat and power plant, a case study. Applied Thermal Engineering, 2843- 2848. Lemmens, S., & Lecompte, S. (2017). Case study of an organic Rankine cycle applied for excess heat recovery: Technical, economic and policy matters. Energy Conversion and Management, 670-685. Oluleye, G., Jobson, M., Smith, R., & Perry, S. (2016). Evaluating the potential of process sites for waste heat recovery. Applied Energy. Lolos, P., & Rogdakis, E. (2009). A Kalina power cycle driven by renewable energy soruces. Energy, 457-464. Lu, H. (2015). Capturing the invisible Resource: R E V I S T A B O L E T Í N R E D I P E 1 0 ( 6 ) : 3 8 2 - 3 9 2 - J U N I O 2 0 2 1 - I S S N 2 2 5 6 - 1 5 3 S T U D Y O F P O T E N T I A L A P P L I C A T I O N O F A L T E R N A T I V E P O W E R C Y C L E S I N C O L O M B I A Paredes Sanchez, J. P., Restrepo Baena, O. J., Álvarez Rodríguez, B., Osorio Correa, A. M., & Restrepo, G. (2015). Using waste energy from the Organic Rankine Cycle cogeneration in the Portland cement industry. DYNA, 15-20. Salazar , S., Muñoz, Y., & Ospino, A. (2017). Analysis of geothermal energy as an alternative source for electricity in Colombia. Salazar et al. Geotherm Energy, 5-27. Tchance , B., Lambrinos, G., Frangoudakis, A., & Papadakis, G. (2011). Low-grade heat conversion into power using organic Rankine cycles - a weview of various applications. Renew Sustain Energy Rev. Tissot, R. (2012). Latin America’s Energy Future. R E V I S T A B O L E T Í N R E D I P E 1 0 ( 6 ) : 3 8 2 - 3 9 2 - J U N I O 2 0 2 1 - I S S N 2 2 5 6 - 1 5 3 6 4. CONCLUSIONS Inter-American Development Bank. TURBODEN clean energy ahead. (n.d.). Retrieved from https://www.turboden.com/ solutions/1053/waste-heat-recovery · 3 9 2 Umwelt bundesamt. (n.d.). Retrieved from https://www.umweltbundesamt.de/en/ indicator-combined-heat-power-chp#at-a- glance UPME. (2016). Plan de acción indicativo de eficiencia energetica. Bogotá. Valdimarsson, P., & Eliasson, L. (2003). Factors influencing the economics of the Kalina power cycle and situacions of superior performance. International Geothermal Conference. Wolpert, J., & Riffat, S. (1996). Solar-Powered Rankine system for domestic applications. Applied Thermal Engineering, 281-289. Yamada, N., Tominaga, Y., & Yoshida, T. (2014). Demostration of 10-Wp micro organic Rankine cycle generator for low-grade heat recovery. Energy, 806-813. R E V I S T A B O L E T Í N R E D I P E 1 0 ( 6 ) : 3 8 2 - 3 9 2 - J U N I O 2 0 2 1 - I S S N 2 2 5 6 - 1 5 3
https://openalex.org/W2808896558	https://thescipub.com/pdf/ajeassp.2018.986.995.pdf	English	null	An Intelligent Control System for an Electrically Power Assisted Cycle (EPAC)	American journal of engineering and applied sciences	2,018	cc-by	5,911	Original Research Paper Original Research Paper Literature Review An effective EPAC system comprises three main components, (a) the hybridized bicycle (b) the rider and (c) the control system. The following section describes the background and the state-of art of the hybrid bicycle detailing energy storage and power-split mechanisms, Biomechanics of cycling and rider comfort, control and verification of the bicycle for rider comfort. Low All-Electric Range (AER) and regular changing of gears to reach the desired balance between torque and speed are some drawbacks with available EPACs. A key problem with electric assistance controlling is that most of the available bicycles are unable to provide the electric assistance timely, as the rider requires it. However, research is lacking on implementing an automatic or semi-automatic transmission method to maximize the energy utilization in EPACs and increase rider comfort. Therefore, the design and development of An Intelligent Control System for an Electrically Power Assisted Cycle (EPAC) Venura Subuddhika Chandraprabha Dissanayake, Risira Erantha Kannangara, Muthukudaarachchige Uvindu Bigumjith Dias, Asitha Lakruwan Kulasekera and Nirosh Jayaweera Department of Mechanical Engineering, University of Moratuwa, Sri Lanka Venura Subuddhika Chandraprabha Dissanayake, Risira Erantha Kannangara, Muthukudaarachchige Uvindu Bigumjith Dias, Asitha Lakruwan Kulasekera and Nirosh Jayaweer Department of Mechanical Engineering, University of Moratuwa, Sri Lanka Article history Received: 14-05-2018 Revised: 25-05-2018 Accepted: 04-06-2018 Abstract: This paper is focused on proposing an enhanced controller for a hybrid drive mechanism in an Electrically Power Assisted Cycle (EPAC) to improve the battery energy utilization while maintaining the rider’s physical comfort. A real time data acquisition system was set up on a conventional bicycle using sensors and interfacing modules to verify the operating parameters. A fuzzy logic based novel control algorithm was developed upon the acquired data, to overcome the limitations in proportional assist control aided EPACs. The fuzzy controller was implemented using a novel ‘iControl’ algorithm. The developed control algorithm was installed on the newly developed EPAC and practically implemented. The developed algorithm showed the capability to improve range via better energy utilization and maintain rider comfort at the same time. Corresponding Author: Nirosh Jayaweera Department of Mechanical Engineering, University of Moratuwa, Sri Lanka Email: niroshj@uom.lk Keywords: Cycle, Hybrid Drive, Intelligent Control, Physical Comfort, Power Assist Keywords: Cycle, Hybrid Drive, Intelligent Control, Physical Comfort, Power Assist an EPAC incorporating an enhanced hybrid drive mechanism to improve the battery energy utilization while maintaining the rider’s physical comfort, will be able to overcome the limitations of available EPACs. To achieve this, the authors propose a novel intelligent control system for an EPAC capable of ensuring the rider’s physical comfort and increasing the battery energy utilization. This includes the system design of the EPAC and the presentation of the intelligent control system. © 2018 Venura Subuddhika Chandraprabha Dissanayake, Risira Erantha Kannangara, Muthukudaarachchige Uvindu Bigumjith Dias, Asitha Lakruwan Kulasekera and Nirosh Jayaweera. This open access article is distributed under a Creative Commons Attribution (CC-BY) 3.0 license. Introduction Bicycles have become a popular transportation mode in modern world with an escalating demand due to the zero emissions, minimal carbon footprint and being a simple solution to urban congestion. In addition, cycling is also a prevalent evening activity and an adventure sport as well. In most tropical countries (i.e., Sri Lanka), many people are hesitant to use bicycles because of the physical discomfort that comes with the effort in pedaling. Electrically Power Assisted Cycles (EPAC) are a good solution for Sri Lankan cyclists, as they make the riding effort low, by providing partial assistance. The significant demand for EPACs in developed countries, have influenced to design and develop of EPACs, consequently there are many types of EPACs readily available in the global market. American Journal of Engineering and Applied Sciences American Journal of Engineering and Applied Sciences Power-Split Mechanism Commercial EPACs usually incorporate three main types of transmission systems: Manual gear shifting with derailleur, hub-gear transmission and Continuously Variable Transmission (CVT) systems. Few other concepts have been presented in literature, which are yet to be implemented in commercial EPACs. Chien and Tseng (2004) has proposed an automatic gear shifting mechanism for conventional bicycles based on comfortable cadence and torque. Tandon et al. (2011) has presented an intelligent transmission system, based on the theory of cadence intervals considering the comfort of the rider, for bicycles with discrete transmission ratios. Among the major sub-systems of EPACs, the energy storage system and the power-split mechanism require special attention. A key feature expected from an improved EPAC is the ability provide seamless combination of battery power and human power. Devices used in this regard are called power-split mechanisms. A series hybrid drive, with a generator in the crank and a motor in the rear wheel hub, which are mechanically isolated is presented in (Dalvi and Ashtagi, 2014). The major disadvantage here is that human power is converted twice before being utilized. Another e-CVT is presented in (Watterson, 2008), where only a portion of the human power is converted to electrical power and the rest is directly driving the wheel. Direct drive motor in a wheel hub Planetary geared hub motor Spur geared hub motor Geared motor driving additional chain sprocket Additional chain drive Spur gear motor drive on crank Tire roller drive Direct drive motor in a wheel hub Planetary geared hub motor Spur geared hub motor Geared motor driving additional chain sprocket Additional chain drive Spur gear motor drive on crank Tire roller drive Crystalyte, Avanti Electra Rabbittool, Tongxin Heinzmann Panasonic US Pro Drive Aprilia Enjoy Sinclair Zeta Crystalyte, Avanti Electra Rabbittool, Tongxin Heinzmann Panasonic US Pro Drive Aprilia Enjoy Sinclair Zeta rapid charging, but the voltage drop during discharging is problematic in EPAC applications. A combination of super-capacitor and battery bank has been presented in (Malan et al., 2014). In a parallel hybrid, human power and motor power are mechanically summed up by use of a power train. The summation of the two sources of power then transferred to a wheel to take the output. In a series hybrid, the energy input given at the pedal directly transferred to a generator which powers up the drive motor. Any excessive power generated is employed to recharge the battery. In this method, as the user power is totally directed to the battery, the motor should be able to deliver the full mechanical torque required because none is available from the pedals (Mi et al., 2011). Series hybrid bicycles are commercially available, because they are simple in theory and manufacturing. Some commercial human-electric hybrid bicycles are described in Table 1 (references omitted for brevity). Energy Storage Systems Electrical energy storage is a major obstacle faced by all hybrid-drive vehicle systems. The major limitation being the limited energy storage density. Recent studies have shown that 20 Wh of electrical energy needs to travel 1.6 km (Andrea and Andrea, 2010). Most common battery type found in commercial EPACs are lead-acid batteries. Their usage is largely hindered by the low energy density and requirement of continued maintenance. Nickel-Metal Hydride (NiMH) and Nickel- Cadmium batteries have a higher energy density compared to the Lead acid at the expense of cycle life (Weinert et al., 2018). Li-ion has a high-energy density which is advantageous in EPAC applications. There are many variants such as Li-Mg, Li-Ni and Li-Co. Li-Co have become of interest in EPACs (Nitta et al., 2015). Li-Ni has been specially developed for HEVs, PHEVs and EVs with higher C-rate, but has lower energy density than Li-Co (Gohm and Cruden, 2012). Lithium ion polymer is another contender, with the distinct advantage of allowing for slimmer battery packs due to easy stack ability (Gohm and Cruden, 2012). Another alternative is super-capacitors (Hatwar et al., 2013), which are still held back by their cost. They provide The Hybrid Bicycle EPACs are often called hybrid bicycles because those are driven by combination of two power sources, human power and battery power. Hybrid bicycles can be classified as parallel or series hybrid, depending on their drive mechanism. Venura Subuddhika Chandraprabha Dissanayake et al. / American Journal of Engineering and Applied Sciences 2018, 11 (2): 986.995 DOI: 10.3844/ajeassp.2018.986.995 Table 1: Commercial EPACs Table 1: Commercial EPACs Biomechanics of Cycling and Rider Comfort The rotational speed of crank is a one parameter that can be used to measure the physical comfort of the rider. The rotational speed that can be applied by a rider over the pedal crank is called as “cadence” (Seabury et al., 1977). The cadence varies from person to person; hence the physical comfort also varies. Several studies have presented desirable parametric values for a comfortable cadence. A cyclist cycling with a cadence of 50-65 rpm consumes the least oxygen (Seabury et al., 1977). It has also been found that most cyclists cycle at a cadence 987 Venura Subuddhika Chandraprabha Dissanayake et al. / American Journal of Engineering and Applied Sciences 2018, 11 (2): 986.995 DOI: 10.3844/ajeassp.2018.986.995 Fig. 1: Data Acquisition system developed for experimentation SD card Wheel rpm sensor Accelerometer Cadence sensor Controller (Arduino Mega) Pulse sensor between 85 and 95 rpm (Hagberg et al., 1981). The optimum cycling cadence, based on a muscle stress- based cost function, has been found to be between 95- 100 rpm (Neptune et al., 1997). It has also been found that the minimum neuromuscular activation, force, stress and endurance occurs at a cadence of 90 rpm (Hull et al., 1988). between 85 and 95 rpm (Hagberg et al., 1981). The optimum cycling cadence, based on a muscle stress- based cost function, has been found to be between 95- 100 rpm (Neptune et al., 1997). It has also been found that the minimum neuromuscular activation, force, stress and endurance occurs at a cadence of 90 rpm (Hull et al., 1988). The output power which exerted by the cyclist is another important parameter. Previous studies have shown that typical steady-state cycling over level ground at ~9 m/s the power required is ~200 W at a cadence of 65 rpm (Hull et al., 1988). More recent studies have shown that world class cyclists generate 7.6 W/kg while an average cyclist would generate 3.7 W/kg (Coggan, 2008). Fig. 1: Data Acquisition system developed for experimentation A test protocol was developed to cover different ride environments so that the system parameters can be identified which is useful in making a robust design. The test has been performed by using a rider of near the average weight (63 kg), on a level and smooth asphalt terrain without hindrances from other vehicles, under three different road conditions in a tropical climate. Biomechanics of Cycling and Rider Comfort The obtained sensor data is used to calculate the output power which is available for drive the bicycle: Control Systems Several control systems have been used in existing EPACs as well as been presented in literature. Reference (Lomonova et al., 2002) presents a control system where a predefined assisted power is provided without considering the rider’s physical condition, so that the comfort and the safety of the rider might not be guaranteed. Most commercial systems such as Bosch and Panasonic mid drive system, provide a motor power proportional to the rider’s input power. In these systems, rider can select the percentage of proportional assist whether 100%, 200%, 300% or 400% (Cai et al., 2010). A method called the ‘Delta learning rule’ has been presented where, the rider’s pedaling power is forecasted using the delta rule, to calculate the weights of neurons in a neural network- based control system (Cai et al., 2010). The essential assisted motor power can be thus calculated from the forecasted pedal power under the preferred comfortable speed. A reinforcement learning based controller is proposed in (Hsu et al., 2012), where the riding comfort is achieved by maintaining a velocity comfort zone. This provides the advantage of reducing the instantaneous forward acceleration which can make the rider feel unsafe if sudden assisted power produced too much acceleration. This is defined as a safe acceleration zone. i. Flat road (00 gradient) – This setting is required to ascertain the rated power requirement to be given by the motor ii. An uphill travel on a road with 50 gradients (measured with an accelerometer with a ±10 tolerance) – most of the common road gradients are less than 50. This value is important to determine the maximum power that the motor can produce iii. A downhill travel on the same road (-50 gradient) – To identify the regenerative capabilities. The data from wheel rpm sensor is used to calculate the cruise speed of the bicycle and it was displayed on an odometer which was placed at the stem of the bicycle. According to the test protocol, the rider had to keep the cruising speed at 10 km/h constant level (with ±1 km/h tolerance). The variation of gradient of the road, cadence, wheel rpm and pulse rate with time is acquired via the above experiment. The gathered results (omitted for brevity) successfully verified the values considered from the literature in the context of an application in a tropical climate. Verification of Cadence for Rider Comfort To setup the experimental foundation for the bio- mechanic and rider comfort parameters, an experimental model and a test protocol was set up using a conventional bicycle with an attached sensor array. Initially, a bicycle was fitted with four sensors; a cadence sensor, a wheel rpm sensor, an accelerometer and a pulse sensor. An Arduino Mega2560 was used to capture sensor data and store them on a SD card as shown in Fig. 1. EPAC Design The EPAC proposed by the authors have been designed for use in tropical climates to provide effective power assist while maintaining rider comfort. The major design criteria are as follows: (i) a conventional bicycle with standard manual derailleur mechanism is used as 988 Venura Subuddhika Chandraprabha Dissanayake et al. / American Journal of Engineering and Applied Sciences 2018, 11 (2): 986.995 DOI: 10.3844/ajeassp.2018.986.995 the controller, so that the motor will be disconnected when the brake lever is pulled. the controller, so that the motor will be disconnected when the brake lever is pulled. the base frame for the build. (ii) to be used on asphalt paved roads with maximum 10% uniform gradient. iii) Maximum speed to be 15 km/h. (iv) maximum susceptible headwind to be 10 m/s. (v) Average rider weight from global average 62 kg (Walpole et al., 2012). (vi) provide manual, fully-electric and hybrid drive with power assist drive capabilities. Traditional derailleur/multi sprocket mechanism is replaced by a cable-actuated hub transmission unit installed on the rear hub (Fig. 3). Fig. 2: Drive motor placement near pedal crank Fig. 3: Cable-actuated hub transmission unit installed on the rear hub Fig. 2: Drive motor placement near pedal crank Mechanical Design / American Journal of Engineering and Applied Sciences 2018, 11 (2): 986.995 DOI: 10.3844/ajeassp.2018.986.995 Nickel Manganese Cobalt Oxide (LiNiMnCoO2 or NMC) was selected due to its higher maximum continuous discharge current (C-rate) and good cycle life (~800 cycles) at a lower cost compared to LiFeO4, LiNiCoAlO2 and LiTiO2. INR18650-29E (Samsung) with a nominal voltage of 3.6 V and 2850 mAh capacity was selected as the battery cell. To obtain 36V at peak current of 10A 10 cells were connected in series and a similar stack was put in parallel, with a total of 20 cells. The available capacity from this battery pack is 5.7 Ah. This is a 14% increase in capacity over most commercial battery packs rated at 5 Ah. The expected range from this 5.7 Ah pack is 16.4 km. Battery Management System To improve the safety and life cycle of battery, a Battery Management System (BMS) is introduced. The main function of battery management system is protecting battery cell when overcharging and discharging. Other than that, temperature monitoring and temperature management is also done by BMS to prevent a possible explosion at elevated temperatures. BMS is integrated with active cell balancing to improve the efficiency of the battery pack and extend the lifecycle of battery cells. Sensing System A 6-axis motion tracking system (MPU 6050, InvenSense TDK) was used to provide road gradient and linear acceleration. The captured signal was conditioned using a Kalman filter to eliminate the noise. The cadence was measured by an encoder placed near the pedal crank. The encoder uses hall sensor and magnet embedded encoder wheel. A 12-magnet encoder wheel was used to obtain a resolution of 5 rpm. A similar encoder was installed between the bicycle fork arm and a spoke to act as the wheel speed sensor. To measure the rider’s pulse rate, photoplethysmograph based pulse sensor (Tamura et al., 2014) was installed on the throttle to be in continuous contact with the rider’s index finger. A current sensor was installed with the battery pack to observe and control the battery temperature via current control. A display unit was mounted on the handle to display the following to the rider: (i) cadence, (ii) speed of the bicycle, (iii) state of charge of the battery pack, (iv) pulse rate, (v) ride mode and (vi) the selected transmission ratio. The layout of these sensors together with other components are displayed in Fig. 4. Mechanical Design Based on the design criteria, total power requirement was calculated as 570 W for a mountain bicycle tire on asphalt road (Engineering ToolBox, 2008). The power capacity of the rider was approximated as 225 W following the work of Coggan (2008) and the required motor output power was estimated as 350 W. To reduce controller cost and wiring complexity the peak current was limited to 10 A. Therefore, a 350 W BLDC of 36 V was selected as the power assisting device (Chan et al., 1999; Park et al., 2011). The other major components of the EPAC are, the motor controller, hub transmission unit and sensor array. The motor and pedal crank is connected by two freewheels, one at the pedal crank and one at the motor output shaft, as shown in Fig. 2. When only one of the two power sources are being used, the other is freewheeling. The torque addition will be taken place only when the two rpms (i.e., pedal and motor) are equal. This combined torque is then transmitted to the rear hub via a chain drive. The transmission ratio will be chosen upon the desired speed and calculated required torque to reach the desired speed. The chosen transmission ratio will be shown on a handle mounted display, so that the rider can shift to the selection. The rider can use a hand throttle in case of an instantaneous power demand. There is an electrical connection between the brake levers and Fig. 2: Drive motor placement near pedal crank Fig. 3: Cable-actuated hub transmission unit installed on the rear hub Fig. 3: Cable-actuated hub transmission unit installed on the rear hub Fig. 3: Cable-actuated hub transmission unit installed on the rear hub Fig. 4: Mechanical and electrical layout of the proposed EPAC Rear brakes Hub transmission unit Battery pack Pulse sensor Cadence sensor Drive motor Pedal crank Brake lever 2 Controller Display Accelerometer Throttle Brake lever 1 Electrical connections Mechanical connections Wheel rpm sensor Front brakes Wheel rpm sensor Front brakes Hub transmission unit Fig. 4: Mechanical and electrical layout of the proposed EPAC 989 Venura Subuddhika Chandraprabha Dissanayake et al. / American Journal of Engineering and Applied Sciences 2018, 11 (2): 986.995 DOI: 10.3844/ajeassp.2018.986.995 Venura Subuddhika Chandraprabha Dissanayake et al. / American Journal of Engineering and Applied Sciences 2018, 11 (2): 986.995 DOI: 10.3844/ajeassp.2018.986.995 Venura Subuddhika Chandraprabha Dissanayake et al. Battery Pack The complete EPAC design is shown in Fig. 5 comprising the drive system, battery pack, BMS and sensor array. Li-ion was selected for the battery pack design, due to its higher energy density. Among the various Li-ion battery chemistries available (Nitta et al., 2015), Lithium Fig. 5: Complete EPAC design Motor controller Main control unit Pulse sensor Accelerometer Plug-in charging port Transmission hub Coupling unit Cadence sensor 36V BLDC motor Speed sensor Throttle Battery pack Main control unit Plug-in charging port Speed sensor Fig. 5: Complete EPAC design 990 Venura Subuddhika Chandraprabha Dissanayake et al. / American Journal of Engineering and Applied Sciences 2018, 11 (2): 986.995 DOI: 10.3844/ajeassp.2018.986.995 Venura Subuddhika Chandraprabha Dissanayake et al. / American Journal of Engineering and Applied Sciences 2018, 11 (2): 986.995 DOI: 10.3844/ajeassp.2018.986.995 iControl. This technology ensures the rider’s physical comfort and a proper battery energy utilization on a ride. The rider’s physical comfort is maintained by monitoring the cadence and pulse rate of the rider and control the motor rpm accordingly. Battery energy utilization is achieved by controlling the Pulse Width Modulation (PWM) to maintain the desired speed of rider. iControl. This technology ensures the rider’s physical comfort and a proper battery energy utilization on a ride. EPAC Controller Design The major advancement of this research is to incorporate an intelligent control system capable of increasing range by improving the energy utilization while maintaining rider comfort. The proposed controller design to allow four ride modes which the rider can switch between at his convenience via a toggle switch. The four ride modes are, (a) no assistance-manual mode (b) fully electric mode (c) motor assistance during traction and (d) pedaling regeneration. Regeneration capability is considered but is not discussed in this study. The rider’s physical comfort is maintained by monitoring the cadence and pulse rate of the rider and control the motor rpm accordingly. Battery energy utilization is achieved by controlling the Pulse Width Modulation (PWM) to maintain the desired speed of rider. Establishing a direct control relationship between cadence, pulse rate and motor speed is difficult as comfortable cadence and pulse rate varies depending on the rider. Therefore, a fuzzy controller is developed to establish an effective controller. The main components in building the fuzzy controller is the development of Membership Functions (MF). Three membership function are created for pulse rate, cadence and motor speed. Discussion Figure 7 verifies the expected operation of the iControl system, such that, during most of the Times, the motor speed lies in step 2 mode (middle plane: The cyan region in Fig. 7), where the motor is in its highest efficiency region. Therefore, a maximum energy utilization can be expected from the motor within a wider operating range. Figure 8 shows that; the peak states of motor speed and cadence do not occur at the same time. This ensures a better energy utilization. Also, when the peak state of the pulse rate occurs, the motor speed retains at a maximum level, which assists in improving the rider’s physical comfort. Results The control algorithm was simulated using the MATLAB using a simulated dataset. The fuzzy toolbox in MALAB was utilized in the development of the control system simulation. A simulated ride pattern was generated and input to the simulation. The simulated controller output of the MATLAB simulation is depicted as a 3D graph between cadence, heart rate and motor speed in Fig. 7. Data collected from an experimental ride using the same volunteer rider, as in the previous experiment, is given in Fig. 9. iControl The above control strategies are implemented alongside full EPAC system control using a control defined as Fig. 6: Membership functions of the fuzzy controller 181 plot points Membership function plots hard easy very hard 1 0.5 0 60 70 80 90 100 110 120 130 140 150 160 60 70 80 90 100 110 120 180 200 220 240 260 280 300 320 340 181 181 hard easy very hard Membership function plots Membership function Motor speed Cadence Pulse rate 1 0.5 0 1 0.5 0 mf1 mf2 mf3 Plot points Plot points 60 70 80 90 100 110 120 180 200 220 240 260 280 300 320 340 181 Membership function Motor speed Cadence 0 1 0.5 0 mf1 mf2 mf3 Plot points Fig. 6: Membership functions of the fuzzy controller 991 Venura Subuddhika Chandraprabha Dissanayake et al. / American Journal of Engineering and Applied Sciences 2018, 11 (2): 986.995 DOI: 10.3844/ajeassp.2018.986.995 Venura Subuddhika Chandraprabha Dissanayake et al. / American Journal of Engineering and Applied Sciences 2018, 11 (2): 986.995 DOI: 10.3844/ajeassp.2018.986.995 Pulse rate MF is designed based on the Borg RPE scale which measure exertion during human work (Borg, 1998). The pulse rate MF is developed with the lower limit at 60-70, upper limit at 120 and very high between 140-170 bpm. Least O2 consumption occurs when the cadence is at 55-65 rpm (Hull and Jorge, 1985) and endurance limit is set to 100 rpm. Therefore, the cadence MF is set to easy 60-65 rpm, hard 90 rpm and very hard 115-120 rpm. The member function for the motor is developed based on the power curve of the motor. The speed of the motor is inversely proportional to the motor torque, assuming the motor power is constant. Therefore, higher the motor speed, lesser the torque provided by the motor. iControl ensures the motor is operating in its optimum operating range, which ultimately contributes to the power efficiency. Low speed is set to 180 rpm, moderate speed between 240-280 rpm and high speed to 340 rpm. The defined MFs are depicted in Fig. 6. Fuzzy Rules The pulse rate is a measurement of physical comfort, so that increasing the pulse rate will result in rider’s discomfort. Therefore, if the pulse rate increases, the motor speed should also be increased. Also, when riding at a higher cadence the rider feels discomfort the motor speed should again be increased. The conditions selected to implement the fuzzy controller is given in Table 2. Table 2: Fuzzy rule set Pulse rate Cadence Motor speed Easy Easy Step 1 Easy Hard Step 2 Easy Very hard Step 2 Hard Very hard Step 2 Very hard Very hard Step 3 the fuzzy controller is given in Table 2. ntrol algorithm works based on the sory data; Tilt angle, Speed of the bicycle, he bicycle, Heat rate of the rider, Throttle ke position, Battery current and Battery ntrol flow chart of the iControl algorithm ig. 8. Table 2: Fuzzy rule set Pulse rate Cadence Motor speed Easy Easy Step 1 Easy Hard Step 2 Easy Very hard Step 2 Hard Very hard Step 2 Very hard Very hard Step 3 Fig. 7: Output characteristic of the fuzzy controller 300 280 260 240 220 120 100 80 60 60 80 100 120 140 160 Cadence Heart rate Motor Speed The iControl algorithm works based on the following sensory data; Tilt angle, Speed of the bicycle, Cadence of the bicycle, Heat rate of the rider, Throttle position, Brake position, Battery current and Battery SOC. The control flow chart of the iControl algorithm is shown in Fig. 8. Fig. 7: Output characteristic of the fuzzy controller 300 280 260 240 220 120 100 80 60 60 80 100 120 140 160 Cadence Heart rate Motor Speed Fig. 7: Output characteristic of the fuzzy controller 300 280 260 240 220 120 100 80 60 60 80 100 120 140 160 Cadence Heart rate Motor Speed Fig. 7: Output characteristic of the fuzzy controller 992 Venura Subuddhika Chandraprabha Dissanayake et al. / American Journal of Engineering and Applied Sciences 2018, 11 (2): 986.995 DOI: 10.3844/ajeassp.2018.986.995 DOI: 10.3844/ajeassp.2018.986.995 Fig. 8: iControl algorithm Fig. Fuzzy Rules 9: Sample results from an experimental run with the iControl algorithm Cadence Pulse rate Speed of the bicycle Gear selection method Tilt angle If cadence >50 Yes Yes Yes Yes Yes No No No No iControl (fuzzy rules) Display required gear Current sensor PI controller Reference motor speed Current >8A Drive motor Throttle position >0 Motor speed feedback Motor speed does not change If temp >60 Battery temp. sensor Reduce motor speed Throttle position Drive motor start Cadence Drive motor off Cadence angle >720 Calculate crank angular displacement (crank θ) If power turned on Power switch status 300 200 100 0 100 80 60 40 20 0 160 140 120 100 80 0 10 20 30 40 50 60 Time (s) Cadence Pulse rate Motor speed If temp >60 Cadence Current sensor Current >8A If cadence >50 If cadence >50 Battery temp. sensor Pulse rate PI controller Drive motor Reduce motor speed iControl (fuzzy rules) Cadence Cadence Drive motor start Reference motor speed Throttle position Throttle position >0 Display required gear Motor speed feedback Drive motor off If power turned on Fig. 8: iControl algorithm Fig. 8: iControl algorithm g g Fig. 9: Sample results from an experimental run with the iControl algorithm 300 200 100 0 100 80 60 40 20 0 160 140 120 100 80 0 10 20 30 40 50 60 Time (s) Cadence Pulse rate Motor speed Pulse rate Fig. 9: Sample results from an experimental run with the iControl algorithm References Andrea, A. and D. Andrea, 2010. Battery management systems for large lithium-ion battery packs. Artech House. Lomonova, E.A., A.J.A. Vandenput, J. Rubacek, B. D’Herripon and G. Roovers, 2002. Development of an improved electrically assisted bicycle. Proceedings of the 37th IAS Annual Meeting Conference Record of the Industry Applications Conference, Oct. 13-18, IEEE Xplore Press, Pittsburgh, PA, USA, pp: 384-389. DOI: 10.1109/IAS.2002.1044116 Borg, G., 1998. Borg’s rating of perceived exertion and pain scales. Human Kinetics. Chan, T.F., L.T. Yan and S.Y. Fang, 1999. Design of a permanent-magnet brushless DC motor drive for an electric bicycle. Proceedings of the IEEE International Conference on Power Electronics and Drive Systems, Jul. 27-29, IEEE Xplore Press, Hong Kong, pp: 714-8. DOI: 10.1109/PEDS.1999.792792 Chien, H.C. and C.H. Tseng, 2004. An automatic transmission for bicycles: A simulation. Int. J. Ind. Ergon., 33: 123-32. DOI: 10.1016/j.ergon.2003.09.002 Chan, T.F., L.T. Yan and S.Y. Fang, 1999. Design of a permanent-magnet brushless DC motor drive for an electric bicycle. Proceedings of the IEEE International Conference on Power Electronics and Drive Systems, Jul. 27-29, IEEE Xplore Press, Hong Kong, pp: 714-8. DOI: 10.1109/PEDS.1999.792792 Malan, K., M. Coutlakis and J. Braid, 2014. Design and development of a prototype super-capacitor powered electric bicycle. Proceedings of the International Energy Conference, May 13-16, IEEE Xplorer Press, Cavtat, Croatia, pp: 1434-1439. DOI: 10.1109/ENERGYCON.2014.6850611 Chien, H.C. and C.H. Tseng, 2004. An automatic transmission for bicycles: A simulation. Int. J. Ind. Ergon., 33: 123-32. DOI: 10.1016/j.ergon.2003.09.002 g , j g Coggan, A., 2008. Power profiling. trainingpeaks.com. Mi, C., M.A. Masrur and D.W. Gao, 2011. Hybrid Electric Vehicles: Principles and Applications with Practical Perspectives. 1st Edn., John Wiley and Sons, Ltd, Chichester, UK, ISBN-10: 0470747730, pp: 468. Dalvi, P.V. and A.R. Ashtagi, 2014. Review of series hybrid drive-system: Advantages for velomobiles how a series hybrid human power drive works. Int. J. Innov. Eng. Res. Technol., 1: 1-6. Engineering ToolBox, 2008. Rolling resistance. Engineering ToolBox. Dalvi, P.V. and A.R. Ashtagi, 2014. Review of series hybrid drive-system: Advantages for velomobiles how a series hybrid human power drive works. Int. J. Innov. Eng. Res. Technol., 1: 1-6. Neptune, R.R., S.A. Kautz and M.L. Hull, 1997. The effect of pedaling rate on coordination in cycling. J. Biomech., 30: 1051-1058. DOI: 10.1016/S0021-9290(97)00071-7 g Engineering ToolBox, 2008. Rolling resistance. Engineering ToolBox. Gohm, C.T. and A. Cruden, 2012. Automated high current cycling test system for supercapacitor characterisation. Acknowledgment The authors gratefully acknowledge the support extended by the staff members of the Department of Mechanical Engineering, University of Moratuwa, Sri Lanka. The authors identify no ethical issues. Cai, L., A.B. Rad and W.L. Chan, 2010. An intelligent longitudinal controller for application in semiautonomous vehicles. IEEE Trans. Ind. Electron., 57: 1487-1497. DOI: 10.1109/TIE.2009.2029571 Conclusion pack and an intelligent battery management system (BMS). This EPAC controlled by a novel ‘iControl’ algorithm. Experimental results show that the system can effectively assist the rider while improving energy utilization and maintain rider comfort. This research presents an intelligent control system for an Electrically Power Assisted Cycle (EPAC) capable of increasing the electric range by improving the energy utilization while maintaining the rider comfort. A prototype EPAC is built using a conventional bicycle and integrating a motor drive, rear hub transmission, an array of sensors including those for cadence, pulse rate and wheel speed, a novel battery The control system is to be further developed by introducing a learning function to adapt to different rider’s characteristics. Inclusion of the regenerative system with a high-power density secondary storage system is the next stage in the EPAC development. 993 Venura Subuddhika Chandraprabha Dissanayake et al. / American Journal of Engineering and Applied Sciences 2018, 11 (2): 986.995 DOI: 10.3844/ajeassp.2018.986.995 Venura Subuddhika Chandraprabha Dissanayake et al. / American Journal of Engineering and Applied Sciences 2018, 11 (2): 986.995 DOI: 10.3844/ajeassp.2018.986.995 Hatwar, N., A. Bisen, H. Dodke, A. Junghare and M. Khanapurkar, 2013. Design approach for electric bikes using battery and super capacitor for performance improvement. Proceedings of the 16th International IEEE Conference on Intelligent Transportation Systems, Oct. 6-9, IEEE Xplore Press, The Hague, Netherlands, pp: 1959-1964. DOI: 10.1109/ITSC.2013.6728516 Author’s Contributions Hsu, R.C., C.T. Liu and D.Y. Chan, 2012. A reinforcement-learning-based assisted power management with QoR provisioning for human– electric hybrid bicycle. IEEE Trans. Ind. Electron., 59: 3350-3359. DOI: 10.1109/TIE.2011.2141092 Venura S.C. Dissanayake and Risira E. Kannangara: Participated in all experiments, coordinated the data-analysis and contributed to the writing of the manuscript. Venura S.C. Dissanayake and Risira E. Kannangara: Participated in all experiments, coordinated the data-analysis and contributed to the writing of the manuscript. M.U.B. Dias: Coordinated the data-analysis and contributed to the writing of the manuscript. Hull, M.L., H.K. Gonzalez and R. Redfield, 1988. Optimization of pedaling rate in cycling using a muscle stress-based objective function. Int. J. Sport Biomech., 4: 1-20. DOI: 10.1123/ijsb.4.1.1 Asitha L. Kulasekera and Nirosh Jayaweera: Designed the research plan and organized the study. Hull, M.L. and M. Jorge, 1985. A method for biomechanical analysis of bicycle pedalling. J. Biomech., 18: 631-644. DOI: 10.1016/0021-9290(85)90019-3 Ethics The authors identify no ethical issues. References Proceedings of the 21st International Symposium on Power Electronics, Electrical Drives, Automation and Motion, Jun. 20-22, IEEE Xplore Press, Sorrento, Italy, pp: 748-53. DOI: 10.1109/SPEEDAM.2012.6264509 Nitta, N., F. Wu, J.T. Lee and G. Yushin, 2015. Li-ion battery materials: Present and future. Mater Today, 18: 252-264. DOI: 10.1016/j.mattod.2014.10.040 Park, S.Z., Y.K. Kim, C.H. Song, J.W. Lee and H.S. Mok, 2011. Operation method of electric bicycle using change of BLDC operation mode and PMSM operation mode. Proceedings of the 8th International Conference on Power Electronics and ECCE Asia, May30-Jun. 3, IEEE Xplore Press, Jeju, South Korea, pp: 2529-2536. DOI: 10.1109/ICPE.2011.5944733 Hagberg, J.M., J.P. Mullin, M.D. Giese and E. Spitznagel, 1981. Effect of pedaling rate on submaximal exercise responses of competitive cyclists. J. Applied Physiol., 51: 447-451. DOI: 10.1152/jappl.1981.51.2.447 994 Venura Subuddhika Chandraprabha Dissanayake et al. / American Journal of Engineering and Applied Sciences 2018, 11 (2): 986.995 DOI: 10.3844/ajeassp.2018.986.995 Walpole, S.C., D. Prieto-Merino, P. Edwards, J. Cleland and G. Stevens et al., 2012. The weight of nations: an estimation of adult human biomass. BMC Public Health, 12: 439-439. DOI: 10.1186/1471-2458-12-439 Seabury, J.J., W.C. Adams and M.R. Ramey, 1977. Influence of pedalling rate and power output on energy expenditure during bicycle ergometry. Ergonomics, 20: 491-498. g DOI: 10.1080/00140137708931658 Watterson, P.A., 2008. An electric assist bicycle drive with automatic continuously variable transmission. Proceedings of the International Conference on Electrical Machines and Systems, Oct. 17-20, IEEE Xplroe Press, Wuhan, China, pp: 2992-2997. Tamura, T., Y. Maeda, M. Sekine and M. Yoshida, 2014. Wearable photoplethysmographic sensors-past and present. Electronics, 3: 282-302. DOI: 10.3390/electronics3020282 Tandon, P., A. Awasthi, B.K. Mishra, P. Rathore and R.K. Shukla, 2011. Design and simulation of an intelligent bicycle transmission system. IEEE/ASME Trans. Mechatron., 16: 509-517. DOI: 10.1109/TMECH.2010.2045431 Weinert, J.X., A.F. Burke and X. Wei, 2018. Lead-acid and lithium-ion batteries for the Chinese electric bike market and implications on future technology advancement. J. Power Sources, 172: 938-945. DOI: 10.1016/j.jpowsour.2007.05.044 10.1109/TMECH.2010.2045431 995
https://openalex.org/W4283377882	https://journal.poltekkesaceh.ac.id/index.php/jand/article/download/48/12	English	null	Phosphorus and calcium intake of stunted toddlers aged 24-59 months: A case-control study in Sinar Bahagia Village, Simeulue	Journal of Applied Nutrition and Dietetic	2,022	cc-by-sa	3,306	Abstract Stunting is still a serious problem in toddlers and is usually caused by insufficient calcium and phosphorus intake, which plays an important role in bone formation. This study aimed to analyze the phosphorus and calcium intake of stunted toddlers aged 24-59 months in Sinar Bahagia Village, Simeulue Barat District, Simeulue Regency. This research was a case- control study. The sample of this study was stunted and non-stunted children. Each case and control group consisted of 62 subjects who were selected using the simple random sampling technique. Food recall and food frequency questionnaire (FFQ) were used to investigate nutrient intake. NutriSurvey was used to analyze the content of nutrient intake. Meanwhile, the chi-square test was used to analyze the effects of calcium and phosphorus intake on the incidence of stunting. Stunted children had lower calcium and phosphorus intakes than non- stunted children (P-value <0.05). Stunted children had 2.879 times lower phosphorus than the control group. Moreover, they had 35 times lower calcium intake than the control group. Serious stunting problems in children require a nutritional counseling program that trains mothers to meet children’s daily food intake, especially high calcium and phosphorus. Keywords: calcium, phosphorus, stunting, toddlers Phosphorus and calcium intake of stunted toddlers aged 24-59 months: A case-control study in Sinar Bahagia Village, Simeulue 1 2* Alda Wati 1 and Suryana 2* 1Nutrition Transfer Program Polytechnic of Health, Aceh Health Ministry 2Department of Nutrition, Polytechnic of Health, Aceh Health Ministry Correspondence email: suryana@poltekkesaceh.ac.id Alda Wati 1 and Suryana 2 1Nutrition Transfer Program Polytechnic of Health, Aceh Health Ministry 2Department of Nutrition, Polytechnic of Health, Aceh Health Ministry *Correspondence email: suryana@poltekkesaceh.ac.id Submitted: 05/04/2021 Published: 30/06/2021 Wati et al. \| Intake of phosphorus and calcium on stunting in toddlers aged 24-59 months Journal of Applied Nutrition and Dietetic Volume 1, Number 1, June 2021 P-ISSN: 2797-7412 Wati et al. \| Intake of phosphorus and calcium on stunting in toddlers aged 24-59 months Journal of Applied Nutrition and Dietetic Volume 1, Number 1, June 2021 P-ISSN: 2797-7412 Wati et al. \| Intake of phosphorus and calcium on stunting in toddlers aged 24-59 months Journal of Applied Nutrition and Dietetic Volume 1, Number 1, June 2021 P-ISSN: 2797-7412 Calcium forms complex bonds with phosphate that can provide strength to bones; thus, phosphorus deficiency can interfere with growth. Meanwhile, prolonged phosphorus deficiency will cause osteomalacia and release calcium from bones (Mikhail WZA et al., 2013). Methods This study employed a case-control design to investigate the relationship between calcium and phosphorus intake as well as the incidence of stunting. The sample of this study was two groups: the case group and the control group. The case group referred to the group of stunted while the control group referred to the group with normal nutritional status. Microtoise was a tool used to measure the height of toddlers anthropometrically. Nutritional status data were based on height/age categories: the short category from -3 SD to <-2 SD and the normal category from -2 SD to +3 SD (WHO, 2019). 1x24 hour food recall and semi-food frequency questionnaire were employed to explore the phosphorus and calcium intakes. The research sample was 62 children aged 24-59 months. The data were collected by interviewing mothers of toddlers in Sinar Bahagia Village. A Chi-square test was used to analyze the effects of phosphorus and calcium intake on the incidence of stunting in under-five-year-old children. Introduction Stunting is a linear growth disorder mostly occurring in children aged two years old or less. Stunting is caused by several factors, such as malnutrition, chronic nutrient intake, and chronic infectious diseases (WHO, 2010). The 2013 Indonesia Health Survey reported that the prevalence of stunting was 37.2%. This number showed an increase from 36.8% in 2007. The prevalence of stunting in Aceh Province is 44.6% while the prevalence of stunted children aged five years in Simuelue Regency is 28.6%. This is a very important number to note. One of the risk factors for stunting is lack of nutritional intake in a long term. Therefore, slow growth can occur and affect nutritional status. Inadequate intake of energy and nutrients as well as infectious diseases are factors that greatly cause stunting. In addition, the Lancet Series describes several pivotal micronutrients to prevent stunting, such as vitamin A, zinc, iron, and iodine (Souganidis E., 2012). Other micronutrients, such as calcium and phosphorus, also play a crucial role in the linear growth of children (Mikhail WZA, 2013). Stunting in children under five years old will affect their health problems, education, and productivity for the long term. Stunted toddlers have difficulty in achieving optimal physical and psychomotor growth and development (Dewey KG and Begum K., 2011). High calcium intake is required for children’s growth and mineralizes new bone deposits and osteoblast dysfunctions (Khairy SAM et al., 2010). Insufficient calcium intake will affect linear growth in toddlers if the calcium content is 50% lower than the normal content. 43 Wati et al. \| Intake of phosphorus and calcium on stunting in toddlers aged 24-59 months Journal of Applied Nutrition and Dietetic Volume 1, Number 1, June 2021 P-ISSN: 2797-7412 Result This study revealed that the data were classified into age, gender, height, and numbers of sibling categories. The data showed that the majority of the respondents, 34 people, were aged > 36 months (54.8%). The data on gender showed that 38 children were male (61.3%). Meanwhile, the data on height showed that most of the children, 52 people, had >90 cm totaling (83.9%). Meanwhile, the data on numbers of siblings showed that 31 respondents had only one sibling (50%). Table 1. Characteristics of Respondents (n = 60) Age (Months) f % > 36 34 54.8 < 36 28 45.2 Gender f % Boy 38 61.3 Girl 24 38.7 Height f % > 90 cm 52 83.9 < 90 cm 10 16.1 Parity f % 1st child 31 50.0 2nd child 18 29.0 3rd child 13 21.0 Table 2 shows the characteristics of the parents of 62 respondents. Most of the sample’s fathers were above 30 years (49 people or 79.0%). Meanwhile, 42 fathers had height for > 170 cm (67.7%). The majority of the fathers works as a farmer (83.9%) and earned high school education (62.9%). The data showed that most respondents’ maternal age was more than 30 years (35 people or 56.5%). Moreover, Table 2 shows the characteristics of the mothers. The highest height of the mothers was more than 150 cm (64.5%). Most of the mothers graduated from high school (67.7%). Meanwhile, the data on family income showed that 51 people earned more than IDR1,000,000 (82.3%). Table 2 shows the characteristics of the parents of 62 respondents. Most of the sample’s fathers were above 30 years (49 people or 79.0%). Meanwhile, 42 fathers had height for > 170 cm (67.7%). The majority of the fathers works as a farmer (83.9%) and earned high school education (62.9%). The data showed that most respondents’ maternal age was more than 30 years (35 people or 56.5%). Moreover, Table 2 shows the characteristics of the mothers. The highest height of the mothers was more than 150 cm (64.5%). Most of the mothers graduated from high school (67.7%). Meanwhile, the data on family income showed that 51 people earned more than IDR1,000,000 (82.3%). Table 2 shows the characteristics of the parents of 62 respondents. Most of the sample’s fathers were above 30 years (49 people or 79.0%). Meanwhile, 42 fathers had height for > 170 cm (67.7%). Result The majority of the fathers works as a farmer (83.9%) and earned high school education (62.9%). The data showed that most respondents’ maternal age was more than 30 years (35 people or 56.5%). Moreover, Table 2 shows the characteristics of the mothers. The highest height of the mothers was more than 150 cm (64.5%). Most of the mothers graduated from high school (67.7%). Meanwhile, the data on family income showed that 51 people earned more than IDR1,000,000 (82.3%). Table 2 shows the characteristics of the parents of 62 respondents. Most of the sample’s fathers were above 30 years (49 people or 79.0%). Meanwhile, 42 fathers had height for > 170 cm (67.7%). The majority of the fathers works as a farmer (83.9%) and earned high school education (62.9%). The data showed that most respondents’ maternal age was more than 30 years (35 people or 56.5%). Moreover, Table 2 shows the characteristics of the mothers. The highest height of the mothers was more than 150 cm (64.5%). Most of the mothers graduated from high school (67.7%). Meanwhile, the data on family income showed that 51 people earned more than IDR1,000,000 (82.3%). 44 Wati et al. \| Intake of phosphorus and calcium on stunting in toddlers aged 24-59 months Journal of Applied Nutrition and Dietetic Volume 1, Number 1, June 2021 P-ISSN: 2797-7412 Table 2. Characteristics of Respondents’ Parents (n = 60) Fathers’ Age (Years) f % > 30 49 79.0 < 30 13 21.0 Height f % > 170 cm 42 67.7 < 170 cm 20 32.3 Fathers’ Occupation f % Civil servants 2 3.2 Farmers 52 83.9 Fishermen 8 12.9 Fathers’ Education f % Bachelor degree 2 3.2 Senior high schools 39 62.9 Junior high/elementary schools 21 33.9 Mothers’ Age (Years) f % > 30 27 43.5 < 30 35 56.5 Mothers’ Height f % > 150 cm 40 64.5 < 150 cm 22 35.5 Mother’s Education f % Bachelor degree 4 6.5 Senior high schools 42 67.7 Junior high/elementary schools 16 25.8 Family income f % > IDR 1,000,000 51 82.3 < IDR 1,000,000 11 17.7 Table 3 shows that the majority of the respondents (42 people or 67.7%) had less sufficient phosphorus intake. In contrast, only 20 children had sufficient phosphorus intake (32.3%). The data also showed that most of the children had less sufficient calcium intake (46 people or 74.2%). Result While the number of sufficient intake of 16 people (25.8%). Wati et al. \| Intake of phosphorus and calcium on stunting in toddlers aged 24-59 months Journal of Applied Nutrition and Dietetic Volume 1, Number 1, June 2021 P ISSN 2797 7412 Table 3 shows that the majority of the respondents (42 people or 67.7%) had less sufficient phosphorus intake. In contrast, only 20 children had sufficient phosphorus intake (32.3%). The data also showed that most of the children had less sufficient calcium intake (46 people or 74.2%). While the number of sufficient intake of 16 people (25.8%). Table 3. Univariate Analysis (n=60) Phosphorus Intake f % Less sufficient 42 67.7 Sufficient 20 32.3 Calcium Intake f % Less sufficient 46 74.2 Sufficient 16 25.8 Sufficient Sufficient Bivariate Analysis of Calcium Intake on Stunting Incidence Calcium Intake Stunting incident OR (95% CI) P-Value Stunting Not Stunting Total n % n % n % Less sufficient 26 86.7 4 13.3 30 100 35 8.477 - 145.330 0.000 Sufficient 5 15.6 27 84.4 32 100 Total 31 102.3 31 97.7 62 100 Table 5 shows that there were more stunted respondents with there were with less sufficient and sufficient calcium intake (102.3%) than non-stunted children (97.7%). The chi- square test obtained a p-value of 0.000 (<0.05). Moreover, this study found that calcium intake significantly affected the incidence of stunting in under-five children. The results of the analysis showed that the OR value was 35. Table 5 shows that there were more stunted respondents with there were with less sufficient and sufficient calcium intake (102.3%) than non-stunted children (97.7%). The chi- square test obtained a p-value of 0.000 (<0.05). Moreover, this study found that calcium intake significantly affected the incidence of stunting in under-five children. The results of the analysis showed that the OR value was 35. Sufficient Table 4 shows that there were more stunted children with less sufficient of phosphorus (42%) and Calcium intakes (46%) than non-stunted children. The chi-square test obtained p- value = 0.075 (<0.05). Moreover, this research revealed that phosphorus intake significantly affected the incidence of stunting in under-five children. Phosphorus intake is a risk factor for stunting in under-five children. The analysis obtained that the OR value was 2.879. This number interprets that toddlers with low phosphorus intake had a 2.879 more risk of 45 Wati et al. \| Intake of phosphorus and calcium on stunting in toddlers aged 24-59 months Journal of Applied Nutrition and Dietetic Volume 1, Number 1, June 2021 P-ISSN: 2797-7412 experiencing stunting than toddlers with adequate phosphorus intake. P-ISSN: 2797-7412 experiencing stunting than toddlers with adequate phosphorus intake. P ISSN experiencing stunting than toddlers with adequate phosphorus intake. Table 4. Bivariate Analysis of Phosphorus Intake on Stunting Incidence Phosphorus Intake Stunting incident OR (95% CI) P-Value Stunting No Stunting Total n % n % n % Less sufficient 20 66.7 10 33.3 32 100 2.879 1.026 – 8.074 0.075 Sufficient 11 34.4 21 65.6 30 100 Total 31 101.1 31 98.9 62 100 Table 5. Bivariate Analysis of Calcium Intake on Stunting Incidence Calcium Intake Stunting incident OR (95% CI) P-Value Stunting Not Stunting Total n % n % n % Less sufficient 26 86.7 4 13.3 30 100 35 8.477 - 145.330 0.000 Sufficient 5 15.6 27 84.4 32 100 Total 31 102.3 31 97.7 62 100 Table 5 shows that there were more stunted respondents with there were with less sufficient and sufficient calcium intake (102.3%) than non-stunted children (97.7%). The chi- square test obtained a p-value of 0.000 (<0.05). Moreover, this study found that calcium intake significantly affected the incidence of stunting in under-five children. The results of the analysis showed that the OR value was 35. Table 4. Bivariate Analysis of Phosphorus Intake on Stunting Incidence Phosphorus Intake Stunting incident OR (95% CI) P-Value Stunting No Stunting Total n % n % n % Less sufficient 20 66.7 10 33.3 32 100 2.879 1.026 – 8.074 0.075 Sufficient 11 34.4 21 65.6 30 100 Total 31 101.1 31 98.9 62 100 Table 4. Bivariate Analysis of Phosphorus Intake on Stunting Incidence St ti i id t Table 5. Bivariate Analysis of Calcium Intake on Stunting Incidence Table 5. Wati et al. \| Intake of phosphorus and calcium on stunting in toddlers aged 24-59 months Journal of Applied Nutrition and Dietetic Volume 1, Number 1, June 2021 P-ISSN: 2797-7412 method. The results showed that stunted children had significantly lower phosphorus and calcium levels than non-stunted children. This difference occurred because various food sources provide high calcium but low phosphorus. method. The results showed that stunted children had significantly lower phosphorus and calcium levels than non-stunted children. This difference occurred because various food sources provide high calcium but low phosphorus. Conclusion The results of this study indicated that calcium and phosphorus intake influenced the nutritional status, and height of children aged 24-59 months and led to stunting. It is necessary to conduct a nutrition education program to train mothers to fulfill their children’s daily food intake, especially calcium and phosphorus. As a result, stunting can be detected quickly, and intervention can be given immediately. Lack of phosphorus and calcium consumption are the causes of stunting in children. In fact, children’s growth needs to be considered better. Discussion This research shows that toddlers with insufficient calcium intake had 35 times more risk of experiencing stunting than toddlers with sufficient calcium intake. Sudiarmanto A., R. (2020) discovered that 7.4% of the research participants had sufficient calcium intake, while 92.6% had less sufficient intake. An average intake of 336.7 ± 326.2 mg/day caused stunting incidence. Meanwhile, Kusuma H. (2018) revealed that 39 samples were stunted children; 72.2% of them had insufficient calcium intake, and 26% of them had an adequate calcium intake. Calcium intake in infancy is necessary for their growth period. Lack of calcium intake in children can lead to bone fractures that disable them to grow optimally (Goulding et al. in Ferani OA, 2019). Calcium is the main mineral needed in the process of bone formation. Calcium can be found in daily food. Dairy products and calcium-processed products are high sources of calcium. Besides, green vegetables, fish, seafood, and soybeans are good sources of calcium. Adequate calcium intake is required to maintain several physiological functions of the body, especially for bone growth and development. Monitoring children’s growth is substantial because their growth in this stage can affect their growth and health conditions in adulthood and future life. The data on average intake of phosphorus and calcium were collected twice a day using the food recall method and the semi-food frequency questionnaire 46 References Asrar M, Hadi H, Boediman D. 2009. Hubungan pola asuh, pola makan, asupan zat gizi dengan status gizi anak balita masyarakat Suku Nuaulu di Kecamatan Amahai Kabupaten Maluku Tengah Provinsi Maluku. Jurnal Gizi Klinik Indonesia. 6(2):84-94. Asrar M, Hadi H, Boediman D. 2009. Hubungan pola asuh, pola makan, asupan zat gizi dengan status gizi anak balita masyarakat Suku Nuaulu di Kecamatan Amahai Kabupaten Maluku Tengah Provinsi Maluku. Jurnal Gizi Klinik Indonesia. 6(2):84-94. Burckhardt P, Dawson-Hughes B, Weaver C. 2010. Nutritional influences on bone health. New York: Springer. Christiany, I., Hakimi, M. and Sudargo, T. 2009. Status gizi, asupan zat gizi mikro (kalsium, magnesium) hubungannya dengan sindroma premenstruasi pada remaja putri SMU Sejahtera di Surabaya’, Jurnal Gizi Klinik Indonesia, pp. 29–34. doi: 10.22146/IJCN.17685. Depkes RI. 2007. Riset Kesehatan Dasar (RISKESDAS) 2007. Badan Penelitian dan Pengembangan Kesehatan. Departemen Kesehatan RI. Jakarta. Dewey KG dan Begum K. 2011. Long-term Consequences Of Stunting In Early Life. Blackwell Publishing Ltd Maternal and Child Nutrition. NCBI. 2011: Vol (7): 5-18 [diakses tanggal 30 Mei 2014] Available from: http://www.ncbi.nlm.nih.gov Endah Mayang Sari, Mohammad Juffrie, Neti Nurani, Mei Neni Sitaresmi. 2016. Asupan protein, kalsium dan fosfor pada anak stunting dan tidak stunting usia 24-59 bulan April 2016 (152-159) Gibson RS, Manger MS, Krittaphol W, Pongcharoen T, Gowachirapant S, Winichagoon P, et al. 2007. Does zinc deficiency play a role in stunting among primary school children in Thailand. Br J Nutr 2007;97(1):167-75. Goulding. 2004. Children Who Avoid Drinking Cows’s Milk at Increased Risk for Prepubertal Bone Factures. Journal of The American Dietetic Association, 104 (2): 250-3. Kemenkes RI. 2013. Angka Kecukupan Gizi yang Dianjurkan Bagi Bangsa Indonesia. Jakarta. Kemenkes RI. 2013. Regulation on Recommended Dietary Allowance of Indonesia (Angka Kecukupan Gizi). Khairy SAM, Mattar MK, Refaat LAM, El-Sherbeny SA. 2010. Plasma micronutrient levels of stunted Egyptian school age children. Kasr El Aini Med J .16(1). 47 Wati et al. \| Intake of phosphorus and calcium on stunting in toddlers aged 24-59 months Journal of Applied Nutrition and Dietetic Volume 1, Number 1, June 2021 P-ISSN: 2797-7412 Larson, L. M., Martorell, R., & Bauer, P. J. 2018. A Path Analysis of Nutrition, Stimulation, and Child Development Among Young Children in Bihar, India. Child Development, 89(5), 1871–1886. doi:10.1111/cdev.13057. Li j, Yuan J, Guo Y, Sun Q, Hu X. 2012. The influence of dietary calcium and phosphorus imbalance on intestinal NaPi-IIb and Calbindin mRNA Expression and tibia parameters of broilers. References Asian-Aust J Anim 2012;25(4):552-8. Mikhail WZA, Sabhy HM, El-sayed HH, Khairy SA, Salem HYHA, Samy MA. 2013. Effect of nutritional status on growth pattern of stunted preschool children in Egypt. Acad J Nutr 2013;2(1):1-9. Peacock M. 2010. Calcium metabolism in health and disease. Clin J Am Soc Nephrol 2010;5(Suppl 1):S23-30. Prentice A, Bates CJ. 1993. An appraisal of the adequacy of dietary mineral intakes in developing countries for bone growth and development in children. Nutr Res Rev 1993;6(1):51-69. Puspita Y. 2014. Hubungan riwayat penyakit infeksi saluran pernafasan akut dengan kejadian stunting pada anak balita di Kabupaten Rejang Lebong Provinsi Bengkulu [Tesis]. Yogyakarta: Universitas Gadjah Mada; 2014 Sudiarmanto AR, dan Sri Sumarmi. 2020. Hubungan Asupan Kalsium dan Zink dengan Kejadian Stunting Pada Siswi SMP Unggulan Bina Insani Surabaya Media Gizi & Kesehatan Masyarakat. WHO. 2010. Nutrition Landscape Information System (NLIS) country Profile Indicators: Interpretation Guide. Swizarland: WHO press ; (2010). Physical status: the use and interpretation of anthropometry. Geneva: WHO Press. WHO. 2019. WHO Child Growth Standars. Geneva: WHO Press. 48
https://openalex.org/W2170488268	https://bmcgenomics.biomedcentral.com/counter/pdf/10.1186/1471-2164-13-708	English	null	Identification of 3′ gene ends using transcriptional and genomic conservation across vertebrates	BMC genomics	2,012	cc-by	12,544	METHODOLOGY ARTICLE Open Access Morgan et al. BMC Genomics 2012, 13:708 http://www.biomedcentral.com/1471-2164/13/708 © 2012 Morgan et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Keywords: pre-mRNA cleavage site, PhyloP, RNA sequencing, TransMap Keywords: pre-mRNA cleavage site, PhyloP, RNA sequencing, TransMap Keywords: pre-mRNA cleavage site, PhyloP, RNA sequencing, TransMap Marcos Morgan1,2, Alessandra Iaconcig1 and Andrés Fernando Muro1 Marcos Morgan1,2, Alessandra Iaconcig1 and Andrés Fernando Muro1 * Correspondence: marcos.morgan@embl.it; muro@icgeb.org 1International Centre for Genetic Engineering and Biotechnology (ICGEB), Padriciano 99, I 34149, Trieste, Italy 2Present address: EMBL Monterotondo Adriano Buzzati-Traverso Campus, Via Ramarini 32, 00015, Monterotondo, Italy Abstract Background: In higher eukaryotes, gene expression is regulated at different levels. In particular, 30UTRs play a central role in translation, stability and subcellular localization of transcripts. In recent years, the development of high throughput sequencing techniques has facilitated the acquisition of transcriptional data at a genome wide level. However, annotation of the 30 ends of genes is still incomplete, thus limiting the interpretation of the data generated. For example, we have previously reported two different genes, ADD2 and CPEB3, with conserved 30UTR alternative isoforms not annotated in the current versions of Ensembl and RefSeq human databases. Results: In order to evaluate the existence of other conserved 30 ends not annotated in these databases we have now used comparative genomics and transcriptomics across several vertebrate species. In general, we have observed that 30UTR conservation is lost after the end of the mature transcript. Using this change in conservation before and after the 30 end of the mature transcripts we have shown that many conserved ends were still not annotated. In addition, we used orthologous transcripts to predict 30UTR extensions and validated these predictions using total RNA sequencing data. Finally, we used this method to identify not annotated 30 ends in rats and dogs. As a result, we report several hundred novel 30UTR extensions in rats and a few thousand in dogs. Conclusions: The methods presented here can efficiently facilitate the identification of not-yet-annotated conserved 30UTR extensions. The application of these methods will increase the confidence of orthologous gene models across vertebrates. Background thousands of not annotated exons or exon extensions [4,5] limiting the interpretation of the data generated. Gene expression in higher eukaryotes is a complex phenomenon which is regulated at different levels. In particular, regulatory sequences present in the messen- ger RNAs (mRNAs) can determine their localization, stability and translational activity. Recent developments in high throughput sequencing have greatly facilitated the acquisition of transcriptional data [1-3]. Nowadays, it is possible to obtain the transcriptome landscape of a tissue or cell line in a few days at a relatively low cost. However, recent results obtained using total RNA se- quencing from humans have shown that there are still Genome annotation is a rather complex process that can be facilitated with computer-based methods but ul- timately requires extensive manual curation. In order to address this challenging problem, different consortia are trying to generate reference databases of different spe- cies transcriptomes. Two of the most popular and rather independent databases currently available for the human genome are the RefSeq and the Ensembl gene models [6,7]. These databases consider all types of long tran- scripts, including non-coding RNAs. They are of high quality as they have been extensively curated in the past and are periodically updated. In addition, they are freely available to the scientific community through accessible web interfaces. * Correspondence: marcos.morgan@embl.it; muro@icgeb.org 1International Centre for Genetic Engineering and Biotechnology (ICGEB), Padriciano 99, I 34149, Trieste, Italy 2Present address: EMBL Monterotondo Adriano Buzzati-Traverso Campus, Via Ramarini 32, 00015, Monterotondo, Italy Page 2 of 13 Morgan et al. BMC Genomics 2012, 13:708 http://www.biomedcentral.com/1471-2164/13/708 Morgan et al. BMC Genomics 2012, 13:708 http://www.biomedcentral.com/1471-2164/13/708 One of the big hurdles in higher eukaryotes genome annotation is that genes generally encode for more than one transcript isoform. For example, the same gene can be transcribed from different promoters, which can be tissue-specific or activated during different phases of development. In addition, exons can be alternatively spliced to generate different mature isoforms. A third way in which different mature transcripts can originate from the same locus is by alternative usage of cleavage and polyadenylation sites, referred hereafter as polyade- nylation sites (PAS). The PAS demarcates the end of the mature transcript and its usage is strongly determined by the presence of a polyA signal (hexanucleotide motif, HexM) and a Downstream Sequence Element (DSE) [8]. The canonical HexM is AAUAAA, but other variants such as AUUAAA are frequently used [9,10]. Background This hexa- nucleotide motif is located 15–25 nucleotides upstream of the end of the mature transcript whilst the DSE is a G/GU rich region located approximately 25 nucleotides downstream of the end of the mature transcripts. These signals are recognized by a well-described protein com- plex that cleaves the transcript precursor [11]. The cleavage is followed by the addition of a polyA tail at the free 30 end. isoform. This brain-specific site is still not annotated in databases although it corresponds to the most abundant ADD2 mRNA species in brain, is highly expressed in mice and humans, and is conserved across vertebrates. The sec- ond case corresponds to the CPEB3 transcript [16]. This gene is expressed in different tissues where usually a prox- imal PAS is used [17]. In brain, however, a more distal PAS is also used, that is abundantly expressed and extremely conserved. Again, this distal, brain specific transcript is still not annotated in the current databases. Therefore, we believe that a more complete annotation of PASs is needed. To this end, we decided to look for evidence of other potential PASs that might not be annotated in the current versions of RefSeq and Ensembl gene predictions. In particular, we concentrated on puta- tive evolutionarily conserved sites, similar to the ones that we previously described for the ADD2 and CPEB3 transcripts, as a strong selective pressure may reflect relevant biological functions. Using these criteria and combining them with transcriptional information from human Expressed Sequence Tags (ESTs), we identified several highly conserved, still not annotated PASs in these databases. We also used a complementary ap- proach in which we evaluated putative PAS used in other species, but not necessarily highly conserved at the genomic level. In this case, we used deep sequencing data from total RNA of eight different human tissues as transcriptional evidence. Finally, we applied our method to identify novel 30 ends in rats and dogs. As we identi- fied hundreds of conserved not annotated PASs in these species we propose that our method can be used to im- prove the annotation of any mammalian genome. Although the mechanisms that determine the pro- moter usage sites and alternative splice sites have been extensively studied much less is known about the alter- native selection of PASs. Nevertheless, in recent years, alternative PAS usage was shown to be very common and tightly regulated during development [12,13]. Background Im- portantly, the selection of a particular PAS determines the length of the 30 UnTranslated Regions (30UTRs) of genes encoding for proteins and, consequently, the regu- latory elements present in these sequences. In practice, the use of a PAS proximal to the stop codon generally gives rise to a short 30UTR transcript whilst use of a more distal PAS generates a longer 30UTR. The func- tional consequences of PAS-site choice is that distinct regulatory elements will be exclusively present in the longer isoform of the transcript as opposed to the shorter isoform. During the last decade it has become evident that the 30UTRs of genes play a central role in gene regulation as they are the natural targets of miR- NAs [14]. Genomic conservation decreases after the polyadenylation site (PAS) It is well known that 30UTRs can be highly conserved [18]. However, we have previously observed that this conservation resulted to be lost immediately down- stream of the PAS for the CPEB transcripts [16] with just one exception. In CPEB3, in fact, we observed an is- land of conservation approximately 2 Kb downstream of the annotated PAS. Interestingly, the end of this island coincided with the end of a cluster of ESTs suggesting the existence of a novel PASs, that we later validated through different biochemical approaches. Similarly to CPEBs, we also observed an important drop in conserva- tion after the distal PAS of the ADD2 gene (data not shown), which was associated with a cluster of ESTs [15]. In this work, therefore, we thought that we could use a similar rationale to detect other not annotated dis- tal PASs at a genome-wide level. We previously reported two instances in which alterna- tive 30 ends of genes were not annotated in the available human genome databases. The first case corresponds to the ADD2 gene [15]. This gene is mainly expressed in erythroid tissues and in the brain. In erythroid tissues, a specific pro- moter is used together with proximal PASs. On the other hand, in brain a different tissue specific promoter is used and a more distal PAS is recognized by the cleavage and polyadenylation machinery. As a result, the brain-specific ADD2 transcript is 5–6 Kb longer than the erythropoietic To estimate the genomic conservation in regions asso- ciated with the transcripts’ extremes, we used PhyloP Morgan et al. BMC Genomics 2012, 13:708 http://www.biomedcentral.com/1471-2164/13/708 Page 3 of 13 scores calculated for 44 vertebrates [19]. The PhyloP scores consider the conservation at the nucleotide level without taking into account the genomic context. To de- termine changes in conservation at the ends of each transcript we calculated the average PhyloP value of the 50 nucleotides present at the 30 (or 50) end of the mature transcript (“T”, see Figure 1A). We then calculated the average PhyloP value of a 50 nucleotide-long interval starting 50 nucleotides from the extreme of the mature transcript (“NT”, see Figure 1A). Finally, we defined a conservation drop index (CDI) as the difference between the average PhyloP values of the “T” region minus the average of the “NT” region. Genomic conservation decreases after the polyadenylation site (PAS) We considered 50 nucleotides upstream of the PAS in order to define an interval containing the well-conserved polyA signal to- wards the middle and then we also included a 50- nucleotide gap to account for the DSE. Therefore, a positive CDI value for the 30 end indicates that the gen- omic region encoding the end of the mature transcript is more conserved than the adjacent downstream se- quence. Instead, a CDI value close to 0 indicates that there are small differences in conservation between the mature transcript end and the adjacent downstream gen- omic sequence. To determine whether the drop in conservation after the PAS was a general phenomenon, we first tested this hypothesis with the annotated genes present in the RefSeq database. To this end, we calculated the CDI for all the PASs annotated in RefSeq. As shown in Figure 1B, PASs had a positive CDI on average and the population was asymmetrically distributed towards posi- tive values. This result indicated that many annotated PASs had an important drop in conservation like that observed for the CPEB genes [16]. In contrast, the distri- bution of transcriptional start sites (TSSs) did not have an extended tail towards the positive values (Figure 1B). This showed that the sharp drop in conservation observed in the 30UTR was rarer at these ends of the transcript. Therefore, we postulated that this difference in conservation before and after the end of the mature transcript could be used to identify not annotated PASs but not TSSs. Figure 1 Genomic conservation falls after the PAS. Panel A. PhyloP conservation values for the genomic region spanning the first exon and first intron of a RefSeq annotated gene (GAPDH) (upper panel) and the last intron and the last exon of the same gene (lower panel). The windows were adapted from the UCSC genome browser [21]. TSS, Transcriptional Start Site; ATG, start codon; stop, stop codon; PAS, polyadenylation site; T, transcribed interval encoding for the mature transcript; NT, interval not encoding for the mature transcript; CDI, Conservation Drop Index. The black arrows indicate the direction of transcription of the gene. Panel B. Absolute frequency of TSS and PAS annotated in the RefSeq database according to their CDI. Genomic conservation sharply decreases in hundreds of putative PASs Next, to determine the presence of distal conserved PASs still not annotated in the RefSeq or Ensembl data- bases, we clustered ESTs finishing at the same position and assigned a CDI to each of them (Figure 2A). We refer to these clusters as Potential mature Transcript Extremes (PTEs). In addition, we classified each of these PTEs in putative TSSs or putative PASs according to the orientation of the most proximal reference gene (see Figure 2B, Materials and Methods Section, and Additional file 1: Figure S1). We did this analysis separ- ately for the RefSeq and Ensembl gene predictions. We observed that, like for the annotated RefSeq genes, the putative PASs showed an asymmetric distribution to- wards high CDIs while this was less evident for TSSs (Figure 2C and D). In this way, the distribution of the putative PASs and TSSs resembled the distribution of the annotated ones, suggesting that many PTEs may cor- respond to real transcript extremes. Figure 1 Genomic conservation falls after the PAS. Panel A. Figure 1 Genomic conservation falls after the PAS. Panel A. PhyloP conservation values for the genomic region spanning the Figure 1 Genomic conservation falls after the PAS. Panel A. PhyloP conservation values for the genomic region spanning the first exon and first intron of a RefSeq annotated gene (GAPDH) (upper panel) and the last intron and the last exon of the same gene (lower panel). The windows were adapted from the UCSC genome browser [21]. TSS, Transcriptional Start Site; ATG, start codon; stop, stop codon; PAS, polyadenylation site; T, transcribed interval encoding for the mature transcript; NT, interval not encoding for the mature transcript; CDI, Conservation Drop Index. The black arrows indicate the direction of transcription of the gene. Panel B. Absolute frequency of TSS and PAS annotated in the RefSeq database according to their CDI. Figure 1 Genomic conservation falls after the PAS. Panel A. PhyloP conservation values for the genomic region spanning the first exon and first intron of a RefSeq annotated gene (GAPDH) (upper panel) and the last intron and the last exon of the same gene (lower panel). The windows were adapted from the UCSC genome browser [21]. TSS, Transcriptional Start Site; ATG, start codon; stop, stop codon; PAS, polyadenylation site; T, transcribed interval encoding for the mature transcript; NT, interval not encoding for the mature transcript; CDI, Conservation Drop Index. Genomic conservation sharply decreases in hundreds of putative PASs Panel B. A PTE in a wider genomic context. The position of the PTE is indicated with a black arrowhead. The last two exons (and the last intron) of the most proximal RefSeq and Ensembl transcripts are shown above. Their direction of transcription is indicated by the black arrow. The positions of the annotated PASs are indicated with open arrowheads. The PTE was classified as a putative PAS for both RefSeq and Ensembl databases because the proximal reference transcripts are transcribed towards the PTE. Below a TransMap transcript that ends in the same position as the PTE is shown (Trans). In this case, the Trans transcript is supporting the PTE. On the bottom, the genomic region covered by total RNA deep sequencing reads (RNA seq). Panel C. Absolute frequency of PTEs, either putative PASs or putative TSS, according to their CDI. The PTEs were obtained using the RefSeq database as reference. Panel D. As in Panel C but using the Ensembl database as reference. PTEs with CDI in between −0.5 and +0.5 were omitted from the graphs shown in Panels C and D. Figure 2 Hundreds of putative PASs show a high CDI. Panel A. PhyloP scores for the genomic region spanning the end of an EST cluster that originated a PTE (black arrowhead). The intervals used to calculate the PTE CDI are indicated below. T, transcribed interval encoding for the mature transcript; NT, interval not encoding for the mature transcript. Panel B. A PTE in a wider genomic context. The position of the PTE is indicated with a black arrowhead. The last two exons (and the last intron) of the most proximal RefSeq and Ensembl transcripts are shown above. Their direction of transcription is indicated by the black arrow. The positions of the annotated PASs are indicated with open arrowheads. The PTE was classified as a putative PAS for both RefSeq and Ensembl databases because the proximal reference transcripts are transcribed towards the PTE. Below a TransMap transcript that ends in the same position as the PTE is shown (Trans). In this case, the Trans transcript is supporting the PTE. On the bottom, the genomic region covered by total RNA deep sequencing reads (RNA seq). Panel C. Absolute frequency of PTEs, either putative PASs or putative TSS, according to their CDI. The PTEs were obtained using the RefSeq database as reference. Panel D. Genomic conservation sharply decreases in hundreds of putative PASs The black arrows indicate the direction of transcription of the gene. Panel B. Absolute frequency of TSS and PAS annotated in the RefSeq database according to their CDI. Determination of the methods’ False Discovery Rate (FDR) In order to evaluate the method, we then estimated the FDR at different CDI cutoffs. To determine whether a Morgan et al. BMC Genomics 2012, 13:708 http://www.biomedcentral.com/1471-2164/13/708 Morgan et al. BMC Genomics 2012, 13:708 Page 4 of 13 http://www.biomedcentral.com/1471-2164/13/708 Figure 2 Hundreds of putative PASs show a high CDI. Panel A. PhyloP scores for the genomic region spanning the end of an EST cluster that originated a PTE (black arrowhead). The intervals used to calculate the PTE CDI are indicated below. T, transcribed interval encoding for the mature transcript; NT, interval not encoding for the mature transcript. Panel B. A PTE in a wider genomic context. The position of the PTE is indicated with a black arrowhead. The last two exons (and the last intron) of the most proximal RefSeq and Ensembl transcripts are shown above. Their direction of transcription is indicated by the black arrow. The positions of the annotated PASs are indicated with open arrowheads. The PTE was classified as a putative PAS for both RefSeq and Ensembl databases because the proximal reference transcripts are transcribed towards the PTE. Below a TransMap transcript that ends in the same position as the PTE is shown (Trans). In this case, the Trans transcript is supporting the PTE. On the bottom, the genomic region covered by total RNA deep sequencing reads (RNA seq). Panel C. Absolute frequency of PTEs, either putative PASs or putative TSS, according to their CDI. The PTEs were obtained using the RefSeq database as reference. Panel D. As in Panel C but using the Ensembl database as reference. PTEs with CDI in between −0.5 and +0.5 were omitted from the graphs shown in Panels C and D. Figure 2 Hundreds of putative PASs show a high CDI Panel A PhyloP scores for the genomic region spanning the end of an EST cluster that Figure 2 Hundreds of putative PASs show a high CDI. Panel A. PhyloP scores for the genomic region spanning the end of an EST cluster that originated a PTE (black arrowhead). The intervals used to calculate the PTE CDI are indicated below. T, transcribed interval encoding for the mature transcript; NT, interval not encoding for the mature transcript. Genomic conservation sharply decreases in hundreds of putative PASs As in Panel C but using the Ensembl database as reference. PTEs with CDI in between −0.5 and +0.5 were omitted from the graphs shown in Panels C and D. way, for every PTE we obtained an associated FDR as shown in Figure 3. For both Ensembl and RefSeq data- bases, we observed that the higher the CDI the lower the FDR. The FDR values for different cutoffs are similar for both databases but the total number of not anno- tated PASs is higher for the RefSeq database. For ex- ample, the FDRs for a CDI of 1.25 are of 0.483 and 0.466 for the Ensembl and RefSeq databases respectively while the numbers of PTEs with a CDI higher than 1.25 are 227 and 416. These results show that conservation signatures can be use to facilitate the identification of not annotated PASs. PTE was a true positive, we looked for transcripts on other databases with the predicted end. For example, we looked if the predicted ends not annotated in the RefSeq database were already annotated in the Ensembl data- base and vice versa. Since predicted ends with high CDIs are highly con- served, we reasoned that orthologous ends are used in other species. Therefore, we also considered other spe- cies transcripts to estimate the FDR. In particular, we examined orthologous transcripts mapped with the TransMap program to the human genome [20]. To assess the FDR for a given CDI cutoff, we consid- ered all PTEs with an equal or higher CDI. Next, for each of these predicted PASs, we assessed if they were represented or not on the above mentioned databases. The FDR was estimated as the number of PTEs not represented on other databases divided by the total number of PTEs. Given that Ensembl annotation is more exhaustive than RefSeq, as it had a lower number of not annotated PASs, we used this database as reference for an exhaustive man- ual annotation. In order to confirm the predicted sites used in other species we performed a case-by-case exam- ination of the putative PASs with CDI higher than 0.75, that were supported by an orthologous transcript (Trans- Map), an orthologous RefSeq transcript (Trans RefSeq), or Given that every PTE has an associated CDI, we calcu- lated the FDR using each of these CDIs as cutoffs. In this Morgan et al. Genomic conservation sharply decreases in hundreds of putative PASs The PTE with the highest CDI is on the first position of the scale, the PTE with the second highest CDI is on the second position, and so on until the last position with the PTE with lowest CDI. Note that the spacing between contiguous PTEs is constant independently of the absolute difference of their CDI. The FDRs obtained using as cutoffs CDIs of 1, 1.25 and 1.75 are indicated. The numbers of PTEs with a CDI equal or higher than the cutoff used are shown between brackets. Panel B. As in A using the RefSeq database as reference to obtain the PTEs. Figure 3 PTEs with high CDI are more likely to be real PASs. Panel A. FDR values calculated using each of the PTEs also not be correctly annotated. For this reason, in order to validate the usage of the PASs identified so far and to look for other not annotated ones, we designed a differ- ent approach using as transcriptional evidence deep se- quencing reads, 32 nucleotide long, from human RNA produced by Wang and collaborators [2]. The strategy was based on the identification of orthologous tran- scripts with gene structures similar to those of the anno- tated genes but with longer 30UTRs (see Figure 4A). Using these extensions as predictions, we calculated the read coverage percentage of the genomic interval span- ning from the annotated PAS to the putative PAS (shown as “Up” in Figure 4A). We also calculated the read coverage percentage of an interval of the same length immediately downstream of the putative PAS (shown as “Down” in Figure 4A). We then defined the coverage difference (CD) as the difference between the reads coverage of the 30UTR extension (Up) and the reads coverage of the interval immediately downstream (Down). Therefore, a CD value of 100 indicates that the proposed 30UTR extension is completely covered by reads and no reads are found in an interval of the same length immediately downstream as expected in the pres- ence of a strong PAS. Instead, a CD value of 0 indicates that there are no differences in the transcriptional levels upstream and downstream the putative PAS. a RefSeq transcript. Genomic conservation sharply decreases in hundreds of putative PASs To this end, we inspected the genomic context of the putative PASs using the graphical interface of the UCSC genome browser [21] and we confirmed that most of these sites appear to be functional, including the ADD2 brain specific isoform (Additional file 2: Table S1) previously described [15]. We also found ~49 PTEs with CDI higher than 1 but without support from TransMap, Trans RefSeq or RefSeq transcripts, that could be functional PASs as well, includ- ing the previously characterized CPEB3 distal PAS [16]. On Additional file 3: Figure S2 we show examples of two of these not supported PTEs downstream of the annotated PASs of the RNF130 and RNF150 genes that could corres- pond to not annotated PASs. Taken together, these obser- vations indicate that the above-calculated FDRs are overestimated. The PhyloP conservation scores, used to calculate the CDI values, were calculated using 44 vertebrates’ genomes. These conservation scores, however, might be too stringent to detect PASs specific to a restricted group of vertebrates such as the mammals. In order to identify not annotated mammalian specific PASs, we repeated the analysis using PhyloP scores calculated across different mammals. We found a strong correlation between the vertebrate and mammalian specific CDIs (Additional file 4: Figure S3). However, we failed to detect any clear population of mammalian-specific PASs. Therefore, we conclude that the method detects mainly ancestral vertebrates 30 ends. Using this approach, we detected several other not annotated PASs (See Additional file 5: Table S2 for a manually curated list of the sites identified, with CD higher than 50%, not annotated in the Ensembl data- base). We also calculated the FDR of the method as described before. The FDR increases as the CD decreases (Figure 4B and C) indicating that the method Genomic conservation sharply decreases in hundreds of putative PASs BMC Genomics 2012, 13:708 http://www.biomedcentral.com/1471-2164/13/708 Page 5 of 13 Figure 3 PTEs with high CDI are more likely to be real PASs. Panel A. FDR values calculated using each of the PTEs CDI as a cutoff. The PTEs were obtained using the Ensembl database as reference and only putative PASs were considered. PTEs were ranked according to their CDI. The PTE with the highest CDI is on the first position of the scale, the PTE with the second highest CDI is on the second position, and so on until the last position with the PTE with lowest CDI. Note that the spacing between contiguous PTEs is constant independently of the absolute difference of their CDI. The FDRs obtained using as cutoffs CDIs of 1, 1.25 and 1.75 are indicated. The numbers of PTEs with a CDI equal or higher than the cutoff used are shown between brackets. Panel B. As in A using the RefSeq database as reference to obtain the PTEs. Figure 3 PTEs with high CDI are more likely to be real PASs. Panel A. FDR values calculated using each of the PTEs CDI as a cutoff. The PTEs were obtained using the Ensembl database as reference and only putative PASs were considered. PTEs were ranked according to their CDI. The PTE with the highest CDI is on the first position of the scale, the PTE with the second highest CDI is on the second position, and so on until the last position with the PTE with lowest CDI. Note that the spacing between contiguous PTEs is constant independently of the absolute difference of their CDI. The FDRs obtained using as cutoffs CDIs of 1, 1.25 and 1.75 are indicated. The numbers of PTEs with a CDI equal or higher than the cutoff used are shown between brackets. Panel B. As in A using the RefSeq database as reference to obtain the PTEs. Figure 3 PTEs with high CDI are more likely to be real PASs. Panel A. FDR values calculated using each of the PTEs CDI as a cutoff. The PTEs were obtained using the Ensembl database as reference and only putative PASs were considered. PTEs were ranked according to their CDI. Identification of not annotated PASs using orthologous transcripts The one with the highest CD is on the first position of the scale, the putative PASs with the second highest CD is on the second position, and so on until the last position with the putative PASs with lowest CD. The FDRs obtained using as cutoffs CDs of 95, 80 and 50 are indicated. The numbers of putative PASs with a CD equal or higher than the cutoff used are shown between brackets. Panel C. As in B using the RefSeq database as reference to obtain the putative PASs. Figure 4 Identification of PASs using orthologous transcripts and total RNA sequencing data. Panel A. Genomic context of a putative PAS associated with a TransMap transcript (Trans). The last two exons (and the last intron) of the Ensembl prediction that could have a larger associated isoform using the putative PAS are shown above (Ensembl). The TransMap transcript and total RNA Seq coverage for the interval is shown below. The intervals used to calculate the Coverage Difference (CD) and the applied formula are indicated. Up, upstream interval; Down, downstream interval. Panel B. FDR values calculated using each of the putative PASs CD as a cutoff. The putative PASs were obtained using the Ensembl database as reference and were ranked according to their CD. The one with the highest CD is on the first position of the scale, the putative PASs with the second highest CD is on the second position, and so on until the last position with the putative PASs with lowest CD. The FDRs obtained using as cutoffs CDs of 95, 80 and 50 are indicated. The numbers of putative PASs with a CD equal or higher than the cutoff used are shown between brackets. Panel C. As in B using the RefSeq database as reference to obtain the putative PASs. Figure 4 Identification of PASs using orthologous transcripts and total RNA sequencing data. Panel A. Genomic c ASs using orthologous transcripts and total RNA sequencing data. Panel A. Genomic context of a putative PAS for examples). To evaluate the performance of the method in this context, we calculated the FDR as we previously did while analyzing the human databases. As shown in Figures 5A and B, the FDR decreased for higher CD cutoffs, thus validating the use of this method on other species. can facilitate the identification of not annotated PASs. Identification of not annotated PASs using orthologous transcripts We also confirmed with this approach that the Ensembl predictions are more complete than the RefSeq models. For example, the number of potential new PASs, with a CD higher than 50, was of 106 and 190 for Ensembl and RefSeq databases with FDR values of 0.288 and 0.215, re- spectively (Figure 4B and C). Next we investigated the RefSeq and Ensembl annota- tions of the dog genome. Using our method, we found a few thousand new potential PASs for the Ensembl models (Additional file 8: Table S4A and see Additional file 9: Figure S5 for examples). Instead, we identified very few new putative 30ends for the RefSeq database in compari- son (Additional file 8: Table S4B). The differences, in this case, can be explained by the limited number of gene models present on the RefSeq databases and not by a more exhaustive annotation of the RefSeq models (data not shown). Nevertheless, the FDR values again decreased for higher CD cutoffs (Figure 5C and D), showing that the method can be used even at early stages of the annotation process. In recent months we observed a major effort to improve the Ensembl models for dogs. To test whether our predictions were in agreement with the new models, we compared our proposed list of proposed gene ends Identification of not annotated PASs using orthologous transcripts Next, we thought that several other conserved tran- scripts with not necessarily high CDI (CDI ≤0.75), might Morgan et al. BMC Genomics 2012, 13:708 http://www.biomedcentral.com/1471-2164/13/708 Page 6 of 13 Figure 4 Identification of PASs using orthologous transcripts and total RNA sequencing data. Panel A. Genomic context of a putative PAS associated with a TransMap transcript (Trans). The last two exons (and the last intron) of the Ensembl prediction that could have a larger associated isoform using the putative PAS are shown above (Ensembl). The TransMap transcript and total RNA Seq coverage for the interval is shown below. The intervals used to calculate the Coverage Difference (CD) and the applied formula are indicated. Up, upstream interval; Down, downstream interval. Panel B. FDR values calculated using each of the putative PASs CD as a cutoff. The putative PASs were obtained using the Ensembl database as reference and were ranked according to their CD. The one with the highest CD is on the first position of the scale, the putative PASs with the second highest CD is on the second position, and so on until the last position with the putative PASs with lowest CD. The FDRs obtained using as cutoffs CDs of 95, 80 and 50 are indicated. The numbers of putative PASs with a CD equal or higher than the cutoff used are shown between brackets. Panel C. As in B using the RefSeq database as reference to obtain the putative PASs. Figure 4 Identification of PASs using orthologous transcripts and total RNA sequencing data. Panel A. Genomic context of a putative PAS associated with a TransMap transcript (Trans). The last two exons (and the last intron) of the Ensembl prediction that could have a larger associated isoform using the putative PAS are shown above (Ensembl). The TransMap transcript and total RNA Seq coverage for the interval is shown below. The intervals used to calculate the Coverage Difference (CD) and the applied formula are indicated. Up, upstream interval; Down, downstream interval. Panel B. FDR values calculated using each of the putative PASs CD as a cutoff. The putative PASs were obtained using the Ensembl database as reference and were ranked according to their CD. Extension of the method to other species The development of deep sequencing techniques made possible to obtain a global and unbiased picture of the whole transcriptome with minimum effort and at a rela- tively low cost. In this way, new RNA-seq datasets from different organisms are being deposited every month in public repositories. Therefore, we envisioned that we could apply the previous method using the new informa- tion available to improve the annotation of other species. To this end, we analyzed different RNA-seq libraries from dogs and rats (see Materials and Methods Section). We first investigated the Ensembl and RefSeq models for rats. We found several hundred genes with potential gene extensions for both databases (Additional file 6: Tables S3A and S3B and see Additional file 7: Figure S4 We first investigated the Ensembl and RefSeq models for rats. We found several hundred genes with potential gene extensions for both databases (Additional file 6: Tables S3A and S3B and see Additional file 7: Figure S4 Morgan et al. BMC Genomics 2012, 13:708 http://www.biomedcentral.com/1471-2164/13/708 Morgan et al. BMC Genomics 2012, 13:708 Page 7 of 13 Page 7 of 13 Figure 5 Identification of PASs in rats and dogs using orthologous transcripts and total RNA sequencing data. FDR values calculated using each of the putative PASs CD as a cutoff are shown as in Figure 4. The putative PASs were obtained using the Ensembl (Panels A and C) and RefSeq (Panels B and D) databases from rats (Panels A and B) and dogs (Panels C and D). Figure 5 Identification of PASs in rats and dogs using orthologous transcripts and total RNA sequencing data. FDR values calculated using each of the putative PASs CD as a cutoff are shown as in Figure 4. The putative PASs were obtained using the Ensembl (Panels A and C) and RefSeq (Panels B and D) databases from rats (Panels A and B) and dogs (Panels C and D). with the PASs of the new transcripts. We observed that more than 500 of our predictions were incorporated into the new models (Additional file 8: Table S4A). This obser- vation indicates that a significant proportion of our predic- tions are in agreement with the recently proposed models. generated by alternative cleavage and polyadenylation. In parallel, we chose these genes because we noticed that they have an extensive coverage of deep sequencing reads obtained from brain RNA (Figures 6A and B). Extension of the method to other species In addition, to investigate the tissue specificity of the genes, we used total RNA samples from brain, testes and heart. Validation of PTEs by Northern blot analysis Although the ann cies showed shorter 30UT coming from RNA-Seq the long isoforms (Add Additional file 11: Figure In conclusion, these ob the existence of long RN region linking the annota In addition, these results species are abundantly ex compared with the anno this observation, the kcn to the 30 end of the singl Similarly, the two RefSeq support the predicted kcn Discussion After the completion of t annotation of the whole s lenge. Most of the initial notation of the coding s Figure 6 Analyses of two mo transcripts, Kcnb1 and Kcnq3 Genomic context of a PTE dow interval shown goes from base chromosome 8 of the human The PTE position is indicated w arrow indicates KCNQ3 directio annotated PAS is indicated wit bottom, the genomic region c reads (RNA seq) from brain (B), Panel A, but for a PTE downstr shown goes from base 47,978, chromosome 20. Panel C. Nort different tissues were tested. B bottom of each panel, the 28S control. The molecular weight expected size of the transcript ~3.7 Kb and the expected size predicted distal PAS is of ~11.2 size of the transcript that uses the expected size of the transc is of ~11.3 Kb. Figure 6 Analyses of two mouse potassium channels transcripts, Kcnb1 and Kcnq3, with putative distal PASs. Panel A. Genomic context of a PTE downstream of the KCNQ3 gene. The interval shown goes from base 133,131,500 to base 133,145,500 of chromosome 8 of the human reference sequence (GRCh37/hg19). The PTE position is indicated with a black arrowhead. The black arrow indicates KCNQ3 direction of transcription. The position of the annotated PAS is indicated with an open arrowhead. On the bottom, the genomic region covered by total RNA deep sequencing reads (RNA seq) from brain (B), testes (T) or heart (H). Panel B. As in Panel A, but for a PTE downstream of the KCNB1 gene. The interval shown goes from base 47,978,000 to base 47,993,000 of chromosome 20. Panel C. Northern blot analysis of total RNA. Three different tissues were tested. B, brain; T, testes; H, heart. On the bottom of each panel, the 28S ribosomal RNA is shown as a loading control. The molecular weight is indicated. Validation of PTEs by Northern blot analysis For Kcnb1 mRNA, the expected size of the transcript that uses the annotated PAS is of ~3.7 Kb and the expected size of the transcript that uses the predicted distal PAS is of ~11.2 Kb. For Kcnq3 mRNA, the expected size of the transcript that uses the annotated PAS is of ~2.9 Kb and the expected size of the transcript that uses the predicted distal PAS is of ~11.3 Kb. correspond to 30UTR extensions of the upstream tran- scripts but to novel large intervening non-coding RNAs (lincRNAs). This is the case for example of the PTE downstream the KCNB1 gene. Our experimental data (Figure 6) favor a model in which the PTE corresponds to the end of a 30UTR extension of the KCNB1 gene, however, both models are not mutually exclusive. In order to gain further evidence for the evolutionary conservation of the KCNB1 and KCNQ3 extended 30UTRs, we investigated whether these isoforms were present in the corresponding rats and dogs ortholog genes. Although the annotated homologs in these spe- cies showed shorter 30UTRs, the transcriptional evidence coming from RNA-Seq data supports the existence of the long isoforms (Additional file 10: Figure S6 and Additional file 11: Figure S7). In conclusion, these observations provide evidence for the existence of long RNA species spanning the 30UTR region linking the annotated transcripts with the PTEs. In addition, these results show that these not annotated species are abundantly expressed in the inspected tissues compared with the annotated ones. In agreement with this observation, the kcnb1 predicted PAS corresponds to the 30 end of the single kcnb1 RefSeq model in mice. Similarly, the two RefSeq models of the gene in humans support the predicted kcnq3 PAS. Validation of PTEs by Northern blot analysis As shown in Figure 6C, we observed for both genes a high molecular weight band of more than 10 Kbs con- sistent with the use of the not annotated PASs. As expected, we also observed high expression in brain but not in other tissues. Interestingly, the high molecular weight band was one of the main species observed in both cases. Similar results were previously reported by other groups for KCNQ3 in mice and humans [22,23], further supporting our results. In order to provide biochemical support for the bioinfor- matics results obtained so far, we decided to investigate some of the transcripts predicted to have long 30UTRs by Northern blot analysis. As shown above, the use of a highly conserved PAS by any vertebrate is a good indica- tion of the use of the orthologous site in humans. Thus, we selected the PTEs associated with the KCNB1 and KCNQ3 genes that were not annotated as PASs, neither in humans nor in mice Ensembl databases, and evalu- ated their expression in mice. In order to detect all the possible 30UTR isoforms, we used probes proximal to the stop codon. In this way, we could simultaneously as- sess the use of the annotated isoforms, the predicted longer isoform, and any other intermediate species In the latest versions of the Ensembl human annota- tions, the models have been merged with the manually annotated Havana transcripts. We observed that some of our predictions were incorporated into the unified data- base (Additional file 2: Table S1 and Additional file 5: Table S2). In most cases, the new gene ends did not Page 8 of 13 Morgan et al. BMC Genomics 2012, 13:708 http://www.biomedcentral.com/1471-2164/13/708 Morgan et al. BMC Genomics 2012, 13:708 correspond to 30UTR ext scripts but to novel large (lincRNAs). This is the downstream the KCNB1 (Figure 6) favor a model to the end of a 30UTR e however, both models are In order to gain furthe conservation of the KC 30UTRs, we investigated present in the correspo genes. Discussion In order to develop new appropriate approaches, dif- ferent aspects of 30 UTRs nature have to be considered. One particular hurdle of 30UTR annotation is that the highly conserved elements that might be present in these sequences that could be used to define them can be separated from one another by other elements such as Alu sequences that are not necessarily conserved. Another difficulty is that untranslated regions, in particular 30UTRs, can be several kilobases long. This is of particular importance because most of the transcriptional informa- tion used for annotation comes from ESTs that typically cover only a few hundred bases, or from incomplete cDNA sequences. Moreover, while coding sequences are spliced and exons junctions can be precisely delimited by short ESTs this cannot be done for long unspliced 30UTRs. In addition, while coding sequences have a reading frame that provides useful information for a correct annotation, there is not such a thing for 30UTRs. Until this moment, the position of the PAS was usually determined by cluster- ing ESTs finishing at the same position and examining for the presence of a polyA tail, not encoded in the genome. In addition, the presence of the canonical HexM approxi- mately twenty bases upstream of the PAS was usually con- sidered a good indicator of a bona fide 30end although we now know that less than 60% of the PASs are associated with a canonical HexM [9,10]. To look for more evidence of a physical link between the predicted extensions and the annotated gene, we then coupled the transcriptional data from other species together with deep sequencing reads from total RNA of different human tissues. Given the elevated number of short tags generated with this technique, it is now pos- sible in many cases to reconstruct almost the entire 30UTR of highly expressed genes. The brain specific iso- form of the ADD2 gene, with a 30UTR of more than 6 Kb, is an example. Although the read-coverage is gen- erally not complete for genes expressed at lower levels these gaps could potentially be filled by increasing the sequencing depth. Importantly, the signal-to-noise ratio of this method allowed us to detect a clear transition in the RNA signal for both high and low expression transcripts. Discussion After the completion of the human genome project, the annotation of the whole sequence became a major chal- lenge. Most of the initial efforts were focused on the an- notation of the coding sequences. Nowadays, however, increasing attention is also given to non-coding RNAs Morgan et al. BMC Genomics 2012, 13:708 http://www.biomedcentral.com/1471-2164/13/708 Page 9 of 13 conserved elements crucial for the 30end processing of the messenger. Consistent with this view, Xie and collea- gues [31] showed that most conserved elements in 50 regions of genes are associated with binding sites for tran- scription factors, which usually fall outside the mature transcript, while most conserved elements in the down- stream region correspond to AU rich elements and bind- ing sites for miRNAs, which fall inside the mature transcript. and untranslated regions. To achieve this aim, different methods have been developed in recent years to anno- tate some of these elements using deep sequencing data [24-26]. For example, a novel approach using histone methylation marks have been successfully used to iden- tify large intervening non-coding RNAs (lincRNAs) [25]. In addition, important progresses have been made on 30UTR annotation of model organisms, such as Caenor- habditis elegans, using high-throughput approaches [27,28]. However, we believe that 30UTRs annotation in vertebrates also requires to be complemented with new and specific methods as the ones we described in this work. In this study, we used ESTs to define not annotated PASs that in general did not overlap with nearby- annotated gene. Thus, they were not direct evidences of a physical link between the proximal annotated gene and the predicted extended 30UTR. However, the CDI value of the PTEs depended on whether they were putative PASs or TSSs, as shown in Figures 1 and 2. In particular, the distribution of the putative PASs resembled the dis- tribution of the annotated PASs. This observation strongly suggests that at least some of these sites corre- sponded to the 30 end of annotated genes. In order to provide evidence for a physical link between the anno- tated genes and their putative extensions, we examined the existence of orthologous transcripts using the pre- dicted distal PAS. We found that for higher CDIs there was an increased probability of finding a transcript from other species supporting the predicted PAS. This result indicates that using this conservation parameter can fa- cilitate the identification of bona fide 30UTR extensions. Discussion From an evolutionarily point of view, however, previ- ous studies also showed that PASs can be conserved be- tween mice and humans [29] and among different vertebrates [30]. In this study, we observed that the drop in genomic conservation after the PAS can be used to identify not annotated 30ends. Interestingly, this change in conservation was not observed for TSSs, possibly due to the strong evolutionary constrains acting upon 30UTRs [18]. In addition, different evolutionary forces shape the proximal regions of PASs and TSSs. For ex- ample, 50UTRs are flanked by promoters that contain transcription factors binding sites which are necessary to recruit the transcriptional machinery to the TSS. On the contrary, apart from the DSE necessary for a correct cleavage reaction, there are no other well-described Although overlapping deep sequencing reads can re- construct a putative extended 30UTR, the possibility in which the locus being considered encodes for different overlapping transcripts not physically linked still exist. Using Northern blot analysis, we showed that this was not the case for two PTEs. In both cases, we observed a high molecular weight band that corresponded to the predicted isoform with an extremely long 30UTR. We previously provided evidence of the existence of high molecular weight molecules by Northern blot analysis Morgan et al. BMC Genomics 2012, 13:708 http://www.biomedcentral.com/1471-2164/13/708 Page 10 of 13 for the not annotated CPEB3 and ADD2 isoforms with similar results [15,16]. Interestingly, we found other examples in the literature, such as the TNR or RORB genes, where very high molecular weight bands were detected by Northern blot for their rodent orthologs [32,33]. These isoforms are longer than the annotated isoforms but they are consistent with the usage of the predicted PTEs identified in this study. Moreover, the majority of the isoforms that we predicted were abun- dantly expressed and might correspond to the most rep- resentative transcripts, if not the only one, used by these genes in specific tissues. In recent updates of the human datasets we noticed the incorporation of new lincRNAs that partially overlap and share the same 30end of our predicted 30UTR extensions. We therefore speculate that some of these lincRNA may be part of the 30UTR of the immediately upstream gene, as we have experimentally shown here for the KCNB1 gene (Figure 6). To evaluate this possibility, Northern blot analyses are required to evaluate each case. laboratories around the world. Discussion However, a recurrent re- sult from high throughput sequencing studies is that a considerable amount of transcribed elements map to not annotated regions, including untranslated regions of genes encoding for proteins [4,5]. For example, this was observed in an early study that investigated the tran- scripts bound by the RNA binding protein Nova, previ- ously known to participate in the regulation of alternative splicing [35]. Unexpectedly, the authors found that many binding sites fall within 30UTRs or a few hun- dred bases downstream of the annotated PASs, presum- ably on not annotated 30UTR extensions. Interestingly, NOVA2, one of the two members of the Nova family, appears to have a highly conserved not annotated 30UTR itself (data not shown). Similar results were recently obtained by another group studying the RNA binding protein TDP-43 [36]. Different cis-acting elements present in 30UTRs can be recognized by RNA-binding protein and/or small RNAs. The effect of some of these trans-acting factors has been demonstrated using high throughput techniques. In par- ticular, it has been elegantly shown using genome wide microarrays that variations in the levels of a miRNA change the stability of the population of mRNAs con- taining the specific recognition motif for that particular small RNA [37,38]. Importantly, many of these regula- tory factors have subtle effects upon their targets. There- fore, the best way (and possibly the only one) to study their function is by analyzing their overall impact on the transcriptome. A better annotation of the 30UTRs will facilitate the identification of all the potential targets for a particular regulator. This, in turn, will lead to an in- crease in the signal-to-noise ratio of the effect of the fac- tor at a genome wide level. Comparative analysis using genomic and transcrip- tomic information from different species revealed that gene structure can be highly conserved across verte- brates [20] and could be used to improve gene annota- tion [34]. This observation, coupled with the presence of evolutionary conserved genomic signatures and high- throughput transcriptomic data, facilitated the design of novel approaches that can be used to improve gene an- notation of extensively curated databases. Indeed, in this work, we identified a few hundred conserved PASs not represented in the current Ensembl human predictions, possibly the most exhaustive annotation of the human genome. Discussion Given that the identification of the PASs is strongly based on conservation across species, we ratio- nalized that the methodology could be applied to other vertebrates. Therefore, we extended our analysis to rats and dogs using total RNA-Seq data recently deposited on the public archives. The annotation of the rat gen- ome is on an advanced state because the species has been extensively used as animal model. Still, we detected several hundred conserved 30UTRs not present on the current databases. The annotation of the dog genome is instead at an early stage. Thus, we found thousands of genes with distal PASs not represented on the existent models. Since the approach could be applied to different mammals, we propose that the method can be incorpo- rated to the annotation pipeline of any species in the phylum. Moreover, we believe that the validation of the predictions in different organisms using species-specific transcriptional data will increase the confidence of the orthologous models. FDR estimation T d i To determine whether a putative PAS obtained analyzing one database (“examined database”) was annotated as a PAS on other database (“validating database”), all tran- scripts in the validating database that had a PAS in the same position as the PTE were considered. Additionally, these transcripts had to share their last intron with any in- tron of any gene on the examined database. A putative PAS was considered to be supported by a validating data- base if any of its transcripts satisfied these conditions. In order to estimate the FDR, we considered all puta- tive PASs with a CDI or CD, depending of the method, higher than a given cutoff value. Next, we determined whether these putative PASs were supported or not by a validating database. The FDR was estimated as the frac- tion of not supported putative PASs. Methods included. In the case of overlapping genes with opposite directions the identity of the PTE was undetermined and it was not further considered. See Additional file 1: Figure S1 for a summary. Conclusions Th The present manuscript examines the current state of gene annotation in reference databases and, in particular, aims to improve the annotation of conserved 30UTRs. The methods presented here can be used to facilitate the iden- tification of not-yet-annotated bona fide 30UTR extensions in vertebrates. We provide evidence that many conserved 30ends are not represented in the commonly used data- bases, and we also make available a list of a few hundreds manually curated 30ends not annotated in the rather ex- haustive Ensembl data set. We believe that the application of these methods will increment the confidence of the orthologous models. We envision that a more complete annotation of the genome will facilitate the analysis of the data obtained from high throughput studies. The work will be of par- ticular benefit to the growing number of investigators using total RNA sequencing and CLIP techniques. The RefSeq and Ensembl databases, among others, are intended to provide a common framework for genome wide analyses, facilitating the communication of different Morgan et al. BMC Genomics 2012, 13:708 http://www.biomedcentral.com/1471-2164/13/708 Morgan et al. BMC Genomics 2012, 13:708 http://www.biomedcentral.com/1471-2164/13/708 Page 11 of 13 Identification of putative PTEs All databases used in this study were downloaded from the UCSC genome browser [21] or the Ensembl web site. The RefSeq and Ensembl databases were down- loaded on March, 2012 or later. ESTs with an extreme at the same chromosomal position or at one nucleotide of distance from one another were clustered. The number of ESTs that gave origin to a cluster is referred as the cluster support. In order to account for the few nucleo- tides variability associated with PASs, when several clus- ters fell at a distance of less than 25 nucleotides, only the most distal cluster was considered. The ESTs orien- tations were not considered in the analysis. Only PTEs with a cluster support of 4 or more ESTs were consid- ered. PTEs falling inside exons of the reference database (RefSeq or Ensembl) were not considered either. How- ever, PTEs inside the reference introns were included in the analysis as they might correspond to the TSSs of al- ternative promoters or to PASs of alternative last exons. PTEs that might correspond to 30 or 50 splice sites (SS) of not annotated exons were also excluded from the ana- lysis. To this end, PTEs falling 25 nucleotides apart from any SS of a spliced EST were filtered out. Determination of the usage of conserved PASs using deep sequencing data In order to evaluate the functionality of putative PASs used in other species, TransMap transcripts [20] from different vertebrates were analyzed. A TransMap tran- script and an Ensembl gene were considered to share a similar structure if they had the same last intron. A TransMap transcript was considered to have an extended last exon if it shared a similar structure with an Ensembl gene but had a more distal PAS. For each of the transcripts with an extended last exon, an upstream interval spanning the transcript extension and a down- stream interval spanning a genomic interval of the same length downstream of the putative PAS were defined (see Figure 4A). The deep reads data generated by Wang and colleges [2] and mapped to the human genome by the group of Dr. Guigó were downloaded form the UCSC genome browser. The 8 tissues/organs considered were adipose, brain, breast, colon, heart, liver, lung and muscle. Each base in the interval was classified into two mutually exclusive categories: covered by a read or not covered. The orientation of the reads was not consid- ered. All the covered bases in the intervals were counted and divided by the length of the intervals times a hun- dred to obtain the percentage covered of each of the intervals. The coverage difference was defined as the subtraction of the coverage percentage of the down- stream interval to the coverage percentage of the inter- val upstream of the PAS. CDI calculation PhyloP scores [19] were used to obtain the CDI of tran- scripts’ extremes. To calculate the conservation of the ma- ture transcript region, an interval of 50 nucleotides starting 50 nucleotides inside the mature transcript and finishing at the 30 (or 50) end of the mature transcript was considered. The conservation of the mature transcript re- gion was defined as the arithmetic media of the PhyloP score of each nucleotide of the interval. To obtain the con- servation of the proximal region, the average conservation score for the 50-nucleotide interval starting 50 nucleotides outside the mature transcript region was calculated. The CDI was calculated subtracting the conservation of the proximal region to the conservation of the mature tran- script region (see Figure 1 for a summary). Additional files Depicts two examples of PTEs with high CDI and no support from other databases. Additional file 4: Figure S3. Shows the correlation between CDI values calculated using the Mammalian and Vertebrates PhyloP scores. Additional file 5: Table S2. List the putative PASs identified with CD higher than 50% in humans. Additional file 6: Table S3. List the putative PASs identified with CD higher than 50% in rats. Additional file 7: Figure S4. Examples of PTEs identified in rats. Additional file 8: Table S4. List the putative PASs identified with CD higher than 50% in dogs. Additional file 9: Figure S5. Examples of PTEs identified in dogs. Additional file 10: Figure S6. Homolog PTEs of the Kcnb1 gene in rats. Additional file 11: Figure S7. Homolog PTEs of the Kcnq3 gene in dogs. Additional file 1: Figure S1. Describes the criteria used to classify PTEs in putative PASs or TSSs. 8. Proudfoot NJ, Brownlee GG: 30 non-coding region sequences in eukaryotic messenger RNA. Nature 1976, 263(5574):211–214. Additional file 2: Table S1. Lists the putative PASs identified with high CDI. y g 9. Beaudoing E, Freier S, Wyatt JR, Claverie JM, Gautheret D: Patterns of variant polyadenylation signal usage in human genes. Genome Res 2000, 10(7):1001–1010. Additional file 3: Figure S2. Depicts two examples of PTEs with high CDI and no support from other databases. 10. Tian B, Hu J, Zhang H, Lutz CS: A large-scale analysis of mRNA polyadenylation of human and mouse genes. Nucleic Acids Res 2005, 33(1):201–212. 11. Shi Y, Di Giammartino DC, Taylor D, Sarkeshik A, Rice WJ, Yates JR 3rd, Frank J, Manley JL: Molecular architecture of the human pre-mRNA 30 processing complex. Mol Cell 2009, 33(3):365–376. 12. Sandberg R, Neilson JR, Sarma A, Sharp PA, Burge CB: Proliferating cells express mRNAs with shortened 30 untranslated regions and fewer microRNA target sites. Science 2008, 320(5883):1643–1647. 13. Ji Z, Lee JY, Pan Z, Jiang B, Tian B: Progressive lengthening of 30 untranslated regions of mRNAs by alternative polyadenylation during mouse embryonic development. Proc Natl Acad Sci U S A 2009, 106(17):7028–7033. 14. Bartel DP: MicroRNAs: target recognition and regulatory functions. Cell 2009, 136(2):215–233. Additional file 11: Figure S7. Homolog PTEs of the Kcnq3 gene in dogs. 15. Costessi L, Devescovi G, Baralle FE, Muro AF: Brain-specific promoter and polyadenylation sites of the beta-adducin pre-mRNA generate an unusually long 30-UTR. Nucleic Acids Res 2006, 34(1):243–253. 16. References 1. Sultan M, Schulz MH, Richard H, Magen A, Klingenhoff A, Scherf M, Seifert M, Borodina T, Soldatov A, Parkhomchuk D, et al: A global view of gene activity and alternative splicing by deep sequencing of the human transcriptome. Science 2008, 321(5891):956–960. 1. Sultan M, Schulz MH, Richard H, Magen A, Klingenhoff A, Scherf M, Seifert M, Borodina T, Soldatov A, Parkhomchuk D, et al: A global view of gene activity and alternative splicing by deep sequencing of the human transcriptome. Science 2008, 321(5891):956–960. 2. Wang ET, Sandberg R, Luo S, Khrebtukova I, Zhang L, Mayr C, Kingsmore SF Schroth GP, Burge CB: Alternative isoform regulation in human tissue transcriptomes. Nature 2008, 456(7221):470–476. 3. Ozsolak F, Goren A, Gymrek M, Guttman M, Regev A, Bernstein BE, Milos PM: Digital transcriptome profiling from attomole-level RNA samples. Genome Res 2010, 20(4):519–525. 4. Pickrell JK, Marioni JC, Pai AA, Degner JF, Engelhardt BE, Nkadori E, Veyrieras JB, Stephens M, Gilad Y, Pritchard JK: Understanding mechanisms underlying human gene expression variation with RNA sequencing. Nature 2010, 464(7289):768–772. 5. van Bakel H, Nislow C, Blencowe BJ, Hughes TR: Most “dark matter” transcripts are associated with known genes. PLoS Biol 2010, 8(5):e1000371. 6. Flicek P, Amode MR, Barrell D, Beal K, Brent S, Chen Y, Clapham P, Coates G, Fairley S, Fitzgerald S, et al: Ensembl 2011. Nucleic Acids Res 2011, 39(Database issue):D800–D806. 6. Flicek P, Amode MR, Barrell D, Beal K, Brent S, Chen Y, Clapham P, Coates G, Fairley S, Fitzgerald S, et al: Ensembl 2011. Nucleic Acids Res 2011, 39(Database issue):D800–D806. Authors’ contributions AFM and MM designed the study, interpreted the data and wrote the manuscript. MM performed most of the experiments. MM and AI performed the Northern blots. All authors read and approved the final manuscripts. Northern blot analysis Northern blots analyses were performed using standard procedures. Briefly, for the Kcnb1 and Kcnq3 mRNA ana- lysis, 5 μg and 25 μg respectively of mouse total RNA from brain, testes and heart were used. The samples were run in a 1.2% denaturating agarose gel at 70 V for 5 hours. The RNA was transferred to a nitrocellulose membrane by capillarity, UV cross-linked and pre-hybridized at 65°C for half an hour. The probes used were amplified from mouse genomic DNA with the following set of primers: 50 CCAGTCTCAACCCATCCTCAA 30 and 50 GTCATCAG TGTCGGTGTCTA 30 for the Kcnb1 probe, and 50 AACT GGACTTCCTCGTGGACA 30 and 50 CATGGAACCAC TGGGTGTGAA 30 for Kcnq3 probe. For each probe, the unique amplified band was then purified from gel and radiolabeled using [α-32P]dCTP. The identity of the band was verified by sequencing. Hybridization was performed overnight at 65°C and the next day the membrane was washed twice with SSC 2X, SDS 0,1% and once with SSC 1X, SDS 0,1%. The membrane was then exposed ON to a pre-blanked Cyclone screen. Acknowledgements We thank the members of the Mouse Molecular Genetics Group, Dr. K. Havas, Dr. Emanuele Buratti and Dr. Y. Ayala for critical reading of the manuscript. Received: 18 June 2012 Accepted: 15 December 2012 Published: 18 December 2012 Received: 18 June 2012 Accepted: 15 December 2012 Published: 18 December 2012 Determination of PTEs putative identity Determination of PTEs putative identity In order to classify the PTEs in putative PASs or TSSs, the orientation and distance of the closest reference gene was considered. If the closest reference gene was tran- scribed towards the PTE, the PTE was classified as a pu- tative PAS. If the closest reference gene was transcribed in the opposite direction, the PTE was classified as a pu- tative TSS. The PTE and the closest transcript had to be less than 1 Mb apart from each other. PTEs that fell in genomic regions with no proximal reference genes were not considered in the analysis. If a PTE fell inside the intron of a reference gene, it was classified according to the orientation of the transcripts in which it was The same method was used to identify conserved PASs in dogs and rats. The following deep sequencing runs were used: SRR388734, SRR388736, SRR388737, SRR388738, Page 12 of 13 Page 12 of 13 Morgan et al. BMC Genomics 2012, 13:708 http://www.biomedcentral.com/1471-2164/13/708 SRR388742, SRR388743, SRR388745, SRR388746, SRR3 88749 and SRR388754, and SRR042499 [39]. The reads were aligned to their respective genomes using Bowtie [40]. Reads mapping only once to the genome and with at most 2 mismatches were considered for subsequent ana- lysis. The reads were visualized using IGV [41]. Transcript extremes; FDR: False discovery rate; CD: Coverage difference; ON: Overnight; EtBr: Ethidium bromide. Competing interests The authors declare that they have no competing interests. Additional files Morgan M, Iaconcig A, Muro AF: CPEB2, CPEB3 and CPEB4 are coordinately regulated by miRNAs recognizing conserved binding sites in paralog positions of their 30-UTRs. Nucleic Acids Res 2010, 38(21):7698–7710. Abbreviations UTR: Untranslated region; mRNA: messenger RNA; PAS: Polyadenylation site; HexM: Hexanucleotide motif (polyA signal); DSE: Downstream sequence element; miRNA: micro RNA; EST: Expressed sequence tags; CDI: Conservation drop index; TSS: Transcription start site; PTE: Potential UTR: Untranslated region; mRNA: messenger RNA; PAS: Polyadenylation site; HexM: Hexanucleotide motif (polyA signal); DSE: Downstream sequence element; miRNA: micro RNA; EST: Expressed sequence tags; Additional files 7. Maglott D, Ostell J, Pruitt KD, Tatusova T: Entrez gene: gene-centered information at NCBI. Nucleic Acids Res 2011, 39(Database issue):D52–D57. Additional file 1: Figure S1. Describes the criteria used to classify PTEs in putative PASs or TSSs. Additional file 2: Table S1. Lists the putative PASs identified with high CDI. Additional file 3: Figure S2. Depicts two examples of PTEs with high CDI and no support from other databases. Additional file 4: Figure S3. Shows the correlation between CDI values calculated using the Mammalian and Vertebrates PhyloP scores. Additional file 5: Table S2. List the putative PASs identified with CD higher than 50% in humans. Additional file 6: Table S3. List the putative PASs identified with CD higher than 50% in rats. Additional file 7: Figure S4. Examples of PTEs identified in rats. Additional file 8: Table S4. List the putative PASs identified with CD higher than 50% in dogs. Additional file 9: Figure S5. Examples of PTEs identified in dogs. Additional file 10: Figure S6. Homolog PTEs of the Kcnb1 gene in rats. Additional file 11: Figure S7. Homolog PTEs of the Kcnq3 gene in dogs. Additional file 1: Figure S1. Describes the criteria used to classify PTEs in putative PASs or TSSs. Additional file 2: Table S1. Lists the putative PASs identified with high CDI. Additional file 3: Figure S2. Depicts two examples of PTEs with high CDI and no support from other databases. Additional file 4: Figure S3. Shows the correlation between CDI values calculated using the Mammalian and Vertebrates PhyloP scores. Additional file 5: Table S2. List the putative PASs identified with CD higher than 50% in humans. Additional file 6: Table S3. List the putative PASs identified with CD higher than 50% in rats. Additional file 7: Figure S4. Examples of PTEs identified in rats. Additional file 8: Table S4. List the putative PASs identified with CD higher than 50% in dogs. Additional file 9: Figure S5. Examples of PTEs identified in dogs. Additional file 10: Figure S6. Homolog PTEs of the Kcnb1 gene in rats. Additional file 11: Figure S7. Homolog PTEs of the Kcnq3 gene in dogs. Additional file 1: Figure S1. Describes the criteria used to classify PTEs in putative PASs or TSSs. Additional file 2: Table S1. Lists the putative PASs identified with high CDI. Additional file 3: Figure S2. Abbreviations 17. Theis M, Si K, Kandel ER: Two previously undescribed members of the mouse CPEB family of genes and their inducible expression in the Page 13 of 13 Morgan et al. BMC Genomics 2012, 13:708 http://www.biomedcentral.com/1471-2164/13/708 38. Manakov SA, Grant SG, Enright AJ: Reciprocal regulation of microRNA and mRNA profiles in neuronal development and synapse formation. BMC Genomics 2009, 10:419. principal cell layers of the hippocampus. Proc Natl Acad Sci U S A 2003, 100(16):9602–9607. principal cell layers of the hippocampus. Proc Natl Acad Sci U S A 2003, 100(16):9602–9607. 18. Siepel A, Bejerano G, Pedersen JS, Hinrichs AS, Hou M, Rosenbloom K, Clawson H, Spieth J, Hillier LW, Richards S, et al: Evolutionarily conserved elements in vertebrate, insect, worm, and yeast genomes. Genome Res 2005, 15(8):1034–1050. 39. Hammer P, Banck MS, Amberg R, Wang C, Petznick G, Luo S, Khrebtukova I, Schroth GP, Beyerlein P, Beutler AS: mRNA-seq with agnostic splice site discovery for nervous system transcriptomics tested in chronic pain. Genome Res 2010, 20(6):847–860. 19. Rhead B, Karolchik D, Kuhn RM, Hinrichs AS, Zweig AS, Fujita PA, Diekhans M, Smith KE, Rosenbloom KR, Raney BJ, et al: The UCSC Genome Browser database: update 2010. Nucleic Acids Res 2010, 38(Database issue):D613–D619 40. Langmead B, Trapnell C, Pop M, Salzberg SL: Ultrafast and memory- efficient alignment of short DNA sequences to the human genome. Genome Biol 2009, 10(3):R25. 20. Zhu J, Sanborn JZ, Diekhans M, Lowe CB, Pringle TH, Haussler D: Comparative genomics search for losses of long-established genes on the human lineage. PLoS Comput Biol 2007, 3(12):e247. 41. Robinson JT, Thorvaldsdottir H, Winckler W, Guttman M, Lander ES, Getz G, Mesirov JP: Integrative genomics viewer. Nat Biotechnol 2011, 29(1):24–26. 41. Robinson JT, Thorvaldsdottir H, Winckler W, Guttman M, Lander ES, Getz G, Mesirov JP: Integrative genomics viewer. Nat Biotechnol 2011, 29(1):24–26. 41. Robinson JT, Thorvaldsdottir H, Winckler W, Guttman M, Lander ES, Getz G, Mesirov JP: Integrative genomics viewer. Nat Biotechnol 2011, 29(1):24–26. 21. Fujita PA, Rhead B, Zweig AS, Hinrichs AS, Karolchik D, Cline MS, Goldman M, Barber GP, Clawson H, Coelho A, et al: The UCSC Genome Browser database: update 2011. Nucleic Acids Res 2011, 39(Database issue):D876–D882. doi:10.1186/1471-2164-13-708 Cite this article as: Morgan et al.: Identification of 30 gene ends using transcriptional and genomic conservation across vertebrates. BMC Genomics 2012 13:708. 22. Abbreviations Tinel N, Lauritzen I, Chouabe C, Lazdunski M, Borsotto M: The KCNQ2 potassium channel: splice variants, functional and developmental expression. Brain localization and comparison with KCNQ3. FEBS Lett 1998, 438(3):171–176. 23. Yang WP, Levesque PC, Little WA, Conder ML, Ramakrishnan P, Neubauer MG, Blanar MA: Functional expression of two KvLQT1-related potassium channels responsible for an inherited idiopathic epilepsy. J Biol Chem 1998, 273(31):19419–19423. 24. Chiang HR, Schoenfeld LW, Ruby JG, Auyeung VC, Spies N, Baek D, Johnston WK, Russ C, Luo S, Babiarz JE, et al: Mammalian microRNAs: experimental evaluation of novel and previously annotated genes. Genes Dev 2010, 24(10):992–1009. 25. Guttman M, Amit I, Garber M, French C, Lin MF, Feldser D, Huarte M, Zuk O, Carey BW, Cassady JP, et al: Chromatin signature reveals over a thousand highly conserved large non-coding RNAs in mammals. Nature 2009, 458(7235):223–227. 26. Ozsolak F, Kapranov P, Foissac S, Kim SW, Fishilevich E, Monaghan AP, John B, Milos PM: Comprehensive polyadenylation site maps in yeast and human reveal pervasive alternative polyadenylation. Cell 2010, 143(6):1018–1029. 27. Mangone M, Manoharan AP, Thierry-Mieg D, Thierry-Mieg J, Han T, Mackowiak SD, Mis E, Zegar C, Gutwein MR, Khivansara V, et al: The landscape of C. elegans 30UTRs. Science 2010, 329(5990):432–435. 28. Jan CH, Friedman RC, Ruby JG, Bartel DP: Formation, regulation and evolution of Caenorhabditis elegans 30UTRs. Nature 2011, 469(7328):97–101. 29. Ara T, Lopez F, Ritchie W, Benech P, Gautheret D: Conservation of alternative polyadenylation patterns in mammalian genes. BMC Genomics 2006, 7:189. 30. Lee JY, Yeh I, Park JY, Tian B: PolyA_DB 2: mRNA polyadenylation sites in vertebrate genes. Nucleic Acids Res 2007, 35(Database issue):D165–D168. 31. Xie X, Lu J, Kulbokas EJ, Golub TR, Mootha V, Lindblad-Toh K, Lander ES, Kellis M: Systematic discovery of regulatory motifs in human promoters and 30 UTRs by comparison of several mammals. Nature 2005, 434(7031):338–345. 32. Andre E, Conquet F, Steinmayr M, Stratton SC, Porciatti V, Becker-Andre M: Disruption of retinoid-related orphan receptor beta changes circadian behavior, causes retinal degeneration and leads to vacillans phenotype in mice. EMBO J 1998, 17(14):3867–3877. 33. Weber P, Bartsch U, Rasband MN, Czaniera R, Lang Y, Bluethmann H, Margolis RU, Levinson SR, Shrager P, Montag D, et al: Mice deficient for tenascin-R display alterations of the extracellular matrix and decreased axonal conduction velocities in the CNS. J Neurosci 1999, 19(11):4245–4262. Morgan et al. BMC Genomics 2012, 13:708 http://www.biomedcentral.com/1471-2164/13/708 37. Grimson A, Farh KK, Johnston WK, Garrett-Engele P, Lim LP, Bartel DP: MicroRNA targeting specificity in mammals: determinants beyond seed pairing. Mol Cell 2007, 27(1):91–105. Abbreviations Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit 34. Stanke M, Diekhans M, Baertsch R, Haussler D: Using native and syntenically mapped cDNA alignments to improve de novo gene finding. Bioinformatics 2008, 24(5):637–644. • Convenient online submission 35. Licatalosi DD, Mele A, Fak JJ, Ule J, Kayikci M, Chi SW, Clark TA, Schweitzer AC, Blume JE, Wang X, et al: HITS-CLIP yields genome-wide insights into brain alternative RNA processing. Nature 2008, 456(7221):464–469. 36. Sephton CF, Cenik C, Kucukural A, Dammer EB, Cenik B, Han Y, Dewey CM, Roth FP, Herz J, Peng J, et al: Identification of Neuronal RNA Targets of TDP-43-containing ribonucleoprotein complexes. J Biol Chem 2011, 286(2):1204–1215. 37. Grimson A, Farh KK, Johnston WK, Garrett-Engele P, Lim LP, Bartel DP: MicroRNA targeting specificity in mammals: determinants beyond seed pairing. Mol Cell 2007, 27(1):91–105. 37. Grimson A, Farh KK, Johnston WK, Garrett-Engele P, Lim LP, Bartel DP: MicroRNA targeting specificity in mammals: determinants beyond seed pairing. Mol Cell 2007, 27(1):91–105.
https://openalex.org/W2915637656	https://hsag.co.za/index.php/hsag/article/download/1088/pdf	English	null	Experiences of women enrolled in a prevention of mother to child transmission of human immunodeficiency virus infection programme in Zimbabwe	Health SA Gesondheid	2,019	cc-by	6,698	Health SA Gesondheid ISSN: (Online) 2071-9736, (Print) 1025-9848 Page 1 of 7 Original Research Original Research Page 1 of 7 Dates: Conclusions: The study concluded that pregnant women living with HIV require empowerment and support to live positively with HIV. Conclusions: The study concluded that pregnant women living with HIV require empowerment and support to live positively with HIV. How to cite this article: Tagutanazvo, O.B., Nolte, A.G.W. & Temane, A., 2019, ‘Experiences of women enrolled in a prevention of mother to child transmission of human immunodeficiency virus infection programme in Zimbabwe’, Health SA Gesondheid 24(0), a1088. https://doi.org/10.4102/ hsag.v24i0.1088 Experiences of women enrolled in a prevention of mother to child transmission of human immunodeficiency virus infection programme in Zimbabwe Background: Prevention of mother-to-child transmission (PMTCT) programmes have been reported to reduce the rate of transmission of human immunodeficiency virus (HIV) infection by 30% – 40% during pregnancy and childbirth. The PMTCT transmission is achieved by offering HIV prophylaxis or initiating antiretrovirals to pregnant women who test HIV positive. Being aware of the experiences of these women will assist in planning and implementing the relevant care and support. The study was conducted in three phases. Aim: This article will address phase 1 which is to explore and describe the experiences of pregnant women living with HIV. Setting: The study setting was a PMTCT site in a Provincial Hospital, in Zimbabwe. Setting: The study setting was a PMTCT site in a Provincial Hospital, in Zimbabwe. Methods: The study design was qualitative, exploratory, descriptive and contextual. In-depth face-to-face interviews were conducted from a purposive sample of 20 pregnant women. Thematic data analysis was performed. Methods: The study design was qualitative, exploratory, descriptive and contextual. In-depth face-to-face interviews were conducted from a purposive sample of 20 pregnant women. Thematic data analysis was performed. Corresponding author: Oslinah Tagutanazvo, oslinah.tagutanazvo@gmail. com Results: Six themes emerged: realities of disclosure, a need for quality of life, perceived stigmatisation, inadequate knowledge on infant feeding, continuity of care, empowerment and support. Dates: Received: 29 Jan. 2018 Accepted: 10 Aug. 2018 Published: 27 Feb. 2019 Copyright: © 2019. The Authors. Licensee: AOSIS. This work is licensed under the Creative Commons Attribution License. Read online: Scan this QR code with your smart phone or mobile device to read online. Read online: Scan this QR code with your smart phone or mobile device to read online. Introduction and background Zimbabwe has an estimated 1.4 million people living with human immunodeficiency virus (HIV), of whom 77 000 are children aged below 15 years who contracted HIV through vertical transmission (UNAID 2015). The HIV prevalence among women and men aged 15–49 years is reported to be about 14% (UNAID 2015). Human immunodeficiency virus responses in Zimbabwe constitute a national emergency, and prevention of HIV infection in infants is an important priority (ICF 2012; MOH & CW 2007; ZIMSTAT 2012). The Zimbabwean government has shown its commitment to the fight against HIV and AIDS through the implementation of the 1999 HIV and AIDS policy, the Zimbabwe National HIV and AIDS Strategic Plan 2006–2010, provision of prevention of mother to child transmission (PMTCT) services and its second edition of the PMTCT policy of 2006 (MOH & CW 2006a). As part of the PMTCT and Paediatric HIV Prevention Treatment and Care National Plan, the latter policy was revised in 2007. Copyright: © 2019. The Authors. Licensee: AOSIS. This work is licensed under the Creative Commons Attribution License. Copyright: Prevention of mother to child transmission programmes have been reported to reduce the transmission rate of HIV infection by 30% – 40% during pregnancy and childbirth (MOH & CW 2008:i). The Southern African Development Community (SADC), of which Zimbabwe is a member country, acknowledges that the problems related to HIV and AIDS differently impact on men and women, are multi-sectorial and multidimensional, and raise the need to develop effective and relevant policies. In the same report, emphasis is placed on developing programmes that bring about collaboration among partners (SADC Strategy and Programme of Action 2003–2006). This is consistent with Both and Van Roosmalen (2010:1448), who indicate that ‘MTCT programmes miss the opportunity to have an overall positive effect on maternal health because of the way the programmes are structured’. Read online: Scan this QR code with your smart phone or mobile device to read online. Read online: Scan this QR code with your smart phone or mobile device to read online. The PMTCT programme in Zimbabwe focuses on primary prevention of HIV in the general population, prevention of unintended pregnancies among women living with HIV, prevention of HIV transmission from women living with HIV to their infants, treatment as well as care and http://www.hsag.org.za Page 2 of 7 Page 2 of 7 Original Research support for women living with HIV, including their infants and families. support for women living with HIV, including their infants and families. Human immunodeficiency virus infection: By damaging the immune system, HIV interferes with the body’s ability to fight the organisms that cause the infection. Human immunodeficiency virus is a sexually transmitted infection. Human immunodeficiency virus infection: By damaging the immune system, HIV interferes with the body’s ability to fight the organisms that cause the infection. Human immunodeficiency virus is a sexually transmitted infection. In Zimbabwe, provider-initiated HIV testing and counselling (PI-HTC) is provided for pregnant women presenting at health care facilities, who are not aware of their HIV status. Midwives provide group HIV pre-test counselling to pregnant women during the initial antenatal visit. The women who test positive for HIV receive individual post-test counselling, which is short and focused on PMTCT with emphasis on the need to disclose their status to their partners, relatives or friends for psychosocial support. Their follow-up care and support is not explicit and is partially vested in the hands of friends, lay community counsellors or support groups (Shetty et al. Research design The research design used in this phase of the study was a qualitative, exploratory approach, descriptive and contextual in nature, to explore and describe the experiences of pregnant women living with HIV. The research approach was chosen because it allows the researcher to collect information from the person experiencing the phenomenon as the focus of the study was on understanding an experience. The purpose of this phase of the research was to explore and describe the experiences of pregnant women living with HIV at a Provincial Hospital, who were enrolled in the PMTCT programme. This study is part of a larger study that led to the development of a holistic care model that will be used as a framework by midwives to facilitate quality of life for pregnant women living with HIV. Research methods Study setting The study was conducted in Zimbabwe at a Provincial Hospital in the maternal and child health (MCH) department. The PMTCT was incorporated in the MCH programme. Population and sampling The accessible population was pregnant women living with HIV who were enrolled in a PMTCT programme at a Provincial Hospital in Zimbabwe. The study was conducted in three phases addressing the following objectives: Phase 1: To explore and describe the experiences of pregnant women living with HIV infection during pregnancy. Phase 2: To develop a holistic care model for use by midwives caring for pregnant women living with HIV who are enrolled in a PMTCT programme. Phase 3: To evaluate the holistic care model for use by midwives caring for pregnant women living with HIV who are enrolled in a PMTCT programme. This article addresses Phase 1 of the study. The site was purposively selected as the researcher has worked as a midwifery lecturer in this setting. The motivation for the study came as a result of the women within the site’s catchment area confiding in the researcher on the challenges they faced as people living with HIV. A purposive sample of pregnant women who tested positive for HIV as a result of PI-HTC in the PMTCT programme was used. The inclusion criteria were as follows: The experiences of the women who were under study and the roles and responsibilities of the midwife as enshrined in the definition of the ‘midwife’ (International Confederation of Midwives [ICM] 2011) were used to identify the relevant care and support the midwives provide. The midwife’s roles and responsibilities include those of carer, counsellor, collaborator and coordinator of care. The midwife works as a coordinator and collaborator of midwifery care in a multidisciplinary team involving obstetricians, nutritionists or dieticians, social workers, paediatricians and traditional birth attendants, in line with the philosophy and model of midwifery care (ICM 2011). • pregnant women who had known their HIV-positive status during pregnancy and were in the second or third trimester, • participants who were aged 18 years and older. In Zimbabwe, 18 is the legal age of majority; thus, this age group is viewed as capable of providing informed consent. The participants were identified by the midwife in charge of the MCH department from a queue of pregnant women who had reported for their scheduled antenatal visits. The identified participants were then each taken to a side room where they were introduced to the researcher. The researcher explained the purpose of the study to each of the participants and Copyright: 2008). However, following disclosure, these women need support and skills to deal with various challenges such as the HIV- related stigma, the unpredicted responses of their partners, dealing with issues related to infant feeding and any other matters relating to HIV infection (Nachega et al. 2012). The pregnant women living with HIV have to start on antiretrovirals (ARVs) as prophylaxis or as treatment for their lifetime. Being aware of these women’s experiences will assist in planning and implementing the relevant care and support. Prevention of mother to child transmission of HIV: The mother to child transmission (MTCT) of HIV refers to the transmission of HIV from an HIV-positive woman to her child during pregnancy, labour, childbirth or breastfeeding. Pregnant woman: A woman who is in the state of carrying a developing embryo or foetus within the female body. Data analysis Ethical clearance was provided by the academic ethical committee of a university in South Africa. Permission to carry out the study was obtained from the Acting Medical Superintendent of the Provincial Hospital, while written informed consent was obtained from the participants. Ethical considerations adhered to in the study were the following: freedom to participate in or withdraw from the study, freedom from harm, the right to full disclosure, benefits of the study, the risk/benefit ratio, the right to privacy, anonymity and confidentiality (ethics approval number AEC39/02-2011, Faculty of Health Sciences, University of Johannesburg). Anonymity was also achieved by not linking the names of the participants to the data collected and by using identifying numbers. Data from the audiotapes and field notes were transcribed and formed a written record of each of the interviews conducted. The researcher engaged an independent coder who carried out an in-depth analysis of the data, while the researcher also analysed the data. The independent coder is a nurse-midwife who holds a doctoral degree and is well versed in qualitative research with an understanding of both languages (Shona and English). Tesch’s data analysis method (in Creswell 1994:154– 156) was used which includes eight steps described as follows: Step 1: The researcher listened to the tapes (recordings) and obtained a sense of the whole, carefully reading through the transcriptions and jotting down some ideas as they came into the researcher’s mind. Data collection Data were collected by the researcher through in-depth individual face-to-face interviews during 2013. The interviews were tape-recorded and conducted privately in side rooms, away from the other women to ensure privacy, anonymity and confidentiality. The interviews were conducted in Shona (one of the vernacular languages in Zimbabwe) and data were also analysed in Shona before being translated into English. Step 6: The researcher made a final decision on the categories that were then merged into themes. Step 7: Data belonging to each category were identified and put together, then a preliminary analysis was performed. One central question was asked, namely: ‘How has it been for you since the time you were informed that you are living with HIV infection?’ Step 8: All the existing data were recorded. Step 8: All the existing data were recorded. Step 8: All the existing data were recorded. Original Research Original Research Step 2: The researcher picked one interview recording at a time and went through it, asking herself what it was about, and thinking about the underlying meaning. The researcher then wrote down her thoughts in the margins. Step 2: The researcher picked one interview recording at a time and went through it, asking herself what it was about, and thinking about the underlying meaning. The researcher then wrote down her thoughts in the margins. highlighted that participation was voluntary. Participants were also free to discontinue the interview at any point without any penalty and also without affecting the services they received. The researcher then obtained an informed verbal and written consent before conducting the interviews. Because of the sensitive nature of living with HIV and the stigma still attached to HIV, access to participants was at the hospital site, instead of participant homes, to maintain their privacy and confidentiality, and to protect them from stigmatisation by society as a whole. Step 3: Upon completion of the task, the researcher made a list of topics. The researcher then clustered together similar topics in typed form and arranged these topics in columns under major topics, unit topics and topics left over that had no similarity. Step 4: The researcher used the list of topics and went back to the data, abbreviated the topics as codes and wrote the codes next to the appropriate segments of the texts. The researcher then made a preliminary organising scheme to see whether new categories and codes emerged. A total of 20 pregnant women participated in the study. Data saturation determined the sample size. According to Polit and Beck (2014), data saturation entails sampling to the point where no new information is obtained and redundancy is achieved. In this study, no new themes or categories emerged from the in-depth interviews after the 20 participants. Step 5: The researcher identified the most descriptive wording for the topics and turned these into categories. Topics that relate to each other were grouped together, thereby reducing the list of categories. The researcher then identified interrelationships between categories. Trustworthiness Trustworthiness was ensured according to the framework by Lincoln and Guba (1985) (credibility, dependability, confirmability and transferability). In this study, each individual in-depth face-to-face interview took about 45 min to 1 h per participant. This includes the time taken to create rapport. Field notes were used by the researcher to support the emerging themes and categories. Definition of concepts Experience: To do or see (something) or have (something) happen to you; to feel or be affected by (something) (Merriam Webster Dictionary). http://www.hsag.org.za Open Access Page 3 of 7 Page 3 of 7 One participant said: HIV, human immunodeficiency virus; ARV, antiretroviral. HIV, human immunodeficiency virus; ARV, antiretroviral. ‘I disclosed to my mother ….she is very supportive. Now she reminds me about taking the ARVs on time’. (Participant 2, female, 26 years old) Another participant echoed: ‘I placed my ANC (antenatal care) card on the headboard on his side of the bed; he said oh…. Is that what they said (referring to midwives)…, it does not matter? We will take the medication together.’ (Participant 14, female, 26 years old) On unsafe disclosure, participants feared for a possible breach of confidentiality by their confidantes in terms of gossip, physical abuse, spousal abandonment and marital breakdown. A participant said: ‘It took me a long time to accept my status; I imagined that I may deliver a baby who is infected with HIV.’ (Participant 10, female, 28 years old) ‘It took me a long time to accept my status; I imagined that I may deliver a baby who is infected with HIV.’ (Participant 10, female, 28 years old) Another participant echoed: ‘Other people are surviving, other people have been living with HIV for years; I will also survive.’ (Participant 1, female, 30 years old) ‘Other people are surviving, other people have been living with HIV for years; I will also survive.’ (Participant 1, female, 30 years old) Theme 2: Realities of disclosure Participants reported both positive and negative experiences following disclosure and these were categorised as safe and unsafe disclosures. On safe disclosure, participants were likely to disclose to their husbands and family members in anticipation of support and confidentiality, and this also included those participants who had realised the benefits of taking ARVs. Pregnant women living with HIV reported midwives as supportive as: ppi • Empowered with knowledge on HIV and available treatment. Giving hope for: Giving hope for: • An improved quality of life. Giving hope for: • An improved quality of life. g p • An improved quality of life. One participant said: ‘I will not disclose to others …because people talk too much and this may affect my health…so I will keep it to myself.’ (Participant 12, female, 18 years old) Another participant claimed (with a relaxed face): One participant had this to say: ‘A.a.a.h, this did not worry me much because most people now are living with HIV so it is no longer scary to live with HIV.’ (Participant 2, female, 26 years old) Research findings I have lost the sexual drive … he does not force me when I say I am tired … because I think he is also afraid.’ (Participant 9, female, 35 years old) This was echoed by another participant as well: ‘We have not yet had sexual activity … I told him that we should use condoms … he said it’s ok but currently I do not have the sexual drive – I am not interested.’ (Participant 15, female, 25 years old) Theme 1: A need for quality of life The need for a quality life, as reported by participants, was described as the acceptance of their own HIV status attributed to the availability and access to ARV drugs, which contribute to the improvement of an individual’s health. Participants who knew someone currently living with HIV and taking ARVs were likely to have hope for survival and accept their HIV-positive status. Another participant claimed (with a relaxed face): ‘I have seen some women who are living with HIV that is why I accepted my situation…’ (Participant 6, female, 29 years old) ‘I have seen some women who are living with HIV that is why I accepted my situation…’ (Participant 6, female, 29 years old) This was echoed by another participant: ‘I cannot even tell my sister because if 1 day we happen to have misunderstandings; she might hurl insults at me…so it is better to keep it to myself.’ (Participant 19, female, 41 years old) Research findings Six themes emerged from the data. The themes and categories that emerged from this study are summarised in Table 1. Open Access http://www.hsag.org.za Page 4 of 7 Original Research Another participant stated: ‘Our life is not the same … these days we rarely indulge in sexual intercourse. I have lost the sexual drive … he does not force me when I say I am tired … because I think he is also afraid.’ (Participant 9, female, 35 years old) TABLE 1: Themes and categories generated from the study. Themes Categories A need for quality life Pregnant women living with HIV expressed the following: • Acceptance of their own HIV status. • A need to postpone sexual activity. Realities of disclosure Pregnant women living with HIV reported both positive and negative experiences in dealing with disclosure: • Positive experience: • Safe disclosure. Hope for better results. Negative experiences: • Lack of trust. • Fear of the unknown. • Mixed spousal or sexual partners’ responses following disclosure. Perceived stigmatisation Pregnant women living with HIV expressed fear of stigma and negativity from: • In-laws following disclosure. • Family, friends and the community. • Lack of privacy for provision of HIV services. Knowledge deficit related to pregnancy and HIV progression in pregnancy Pregnant women living with HIV expressed a lack of knowledge, including: • Fear of transmitting HIV to the infant during breastfeeding. • Inadequate knowledge on infant feeding options. • How ARVs work in their bodies regarding exclusive breastfeeding. Knowledge deficit related to continuity of care and support Pregnant women living with HIV expressed: • Inadequate knowledge in relation to how to access ARVs. • Inadequate knowledge in relation to care by midwives while in labour. Pregnant women living with HIV also reported the lack of psychosocial or emotional support as a challenge in relation to: • Inadequate knowledge of support groups. • An inadequate supportive environment. Empowerment by midwives Pregnant women living with HIV reported midwives as supportive as: • Empowered with knowledge on HIV and available treatment. Giving hope for: • An improved quality of life. HIV, human immunodeficiency virus; ARV, antiretroviral. Another participant stated: ‘Our life is not the same … these days we rarely indulge in sexual intercourse. One participant had this to say: ‘The challenge is that we are all seen in the same room for all services i.e. those who are having blood collected for CD4 count, exposed children who have come for DBS and those collecting medications for prophylaxis for the mother and baby. Masvingo is a very small town; you may be staying in Rujeko or Mucheke Township; so these are the people who will tell others about your HIV positive status.’ (Participant 11, female, 33 years old) ‘… I was informed about support groups but I am not sure of how I can gain access to the support groups … The midwife just told me to go on my own.’ (Participant 18, female, 27 years old) Another participant said: Participants had concerns about how ARVs work during breastfeeding. A participant said: ‘My life is the same following accepting my condition … these days it is not scary because now I know that for the rest of my life I will be taking ARVs … I have seen many people who have been sick; and after taking ARVs they are now looking healthy … you can’t even tell that they are living with HIV.’ (Participant 12, female, 18 years old) ‘… I am going to breast feed my baby because I was informed by the midwives that the ARV prophylaxis only works in babies who are breastfeeding exclusively.’ (Participant 12, female, 18 years old) ‘… I am going to breast feed my baby because I was informed by the midwives that the ARV prophylaxis only works in babies who are breastfeeding exclusively.’ (Participant 12, female, 18 years old) Another participant echoed: ‘I have heard about HIV and AIDS but I do not really understand what it is.’ (Participant 4, female, 25 years old) ‘I have heard about HIV and AIDS but I do not really understand what it is.’ (Participant 4, female, 25 years old) ‘If I follow the advice I received from the midwives it will be possible for me to survive.’ (Participant 1, female, 30 years old) Participants had concerns about how ARVs work during breastfeeding. A participant said: Participants had concerns about how ARVs work during breastfeeding. A participant said: Another participant responded with a question: Another participant responded with a question: ‘Do you think that the midwives will be free to touch me during labour?’ (Participant 12, female, 18 years old) ‘Do you think that the midwives will be free to touch me during labour?’ (Participant 12, female, 18 years old) Participants expressed inadequate knowledge about support groups and an inadequate supportive environment as they reported not being aware of where to find support groups, or having fear of accessing them on their own. Thus, participants did not attend the meetings, abandoned the meetings or stopped attending subsequent support group meetings. One participant had this to say: Another participant echoed: ‘You should be happy that a timely diagnosis has been made because you will get help to start taking ARVs early and you will remain healthy and if you are sick you will actually recover well.’ (Participant 20, female, 21 years old) ‘You should be happy that a timely diagnosis has been made because you will get help to start taking ARVs early and you will remain healthy and if you are sick you will actually recover well.’ (Participant 20, female, 21 years old) Participants expressed inadequate knowledge about how to access ARVs. One participant claimed: ‘… I am not worried about my status because I am already taking ARVs … but I am not sure of whether I will continue taking ARVs.’ (Participant 17, female, 25 years old) Another participant mentioned: ‘I have been to one support group but on the day I attended the meeting; no one noticed my presence and what was being discussed there did not affect me so I left before the end of the meeting … since then I have not attended any other support group meeting.’ (Participant 19, female, 41 years old) ‘I have been to one support group but on the day I attended the meeting; no one noticed my presence and what was being discussed there did not affect me so I left before the end of the meeting … since then I have not attended any other support group meeting.’ (Participant 19, female, 41 years old) Participants expressed a lack of knowledge in relation to pregnancy and HIV progression in pregnancy, as highlighted by a persistent fear of transmitting HIV to their babies and inadequate knowledge about infant feeding options in relation to HIV and ARVs. One participant stated: Theme 6: Empowerment by midwives ‘The midwives said that we should breastfeed only …. the reason is that the medications which are administered to exposed babies as prophylaxis against HIV are only effective in children feeding on breast milk only … I want to protect my baby from HIV infection.’ (Participant 15, female, 25 years old) Participants reported midwives being supportive and they were hopeful for an improved quality of life attributed to taking ARVs and their ability to make informed decisions on issues related to living positively with HIV. Another participant echoed: Another participant said: ‘I am afraid to tell people because I do not know what the people will say about me when they get to know that I am living with HIV.’ (Participant 12, female, 18 years old) ‘I am not sure of how I will be treated by midwives when they know that they are conducting a delivery on a patient living with HIV... I am not sure of how I can address this.’ (Participant 11, female, 33 years old) The participants indicated that the environment did not provide adequate privacy, because all clients collecting ARVs for prophylaxis, women who bring their HIV exposed babies for dry blood testing (DBS) at 6 weeks, and pregnant women who recently tested positive were served in the same room which was crowded and did not allow for privacy. Theme 3: Perceived stigmatisation Participants reported lifestyle adjustments ranging from engaging in protected intimacy to the postponement of intimacy. A participant said: Participants expressed a fear of stigma and negativity emanating from their in-laws and the community. One participant revealed: ‘We were advised by midwives at the clinic to use condoms when indulging in sexual intercourse….’ (Participant 4, female, 25 years old) ‘I cannot disclose to my in laws…I am afraid of being labelled and stigmatised by my in-laws.’ (Participant 2, female, 26 years old) http://www.hsag.org.za Open Access Page 5 of 7 Page 5 of 7 Original Research Participants also expressed a fear of neglect by midwives when they report to the hospital in labour. One participant said: Another participant said: Discussion Another participant agreed: Another participant agreed: Another participant agreed: The study findings indicate that most participants reported acceptance of their HIV status. Their acceptance of their positive HIV status was mainly based on the knowledge that ‘… I do not know where I will get the ARVs to continue with my treatment.’ (Participant 4, female, 25 years old) Open Access http://www.hsag.org.za Page 6 of 7 Original Research Page 6 of 7 Original Research Page 6 of 7 and sero-status disclosure, Nachega et al. (2012:176) claim that participants stated that HIV-related stigma was still rife and affects the quality of life for people living with HIV, including their determinants to disclose for fear of discrimination. treatment is now available to control opportunistic infections, thereby making a contribution to an improved quality of life. Participants also reported that they had observed some people with advanced disease who responded well to ARVs. As a result, participants who had knowledge of someone living with HIV were likely to accept their status and have hope for survival. Nam et al. (2008) reported that having confidence from relatives, friends as well as clinicians and one’s religious grouping contributes to promoting hope and acceptance of an HIV-positive status, thereby enabling patients to develop a positive therapeutic relationship with their ARVs and make lifestyle changes that promote adherence. Mucheto et al. (2011) indicate that women in the PMTCT programme following HIV counselling and testing reported having not been referred for any form of support after being tested. This is in agreement with Bharat and Mahendra (2007:119), who conveyed that HIV-positive people are still under-utilising care and support services. According to these authors, persons living with HIV and AIDS (PLWHAs) can be more effective counsellors for recently diagnosed individuals to confront sexual and reproductive health issues and reduce HIV- and AIDS-related shame and stigma. This is consistent with Muchtler et al. (2011:79), who indicate that treatment advocacy has been reported to complement medical and other social services to prepare people living with HIV on how to access appropriate health care services. Pregnant women living with HIV reported both positive and negative experiences in dealing with disclosure. Positive disclosure beliefs have been reported as significantly associated with lower stigma, greater self-esteem, lower depressive symptoms and better quality of life (Patel et al. 2011:364). The findings in this study indicated that positive experiences following disclosure ranged from safe disclosure, hope for better results and hope in religion. Limitations of the study The study was contextual and the results cannot be generalised. Data were collected through in-depth face-to-face interviews, which were audio-taped. This can contribute to participants providing socially desirable responses. Alternatively, they could also have found the use of a voice recorder as intimidating. Both in-depth interviews and naïve sketches could have been used to generate rich data about the phenomenon under investigation. The researcher addressed the latter by adhering to the ethical principles that were observed. Pregnant women living with HIV expressed a fear of being stigmatised as well as the possibility of breach of confidentiality following disclosure to family, in-laws, friends and the community. Nachega et al. (2012:176) indicate that perceptions of HIV-infected individuals show that people in settings with either a high or low HIV prevalence rate continue to perceive HIV-related stigma. This impacts on their willingness to disclose their HIV status to others and their quality of life. The same authors further report that about three-quarters to a third of people living with HIV experience depression, which is attributed to self- stigmatisation and fear of discrimination. All interviews were conducted and analysed in vernacular Shona and then translated into English. To minimise loss of information during the translation process, verbatim quotes were used, and both an independent coder and a language person well versed in Shona and English were engaged. Another participant agreed: Alternatively, negative experiences ranged from lack of trust, non-decisiveness to disclose, the ‘waiting game’ or waiting period hoping for a better cluster of differentiation 4 (CD4) count, to mixed spousal and sexual partners’ responses following disclosure. CD4 cells are white blood cells which are destroyed by he HIV hence the need to count them in people infected by HIV as they are likely to drop due to destruction by the HIV virus. References Bharat, S. & Mahendra, V.S., 2007, ‘Meeting the sexual and reproductive health needs of people living with HIV: Challenges for health care providers’, Reproductive Health Matters 25(29), 93–112. https://doi.org/10.1016/S0968-8080(07)29030-5 Both, J.M.C. & Van Roosmalen, J., 2010, ‘The impact of prevention of mother to child transmission programmes on maternal health care in resource – Poor settings: Looking beyond the PMTCT programme – A systemic review’, An International Journal of Obstetrics and Gynaecology 117, 1444–1450. https://doi.org/10.1111/ j.1471-0528-2010.02692.x The intention of the bigger study was to develop a holistic care model for use by midwives to provide holistic care to pregnant women who become aware of their HIV-positive status during pregnancy as a result of PMTCT. In this study, the researcher defined holistic care as the care that addresses the physical, psychological, sexual and social needs of the pregnant women who test positive for HIV during pregnancy. This implies that as the midwife provides care related to HIV infection in pregnancy, the midwife should identify the physical, psychological, social, mental and sexual needs of the pregnant woman living with HIV and address these needs. Creswell, J.W., 1994, Research design quantitative and qualitative approaches, Sage, California. Inner City Fund International (ICF), 2012, HIV prevalence estimates from the demographic and health surveys, ICF International, Calverton, MD, viewed 12 September 2013, from http://www.measuredhs.com/pubs/pdf/OD65/OD65.pdf International Confederation of Midwives (ICM), 2011, International definition of the midwife, viewed 14 August 2013, from http://www.internationalmidwives.org/ who-we-are/policy-and-parctice/icm-international/definition Lincoln, Y.S. & Guba, E.G., 1985, Naturalistic enquiry, Sage, Beverly Hills, CA. McDonald, K. & Kirkman, M., 2011, ‘HIV positive women in Australia explain their use and non-use of antiretroviral therapy in preventing mother-to-child transmission’, AIDS Care: Psychological and Socio-medical Aspects of AIDS/HIV 23(5), 578–584. https://doi.org/10.1080/09540121.2010.482124 ‘Facilitating quality of life through empowerment and support’ was regarded as the main concept of the proposed model for use by midwives to provide holistic care to pregnant women who test positive for HIV during pregnancy. The midwife facilitates the process for the pregnant woman to be constantly engaging her ‘self’ to seek appropriate health care resources and adopt appropriate health-seeking behaviours to keep herself healthy after knowing her HIV-positive status. Such self-care by the pregnant women living with HIV ultimately translates to empowerment. Competing interests Shetty, A.K., Marangwanda, C., Stranix-Chibanda, L., Chandisarewa, W., Chirapa, E., Mahomva, A. et al., 2008, ‘The feasibility of preventing mother to child transmission of HIV using peer counselors in Zimbabwe’, AIDS Research and Therapy 5, 17, viewed 25 July 2011, from http://www.ncbi.nlm.nih.gov/pmc/ articles/PMC2517064/ The authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. Southern African Development Community (SADC), Regional BLTS HTA (CBS/HTS)STI/ HIV and AIDS, Strategy and program of action, September 2003–2006. UNAID, 2016, Global AIDS update, viewed n.d., from http://www.unaids.org/en/ regionscountries/countries/zimbabwe/ Conclusions and recommendations Empowerment by midwives was reported to contribute to behaviour change and hope for a quality life. Participants realised that there was hope for a better quality of life beyond a diagnosis of HIV infection, though most of the participants were still processing information in an effort to promote their health. Some of the participants postponed sexual intercourse or stopped having sexual intercourse, while others used condoms. Mcdonald and Kirkman (2011:580), in a study determining use and non-use of ARVs for PMTCT in Australia, reported that the women who participated in the study revealed that taking ARVs was quite distressing and ‘a daily reminder of a serious, stigmatising illness’. However, these same women found it helpful when they re-framed the treatment as helpful to ensure an HIV-negative baby. This is consistent with Nachega et al. (2012:175), who cited five common HIV- or AIDS-related stigmas affecting respondents, including: Lack of stigmatisation and increasing access to ARVs have contributed to an improved health status in people living with HIV. Participants realised the benefit of ARVs and managed to surpass the stigma attached to HIV, even though some of the participants had a fear of stigmatisation from their relatives, friends and in-laws, which contributed to delayed disclosure. people living with HIV/AIDS do not live a long time, people living with HIV/AIDS should be avoided, people living with HIV/AIDS look different and HIV/AIDS is easily transmitted through normal everyday life. Fear of the care participants will receive from the midwives during labour was evident among participants. This implies that pregnant women living with HIV should be given Roopnaraine et al. (2011:652) state that there is stigma attached to joining a group consisting of people living with HIV infection. In a global survey on HIV-related stigma, isolation http://www.hsag.org.za Page 7 of 7 Original Research Page 7 of 7 relevant information explaining the care they will receive in the maternity unit during the process of childbirth to dispel misconceptions. References These women are empowered to reflect on self, basing their reflections on the information and resources that are available to them to facilitate the adoption of appropriate health promotion behaviours which will help them to achieve optimum health. Ministry of Health and Child Welfare (MOH & CW), 2006a, The Zimbabwe programme for prevention of mother to child transmission of HIV, Annual report, 2006, Government Printers, Harare. Ministry of Health and Child Welfare (MOH & CW), 2006b, Trainer’s manual for the integrated approach to HIV and AIDS prevention, care, treatment and follow up for pregnant women, their babies and families: Prevention of mother to child transmission of HIV in Zimbabwe, 2nd edn., Government Printers, Harare. Ministry of Health and Child Welfare (MOH & CW), 2007, The Zimbabwe programme for prevention of mother to child transmission of HIV, Annual Report, 2007, Government Printers, Harare. Ministry of Health and Child Welfare (MOH & CW), 2008, A trainer’s manual for the integrated approach to HIV and AIDS prevention, care, treatment and follow up for pregnant women their babies and families: Module 9: More efficacious ARV prophylaxis for the prevention of mother to child transmission programme in Zimbabwe, Government of Zimbabwe, Harare. Mucheto, P., Chadambuka, A., Shambira, G., Tshimanga, M., Notion, G. & Nyamayaro, W., 2011, ‘Determinants of disclosure of HIV status among women attending the prevention of mother to child transmission programme, Makonde district, Zimbabwe’, Pan African Medical Journal 8(51), viewed 20 January 2013, from http://www.ncbi.nlm.nih./gov/pmc/articles/PMC3201613 Muchtler, M.G., Wagner, G., Cowgill, B.O., McKay, T., Risley, B. & Bogart, L.M., 2011, ‘Improving HIV/AIDS care through treatment and advocacy: Going beyond client education to empowerment by facilitating client provider relationships’, AIDS Care: Psychological and Socio-Medical Aspects of AIDS/HIV 23(1), 79–90. https:// doi.org/10.1080/09540121.2010.496847 Acknowledgements Nachega, J.M., Morroni, C., Zuniga, J.M., Sherer, R., Beyrer, C., Solomon, S. et al., 2012, ‘HIV related stigma, isolation, discrimination and serostatus disclosure: A global survey of 2035 HIV–Infected adults’, Journal of the International Association of Physicians in AIDS Care (JIAPAC) 11(3), 172–178, viewed 20 June 2012, from http://jia.sagepub.com/content/11/3/172 The authors would like to acknowledge the management and staff of the provincial hospital particularly the Acting Medical Superintendent and staff from the Maternal Child Department and PMTCT unit for allowing access to the study site, the Sister in Charge of the MCH department and the PMTCT unit for assisting with identifying the participants. Great appreciation goes to the pregnant women for sharing their most intimate and sensitive information with us with regard to their private lives. Nam, S.L., Fielding, K., Avalos, A., Dikinson, D., Gaolathe, T. & Geissler, P.W., 2008, ‘The relationship of acceptance or denial of HIV-status to antiretroviral adherence among adult HIV patients in urban Botswana’, Social Science Medicine 67(2), 301–310, viewed 13 May 2012, from http//www.ncbi.nlm.nih.gov/pubmed/18455285 Patel, R., Ratner, J., Gore-Felton, C., Kadzirange, G., Woelk, G. & Katzenstein, D., 2011, ‘HIV disclosure patterns, predictors and psychosocial correlates among HIV positive women in Zimbabwe’, AIDS Care: Psychological and Socio-medical Aspects of AIDS/HIV 24(3), 358–368. https://doi.org/10.1080./09540121.2011.608786 Roopnaraine, T., Rawat, R., Babirye, F., Ochai, R. & Kadiyala, S., 2011, ‘“The group” in integrated HIV and livelihoods programming: Opportunity or challenge?’, AIDS Care: Psychological and Socio-medical Aspects of AIDS/HIV 24(5), 649–657. https://doi.org/10.1080/09540121.2011.630349 Authors’ contributions Zimbabwe National Statistics Agency (ZIMSTAT), 2012, Census. National report, Population Census Office, Harare, Zimbabwe, viewed 12 September 2013, from http://www.zimstat.co.zw/index.php?option=com_content&view=article&id=65: census This article is based on the PhD study of the first author, O.B.T. A.G.W.N. and A.T. supervised the study. http://www.hsag.org.za Open Access
https://openalex.org/W3083613115	https://ejurnal.ars.ac.id/index.php/jti/article/download/272/226	Indonesian	null	ANALISIS KEPUASAN PENGGUNA SHOPEEPAYLATER MENGGUNAKAN MODEL DELONE & MCLEAN SEBAGAI MEDIA PENGAJUAN KREDIT ONLINE	Jurnal Responsif : Riset Sains dan Informatika	2,020	cc-by-sa	2,787	1Universitas Adhirajasa Reswara Sanjaya email: ali@ars.ac.id 2Universitas Adhirajasa Reswara Sanjaya email: dedekartikadewi@gmail.com 2Universitas Adhirajasa Reswara Sanjaya email: dedekartikadewi@gmail.com 3Universitas Adhirajasa Reswara Sanjaya email: iedam@ars.ac.id 3Universitas Adhirajasa Reswara Sanjaya email: iedam@ars.ac.id Abstrak Industri keuangan tradisional memiliki keterbatasan karena memiliki aturan yang ketat dalam melayani masyarakat di daerah tertentu. Sebagai konsikuensinya masyarakat mencari alternatif pendanaan selain jasa industri tradisional seperti bank, yang lebih transparan dan memiliki layanan keuangan yang efisien serta dapat menjangkau berbagai level. Dengan hadirnya ShopeePayLater diharapkan dapat memudahkan para pengguna yang sedang melakukan transaksi di Shopee. Tujuan Penelitian ini untuk melihat hubungan antara variabel-variabel terhadap kepuasan pengguna ShopeePayLater di Kota Bandung menggunakan model DeLone & McLean. Pada penelitian ini, peneliti hanya menggunakan 5 variabel yaitu, Kualitas Sistem (System Quality), Kualitas Informasi (Information Quality), Kualitas Layanan (Service Quality), Penggunaan (Use), dan Kepuasan Pengguna (User Satisfaction). Teknik pengambilan sampel yang digunakan adalah pendekatan convinience sampling. Hasil penelitian yang diperoleh adalah variabel Kualitas Sistem (System Quality) (X1), Kualitas Informasi (Information Quality) (X2), Penggunaan Pengunaan (Use) (X3), dan Kualitas Layanan (Service Quality) (X4) secara bersama-sama atau simultan berpengaruh terhadap Kepuasan Pengguna (User satisfaction) (Y). Kata Kunci: Kepuasan Pengguna, Model DeLone & McLean, PayLater, ShopeePayLater Keywords: User Satisfaction, DeLone & McLean Model, PayLater, ShopeePayLater JURNAL RESPONSIF, Vol. 2 No.2 Agustus 2020, pp. 191~197 E ISSN: 2685 6964 JURNAL RESPONSIF, Vol. 2 No.2 Agustus 2020, pp. 191~197 E ISSN: 2685 6964 191 Abstract The traditional financial industry has limitations because it has strict rules in serving the community in certain areas. As a consequence the community is looking for alternative funding in addition to traditional industrial services such as banks, which are more transparent and have efficient financial services and can reach various levels. The presence of ShopeePayLater is expected to make it easier for users who are conducting transactions at Shopee. The purpose of this study was to look at the relationship between variables on ShopeePayLater user satisfaction in the city of Bandung using the DeLone & McLean model. In this study the researchers only used 5 variables, namely, System Quality, Information Quality, Service Quality, Use, and User Satisfaction. The sampling technique used is convinience sampling approach. The results obtained are variable System Quality (X1), Information Quality (X2), Use (X3), and Service Quality (X4) together or simultaneous effect on User Satisfaction (Y). Naskah diterima 29 Juli 2020; direvisi 5 Agustus 2020; diterbitkan 31 Agustus 2020 192 Theory of Acceptance and use of Technology (UTAUT) juga merupakan hasil sintesis beberapa teori perilaku penerima, motivasi dan penggunaan teknologi dimana pengaruh sosial memiliki pengaruh terhadap variabel- variabelnya (Darmawan, et al, 2019). 1. Pendahuluan Financial Technology atau biasa dikenal dengan nama Fintech, sebagai teknologi yang menjadi perantara dan penghubung antara masyarakat umum dan sektor finansial. Fintech menjadi salah satu solusi atas permasalahan industri keuangan tradisional yang tidak dapat melayanai masyarakat secara menyeluruh. Industri keuangan tradisional memiliki keterbatasan karena memiliki aturan yang ketat dalam melayani masyarakat di daerah tertentu. Sebagai konsikuensinya masyarakat mencari alternatif pendanaan selain jasa industri tradisional seperti bank, yang lebih transparan dan memiliki layanan keuangan yang efisien serta dapat menjangkau berbagai level (Adiningsih, et al, 2019). Model Delone & McLean digunakan untuk melakukan penelitian yang bertujuan untuk mengetahui faktor- faktor yang mempengaruhi kepuasan pengguna Layanan ShopeePayLater dengan menganalisis hubungan antara kualitas sistem dengan kepuasan pengguna, kualitas informasi dengan kepuasan pengguna, kualitas layanan terhadap pengguna, pengguna terhadap kepuasan pengguna. p p gg Secara Skematis, kerangka berfikir dalam penelitian ini dapat digambarkan sebagai berikut : , ) Shopee sebagai aplikasi mobile platform e-commerce 3 teratas di indonesia, didorong untuk selalu melakukan terobosan terbaru terhadap fitur-fitur yang mereka sediakan termasuk di bidang sistem pembayaran. Layanan Shopee juga menyediakan fasilitas transaksi non-tunai yakni fitur ShopeePayLater. ShopeePayLater ini memiliki sistem yang fungsi dan manfaat yang sama persis dengan kartu kredit. Konsep utama dari fitur pembayaran ini adalah 'beli sekarang bayar nanti'. Layanan ShopeePayLater bertujuan untuk memudahkan para pelanggan yang sedang melakukan kegiatan berbelanja atau berjualan di Shopee (Hadijah, 2019). Gambar 1. Kerangka Berfikir Gambar 1. Kerangka Berfikir Gambar 1. Kerangka Berfikir Pada gambar 1 dapat dilihat kerangka berfikir dari penelitian ini sebagai berikut : ( j ) Kepuasan Individu dalam pengunaan layanan ShopeePayLater mampu diukur dengan menggunakan teori yang dapat mengukur tingkat kepuasan pengguna terhadap suatu teknologi. Model DeLone & McLean dipilih oleh peneliti dalam penelitian ini karena pada model ini menguji kepuasan pengguna terhadap kualitas sistem, kualitas informasi, kualitas layanan, dan pengguna. Sementara model lain tidak memiliki kontruksi tersebut (Trihandayani, et al, 2018). Seperti model Technology Acceptance Model (TAM) yang meneliti persepsi pengguna terhadap teknologi dapat mempengaruhi sikapnya dalam penerimaan penggunaan teknologi, tetapi tidak bisa mengukur kepuasan pengguna terhadap sistem (Destianti, et al, 2019). Model unified g 1. Variabel kualitas sistem (X1) akan berpengaruh positif dan signifikan terhadap variabel kepuasan pengguna (Y). p gg ( ) 2. Variabel kualitas informasi (X2) akan berpengaruh positif dan signifikan terhadap variabel kepuasan pengguna (Y) p gg ( ) 3. Variabel penggunaan (X3) akan berpengaruh positif dan signifikan terhadap variabel kepuasan pengguna (Y) p gg 4. Variabel kualitas layanan (X4) akan berpengaruh positif dan signifikan terhadap variabel kepuasan pengguna (Y) p gg ( ) 5. Variabel kualitas informasi (X2), kualitas informasi (X2), penggunaan http://ejurnal.ars.ac.id/index.php/jti 193 (X3), dan kualitas layanan (X4) secara bersama-sama atau simultan akan berpengaruh positif dan signifikan terhadap variabel kepuasan pengguna (Y) (X3), dan kualitas layanan (X4) secara bersama-sama atau simultan akan berpengaruh positif dan signifikan terhadap variabel kepuasan pengguna (Y) dan dianalisis. Dari hasil pengolahan data yang telah dilakukan maka data dianalisis lebih lanjut dengan menggunakan alat-alat analisis yang sesuai dengan tujuan riset agar menghasilkan kajian yang cukup akurat, mendalam dan luas. 2. Metode Penelitian Hasil kajian ini dilengkapi dengan penjelasannya. Alat yang digunakan oleh peneliti adalah analisis deskriptif dan analisis statistik. Setelah peneliti melakukan analisis data terhadap objek penelitian, maka peneliti membuat kesimpulan dan saran. Dimana kesimpulan yang ditarik berdasarkan acuan hipotesis yang telah dibuat sebelumnya. Saran yang disajikan oleh peneliti mengacu pada hasil akhir ppenelitian. Karena penelitian yang dilakukan memiliki keterbatasan- keterbatasan atau asumsi-asumsi. Penelitian ini diawali dengan Peneliti mengidentifikasi dan merumuskan masalah dengan bentuk pertanyaan yang dijawab pada maksud penelitian. Permasalahan yang diangkat pada penelitian ini yaitu anaslisis kepuasan pengguna ShopeePayLater dengan tujuan untuk mengetahui tingkat kepuasan pengguna ShopeePayLater sebagai media kredit online menggunakan Model DeLone & McLean. Setelah itu peneliti merumuskan hipotesis. Hipotesis dalam penelitian ini bersifat dugaan sementara terhadap objek penelitian pengguna ShopeePayLater di Kota Bandung, dimana hipotesis yang diambil dalam penelitian ini didasarkan pada penggunaan variabel seperti kualitas sistem (system quality), kualitas informasi (information quality), kualitas layanan (service quality), penggunaan (use), dan kepuasan pengguna (user satisfiction) yang terdapat dalam Model DeLone & McLean. Berikut Gambar dari tahapan penelitian secara keseluruhan dapat dilihat pada gambar 2: Gambar 2. Tahapan Penelitian Peneliti mengumpulkan data untuk bahan pengolahan data penelitian berupa data primer seperti kuisioner berbentuk google form yang disebarkan kepada responden melalui link tautan alamat kuisioner https://forms.gle/5mwNodWn4FJE4rVB6 tersebut untuk mendapatkan tanggapan dari responden terkait indikator-indikator dari masing-masing variabel Model DeLone & McLean yang digunakan diantaranya kualitas sistem (system quality), kualitas informasi (information quality), kualitas layanan (service quality), penggunaan (use), dan kepuasan pengguna (user satisfiction) terhadap responden pengguna ShopeePayLater di Kota Bandung. Gambar 2. Tahapan Penelitian Populasi pada penelitian ini adalah pengguna layanan ShopeePayLater di kota bandung. Teknik yang digunakan untuk pengambilan sampel minimun adalah teknik pendekatan Non Probability Sampilng dengan menggunakan teknik convenience sampling. Metode ini dipilih oleh penulis karena jumlah populasi yang tidak diketahui sehingga peneliti memiliki kebebasan untuk memilih sampel yang paling cepat. Maka dari itu, dalam penentuan jumlah sampel peneliti menggunakan rumus lameshow, yaitu: Langkah selanjutnya setelah mengumpulkan data adalah mengolah dan menyajikan data. Data yang telah terkumpul diolah sehingga menghasilkan informasi dari objek penelitian. Setelah data diolah maka peneliti menyajikan data atau menyusun data agar lebih teratur sehingga mudah dibaca, dipahami http://ejurnal.ars.ac.id/index.php/jti 194 Keakuratan Informasi Informasi pembayaran yang dihasilkan setelah menggunakan ShopeePayLater akurat dan bebas kesalahan. Kelengkapan Informasi Informasi yang dihasilkan dari transaksi dengan menggunakan ShopeePayLater lengkap dan jelas. Penggunaan (use) Penggunaan Sehari-hari Menggunakan ShopeePayLater setiap transaksi. http://ejurnal.ars.ac.id/index.php/jti A. Uji validitas Uji validitas dilakukan yang dalam penelitian ini menggunakan rumus korelasi Bivariate pearson dimana data pertanyaan dikatakan valid jika memilki r hitung > r tabel. Nilai r tabel pada penelitian ini adalah 0,195. Gambar 3. Hasil Uji Normalitas , Tabel 2. Hasil Uji Validitas j Kode r hitung Keterangan X1.1 0,816 Valid X1.2 0,860 Valid X2.1 0,615 Valid X2.2 0,770 Valid X2.3 0,718 Valid X3.1 0,887 Valid X3.2 0,893 Valid X3.3 0,821 Valid X4.1 0,796 Valid X4.2 0,863 Valid Y1.1 0,745 Valid Y1.2 0,888 Valid Y1.3 0,844 Valid Y1.4 0,873 Valid 3.3 Uji Asumsi Klasik A. Uji Normalitas Hasil dan pembahasan merupakan bagian yang membahas dan menjelaskan data yang terkumpul dari hasil penelitian yang meliputi uji instrumen: uji validitas dan uji reliabilitas, uji asumsi klasik: uji normalitas, uji autokolerasi, uji multikolinearitas, dan uji heteroskedastisitas, dan regresi linear berganda: uji hipotesis. Uji normalitas digunakan untuk menguji apakah data penelitian terdistribusi norma atau tidak. Data akan dinyatakan normal apabila titik-titik pada grafik menyabar disekitar garis dan mengikuti garis diagonal. Gambar 3. Hasil Uji Normalitas B. Uji Autokolerasi Uji autokolerasi bertujuan untuk mencari tahu apakah suatu data pada periode tertentu berkolerasi dengan periode lainnya. Metode pengujian yang digunakan adalah uji Durbin-Watson (uji DW) dengan ketentuan jika nilai 1 < DW > 3 tidak terjadi autokolerasi. j Tabel 4. Hasil Uji Autokolerasi Model Summaryb Model R R Square Durbin- Watson 1 ,773a ,597 1,880 a. Predictors: (Constant), Total X4, Total X3, Total X2, Total X1 b. Dependent Variable: Total Y B. Uji Reliabilitas Uji reliabilitas menggunakan teknik Cronbach’s alpha untuk menentukaan reliabilitas instrumen. Instrumen dikatakan reliabel jika nilai alpha > nilai r tabel. B. Uji Reliabilitas Uji reliabilitas menggunakan teknik Cronbach’s alpha untuk menentukaan reliabilitas instrumen. Instrumen dikatakan reliabel jika nilai alpha > nilai r tabel. Tabel 3. Hasil Uji Reliabilitas Tabel 3. Hasil Uji Reliabilitas No. Item r hitung r tabel 5% (100) Keterangan X1 0,575 0,195 Reliabel X2 0,545 0,195 Reliabel X3 0,825 0,195 Reliabel X4 0,544 0,195 Reliabel Y 0,846 0,195 Reliabel 2. Metode Penelitian Frekuensi penggunaan Dalam satu bulan sering menggunakan ShopeePayLater Sifat pengguna Saya sering menggunakan ShopeePayLater untuk mempermudah pembayaran saat transaksi berlangsung. Kualitas Layanan (service quality) Resposif Menampilkan informasi transaksi sesuai dengan yang pengguna perlukan secara cepat dan tepat. Jaminan Selama menggunakan ShopeePayLater, pengguna merasa nyaman dan aman dalam melakukan transaksi. Kepuasan Pengguna (user satisfication) Konten Rasa puas terhadap ShopeePayLater karena Komponen dan isi sistem secara menyeluruh. Akurasi Rasa puas terhadap ShopeePayLater karena ketepatan sistem pembayaran dalam menerima input hingga mengeluarkan output. Kemudahan Rasa puas terhadap ShopeePayLater karena langkah- langkah pengajuan kredit dan penggunaannya mudah untuk dimengerti dan dilakukan. waktu Rasa puas terhadap ShopeePayLater karena efektivitas dan efisiensi yang dihasilkan melalui waktu yang dibutuhkan dalam proses transaksi. Sumber : Stanley Lameshow Sumber : Stanley Lameshow Keterangan: n = Jumlah Sampel z = Skor z pada kepercayaan 95%= 1,96 p = maksimal estimasi = 0,5 d = alpa (0,10) atau sampling= 10% Keterangan: n = Jumlah Sampel z = Skor z pada kepercayaan 95%= 1,96 p = maksimal estimasi = 0,5 d = alpa (0,10) atau sampling= 10% p , d = alpa (0,10) atau sampling= 10% Menghitung Sampel Penelitian : n= n= n= n= 96,04 = 100 Sehingga jika berdasarkan rumus diatas maka n yang didapatkan adalah 96,04= 100 orang. Sehingga pada penelitian ini penulis harus mengambil data dari sampel setidaknya sekurang-kurangnya 100 orang. Pengolahan hasil penelitian ini dimulai dengan melakukan uji coba instrumen dengan sample penelitian sebanyak 100 orang responden. Kuisioner berisikan pertanyaan berupa pernyataan yang tersusun dari variabel kualitas sistem (X1), kualitas informasi (X2), penggunaan (X3), kualitas layanan (X4), dan kepuasan pengguna (Y) yang setiap indikatornya merupakan referensi dari penelitian sebelumnya. Tabel 1. Operasionalisasi Variabel http://ejurnal.ars.ac.id/index.php/jti 195 g j p 3.1. Pengumpulan Data g j p 3.1. Pengumpulan Data Penelitian ini menggunakan data hasil penelitian yang diperoleh dari penyebaran kuisioner yang berupa google form. Responden terbayak jika dilihat berdasarkan jenis kelamin adalah perempuan sebesar 67%, sedangkan laki-laki sebesar 33%. 3.2. Uji Instrumen A. Uji validitas C. Uji Multikolinearitas C. Uji Multikolinearitas Uji multikolinearitas digunakan untuk menguji ada atau tidaknya multikolinearitas yang dapat dilihat melalui Variance Inflation Factor (VIF). Nilai VIF < 10 dan nilai tolerance > 0,1 maka dapat disimpulkan tidak terjadi multikolinearitas. http://ejurnal.ars.ac.id/index.php/jti 196 Tabel 5. Hasil Uji Multikolinearitas Coefficientsa Model Sig. Collinearity Statistics Tolerance VIF (Constant) ,505 Total X1 ,000 ,625 1,601 Total X2 ,000 ,755 1,324 Total X3 ,014 ,783 1,278 Total X4 ,121 ,521 1,918 a. Dependent Variable: Total Y Tabel 5. Hasil Uji Multikolinearitas Coefficientsa D. Uji Heteroskedastisitas heteroskedastisitas ini apabila titik-titik menyebar diatas dan bawah angka 0 pada sumbu y sehingga dapat disimpulkan bahwa tidak terjadi masalah heteroskedastisitas. C. Uji Koefisien Determinasi http://ejurnal.ars.ac.id/index.php/jti C. Uji Koefisien Determinasi Uji keofisien determinasi bertujuan untuk mengetahui besarnya sumbangan atau kontribusi dari variabel bebas terhadap variabel terkait. Gambar 4. Hasil Uji Heteroskedastisitas Gambar 4. Hasil Uji Heteroskedastisitas Tabel 8. Persentase hasil uji koefisien determinasi Tabel 8. Persentase hasil uji koefisien determinasi No Nama Variabel Presentase 1 X1 (KS) 42,3% 2 X2 (KI) 32% 3 X3 (P) 22,4% 4 X4 (KL) 36,3% No Nama Variabel Presentase 1 X1 (KS) 42,3% 2 X2 (KI) 32% 3 X3 (P) 22,4% 4 X4 (KL) 36,3% Gambar 4. Hasil Uji Heteroskedastisitas D. Uji Heteroskedastisitas Ketentuan dalam uji D. Uji Heteroskedastisitas Ketentuan dalam uji B. Uji F U Uji F digunakan untuk menguji bagaimana pengaruh semua variabel bebas secara bersama-sama terhadap variabel terkait. Nilai sig < 0,05 atau f hitung > f tabel maka terdapat pengaruh variabel X terhadap variabel Y. p Tabel 7. Hasil Uji F ANOVAa Model F Sig. Regression 35,161 ,000b Residual Total a. Dependent Variable: Total Y b. Predictors: (Constant), Total X4, Total X3, Total X2, Total X1 p Tabel 7. Hasil Uji F p Tabel 7. Hasil Uji F a. Dependent Variable: Total Y Saran Hadijah, S. (2019, Oktober 16). Aplikasi Layanan Pay Later Makin Diminati, Yuk Cek Keuntungan dan Kerugiannya. Dipetik Desember 6, 2019, dari Cermati.Com: www.cermati.com Berdasarkan hasil penelitian yang telah dilakukan peneliti memberikan beberapa saran sebagai berikut : 1. Bagi pihak Shopee, dapat meningkatkan kualitas layanan dapat ditingkatkan agar perangkat lebih dapat diandalkan oleh pengguna. Selain itu juga diharapkan agar terus berinovasi dengan menghadirkan berbagai fitur yang dapat mendorong minat pengguna untuk menggunakan fiur ShopeePayLater pada aplikasi Shopee. Trihandayani, L. H., Aknuranda, I., & Mursityo, Y. T. (2018). Penerapan Model Kesuksesan Delone dan Mclean pada Website Fakultas Ilmu Komputer (FILKOM) Universitas Brawijaya. Jurnal Pengembangan Teknologi Informasi dan Ilmu Komputer, Vol. 2, No. 12, Desember, 7074- 7082. 2. p 2. Bagi penelitian selanjutnya, dapat menambahkan variabel-variabel dan model/metode lain selain variabel dan metode yang telah diuji pada penelitian ini guna memperolah perbandingan hasil kesimpulan. j A. Uji T p Dari hasil analisis dengan menggunakan model DeLone & McLean maka dapat dibuat kesimpulan: j Uji T aatu uji parsial digunakan untuk menguji bagaimana pengaruh masing- masing variabel bebas terhadap variabel terikat. Nilai sig < 0,05 atau t hitung < t tabel maka terdapat pengaruh variabel X terhadap variabel Y. 1. Variabel Kualitas Sistem (System Quality) secara parsial memiliki pengaruh positif signifikan terhadap Kepuasan Pengguna (User satisfaction). Tabel 6. Hasil Uji T Tabel 6. Hasil Uji T ) 2. Variabel Kualitas Informasi (Information Quality) secara parsial memiliki pengaruh positif signifikan terhadap Kepuasan Pengguna (User satisfaction). ) 3. Variabel Pengunaan (Use) secara parsial memiliki pengaruh positif signifikan terhadap Kepuasan Pengguna (User satisfaction). 4. Variabel Kualitas Layanan (Service Quality) secara parsial tidak memiliki pengaruh positif signifikan terhadap Kepuasan Pengguna (User satisfaction). http://ejurnal.ars.ac.id/index.php/jti 197 Beharvior terhadap Minat Pengguna Produk EMoney (Go- Pay) (Studi Kasus pada Mahasiswa Fakultas Syariah Prodi Hukum Ekonomi Syariah Fakultas Syariah Angkatan 2015- 2016 Universi. Keuangan dan Perbankan Syariah, Volume 5, No. 2, 312-319. Beharvior terhadap Minat Pengguna Produk EMoney (Go- Pay) (Studi Kasus pada Mahasiswa Fakultas Syariah Prodi Hukum Ekonomi Syariah Fakultas Syariah Angkatan 2015- 2016 Universi. Keuangan dan Perbankan Syariah, Volume 5, No. 2, 312-319. 5. Variabel Kualitas Sistem (System Quality) (X1), Kualitas Informasi (Information Quality) (X2), Penggunaan Pengunaan (Use) (X3), Kualitas Layanan (Service Quality) (X4) secara bersama-sama atau simultan berpengaruh terhadap Kepuasan Pengguna (User satisfaction) (Y). S 5. Variabel Kualitas Sistem (System Quality) (X1), Kualitas Informasi (Information Quality) (X2), Penggunaan Pengunaan (Use) (X3), Kualitas Layanan (Service Quality) (X4) secara bersama-sama atau simultan berpengaruh terhadap Kepuasan Pengguna (User satisfaction) (Y). S Referensi Adiningsih, S., Lokollo, E. M., Setiaji, S. N., Ardiansyah, S. R., Islam, M., & Rahmawaty, U. F. (2019). TRANSFORMASI EKONOMI BERBASIS DIGITAL DI INDONESIA: LahirnyaTren Baru Teknologi, Bisni, Ekonomi, dan Kebijakan di Indonesia. Jakarta: PT Granedia Pustaka Utama. Darmawan, P. F., Pradnyana, I. A., & Divayana, G. H. (2019). Analisis Penerimaan Pengguna Aplikasi Cerdas Layanan Perizinan Terpadu Untuk Publik (Sicantik) Pada Dinas Penanaman Modal dan Pelayanan Perizinan Terpadu Satu Pintu (Dpmpptsp) Menggunakan Pendekatan Utaut. KARMAPATI, Vol 8, NO.2, 379-393. Destianti, A. E., Hidayat, A. R., & Srisusilawati, P. (2019). Analisis Faktor Pengaruh Teori Technology Acceptance Model dan Theory Of Planned http://ejurnal.ars.ac.id/index.php/jti
https://openalex.org/W4233345066	https://www.researchsquare.com/article/rs-51053/latest.pdf	English	null	Analysis of MDR1 Polymorphisms Among HIV-Infected Individuals on ARV Therapy from Western India	Research Square (Research Square)	2,020	cc-by	10,788	Analysis of MDR1 Polymorphisms Among HIV- Infected Individuals on ARV Therapy from Western India HariOm Singh (  hariomsgpgims@gmail.com ) National AIDS Research Institute Dharmesh Samani National AIDS Research Institute Vijay Chauware National AIDS Research Institute T.N Dhole Sanjay Gandhi Post Graduate Institute of Medica Abstract Background: MDR1 is involved in the transport of numerous drugs. Polymorphism of MDR1 is linked with the treatment outcome. ARV regimen is being used to manage the progression of HIV infection. Ethnic disparities have been observed in the distribution of MDR1 genotypes. Methods: MDR1 polymorphism (1236 C/T, 3435 C/T) was genotyped in 34 individuals with ARV- associated hepatotoxicity, 131 HIV-infected, and one-fifty-five healthy by utilization of PCR-RFLP. Results: Haplotype TC exposed the greater risk for hepatotoxicity severity when compared between individuals with hepatotoxicity and HIV infected (OR=1.96, P=0.06). While haplotypes TT and CC bared a reduce risk for hepatotoxicity severity (OR= 0.16, P=0.006; OR= 0.46, P=0.06). Haplotype TT and CC displayed a decrease risk of hepatotoxicity severity while compared between individuals with hepatotoxicity and healthy (OR=0.09, P=0.003; OR=0.34, P=0.03). A higher occurrence of MDR1 1236TT genotype was seen among patients with hepatotoxicity who consumed alcohol (28.6% versus 14.8%, OR=1.50). In patients with hepatotoxicity taking nevirapine, there was an increased incidence of MDR1 1236TT genotype in contrast with efavirenz (21.7% versus 9.1%, OR=2.11). In HIV-infected people taking nevirapine, MDR1 1236CT, 1236TT genotypes found to be increased compared with efavirenz (43.7% versus 33.3%, OR=1.66; 12.6% versus 8.3%, OR=1.96). A higher occurrence of MDR1 1236TT genotype has happened in hepatotoxicity cases having both alcohol and nevirapine (40.0% versus 16.67%, OR=2.21). Conclusion: MDR1 haplotypes may have an influence on the severity of hepatotoxicity. Individual utilizing nevirapine and alcohol with MDR1 1236TT and 3435CT genotypes may have combined effect on vulnerability of severity of hepatotoxicity and progression of HIV infection Research article Page 1/29 Page 1/29 1. Background Antiretroviral (ARV) regimen is being extensive utilized for the management of HIV patients, the death rate among individuals with HIV is constantly increasing. Long-term efficacy and toxicity are significant issues of worry with the ARV regimen. Hepatotoxicity is an adverse outcome of ARV drugs in HIV-infected individuals [1, 2]. A higher occurrence of hepatotoxicity was seen with the utilization of nevirapine-based regimen than the efavirenz-based regimen [3]. The shown occurrence of hepatotoxicity because of nevirapine was 3.19% [4]. In another investigation, the stated occurrence of grade 3 or 4 hepatotoxicity had 10.8% in the patients treated with efavirenz and 8.9% in patients treated with nevirapine (5). ABCB1 is one of the universal adenosine triphosphate (ATP)-binding cassette (ABC) genes is responsible for cell homeostasis [6–8]. ABCB1 gene is situated on chromosome 7q21. It is a part of the MDR subfamily [8]. ABCB1 protein is expressed in a few tissues including epithelial cells of the blood-brain barrier [9, 10] and has appeared to transport numerous drugs [11]. P-glycoprotein (P-GP), a transmembrane transporter protein, is encoded by the MDR1.P-gp system of ATP- subordinate efflux transports outside particles including drugs from Page 2/29 Page 2/29 intracellular to the extracellular matrix [12–14]. The variation in P-gp expression may vary the function, thus affect the transport of nevirapine (NVP) [15]. The absorption and penetration of efavirenz (EFV) and NVP are impacted by the P-gp expression [16]. Chelule et al., (2003) have shown the commonness of MDR1 3435CC genotype had 85.9% in Africans, 41.70% in Indians, and 35.7% in whites of Kwazulu-Natal, South Africa, respectively. In the African population, the predominance of the MDR1 3435CC genotype showed overexpression of P-glycoprotein, while in patients with TT genotype it was demonstrated a lower expression of P-GP [17, 18]. Studies have been reported a significant increase of CD4 cell counts with MDR1 3435TT genotype in HIV patients [19, 20]. Haas et al., (2005) proposed that MDR1 3435C/T polymorphism was not linked to efavirenz exposure [19]. Salem et al., (2014) suggested that MDR1 3435C/T polymorphism had no impact on efavirenz clearance [21]. Zhu et al., (2013) proposed that polymorphisms of MDR1 3435T/C and 2677T/G was linked to the response of nevirapine treatment (P = 0.031, P = 0.001) and could help to predict the drug response in HIV patients [22]. 1. Background MDR1 3435TT genotype among individuals treated with EFV or nelfinavir was connected with a more elevated level of CD4 + count than the CT/CC genotypes [22]. Leschziner et al., (2007) recommended that MDR1 3435 TT genotype was linked with higher adverse outcome of 3TC and NVP treatment than EFV treatment [23]. Ritchie et al., (2006) indicated a significant reduced risk of hepatotoxicity with NNRTI-containing regimens in the presence of MDR1 3435C/T polymorphism [24]. The link between polymorphism of MDR1 3435C/T and nevirapine induced hepatotoxicity was documented by studies [24, 25]. Few studies have shown a link between MDR1 polymorphism and adverse outcome of ARV drug and other studies do not suggest the link. Moreover, the link between MDR1 polymorphism and ARV-related hepatotoxicity has not been examined from India. Thus, we analyzed the relationship between MDR1 (1236C/T and 3435C/T) polymorphism and hepatotoxicity induced by ARV regimen. 2.2 Extraction of DNA The collection of 2 ml peripheral blood was done from all subjects and put away at − 700C until DNA extraction. The DNA extraction has been completed from blood leukocytes pellets utilizing the QIAamp DNA Mini Kit according to the guidance of the company. 2.0 Methods 2.1 Subjects A case-control design was undertaken between August 2014 to September 2017. The number of individuals with HIV infection experienced a liver function test (LFT) was one hundred sixty-five. Out of which, thirty-four individuals of hepatotoxicity and one thirty-one individual with no hepatotoxicity were affirmed and one hundred fifty-five healthy people age-matched were recruited. The patients were enlisted from the clinic of the National Institute of AIDS Research, Pune. Tuberculosis, Hepatitis B and C, immune reconstitution syndrome, untreated opportunistic infections, and other known hepatotoxic medications have been excluded from the hepatotoxicity case group. At the same time, one hundred fifty-five people (those from a similar family have been excluded) lacking diseases for example, TB, Hepatitis B, C, and HIV, have been recruited as controls. Clinical information was noted through the reviews of case records, questionnaires, and personal interviews. The status of the liver enzymes was assessed by the utilization of LFT. In patients with hepatotoxicity, males with SGOT > 93.8 U/ml, Alkaline phosphatase > 550.8 U/ml, total bilirubin > 3. 22 mg /ml, SGPT > 229.5 U/ml and females with SGOT > 163.2 U/ml, Alkaline phosphatase > 550.8 U/ml, total bilirubin > 3. 22 mg /ml, SGPT > 173.4 U/ml were considered. In HIV- Page 3/29 Page 3/29 infected male and female controls, SGOT < 32 U/ml, Alkaline phosphatase < 108 U/ml, total bilirubin < 1. 24 mg /ml, and SGPT < 34 U/ml were considered. FACS analysis was utilized to estimate the CD4 + count. The stages of patients were characterized based on current CD4 status. CD4 + cell count ranges from < 200 cells/mm3, 201–350 cells/mm3, and > 350cells /mm3 were considered as advanced, intermediate, and early stage of HIV infection. HBsAg and hepatitis C testing was completed by ELISA with the Ortho HCV ELISA test system. A questionnaire was utilized to record the usage of tobacco and alcohol. The ethical endorsement was taken from the Ethics Committee, National AIDS Research Institute, Pune, India (Reference number: August 28, 2013, EC/NARI/Genetic Susceptibility/13–14/146) and written consents has been taken from every single qualified subject. 2.3 Genotyping Restriction fragment length polymorphism (RFLP) analysis was utilized to genotype the MDR1 (1236 C/T and 3435 C/T) polymorphism. Primers to amplify the MDR1 (C1236T and C3435T) polymorphism were utilized as designated by the previous report [26]. PCR was performed in a volume of 20 µl. The PCR conditions for amplification of MDR1 C1236T and C3435T polymorphisms were used as described previously [27]. A thermocycler was utilized to complete all the reactions. PCR products were visualized by ethidium bromide staining. The PCR products of MDR1 C1236T and C3435T polymorphism was digested by utilization of restriction enzymes HaeIII and MboI at 37 °C for 16 hours separately. 10% polyacrylamide gel along with molecular weight markers was utilized to the genotype of MDR1 C1236T and C3435T polymorphism. The sequences and location of SNP were employed for genotyping of MDR1 C1236T and C3535T polymorphisms. Genotypes were: for MDR1 C1236T: 93 and 87 bp for CC, 87, 58, and 35 bp for TT, and 93, 87, 58, and 35 bp for CT, for MDR1 C3435T: 130 and 76 bp for CC, 206 bp for TT and 206, 130, and 76 bp for CT. Additional staff of the laboratory was done re-genotyping in 20% samples to check the disparities in genotyping. The genotyping mistake was cross-verified by DNA sequencing in 10% of samples. 2.4 Statistical examination The age variable was communicated as mean ± standard deviation (SD). Hardy-Weinberg equilibrium was examined by the utilization of Chi-Square Goodness - of - fit test in healthy people. Fisher's exact test was utilized to analyze the genotype frequencies between groups. Logistic regression was utilized to compute the odds ratios (ORs) and 95% confidence interval (CI). SPSS (SPSS Inc) programming form 17.0 was utilized for statistical examination and the two-sided value was taken as a test of statistical significance. A p-value under ≤ 0.05 was considered for significance. SNPStats online software was utilized to compare the frequency of haplotypes among groups (25). Linkage disequilibrium (LD) was Page 4/29 Page 4/29 analyzed between both the loci by computing the relative LD value (D') as D' = Dij/ Dmax (28). The Dij value was compared between various groups by the comparison of confidence intervals. 3.0 analyzed between both the loci by computing the relative LD value (D') as D' = Dij/ Dmax (28). The Dij value was compared between various groups by the comparison of confidence intervals. 3.0 analyzed between both the loci by computing the relative LD value (D') as D' = Dij/ Dmax (28). The Dij value was compared between various groups by the comparison of confidence intervals. 3.0 analyzed between both the loci by computing the relative LD value (D') as D' = Dij/ Dmax (28). The Dij value was compared between various groups by the comparison of confidence intervals. 3.0 3.0 Results An aggregate of 165 individuals of HIV infection incorporating thirty-four individuals with hepatotoxicity, one-thirty-one HIV-infected and one- fifty-five healthy were used to analysis the polymorphism of MDR1. The mean age ± SD of individuals with hepatotoxicity, HIV infected, and healthy have been 37.24 ± 3.29, 40.27 ± 2.45, and 37.25 ± 6.30 years. The demographic profiles of the participants are outlined in Table 1. Page 5/29 Table 1 Characteristics of individuals of ARV related hepatotoxicity, HIV infected and healthy controls Subjects Individual of ARV- related hepatotoxicity (Grade III and IV) Individual of HIV infection Healthy controls Number N = 34 N = 131 N = 155 Mean Age (Range) 35.14 ± 8.96 39.29 ± 1.34 36.75 ±8.50 Females 16(47.05) 44(33.58) 40(25.80) Males 18(52.94) 84(64.12) 112(72.25) NNRTI Regimen Efavirenz N = 23 11 (32.35) 12 (9.16) Not applicable (NA) Nevirapine N = 142 23(67.64) 119 (90.83) Not applicable Alcohol habit User N = 51 7(20.58) 44(33.58) 0 Non user N = 114 27(79.41) 87(66.41) 0 Tobacco habit User N = 50 23(67.64) 27(20.61) 0 Non user N = 115 11(32.35) 104(79.38) 0 CD4 + Status 0-200(N = 95) 16(16.84) 79(83.16) NA 201–350 (N = 50) 17(50) 33(25.19) NA > 350(N = 20) 1(2.94) 19(14.50) NA Abbreviations: NNRTI, Non-nucleoside reverse-transcriptase inhibitors; NA, Not applicable; N, total number of subject participants; 0, Alcohol and tobacco status was not reported 3.1 MDR1 polymorphism and ARV-associated hepatotoxicity Table 1 dividuals of ARV related hepatotoxicity, HIV infected and healthy controls Page 6/29 Page 6/29 The incidence of polymorphism of MDR1 in individuals of hepatotoxicity and HIV infected has appeared in Table 2. MDR1 polymorphisms has not been distinct between sufferers with hepatotoxicity and HIV infected. In hepatotoxicity cases, the predominance of MDR1 1236TT genotype was increased (17.6% versus 12.2%, OR = 1.38, 95%CI: 0.45–4.12, P = 0.57). MDR1 3435TT genotype and T allele were underrepresented in hepatotoxicity cases when contrasted with HIV-infected people (35.3% versus 43.5%, OR = 0. 56, 95%CI: 0. 20- 1.59, P = 0. 28 and 55.88% versus 62.59%, OR = 0.65, P = 0.13). MDR1 polymorphism were not significantly varied between hepatotoxicity cases and healthy people. The incidence of polymorphism of MDR1 in individuals of hepatotoxicity and HIV infected has appeared in Table 2. MDR1 polymorphisms has not been distinct between sufferers with hepatotoxicity and HIV infected. 3.0 Results In hepatotoxicity cases, the predominance of MDR1 1236TT genotype was increased (17.6% versus 12.2%, OR = 1.38, 95%CI: 0.45–4.12, P = 0.57). MDR1 3435TT genotype and T allele were underrepresented in hepatotoxicity cases when contrasted with HIV-infected people (35.3% versus 43.5%, OR = 0. 56, 95%CI: 0. 20- 1.59, P = 0. 28 and 55.88% versus 62.59%, OR = 0.65, P = 0.13). MDR1 polymorphism were not significantly varied between hepatotoxicity cases and healthy people. Page 7/29 Table 2 Incidence of MDR1 (1236 C/T and 3435 C/T) genotypes/alleles in individuals of ARV related hepatotoxicity and HIV infected Genotype MDR1 (1236 C/T) Individual of ARV- related hepatotoxicity N = 34 (%) Individual of HIV infection N = 131 (%) P- Value OR( 95%CI) CC 16 (47.1%) 59 (45.0%) 1(Reference) CT 12 (35.3%) 56 (42.7%) 0.37 0.68 (0.29– 1.60) TT 6 (17.6%) 16 (12.2%) 0.57 1.37 (0.45– 4.12) CT + TT 18(52.94) 72(54.96) 0.63 0.82 (0.38– 1.79) MDR1 (1236 C/T) Allele Individual of ARV- related hepatotoxicity N = 68 (%) Individual of HIV infection N = 262 (%) P- Value OR( 95%CI) C 44 (64.71%) 174 (66.41%) 1(Reference) T 24 (35.29%) 88 (33.59%) 0.91 1.03 (0.59– 1.82) Genotype MDR1 (3435 C/T) Individual of ARV- related hepatotoxicity N = 34 (%) Individual of HIV infection N = 131 (%) P- Value OR( 95%CI) CC 8 (23.5%) 24 (18.3%) - 1(Reference) CT 14 (41.2%) 50 (38.2%) 0.70 0.82 (0.30– 2.25) TT 12 (35.3%) 57 (43.5%) 0.28 0.56 (0.20– 1.59) CT + TT 26(74.47%) 107(81.67%) 0.42 0.68 (0.27– 1.71) MDR1 (3435 C/T) Allele Individual of ARV- related hepatotoxicity N = 68 (%) Individual of HIV infection N = 262 (%) P- Value OR( 95%CI) C 30 (44.12%) 98 (37.40%) - 1(Reference) T 38 (55.88%) 164 (62.59%) 0.13 0.65 (0.37– 1.14) Genotype MDR1 (1236 C/T) Individual of ARV- related hepatotoxicity N = 34 (%) Healthy control N = 155(%) P- Value OR( 95%CI) N, total number of Individual of ARV-related hepatotoxicity (34), HIV infection (131) and healthy controls (155). OR and 95% CIs were derived from logistic regression model comparing the homozygous wild-type genotype/allele (CC genotype and C allele for MDR1 1236 C/T and 3435 C/T) N, total number of Individual of ARV-related hepatotoxicity (34), HIV infection (131) and healthy controls (155). 3.0 Results OR and 95% CIs were derived from logistic regression model comparing the homozygous wild-type genotype/allele (CC genotype and C allele for MDR1 1236 C/T and 3435 C/T) with other genotypes/alleles. N, total number of Individual of ARV-related hepatotoxicity (34), HIV infection (131) and healthy controls (155). OR and 95% CIs were derived from logistic regression model comparing the homozygous wild-type genotype/allele (CC genotype and C allele for MDR1 1236 C/T and 3435 C/T) with other genotypes/alleles. Page 8/29 Page 8/29 Genotype MDR1 (1236 C/T) Individual of ARV- related hepatotoxicity N = 34 (%) Individual of HIV infection N = 131 (%) P- Value OR( 95%CI) CC 16 (47.1%) 69 (44.52%) 1 (Reference) CT 12 (35.3%) 65 (41.94%) 0.45 0.70 (0.27– 1.79) TT 6 (17.6%) 21 (13.54%) 0.98 0.99 (0.29– 3.38) CT + TT 18(52.94) 86(55.48) 0.55 0.77 (0.33– 1.82) MDR1 (1236 C/T) Allele Individual of ARV- related hepatotoxicity N = 68 (%) Healthy Control N = 310 P- Value OR( 95%CI) C 44 (64.71%) 203 (65.48%) - 1(Reference) T 24 (35.29%) 107 (34.52%) 0.77 0.91 (0.49– 1.71) Genotype MDR1 (3435 C/T) Individual of ARV- related hepatotoxicity N = 34 (%) Healthy Control N = 155 P- Value OR( 95%CI) CC 8 (23.5%) 34 (21.94%) - 1(Reference) CT 14 (41.2%) 67 (43.23%) 0.24 0.52 (0.17– 1.58) TT 12 (35.3%) 54 (34.83%) 0.28 0.53 (0.16– 1.69) CT + TT 26(76.47) 121(78.06) 0.22 0.52 (0.19– 1.46) MDR1 (3435 C/T) Allele Individual of ARV- related hepatotoxicity N = 68 (%) Healthy Control N = 310 P- Value OR( 95%CI) C 30 (44.12%) 135 (43.54%) - 1(Reference) T 38 (55.88%) 175 (56.45%) 0.39 0.76 (0.41– 1.41) N, total number of Individual of ARV-related hepatotoxicity (34), HIV infection (131) and healthy controls (155). OR and 95% CIs were derived from logistic regression model comparing the homozygous wild-type genotype/allele (CC genotype and C allele for MDR1 1236 C/T and 3435 C/T) with other genotypes/alleles. N, total number of Individual of ARV-related hepatotoxicity (34), HIV infection (131) and healthy controls (155). OR and 95% CIs were derived from logistic regression model comparing the homozygous wild-type genotype/allele (CC genotype and C allele for MDR1 1236 C/T and 3435 C/T) with other genotypes/alleles. 3.2 MDR1 polymorphism in HIV-infected people The occurrence of polymorphisms of MDR1 (1236C/T, 3435C/T) in people with HIV infection and healthy is introduced in Table 3. The deviation from Hardy- Weinberg equilibrium in healthy people was Page 9/29 Page 9/29 investigated for MDR1 polymorphism, we found that it was followed (P = 0.36, 0.13). The distribution of MDR1 polymorphism was alike between HIV-infected and healthy people. HIV-infected people had more occurrence of MDR1 3435TT genotype than healthy people (43.5% versus 34.83%, OR = 1.24, 95%CI: 0.59–2.61, P = 0.57). The dispersion of other genotypes and alleles of MDR1 polymorphisms were comparable between both groups. investigated for MDR1 polymorphism, we found that it was followed (P = 0.36, 0.13). The distribution of MDR1 polymorphism was alike between HIV-infected and healthy people. HIV-infected people had more occurrence of MDR1 3435TT genotype than healthy people (43.5% versus 34.83%, OR = 1.24, 95%CI: 0.59–2.61, P = 0.57). The dispersion of other genotypes and alleles of MDR1 polymorphisms were comparable between both groups. investigated for MDR1 polymorphism, we found that it was followed (P = 0.36, 0.13). The distribution of MDR1 polymorphism was alike between HIV-infected and healthy people. HIV-infected people had more occurrence of MDR1 3435TT genotype than healthy people (43.5% versus 34.83%, OR = 1.24, 95%CI: 0.59–2.61, P = 0.57). The dispersion of other genotypes and alleles of MDR1 polymorphisms were comparable between both groups. Page 10/29 Table 3 Incidence of MDR1 (1236 C/T and 3435 C/T) genotypes/alleles in individual of HIV infection and healthy controls. 3.2 MDR1 polymorphism in HIV-infected people Genotype MDR1 (1236 C/T) Individual of HIV infection N = 131 (%) Healthy controls N = 155(%) P- Value OR( 95%CI) CC 59 (45.0%) 69 (44.52%) - 1(Reference) CT 56 (42.7%) 65 (41.94%) 0.65 0.87 (0.48– 1.59) TT 16 (12.2%) 21 (13.54%) 0.39 0.69 (0.29– 1.61) CT + TT 72(54.96) 86(55.48) 0.49 0.82 (0.47– 1.43) MDR1 (1236 C/T) Allele Individual of HIV infection N = 262 (%) Healthy controls N = 310 P- Value OR( 95%CI) C 174 (66.41%) 203 (65.48%) - 1(Reference) T 88 (33.59%) 107 (34.52%) 0.37 0.83 (0.55– 1.25) Genotype MDR1 (3435 C/T) Individual of HIV infection N = 131 (%) Healthy controls N = 155 P- Value OR( 95%CI) CC 24 (18.3%) 34 (21.94%) - 1(Reference) CT 50 (38.2%) 67 (43.23%) 0.53 0.79 (0.37– 1.66) TT 57 (43.5%) 54 (34.83%) 0.57 1.24 (0.59– 2.61) CT + TT 107(81.67) 121(78.06) 0.97 0.99 (0.50– 1.94) MDR1 (3435 C/T) Allele Individual of HIV infection N = 262 (%) Healthy controls N = 310 P- Value OR( 95%CI) C 98 (37.40%) 135 (43.54%) - 1(Reference) N, total number of individual of HIV infection (131) and healthy controls (155). OR and 95% CIs were derived from logistic regression model comparing the homozygous wild-type genotype/allele (CC 164 (62.59%) N, total number of individual of HIV infection (131) and healthy controls (155). OR and 95% CIs were derived from logistic regression model comparing the homozygous wild-type genotype/allele (CC genotype and C allele for MDR1 1236 C/T and 3435 C/T) with other genotypes/alleles. N, total number of individual of HIV infection (131) and healthy controls (155). OR and 95% CIs were derived from logistic regression model comparing the homozygous wild-type genotype/allele (CC genotype and C allele for MDR1 1236 C/T and 3435 C/T) with other genotypes/alleles. N, total number of individual of HIV infection (131) and healthy controls (155). OR and 95% CIs were derived from logistic regression model comparing the homozygous wild-type genotype/allele (CC genotype and C allele for MDR1 1236 C/T and 3435 C/T) with other genotypes/alleles. Page 11/29 Page 11/29 Genotype MDR1 (1236 C/T) Individual of HIV infection N = 131 (%) Healthy controls N = 155(%) P- Value OR( 95%CI) T 164 (62.59%) 175 (56.45%) 0.41 1.18 (0.80– 1.74) N, total number of individual of HIV infection (131) and healthy controls (155). OR and 95% CIs were derived from logistic regression model comparing the homozygous wild-type genotype/allele (CC genotype and C allele for MDR1 1236 C/T and 3435 C/T) with other genotypes/alleles. Genotype MDR1 (1236 C/T) Individual of HIV infection N = 131 (%) Healthy controls N = 155(%) P- Value OR( 95%CI) T 164 (62.59%) 175 (56.45%) 0.41 1.18 (0.80– 1.74) 3.3 Haplotypes distribution We have likewise investigated the occurrence of MDR1 haplotypes among people of hepatotoxicity, HIV- infected, and healthy people. Haplotype CT (1236C/3435T) was considered as a reference. Haplotype occurrence has appeared in Table 4. The incidence of TT haplotype (1236T/3435T) had reduced in individuals with hepatotoxicity than HIV-infected (0.05% versus 0.22%, OR = 0.16, 95%CI: 0.04–0.059, P = 0.0065). However, in a similar investigation, the incidence of TC haplotype (1236T/3435C) was increased in individuals with hepatotoxicity than HIV-infected (0.30% versus 0.11%, OR = 1.96, 95%CI: 0.98–3.94, P = 0.06). The commonness of CC (1236C/3435C) and TT (1236T/3435T) haplotypes was reduced in peoples with hepatotoxicity than healthy (0.14% versus 0.30%, OR = 0.34, 95%CI: 0.12– 0.94, P = 0.039, 0.05% versus 0.22%, OR = 0.09, 95%CI: 0.02–0.44, P = 0.0032). The incidence of TC (1236T/3435C) haplotype was predominantly higher in patients with hepatotoxicity when contrasted with healthy (0.30% versus 0.13%, OR = 1. 94, 95%CI: 0. 87-4.37, P = 0.11). The incidence of MDR1 haplotypes was not distinguished between individuals of HIV contamination and healthy. Page 12/29 Page 12/29 Page 12/29 Table 4 Incidence of MDR1 haplotypes (1236C/T and 3435C/T) in individual of ARV- related hepatotoxicity, HIV infection and healthy controls Haplotype MDR1 (1236 C/T and 3435 C/T) Individual of ARV- related hepatotoxicity (N = 68) Individual of HIV infection (N = 262) P- Value OR( 95%CI) CT 0.51 0.41 - 1(Reference) CC 0.14 0.26 0.062 0.46 (0.20– 1.03) TT 0.05 0.22 0.006 0.16(0.04– 0.59) TC 0.30 0.11 0.06 1.96 (0.98– 3.94) Haplotype MDR1 (1236 C/T and 3435 C/T) Individual of ARV- related hepatotoxicity (N = 68) Healthy controls (N = 310) P- Value OR( 95%CI) CT 0.51 0.35 - 1(Reference) CC 0.14 0.30 0.03 0.34 (0.12– 0.94) TT 0.05 0.22 0.003 0.09 (0.02– 0.44) TC 0.30 0.13 0.11 1.94 (0.87– 4.37) Haplotype MDR1 (1236 C/T and 3435 C/T) Individual of HIV infection (N= 262) Healthy controls (N = 310) P- Value OR( 95%CI) CT 0.41 0.35 - 1(Reference) CC 0.26 0.30 0.31 0.77 (0.46– 1.28) TT 0.22 0.22 0.31 0.75 (0.43– 1.31) TC 0.11 0.13 0.43 0.77 (0.40– 1.48) N, total number of allele in HIV- patients with hepatotoxicity (68), without hepatotoxicity (262) and healthy controls (310). OR and 95% CIs were derived from logistic regression model comparing the haplotype CT with other haplotypes. 3.4 MDR1 polymorphisms and stages of HIV-1 The incidence of MDR1 polymorphism between stages of HIV infection and healthy people was examined as outlined in table-5. A reduced frequency of MDR1 1236TT genotype was found in individuals with an intermediate stage of HIV than healthy (24.2% versus 41.94%, OR = 0.43, P = 0.09). The incidence of MDR1 3435CT and 3435TT genotypes did not vary between individuals of stages of HIV infection and healthy. 3.3 Haplotypes distribution Significant P values are shown in bold (P < 0.05) N, total number of allele in HIV- patients with hepatotoxicity (68), without hepatotoxicity (262) and healthy controls (310). OR and 95% CIs were derived from logistic regression model comparing the haplotype CT with other haplotypes. Significant P values are shown in bold (P < 0.05) Page 13/29 Table 5 Occurance of MDR1 (1236 C/T and 3435 C/T) genotypes in different HIV stages of HIV and healhty controls Genotype MDR1 (1236 C/T) Healthy controls N = 155 (%) Early stage of HIV Intermediate stage of HIV Advanced stage of HIV N = 19 (%) OR (P) N = 33 (%) OR (P) N = 79 (%) OR (P) CC 69 (44.52%) 7 (36.8%) 1 (Reference) 18 (54.5%) 1 (Reference) 34 (43.0%) 1 (Reference) CT 65 (41.94%) 10 (52.6%) 1.67 (0.36) 8 (24.2%) 0.43 (0.09) 38 (48.1%) 1.03 (0.93) TT 21 (13.54%) 2 (10.5%) 0.77 (0.77) 7 (21.2%) 0.93 (0.90) 7 (8.9%) 0.58 (0.32) Genotype MDR1 (3435 C/T) Healthy controls N = 155(%) Early stage of HIV Intermediate stage of HIV Advanced stage of HIV N = 19 (%) OR (P) N = 33 (%) OR (P) N = 79 (%) OR (P) CC 34 (21.94%) 4 (21.1%) 1 (Reference) 5 (15.2%) 1 (Reference) 15 (19.0%) 1 (Reference) CT 67 (43.23%) 6 (31.6%) 0.41 (0.23) 12 (36.4%) 0.80 (0.72) 32 (40.5%) 0.83 (0.66) TT 54 (34.83%) 9 (47.4%) 0.93 (0.92) 16 (48.5%) 1.56 (0.45) 32 (40.5%) 1.11 (0.81) N = number of subjects, (%) = frequency of subjects, OR and 95% CIs were derived from logistic regression model comparing the homozygous wild-type genotype/allele (CC genotype and C allele for MDR1 1236 C/T and 3435 C/T) with other genotypes. N = number of subjects, (%) = frequency of subjects, OR and 95% CIs were derived from logistic regression model comparing the homozygous wild-type genotype/allele (CC genotype and C allele for MDR1 1236 C/T and 3435 C/T) with other genotypes. 3.5 Gene-environment interaction The dispersion of MDR1 polymorphism between individuals with hepatotoxicity and HIV infected utilizing tobacco, alcohol, and NNRTI regimen and nonusers appeared in Tables 6–9. The occurrence of polymorphisms of MDR1 (1236C/T and 3435C/T) was not varied between tobacco utilizing people with hepatotoxicity, HIV infected versus nonusers. MDR1 1236TT genotype was overrepresented in tobacco utilizing hepatotoxicity patients than nonusers (28.6% versus 14.8%) (Table-6). The incidence of Page 14/29 Page 14/29 Page 14/29 MDR11236TT genotype was higher in alcohol devouring people with hepatotoxicity than nonusers (28.6% versus 14.8%, OR = 1.50, 95% CI: 0.13–17.35, P = 0.88). Moreover, an increased incidence of 3435CT genotype of MDR1 was observed in HIV-infected people who consumed alcohol than nonusers (50.0% versus 32.2%, OR = 2. 47, 95%CI: 0.79–7. 70, P = 0.12) (Table-7). The genotype 1236TT of MDR1 was greater in nevirapine taking HIV-contaminated individuals than efavirenz users (14.1%versus 8. 7%, OR = 1. 93, 95%CI: 0. 39- 9.45, P = 0.42). MDR1 1236TT genotype distribution was increased in nevirapine taking patients with hepatotoxicity than efavirenz users (21.7% versus 9. 1%, OR = 2.11, 95%CI: 0. 18– 24.66, P = 0.55). The occurrence of MDR1 1236CT 1236TT genotypes was higher in HIV-infected people taking nevirapine than efavirenz users (43.7% versus 33 .3%, OR = 1.66, 95%CI: 0. 44–6.24, P = 0. 45 and 12.6% versus 8.3%, OR = 1. 96, 95%CI: 0. 22–17.42, P = 0. 55) (Table-8). In both alcohol and nevirapine taking hepatotoxicity patients, the dispersion of MDR1 1236TT genotype appeared greater when contrasted to alcohol nonusers and nevirapine users (40.0% versus 16.67%, OR = 2.21, 95%CI: 0. 17-29.21, P = 0.55). MDR11236TT genotype was higher in alcohol devouring people with hepatotoxicity than nonusers (28.6% versus 14.8%, OR = 1.50, 95% CI: 0.13–17.35, P = 0.88). Moreover, an increased incidence of 3435CT genotype of MDR1 was observed in HIV-infected people who consumed alcohol than nonusers (50.0% versus 32.2%, OR = 2. 47, 95%CI: 0.79–7. 70, P = 0.12) (Table-7). The genotype 1236TT of MDR1 was greater in nevirapine taking HIV-contaminated individuals than efavirenz users (14.1%versus 8. 7%, OR = 1. 93, 95%CI: 0. 39- 9.45, P = 0.42). MDR1 1236TT genotype distribution was increased in nevirapine taking patients with hepatotoxicity than efavirenz users (21.7% versus 9. 1%, OR = 2.11, 95%CI: 0. 18– 24.66, P = 0.55). 3.5 Gene-environment interaction The occurrence of MDR1 1236CT 1236TT genotypes was higher in HIV-infected people taking nevirapine than efavirenz users (43.7% versus 33 .3%, OR = 1.66, 95%CI: 0. 44–6.24, P = 0. 45 and 12.6% versus 8.3%, OR = 1. 96, 95%CI: 0. 22–17.42, P = 0. 55) (Table-8). In both alcohol and nevirapine taking hepatotoxicity patients, the dispersion of MDR1 1236TT genotype appeared greater when contrasted to alcohol nonusers and nevirapine users (40.0% versus 16.67%, OR = 2.21, 95%CI: 0. 17-29.21, P = 0.55). Page 15/29 Table 6 Incidence of MDR1 (1236 C/T and 3435 C/T) genotypes in individuals of ARV related hepatotoxicity and HIV infected utilizing tobacco and non-users Genotype MDR1 (1236 C/T) Tobacco users N = 7 (%) Tobacco non- users N = 27 (%) P- Value OR( 95%CI) Individuals of ARV related hepatotoxicity CC 4 (57.1%) 12 (44.4%) - 1 (Reference) CT 1 (14.3%) 11 (40.7%) 0.30 0.28 (0.024– 3.18) TT 2 (28.6%) 4 (14.8%) 0.88 1.50 (0.13– 17.35) Genotype MDR1 (3435 C/T) Tobacco users N = 7 (%) Tobacco non- users N = 27 (%) P- Value OR( 95%CI) CC 3 (42.9%) 5 (18.5.0%) - 1 (Reference) CT 2 (28.6%) 12 (44.4%) 0.37 0.37 (0.042– 3.25) TT 2 (28.6%) 10 (37.0%) 0.59 0.53 (0.051– 5.40) Individuals of HIV infection Genotype MDR1 (1236 C/T) Tobacco users N = 43 (%) Tobacco non- user N = 88 (%) P- Value OR( 95%CI) CC 19 (44.2%) 40 (45.5%) - 1 (Reference) CT 19(44.2%) 37(42.0%) 0.44 1.39 (0.60– 3.20) TT 5 (11.6%) 11 (12.5%) 0.90 1.08 (0.31– 3.75) Genotype MDR1 (3435 C/T) Tobacco users N = 43 (%) Tobacco non- user N = 88 (%) P- Value OR( 95%CI) CC 7 (16.3%) 17 (19.3%) - 1 (Reference) N = number of subjects, (%) = frequency of subjects, OR and 95% CIs were derived from logistic regression model comparing the homozygous wild-type genotype/allele (CC genotype and C allele for MDR1 1236 C/T and 3435 C/T) with other genotypes. N = number of subjects, (%) = frequency of subjects, OR and 95% CIs were derived from logistic regression model comparing the homozygous wild-type genotype/allele (CC genotype and C allele for MDR1 1236 C/T and 3435 C/T) with other genotypes. 3.5 Gene-environment interaction Page 16/29 Page 16/29 Genotype MDR1 (1236 C/T) Tobacco users N = 7 (%) Tobacco non- users N = 27 (%) P- Value OR( 95%CI) CT 17 (39.5%) 33(37.5%) 0.74 0.80 (0.28– 2.46) TT 19 (44.2%) 38 (43.2%) 0.98 1.006 (0.43– 2.32) N = number of subjects, (%) = frequency of subjects, OR and 95% CIs were derived from logistic regression model comparing the homozygous wild-type genotype/allele (CC genotype and C allele for MDR1 1236 C/T and 3435 C/T) with other genotypes. N = number of subjects, (%) = frequency of subjects, OR and 95% CIs were derived from logistic regression model comparing the homozygous wild-type genotype/allele (CC genotype and C allele for MDR1 1236 C/T and 3435 C/T) with other genotypes. Page 17/29 Table 7 Frequency distribution of MDR1 (1236 C/T and 3435 C/T) genotypes in hepatotoxicity and HIV pateints with alcohol users and non-users Genotype MDR1 (1236 C/T) Alcohol users N = 7 (%) Alcohol non-users N = 27 (%) P- Value OR( 95%CI) Individuals of ARV related hepatotoxicity CC 4(57.1%) 12 (44.4%) - 1(Reference) CT 1 (14.3%) 11 (40.7%) 0.33 0.29 (0.025– 3.43) TT 2 (28.6%) 4 (14.8%) 0.88 1.50 (0.13– 17.35) Genotype MDR1 (3435 C/T) Alcohol users N = 7 (%) Alcohol non-users N = 27 (%) P- Value OR( 95%CI) CC 3(42.9) 5 (18.5%) - 1(Reference) CT 3(42.9) 11 (40.7%) 0.63 0.60 (0.077– 4.73) TT 1(14.3) 11 (40.7%) 0.32 0.26 (0.018– 3.76) Individuals of HIV infection Genotype MDR1 (1236 C/T) Alcohol users N = 44 (%) Alcohol non-users N = 87 (%) P- Value OR( 95%CI) CC 23(52.3%) 36 (41.4%) - 1(Reference) CT 18 (40.9%) 38 (43.7%) 0.95 1.02 (0.45– 2.35) TT 3 (6.8%) 13 (14.9%) 0.20 0.40 (0.098– 1.64) Genotype MDR1 (3435 C/T) Alcohol users N = 44 (%) Alcohol non-users N = 87 (%) P- Value OR( 95%CI) CC 6 (13.6%) 18(20.7%) - 1(Reference) CT 22 (50.0%) 28 (32.2%) 0.12 2.47 (0.79– 7.70) TT 16 (36.4%) 41 (47.1%) 0.81 1.15 (0.37– 3.59) N = number of subjects, (%) = frequency of subjects, OR and 95% CIs were derived from logistic regression model comparing the homozygous wild-type genotype/allele (CC genotype and C allele for MDR1 1236 C/T and 3435 C/T) with other genotypes. 3.5 Gene-environment interaction N = number of subjects, (%) = frequency of subjects, OR and 95% CIs were derived from logistic regression model comparing the homozygous wild-type genotype/allele (CC genotype and C allele for MDR1 1236 C/T and 3435 C/T) with other genotypes. Table 8 Incidence of MDR1 (1236 C/T and 3435 C/T) genotypes in individuals of ARV- related hepatotoxicity and HIV infected with NNRTIs regimen users and non-users Genotype MDR1 (1236 C/T) Total Individuals of HIV infection with nevirapine users N = 142 (%) Total Individuals of HIV infection with efavirenz users N = 23 (%) P- Value OR( 95%CI) CC 63 (44.4) 12 (52.2) 1 Reference CT 59 (41.5) 9 (39.1) 0.56 1.33 (0.51– 3.47) TT 20 (14.1) 2 (8.7) 0.42 1.93 (0.39– 9.45) Genotype MDR1 (3435 C/T) Total Individuals of HIV infection with with nevirapine users N = 142 (%) Total Individuals of HIV infection with with efavirenz users N = 23 (%) P- Value OR( 95%CI) CC 31 (21.8) 1 (4.3) 1 Reference CT 52 (36.6) 12 (52.2) 0.063 0.14 (0.017– 1.11) TT 59 (41.5) 10 (43.5) 0.14 0.20 (0.025– 1.68) Individuals of ARV- related hepatotoxicity Genotype MDR1 (1236 C/T) Nevirapine users N = 23 (%) Efavirenz users N = 11 (%) P- Value OR( 95%CI) CC 11 (47.8%) 5 (45.5%) - 1(Reference) CT 7 (30.4%) 5 (45.5%) 0.64 0.69 (0.14– 3.35) TT 5 (21.7%) 1 (9.1%) 0.55 2.11 (0.18– 24.66) NS, not significant. N = number of subjects, (%) = frequency of subjects, OR and 95% CIs were derived from logistic regression model comparing the homozygous wild type genotype/allele (CC genotype NS, not significant. N = number of subjects, (%) = frequency of subjects, OR and 95% CIs were derived from logistic regression model comparing the homozygous wild-type genotype/allele (CC genotype and C allele for MDR1 1236 C/T and 3435 C/T) with other genotypes. NS, not significant. N = number of subjects, (%) = frequency of subjects, OR and 95% CIs were derived from logistic regression model comparing the homozygous wild-type genotype/allele (CC genotype and C allele for MDR1 1236 C/T and 3435 C/T) with other genotypes. NS, not significant. N = number of subjects, (%) = frequency of subjects, OR and 95% CIs were derived from logistic regression model comparing the homozygous wild-type genotype/allele (CC genotype and C allele for MDR1 1236 C/T and 3435 C/T) with other genotypes. 3.5 Gene-environment interaction Page 19/29 Page 19/29 Genotype MDR1 (1236 C/T) Total Individuals of HIV infection with nevirapine users N = 142 (%) Total Individuals of HIV infection with efavirenz users N = 23 (%) P- Value OR( 95%CI) Genotype MDR1 (3435 C/T) Nevirapine users N = 23 (%) Efavirenz users N = 11 (%) P- Value OR( 95%CI) CC 7 (30.4%) 1 (9.1%) - 1(Reference) CT 8 (34.8%) 6 (54.5%) 0.18 0.19 (0.017– 2.11) TT 8 (34.8%) 4 (36.4%) 0.43 0.37 (0.030– 4.49) Individuals of HIV infection Genotype MDR1 (1236 C/T) Nevirapine users N = 119 (%) Efavirenz users N = 12 (%) P- Value OR( 95%CI) CC 52(43.7%) 7 (58.3%) - 1(Reference) CT 52 (43.7%) 4 (33.3%) 0.45 1.66 (0.44– 6.24) TT 15 (12.6%) 1 (8.3%) 0.55 1.96 (0.22– 17.42) Genotype MDR1 (3435 C/T) Nevirapine users N = 119 (%) Efavirenz users N = 12 (%) P- Value OR( 95%CI) CC 24 (20.2%) 0 (0.0%) NS - CT 44 (37.0%) 6 (50.0%) - 1(Reference) TT 51 (42.9%) 6(50.0%) 0.81 1.16 (0.34– 3.12) NS, not significant. N = number of subjects, (%) = frequency of subjects, OR and 95% CIs were derived from logistic regression model comparing the homozygous wild-type genotype/allele (CC genotype and C allele for MDR1 1236 C/T and 3435 C/T) with other genotypes. TT 8 (34.8%) 4 (36.4%) 0.43 0.37 (0.030– 4.49) Individuals of HIV infection Genotype MDR1 (1236 C/T) Nevirapine users N = 119 (%) Efavirenz users N = 12 (%) P- Value OR( 95%CI) CC 52(43.7%) 7 (58.3%) - 1(Reference) CT 52 (43.7%) 4 (33.3%) 0.45 1.66 (0.44– 6.24) TT 15 (12.6%) 1 (8.3%) 0.55 1.96 (0.22– 17.42) Genotype MDR1 (3435 C/T) Nevirapine users N = 119 (%) Efavirenz users N = 12 (%) P- Value OR( 95%CI) CC 24 (20.2%) 0 (0.0%) NS - CT 44 (37.0%) 6 (50.0%) - 1(Reference) TT 51 (42.9%) 6(50.0%) 0.81 1.16 (0.34– 3.12) NS, not significant. N = number of subjects, (%) = frequency of subjects, OR and 95% CIs were derived from logistic regression model comparing the homozygous wild-type genotype/allele (CC genotype and C allele for MDR1 1236 C/T and 3435 C/T) with other genotypes. NS, not significant. N = number of subjects, (%) = frequency of subjects, OR and 95% CIs were derived from logistic regression model comparing the homozygous wild-type genotype/allele (CC genotype and C allele for MDR1 1236 C/T and 3435 C/T) with other genotypes. 3.5 Gene-environment interaction Table 9 Incidence of MDR1 (1236 C/T and 3435 C/T) genotypes in individuals of ARV- related hepatotoxicity and HIV infected with alcohol and NNRTI regimen users and non-users Genotype MDR1 (1236 C/T) Alcohol+ Nevirapine users N = 5 (%) Nevirapine users + Alcohol non-user N = 18 (%) P- Value OR( 95%CI) Individuals of ARV- related hepatotoxicity CC 3 (60.0%) 8 (44.44%) - 1(Reference) CT 0 (0.0%) 7 (38.89%) NS - TT 2 (40.0%) 3 (16.67%) 0.55 2.21 (0.17– 29.21) Genotype MDR1 (3435 C/T) Alcohol+ Nevirapine users N = 5 (%) Nevirapine users + Alcohol non-user N = 18 (%) P- Value OR( 95%CI) CC 3 (60.0%) 4 (22.22%) - 1(Reference) CT 1(20.0%) 7 (38.39%) 0.38 0.27 (0.015– 5.01) TT 1 (20.0%) 7 (38.39%) 0.75 0.61 (0.031– 12.05) Genotype MDR1 (1236 C/T) Alcohol + Efavirenz users N = 2(%) Efavirenz users + Alcohol non- users N = 9(%) P- Value OR( 95%CI) CC 1 (50.0%) 4 (44.44%) - 1(Reference) CT 1 (50.0%) 4 (44.44%) 0.85 0.73 (0.028– 18.97) TT 0 (0.0%) 1 (11.12%) NS - Genotype MDR1 (3435 C/T) Alcohol + Efavirenz users N = 2(%) Efavirenz users + Alcohol non- users N = 9(%) P- Value OR( 95%CI) CC 0 (0.0%) 1 (11.12%) NS - CT 2 (100%) 4 (44.44%) - 1(Reference) NS, not significant. N = number of subjects, (%) = frequency of subjects, OR and 95% CIs were derived from logistic regression model comparing the homozygous wild-type genotype/allele (CC genotype and C allele for MDR1 1236 C/T and 3435 C/T) with other genotypes. 3.5 Gene-environment interaction The incidence of 3435CT genotype of MDR1 was greater in nevirapine and alcohol consumed HIV- infected individuals than nevirapine users and alcohol nonusers (44.74% versus 33.33%, OR = 2.04, 95%CI: 0. 64–6.53, P = 0. 23) (Table 9). Genotype MDR1 (1236 C/T) Alcohol+ Nevirapine users N = 5 (%) Nevirapine users + Alcohol non-user N = 18 (%) P- Value OR( 95%CI) CT 5 (83.33%) 1 (16.67%) - 1(Reference) TT 1 (16.67%) 5 (83.33%) 0.04 0.04 (0.002– 0.83) NS, not significant. N = number of subjects, (%) = frequency of subjects, OR and 95% CIs were derived from logistic regression model comparing the homozygous wild-type genotype/allele (CC genotype and C allele for MDR1 1236 C/T and 3435 C/T) with other genotypes. NS, not significant. N = number of subjects, (%) = frequency of subjects, OR and 95% CIs were derived from logistic regression model comparing the homozygous wild-type genotype/allele (CC genotype and C allele for MDR1 1236 C/T and 3435 C/T) with other genotypes. The incidence of 3435CT genotype of MDR1 was greater in nevirapine and alcohol consumed HIV- infected individuals than nevirapine users and alcohol nonusers (44.74% versus 33.33%, OR = 2.04, 95%CI: 0. 64–6.53, P = 0. 23) (Table 9). The incidence of 3435CT genotype of MDR1 was greater in nevirapine and alcohol consumed HIV- infected individuals than nevirapine users and alcohol nonusers (44.74% versus 33.33%, OR = 2.04, 95%CI: 0. 64–6.53, P = 0. 23) (Table 9). 3.5 Gene-environment interaction Page 21/29 Page 21/29 Genotype MDR1 (1236 C/T) Alcohol+ Nevirapine users N = 5 (%) Nevirapine users + Alcohol non-user N = 18 (%) P- Value OR( 95%CI) TT 0 (0.0%) 4 (44.44%) NS - individuals of HIV infected Genotype MDR1 (1236 C/T) Alcohol + Nevirapine users N = 38 (%) Nevirapine users + Alcohol non-users N = 81 (%) P- Value OR( 95%CI) CC 18 (47.37%) 34 (41.98%) - 1(Reference) CT 17 (44.74%) 35 (43.20%) 0.68 1.20 (0.50– 2.89) TT 3 (7.89%) 12 (14.82%) 0.40 0.54 (0.13– 2.26) Genotype MDR1 (3435 C/T) Alcohol + Nevirapine users N = 38 (%) Nevirapine users + Alcohol non-users N = 81 (%) P- Value OR( 95%CI) CC 6 (15.78%) 18 (22.22%) - 1(Reference) CT 17 (44.74%) 27 (33.33%) 0.23 2.04 (0.64– 6.53) TT 15 (39.47%) 36 (44.45%) 0.74 1.22 (0.39– 3.84) Genotype MDR1 (1236 C/T) Alcohol + Efavirenz users N = 6(%) Efavirenz users + Alcohol non- user N = 6(%) P- Value OR( 95%CI) CC 5 (83.33%) 2 (33.33%) - 1(Reference) CT 1 (16.67%) 3 (50.0%) 0.16 0.13 (0.008– 2.18) TT 0 (0.0%) 1 (16.67%) NS - Genotype MDR1 (3435 C/T) Alcohol + Efavirenz users N = 6(%) Efavirenz users + Alcohol non- user N = 6(%) P- Value OR( 95%CI) CC 0 (0.0%) 0 NS - NS, not significant. N = number of subjects, (%) = frequency of subjects, OR and 95% CIs were derived from logistic regression model comparing the homozygous wild-type genotype/allele (CC genotype and C allele for MDR1 1236 C/T and 3435 C/T) with other genotypes. NS, not significant. N = number of subjects, (%) = frequency of subjects, OR and 95% CIs were derived from logistic regression model comparing the homozygous wild-type genotype/allele (CC genotype and C allele for MDR1 1236 C/T and 3435 C/T) with other genotypes. Page 22/29 Page 22/29 Genotype MDR1 (1236 C/T) Alcohol+ Nevirapine users N = 5 (%) Nevirapine users + Alcohol non-user N = 18 (%) P- Value OR( 95%CI) CT 5 (83.33%) 1 (16.67%) - 1(Reference) TT 1 (16.67%) 5 (83.33%) 0.04 0.04 (0.002– 0.83) NS, not significant. N = number of subjects, (%) = frequency of subjects, OR and 95% CIs were derived from logistic regression model comparing the homozygous wild-type genotype/allele (CC genotype and C allele for MDR1 1236 C/T and 3435 C/T) with other genotypes. 4.0 Discussion We analyzed the relationship between MDR1 polymorphism and ARV-related hepatotoxicity from Western India. MDR1 encodes for the ATP- dependent membrane efflux transporter (14). The genetic variants that impact patients drugs are substrates of P-glycoproteins. The occurrence of MDR1 polymorphism changes from the population to the population [17]. We analyzed the MDR1 genotypes and haplotypes between individuals infected with HIV and hepatotoxicity. The occurrence of MDR1 3435C/T polymorphism in our healthy people was identical to the investigations done in European, North India, Turkish, and Asian populations [29–34] and contrasted with the examinations did in Chinese, Iranian and Thailand populations [26, 32, 35]. The genotypic dispersal of MDR1 1236C/T polymorphism in our healthy people was almost alike to the investigation announced from North India [34], however, contrasted with the examinations shown from Indians, Mexicans, Chinese and South Africans populations [18, 30, 37, 38]. We have done the genotype-phenotype analysis and found that the MDR11236TT genotype was displayed a hazard for hepatotoxicity severity (OR = 1.37, P = 0.57). However, due to the small sample size in the hepatotoxicity group, the risk could not reach statistical significance. The low phenotypic expression is dictated by common polymorphisms inside P- gp. People with 3435TT genotype were connected with lower levels of P-gp than CC and CT genotype. MDR1 3435C/T polymorphism was related with the reduced risk of NNRTI-induced liver toxicity [24]. We have examined the relationship of gene-gene interaction to understand the additive impact of MDR1 polymorphism on ARV-related hepatotoxicity. The gene-gene collaboration had a greater effect on gene expression than a single gene [39]. In our investigation, haplotype TC was shown with a greater risk for the severity of hepatotoxicity (OR = 1.96, P = 0.06). While, haplotypes TT and CC were linked with reduced risk of severity of hepatotoxicity (OR = 0.16, P = 0.006; OR = 0.46, P = 0.06; OR = 0.09, P = 0.003; OR = 0.34, Page 23/29 Page 23/29 P = 0.03). It is likely that individuals with haplotype TC may have prone to the severity of hepatotoxicity, whereas haplotypes TT and CC may have reduced risk for hepatotoxicity severity. P = 0.03). It is likely that individuals with haplotype TC may have prone to the severity of hepatotoxicity, whereas haplotypes TT and CC may have reduced risk for hepatotoxicity severity. Likewise, we analyzed the relationship between the MDR1 genotype and the stage of HIV infection. 4.0 Discussion Haas et al., (2005) recommended no significant relationship between ABCB1 variations and plasma EFV concentrations [19]. Individuals with HIV infection and utilizing nevirapine with MDR1 1236CT, 1236TT genotypes exposed a risk for the progression of HIV infection (OR = 1.66, P = 0.45; OR = 1.96, P = 0.55). In people with hepatotoxicity who took both alcohol and nevirapine, MDR1 1236TT genotype exposed the higher vulnerability for hepatotoxicity severity (OR = 2.21, P = 0.55). In people having an infection of HIV with MDR1 3435CT genotype and taking both nevirapine and alcohol showed a vulnerability for the progression of HIV infection (OR = 2.04, P = 0.23). This suggests that individuals with MDR1 1236TT and 3435CT genotypes having HIV infection or ARV-related hepatotoxicity have an additive effect on vulnerability of hepatotoxicity severity and progression of HIV disease. Individuals with 3435 T allele having infection of HIV taking nevirapine were connected with the reduced risk of hepatotoxicity [25]. People with MDR1 1236T and 1235T alleles were related to diminished plasma NNRTI concentration influencing the virological response to HAART [21]. Haas et al., (2005) recommended no significant relationship between ABCB1 variations and plasma EFV concentrations [19]. This work has a few limit points, it can just assess association and not indicate causation. In the beginning, the present investigation was planned for a 1:4 proportion of case controls. However, we were not able to complete to select a similar proportion of controls. Though, we recruited a 1:3 proportion which may be sufficient. 4.0 Discussion In our investigation, the prevalence of MDR1 genotypes did not significantly vary between people of various stages of HIV and healthy. MDR1 1236CT, 1236TT, and 3435CT genotypes were correlated with the HIV disease progression, however, these polymorphisms did not regulate the susceptibility of HIV-1 [40]. Following the treatment of a half year, patients with 3435 TT genotype had raised the CD4 + count [22]. Analysis of the relationship of gene environment was done to take a look at the impact on the etiology of the disease [41, 42]. We had selected a case-only analysis. We did not examine the case-control in light of the fact that in the case-control investigation, cases must be coordinated with the controls in the population [43]. Hence, we utilized the case-only analysis. HIV patient’s naïve ART utilizing alcohol has demonstrated a reduction in CD4 + cell count [44]. In women with HIV disease and tobacco utilization, ART response was seen to be diminished [45]. In our examination, the patients of hepatotoxicity utilizing alcohol with MDR11236TT genotype exposed a hazard for severity of hepatotoxicity (OR = 1.50, P = 0.88), while individuals of HIV contamination using alcohol with 3435CT genotype were at higher danger of HIV disease progression (OR = 2.47, P = 0.12). In hepatotoxic patients utilizing nevirapine and MDR1, 1236TT genotype demonstrated a higher threat for the hepatotoxicity severity (OR = 2.11, P = 0.55). Individuals with HIV infection and utilizing nevirapine with MDR1 1236CT, 1236TT genotypes exposed a risk for the progression of HIV infection (OR = 1.66, P = 0.45; OR = 1.96, P = 0.55). In people with hepatotoxicity who took both alcohol and nevirapine, MDR1 1236TT genotype exposed the higher vulnerability for hepatotoxicity severity (OR = 2.21, P = 0.55). In people having an infection of HIV with MDR1 3435CT genotype and taking both nevirapine and alcohol showed a vulnerability for the progression of HIV infection (OR = 2.04, P = 0.23). This suggests that individuals with MDR1 1236TT and 3435CT genotypes having HIV infection or ARV-related hepatotoxicity have an additive effect on vulnerability of hepatotoxicity severity and progression of HIV disease. Individuals with 3435 T allele having infection of HIV taking nevirapine were connected with the reduced risk of hepatotoxicity [25]. People with MDR1 1236T and 1235T alleles were related to diminished plasma NNRTI concentration influencing the virological response to HAART [21]. Conclusions Page 24/29 MDR1 haplotypes may have impact on hepatotoxicity severity. Individual with MDR1 1236TT and 3435CT genotypes in presence of alcohol and nevirapine had an additive effect for vulnerability of Page 24/29 severity of hepatotoxicity and progression of HIV disease. MDR1 is associated with drug clearance. MDR1 expression differed in response to NVP and EFV administration. Hence, further examination of the relationship between MDR1 polymorphism and plasma drug concentration would be done with a bigger sample size in different populations. In addition, the correlation of polymorphisms of other drug transporter genes with plasma drug levels is required to comprehend the effect of genetic variants on treatment effect. Abbreviation: HIV, Human immunodeficiency virus; MDR-1; Multidrug-resistant-1, ABCB1, ATP binding cassette subfamily B member 1; NNRTIs, Non-nucleoside reverse transcriptase inhibitors; ARV, Antiretroviral; ART, Antiretroviral therapy; NVP, Nevirapine; EFV, Efavirenz; SNP, Single nucleotide polymorphism; PCR-RFLP, Polymerase chain reaction- Restriction fragment length polymorphisms; DILI, drug-induced liver injury; ADR, Adverse drug reaction; LFT, Liver function test; SGOT, Serum glutamic oxaloacetic transaminase; SGPT, Serum glutamic pyruvic transaminase; ELISA, Enzyme-linked immunosorbent assay; ABC, ATP- binding cassette Acknowledgment We greatly appreciate and acknowledge the clinic In-charges and other supporting staff of NARI for the recruitment of study participants. I would like to thank Asha Krishnaraj for helping in editing the manuscript. Availability of data and material: On request by email to Corresponding Author Consent for publication: Yes Page 25/29 Source of funding: study was not supported by any extramural funds. Competing interests: None Conflict of interests: Nil Ethical approval: NARI/EC/ICF version 1.0, dated 28 August 2013 Consent to Participate: Taken Author’s contribution HS: Overall supervision DS: Experimental work Source of funding: study was not supported by any extramural funds Competing interests: None Author’s contribution Page 25/29 VC: Manuscript writing TN D: Clinical input and Critical Review of manuscript References 1. O'Brien ME, Clark RA, Besch CL, Myers L, Kissinger P. (2003). Patterns and correlates of discontinuation of the initial HAART regimen in an urban outpatient cohort. J Acquir Immune. 2. Defic Syndr, 34:407 – 14. 1. O'Brien ME, Clark RA, Besch CL, Myers L, Kissinger P. (2003). Patterns and correlates of discontinuation of the initial HAART regimen in an urban outpatient cohort. J Acquir Immune. 2. Defic Syndr, 34:407 – 14. 1. O'Brien ME, Clark RA, Besch CL, Myers L, Kissinger P. (2003). Patterns and correlates of discontinuation of the initial HAART regimen in an urban outpatient cohort. J Acquir Immune. 2. Defic Syndr, 34:407 – 14. 1. O'Brien ME, Clark RA, Besch CL, Myers L, Kissinger P. (2003). Patterns and correlates of discontinuation of the initial HAART regimen in an urban outpatient cohort. J Acquir Immune. 2. Defic Syndr, 34:407 – 14. 3. Van Dyke RB, Wang L, Williams PL, for the Pediatric ACTGCT. Toxicities Associated with Dual Nucleoside Reverse-Transcriptase Inhibitor Regimens in HIV-Infected Children. The Journal of Infectious Diseases. 2008;198:1599–608. 3. Van Dyke RB, Wang L, Williams PL, for the Pediatric ACTGCT. Toxicities Associated with Dual Nucleoside Reverse-Transcriptase Inhibitor Regimens in HIV-Infected Children. The Journal of Infectious Diseases. 2008;198:1599–608. 3. Van Dyke RB, Wang L, Williams PL, for the Pediatric ACTGCT. Toxicities Associated with Dual Nucleoside Reverse-Transcriptase Inhibitor Regimens in HIV-Infected Children. The Journal of Infectious Diseases. 2008;198:1599–608. 4. Minzi OM, Irunde H, Moshiro C. HIV patients presenting common adverse drug events caused by highly active antiretroviral therapy in Tanzania. Tanzan J Health Res. 2009;11:5–10. 4. Minzi OM, Irunde H, Moshiro C. HIV patients presenting common adverse drug events caused by highly active antiretroviral therapy in Tanzania. Tanzan J Health Res. 2009;11:5–10. 5. Nagpal M, Tayal V, Kumar S, Gupta U. (2010). Adverse drug reactions to antiretroviral therapy in AIDS patients at a tertiary care hospital in India: A prospective observational study. Indian. 6. J Med Sci. 64:245–52. 5. Nagpal M, Tayal V, Kumar S, Gupta U. (2010). Adverse drug reactions to antiretroviral therapy in AIDS patients at a tertiary care hospital in India: A prospective observational study. Indian. 6. J Med Sci. 64:245–52. 7. Mascolini M. (2001). HIV news from Buenos Aires: Part 1-the Zahir and the band-aid. 1st IAS conference on HIV pathogenesis and treatment; July 8–11; Buenos Aires: APAC Mon, 274 – 89. 8. Croop JM. P-glycoprotein structure and evolutionary homologies. Cytotechnology. 1993;12:1–32. 9. References Jones PM, George AM. The ABC transporter structure and mechanism: perspectives on recent research. Cell Mol Life Sci. 2004;61:682–99. 9. Jones PM, George AM. The ABC transporter structure and mechanism: perspectives on recent research. Cell Mol Life Sci. 2004;61:682–99. 10. Rosenberg MF, Callaghan R, Ford RC, Higgins CF. Structure of the multidrug resistance P-glycoprotein to 2.5 nm resolution determined by electron microscopy and image analysis. J Biol Chem. 1997;272:10685–94. 10. Rosenberg MF, Callaghan R, Ford RC, Higgins CF. Structure of the multidrug resistance P-glycoprotein to 2.5 nm resolution determined by electron microscopy and image analysis. J Biol Chem. 1997;272:10685–94. 11. Cordon-Cardo C, O'Brien JP, Casals D, Rittman-Grauer L, Biedler JL, Melamed MR, Bertino JR. Multidrug-resistance gene (P-glycoprotein) is expressed by endothelial cells at blood-brain barrier sites. Proc Natl Acad Sci U S A. 1989;86:695–8. 11. Cordon-Cardo C, O'Brien JP, Casals D, Rittman-Grauer L, Biedler JL, Melamed MR, Bertino JR. Multidrug-resistance gene (P-glycoprotein) is expressed by endothelial cells at blood-brain barrier sites. Proc Natl Acad Sci U S A. 1989;86:695–8. 12. Pileri SA, Sabattini E, Falini B, Tazzari PL, Gherlinzoni F, Michieli MG, Damiani D, Zucchini L, Gobbi M, Tsuruo T, et al. Immunohistochemical detection of the multidrug transport protein P170 in human normal tissues and malignant lymphomas. Histopathology. 1991;19:131–40. 13. Sakaeda T, Nakamura T, Okumura K. Pharmacogenetics of MDR1 and its impact on the pharmacokinetics and pharmacodynamics of drugs. Pharmacogenomics. 2003;4:397–410. 14. Ambudkar SV, Dey S, Hrycyna CA, Ramachandra M, Pastan I, Gottesman MM. Biochemical, cellular, and pharmacological aspects of the multidrug transporter. Annu Rev Pharmacol Toxicol. 1999;39:361–98. Page 26/29 15. Sugawara I, Kataoka I, Morishita Y, Hamada H, Tsuruo T, Itoyama S, Mori S. Tissue distribution of P- glycoprotein encoded by a multidrug-resistant gene as revealed by a monoclonal antibody, MRK 16. Cancer Res. 1988;48:1926–9. 16. Thiebaut F, Tsuruo T, Hamada H, Gottesman MM, Pastan I, Willingham MC. (1987) Cellular localization of the multidrug-resistance gene product P-glycoprotein in normal human tissues. l d S i S 8 3 8 16. Thiebaut F, Tsuruo T, Hamada H, Gottesman MM, Pastan I, Willingham MC. (1987) Cellular localization of the multidrug-resistance gene product P-glycoprotein in normal human tissues. 17. Proc. Natl Acad Sci U S A 84:7735–8. 18. Stormer E, von Moltke LL, Perloff MD, Greenblatt DJ.(2002). Differential modulation of Pglycoprotein expression and activity by non-nucleoside HIV-1 reverse transcriptase inhibitors. 19. in cell culture. Pharm Res, 19:1038-45. 19. in cell culture. References Pharm Res, 19:1038-45. 20. Tozzi V. Pharmacogenetics of antiretrovirals. Antiviral Res. 2010;85(1):19 20. Tozzi V. Pharmacogenetics of antiretrovirals. Antiviral Res. 2010;85(1):190–200. 21. Chelule PK, Gordon M, Palanee T, Page T, Mosam A, Coovadia HM, Cassol S. MDR1 and CYP3A4 polymorphisms among African, Indian, and white populations in KwaZulu-Natal, South Africa. Clin Pharmacol Ther. 2003;74:195–6. 22. Dong Q, Xu B, Tan Y, Liu Z, Tian L, Zhang B, Lin CK, Kung HF, Sung JJ, He ML. The genetic variability of MDR1 C3435T polymorphisms in four Southern Chinese populations. Biomed Pharmacother. 2009;63:658–62. 23. Haas DW, Smeaton LM, Shafer RW, Robbins GK, Morse GD, Labbe L, Wilkinson GR, Clifford DB, D'Aquila RT, De Gruttola V, Pollard RB, Merigan TC, Hirsch MS, George AL Jr, Donahue JP, Kim RB. Pharmacogenetics of long-term responses to antiretroviral regimens containing Efavirenz and/or Nelfinavir: an Adult Aids Clinical Trials Group Study. J Infect Dis. 2005;192:1931–42. 24. Li YH, Wang YH, Li Y, Yang L. MDR1 gene polymorphisms and clinical relevance. Acta Genetica Sinica. 2006;33:93–104. 25. Salem AH, Fletcher CV, Brundage RC. (2014). Pharmacometric characterization of efavirenz developmental pharmacokinetics and pharmacogenetics in HIV-infected children. Antimicrob. 26. Agents Chemother, 58:136 – 43. 25. Salem AH, Fletcher CV, Brundage RC. (2014). Pharmacometric characterization of efavirenz developmental pharmacokinetics and pharmacogenetics in HIV-infected children. Antimicrob. developmental pharmacokinetics and pharmacogenetics in HIV infected children. Antimicrob. 26. Agents Chemother, 58:136 – 43. 26. Agents Chemother, 58:136 – 43. 27. Fellay J, Marzolini C, Meaden ER, Back DJ, Buclin T, Chave JP, Decosterd LA, Furrer H, Opravil M, Pantaleo G, Retelska D, Ruiz L, Schinkel AH, Vernazza P, Eap CB, Telenti A;Swiss HIV Cohort Study. (2002). Response to antiretroviral treatment in HIV-1-infected individuals with allelic variants of the multidrug resistance transporter 1: a pharmacogenetics study. Lancet, 359:30–6. 27. Fellay J, Marzolini C, Meaden ER, Back DJ, Buclin T, Chave JP, Decosterd LA, Furrer H, Opravil M, Pantaleo G, Retelska D, Ruiz L, Schinkel AH, Vernazza P, Eap CB, Telenti A;Swiss HIV Cohort Study. (2002). Response to antiretroviral treatment in HIV-1-infected individuals with allelic variants of the multidrug resistance transporter 1: a pharmacogenetics study. Lancet, 359:30–6. 28. Leschziner GD, Andrew T, Pirmohamed M, Johnson MR. (2007). ABCB1 genotype and PGP expression, function and therapeutic drug response: a critical review and recommendations for. 29. future research. Pharmacogenomics J.7:154 – 79. 28. Leschziner GD, Andrew T, Pirmohamed M, Johnson MR. (2007). References Am J Hum Genet. 1998;62:1180–8. 35. Ghodke Y, Chopra A, Shintre P, Puranik A, Joshi K, Patwardhan B. Profiling single nucleotide polymorphisms (SNPs) across intracellular folate metabolic pathway in healthy Indians. Indian J Med Res. 2011;133:274–9. 36. Lakhan R, Misra UK, Kalita J, Pradhan S, Gogtay NJ, Singh MK, Mittal B. No association of ABCB1 polymorphisms with drug-refractory epilepsy in a north Indian population. Epilepsy Behav. 2009;14:78–82. 37. Rubis B, Holysz H, Barczak W, Gryczka R, Lacinski M, Jagielski P, et al. Study of ABCB1 polymorphism frequency in breast cancer patients from Poland. Pharmacol Rep. 2012;64(6):1560–6. 37. Rubis B, Holysz H, Barczak W, Gryczka R, Lacinski M, Jagielski P, et al. Study of ABCB1 polymorphism frequency in breast cancer patients from Poland. Pharmacol Rep. 2012;64(6):1560–6. 38. Shi NJ, Zhang WX, Zhang N, Zhong LN, Wang LP. Correlation of MDR1 gene polymorphisms with anesthetic effect of sevoflurane-remifentanil following pediatric tonsillectomy. Medicine. 2017;96:e7002. 39. Tan EK, Chan DK, Ng PW, Woo J, Teo YY, Tang K, et al. Effect of MDR1 haplotype on risk of Parkinson disease. Arch Neurol. 2005;62(3):460–4. 39. Tan EK, Chan DK, Ng PW, Woo J, Teo YY, Tang K, et al. Effect of MDR1 haplotype on risk of Parkinson disease. Arch Neurol. 2005;62(3):460–4. 40. Rustemoglu A, Gumus-Akay G, Yigit S, Tasliyurt T. Analysis of common MDR1 (ABCB1) gene C1236T and C3435T polymorphisms in Turkish patients with familial Mediterranean fever. Genet Mol Res. 2011;10:3411–20. 40. Rustemoglu A, Gumus-Akay G, Yigit S, Tasliyurt T. Analysis of common MDR1 (ABCB1) gene C1236T and C3435T polymorphisms in Turkish patients with familial Mediterranean fever. Genet Mol Res. 2011;10:3411–20. 41. Saidijam M, Mahjub H, Shabab N, Yadegarazari R. (2015).Simultaneous analysis of multidrug resistance 1(MDR1) C3435T, G2677T/A, and C1236T genotypes in Hamadan City population. 41. Saidijam M, Mahjub H, Shabab N, Yadegarazari R. (2015).Simultaneous analysis of multidrug resistance 1(MDR1) C3435T, G2677T/A, and C1236T genotypes in Hamadan City population. 42 West of Iran Iran Biomed J 19:57 62 42. West of Iran. Iran Biomed J, 19:57–62. 43. Jafar T, Prasad N, Agarwal V, Mahdi A, Gupta A, Sharma RK, et al. (2011). MDR-1 gene polymorphisms in steroid-responsive versus steroid-resistant nephrotic syndrome in children. 43. Jafar T, Prasad N, Agarwal V, Mahdi A, Gupta A, Sharma RK, et al. (2011). MDR-1 gene polymorphisms in steroid-responsive versus steroid-resistant nephrotic syndrome in children. 44. Nephrol. Dial Transplant, 26:3968–74. 45. References ABCB1 genotype and PGP expression, function and therapeutic drug response: a critical review and recommendations for. 28. Leschziner GD, Andrew T, Pirmohamed M, Johnson MR. (2007). ABCB1 genotype and PGP expression, function and therapeutic drug response: a critical review and recommendations for. 29. future research. Pharmacogenomics J.7:154 – 79. 29. future research. Pharmacogenomics J.7:154 – 79. 30. Ritchie MD, Haas DW, Motsinger AA, Donahue JP, Erdem H, Raffanti S, Rebeiro P, George AL, Kim RB, Haines JL, Sterling TR. Drug transporter and metabolizing enzyme gene variants and nonnucleoside reverse-transcriptase inhibitor hepatotoxicity. Clin Infect Dis. 2006;43:779–82. 30. Ritchie MD, Haas DW, Motsinger AA, Donahue JP, Erdem H, Raffanti S, Rebeiro P, George AL, Kim RB, Haines JL, Sterling TR. Drug transporter and metabolizing enzyme gene variants and nonnucleoside reverse-transcriptase inhibitor hepatotoxicity. Clin Infect Dis. 2006;43:779–82. 31. Haas DW, Bartlett JA, Andersen JW, Sanne I, Wilkinson GR, Hinkle J, Rousseau F, Ingram CD, Shaw A, Lederman MM, Kim RB, Adult AIDS, Clinical Trials Group. Pharmacogenetics of nevirapine- 31. Haas DW, Bartlett JA, Andersen JW, Sanne I, Wilkinson GR, Hinkle J, Rousseau F, Ingram CD, Shaw A, Lederman MM, Kim RB, Adult AIDS, Clinical Trials Group. Pharmacogenetics of nevirapine- 31. Haas DW, Bartlett JA, Andersen JW, Sanne I, Wilkinson GR, Hinkle J, Rousseau F, Ingram CD, Shaw A, Lederman MM, Kim RB, Adult AIDS, Clinical Trials Group. Pharmacogenetics of nevirapine- Page 27/29 Page 27/29 associated hepatotoxicity: an Adult AIDS Clinical Trials Group collaboration. Clin Infect Dis. 2006;43:783–6. associated hepatotoxicity: an Adult AIDS Clinical Trials Group collaboration. Clin Infect Dis. 2006;43:783–6. 32. Pongstaporn W, Pakakasama S, Chaksangchaichote P, Pongtheerat T, Hongeng S, Permitr S. MDR1 C3435T and C1236T polymorphisms: association with high-risk childhood acute lymphoblastic leukemia. Asian Pac J Cancer Prev. 2015;16:2839–43. 33. Sole X, Guino E, Valls J, Iniesta R, Moreno V. SNPStats: a web tool for the analysis of association studies. Bioinformatics. 2006;22:1928–9. 33. Sole X, Guino E, Valls J, Iniesta R, Moreno V. SNPStats: a web tool for the analysis of association studies. Bioinformatics. 2006;22:1928–9. 34. Cox A, Camp NJ, Nicklin MJ, di Giovine FS, Duff GW. An analysis of linkage disequilibrium in the interleukin-1 gene cluster, using a novel grouping method for multiallelic markers. Am J Hum Genet. 1998;62:1180–8. 34. Cox A, Camp NJ, Nicklin MJ, di Giovine FS, Duff GW. An analysis of linkage disequilibrium in the interleukin-1 gene cluster, using a novel grouping method for multiallelic markers. References Gutierrez-Rubio SA, Quintero-Ramos A, Duran-Cardenas A, Franco-Topete RA, Castro-Cervantes JM, Oceguera-Villanueva A, et al. 1236 C/T and 3435 C/T polymorphisms of the ABCB1 gene in Mexican breast cancer patients. Genet Mol Res. 2015;14:1250–9. 46. Masebe TM, Bessong PO, Nwobegahay J, Ndip RN, Meyer D. Prevalence of MDR1 C3435T and CYP2B6 G516T polymorphisms among HIV-1 infected South African patients. Dis Markers. 46. Masebe TM, Bessong PO, Nwobegahay J, Ndip RN, Meyer D. Prevalence of MDR1 C3435T and CYP2B6 G516T polymorphisms among HIV-1 infected South African patients. Dis Markers. Page 28/29 Page 28/29 2012;32:43–50. 47. Palmer LJ, Cardon LR. Shaking the tree: mapping complex disease genes with linkage disequilibrium. Lancet. 2005;366:1223–34. 48. Bellusci CP, Rocco CA, Aulicino PC, Mecikovsky D, Bologna R, Sen L, et al. MDR1 3435T and 1236T alleles delay disease progression to pediatric AIDS but have no effect on HIV-1 vertical transmission. AIDS. 2010;24:833–40. 49. Deng Y, Newman B, Dunne MP, Silburn PA, Mellick GD. Case-only study of interactions between genetic polymorphisms of GSTM1, P1, T1 and Z1 and smoking in Parkinson's disease. Neurosci Lett. 2004;366:326–31. 50. Hunter DJ, Hankinson SE, Hough H, Gertig DM, Garcia-Closas M, Spiegelman D, et al. (1997). A prospective study of NAT2 acetylation genotype, cigarette smoking, and risk of breast. 51. cancer. Carcinogenesis, 18:2127–32. 52. Greenland S. The effect of misclassification in the presence of covariates. Am J Epidemiol. 1980;112:564–9. 53. Samet JH, Cheng DM, Libman H, Nunes DP, Alperen JK, Saitz R. Alcohol consumption and HIV disease progression. J Acquir Immune Defic Syndr. 2007;46:194–9. 53. Samet JH, Cheng DM, Libman H, Nunes DP, Alperen JK, Saitz R. Alcohol consumption and HIV disease progression. J Acquir Immune Defic Syndr. 2007;46:194–9. 54. Feldman JG, Minkoff H, Schneider MF, Gange SJ, Cohen M, Watts DH, et al. Association of cigarette smoking with HIV prognosis among women in the HAART era: a report from the women's interagency HIV study. Am J Public Health. 2006;96(6):1060–5. Page 29/29 Page 29/29 Page 29/29
https://openalex.org/W2016241400	https://www.jstage.jst.go.jp/article/jea1991/12/2/12_2_179/_pdf	English	null	Estimates of Cancer Mortality in Hanoi and Ho Chi Minh City, Viet Nam in the 1990s.	Journal of epidemiology	2,002	cc-by	4,836	Received October 10, 2001; accepted December 21, 2001. Department of Clinical Epidemiology, Institute of Industrial Ecological Sciences, University of Occupational and Environmental Health, Japan. Address for correspondence : Le Tran Ngoan, Department of Clinical Epidemiology, Institute of Industrial Ecological Sciences, University of Occupational and Environmental Health, 1-1 Iseigaoka, Yahatanishi-ku, Kitakyushu 807-8555, Japan. BACKGROUND Therefore, a study regarding the real problem of cancer mortal- ity is timely and urgent in Viet Nam. The aim of the present study was to calculate cancer mortality in the two cities of Hanoi and Ho Chi Minh in the 1990s. Mortality statistics are commonly obtained from death cer- tificate, however, death certificate are not available in Viet Nam at present 1,2). From 1995-98, the average annual number of deaths that occurred in hospitals from cancer was only 489 throughout the country (hospital based cancer registry). From these registered numbers, the proportion of cancer deaths was 0.1% of all causes of deaths and 1.5% among new cases of cancer 3-6). This proportion of deaths from cancer in Viet Nam was much lower than that of the estimated data for developing countries (0.1% VS. 9.0%) in the 1990s 7). The reason for this uncommon observation data for cancer mortality is that, only a small number of deaths occurred in hospitals nationwide (Annual cancer death number 489) and this caused serious biased data of cancer mortality in Viet Nam 8,7). This underesti- mation of cancer mortality dose not reflect the real public health problems of cancer morbidity and mortality nationwide. Le Tran Ngoan, Tetsuya Mizoue and Takesumi Yoshimura As cancer mortality data is not available, a study regarding the real problem of cancer mortality is timely and urgent in Viet Nam. Therefore the aim of the present study was to calculate cancer mortality in the city of Hanoi and Ho Chi Minh. The correlation between cancer mortality to incidence ratios and relative survival probabilities for 23 cancer sites was estimated according to SEER (1973-97), then cancer mortality was calculated from the cancer incidence and cancer survival for 25 cancer sites in each city. Cancer mortality rate for all cancer sites except skin (ASR per 100,000) was 103.9 for males and 52.4 for females in Hanoi, and 93.7 for males and 60.7 for females in Ho Chi Minh. For males, the five most common cancer deaths were cancers of the lung, liver, stomach, colon/rectum, and nasopharynx in both Hanoi and Ho Chi Minh. For females, cancer death in the cervix was uncommon in Hanoi but the most common site in Ho Chi Minh (ASR 2.2 VS. 14.2 per 100,000). The present findings are the first results of cancer mortality from Viet Nam and should be useful for further cancer control programs there. J Epidemio/, 2002 ; 12 : 179-187 Journal of Eoidemiology Journal of Eoidemiology Vol. 12, No. 2 March Estimates of Cancer Mortality in Hanoi and Ho Chi Minh City, Viet Nam in the 1990s Le Tran Ngoan, Tetsuya Mizoue and Takesumi Yoshimura Viet Nam, cancer mortality, survival, population-based-cancer-registry MATERIALS AND METHODS The geographical unit in the present study was two cities: Hanoi and Ho Chi Minh. Cancer mortality was calculated based on 7 age groups from 0-14 to 65+ for given cancer sites. The number of cancer sites examined in the present study was 25: oral cavity (ICD-9 140-5), nasopharynx (147), other phar- ynx (146, 8, 9), oesophagus (150), stomach (151), colon/rec- tum (153-4), liver (155), pancreas (157), larynx (161), bronchus and lung (162), connective tissue (171), melanoma of skin (172), breast (174), cervix uteri (180), corpus uteri (182), ovary (183), prostate (185), testis (186), penis (187), bladder (188), brain/nervous system (191-2), thyroid (193), Hodgkin's 179 180 Cancer Incidence Data from Viet Nam: Cancer Incidence Data from Viet Nam: Population based cancer registries have been in existence since 1987 in Hanoi City and 1990 in Ho Chi Minh City . The general population covered by the two cancer registries was numbered at 2,115,673 in Hanoi and 4 ,820,131 in Ho Chi Minh. The initial cancer incidence from Viet Nam were report- ed from 1991-93 in Hanoi City and from 1995-96 in Ho Chi Minh City 2, 10). These data were the source for cancer inci- dences in the present study. To calculate age standardized inci- dence and mortality rate (ASR) per 100,000 person-year, we use the world population in the present study. Estimates of Cancer Mortality in Hanoi and Ho Chi Minh disease (201), Non-Hodgkin's lymphomas (200 , 2), and leukemia (204-8). Cancer sites (other than 25 listed above except for skin) were grouped under "others except for skin." The number of cancer deaths for a specific site was calculated first, then summed up under "all cancer sites except for skin" . sites except for skin was referenced from previous results for developing countries and applied to the 5-year survival rate of the 13 cancer sites in both Hanoi and Ho Chi Minh 11) Regarding the relative cancer survival between males and females, the estimated 5-year relative cancer survival for developing countries was seen to be slightly higher in males for cancers of the mouth and pharynx, stomach, larynx, and leukemia in comparison with those in females. That was about equal for cancers of the colon/rectum, melanoma of skin, blad- der, kidney, and lymphoma but slightly lower for cancers of the esophagus, liver, pancreas, and lung when compared to those in females 12). Therefore, we decided to use the 5-year rel- ative survival for both sexes to estimate cancer mortality in both males and females in the present study. For the age spe- cific group, 5-year relative survival was found to be almost the same, 70.4% to 69.4%, for the age groups 45 or less, 45-54, 55-64 and 65-74 for all cancer sites excluding lung and bronchus. A fairly lower 5-year relative survival (59.4%) was seen for the age group 75+ when compared to that in the above younger age groups 13). However, cancer patients aged 75 or over were reported to be the lowest proportion (4.6%) of all cancer patients in Viet Nam 14). For all cancer sites, the 5-year relative survival rates were observed to be slightly decreased with increased ages (63.3% and 60.3% for the age group 45-54 and 65-74, respectively). Therefore, we decided to use the sur- vival rate for all ages to estimate cancer mortality for 7 age groups from 1-14 to 65+ in the present study. The number of follow-up subjects lost for (1): 642, (2): 39, (3): 31, and (4): 113 that was already excluded from the present analysis. For the other remaining cancer sites, the number of follow-up subjects lost was not stated. Cancer Survival Data from Viet Nam: Data for hospital based cancer survival for 12 cancer sites is available in hospitals in Hanoi and Ho Chi Minh. We decided to use this data from Viet Nam for the present study because the population based cancer survival and relative cancer sur- vival is not available there at present (Table 1). In the city of Ho Chi Minh, the cancer survival data was available for cervi- cal cancer only, therefore, the survival rate for the other 11 cancer sites was referenced from the results in Hanoi and applied to the survival rate in Ho Chi Minh. The 5-year sur- vival rate for the remaining 13 cancer sites and other cancer Table 1. Hospital based cancer survival data in Viet Nam. (#) Source: (#) 16.24-35) HCM: Ho Chi Minh City; HN: Hanoi City The number of follow-up subjects lost for (1): 642, (2): 39, (3): 31, and (4): 113 that was already excluded from the present analysis. For the other remaining cancer sites, the number of follow-up subjects lost was not stated Table 1. Hospital based cancer survival data in Viet Nam. (#) ( ) ) HCM: Ho Chi Minh City; HN: Hanoi City Le Tran Ngoan, et al. 181 Statistical Methods: The strength of the relationship between cancer mortality to incidence ratios and relative survival probabilities after each given time period, 5, 3, and 1 year since the date of diagnosis was measured. Pearson Correlation Coefficients were calculat- ed by using SAS software on a personal computer 15). The rela- tionship between variables (M / I ratios and relative survivals: Si) was obtained as (M / I ratios) = a + b * (Si), then we have M = I * [(a + b * (Si)], where "a" and "b" was the Parameter Estimate of the intercept and the survival, respectively. In the male population of Hanoi City during the 3-year peri- od from 1991-93, the number of cancer deaths was 2,506, and the annual mortality rates were 80.3 and 103.9 per 100,000 (crude and ASR rate, respectively). More than one-fourth of cancer deaths was due to lung cancer (26.7%). The second most common cancer death was liver cancer (16.9%), followed by stomach cancer (14.7%) and nasopharygeal cancer (7.1%) (Table 2). For females, the number of cancer deaths was 1,513, and the annual mortality rates were 46.9 and 52.4 per 100,000 (crude and ASR, respectively), the most common cancer death was stomach cancer (15.1%), followed by breast cancer (14.1%), lung cancer (9.8%), and liver cancer (7.9%) (Table 2). In the male population of Ho Chi Minh City during the 2- year period from 1995-96, the number of cancer deaths was 2,960, and the annual mortality rates were 64.3 and 93.7 per 100,000 (crude and ASR rate, respectively). The most com- mon cancer death was liver cancer (23.7%), followed by lung cancer (20.9%), stomach cancer (12.8%), and colon/rectum cancer (7.2%). For females, the number of cancer deaths was 2,632, and the annual mortality rates were 52.3 and 60.7 per 100,000 (crude and ASR rate, respectively). Nearly one-fourth of cancer deaths was due to cervical cancer (22.2%), followed by lung cancer (9.3%), stomach cancer (9.2%), colon/rectum cancer (9.1%), breast cancer (8.4%), and liver cancer (8.4%). In the male population of Hanoi City during the 3-year peri- od from 1991-93, the number of cancer deaths was 2,506, and the annual mortality rates were 80.3 and 103.9 per 100,000 (crude and ASR rate, respectively). More than one-fourth of cancer deaths was due to lung cancer (26.7%). Statistical Methods: The second most common cancer death was liver cancer (16.9%), followed by stomach cancer (14.7%) and nasopharygeal cancer (7.1%) (Table 2). For females, the number of cancer deaths was 1,513, and the annual mortality rates were 46.9 and 52.4 per 100,000 (crude and ASR, respectively), the most common cancer death was stomach cancer (15.1%), followed by breast cancer (14.1%), lung cancer (9.8%), and liver cancer (7.9%) (Table 2). The Correlation between the Cancer Incidence and Mortality: The Correlation between the Cancer Incidence and Mortality: The present method of estimates of cancer mortality from incidence has been introduced in previous studies 11,12). We have applied this method by using the latest data of SEER from 1973-97 13). Since the survival rate was available in Hanoi and Ho Chi Minh at 5, 3, and 1 year, the scatter diagram of data and a trend line for the relationship between mortality per incidence ratios and 5, 3, 1 year relative survival probabilities was calculated, where y = M : I ratio, x = S(5), S(3), and S(1), respectively. The equations are Y = -0.8717 * X + 0.9219 (R2 = 0.9533), Y = -0.8709 * X + 0.9648 (R2 = 0.9574), and Y = -0.9554 * X + 1.1468 (R2 = 0.9229) for 5, 3, and 1 year relative survival probabilities, respectively. Figure 1 shows the scatter diagram of data and a trend line for the relationship between mortality per incidence ratios and 1-year relative survival prob- abilities. In the male population of Ho Chi Minh City during the 2- year period from 1995-96, the number of cancer deaths was 2,960, and the annual mortality rates were 64.3 and 93.7 per 100,000 (crude and ASR rate, respectively). The most com- mon cancer death was liver cancer (23.7%), followed by lung cancer (20.9%), stomach cancer (12.8%), and colon/rectum cancer (7.2%). For females, the number of cancer deaths was 2,632, and the annual mortality rates were 52.3 and 60.7 per 100,000 (crude and ASR rate, respectively). Nearly one-fourth of cancer deaths was due to cervical cancer (22.2%), followed by lung cancer (9.3%), stomach cancer (9.2%), colon/rectum cancer (9.1%), breast cancer (8.4%), and liver cancer (8.4%). 1-year relative survival probabilities Figure 1. Scatter diagram of data and a trend line for the relationship between mortality/incidence ratios and 1-year relative survival probabilities of SEER for 23 cancer sites. 1-year relative survival probabilities Figure 1. Scatter diagram of data and a trend line for the relationship between mortality/incidence ratios and 1-year relative survival probabilities of SEER for 23 cancer sites. Estimates of Cancer Mortality in Hanoi and Ho Chi Minh 182 Table 2. Number of cancer deaths by age groups in males and females in Hanoi from 1991-93. * World population Table 2. Number of cancer deaths by age groups in males and females in Hanoi from 1991-93. The Correlation between the Cancer Incidence and Mortality: er of cancer deaths by age groups in males and females in Hanoi from 1991-93. Le Tran Ngoan, et al. Le Tran Ngoan, et al. 183 (ASR 4.2 and 5.1 per 100,000, respectively) (Table 2, Table 3). Sex ratio (M : F) was found to be about 4.0 both in Hanoi and Ho Chi Minh. For both sexes, death from liver cancer was the third most common cancer in Hanoi and the most common cancer in Ho Chi Minh (13.5% and 16.5% of all deaths in each city, respectively). All cancer sites except for skin: Cancer mortality was higher among males (ASR 103.9 VS. 93.7 per 100,000) but lower among females in Hanoi (ASR 52.4 VS. 60.7 per 100,000) when compared to those in Ho Chi Minh. A fairly higher annu- al mortality rate was observed for the male age groups 35-44, 45-54, and 55-64 but a much lower annual mortality rate was observed for the female age groups 55-64 and 65+ in Hanoi when compared to those in Ho Chi Minh (Figure 2). However, for both sexes, cancer mortality for all sites except for skin was seen to be similar, that is, about 75 per 100,000 (ASR) and 60 per 100,000 (crude) in both Hanoi and Ho Chi Minh. Stomach cancer: Stomach cancer mortality rate was the third most frequent cancer in both Hanoi and Ho Chi Minh in males, (ASR 15.8 and 12.5 per 100,000, respectively). For females, stomach cancer death was the most frequent cancer death in Hanoi and the third most frequent cancer in Ho Chi Minh (ASR 7.9 and 5.7 per 100,000, respectively) (Table 2, Table 3). Sex ratio (M : F) was about 2.0 in both Hanoi and Ho Chi Minh. For both sexes, death from stomach cancer was the sec- ond most common cause of death from cancer in Hanoi and the third most common cause in Ho Chi Minh (14.9% and 11.1% of all deaths in each city, respectively). p , ( ) Lung cancer: For males, lung cancer was the most common cause of death from cancer in Hanoi, the second most common cause in Ho Chi Minh (ASR 29.3 and 20.8 per 100,000, respectively). The Correlation between the Cancer Incidence and Mortality: For females, lung cancer was the third most common cause of death from cancer in Hanoi and the second most common cause in Ho Chi Minh (ASR 5.3 and 5.8 per 100,000, respectively (Table 2, Table 3). Sex ratio (M: F) was 5.5 in Hanoi and 3.6 in Ho Chi Minh. For both sexes, cancer death from lung cancer was the most common cause of death from cancer in Hanoi and the second most common cause in Ho Chi Minh (20.3% and 16.5% of all cancer sites in each city, respectively. Nasopharygeal cancer: For males, nasopharygeal cancer was the fourth most common cause of death from cancer in Hanoi and the fifth most common cause of death from cancer in Ho Chi Minh (ASR 7.1 and 3.6 per 100,000, respectively). For females, it was the sixth most common cause of death from cancer in Hanoi and an uncommon cause of death from cancer in Ho Chi Minh, (ASR 3.0 and 1.0 per 100,000, respec- tively) (Table 2, Table 3). Sex ratio (M : F) was lower in Hanoi (2.4) than in Ho Chi Minh (3.6). Liver cancer: For males, liver cancer was ranked as the sec- ond most frequent cancer in Hanoi but the most frequent can- cer in Ho Chi Minh (ASR 17.3 and 21.8 per 100,000, respec- tively). For females, it was the fourth most frequent cancer in Hanoi and the sixth most frequent cancer in Ho Chi Minh ( ) ( ) Colon/rectum cancer: In Hanoi, Colon/rectum cancer was the fifth most common cause of death from cancer in both Age groups Figure 2. Annual cancer mortality rate by age group in Hanoi and HCM for all cancer sites except for skin in males and females Figure 2. Annual cancer mortality rate by age group in Hanoi and HCM for all cancer sites except for skin in males and females Estimates of Cancer Mortality in Hanoi and Ho Chi Minh 184 Table 3. Number of cancer deaths by age group in males and females in HCM from 1995-96 . * World population Table 3. Number of cancer deaths by age group in males and females in HCM from 1995-96 . Le Tran Ngoan, et al . 185 Age groups Figure 3. Annual cancer mortality rate by age group in Hanoi and HCM for breast and cervical cancers . sexes (ASR 6.8 and 3.8 per 100,000, respectively). The Correlation between the Cancer Incidence and Mortality: In Ho Chi Minh, it was the fourth most common cause of death from can- cer in both males and females (ASR 6.7 and 5.6 per 100,000, respectively) (Table 2, Table 3). help us to compare cancer mortality between Hanoi and Ho Chi Minh. Since the 1950s in both Hanoi and Ho Chi Minh City, can- cer treatment and a follow-up for cancer survival in general and cervical cancers in particular has been established 16, 17) Following the experiences of these researchers (Hien and Hoanh et al), cancer treatment and a follow-up has been done well for the 12 cancer sites used in the present study. Breast and cervical cancer: Breast cancer was the second and fifth most frequent cause of death from cancer in Hanoi and Ho Chi Minh, ASR 7.6 and 5.1 per 100,000, respectively. Cancer death from cervical cancer was an uncommon cause of death from cancer in Hanoi but it was the most frequent cause of death from cancer in females in Ho Chi Minh (ASR 2.2 and 14.2 per 100,000, respectively) (Table 2, Table 3). A much lower annual mortality rate in Hanoi for all specific age groups than in Ho Chi Minh was seen and the highest annual mortality rate in Ho Chi Minh was seen for the age group 55-64 for cer- vical cancer (Figure 3). Among selected Asian countries where cancer mortality data have been available, for both sexes, the proportion of deaths from cancers in the population of Hanoi from 1991-93 (10.2% of all causes of deaths) was much lower than that in urban China in 1992 (21.8%) 18). This proportion was also much lower than that in the general population of Japan in 1992 (27.0%), the general population of Hong Kong in 1991 (30.8%), the general population of Singapore in 1991 (24.2%) 19-21 This proportion of deaths from cancer was fairly higher than that in the general population of the Philippines in 1993 (7.9%) and in the general population of Thailand in 1994 (9.3%) 22,23). Estimates of Cancer Mortality in Hanoi and Ho Chi Minh 186 3. Ministry of Health Vietnam. Health statistics yearbook. Vietnam Ministry of Health, 1995. tions. The relative survival rate was always larger than the observed rate for the same group of patients 13). Therefore, the number of cancer deaths estimated from observed survival may be, at least in part, overestimated for 12 cancer sites in the present study, which were calculated based on the data of hos- pital based cancer survival from Viet Nam. These data of observed survival were calculated on the basis of inpatient medical records. The patients with a serious advanced stage of incurable cancer that was diagnosed at outpatient clinics may not be included. Another limitation is data of hospital based cancer survival for 12 cancer sites from Viet Nam and the esti- mated data for another 13 cancer sites referenced from devel- oping countries. Accuracy of hospital based cancer survival data in Viet Nam is very limited due to the small number of study subjects (Cancers of ovary, testis) and large number of follow-up subjects lost (Cancers of cervical uteri in Ho Chi Minh and penis) (Table 1). In addition estimated data from developing countries that is applied in the present study may not reflect the real problem in Viet Nam. We believe that our population based cancer survival study will provide a better database to calculate cancer mortality in the near future. 4. Ministry of Health Vietnam. Health statistics yearbook. Vietnam Ministry of Health, 1996. 5. Ministry of Health Vietnam. Health statistics yearbook. Vietnam Ministry of Health, 1997. 6. Ministry of Health Vietnam. Health statistics yearbook. Vietnam Ministry of Health, 1998. 7. Murray CJ, Lopez AD. Mortality by cause for eight regions of the world: Global Burden of Disease Study. Lancet, 1997; 349: 1269-1276. 8. Matsuda S. An introduction to the health system in Viet Nam. Environ Health Prev Med, 1997; 2: 99-104. 9. Lam LH. Health issues and challenge in Vietnam. J Natl Inst Public Health, 1999; 48: 121-133. 10. Anh PTH, Duc NB, Khang HX, Truong TH, Nga NH. Viet Nam, Hanoi 1991-1993. In; Parkin DM, Whelan SL, Ferlay J, Raymond L, Young J, eds. Cancer incidence in five continents Vol VII, IARC Scientific Publications No. 143, Lyon, IARC, WHO, IACR, 1997: 442-445. 11. Pisani P, Parkin DM, Bray F, Ferlay J. Estimates of the worldwide mortality from 25 cancers in 1990. Int J Cancer, 1999; 83: 18-29. Estimates of Cancer Mortality in Hanoi and Ho Chi Minh In spite of the limitations of the data sources, the present findings have indicated cancer mortality data in the city of Hanoi and Ho Chi Minh that should be useful for further can- cer control programs in Viet Nam in particular and in Asia regions in general. 12. Pisani P, Parkin DM, Ferlay J. Estimates of the world- wide mortality from eighteen major cancers in 1985. Implications for prevention and projections of future bur- den. Int J Cancer, 1993; 55: 891-903. ACKNOWLEDGMENTS 13. Ries LAG, Eisner MP, Kosary CL et al. SEER cancer sta- tistics review, 1973-1997. National Cancer Institute, Bethesda, Maryland, 2000. We especially thank Dr. Nguyen Ba Duc, the Director of Hanoi Cancer Hospital and Prof. Nguyen Chan Hung, the Director of Ho Chi Minh City Oncology Center for their kind assistance. We are grateful to Dr. D. M. Parkin for his encour- agement to study the problem of cancer in Viet Nam. We thank Dr. Pham Hoang Anh and Dr, Nguyen Manh Quoc for their kind assistance in gathering information from the cancer registries in the city of Hanoi and Ho Chi Minh. We also espe- cially thank the staff members of the Dept. of Clinical Epidemiology, Inst. of Industrial Ecological Sciences, Univ. of Occupational and Environmental Health, Japan, for their assis- tance and useful comments. 14. Truong LT. Viet Nam, Ho Chi Minh City, 1976-81. In; Parkin DM, ed. Cancer occurrence in developing coun- tries. IARC Scientific Publications No. 75, Lyon, France, 1986: 309-311. 15. The SAS System for Windows [program]. Release 6.12 version. SAS Institute Inc, Noth Carolina, USA, 1996. 16. Hien DB. Analyse de 615 cas de cancer du col uterin vus & traites a L'Institue Radium Hanoi (1955-1961). Med Sci Vietnam, 1962; 4: 72-85. (in Vietnamese with France abstract) 17. Hoanh DD, Tam PB, Vien NL, Can NH. Cancer of the cervix in South Vietnam. Gann Monogr Cancer Res, 1976; 18: 167-175. This work was partly supported by the Japanese Ministry of Health and Welfare, (Marui study group) and by the Japanese Government (Monbusho) Scholarship for Research Students. 18. WHO. China: selected rural and urban areas, 1992. World Health Statist Annu, 1994; 1994: B322-B329. DISCUSSION The present findings are the first results of cancer mortality from Viet Nam for the population of Hanoi and Ho Chi Minh in the 1990s. The annual crude mortality from all causes in Hanoi was about 620 per 100,000 in 1991-93 for both sexes 10) Also for both sexes, the present results have shown that the crude annual mortality rate from cancers at the same time in Hanoi was 63.3 per 100,000. Therefore, the proportion of deaths from cancers was 10.2% of all causes of deaths. The present study findings also present the number of deaths from cancer and its mortality rate for 25 cancer sites in both Hanoi and Ho Chi Minh in males and females. These findings also The sex ratios of crude rate and ASR for all cancer sites were somewhat higher in Hanoi than those in Ho Chi Minh (Crude rate: 1.7 VS. 1.2, ASR: 2.0 VS. 1.5) (Table 1, Table 2). These differences may, at least in part, be due to a lower 3-year survival rate for cervical cancer in Ho Chi Minh (16.3%, data not shown) than that in Hanoi (69.9%). Cervical cancer mortal- ity was predominantly high in Ho Chi Minh, comprising about 22.2% of all deaths from cancer, but it was only 4.0% in Hanoi. However, the present study may have some potential limita- Estimates of Cancer Mortality in Hanoi and Ho Chi Minh REFERENCES 19. WHO. Hong Kong 1991. World Health Statist Annual, 1993; 1993: D366-D369. 20. WHO. Japan 1992. World Health Statist Annual, 1993; 1993: D370-D373. 1. Anh PT, Parkin DM, Hanh NT, Duc NB. Cancer in the population of Hanoi, Vietnam, 1988-1990. Br J Cancer, 1993; 68: 1236-1242. 21. WHO. Singapore 1991. World Health Statist Annual, 1993; 1993: D398-D401. 2. Nguyen MQ, Nguyen CH, Parkin DM. Cancer incidence in Ho Chi Minh City, Viet Nam, 1995-1996. Int J Cancer, 1998; 76: 472-479. 22. WHO. Philippines 1993. World Health Statist Annu, 1996; 1996: B680-B683. Le Tran Ngoan, et al. 187 (Special issue of oncology in Vietnamese with English abstract) 23. WHO. Thailand 1994. WHO statistic information system (http://www.who.int/whosis/) 2001; Mortality Data. 30. Loan DP, Duy KV. Breast cancer among residents of Hanoi City: 5-year survival. Med Sci Vietnam, 1993; 173(7): 107-109. (Special issue of oncology in Vietnamese with English abstract) ( p ) y 24. Minh LV, Thinh LP, Dat NV, Phuong LA, Phu TT, Hung NC. Review of 5,034 cases of cervical cancer treated in Cancer Center of HCMC in 5 years 1990-1994. Medical Science of Ho Chi Minh City 1997, Special issue of oncology in 1997. 267-273. (in Vietnamese with English Abstract) 31. Dinh NV, Thuan TV, Phuc ND. Diagnosis and treatment of ovarian cancer in K Hospital 1996-98. J Med Sci Pharm Inf, 1999; 169-171. (Special issue of oncology in Vietnamese with English abstract) 25. Thoi NH. Facteurs prognostiques principaux du cancer du nasopharynx. J Med Pract Vietnam, 1995; 11-1995: 17- 19. (Special issue of oncology in Vietnamese with France abstract) 32. Xuan VV, Hieu NV. Diagnostic et traitement des cancers du testicule, Hospital K Hanoi (1979-88). Med Sci Vietnam, 1993; 173(7): 113-117. (Special issue of oncol- ogy in Vietnamese with France abstract) 26. Huan PD, Van DD. La chirurgie D'exerese dans les can- cers de L'oesophage thoracique. J Med Sci Pharm Inf, 1999; 57-59. (Special issue of oncology in Vietnamese with France abstract) 33. Nghi DH, Men V, Ha BM. Survival of 5 years of penile cancers in Hospital K. J Med Sci Pharm Inf, 1999; 172- 177. (Special issue of oncology in Vietnamese with English abstract) 27. Van DD. Traitment chrurgical des cancers gastrique a L'hospital Viet Duc (1970-1992). Med Sci Vietnam, 1993; 7: 45-50. (Special issue of oncology in Vietnamese with France abstract) 34. Trieu NB, Ky N, Hong NP, Ca VNH, Cu NQ. 29. Nghi DH, Dong DV. Soft tissue sarcoma in Hospital K, 1977-95. J Med Pract Vietnam, 1995; 11-1995: 54-58. REFERENCES Contribution au diagnostic precoce des tumeurs dela vessie. J Med Pract Vietnam, 1995; 11-1995: 93-95. (Special issue of oncology in Vietnamese with France abstract) 28. Hung NX, Hung PV, Van DD. Les auteurs raportent une serie retrospective de 206 cancers du rectum, traites a L'hospital Viet Duc, de 1989 a 1996. J Med Sci Pharm Inf, 1999; 79-82. (Special issue of oncology in Vietnamese with France abstract) gy 35. Duc NB, Mo QT. Non Hodgkin's lymphomas at Hospital K: clinical presentations and valua of diagnostic methods. J Med Pract Vietnam, 1995; 11-1995: 85-90. (Special issue of oncology in Vietnamese with English abstract) 29. Nghi DH, Dong DV. Soft tissue sarcoma in Hospital K, 1977-95. J Med Pract Vietnam, 1995; 11-1995: 54-58.
https://openalex.org/W2781780577	https://europepmc.org/articles/pmc5756353?pdf=render	English	null	A comparison of misoprostol vaginal insert and misoprostol vaginal tablets for induction of labor in nulliparous women: a retrospective cohort study	BMC pregnancy and childbirth	2,018	cc-by	6,388	Marsdal et al. BMC Pregnancy and Childbirth (2018) 18:11 DOI 10.1186/s12884-017-1647-3 Marsdal et al. BMC Pregnancy and Childbirth (2018) 18:11 DOI 10.1186/s12884-017-1647-3 Open Access © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. A comparison of misoprostol vaginal insert and misoprostol vaginal tablets for induction of labor in nulliparous women: a retrospective cohort study Kjersti Engen Marsdal1,2, Ingvil Krarup Sørbye1, Lise C. Gaudernack1 and Mirjam Lukasse2* ersti Engen Marsdal1,2, Ingvil Krarup Sørbye1, Lise C. Gaudernack1 and Mirjam Lukasse2* Abstract Background: Since Misoprostol Vaginal Insert (MVI - Misodel ®) was approved for labor induction in Europe in 2013, to date, no study has been published comparing MVI to Misoprostol vaginal tablets (MVT). The aim of this study, performed as part of a quality improvement project, was to compare the efficacy and safety of 200 μg MVI versus 25 μg MVT for labor induction in nulliparous women. Methods: This retrospective cohort study included 171 nulliparous singleton term deliveries induced with MVI (n = 85) versus MVT (n = 86) at Oslo University Hospital Rikshospitalet, Norway, from November 2014 to December 2015. Primary outcomes were time from drug administration to delivery in hours and minutes and the rate of cesarean section (CS). Results were adjusted for Bishop Score and pre-induction with balloon catheter. Results: Median time from drug administration to delivery was shorter in the MVI group compared to the MVT group (15 h 43 min versus 19 h 37 min, p = 0.011). Adjusted for confounding factors, mean difference was 6 h 3 min (p = 0.002). The risk of CS was 67% lower in the MVI group compared to the MVT group (11.8% versus 23.3%, OR = 0.33; adjusted 95% CI 0.13–0.81). Adverse neonatal outcomes did not differ between the groups. Conclusions: In a setting of routine obstetric care, MVI seems to be a more efficient labor induction agent than MVT, and with a lower CS rate and no increase in adverse infant outcomes. Keywords: Labor induction, Cervical ripening, Misoprostol, Nulliparity, Cesarean section Keywords: Labor induction, Cervical ripening, Misoprostol, Nulliparity, Cesarean section Background d f l Whereas induction of multiparous women has a high success rate, the induction of nulliparous women poses a particular obstetrical problem. Inductions in nulliparous women with an unfavorable or unripe cervix carry an increased risk of dystocia and protracted labor [3, 4]. Con- versely, induction of labor also poses a risk of uterine tachysystole and subsequent fetal distress [5]. Protracted labor and fetal distress are the two main indications for CS in Norway [6]. CS in the first delivery also has conse- quences for subsequent labors, as the repeat CS rate in Norway is 50% [6]. Thus it is of clinical importance to determine the safety and efficacy of new methods for induction of labor for nulliparous women in particular. Induction of labor is one of the most frequently performed obstetrical interventions. The decision to induce labor is made if ending the pregnancy is considered more benefi- cial for the mother or the baby than awaiting spontaneous onset of labor. Induction of labor has increased over the last decades across Europe. In 2010, in 15 of 25 countries in Europe, more than 20% of the labors were induced [1]. In Norway, the induction rate increased from 12.5% in 2003 to 20.3% in 2013. The most common indications for induction of labor were pre-labor rupture of the mem- branes (PROM) and post-term pregnancy [2]. Misoprostol is a synthetic prostaglandin E1 analog and has been used off-label for cervical ripening and labor induction since the 1980s [7]. For labor induction in women with an * Correspondence: mirjam.lukasse@hioa.no 2Oslo and Akershus University College, Faculty of Health Sciences, Department of Nursing and Health Promotion, P.O. Box 4, 0130 Oslo, Norway Full list of author information is available at the end of the article Marsdal et al. BMC Pregnancy and Childbirth (2018) 18:11 Page 2 of 8 Page 2 of 8 unfavorable cervix, Misoprostol is more effective than other methods such as oxytocin, Dinoprostone and placebo, with no differences in adverse perinatal or maternal outcomes [7]. In Norway, Misoprostol 25 μg tablets administered vaginally every 4–6 h, has been the most commonly used method for inducing labor with an unfavorable cervix [2]. nulliparous women. During this period, our obstetrical unit improved our protocols for selecting women for induction of labor and improved adherence to the proto- cols for the induction procedures. Background d f l As part of the project, MVI was introduced as an alternative to MVT in nulliparous women from November 2014 onwards. g In 2013 a 200 μg Misoprostol vaginal insert (MVI – Misodel ®) received approval in Europe [8, 9]. In a phase III trial, MVI was compared to a Dinoprostone vaginal insert, a prostaglandin E2 analog. This trial reported sig- nificantly reduced times to delivery and no evidence of differences in maternal or neonatal safety outcomes [10]. Since the phase III trial, only three studies have com- pared MVI to other induction methods in terms of de- livery outcomes [11–13]. Neither of these studies have presented data from nulliparous women exclusively, and no studies have compared MVI with MVT. In this study we included induced nulliparous women that delivered at Oslo University Hospital Rikshospitalet, Norway, from November 2014 through December 2015. The unit is a tertiary obstetrical unit with around 2800 deliveries annually. Women were included if their labors were induced with MVI or MVT, if they had no previous uterine surgery or other uterine abnormality and gave birth to a single fetus, in cephalic presentation, at gesta- tional age of 37 weeks or more. This corresponds to Robson group 2a in the 10-group classification system [14]. As the study was a part of a quality improvement project conducted within a routine care setting, no randomization was performed. Both MVI and MVT were used for induction of labor during the whole study period and the choice of method was decided usually jointly by the obstetric consultant and the midwife on call. During the study period, 174 nulliparous women were initially included. Of these, three women, who received MVT after the MVI was accidentally removed, were excluded from the study. In a daily obstetric practice, individual care might lead to deviation from protocol. Thus, results from experi- mental studies are not always valid for obstetric care. The aim of the present study was to compare efficiency and safety of MVI versus MVT for labor induction in nulliparous women within a routine care setting. Our primary outcomes were time from drug administration to delivery and the rate of CS. Statistical analysis Maternal characteristics and indications for labor induc- tion were compared between the two groups using Stu- dent’s t-test for continuous variables and chi-square test for dichotomous variables. For all outcomes, we identi- fied potential confounding variables a priori according to previous knowledge of factors that could affect the likelihood of successful induction of labor using MVI or MVT [15–18]. Potential confounding factors included maternal age, body mass index, gestational age, birth weight, Bishop Score, PROM and pre-induction with balloon catheter. As a higher proportion of women in the MVI groups were induced due to hypertension/pre- eclampsia, the reason for induction was also considered a potential confounding factor. True confounders were defined as confounders that changed the results with more than 10%. For time outcomes, we used Student’s t- test and Mann-Whitney U test to compare the two groups. Bishop Score and balloon catheter were identi- fied as true confounding factors and were included in linear regression models with the forced entry method for the time outcomes. Due to skewed distributions, we also performed analyses with log transformed time vari- ables in the model. To evaluate if labors interrupted by CS influenced the results, we also performed Cox regres- sion with log rank test, censoring CS and with adjust- ment for Bishop Score and pre-induction with balloon catheter. In terms of delivery mode outcomes, we calculated crude and adjusted odds ratios (OR) with 95% confidence intervals (CI) in logistic regression models. In these analyses Bishop Score and balloon catheter were identified as true confounding factors and included in the models. To evaluate if fetal distress led to more operative deliveries in one of the groups, the proportion of CS and operative vaginal deliveries due to fetal distress were compared between the groups. Less than 2% of the data were missing. Missing data were excluded pairwise. A p-value of <0.05 was considered to indicate statistical significance. Statistical analyses were performed with IBM SPSS Statistics for Windows, Version 21 Armonk, NY: IBM Corp. Our primary outcome regarding efficiency was time from drug administration to delivery. Secondary out- comes included time from drug administration to onset of the active phase of labor and labor duration. As to safety, our primary outcome was the rate of CS. Marsdal et al. BMC Pregnancy and Childbirth (2018) 18:11 Page 3 of 8 Page 3 of 8 Marsdal et al. BMC Pregnancy and Childbirth (2018) 18:11 MVI is a removable vaginal insert with a reservoir of 200 μg Misoprostol, released at a mean rate of approxi- mately 7 μg per hour over a period of 24 h. MVI was inserted once, while the insertion of a 25 μg MVT was repeated every 4 h. After insertion of MVI or MVT, the women remained in bed with continuous cardiotocogra- phy (CTG) for one hour. A CTG was performed every 4–6 h; once regular contractions were established or ac- cording to the department’s procedures. In women with a non-reassuring CTG during the induction process, the insert was withdrawn or tablet removal was attempted. The MVI was removed when the midwife considered that labor was established; if the CTG showed a non- reassuring pattern or if the 24-h dosing period was com- pleted. For women not in labor after the dosing period, artificial rupture of membranes was performed, followed by oxytocin infusion. The oxytocin infusion was started at 5 mU/min and increased by 5 mU/min every 30 min until adequate uterine activity, defined as 4–5 contrac- tions per 10 min. The maximum infusion rate was 30 mU/min. The oxytocin infusion was stopped or decreased if the woman had more than 5 contractions per 10 min. For inductions with MVT, artificial rupture of the membranes was performed when the Bishop Score reached 6 or more, followed by oxytocin infusion as described. At the end of day 3 or beginning of day 4, artificial rupture of the membranes was attempted even if the Bishop Score was below 6. The administration of MVT could be postponed if the woman was having regular contractions, if the woman needed to rest at night due to a long induction process and in rare cases due to logistic considerations. Inductions could be started any time during the day. No progress of labor and failed induction was according to the de- partments protocol defined as no progression after 6 h with the maximum dose of oxytocin. Individual assessments on labor progression were made by the obstetric consultant on call. led to a change in the cervix. PROM was defined as rup- tured membranes without contractions. In this group, labor was induced after 24–48 h, and signs of infection were monitored until delivery. Statistical analysis Second- ary outcomes included the proportion of operative and spontaneous vaginal deliveries, the use of oxytocin stimulation, the proportion of Apgar Score < 7 after 5 min and the rate of CS and operative vaginal deliveries due to fetal distress. All CTG-registrations from labors ending with operative delivery due to fetal distress were investigated for uterine tachysystole, defined as >5 con- tractions in 10 min. Marsdal et al. BMC Pregnancy and Childbirth (2018) 18:11 In women with PROM and meconium-stained amnion fluid, induction was started without delay. Methods In Norway, there is no standardized protocol for induction of labor. The department’s protocol for induc- tion of labor in nulliparous women during the study period is presented in Fig. 1. During 2014–2015 a national obstetric quality improve- ment project on CS was launched in Norway. One of the preselected focus areas was induction of labor in Nullipara Cervix unripe Bishop score < 6 Misoprostol Vaginal Insert (MVI) Ruptured membranes Cervix ripe Bishop score ≥ 6 Cervix ripe Bishop score ≥ 6 Oxytocin Balloon for 24-36 hours Amniotomy Oxytocin Cervix unripe Bishop score < 6 Misoprostol Vaginal Tablets (MVT) Intact membranes Fig. 1 Flow chart of the protocol for induction of labor in nulliparous women Nullipara Cervix ripe Bishop score ≥ 6 Cervix ripe Bishop score ≥ 6 Cervix unripe Bishop score < 6 Cervix unripe Bishop score < 6 Oxytocin Amniotomy Oxytocin Balloon for 24-36 hours Misoprostol Vaginal Tablets (MVT) Misoprostol Vaginal Insert (MVI) Fig. 1 Flow chart of the protocol for induction of labor in nulliparous women Fig. 1 Flow chart of the protocol for induction of labor in nulliparous women Results A total of 171 women were included in the study. Of these, 85 (49.7%) received MVI and 86 (50.3%) received MVT. Maternal and pregnancy characteris- tics were comparable in the two groups except for the mean Bishop Score, which was lower in the MVI group (Table 1). As to delivery mode, women induced with MVI were less likely to be delivered by CS, compared to those in- duced with MVT (11.8% versus 23.3%), see Table 4. In models adjusting for Bishop Score and pre-induction with balloon catheter, there was a 67% reduced risk of CS in the MVI group compared to the MVT group (adjusted OR 0.33; 95% CI 0.13–0.81, p = 0.016). The results did not change in sensitivity analyses where we stratified for other potential confounders. As for the primary indication for induction, more women in the MVI group were induced due to preeclampsia/hypertension than in the MVT group, whereas other indications were similarly distributed. The time interval from drug administration to delivery showed a skewed distribution with a right tail in both the MVI and the MVT groups (Fig. 2). The average time from drug administration to delivery was significantly shorter in the MVI group compared to the MVT group (median time 15 h 43 min versus 19 h 37 min, p = 0.011), see Table 2. In regression models adjusting for Bishop Score and pre-induction with balloon catheter, the mean difference was 6 h 3 min (p = 0.002). The result did not change in models with log transformed outcomes (p = 0.001, data not shown). We conducted sensitivity analyses where we adjusted for additional potential confounders; however, the results did not change. We found no difference in the rate of women deliv- ered by CS due to fetal distress in the MVI versus the MVT groups; however, numbers were few (n = 5 (5.9%) versus n = 8 (9.3%)). Similarly we found no difference in the proportion of operative vaginal deliveries due to fetal distress in the two groups (MVI: n = 8 (9.4%) versus MVT: n = 11 (12.8%)). The number of labors diagnosed with uterine tachysystole and that ended with operative delivery due to fetal distress were few in both groups (MVI n = 4, MVT n = 6). Three neonates had an Apgar Score < 7 after 5 min; two in the MVI group and one in the MVT group. Ethical considerations The study was approved by the Oslo University Hospital Data Protection Official for Research (2012/9668). The study was also evaluated by the Regional Committee for medical and health research ethics (REC South East in Labor onset was defined as when the partogram was started by the attending midwife. Onset of active phase of labor was defined as regular, painful contractions that Page 4 of 8 Page 4 of 8 Marsdal et al. BMC Pregnancy and Childbirth (2018) 18:11 Norway); however, as the study was limited to observa- tions during standard clinical care, written informed consent was waivered. Norway); however, as the study was limited to observa- tions during standard clinical care, written informed consent was waivered. of 2.1 in the MVI group compared to the MVT group, thus confirming the shorter time interval from drug administration to delivery in the former (see Fig. 3 and Table 3). In 9 women, the inductions were started in the evening and the 2nd dose of MVT was delayed so that the woman could rest at night. Excluding these women from the analyses did not change the results for the primary outcomes; time from drug administration to delivery and rate of CS. Results None of the neonates were diagnosed with metabolic acidosis (defined as umbilical artery pH In the Cox model, where we censored deliveries inter- rupted by CS, the hazard ratio was increased by a factor Table 1 Maternal characteristics and indications for labor induction in nulliparous women induced with Misoprostol Vaginal Insert (MVI) compared to Misoprostol Vaginal Tablets (MVT) teristics and indications for labor induction in nulliparous women induced with Misoprostol Vaginal Insert prostol Vaginal Tablets (MVT) Table 1 Maternal characteristics and indications for labor induction in nulliparous women induced with Miso (MVI) compared to Misoprostol Vaginal Tablets (MVT) (MVI) compared to Misoprostol Vaginal Tablets (MVT) MVI, n = 85 MVT, n = 86 mean or n SD or % mean or n SD or % p-value Maternal age in yearsa 32.5 4.8 32.9 6.0 0.64 Body Mass Indexb 24.6 5.5 24.4 4.8 0.807 Gestational age in daysa 281 9.6 282 9.8 0.393 Bishop scorec 3.1 1.2 3.6 1.5 0.016 Birthweight 3454 484 3485 520 0.692 Preinduction with balloon catheter 44 51.8 40 47.1 0.645 Primary indication for induction - Pre labor ruptures of membranes (PROM) 21 24.7 23 27.1 0.861 - Preeclampsia/hypertension 25 29.4 12 14.1 0.026 - Fetal concerns 16 18.8 18 21.2 0.848 - Postterm pregnancyd 8 9.4 13 15.3 0.351 - Maternal concerns 10 11.8 9 10.6 1.000 - Other 5 5.9 10 11.6 0.279 aAt date of delivery bFrom first prenatal visit cAt insertion of MVI or first MVT d≥42 + 0 weeks, ≥41 + 2 if maternal age ≥40 years Marsdal et al. BMC Pregnancy and Childbirth (2018) 18:11 Page 5 of 8 Page 5 of 8 Frequency Frequency Hours from drug administration to delivery Misoprostol Vaginal Insert (MVI) Misoprostol Vaginal Tablets (MVT) Fig. Results 2 Time from drug administration to delivery in women induced with Misoprostol Vaginal Insert (MVI) (n = 85) compared to Misoprostol Vaginal Tablets (MVT) (n = 86) Table 2 Time outcomes in nulliparous women induced with Misoprostol Vaginal Insert (MVI) compared to Misoprostol Vaginal Tablets (MVT) MVI MVT Difference p-value Time intervals n = 85 n = 86 Time from drug administration to delivery Median (IQR) 15:43 (12:29) 19:37 (14:30) 3:54 0.011 Crude mean (SD) 18:39 (10:23) 23:42 (14:29) 5:03 0.010 Adjusted mean difference (CI; 95%)a 6:03 (2:20–9:46) 0.002 Time from drug administration to onset of active labor Median (IQR) 10:37 (10:42) 11:28 (12:13) 0:51 0.215 Crude mean (SD) 13:04 (8:32) 16:20 (13:27) 3:16 0.061 Adjusted mean difference (CI; 95%)a 4:16 (0:54–7:38) 0.013 Time from onset of active labor to delivery Median (IQR) 4:06 (6:54) 6:46 (5:50) 2:40 0.002 Crude mean (SD) 5:35 (4:36) 7:22 (4:09) 1:47 0.009 Adjusted mean difference (CI; 95%)a 1:47 (0:28–3:06) 0.008 Presented as hours:minutes aAdjusted for Bishop Score and pre-induction with balloon catheter Frequency Misoprostol Vaginal Insert (MVI) Table 2 Time outcomes in nulliparous women induced with Misoprostol Vaginal Insert (MVI) compared to Misoprostol Vaginal Tablets (MVT) Frequency Hours from drug administration to delivery Misoprostol Vaginal Tablets (MVT) Fig. 2 Time from drug administration to delivery in women induced Frequency with MVI had a lower risk of CS compared to women induced with MVT. There were no differences between the groups for the proportion of operative deliveries due to fetal distress or Apgar Score < 7 after 5 min. To our knowledge, this is the first study comparing MVI to MVT. After the European approval on MVI, three studies comparing MVI other induction methods have been published [11–13]. Two studies comparing MVI to Dinoprostone insert [11, 13] did not find the same benefits for MVI over Dinoprostone insert as the phase III trial [10]. One study comparing MVI to Oral Misoprostol found shorter time from drug administra- tion to delivery and a higher CS rate in the MVI group compared to Oral Misoprostol [12]. However, numbers for nulliparous women were not reported separately in any of the studies. Given the strong predictive value of parity on successful induction of labor, the results cannot directly be compared to our findings [15, 17]. Hours from drug administration to delivery Fig. Results 2 Time from drug administration to delivery in women induced with Misoprostol Vaginal Insert (MVI) (n = 85) compared to Misoprostol Vaginal Tablets (MVT) (n = 86) <7.00 and/or base deficit ≥12). Forty five (53%) women in the MVI group needed oxytocin during labor, com- pared to 63 (73%) in the MVT group (p = 0.006). Discussion We regard the mean adjusted difference in time from drug administration to delivery of 6 h 3 min as of clin- ical relevance, demonstrating MVI as the most effective induction agent. One contributory factor to the differ- ences in efficiency could be deviation from the protocol in the MVT group, compared to the MVI group. Com- pliance with procedure is more likely with the MVI as In this study, in a setting of routine obstetric care, in- duction of labor with MVI was associated with a shorter time from drug administration to delivery compared to MVT. Both time from drug administration to onset of active labor and labor duration were shorter in the MVI group compared to the MVT group. Women induced Marsdal et al. BMC Pregnancy and Childbirth (2018) 18:11 Page 6 of 8 Fig. 3 Survival plot for time from drug administration to delivery in nulliparous women induced with Misoprostol vaginal insert (MVI) compared to Misoprostol vaginal tablets (MVT) Fig. 3 Survival plot for time from drug administration to delivery in nulliparous women induced with Misoprostol vaginal insert (MVI) compared to Misoprostol vaginal tablets (MVT) Fig. 3 Survival plot for time from drug administration to delivery in nulliparous women induced with Misoprostol vaginal insert (MVI) compared to Misoprostol vaginal tablets (MVT) MVI in previous studies. However, in these studies MVI was compared to Dinoprostone [10, 21]. Previous studies of 25 μg MVT have also reported longer drug adminis- tration to delivery intervals compared to our findings, also in nulliparous women [22–24]. Median time has been reported to be 23.0 h [22], compared to 19.6 h in our study, and mean time 28.0 and 28.2 h [23, 24], com- pared to 23.7 h. In terms of CS, the rate found in our study is lower compared to previous studies on nullipar- ous women. In the MVI group, 11.8% underwent a CS in our study, compared to 32.9% and 34.5% in previous studies [10, 21]. For MVT, previous studies show more divergent results, from 20% to 42% [22, 25], compared to our result of 23.3%. These differences in efficiency and CS rate between the cited studies and our study may reflect provider-preference and a differential induc- tion- and labor management policy. The overall CS rate in Norway and Scandinavia is low compared to other high income countries. From Log rank test Discussion In 2014, 16.6% were delivered by CS in Norway, compared to 32.2% in USA and 26.2% in UK [26–28]. A high proportion of pre-induction with balloon catheter (49.1%) and PROM (25.7%) might have contributed to increased efficiency and lower CS rate. non-compliance would require removal of the insert, while non-compliance with the MVT is “just waiting a bit” with the next tablet. As prolonged latency and labor might lead to an increased risk of emergency CS and ad- verse maternal and neonatal outcome, an induction agent that is effective in the everyday routines of the de- partment is preferable [6, 19, 20]. On the other hand, uterine tachysystole is a matter of considerable safety concern, especially for the fetus, when inducing labor [5]. The 5 min Apgar Score < 7 did not differ between the groups, neither did the proportion of operative deliv- eries due to fetal distress; however, cases were few. The lower rate of CS does however suggest that MVI is a safer alternative for the mother compared to MVT. Compared to other studies, women in our study, both in the MVI and in the MVT groups, had more efficient induction processes and a lower CS rate. The median time from drug administration to delivery of 15.7 h in the MVI group is considerably shorter than the 25.9 h and 29.2 h reported on nulliparous women induced with Table 3 Adjusted hazard ratio in relation to time from drug administration to delivery in nulliparous women induced with Misoprostol Vaginal Insert (MVI) compared to Misoprostol Vaginal Tablets (MVT) Misoprostol Vaginal Insert (MVI) compared to Misoprostol Vaginal Tablets (MVT) Adjusted Hazard ratio 95% CI p-value Misoprostol vaginal insert (MVI) 2.11 1.48–3.02 0.001 Misoprostol vaginal tablets (MVT) 1 Reference Reference *From Log rank test This study has several limitations. First, this retro- spective cohort study was a part of a quality improve- ment project, focusing on induction in nulliparous women. Thus, there was no attempt of randomization. One could hypothesize that high risk pregnancies more often were induced using MVT, especially in the begin- ning of the study period, as the doctors and midwives were more familiar with this method. This might have Marsdal et al. Competing interests Kjersti Engen Marsdal, Ingvil Krarup Sørbye, Lise C Gaudernack, Mirjam Lukasse. The authors declare that they have no competing interests. Authors’ contributions Kjersti Engen Marsdal (KEM), Ingvil Krarup Sørbye (IKS), Lise C Gaudernack (LCG) and Mirjam Lukasse (ML) conceived the idea of the study. KEM and LCG collected the data. KEM, IKS, LCG and ML were involved in developing the analyses strategy, the final analyses strategy was approved by all authors. KEM and IKS performed the analyses. KEM, IKS, LCG and ML were involved in interpreting the results and writing the manuscript, all authors approved the final draft. Ethics approval and consent to participate The study was approved by the Oslo University Hospital Data Protection Official for Research (2012/9668). The study was also evaluated by the Regional Committee for medical and health research ethics (REC South East in Norway); however, as the study was limited to observations during standard clinical care, written informed consent was waivered. Consent for publication Not applicable. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Availability of data and materials h d l d d h The datasets analysed during the current study are available from the corresponding author on reasonable request and with permission of the local Data Protection Official for Research. Abbreviations CI: Confidence interval; CS: Cesarean section; CTG: Cardio-tocographic monitoring; MVI: Misoprostol vaginal insert; MVT: Misoprostol vaginal tablet; OR: Odds ratio; PROM: Pre-labor rupture of the membranes; SD: Standard deviation References 1. European perinatal health report: Health and Care of Pregnant Women and Babies in Europe in 2010. Euro-Peristat. http://www.europeristat.com/ reports/european-perinatal-health-report-2010.html. Accessed 03 Sept 2016. 1. European perinatal health report: Health and Care of Pregnant Women and Babies in Europe in 2010. Euro-Peristat. http://www.europeristat.com/ reports/european-perinatal-health-report-2010.html. Accessed 03 Sept 2016. 2. Dögl M, Vanky E, Heimstad R. Changes in induction methods have not influenced cesarean section rates among women with induced labor. Acta Obstet Gynecol Scand. 2016;95(1):112–5. Discussion BMC Pregnancy and Childbirth (2018) 18:11 Page 7 of 8 Page 7 of 8 Table 4 Delivery mode in nulliparous women induced with Misoprostol Vaginal Insert (MVI) compared to Misoprostol Vaginal Tablets (MVT) MVI, n = 85 MVT, n = 86 Crude, n = 171 Adjusteda, n=169b n % n % OR 95% CI p-value OR 95% CI p-value Cesarean section 10 11.8 20 23.3 0.44 0.19–1.01 0.052 0.33 0.13–0.81 0.016 Operative vaginal delivery 19 22.4 18 20.9 1.09 0.53–2.25 0.821 1.14 0.54–2.39 0.733 Spontaneous vaginal delivery 56 65.9 48 55.8 1.45 0.78–2.69 0.236 1.60 0.84–3.04 0.150 aAdjusted for Bishop score and pre-induction with balloon catheter b2 excluded due to missing data on Bishop score Insert (MVI) compared to Misoprostol Vaginal Tablets (MVT aAdjusted for Bishop score and pre-induction with balloon catheter b2 excluded due to missing data on Bishop score contributed to a higher CS rate in the MVT group. How- ever, as the maternal characteristics and the indications for induction of labor did not show major differences, this selection bias is unlikely to have had a major impact on our results. Conversely, the average Bishop Score was higher in the MVT group, which should correspond to more favorable cervix status. Furthermore fewer women had preeclampsia/hypertension in the MVT group which is a known risk factor for CS [29]. Although the lack of randomization could be considered a weakness, the results reflect what happened when MVI was introduced to a ma- ternity department, without any adjustments in the treat- ment due to research considerations. Our results were robust across several different statistical models. Second, labor onset in this study was defined as when the midwife present defined active labor and started the partogram. This subjective assessment will differ between midwives. However, this is unlikely to represent a differential bias and is unlikely to influence the time from drug adminis- tration to delivery. Finally, the women in our study were not asked about their birth experience. As a negative birth experience is associated with an increased risk of postpar- tum depression, subsequent fear of childbirth and request for elective CS [6, 30], this would have added valuable information to the study. Funding Funder: Oslo and Akershus University College. Conclusions 1Department of Obstetrics, Oslo University Hospital Rikshospitalet, P.O. Box 4956 Nydalen, 0424 Oslo, Norway. 2Oslo and Akershus University College, Faculty of Health Sciences, Department of Nursing and Health Promotion, P.O. Box 4, 0130 Oslo, Norway. In this study among nulliparous women in a routine care setting we found 200 μg MVI to be a more efficient and safe labor induction agent compared to 25 μg MVT. The time from drug administration to delivery was signifi- cantly shorter and the CS rate reduced in women induced with MVI, compared with MVT. Future studies comparing methods for labor induction should acknow- ledge the particular status of nulliparous women. Received: 20 September 2017 Accepted: 27 December 2017 Received: 20 September 2017 Accepted: 27 December 2017 Abbreviations CI C fid i 3. Yeast JD, Jones A, Poskin M. Induction of labor and the relationship to cesarean delivery: a review of 7001 consecutive inductions. Am J Obstet Gynecol. 1999;180(3 Pt 1):628–33. 4. Vahratian A, Zhang J, Troendle JF, Sciscione AC, Hoffman MK. Labor progression and risk of cesarean delivery in electively induced nulliparas. Obstet Gynecol. 2005;105(4):698–704. Acknowledgements Not applicable Acknowledgements Not applicable Marsdal et al. BMC Pregnancy and Childbirth (2018) 18:11 Page 8 of 8 Page 8 of 8 5. Stewart RD, Bleich AT, Lo JY, Alexander JM, McIntire DD, Leveno KJ. Defining uterine tachysystole: how much is too much? Am J Obstet Gynecol. 2012;207(4):290–6. 6. Kolås T, Hofoss D, Daltveit AK, Nilsen ST, Henriksen T, Häger R, Ingemarsson I, Øian P. Indications for cesarean deliveries in Norway. Am J Obstet Gynecol. 2003;188(4):864–70. 6. Kolås T, Hofoss D, Daltveit AK, Nilsen ST, Henriksen T, Häger R, Ingemarsson I, Øian P. Indications for cesarean deliveries in Norway. Am J Obstet Gynecol. 2003;188(4):864–70. 7. Hofmeyr GJ, Gülmezoglu AM, Pileggi C. Vaginal misoprostol for cervical ripening and induction of labour. Cochrane Database Syst Rev. 2010;10 8. Misodel Summary of Product Characteristics. https://www.ferring.com/en/ media/press-releases/2013/misodel-17oct13/. Accessed 01 Oct 2016. y 7. Hofmeyr GJ, Gülmezoglu AM, Pileggi C. Vaginal misoprostol for cervical ripening and induction of labour. Cochrane Database Syst Rev. 2010;10 8. Misodel Summary of Product Characteristics. https://www.ferring.com/en/ media/press-releases/2013/misodel-17oct13/. Accessed 01 Oct 2016. 9. Heads of Medicines Agencies. Misoprostol gynaecological indication PSUR SAR. 2015. http://www.hma.eu/search.html?id=6&q=misodel&L=0. Accessed 28 Sept 2016. 10. Wing DA, Brown R, Plante LA, Miller H, Rugarn O, Powers BL. Misoprostol vaginal insert and time to vaginal delivery: a randomized controlled trial. Obstet Gynecol. 2013;122(2 Pt 1):201–9. 11. Mayer RB, Oppelt P, Shebl O, Pömer J, Allerstorfer C, Weiss C. Initial clinical experience with a misoprostol vaginal insert in comparison with a dinoprostone insert for inducing labor. Eur J Obstet Gynecol Reprod Biol. 2016;200:89–93. 12. Dobert M, Brandstetter A, Henrich W, Rawnaq T, Hasselbeck H, Dobert TF, Hinkson L, Schwaerzler P. The misoprostol vaginal insert compared with oral misoprostol for labor induction in term pregnancies: a pair-matched case- control study. J of. Perinat Med. 2017; 13. Gornisiewicz T, Jaworowski A, Zembala-Szczerba M, Babczyk D, Huras H. Analysis of intravaginal misoprostol 0.2 mg versus intracervical dinoprostone 0.5 mg doses for labor induction at term pregnancies. Ginekologia pol. 2017;88(6):320–4. 14. Robson M, Murphy M, Byrne F. 30. Righetti-Veltema M, Conne-Perréard E, Bousquet A, Manzano J. Risk factors and predictive signs of postpartum depression. J Affect Disord. 1998;49(3):167–80. 29. Kim LH, Cheng YW, Delaney S, Jelin AC, Caughey ABI. Preeclampsia associated with an increased risk of cesarean delivery if labor is induced? J Matern Fetal Med. 2010;23(5):383–8. 28. NHS Digital. NHS Maternity Statistics - England, 2013–14. https://digital.nhs. uk/catalogue/PUB16725. Accessed 19 Oct 2016. 29. Kim LH, Cheng YW, Delaney S, Jelin AC, Caughey ABI. Preeclampsia associated with an increased risk of cesarean delivery if labor is induced? J Matern Fetal Med. 2010;23(5):383–8. 30. Righetti-Veltema M, Conne-Perréard E, Bousquet A, Manzano J. Risk factors and predictive signs of postpartum depression. J Affect Disord. 1998;49(3):167–80. 28. NHS Digital. NHS Maternity Statistics - England, 2013–14. https://digital.nhs. uk/catalogue/PUB16725. Accessed 19 Oct 2016. Abbreviations CI C fid i Submit your next manuscript to BioMed Central and we will help you at every step: Abbreviations CI C fid i Quality assurance: the 10-group classification system (Robson classification), induction of labor, and cesarean delivery. Int J Gynaecol Obstet. 2015;131:23–7. 15. Pevzner L, Rayburn WF, Rumney P, Wing DA. Factors predicting successful labor induction with dinoprostone and misoprostol vaginal inserts. Obstet Gynecol. 2009;114(2 Pt 1):261–7. 16. Wing DA, Tran S, Paul RH. Factors affecting the likelihood of successful induction after intravaginal misoprostol application for cervical ripening and labor induction. Am J Obstet Gynecol. 2002;186(6):1237–40. y 17. Crane JMG, Delaney T, Butt KD, Bennett KA, Hutchens D, Young DC. Predictors of successful labor induction with oral or vaginal misoprostol. J Matern Fetal Med. 2004;15(5):319–23. 18. Chen W, Xue J, Gaudet L, Walker M, Wen SW. Meta-analysis of Foley catheter plus misoprostol versus misoprostol alone for cervical ripening. Int J Gynaecol Obstet. 2015;129(3):193–8. 18. Chen W, Xue J, Gaudet L, Walker M, Wen SW. Meta-analysis of Foley catheter plus misoprostol versus misoprostol alone for cervical ripening. Int J Gynaecol Obstet. 2015;129(3):193–8. 19. Laughon SK, Berghella V, Reddy UM, Sundaram R, Lu Z, Hoffman MK. Neonatal and maternal outcomes with prolonged second stage of labor. Obstet Gynecol. 2014;124(1):57–67. 19. Laughon SK, Berghella V, Reddy UM, Sundaram R, Lu Z, Hoffman MK. Neonatal and maternal outcomes with prolonged second stage of labor. Obstet Gynecol. 2014;124(1):57–67. y 20. Maghoma J, Buchmann EJ. Maternal and fetal risks associated with prolonged latent phase of labour. J Obstet Gynaecol. 2002;22(1):16–9. 20. Maghoma J, Buchmann EJ. Maternal and fetal risks associated with prolonged latent phase of labour. J Obstet Gynaecol. 2002;22(1):16–9. 21. Wing DA, Miller H, Parker L, Powers BL, Rayburn WF. Misoprostol vaginal insert for successful labor induction: a randomized controlled trial. Obstet Gynecol. 2011;117(3):533–41. 21. Wing DA, Miller H, Parker L, Powers BL, Rayburn WF. Misoprostol vaginal insert for successful labor induction: a randomized controlled trial. Obstet Gynecol. 2011;117(3):533–41. 22. Gregson S, Waterstone M, Norman I, Murrells TA. Randomised controlled trial comparing low dose vaginal misoprostol and dinoprostone vaginal gel for inducing labour at term. BJOG. 2005;112(4):438–44. 22. Gregson S, Waterstone M, Norman I, Murrells TA. Randomised controlled trial comparing low dose vaginal misoprostol and dinoprostone vaginal gel for inducing labour at term. BJOG. 2005;112(4):438–44. 23. Calder AA, Loughney AD, Weir CJ, Barber JW. Induction of labour in nulliparous and multiparous women: a UK, multicentre, open-label study of intravaginal misoprostol in comparison with dinoprostone. BJOG. 2008;115(10):1279–88. Submit your next manuscript to BioMed Central and we will help you at every step: Submit your next manuscript to BioMed Central and we will help you at every step: • We accept pre-submission inquiries • Our selector tool helps you to ﬁnd the most relevant journal • We provide round the clock customer support • Convenient online submission • Thorough peer review • Inclusion in PubMed and all major indexing services • Maximum visibility for your research Submit your manuscript at www.biomedcentral.com/submit and we will help you at every step: 24. Wing DA, Ham D, Paul RHA. Comparison of orally administered misoprostol with vaginally administered misoprostol for cervical ripening and labor induction. Am J Obstet Gynecol. 1999;180(5):1155–60. 24. Wing DA, Ham D, Paul RHA. Comparison of orally administered misoprostol with vaginally administered misoprostol for cervical ripening and labor induction. Am J Obstet Gynecol. 1999;180(5):1155–60. 25. van Gemund N, Scherjon S, LeCessie S, van Leeuwen JHS, van Roosmalen J, Kanhai HHH. A Randomised trial comparing low dose vaginal misoprostol and dinoprostone for labour induction. BJOG 2004; 111(1):42–49. 25. van Gemund N, Scherjon S, LeCessie S, van Leeuwen JHS, van Roosmalen J, Kanhai HHH. A Randomised trial comparing low dose vaginal misoprostol and dinoprostone for labour induction. BJOG 2004; 111(1):42–49. 26. Norwegian Institute of Public Health. Medical Birth Registry of Norway. http://statistikk fhi no/mfr/ Accessed 18 Oct 2016 25. van Gemund N, Scherjon S, LeCessie S, van Leeuwen JHS, van Roosmalen J, Kanhai HHH. A Randomised trial comparing low dose vaginal misoprostol and dinoprostone for labour induction. BJOG 2004; 111(1):42–49. 26. Norwegian Institute of Public Health. Medical Birth Registry of Norway. http://statistikk.fhi.no/mfr/. Accessed 18 Oct 2016. 26. Norwegian Institute of Public Health. Medical Birth Registry of Norway. http://statistikk.fhi.no/mfr/. Accessed 18 Oct 2016. 27. European Medicines Agency. List of nationally authorised medical products Active substance: Misoprostol (gynaecological indication - labour induction). http://www.ema.europa.eu/docs/en_GB/document_library/Periodic_safety_ update_single_assessment/2017/03/WC500223582.pdf. Accessed 02 Jan 2018. 27. European Medicines Agency. List of nationally authorised medical products Active substance: Misoprostol (gynaecological indication - labour induction). http://www.ema.europa.eu/docs/en_GB/document_library/Periodic_safety_ update_single_assessment/2017/03/WC500223582.pdf. Accessed 02 Jan 2018.
https://openalex.org/W2567925291	http://discovery.ucl.ac.uk/1553943/1/ddx004.pdf	English	null	High dietary folate in pregnant mice leads to pseudo-MTHFR deficiency and altered methyl metabolism, with embryonic growth delay and short-term memory impairment in offspring	Human molecular genetics online/Human molecular genetics	2,017	cc-by	9,491	High dietary folate in pregnant mice leads to pseudo-MTHFR deﬁciency and altered methyl metabolism, with embryonic growth delay and short-term memory impairment in offspring Renata H. Bahous1, Naﬁsa M. Jadavji2, Liyuan Deng1, Marta Cosın-Tomas3, Jessica Lu1, Olga Malysheva4, Kit-Yi Leung5, Ming-Kai Ho6, Merce` Pallas3, Perla Kaliman7,8, Nicholas D.E. Greene5, Barry J. Bedell6, Marie A. Caudill4 and Rima Rozen1,* 1Departments of Human Genetics and Pediatrics, Research Institute of the McGill University Health Center, Montreal, Quebec, Canada, 2Department of Neuroscience, Carleton University, Ottawa, Ontario, Canada, 3Pharmacology Unit, Faculty of Pharmacy, Institut de Neurocie`ncia Universitat de Barcelona (IBUB), Nucli Universitari de Pedralbes, Barcelona, Spain, 4Division of Nutritional Sciences and Genomics, Cornell University, Ithaca, NY, USA, 5Developmental Biology and Cancer Programme, Great Ormond Street Institute of Child Health, University College London, London, United Kingdom, 6Department of Neurology and Neurosurgery, McGill University, Montreal, Quebec, Canada, 7Institute of Biomedical Investigation of Barcelona, Spanish National Research Council, Barcelona, Spain and 8Center for Mind and Brain, University of California Davis, Davis, CA, USA To whom correspondence should be addressed at: Research Institute of the McGill University Health Centre, 1001 De´carie Boulevard, Room EM0.3211, Montreal, Quebec H4A 3J1, Canada. Tel: +5149341934 ext. 23839; Fax: +514-933-4673; Email: rima.rozen@mcgill.ca Received: October 24, 2016. Revised: December 6, 2016. Accepted: January 3, 2017 V C The Author 2017. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. To whom correspondence should be addressed at: Research Institute of the McGill University Health Centre, 1001 De´carie Boulevard, Room EM0.3211, Montreal, Quebec H4A 3J1, Canada. Tel: +5149341934 ext. 23839; Fax: +514-933-4673; Email: rima.rozen@mcgill.ca Human Molecular Genetics, 2017, Vol. 26, No. 5 888–900 Human Molecular Genetics, 2017, Vol. 26, No. 5 888–900 Human Molecular Genetics, 2017, Vol. 26, No. 5 888–900 doi: 10.1093/hmg/ddx004 Advance Access Publication Date: 6 January 2017 Original Article Introduction 4–5 mg/day, primarily due to the increased use of vitamin sup- plements (17,18). Concerns have emerged about the possible consequences of high folic acid intake; these concerns include increased growth of sub-clinical tumors, the possibility of mask- ing B12 deﬁciency and impaired immune function (16,19). We have been investigating potential adverse outcomes of high fo- lic acid intake in mice. We found that adult male mice fed a folic acid supplemented diet (FASD, 10 the recommended amount for rodents) for 6 months developed a pseudo-MTHFR deﬁ- ciency, with hepatic injury (20). Folate derivatives are critical for brain development and neuro- transmitter synthesis because they provide one-carbon units for nucleotide synthesis and methylation reactions (1). One of the most studied enzymes in the folate metabolic pathway is methylenetetrahydrofolate reductase (MTHFR, EC 1.5.1.20), a ubiquitous enzyme which generates 5-methyltetrahydrofolate (5-methylTHF) for remethylation of homocysteine to methio- nine (2). Methionine is the precursor of S-adenosylmethionine (SAM), the primary methyl donor in most mammalian methyla- tion reactions. An alternate folate-independent pathway for methionine synthesis is present primarily in liver and kidney; it uses the choline metabolite betaine as a methyl donor. Disturbances in folate metabolism can be genetic and/or dietary and often result in compensatory disturbances in choline me- tabolism. Choline is required to synthesize the neurotransmit- ter acetylcholine and critical phospholipids for membrane integrity (3,4). The use of vitamin supplements is particularly important for women of childbearing age, although high-dose supplementa- tion with folic acid raises questions about possible negative ef- fects on their offspring, particularly on brain development (21). Therefore, in this study we aimed to determine whether high maternal folic acid intake in mice affected brain function or me- tabolism in their pups and embryos. Towards this end, we fed wild-type female mice a control diet (CD) or FASD for 5 weeks prior to mating, and maintained the diets throughout preg- nancy and lactation, to assess brain development in offspring. We found that offspring of mothers fed FASD had intra-uterine growth delay, short-term memory impairment and altered brain development. Maternal and offspring pseudo-MTHFR deﬁ- ciency with disturbances in choline/methyl metabolism are likely to have contributed to these outcomes. Relatively rare severe deﬁciencies in MTHFR (usually <20% of enzyme activity) are a cause of homocystinuria, an inborn er- ror of metabolism characterized by a variety of neurologic symptoms including developmental delays, motor disturbances and brain atrophy (5). Introduction A mild deﬁciency in MTHFR, due to the 677C > T (A222V) polymorphism, is present in the homozygous state in 10–15% of many Caucasian populations and encodes a thermolabile enzyme with 30% residual activity (2). This SNP has been reported to increase risk for neural tube defects, preg- nancy complications and neuropsychiatric diseases such as schizophrenia and autism (3,6–8). To study these complex traits, we developed mouse models that mimic these MTHFR deﬁcien- cies (9). Mthfr-/- mice, a model for homocystinuria, have short- term memory impairment, altered hippocampal morphology and disturbed acetylcholine metabolism (10). Heterozygous MTHFR deﬁciency in dams (Mthfrþ/- mice) resulted in memory impairment in their pups (11). Abstract Methylenetetrahydrofolate reductase (MTHFR) generates methyltetrahydrofolate for methylation reactions. Severe MTHFR deﬁciency results in homocystinuria and neurologic impairment. Mild MTHFR deﬁciency (677C > T polymorphism) increases risk for complex traits, including neuropsychiatric disorders. Although low dietary folate impacts brain development, recent concerns have focused on high folate intake following food fortiﬁcation and increased vitamin use. Our goal was to determine whether high dietary folate during pregnancy affects brain development in murine offspring. Female mice were placed on control diet (CD) or folic acid-supplemented diet (FASD) throughout mating, pregnancy and lactation. Three-week- old male pups were evaluated for motor and cognitive function. Tissues from E17.5 embryos, pups and dams were collected for choline/methyl metabolite measurements, immunoblotting or gene expression of relevant enzymes. Brains were exam- ined for morphology of hippocampus and cortex. Pups of FASD mothers displayed short-term memory impairment, de- creased hippocampal size and decreased thickness of the dentate gyrus. MTHFR protein levels were reduced in FASD pup 888 Human Molecular Genetics, 2017, Vol. 26, No. 5 889 livers, with lower concentrations of phosphocholine and glycerophosphocholine in liver and hippocampus, respectively. FASD pup brains showed evidence of altered acetylcholine availability and Dnmt3a mRNA was reduced in cortex and hippo- campus. E17.5 embryos and placentas from FASD dams were smaller. MTHFR protein and mRNA were reduced in embryonic liver, with lower concentrations of choline, betaine and phosphocholine. Embryonic brain displayed altered development of cortical layers. In summary, high folate intake during pregnancy leads to pseudo-MTHFR deﬁciency, disturbed choline/methyl metabolism, embryonic growth delay and memory impairment in offspring. These ﬁndings highlight the unintended nega- tive consequences of supplemental folic acid. Three-week-old pups born to FASD mothers had short-term memory impairment and reduced hippocampal area There were no differences in body weights of pups between the two dietary groups (Supplementary Material, Fig. S1A). Behavioral testing was performed on 3-week-old male pups to assess brain function. In the ladder beam test for measurement of gait, there were no differences in percent error or movement score (Supplementary Material, Fig. S1B). In the open ﬁeld test, which evaluates anxiety, there were no differences in the activ- ity pattern or number of ﬁeld entries (Supplementary Material, Fig. 1C). To assess memory, the novel object recognition test (NOR) and the Y-maze test were performed. No differences were observed in the Y-maze test suggesting there were no differ- ences in spatial memory (Supplementary Material, Fig. 1D). However, mice from FASD mothers had a negative discrimina- tion index in the NOR, i.e. these pups spent signiﬁcantly less time with the novel object than with the familiar object (Fig. 1A; P < 0.005, unpaired t-test). These ﬁndings suggest that pups from FASD mothers have short-term memory impairment, a phenotype that was also observed in Mthfr-/- mice (10). Since we had previously observed that Mthfr-/- mice had reduced hippo- campal volume, we measured the area of the hippocampus and the thickness of the dentate gyrus (DG) in sagittal brain sections Folate cannot be synthesized by humans and must be ac- quired from the diet (12). Low maternal folate intake during pregnancy is a risk factor for neural tube defects and other birth defects, as well as other pregnancy complications including in- trauterine growth restriction (3). Low maternal folate intake has also been associated with disturbances in offspring brain func- tion, since the developing brain is sensitive to nutrient deple- tion during gestation and early development. In both humans and rodents, folate deﬁciency during pregnancy has been asso- ciated with cognitive impairment, increased anxiety and de- pression (13,14). In mice, maternal folate-deﬁcient or choline- deﬁcient diets lead to increased apoptosis in fetal brain, particu- larly the hippocampus (11,15). In a public health effort to reduce the incidence of neural tube defects, grains have been fortiﬁed with folic acid in over 50 countries. The recommended daily intake for adults is 0.4 mg with a recommended tolerable upper intake level of 1 mg (16). However, there are groups in the population that are consuming Human Molecular Genetics, 2017, Vol. 26, No. 5 890 1. Maternal FASD during pregnancy and lactation led to pseudo-MTHFR deﬁciency in offspring liver We previously reported that FASD leads to pseudo-MTHFR deﬁ- ciency in adult male liver (20). We therefore measured MTHFR protein levels in liver of 3-week-old offspring. We found a 3-fold decrease in total MTHFR protein (Fig. 2A and C; P < 0.05, un- paired t-test) and a 4-fold increase in the ratio of the phosphory- lated to non-phosphorylated isoform of MTHFR (Fig. 2B and C; P < 0.005, unpaired t-test). Decreased phosphorylation has been reported to increase MTHFR activity by 1.2 fold in vitro; the effect of phosphorylation in vivo has not been examined (22). No changes were detected at the mRNA level (data not shown). These data are consistent with our previous report and suggest Figure 1. Pups born to mothers fed high folate diet had short-term memory impairment and reduced hippocampal area. (A) Three-week-old male mice were tested us- ing the NOR. The discrimination index is the ratio of time spent with the novel object versus time with the familiar object; a negative discrimination index indicates short-term memory impairment. Data from two independent experiments are shown (n ¼ 15–20/group). (B) The average area of the hippocampus and (C) the average thickness of the dentate gyrus (DG) were reduced in FASD pups (n ¼ 5/group). Representative images of hippocampus and DG from CD pup (D and F, respectively) and FASD pup (E and G, respectively) are shown. Arrows in the DG indicate where the thickness measurements were performed. Values are means 6 SEM; P < 0.05, *P < 0.005. of pups; both were reduced due to FASD (P < 0.005 and P < 0.05, respectively, unpaired t-test) (Fig. 1B–G). that offspring born to FASD mothers have pseudo-MTHFR deﬁciency. We also measured MTHFR levels in the cortex and hippo- campus, two brain regions that impact performance in the NOR test, but no differences in total immunoreactive MTHFR levels were detected (data not shown). Consistent with this observa- tion, there were no differences in the percentages of THF and its one-carbon substituted derivatives in the hippocampus between the two dietary groups (Supplementary Material, Table S1). Three-week-old pups born to FASD mothers had short-term memory impairment and reduced hippocampal area Pups born to mothers fed high folate diet had short-term memory impairment and reduced hippocampal area. (A) Three-week-old male mice were tested us- e NOR. The discrimination index is the ratio of time spent with the novel object versus time with the familiar object; a negative discrimination index indicates term memory impairment. Data from two independent experiments are shown (n ¼ 15–20/group). (B) The average area of the hippocampus and (C) the average ess of the dentate gyrus (DG) were reduced in FASD pups (n ¼ 5/group). Representative images of hippocampus and DG from CD pup (D and F, respectively) ASD pup (E and G, respectively) are shown. Arrows in the DG indicate where the thickness measurements were performed. Values are means 6 SEM; P < 0.05, 0.005. FASD resulted in pseudo-MTHFR deﬁciency and altered folate and choline metabolism in lactating mothers provision of methyl donors for methionine/SAM synthesis (10,20,23). We therefore measured SAM, SAH, choline and related metabolites in offspring liver, the primary organ of one-carbon metabolism. We did not observe differences in SAM, SAH, choline, betaine, glycerophosphocholine (GPC), phosphatidylcholine, sphingomyelin or lysophosphatidylcho- line (Supplementary Material, Table S2). However, there was a signiﬁcant 30% decrease in phosphocholine (PCho), the primary storage form of choline (Fig. 3A; P < 0.05, unpaired t-test). There were no differences in maternal body weights between groups (CD: 24.86 1.8; FASD: 26.56 1.7). As seen in offspring, there was a decrease in total MTHFR protein in maternal liver (Fig. 5A; P< 0.05, unpaired t-test) and an increased ratio of phosphorylated to non-phosphorylated MTHFR (Fig. 5B; P< 0.005, unpaired t-test). We measured mRNA expression of other important enzymes in one-carbon metabolism. Methionine synthase (Mtr) is the en- zyme responsible for remethylating homocysteine to methio- nine using 5-methylTHF generated by MTHFR. In maternal liver, Mtr mRNA levels signiﬁcantly decreased due to diet (Fig. 5C; P < 0.005, unpaired t-test). mRNA for methionine synthase re- ductase (Mtrr), the enzyme required to activate Mtr, was in- creased (Fig. 4D; P < 0.005, unpaired t-test), possibly as a compensatory mechanism for reduced Mtr. We also measured choline metabolites in hippocampus (Supplementary Material, Table S2) and observed a trend for de- creased levels of GPC in offspring (Fig. 3B; P ¼ 0.06, unpaired t- test), suggesting an increased conversion of GPC to choline to replenish choline pools. Choline is a precursor of acetylcholine (Ach), a neurotrans- mitter that plays a role in memory (4). mRNA levels of acetyl- cholinesterase (AchE), which cleaves acetylcholine at the synaptic cleft, were measured in cortex and hippocampus. AchE mRNA was signiﬁcantly reduced in the FASD group in cortex (Fig. 3C; P < 0.05, unpaired t-test with Welsh’s correction). Cortical mRNA levels of the nicotinic receptor of acetylcholine, Chrna7, were decreased in the FASD group but did not reach sta- tistical signiﬁcance (data not shown). However, we observed a signiﬁcant correlation between AchE and Chrna7 levels in cortex (Fig. 3D; r ¼ 0.894, P < 0.05). AchE and Chrna7 mRNA levels did not change in hippocampus (data not shown). Phosphatidylethanolamine methyltransferase (Pemt) converts phosphatidylethanolamine into phosphatidylcholine, the precur- sor of GPC (26). We observed increased Pemt mRNA levels due to diet (Fig. 5E; P< 0.005, unpaired t-test). Choline metabolites were measured in maternal liver and plasma. FASD resulted in pseudo-MTHFR deﬁciency and altered folate and choline metabolism in lactating mothers No changes were ob- served in liver or plasma for choline, betaine, phosphatidylcholine, PCho, sphingomyelin or lysophosphatidylcholine (Supplementary Material, Table S3). However, there was a trend towards reduced GPC concentrations in maternal plasma (Fig. 5F; P¼ 0.06, unpaired t-test). The increase in Pemt may be a compensatory mechanism to increase circulating GPC for the generation of choline. We also measured immunoreactive protein levels of choline acetyltransferase (ChAT), the enzyme that synthesizes Ach from choline; there were no differences in cortical or hippocam- pal extracts (data not shown). However, when we stained for cholinergic neurons in the Substantia Innominata (SI), which project to the cortex, we found a trend for reduced ChAT stain- ing in FASD pups (Fig. 3E; P ¼ 0.07, unpaired t-test). There was no signiﬁcant difference in the number of cholinergic neurons (Fig. 3F). Embryos and placentas of FASD mothers were smaller at E17.5 To understand the effects of FASD during early brain develop- ment, we collected maternal and embryonic tissues at E17.5 from mothers fed CD and FASD. There were no differences in maternal body weights (CD: 29.9 6 1.7; FASD: 30.1 6 2.1) or litter sizes (CD: 6 6 1.5; FASD: 8.3 6 0.3) between groups. However, av- erage fetal body weight per litter was signiﬁcantly reduced in the FASD group (Fig. 6A; P < 0.05, unpaired t-test) and there was a trend towards reduced placental weight per litter (Fig. 6B; P ¼ 0.08, unpaired t-test), suggesting that a high folate diet dur- ing pregnancy leads to growth delay in embryos. Choline metabolism was disturbed in liver and brain of FASD pups In several other studies, we have shown that disturbances in fo- late metabolism are often accompanied by disturbances in cho- line metabolism, because these two pathways intersect in the 891 Human Molecular Genetics, 2017, Vol. 26, No. 5 \| 891 Figure 2. Pups born to mothers fed high folate diet had pseudo-MTHFR deﬁciency in liver. (A) Total immunoreactive MTHFR protein was reduced in livers of FASD pups. (B) The ratio of the phosphorylated isoform (p-MTHFR, less active) to the non-phosphorylated isoform (MTHFR) was increased in FASD pups. (C) Representative Western blot of MTHFR in offspring liver. Values are means 6 SEM from 5 mice/group; P<0.05, *P<0.005. Figure 2. Pups born to mothers fed high folate diet had pseudo-MTHFR deﬁciency in liver. (A) Total immunoreactive MTHFR protein was reduced in livers of FASD pups. (B) The ratio of the phosphorylated isoform (p-MTHFR, less active) to the non-phosphorylated isoform (MTHFR) was increased in FASD pups. (C) Representative Western blot of MTHFR in offspring liver. Values are means 6 SEM from 5 mice/group; P<0.05, **P<0.005. FASD pups had reduced expression of Dnmt3a Maternal high folic acid intake can result in methylation changes in the brain of offspring (24), and DNA methyltransfer- ases have been shown to be involved in memory (25). We there- fore measured mRNA levels of DNA methyltransferases in offspring cortex and hippocampus. We observed a signiﬁcant decrease in Dnmt3a mRNA in cortex and hippocampus (Fig. 4A and B, respectively; P < 0.05, unpaired t-test). We did not observe changes in Dnmt1 (Fig. 4C) or Dnmt3b (Fig. 4D) in cortex, suggest- ing that this effect is speciﬁc to the de novo methyltransferase Dnmt3a. FASD embryos showed reduced expression of one-carbon enzymes in liver, reduced hepatic choline metabolites and altered brain development At E17.5 the embryo is metabolically active and relies less on maternal metabolism (27). In the FASD group, we observed a 892 \| Human Molecular Genetics, 2017, Vol. 26, No. 5 892 Figure 3. Pups born to mothers fed high folate diet had disturbances in choline and acetylcholine metabolism. (A) The concentration of phosphocholine (PCho), the storage form of choline, was signiﬁcantly decreased in liver (n ¼ 6/group). (B) In hippocampus, there was a trend towards reduced glycerophosphocholine (GPC) in FASD pups (n ¼ 7/group, #P ¼ 0.06). (C) mRNA expression of acetylcholinesterase (AchE) was reduced in cortex of FASD pups (n ¼ 7/group). (D) mRNA levels of the nicotinic re- ceptor of acetylcholine, Chrna7 and AchE in cortex were signiﬁcantly positively correlated (n ¼ 7/group; r ¼ 0.894, P < 0.05). ChAT staining to measure (E) staining inten- sity and (F) numbers of cholinergic neurons in the Substantia Innominata (SI). There was a trend towards reduced intensity of ChAT staining in the SI of FASD pups (n ¼ 5/group, #P ¼ 0.07) but no signiﬁcant difference in number of cholinergic neurons. Values are means 6 SEM; P < 0.05. 892 \| Human Molecular Genetics, 2017, Vol. 26, No. 5 Figure 3. Pups born to mothers fed high folate diet had disturbances in choline and acetylcholine metabolism. (A) The concentration of phosphocholine (PCho), the storage form of choline, was signiﬁcantly decreased in liver (n ¼ 6/group). (B) In hippocampus, there was a trend towards reduced glycerophosphocholine (GPC) in FASD pups (n ¼ 7/group, #P ¼ 0.06). (C) mRNA expression of acetylcholinesterase (AchE) was reduced in cortex of FASD pups (n ¼ 7/group). (D) mRNA levels of the nicotinic re- ceptor of acetylcholine, Chrna7 and AchE in cortex were signiﬁcantly positively correlated (n ¼ 7/group; r ¼ 0.894, P < 0.05). ChAT staining to measure (E) staining inten- sity and (F) numbers of cholinergic neurons in the Substantia Innominata (SI). There was a trend towards reduced intensity of ChAT staining in the SI of FASD pups (n ¼ 5/group, #P ¼ 0.07) but no signiﬁcant difference in number of cholinergic neurons. Values are means 6 SEM; P < 0.05. decrease in total MTHFR protein levels in embryonic liver (Fig. 7A; P < 0.05, unpaired t-test), with no differences in isoform ratio (data not shown). These results indicate that the pseudo- MTHFR deﬁciency is present prenatally. Discussion High folate intake is commonly observed in several population sub-groups, including pregnant women, raising concerns about the possible negative consequences on the child’s development (29). We investigated the effect of high folate consumption dur- ing pregnancy and lactation on offspring brain function. Three- week-old pups born to FASD mothers had short-term memory impairment. Memory impairment was also observed in our ear- lier report of Mthfr-/- mice (10). This similarity is particularly in- teresting because we found that the pups of FASD mothers had pseudo-MTHFR deﬁciency. Total MTHFR protein levels were re- duced and there was an increased ratio of the phosphorylated (less active) to non-phosphorylated isoform. Our ﬁrst ﬁnding of pseudo-MTHFR deﬁciency with high folate intake was in the liver of adult male mice maintained on FASD for 6 months (20). The observations in this study of reduced MTHFR protein in liv- ers of pups, embryos and lactating dams suggest that this phe- nomenon is seen in various ages and sexes, at least for liver. FASD embryos showed reduced expression of one-carbon enzymes in liver, reduced hepatic choline metabolites and altered brain development Of additional interest was our ﬁnding of decreased Mthfr mRNA levels (Fig. 7B; P < 0.05, unpaired t-test). Changes in Mthfr mRNA were not ob- served due to FASD in the 3-week-old pups or in our earlier study of adult mice (20), suggesting a different regulatory mech- anism for MTHFR at the embryonic stage. Key enzymes in the synthesis and utilization of SAM were also decreased by FASD. Expression of methionine adenosyltransfer- ase 1A (Mat1a) (Fig. 7D), the enzyme that synthesizes SAM from methionine, and of Pemt (Fig. 7E), a major consumer of SAM for phosphatidylcholine synthesis, were reduced (P < 0.005, un- paired t-test). Several of choline/methyl metabolites measured in embry- onic liver were not affected by diet (GPC, phosphatidylcholine, sphingomyelin, lysophosphatidylcholine, SAM and SAH) (Supplementary Material, Table S4). However, in FASD liver, betaine was signiﬁcantly decreased (Fig. 8A; P < 0.05, unpaired t-test), there was a trend towards decreased choline (Fig. 8B; P ¼ 0.06, unpaired t-test) and PCho signiﬁcantly We also found a trend towards reduced betaine homocyste- ine methyltransferase (Bhmt) mRNA in FASD embryonic liver (Fig. 7C; P ¼ 0.08, unpaired t-test). This enzyme utilizes the cho- line derivative betaine for methionine and SAM synthesis. 893 Human Molecular Genetics, 2017, Vol. 26, No. 5 \| 893 Figure 4. Pups of FASD mothers had reduced expression of Dnmt3a mRNA in cortex and hippocampus. There was a signiﬁcant decrease of Dnmt3a mRNA due to FASD in (A) cortex (n ¼ 7/group) and (B) hippocampus of FASD pups (n ¼ 5/group). (C) Dnmt1 and (D) Dnmt3b mRNA were not changed by diet in cortex. Values are means 6 SEM; P < 0.05. Figure 4. Pups of FASD mothers had reduced expression of Dnmt3a mRNA in cortex and hippocampus. There was a signiﬁcant decrease of Dnmt3a mRNA due to FASD in (A) cortex (n ¼ 7/group) and (B) hippocampus of FASD pups (n ¼ 5/group). (C) Dnmt1 and (D) Dnmt3b mRNA were not changed by diet in cortex. Values are means 6 SEM; P < 0.05. (Supplementary Material, Fig. S2B and C). Consequently, we did not observe any changes in choline metabolites or SAH in pla- centa or maternal liver. There was a trend for increased SAM in the placenta (P ¼ 0.07, unpaired t-test); however this small dif- ference may not have biological relevance (Supplementary Material, Table S5). FASD embryos showed reduced expression of one-carbon enzymes in liver, reduced hepatic choline metabolites and altered brain development These ﬁndings suggest that embryonic fo- late metabolism may be independent of maternal metabolism at this stage and that FASD has more dramatic consequences on embryos than on dams. decreased (Fig. 8C; P < 0.005, unpaired t-test). These observa- tions suggest that the choline/betaine-dependent remethyla- tion pathway is being extensively utilized when folate- dependent remethylation is blocked, as we have previously ob- served (10,23,28). To examine cortical and hippocampal development in E17.5 embryos, we examined H&E stained coronal brain sections. The cortical layers appeared to be more deﬁned in FASD embryos (Fig. 9B and D) compared with CD embryos (Fig. 9A and C), and the cortical cells appeared to be more developed due to their elongated shape. These observations suggest different rates of cortical development due to diet. We also observed increased thickness of the SVZ in FASD embryos (Supplementary Material, Figure S3). Additional experimentation is required to under- stand the nature of these developmental differences. Pregnant mothers at E17.5 showed relatively minor changes in one-carbon metabolism Total hepatic MTHFR protein was not changed by diet in the pregnant females, although there was an increase in the ratio of phosphorylated to non-phosphorylated MTHFR in the FASD group (P < 0.05, unpaired t-test, data not shown). Similar to our results in lactating mothers, there was a decrease in Mtr mRNA (P < 0.05, unpaired t-test) due to FASD in livers of pregnant fe- males (Supplementary Material, Fig. S2A). The placenta, which has signiﬁcantly higher levels of MTHFR compared with embryonic or maternal liver, did not show differences in MTHFR protein levels due to diet Human Molecular Genetics, 2017, Vol. 26, No. 5 894 ure 5. High folic acid intake altered folate metabolism in liver of lactating mothers and led to changes in choline metabolism. (A) Total MTHFR protein was reduced ASD mothers (n ¼ 5/group). (B) The ratio of the phosphorylated MTHFR isoform (less active) to the non-phosphorylated isoform was increased in the FASD group 5/group). mRNA expression of (C) methionine synthase (Mtr) was reduced, (D) methionine synthase reductase (Mtrr) was increased and (E) Pemt was increased due ASD (n ¼ 7/group). (F) There was a trend towards lower GPC due to diet in maternal plasma, (n ¼ 7/group, #P ¼ 0.06). Values are means 6 SEM; P < 0.05, **P < 0.005. Figure 5. High folic acid intake altered folate metabolism in liver of lactating mothers and led to changes in choline metabolism. (A) Total MTHFR protein was reduced in FASD mothers (n ¼ 5/group). (B) The ratio of the phosphorylated MTHFR isoform (less active) to the non-phosphorylated isoform was increased in the FASD group (n ¼ 5/group). mRNA expression of (C) methionine synthase (Mtr) was reduced, (D) methionine synthase reductase (Mtrr) was increased and (E) Pemt was increased due to FASD (n ¼ 7/group). (F) There was a trend towards lower GPC due to diet in maternal plasma, (n ¼ 7/group, #P ¼ 0.06). Values are means 6 SEM; P < 0.05, **P < 0.005. Figure 6. Embryos and placentas from FASD mothers were smaller at E17.5. Weights of embryos and placentas were measured at sacriﬁce. (A) FASD led to lower mean body weights/litter of embryos. (B) There was a trend toward lower placental weights/litter (#P ¼ 0.08). Values are means 6 SEM of 6 l/group; P < 0.05. Figure 6. Embryos and placentas from FASD mothers were smaller at E17.5. Pregnant mothers at E17.5 showed relatively minor changes in one-carbon metabolism Weights of embryos and placentas were measured at sacriﬁce. (A) FASD led to lower mean body weights/litter of embryos. (B) There was a trend toward lower placental weights/litter (#P ¼ 0.08). Values are means 6 SEM of 6 l/group; P < 0.05. 895 Human Molecular Genetics, 2017, Vol. 26, No. 5 \| 895 Figure 7. Maternal FASD altered expression of one-carbon enzymes in liver of E17.5 embryos. (A) Total MTHFR protein and (B) Mthfr mRNA was reduced in liver of E17.5 FASD embryos. (C) There was a trend towards reduced Bhmt mRNA in embryonic liver (#P ¼ 0.08). (D) Mat1a and (E) Pemt mRNA were both signiﬁcantly decreased due to diet in embryonic liver. Values are means 6 SEM of 7–8 embryos/group; P < 0.05, **P < 0.005. Figure 7. Maternal FASD altered expression of one-carbon enzymes in liver of E17.5 embryos. (A) Total MTHFR protein and (B) Mthfr mRNA was reduced in liver of E17.5 FASD embryos. (C) There was a trend towards reduced Bhmt mRNA in embryonic liver (#P ¼ 0.08). (D) Mat1a and (E) Pemt mRNA were both signiﬁcantly decreased due to diet in embryonic liver. Values are means 6 SEM of 7–8 embryos/group; P < 0.05, **P < 0.005. Figure 8. Choline metabolites decreased in E17.5 embryonic liver of FASD mothers. (A) Betaine concentration was signiﬁcantly decreased. (B) There was a trend towards reduced choline (#P ¼ 0.06) (C) Pcho concentration was signiﬁcantly decreased. Values are means 6 SEM of 6 embryos/group; P < 0.05, **P < 0.005. Figure 8. Choline metabolites decreased in E17.5 embryonic liver of FASD mothers. (A) Betaine concentration was signiﬁcantly decreased. (B) There was a trend towards reduced choline (#P ¼ 0.06) (C) Pcho concentration was signiﬁcantly decreased. Values are means 6 SEM of 6 embryos/group; P < 0.05, ***P < 0.005. cortex. Chrna7 mRNA levels also tended to decrease in FASD pup cortex; the difference was not signiﬁcant although AchE and Chrna7 mRNAs were highly positively correlated. Chrna7 mRNA levels were found to be decreased in post-mortem brains of schizophrenia patients which was likely due to increased ex- pression of the transcription factor Activating Protein-2a (AP- 2a), a negative regulator of AchE (30). Therefore, it is possible that the observed correlation is due to the co-regulation of these genes by AP-2a. Pregnant mothers at E17.5 showed relatively minor changes in one-carbon metabolism Altered brain development in embryos of FASD mothers. Coronal sections of embryonic brain were stained with H & E. Representative images from embryos in the CD (A and C) and FASD (B and D) groups are depicted. Images show the cortical layers, the intermediate zone (IZ) and subventricular zone (SVZ). Cortical layers from FASD embryos appeared to be more organized and developed. signiﬁcant disturbances in one-carbon metabolism and their Pemt levels were increased in liver, possibly to maintain choline levels. Maternal metabolism during lactation is critical for maintaining nutrients that are transferred to pups through milk. Nonetheless, we cannot exclude the possibility that the differences between pregnant and lactating mothers are due to the longer period of exposure to FASD and decreased MTHFR protein in lactating dams, instead of a differential response dur- ing pregnancy or lactation. proliferation and increased apoptosis in hippocampus (33), and altered neurogenesis and cortical layering, particularly in later born neurons (34). We examined proliferation in hippocampus and cortex at E17.5 using Ki67 (data not shown) but did not ob- serve any differences. More extensive marker analyses at vari- ous time points would be useful to understand developmental differences. Reduced choline availability can also modulate the expres- sion of DNA and histone methyltransferases (35,36). We ob- served signiﬁcant disturbances in choline metabolites in FASD embryos, and decreased Dnmt3a mRNA in both cortex and hip- pocampus of FASD pups. Dnmt3a plays an important role in brain development and neuronal maturation (37). Mice with a conditional knockout of Dnmt3a performed poorly on memory tasks, particularly the NOR (25). Decreased Dnmt3a expression would be expected to alter DNA methylation and consequently expression of genes that may be essential for brain develop- ment. Increased maternal folic acid intake has been shown to alter methylation and expression of neurodevelopmental genes in a sex-speciﬁc manner in newborn pups (24,38). The differ- ences in rate of brain development in this study may be linked to altered Dnmt3a expression. Our ﬁndings are in line with recent human studies that ex- amined the effects of excess maternal folate intake on offspring health. Mothers who consumed the highest amounts of folic acid had children with reduced embryonic size (41). Pregnant mothers at E17.5 showed relatively minor changes in one-carbon metabolism These changes suggest that there is a distur- bance in acetylcholine metabolism, which could be contributing to memory impairment in offspring. The hippocampus and the cortex are critical regions for memory and learning (31). The re- duced area of the hippocampus in general and the reduced thickness of the dentate gyrus in particular in FASD pups is con- sistent with the impaired memory and with our earlier work in which we also found decreased hippocampal volume and mem- ory impairment in Mthfr-/- mice (10). Pregnant dams exhibited increased MTHFR phosphorylation, which would be expected to reduce MTHFR activity (22), but to- tal MTHFR protein was not altered. It is possible that the reduc- tion in total protein requires a longer exposure to the folate- supplemented diet, since we observed both inhibitory mecha- nisms in lactating mothers. We did not observe changes in MTHFR protein in cortex, hippocampus or placenta due to high folate. Altered expression of liver MTHFR is known to lead to he- patic choline disturbances. Choline is an alternate methyl donor for SAM synthesis, through its metabolite betaine; this alternate pathway is usually upregulated when folate-dependent SAM synthesis is inhibited (20,28). This upregulation may decrease circulating critical choline metabolites and can ultimately affect brain choline utilization for Ach production. Phosphocholine, the primary storage form of choline, was reduced in liver of 3-week old pups. There was a trend for re- duced GPC in pup hippocampus; this decrease could be due to the increased conversion of GPC to choline in order to maintain choline pools (23). Importantly, choline is the precursor of Ach. We found a trend toward decreased ChAT staining in choliner- gic neurons in the SI, and a decrease in mRNA levels of AchE in At E17.5, the hippocampus and upper layers of the cortex are rapidly developing (32) and reduced choline availability may have adverse consequences on embryonic brain development. Maternal choline-deﬁcient diets in mice resulted in decreased 896 \| Human Molecular Genetics, 2017, Vol. 26, No. 5 Figure 9. Altered brain development in embryos of FASD mothers. Coronal sections of embryonic brain were stained with H & E. Representative images from embryos in the CD (A and C) and FASD (B and D) groups are depicted. Images show the cortical layers, the intermediate zone (IZ) and subventricular zone (SVZ). Cortical layers from FASD embryos appeared to be more organized and developed. Figure 9. Pregnant mothers at E17.5 showed relatively minor changes in one-carbon metabolism The mem- ory impairment in newborn pups from FASD dams in this study is consistent with ﬁndings in a Spanish study demonstrating that the children whose mothers consumed over 5mg/day of fo- lic acid periconceptionally had delayed psychomotor develop- ment (21). In addition, there is a recent report that high maternal plasma folate was associated with an increased risk of autism spectrum disorder in offspring (42). In summary, we have shown that high folic acid intake in pregnant mice leads to intrauterine growth delay with altered brain development and impaired memory in offspring. We sug- gest that these outcomes may be due to pseudo-MTHFR deﬁ- ciency in both offspring and mothers, with consequent disturbances in choline and methyl metabolism. This study adds to the increasing evidence that over-consumption of folic acid during pregnancy may have negative health consequences and that determining a safe upper limit is critical. The average weight of embryos per litter was reduced in the FASD group. The observed phenotype is reminiscent of the in- trauterine growth restriction observed in low folate conditions (39,40). Therefore, high folate consumption may have similar negative effects to those seen with low folate, particularly since high folate inhibits MTHFR, a critical folate enzyme, and could be inhibiting other folate-dependent enzymes or transporters. Embryos from FASD mothers had pseudo-MTHFR deﬁciency with signiﬁcant disturbances in folate and choline metabolism. The embryo is a system that is rapidly proliferating and devel- oping with high nutrient demands. There were no dramatic changes in metabolites of pregnant mice. These observations suggest that at this stage of development, embryonic folate and choline metabolism is working relatively independently of ma- ternal metabolism. Animal experimentation and diets For embryo collection, the mice were sacriﬁced at embry- onic day (E) 17.5 of gestation, and maternal and embryonic tissues were collected. In a second group, female mice were maintained on the diets through pregnancy and lactation (until sacriﬁce). Maternal tissues and tissues from 3-week-old male pups were collected after completion of behavioral testing of pups. Brain sections and immunohistochemistry During embryo sacriﬁce, whole heads were collected and ﬁxed in 4% PFA overnight. They were then parafﬁn embedded and cut along the coronal axis at 5-lm thickness; mounted sections were 10 sections apart. For the pups, the brain was cut along the sagittal axis and the right hemisphere of the brain was ﬁxed in 4% PFA. Sections were cut at 5-lm thickness. Hematoxylin and eosin (H&E) staining was performed to assess brain morphol- ogy. Slides were scanned using the Zeiss Axio Scan.Z1 (Oberkochen, Germany). The examined sections were chosen to be at the same or similar levels between mice. Measurements of area and thickness in the hippocampus were performed using the Zen Lite 2 (Blue Edition) (Zeiss Microscopy, Oberkochen, Germany) by two investigators blinded to groups. The hippo- campal area value is an average of four sections per mouse. The thickness values for dentate gyrus are the average of three mea- surements per section, for four sections per mouse. Cholinergic staining was done as previously described (44) using a ChAT polyclonal goat antibody (Millipore, Billerica, USA). Positive cho- linergic neurons were counted in 3–4 sections per animal (5 ani- mals per group) in the Substantia Innominata (SI) at a magniﬁcation of 400. Intensity of staining was measured using ImageJ software. Open ﬁeld test Mice were placed in the middle of an open ﬁeld box and allowed to explore for 5 min. The bottom of the open ﬁeld was divided into 16 equal squares. Recorded videos were analyzed for activ- ity (total number of squares crossed), number of entries into in- ner squares and outer squares and % time spent in each. Choline metabolites Choline, betaine, acetylcholine (Ach), glycerophosphocholine (GPC), phosphatidylcholine (PtdCho), sphingomyelin (SM), lyso- phosphatidylcholine (LPC) and phosphocholine (PCho) were iso- lated from frozen liver, brain, plasma and placenta using liquid– liquid extraction and SPE puriﬁcation (45). Stable isotope- dilution liquid chromatography–electrospray ionization tandem mass spectrometry was used for metabolite quantiﬁcation as previously described (45) with adaptations based on our in- strumentation (46). Calibration curves for each metabolite were prepared via serial dilutions of a 100 mM standard stock solu- tion. Ranges were linear between 1 and 100 nmol/extract for choline, betaine, PCho and GPC; 1–100 pmol/extract for Ach; 150–2500 nmol/extract for PtdCho; 6-100 nmol/extract for SM; and 3–50 nmol/extract LPC. Stable isotope-dilution liquid chromatography–electrospray ionization tandem mass spec- trometry was also used to measure SAM and SAH concentra- tions in placenta, liver and brain as previously described (47,48). Calibration curves for SAM and SAH were prepared via serial dilutions of 100 mM stock solutions and ranges were linear be- tween 0.2 and 7.5 nmol/extract for SAM and 0.04–1.2 nmol/ex- tract for SAH. Quality control for all metabolites was monitored using duplicate samples of in-house control materials (i.e. ho- mogenized pooled liver, brain and human placental tissues). The assay imprecision (coefﬁcient of variation) was <5% for be- taine; <10% for free choline, SAM, SAH, GPC, PCho, PtdCho, SM, LPC; and <16% for Ach. Behavioral testing All behavioral tests were performed as described in Ref. (10). Brief descriptions of the methods of individual tests are as follows: Ladder beam test The apparatus consisted of two Plexiglass walls and metal bars between walls arranged in an irregular pattern, and was placed on top of a mouse housing cage. Recordings were obtained from each mouse and analyzed frame-by-frame. The error score was the combination of total number of errors and the number of steps for each limb. Novel object recognition test Pups were allowed to habituate to the room for 30 min and then to the apparatus (open ﬁeld) for 10 min. Animals explored two identical copies of object 1 placed at opposite corners in an open ﬁeld for 8 min (trial phase), then returned to their cages for one hour. Short-term memory was assessed by allowing ani- mals to explore a familiar object (object 1) and a novel object (object 2) for 5 min (test phase). Both the trial and test phase were recorded and the amount of time exploring objects was as- sessed from the video recordings by two independent re- searchers. The discrimination index (DI) was calculated as the ratio of the amount of time animals spent with the novel versus the familiar object. A positive value indicates that the animal preferred to explore the novel object. Animal experimentation and diets Animal experimentation was conducted according to the guide- lines of the Canadian Council on Animal Care and approved by the Animal Care Committee of the Research Institute of the McGill University Health Center. Mice were housed in speciﬁc pathogen free facilities with 12 h light/dark cycles with food and In contrast to pregnant females, the lactating FASD mice had more marked changes due to diet: reduced MTHFR protein, 897 Human Molecular Genetics, 2017, Vol. 26, No. 5 \| 897 arms. The number of alternations was recorded and calculated as a percentage of the total arm entries. water ad libitium. At weaning, wild-type C57BL/6 female mice were placed on amino acid deﬁned Control Diet (CD) (2 mg folic acid/kg diet, recommended level for rodents (43) (TD.01369, Harlan Laboratories, Inc., Madison, WI, USA) or Folic Acid Supplemented Diet (FASD) which contained 10 times the amount of folic acid (20 mg folic acid/kg diet, TD.09258, Harlan Laboratories, Inc., Madison, WI, USA). Both diets contained 2.5 g/kg choline bitrate, 3.3 g/kg L-methionine and 1% succinyl- sulfathiazole, an antibiotic that inhibits folate synthesis by in- testinal ﬂora. Five weeks after the start of the diets, females were mated with wild-type C57BL/6 males. The morning of the presence of a vaginal plug was considered as day 0.5 of gesta- tion. For embryo collection, the mice were sacriﬁced at embry- onic day (E) 17.5 of gestation, and maternal and embryonic tissues were collected. In a second group, female mice were maintained on the diets through pregnancy and lactation (until sacriﬁce). Maternal tissues and tissues from 3-week-old male pups were collected after completion of behavioral testing of pups. water ad libitium. At weaning, wild-type C57BL/6 female mice were placed on amino acid deﬁned Control Diet (CD) (2 mg folic acid/kg diet, recommended level for rodents (43) (TD.01369, Harlan Laboratories, Inc., Madison, WI, USA) or Folic Acid Supplemented Diet (FASD) which contained 10 times the amount of folic acid (20 mg folic acid/kg diet, TD.09258, Harlan Laboratories, Inc., Madison, WI, USA). Both diets contained 2.5 g/kg choline bitrate, 3.3 g/kg L-methionine and 1% succinyl- sulfathiazole, an antibiotic that inhibits folate synthesis by in- testinal ﬂora. Five weeks after the start of the diets, females were mated with wild-type C57BL/6 males. The morning of the presence of a vaginal plug was considered as day 0.5 of gesta- tion. Supplementary Material is available at HMG online. 11. Jadavji, N., Deng, L., Malysheva, O., Caudill, M.A. and Rozen, R. (2015) MTHFR deﬁciency or reduced intake of folate or choline in pregnant mice results in impaired short-term memory and increased apoptosis in the hippocampus of wild-type offspring. Neuroscience, 300, 1–9. Statistical analysis 8. Rai, V. (2016) Association of methylenetetrahydrofolate re- ductase (MTHFR) gene C677T polymorphism with autism: ev- idence of genetic susceptibility. Metab. Brain. Dis., 31, 727–735. Statistical analysis was performed with Graph Pad software package 5.01 (GraphPad Software, La Jolla, USA, 1995). All data are presented as mean 6 standard error of the mean (SEM). Statistical outliers, determined by using Grubb’s test (GraphPad Software), were removed from the analysis. Data were analyzed using an unpaired t-test. If the variance was statistically signiﬁ- cant, Welsh’s correction was applied. Results were considered statistically signiﬁcant at P 0.05. 9. Chen, Z., Karaplis, A.C., Ackerman, S.L., Pogribny, I.P., Melnyk, S., Lussier-Cacan, S., Chen, M.F., Pai, A., John, S.W., Smith, R.S., et al. (2001) Mice deﬁcient in methylenetetrahy- drofolate reductase exhibit hyperhomocysteinemia and decreased methylation capacity, with neuropathology and aortic lipid deposition. Hum. Mol. Genet., 10, 433–443. 10. Jadavji, N.M., Deng, L., Leclerc, D., Malysheva, O., Bedell, B.J., Caudill, M.A. and Rozen, R. (2012) Severe methylenetetrahy- drofolate reductase deﬁciency in mice results in behavioral anomalies with morphological and biochemical changes in hippocampus. Mol. Gen. Metab., 106, 149–159. References 1. Bottiglieri, T. (1996) Folate, vitamin B12, and neuropsychiat- ric disorders. Nutr. Rev., 54, 382–390. 2. Frosst, P., Blom, H.J., Milos, R., Goyette, P., Sheppard, C.A., Matthews, R.G., Boers, G.J., den Heijer, M., Kluijtmans, L.A., van den Heuvel, L.P., et al. (1995) A candidate genetic risk factor for vascular disease: a common mutation in methylenetetrahydrofolate reductase. Nat. Genet., 10, 111–113. 3. Bailey, L.B., Stover, P.J., McNulty, H., Fenech, M.F., Gregory, J.F., Mills, J.L., Pfeiffer, C.M., Fazili, Z., Zhang, M., Ueland, P.M., et al. (2015) Biomarkers of nutrition for development— folate review. J. Nutr., 145, 1636S–1680S. Western blotting Protein extracts were prepared as before (20) and the concentra- tion was determined by Bradford Assay with a bovine serum al- bumin standard. Western blots were performed as previously described (20). Primary antibodies were against MTHFR (2), ChAT (Millipore, Billerica, USA), GAPDH (Cell Signaling Technology, Boston, USA) and b-actin (Sigma-Aldrich). Secondary antibodies were horseradish peroxidase (HRP)-conju- gated donkey anti-rabbit IgG (GE Healthcare, Mississauga, Canada) and HRP-conjugated donkey anti-goat IgG (Santa Cruz Biotechnology, Santa Cruz, USA), as appropriate. Detection was achieved using ECL Prime Western Blotting Detection Reagent (GE Healthcare, Mississauga, Canada). Bands were quantiﬁed by densitometry using Quantity One 4.1.0 (Bio-Rad) and normal- ized to loading control (GAPDH or b-actin). 4. Zeisel, S.H. (2006) Choline: critical role during fetal develop- ment and dietary requirements in adults. Annu. Rev. Nutr., 26, 229–250. 5. Whitehead, V.M. (2006) Acquired and inherited disorders of cobalamin and folate in children. Br. J. Haematol., 134, 125–136. 6. Nurk, E., Tell, G.S., Refsum, H., Ueland, P.M. and Vollset, S.E. (2004) Associations between maternal methylenetetrahy- drofolate reductase polymorphisms and adverse outcomes of pregnancy: the Hordaland Homocysteine Study. Am. J. Med., 117, 26–31. 7. Mitchell, E.S., Conus, N. and Kaput, J. (2014) B vitamin poly- morphisms and behavior: evidence of associations with neu- rodevelopment, depression, schizophrenia, bipolar disorder and cognitive decline. Neurosci. Biobehav. Rev., 47, 307–320. Funding RNA was extracted from frozen liver (15 mg) using the RNeasy Mini Kit (Qiagen, Toronto, Canada). RNA from brain was extracted using the AllPrep DNA, RNA and miRNA Universal kit (Qiagen, Toronto, Canada). cDNA synthesis was done using the iScript cDNA Synthesis kit (BioRad, Quebec, Canada) and quantitative real-time PCR reactions were done using the SSoAdvanced Universal SYBR Green Supermix. For all tissues, the following reference genes were tested: glyceraldehyde-3- phosphate dehydrogenase (Gapdh), b-2-microglobulin (B2m), b – actin (Actb) and tyrosine 3-monooxygenase/tryptophan 5-moox- ygenase activation protein, zeta polypeptide (Ywhaz). The nor- malization factor was calculated for each tissue using the two most stably expressed genes by geNorm v.3.4 (Ghent University Hospital Center for Medical Genetics) (50). Primers were designed using the Primer-BLAST (National Center for Biotechnology Information) (51). A complete list of primer se- quences and reactions conditions is included in Supplementary Material, Table S6. Canadian Institutes of Health Research (MOP-43232 to R.R.); Medical Research Council (N003713 to N.G.); Doctoral Award from the Fonds de Recherche du Que´bec-Sante´ (to R.H.B.); Fonds de Recherche du Que´bec-Sante´ (grant to McGill University Health Centre). Funding to pay the Open Access publication charges for this article was provided by Medical Research Council (N003713 to NG). Folates Pups were allowed to habituate to the room for 30 min and then placed at the end of the long arm of the maze. Animals were al- lowed to move through the maze for 8 min. The test was re- corded and the series of arm entries were recorded. An alternation is considered as successive entries into the three Quantiﬁcation of the major folates (folic acid, dihydrofolate, tet- rahydrofolate (THF), methenylTHF, methyleneTHF, methylTHF and formylTHF) in mono- and polyglutamated (1–7 glutamates) forms was determined in hippocampus samples by liquid Human Molecular Genetics, 2017, Vol. 26, No. 5 898 chromatography–electrospray ionization tandem mass spec- trometry, as previously described (49). The relative abundance of individual folate forms as a proportion of total folate was compared between groups. University) for assistance with methodologies and Dr. Jason Karamchandani (McGill University) for reviewing embryonic sections. Conﬂict of Interest statement. None declared. Supplementary Material Supplementary Material is available at HMG online. Acknowledgements We thank Drs Nancy Le´vesque and Karen Christensen for tech- nical advice and critical review of the manuscript. We also thank members of Dr. Guillaume Se´bire’s laboratory (McGill Human Molecular Genetics, 2017, Vol. 26, No. 5 \| 899 12. Berry, R.J., Bailey, L., Mulinare, J., Bower, C. and Dary, O. (2010) Fortiﬁcation of ﬂour with folic acid. Food. Nutr. Bull., 31, 22S–35S. dehydrogenase inﬂuence biomarkers of choline metabolism when folate intake is restricted. J. Am. Diet. Assoc., 109, 313–318. 27. Tibbetts, A.S. and Appling, D.R. (2010) Compartmentalization of Mammalian folate-mediated one-carbon metabolism. Ann. Rev. Nutr., 30, 57–81. 13. Anjos, T., Altm€ae, S., Emmett, P., Tiemeier, H., Closa- Monasterolo, R., Luque, V., Wiseman, S., Pe´rez-Garcıa, M., Lattka, E. and Demmelmair, H., et al. (2013) Nutrition and neurodevelopment in children: focus on NUTRIMENTHE project. Eur. J. Nutr., 52, 1825–1842. 28. Caudill, M.A. (2009) Bailey, L.B. (ed.), In Folate in Health and Disease. CRC Press, New York, pp. 449–465. 14. Steenweg–de Graaff, J., Roza, S.J., Steegers, E.A., Hofman, A., Verhulst, F.C., Jaddoe, V.W. and Tiemeier, H. (2012) Maternal folate status in early pregnancy and child emotional and be- havioral problems: the Generation R Study. Am. J. Clin. Nutr., 95, 1413–1421. 29. Plumptre, L., Masih, S.P., Ly, A., Aufreiter, S., Sohn, K.J., Croxford, R., Lausman, A.Y., Berger, H., O’Connor, D.L. and Kim, Y.I. (2015) High concentrations of folate and unmeta- bolized folic acid in a cohort of pregnant Canadian women and umbilical cord blood. Am. J. Clin. Nutr., 102, 848–857. 15. Craciunescu, C.N., Johnson, A.R. and Zeisel, S.H. (2010) Dietary choline reverses some, but not all, effects of folate deﬁciency on neurogenesis and apoptosis in fetal mouse brain. J. Nutr., 140, 1162–1166. 30. Finlay-Schultz, J., Canastar, A., Short, M., El Gazzar, M., Coughlan, C. and Leonard, S. (2011) Transcriptional repres- sion of the a7 nicotinic acetylcholine receptor subunit gene (CHRNA7) by activating protein-2a (AP-2a). J. Biol. Chem., 286, 42123–42132. 16. Crider, K.S., Bailey, L.B. and Berry, R.J. (2011) Folic acid food fortiﬁcation—its history, effect, concerns, and future direc- tions. Nutrients, 3, 370–384. 31. Antunes, M. and Biala, G. (2012) The novel object recognition memory: neurobiology, test procedure, and its modiﬁca- tions. Cogn. Process., 13, 93–110. 17. Wilson, R.D., Johnson, J.A., Wyatt, P., Allen, V., Gagnon, A., Langlois, S., Blight, C., Audibert, F., Desilets, V., Brock, J.A., et al. Acknowledgements (2007) Pre-conceptional vitamin/folic acid supplemen- tation 2007: the use of folic acid in combination with a multi- vitamin supplement for the prevention of neural tube defects and other congenital anomalies. J. Obstet. Gynaecol. Can., 29, 1003–1013. 32. Finlay, B.L. and Darlington, R.B. (1995) Linked regularities in the development and evolution of mammalian brains. Science, 268, 1578–1584. 33. Craciunescu, C.N., Albright, C.D., Mar, M.H., Song, J. and Zeisel, S.H. (2003) Choline availability during embryonic de- velopment alters progenitor cell mitosis in developing mouse hippocampus. J. Nutr., 133, 3614–3618. 18. Obeid, R., Kirsch, S.H., Dilmann, S., Klein, C., Eckert, R., Geisel, J. and Herrmann, W. (2015) Folic acid causes higher prevalence of detectable unmetabolized folic acid in serum than B-complex: a randomized trial. Eur. J. Nutr., 55, 1021–1028. 34. Wang, Y., Surzenko, N., Friday, W.B. and Zeisel, S.H. (2016) Maternal dietary intake of choline in mice regulates devel- opment of the cerebral cortex in the offspring. FASEB J., 30, 1566–1578. 19. Selhub, J. and Rosenberg, I.H. (2016) Excessive folic acid in- take and relation to adverse health outcome. Biochimie, 126, 71–78. 35. Mehedint, M.G., Niculescu, M.D., Craciunescu, C.N. and Zeisel, S.H. (2010) Choline deﬁciency alters global histone methylation and epigenetic marking at the Re1 site of the calbindin 1 gene. FASEB J., 24, 184–195. 20. Christensen, K.E., Mikael, L.G., Leung, K.Y., Le´vesque, N., Deng, L., Wu, Q., Malysheva, O.V., Best, A., Caudill, M.A., Greene, N.D. and Rozen, R. (2015) High folic acid consump- tion leads to pseudo-MTHFR deﬁciency, altered lipid metab- olism, and liver injury in mice. Am. J. Clin. Nutr., 101, 646–658. 36. Krzysztof Blusztajn, J. and J Mellott, T. (2012) Choline nutri- tion programs brain development via DNA and histone methylation. Cent. Nerv. Syst. Agents. Med. Chem., 12, 82–94. 37. Irwin, R.E., Pentieva, K., Cassidy, T., Lees-Murdock, D.J., McLaughlin, M., Prasad, G., McNulty, H. and Walsh, C.P. (2016) The interplay between DNA methylation, folate and neurocognitive development. Epigenomics, 8, 863–879. 21. Valera-Gran, D., de la Hera, M.G., Navarrete-Mu~noz, E.M., Fernandez-Somoano, A., Tardon, A., Julvez, J., Forns, J., Lertxundi, N., Ibarluzea, J.M., Murcia, M., et al. (2014) Folic acid supplements during pregnancy and child psychomotor development after the ﬁrst year of life. JAMA Pediatr., 168, e142611-e142611. 38. Barua, S., Kuizon, S., Chadman, K.K., Flory, M.J., Brown, W.T. and Junaid, M.A. (2014) Single-base resolution of mouse off- spring brain methylome reveals epigenome modiﬁcations caused by gestational folic acid. Epigenet. Chromat., 7, 3. Acknowledgements 22. Yamada, K., Strahler, J.R., Andrews, P.C. and Matthews, R.G. (2005) Regulation of human methylenetetrahydrofolate re- ductase by phosphorylation. Proc. Natl Acad. Sci. U S A., 102, 10454–10459. 39. Hoyo, C., Daltveit, A.K., Iversen, E., Benjamin-Neelon, S.E., Fuemmeler, B., Schildkraut, J., Murtha, A.P., Overcash, F., Vidal, A.C., Wang, F., et al. (2014) Erythrocyte folate concen- trations, CpG methylation at genomically imprinted do- mains, and birth weight in a multiethnic newborn cohort. Epigenetics, 9, 1120–1130. 23. Christensen, K.E., Wu, Q., Wang, X., Deng, L., Caudill, M.A. and Rozen, R. (2010) Steatosis in mice is associated with gen- der, folate intake, and expression of genes of one-carbon metabolism. J. Nutr., 140, 1736–1741. 40. Furness, D., Fenech, M., Dekker, G., Khong, T.Y., Roberts, C. and Hague, W. (2013) Folate, vitamin B12, vitamin B6 and ho- mocysteine: impact on pregnancy outcome. Matern. Child Nutr., 9, 155–166. 24. Barua, S., Kuizon, S., Chadman, K.K., Brown, W.T. and Junaid, M.A. (2015) Microarray analysis reveals higher gesta- tional folic acid alters expression of genes in the cerebellum of mice offspring—a pilot study. Brain. Sci., 5, 14–31. 41. van Uitert, E., van Ginkel, S., Willemsen, S., Lindemans, J., Koning, A., Eilers, P., Exalto, N., Laven, J., Steegers, E. and Steegers-Theunissen, R. (2014) An optimal periconception maternal folate status for embryonic size: the Rotterdam Predict study. BJOG, 7, 821–829. 25. Morris, M.J., Adachi, M., Na, E.S. and Monteggia, L.M. (2014) Selective role for DNMT3a in learning and memory. Neurobiol. Learn. Mem., 115, 30–37. 26. Ivanov, A., Nash-Barboza, S., Hinkis, S. and Caudill, M.A. (2009) Genetic variants in phosphatidylethanolamine N-methyltransferase and methylenetetrahydrofolate 42. Raghavan, R., Fallin, M.D. and Wang, X. (2016) Maternal plasma folate, vitamin B12 levels and multivitamin Human Molecular Genetics, 2017, Vol. 26, No. 5 900 supplementation during pregnancy and risk of Autism Spectrum Disorder in the Boston Birth Cohort. FASEB J., 30, 151.6. quantiﬁcation of nitrogen-containing metabolites in Arabidopsis thaliana T87 cells using in vivo (15)N-isotope enrichment. Biosci. Biotechnol. Biochem., 69, 1331–1340. 43. Reeves, P.G. (1997) Components of the AIN-93 diets as im- provements in the AIN-76A diet. J. Nutr., 127, 838S–841S. 48. Jiang, X., Yan, J., West, A.A., Perry, C.A., Malysheva, O.V., Devapatla, S., Pressman, E., Vermeylen, F. and Caudill, M.A. (2012) Maternal choline intake alters the epigenetic state of fetal cortisol-regulating genes in humans. FASEB J., 26, 3563–3574. 44. Jadavji, N.M., Bahous, R.H., Deng, L., Malysheva, O., Grand’maison, M., Bedell, B.J., Caudill, M.A. Acknowledgements and Rozen, R. (2014) Mouse model for deﬁciency of methionine synthase re- ductase exhibits short-term memory impairment and distur- bances in brain choline metabolism. Biochem. J., 461, 205–212. 49. Pai, Y.J., Leung, K.Y., Savery, D., Hutchin, T., Prunty, H., Heales, S., Brosnan, M.E., Brosnan, J.T., Copp, A.J. and Greene, N.D. (2015) Glycine decarboxylase deﬁciency causes neural tube defects and features of non-ketotic hyperglyci- nemia in mice. Nat. Commun., 6, 6388. 45. Koc, H., Mar, M.H., Ranasinghe, A., Swenberg, J.A. and Zeisel, S.H. (2002) Quantitation of choline and its metabolites in tis- sues and foods by liquid chromatography/electrospray ionization-isotope dilution mass spectrometry. Anal. Chem., 74, 4734–4740. 50. Vandesompele, J., De Preter, K., Pattyn, F., Poppe, B., Van Roy, N., De Paepe, A. and Speleman, F. (2002) Accurate nor- malization of real-time quantitative RT-PCR data by geomet- ric averaging of multiple internal control genes. Genome. Biol., 3, research0034. 46. Yan, J., Jiang, X., West, A.A., Perry, C.A., Malysheva, O.V., Devapatla, S., Pressman, E., Vermeylen, F., Stabler, S.P., Allen, R.H., et al. (2012) Maternal choline intake modulates maternal and fetal biomarkers of choline metabolism in hu- mans. Am. J. Clin. Nutr., 95, 1060–1071. 51. Ye, J., Coulouris, G., Zaretskaya, I., Cutcutache, I., Rozen, S. and Madden, T.L. (2012) Primer-BLAST: a tool to design target-speciﬁc primers for polymerase chain reaction. BMC Bioinformatics, 13, 134. 47. Kim, J.K., Harada, K., Bamba, T., Fukusaki, E. and Kobayashi, A. (2005) Stable isotope dilution-based accurate comparative
W3008398058.txt	https://ojs.unud.ac.id/index.php/sakura/article/download/54317/33829	en		Pembentukan dan Makna Kata Majemuk antara Onomatope dan Bagian Tubuh Manusia dalam Bahasa Jepang di Media Sosial Twitter	Jurnal Sakura	2,020	cc-by	3,093	SAKURA VOL. 2. No. 1 Pebruari 2020 DOI : 10.24843/JS.2020.v02.i01.p03 p-ISSN: 2623-1328 e-ISSN :2623-0151 Pembentukan dan Makna Kata Majemuk dari Onomatope dan Bagian Tubuh Manusia dalam Bahasa Jepang di Media Sosial Twitter Ni Nyoman Ayu Devi Pragasuri, Ngurah Indra Pradhana, I Made Budiana PS. Sastra Jepang, FIB, Universitas Udayana, Bali, Indonesia [devipragasuri.dp@gmail.com], [indra_pradana@unud.ac.id], [budi.hybrid@gmail.com] ABSTRACT The title of this research is “Formation and Meaning of Compound Words from Onomatopoeia and Human Body Parts in Japanese on Social Media Twitter”. This research focuses on the forming process and meaning of compound words from onomatopoeia and human body parts in Japanese on social media twitter. This research was analysed by using formal and informal method. Compound words formation analysis used word formation theory from Kageyama and Kishimoto (2016) and the meaning of compound words analysis used semantic theory from Chaer (2012) and characteristic of Japanese onomatopoeia from Akimoto (2002). The result of this research is on the 25 data was analyzed, there are 14 data formed from the composition process and 11 data formed from the composition process followed by the shortening process of the words and part of speech classification in all data has changed. For the meaning, 22 data have grammatical meaning and 3 data have lexical meaning and all data have derivative meaning from the basic onomatopoeia. Keywords: compound words, onomatopoeia, composition, meaning, twitter ABSTRAK Penelitian ini berjudul “Pembentukan dan Makna Kata Majemuk dari Onomatope dan Bagian Tubuh Manusia dalam Bahasa Jepang di Media Sosial Twitter”. Penelitian ini berfokus pada proses pembentukan dan makna kata majemuk dari onomatope dan bagian tubuh manusia yang termuat dalam postingan di pengguna twitter di Jepang. Penelitian ini dianalisis menggunakan metode formal dan informal. Pembentukan kata majemuk dianalisis dengan teori pembentukan kata dari Kageyama dan Kishimoto (2016) dan makna kata majemuk dianalisis dengan teori semantik dari Chaer (2012) dan teori karakteristik Onomatope bahasa Jepang dari Akimoto (2002). Hasil yang diperoleh dalam penelitian ini adalah bahwa dari 25 data yang dianalisis terdapat 14 data terbentuk dari proses komposisi dan 11 data terbentuk dari proses komposisi yang dilanjutkan dengan proses pemendekan kata serta semua data mengalami perubahan kelas kata. Untuk maknanya, 22 data memiliki makna gramatikal dan 3 data memiliki makna leksikal serta semua data memiliki makna turunan dari kata onomatope dasarnya. Kata Kunci : kata majemuk, onomatope, komposisi, makna, twitter 1. Latar Belakang Negara Jepang merupakan salah satu negara yang sering menggunakan onomatope dalam kehidupan sehari-hari. Onomatope ini merupakan penggunaan kata-kata yang menarik dari bahasa Jepang karena dapat mengekspresikan berbagai arti dalam banyak situasi sehingga sangat perlu memahami onomatope agar dapat berbicara dan mengerti 24 SAKURA VOL. 2. No. 1 Pebruari 2020 DOI : 10.24843/JS.2020.v02.i01.p03 p-ISSN: 2623-1328 e-ISSN :2623-0151 maksud yang disampaikan dalam bahasa Jepang dengan benar. Adapun pengertian dari kata onomatope adalah suatu kata yang subyektif dan berkenaan dengan panca indera karena mewakili makna yang diperoleh dari nada suara dasar (Tamori, 2010:iii). Onomatope bahasa Jepang dibagi menjadi beberapa macam istilah, diantaranya adalah giongo, gitaigo dan giseigo yang merupakan kelas kata khusus yang bentuk atau bunyinya serta maknanya terkait dengan ikonik (kemiripan antara bentuk dan makna) atau sesuai dengan simbol suara aslinya (Iwasaki, 2017:1). Penggunaan onomatope dalam bahasa Jepang tidak hanya berdiri sendiri ataupun menjadi kata keterangan (adverbia) yang menjelaskan kata lainnya dalam suatu kalimat, namun ada juga penggunaan onomatope yang berkomposisi dengan kata lain dan membentuk suatu kata majemuk dalam bahasa Jepang. Kemenarikan bentuk kata majemuk dari penggabungan suatu kata dan onomatope inilah yang akhirnya menjadi dasar untuk melakukan suatu penelitian ini. Agar penelitian tidak meluas maka penulis hanya meneliti kata majemuk yang terbentuk dari penggabungan kata onomatope dengan kata-kata yang berasal dari bagian tubuh manusia. Selain itu, manusia setiap hari menggerakan tubuh mereka untuk bergerak, bekerja, dan melakukan sesuatu sehingga kata majemuk yang terbentuk dari penggabungan kata onomatope dengan kata-kata yang berasal dari bagian tubuh manusia lebih banyak terbentuk dan lebih bervariasi. Penggunaan bentuk kata majemuk ini tidak hanya terdapat dalam percakapan sehari-hari, namun juga muncul dalam pesanpesan yang dimuat dalam media sosial. Oleh karena itu, media sosial dijadikan sumber data dalam penelitian ini, khususnya media sosial Twitter karena Twitter termasuk ke dalam 3 besar media sosial yang sering digunakan di dunia (Badri, 2011:132).. Penelitian ini hanya berfokus pada pembentukan dan makna kata majemuk dari onomatope dan kata lainnya dalam bahasa Jepang di media sosial Twitter. Penelitian yang sudah pernah dilakukan sebelumnya mengenai pembentukan dan makna kata majemuk dalam bahasa Jepang diantaranya Kwarini (2016), Wedayanti (2015), dan Primawan (2015), namun dengan sumber data dan pokok permasalahan yang berbeda. 2. Pokok Permasalahan Berdasarkan latar belakang yang telah dipaparkan tersebut, maka yang dibahas dalam penelitian ini dapat dirumuskan sebagai berikut: a) Bagaimanakah pembentukan kata majemuk dari onomatope dan bagian tubuh manusia dalam bahasa Jepang di media sosial Twitter ? 25 SAKURA VOL. 2. No. 1 Pebruari 2020 DOI : 10.24843/JS.2020.v02.i01.p03 p-ISSN: 2623-1328 e-ISSN :2623-0151 b) Bagaimanakah makna kata majemuk dari onomatope dan bagian tubuh manusia dalam bahasa Jepang di media sosial Twitter ? 3. Tujuan Penelitian Tujuan penelitian ini dibagi menjadi dua bagian, yaitu tujuan umum dan tujuan khusus.Secara umum penelitian yang dilakukan ini bertujuan untuk menambah wawasan serta pengetahuan para pembaca tentang kajian bidang ilmu linguistik, yaitu bidang morfologi dan semantik. Secara khusus penelitian ini bertujuan untuk mengetahui pembentukan dan makna kata majemuk dari onomatope dan bagian tubuh manusia dalam bahasa Jepang di media sosial Twitter. 4. Metode Penelitian Data berupa kata majemuk dari onomatope dan bagian tubuh manusia diperoleh melalui media sosial Twitter dengan menggunakan metode simak dan teknik catat (Sudaryanto, 1993:133). Selanjutnya dianalisis dengan metode agih dan metode padan. Analisis pertama dilakukan untuk mengetahui proses pembentukan kata majemuk dari onomatope dan bagian tubuh manusia tersebut dengan menggunakan teori pembentukan kata oleh Kageyama dan Kishimoto (2016). Analisis kedua dilakukan untuk mengetahui makna yang terdapat dalam kata majemuk dari onomatope dan bagian tubuh manusia tersebut dengan menggunakan teori semantik dari Chaer (2012) yang didukung oleh teori karakteristik onomatope dari Akimoto (2002). Awalnya menganalisis makna leksikal dari setiap kata pembentuk dari kata majemuk tersebut. Kemudian, setelah makna leksikal selesai dianalisis maka selanjutnya menganalisis makna gramatikal dari kata majemuk yang telah mengalami proses gramatikal. Selanjutnya hasil analisis disajikan dengan metode formal dan informal (Zaim, 2014:114). 5. Hasil dan Pembahasan 26 SAKURA VOL. 2. No. 1 Pebruari 2020 DOI : 10.24843/JS.2020.v02.i01.p03 p-ISSN: 2623-1328 e-ISSN :2623-0151 5.1 Pembentukan dan Makna Kata Majemuk dari Onomatope dengan Kepala Gambar (1) (1) @PiT_produce ミニに頭ポンされてる Mini ni atamapon sareteru Mini PAR tepukan kepala dilakukan のかわいすぎ no kawaisugi PAR sangat lucu „Lucu sekali kepalanya ditepuk (atamapon) oleh Mini‟ [30/09/19 diakses dari Twitter] atama + pon → atamapon (N) (Adv) (N) (proses komposisi) Atamapon merupakan kata majemuk yang terbentuk akibat proses komposisi karena merupakan gabungan 2 buah kata dari atama yang merupakan kelas kata nomina dan pon yang merupakan kelas kata adverbia. Setelah mengalami proses komposisi, kedua kata tersebut bergabung menjadi atamapon dan kelas katanya berubah menjadi kelas kata nomina. Selain itu, atamapon juga bisa ditambahkan sufiks –suru melalui proses afiksasi sehingga kata majemuk atamapon ini merupakan kelas kata nomina khususnya verba nomina karena atamapon meskipun nomina tetap bisa diubah ke dalam bentuk kata kerja dengan penambahan sufiks –suru. Pada contoh postingan di atas, atamapon berafiksasi dengan verba bantuk pasif (ukemi), yaitu –sareru. Verba bantu –sareru sendiri berasal dari bentuk verba sufiks –suru. Atama merupakan kata pembentuk atamapon yang artinya kepala dan dalam kata majemuk ini memiliki makna leksikal, yaitu bagian tubuh yang ada di atas wajah manusia. Sedangkan, pon merupakan bentuk onomatope berjenis gitaigo dengan bentuk penasalan suara karena berakhiran –n untuk menunjukkan sesuatu kejadian dengan suara menggema dari benturan benda yang ringan. Pon memiliki 5 makna leksikal, yaitu 1) suara benda meledak atau jatuh dengan kuat; 2) suara ringan menepuk sesuatu; 3) keadaan benda yang dilempar, terbang dan kemudian memantul; 4) keadaan 27 SAKURA VOL. 2. No. 1 Pebruari 2020 DOI : 10.24843/JS.2020.v02.i01.p03 p-ISSN: 2623-1328 e-ISSN :2623-0151 merespon dengan cepat; 5) keadaan ketika tidak bisa memikirkan apapun (Masahiro, 2007:464). Makna gramatikal atamapon adalah tindakan menepuk kepala dengan ringan. Kata onomatope pon dalam atamapon tidak menunjukkan suara menepuk seperti salah satu makna leksikalnya, melainkan tindakan menepuk sesuatu dengan ringan dan yang menjadi objeknya adalah kepala. 5.1.2 Pembentukan dan Makna Kata Majemuk dari Onomatope dengan Rambut Gambar (2) (2) @marynemui ユニクロユー Yunikuroyuu Uniqlo U のニットかわいい no nitto kawaii PAr pakaian rajutan lucu それにしても意外と sorenishitemo igai to namun demikian tak terduga 緑似合う midori niau hijau cocok わたし watashi saya なあ、、、、髪ボサ naa kamibosa yaa rambut kering „Pakaian rajutan Uniqlo U lucu. Namun demikian, di luar dugaan ternyata saya cocok dengan hijau yaa,,, rambut kering (kamibosa)‟ [02/10/19 diakses dari Twitter] kami + bosabosa → kamibosabosa (N) (Adv) (proses komposisi) kamibosabosa → kamibosa (N) (back-clipping) (proses kliping) Kamibosa merupakan kata majemuk yang terbentuk akibat proses komposisi karena merupakan gabungan 2 kata dari kami yang merupakan kelas kata nomina dan bosabosa yang merupakan kelas kata adverbia. Setelah mengalami komposisi, awalnya menjadi kamibosabosa lalu selanjutnya mengalami proses pemendekan kata khususnya backclipping karena yang mengalami pemendekan adalah morfem {bosa} yang ada di 28 SAKURA VOL. 2. No. 1 Pebruari 2020 DOI : 10.24843/JS.2020.v02.i01.p03 p-ISSN: 2623-1328 e-ISSN :2623-0151 belakang kata sehingga menjadi kamibosa. Kelas kata dari kata majemuk kamibosa ini pun akhirnya menjadi nomina. Kata majemuk ini hanyalah nomina biasa bukan termasuk verba nomina karena tidak dapat berafiksasi dengan sufiks –suru. Hal ini dikarenakan faktor dari fungsi gramatikal dari onomatope bosabosa itu sendiri. Penggunaan onomatope bosabosa yang secara khusus berkaitan dengan rambut memiliki fungsi gramatikal sebagai nomina karena dalam penggunaannya diikuti oleh partikel no untuk menjelaskan kata benda setelahnya. Dalam kata majemuk ini karena berkaitan dengan rambut maka bosabosa pun memiliki fungsi gramatikal sebagai nomina. Setelah mengalami proses komposisi dengan kata kami yang juga merupakan kata nomina, maka kamibosa kelas katanya tetap nomina. Kami merupakan kata pembentuk kami yang artinya rambut dan dalam kata majemuk ini memiliki makna leksikal, yaitu bulu yang tumbuh pada tubuh manusia terutama di kepala. Sedangkan, bosabosa merupakan bentuk onomatope berjenis gitaigo dengan bentuk pengulangan yang menunjukkan suatu keadaan yang berkelanjutan. Bosabosa ini memiliki 3 makna lesikal, yaitu 1) keadaan seperti orang dungu dan tidak melakukan apapun; 2) keadaan rambut yang kacau seperti tidak berminyak atau kering; 3) keadaan seperti kertas yang sangat lembut (Masahiro, 2007:441-442). Makna gramatikal kamibosa sama dengan makna leksikal dari onomatope bosabosa. Meskipun telah mengalami proses gramatikal, kata majemuk kamibosa tetap memiliki makna keadaan rambut yang kacau seperti tidak berminyak atau kering. 5.1.3 Pembentukan dan Makna Kata Majemuk dari Onomatope dengan Dahi Gambar (3) (3) @starm79958931 : 親愛な意味を Shinai na imi wo Sayang adj arti PAR 込めて komete memuat する黒猫兄妹 suru kuroneko kyoudai melakukan kucing hitam saudara おでこコツン odekokotsun benturan dahi を wo PAR 29 描きました kakimashita menggambar SAKURA VOL. 2. No. 1 Pebruari 2020 DOI : 10.24843/JS.2020.v02.i01.p03 p-ISSN: 2623-1328 e-ISSN :2623-0151 „Saya telah menggambar kucing hitam bersaudara yang membenturkan dahi (odekokotsun) dengan penuh kasih sayang‟ [23/09/19 diakses dari Twitter] odeko + kotsun → (N) (Adv) odekokotsun (N) (proses komposisi) Odekokotsun merupakan kata majemuk yang terbentuk akibat proses komposisi karena merupakan gabungan 2 buah kata dari odeko yang merupakan kelas kata nomina dan kotsun yang merupakan kelas kata adverbia. Setelah mengalami proses komposisi, kedua kata tersebut bergabung menjadi odekokotsun dan kelas katanya berubah menjadi kelas kata nomina. Selain itu, odekokotsun juga bisa ditambahkan morfem sufiks –suru melalui proses afiksasi sehingga kata majemuk odekokotsun ini merupakan kelas kata nomina khususnya verba nomina karena odekokotsun bisa diubah ke bentuk kata kerja dengan penambahan morfem sufiks –suru. Odeko merupakan kata pembentuk odekokotsun yang artinya dahi atau kening dan dalam kata majemuk ini memiliki makna leksikal, yaitu bagian kepala sebelah depan atas yang sedikit menonjol antara rambut dan alis. Sedangkan, kotsun merupakan bentuk onomatope berjenis gitaigo dengan bentuk penasalan suara karena berakhiran –n untuk menunjukkan sesuatu kejadian yang dengan suara menggema dari benturan benda yang ringan. Kotsun memiliki makna leksikal, yaitu suara benturan ringan ketika mengenai benda keras (Masahiro, 2007:131). Makna gramatikal odekokotsun adalah tindakan membenturkan antara dahi dan benda keras dengan ringan atau tidak keras. Pengertian odekokotsun dilihat dari gambar yang disisipkan di atas adalah membenturkan dahi dengan dahi. Dahi memiliki tekstur yang keras sehingga dapat menggunakan kata onomatope kotsun ini. 5.1.4 Pembentukan dan Makna Kata Majemuk dari Onomatope dengan Dagu Gambar (4) (4) @ugosho_honyo : しっかり顔見せてって 30 言った SAKURA VOL. 2. No. 1 Pebruari 2020 DOI : 10.24843/JS.2020.v02.i01.p03 Shikkari kao Dengan benar wajah あとに atoni setelah 戸惑う tomadou bingung p-ISSN: 2623-1328 e-ISSN :2623-0151 misete tte itta memperlihatkan PAR mengatakan 突然顎クイされて totsuzen agokui sarete tiba-tiba tarikan dagu dilakukan なぁちゃん naachan naachan „Naachan yang kebingungan karena ditarik dagunya (agokui) secara tibatiba setelah disuruh memperlihatkan wajahnya dengan benar‟ [01/09/2019 diakses dari Twitter] Agokui merupakan kata majemuk yang terbentuk akibat proses komposisi karena merupakan gabungan 2 buah kata dari ago yang merupakan kelas kata nomina dan kui yang merupakan kelas kata adverbia. Setelah mengalami proses komposisi, kedua kata tersebut bergabung menjadi agokui dan kelas katanya berubah menjadi kelas kata nomina. Selain itu, agokui juga bisa ditambahkan morfem sufiks –suru melalui proses afiksasi sehingga kata majemuk agokui ini merupakan kelas kata nomina khususnya verba nomina karena agokui bisa diubah ke bentuk kata kerja dengan penambahan morfem sufiks –suru. Pada contoh postingan di atas, agokui berafiksasi dengan verba bantuk pasif (ukemi), yaitu –sareru. Verba bantu –sareru sendiri berasal dari bentuk verba sufiks –suru. Ago merupakan kata pembentuk agokui yang artinya dagu dan dalam kata majemuk ini memiliki makna leksikal, yaitu bagian di bawah mulut bagian bawah. Sedangkan, kui merupakan onomatope dari kata dasar yang tidak mengalami reduplikasi yang memiliki makna bahwa suatu hal terjadi sekali. Kui memiliki 2 makna leksikal, yaitu 1) keadaan menarik, mendorong, atau membelokkan sesuatu dengan ringan; 2) keadaan meminum minuman seperti osake dalam satu tegukan (Masahiro, 2007:82). Makna gramatikal agokui adalah tindakan menarik dagu. Kata onomatope kui dalam agokui tidak hanya menunjukkan keadaan menarik sesuatu seperti makna leksikalnya, melainkan juga menunjukkan tindakan menarik sesuatu dan yang menjadi objeknya adalah dagu. Pengertian agokui dilihat dari gambar yang disisipkan di atas adalah menarik dagu orang lain. 5.1.5 Pembentukan dan Makna Kata Majemuk dari Onomatope dengan Lutut 31 SAKURA VOL. 2. No. 1 Pebruari 2020 DOI : 10.24843/JS.2020.v02.i01.p03 p-ISSN: 2623-1328 e-ISSN :2623-0151 Gambar (5) (5) @vope41 朝から Asa kara Pagi dari 泣きそう nakisou menangis 膝ガクってそりゃそうだよね hizagaku tte sorya sou dayone lutut tertekuk PAR itu terlihat SHUU あんなにもカッピング annani mo kappingu sebegitunya juga likukan も色濃いし mo iro koishi juga warna pekat の跡 no ato PAR tanda 疲れてるよね tsukareteru yone lelah SHUU 帰国したらゆっくり kikokushitara yukkuri setelah pulang perlahan 休めますように yasumemasu youni bisa beristirahat semoga „Dari pagi rasanya ingin menangis. Itu memang terlihat seperti lutut tertekuk (hizagaku) ya. Warna raut wajah pun gelap dan tanda menekuk pun sampai sebegitunya pasti sedang lelah ya. Setelah pulang ke negaramu, semoga bisa beristirahat dengan baik‟ [4/03/2019 diakses dari Twitter] Hizagaku merupakan kata majemuk yang terbentuk akibat proses komposisi karena merupakan gabungan kata dari hiza yang merupakan kelas kata nomina dan gakutt yang merupakan kelas kata adverbia. Setelah mengalami komposisi, awalnya menjadi hizagakutt lalu selanjutnya mengalami proses pemendekan kata khususnya backclipping karena yang mengalami pemendekan adalah morfem {tt} yang ada di belakang kata sehingga menjadi hizagaku. Kelas kata dari kata majemuk hizagaku ini pun akhirnya menjadi nomina, khususnya verba nomina. Hal ini dikarenakan hizagaku bisa berafiksasi dengan sufiks –suru sehingga menjadi nomina yang dapat berubah menjadi verba. Hiza merupakan kata pembentuk hizagaku yang artinya lutut dan dalam kata majemuk ini memiliki makna leksikal, yaitu bagian depan sendi yang menghubungkan paha dengan betis. Sedangkan, gakutt merupakan bentuk onomatope berjenis gitaigo dengan bentuk pemadatan suara untuk menunjukkan sesuatu yang terjadi secara tibatiba dan cepat. Gakutt ini memiliki makna lesikal, yaitu keadaan sesuatu yang dijaga agar lurus tiba-tiba membengkok (Masahiro, 2007:24). Sedangkan, makna gramatikal 32 SAKURA VOL. 2. No. 1 Pebruari 2020 DOI : 10.24843/JS.2020.v02.i01.p03 p-ISSN: 2623-1328 e-ISSN :2623-0151 dari hizagaku adalah keadaan lutut yang lurus saat berdiri tiba-tiba membengkok. Pengertian hizagaku dilihat dari gambar yang disisipkan di atas adalah seseorang yang lututnya tiba-tiba membengkok karena kelelahan. Lutut manusia saat berdiri dijaga agar tetap lurus, namun saat kelelahan atau tersandung tidak jarang lutut tiba-tiba menjadi membengkok dan disinilah penggunaan kata onomatope gakutt digunakan sehingga berkomposisi dengan kata hiza menjadi kata majemuk hizagaku. 6. Simpulan Berdasarkan analisis yang telah dilakukan, dapat ditarik beberapa kesimpulan mengenai pembentukan dan makna kata majemuk antara onomatope dan bagian tubuh manusia dalam bahasa Jepang di media sosial Twitter. 1) Dari 25 data yang dianalisis, kata majemuk yang terbentuk dari proses komposisi sebanyak 14 data dan kata majemuk yang terbentuk dari proses komposisi yang kemudian dilanjutkan dengan proses pemendekan kata sebanyak 11 data. Kata majemuk yang terbentuk dari kata onomatope berupa kata dasar dan berakhiran ~n selalu mengalami proses komposisi saja, sedangkan sisanya ada yang mengalami prosess pemendekan kata. Setelah mengalami proses pembentukan kata, kata majemuk yang memiliki kelas kata nomina biasa sebanyak 2 data, sedangkan kata majemuk yang memiliki kelas kata nomina namun bisa berafiksasi dengan sufiks –suru atau verba nomina sebanyak 22 data. Lalu, kata majemuk yang memiliki kelas kata adjektiva khususnya adjektiva –na sebanyak 1 data. 2) Setelah mengalami proses pembentukan kata, kata majemuk yang memiliki makna gramatikal sebanyak 22 data, sedangkan kata majemuk yang maknanya tidak berubah dan sama seperti makna leksikalnya sebanyak 3 data dengan karakteristik kata onomatope pembentuknya berupa kata dasar, pengulangan, pemadatan suara, penasalan suara, pemanjangan suara, dan penambahan morfem {ri}. Setiap kata majemuk yang terbentuk memiliki makna turunan dari kata onomatope dasarnya. Kata majemuk ini muncul untuk menjelaskan suatu tindakan maupun suatu keadaan dengan satu kata saja, tanpa perlu panjang lebar menjelaskan hal tersebut sehingga lebih efesien digunakan. 7. Daftar Pustaka 33 SAKURA VOL. 2. No. 1 Pebruari 2020 DOI : 10.24843/JS.2020.v02.i01.p03 p-ISSN: 2623-1328 e-ISSN :2623-0151 Akimoto, Miharu. 2002. Yoku Wakaru Goi. Tokyo: ALC. Badri, Muhammad. 2011. Corporate and Marketing Communication. Jakarta: Universitas Mercu Buana Chaer, Abdul. 2012. Linguistik Umum. Jakarta: Rineka Cipta Ikuhiro, Tamori. 2010. Onomatope Gion Gitaigo wo Tanoshimu. Tokyo: Iwanami Shoten Iwasaki, Noriko, dkk. 2017. The Grammar of Japanese Mimetics. New York : Routledge Kageyama, T and Kishimoto, H. (2016). “Handbook of Japanese Lexicon and Word Formation”. Berlin: Mouton De Gruyter Kwarini, Ida Ayu Putri. 2016. “Pembentukan dan Makna Gairaigo dalam buku Ichinichi Sen En De Asoberu Minami No Shima Karya Hayashi Kazuyo” (skripsi). Denpasar: Universitas Udayana. Masahiro, Ono. 2007. Nihongo Onomatope Jiten: Giongo Gitaigo 4500. Tokyo: Shogakkan. Primawan, Randa Arkie. 2015. “Analisis Makna dan Asal Pembentukan Wakamono no Kotoba dalam Sosial Media Twitter” (skripsi). Jakarta: Universitas Bina Nusantara. Surdayanto. 1993. Metode dan Aneka Teknik Analisis Bahasa. Yogyakarta: Duta Wacana Utama Press. Wedayanti, N. P. L. (2015). PRODUKTIVITAS KATA BERKOMPOSISI DALAM BAHASA JEPANG. PRASI, 10(19) HARASSMENT Zaim, M. (2014). Metode Penelitian Bahasa: Pendekatan Struktural. Padang: FBS UNP Press 34
W3171511747.txt	https://www.repository.cam.ac.uk/bitstream/1810/323864/1/20-269308.pdf	ca		Evidence for factors associated with diet and physical activity in African and Caribbean countries	Bulletin of the World Health Organization	2,021	cc-by	10,922	Systematic reviews Evidence for factors associated with diet and physical activity in African and Caribbean countries Eleanor Turner-Moss,a Ahmed Razavi,b Nigel Unwina & Louise Foleya on behalf of the Global Diet and Activity Research Group and Network Objective To identify and describe summarized evidence on factors associated with diet and physical activity in low- and middle-income countries in Africa and the Caribbean by performing a scoping review of reviews. Methods We searched the Medline®, LILACS, Scopus, Global Health and Web of Science databases for reviews of factors associated with diet or physical activity published between 1998 and 2019. At least 25% of studies in reviews had to come from African or Caribbean countries. Factors were categorized using Dahlgren and Whitehead’s social model of health. There was no quality appraisal. Findings We identified 25 reviews: 13 on diet, four on physical activity and eight on both. Eighteen articles were quantitative systematic reviews. In 12 reviews, 25–50% of studies were from Africa or the Caribbean. Only three included evidence from the Caribbean. Together, the 25 reviews included primary evidence published between 1926 and 2018. Little of the summarized evidence concerned associations between international health or political factors and diet or associations between any factor and physical activity across all categories of the social model of health. Conclusion The scoping review found a wide range of factors reported to be associated with diet and physical activity in Africa and the Caribbean, but summarized evidence that could help inform policies encouraging behaviours linked to healthy diets and physical activity in these regions were lacking. Further reviews are needed to inform policy where the evidence exists, and to establish whether additional primary research is needed. Introduction Almost three quarters of deaths from noncommunicable disease occur in low- and middle-income countries, particularly in Africa and the Caribbean.1 Moreover, the burden of noncommunicable disease in the World Health Organization’s (WHO) African Region is expected to exceed that of communicable disease by 2030.2 Premature death from noncommunicable disease in these regions is relatively common; for example, the probability of dying between the ages of 30 and 70 years from noncommunicable disease is 12% in the United Kingdom of Great Britain and Northern Ireland, whereas, in Kenya, Cameroon and South Africa, it is 18%, 20% and 27%, respectively, and in the Caribbean, it ranges from 17% in Jamaica to 37% in Guyana.1 Studies consistently show that an unhealthy diet and physical inactivity are the leading modifiable behavioural risk factors for the four primary noncommunicable diseases: type 2 diabetes, cardiovascular disease, cancer and chronic respiratory disease.3 Clear recommendations have been made by WHO for a healthy diet (i.e. high intake of fruit, vegetables and fibre and low intake of fat, sugar and salt) and physical activity (e.g. at least 150 minutes of moderate-intensity activity per week for adults).4,5 According to 2019 Global Burden of Disease data,6 the percentage of deaths from noncommunicable disease directly attributable to diet was 15.6% in Africa and 15.3% in the Caribbean; the percentage directly attributable to low physical activity was 2.2% in Africa and 3.7% in the Caribbean. In both Africa and the Caribbean there are ongoing regional and national policy initiatives on noncommunicable disease, consistent with WHO’s Global action plan for the prevention and control of noncommunicable diseases 2013–2020.4 In Africa, these include the regional 2011 Brazzaville Declaration and national policy initiatives.7 In the Caribbean, the 2007 Port of Spain Declaration on noncommunicable diseases was a first for lower-middle-income regions. This declaration provided a framework for the development and implementation of policies on the prevention and control of noncommunicable disease, both regionally and nationally. An evaluation of the Port of Spain Declaration in 2018 found that taking effective measures to address the distal (or upstream) determinants of an unhealthy diet and physical inactivity (e.g. cultural and environmental conditions) remained challenging,8 although new initiatives, such as taxing sugar-sweetened beverages, were being implemented. Behaviours associated with a healthy diet and physical activity are core contributors to good health and, thus, the ability to participate in these behaviours can be viewed as a universal right. These behaviours are shaped by a range of factors, including: (i) international policies and politics; (ii) socioeconomic, cultural and environmental conditions; (iii) living and working conditions; (iv) social and community networks; and (v) more proximal individual factors (e.g. age and sex).4,5,9 Evidence on factors associated with these behaviours, on their distribution across different population groups and on whether they are modifiable is important for understanding the drivers of disease burden, for predicting future trends and for identifying targets for interventions and policy changes. Most existing research summaries on the determinants of diet and physical activity come from high-income countries. MRC Epidemiology Unit, University of Cambridge School of Clinical Medicine, Box 285, Institute of Metabolic Science, Cambridge Biomedical Campus, Cambridge, CB2 0QQ, England. b Global Public Health Division, Public Health England, London, England. Correspondence to Louise Foley (email: louise.foley@mrc-epid.cam.ac.uk). (Submitted: 5 June 2020 – Revised version received: 28 January 2021 – Accepted: 11 February 2021 – Published online: 1 April 2021 ) a 464 Bull World Health Organ 2021;99:464–472I \| doi: http://dx.doi.org/10.2471/BLT.20.269308 Systematic reviews Diet and physical activity in Africa and the Caribbean Eleanor Turner-Moss et al. Consequently, the generalizability of their findings to Africa and the Caribbean is questionable and evidence is needed from low- and middle-income countries to inform research, interventions and policy development.10,11 Scoping reviews adopt a systematic approach to map published evidence on a topic, summarize the main themes and highlight knowledge gaps. 12 We chose to conduct a scoping review of reviews because systematic reviews and meta-analyses provide the highest level of evidence on which to draw evidencebased conclusions. The principle aim of our study was to identify and summarize existing reviews on a broad range of factors associated with diet and physical activity in low- and middle-income countries in Africa and the Caribbean. A secondary aim was to identify gaps in the current evidence. Our review was conducted as part of an initial scoping exercise for the Global Diet and Activity Research Network,13 which is a collaboration of researchers in the Caribbean, Cameroon, Kenya, South Africa and the United Kingdom. The overall goal of the network is to generate evidence on the determinants of diet and physical activity to inform noncommunicable disease prevention in Africa and the Caribbean. Methods This scoping review of reviews was conducted according to a previously described method12 and followed reporting guidance in the preferred reporting items for systematic reviews and metaanalyses extension for scoping reviews.14 A review protocol was developed beforehand and was consistent with the scoping review method.15 The review question and the study selection criteria were developed iteratively as familiarity with the literature increased. We searched the Medline®, LILACS, Scopus, Global Health and Web of Science databases for reviews of factors associated with physical activity and dietary behaviour in Africa and the Caribbean that were published between January 1998 and December 2019. A search was carried out in April 2018 and, again, in December 2020 to include literature to the end of 2019. No author was contacted to provide additional information and no grey literature was included because our aim was to identify peer-reviewed evidence syntheses. Our full search strategy is detailed in Box 1 (available at: http://www.who .int/bulletin/volumes/99/6/20-269308). In brief, we combined search terms in sets: (i) diet (e.g. diet, nutrition, food intake, fruit, vegetables, fat, sugar, salt and junk food); (ii) physical activity (e.g. walking, manual labour and screen time); (iii) determinants (e.g. risk factors, correlations and demographic factors); (iv) low- and middle-income countries, with specific terms for African and Caribbean countries; and (v) reviews (i.e. reviews of quantitative or qualitative studies). Reviews were eligible for inclusion if they provided summaries of primary research on factors associated with diet and physical activity and at least 25% of studies included were conducted in lowor middle-income African or Caribbean countries. Reviews could include quantitative or qualitative evidence from observational or interventional studies. We excluded literature reviews that: (i) explored how diet or physical activity shaped health outcomes or disease burden; (ii) reported only health outcomes; (iii) dealt primarily with health-system care models; (iv) focused on migrant groups or ethnic minorities in high-income countries; (v) related to humanitarian crises or natural disasters; (vi) considered only nutritional biomarkers, without an accompanying assessment of diet; (vii) addressed breastfeeding as a determinant of diet in infants; or (viii) were not published in English. Citations identified by the search strategy were first imported into Rayyan QCRI systematic review software (Qatar Computing Research Institute, Doha, Qatar) and any duplicates were removed. Working in two pairs, we double-screened the titles and abstracts of all citations. If there was a conflict, all authors conferred, with two authors acting as arbiters. Then, the full texts of selected reviews were retrieved and, again working in two pairs, we doublescreened all texts. Any disagreement was discussed with reference to the eligibility criteria, with any one of three authors acting as arbiter. We examined the final set of selected reviews to determine which data items should be abstracted and their format. The abstraction fields identified included: (i) the citation; (ii) the type of review; (iii) the number and type of studies in the review, including spe- Bull World Health Organ 2021;99:464–472I\| doi: http://dx.doi.org/10.2471/BLT.20.269308 cifically the number and type of studies conducted in Africa and the Caribbean; (iv) the review setting (e.g. urban or rural); (v) the target population group (e.g. children or adults); (vi) factors associated with diet or physical activity; (vii) outcomes; and (viii) the main findings. Three authors entered data into a pretested data abstraction form. Where possible, data and conclusions specifically relevant to African and Caribbean countries were abstracted separately. We used Dahlgren and Whitehead’s social model of health to categorize and conceptualize both distal factors (e.g. international policies and politics, and socioeconomic, cultural and environmental conditions) and proximal factors (e.g. living and working conditions, social and community networks and individual factors such as age and sex).9 The abstracted data were summarized and tabulated. Our scoping review of reviews describes the results, discussions and conclusions of the selected reviews, not of the primary studies underlying them. Moreover, as is common in scoping reviews, there was no appraisal of the quality of the reviews. Hypothesized or putative explanations for relationships identified in the reviews were included in our summary only if supported by a synthesis of the underlying primary studies. Results The database searches identified 1652 unique citations whose titles and abstracts were screened (Fig. 1). Of these, 199 were selected for full text screening and, finally, 25 reviews were included in our scoping review.16–40 The number of papers identified increased markedly over time (Fig. 2), with nine of the 25 selected reviews being published first in 2019.25–28,36–40 The detailed characteristics of the 25 reviews are listed in Table 1 (available at: http://www.who .int/bulletin/volumes/99/6/20-269308). Th i r te e n re v i e w s c ons i d e re d diet, 17,23,25,29–32,34–38,40 four considered physical activity,16,18–20 and eight considered both.21,22,24,26–28,33,39 Eighteen reviews summarized quantitative evidence only, 16,18–21,24–30,32–34,38–40 including four that conducted meta-analyses,18,21,29,38 and one that used modelling techniques. 32 Four reviews incorporated both quantitative and qualitative evidence,22,31,35,37 including one that used a 465 Systematic reviews Eleanor Turner-Moss et al. Diet and physical activity in Africa and the Caribbean Fig. 1. Selection of reviews for the scoping review of factors associated with diet and physical activity in Africa and the Caribbean, 1998–2019 2419 records identified through database search: 1145 Medline®; 901 Web of Science™; 76 LILACS; 200 Global Health; and 97 Scopus No additional records identified through other sources After duplicates were removed, 1652 titles and abstracts of papers screened 1453 papers excluded: • 18 reported wrong exposure; • 1387 reported wrong outcome; • 216 studied wrong population; • 101 had wrong study design; or • 24 were not in English a The full text of 199 papers assessed for eligibility 25 reviews included in scoping review a 174 papers excluded: • 70 reported wrong outcome; • 10 had wrong study design; • 80 studied wrong population; and • 14 full text not available Records could be excluded for several reasons. No. of papers identified through search Fig. 2. No. of papers identified in search for reviews of factors associated with diet and physical activity in Africa and the Caribbean, by year of publication, 1998–2019 600 500 400 300 200 100 0 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 2014 2015 2016 2017 2018 2019 Year of publication Note: Duplicates were removed. meta-ethnographic approach.22 The remaining three reviews presented a narrative summary of data and the design of the studies included was unclear.17,23,36 In 13 reviews, 16–28 at least 50% of studies included were conducted in Africa or the Caribbean: nine focused on specific African regions or countries, 16–20,23,25,26,28 whereas only one focused on the Caribbean. 21 In the remaining 12 reviews, 29–40 only 25–50% of studies came from Africa or the Caribbean – they tended to focus on low- and middle-income countries. A small number of countries were over-represented in the primary evidence: Kenya, 17,19,20,24,29,30,33–35,37 Ni466 geria 16,18–20,24,33–35,37,39 and South Africa, 19,20,22,23,26–28,30,32,37 featured in 10 reviews each and Jamaica featured in all three reviews that included Caribbean countries.21,29,30 Of the 25 reviews, eight summarized evidence from both children and adults, 17,21,23,24,31,33,36,37 six summarized evidence from adults only,16,18,27,30,35,38 and 11 summarized evidence from children only,19,20,22,25,26,28,29,32,34,39,40 including four related to infants.25,32,34,40 In addition, several reviews focused on specific settings or population groups, such as rural settings, 23,31 socioeconomically disadvantaged areas,29 or pregnant or lactating women.30,35 Overall, the reviews summarized evidence from primary studies published between 1926 and 2018 – a 92year time period. Fourteen reviews included only more recent studies (e.g. the past 20 years),21,23–28,30,32,34,35,38–40 whereas the other 11 either did not set a time period, or did not report time-limits, for the primary evidence. Outcomes Dietar y outcomes summarized in the reviews included: (i) subsistence skills, such as food gathering, hunting and food preparation;22 (ii) child feeding complementary to breastfeeding;25,32,34,40 (iii) school meals or nutrition policies;26,29 (iv) access to and choice of food;35 (v) food security;17,23,37,38 (vi) diet diversity or quality;17,21 (vii) adherence to a prescribed diet;27 (viii) calorie or food group consumption (e.g. fruit and vegetables, animal protein or processed food);21,24,28,33,36,39 and (ix) macro- and micro-nutrient intake.30,32 Physical activity outcomes included: (i) active travel (e.g. walking or cycling for transport);19 (ii) total physical activity; (iii) domains of physical activity (e.g. occupational or leisure);20,26,28 (iv) total sedentary behaviour; (v) domains of sedentary behaviour (e.g. television watching);20,26 and (vi) physical inactivity (e.g. not meeting physical activity guidelines).16,18,21,33 Several reviews also reported physical fitness.20,28 The reviews reported a range of hypothesized and demonstrated relationships between various factors and diet and physical activity. These were categorized using Dahlgren and Whitehead’s social model of health (Table 2). Little of the summarized evidence was related to distal factors in the category of international health, policy and politics in the social model of health and there were relatively few reported associations with physical activity in any category.9 A wide range of associations were described, particularly for diet (Table 2). Several reviews reported that the shift to an urban, westernized lifestyle and diet and the threat of a competitive, globalized market were permeating influences.23,24,29 On diet, reviews that considered factors in the category of international health, policy and politics mentioned: the historic influence of colonization; humanitarian and development aid; the epidemiological transition; the transition to a western lifestyle and diet; the dual burdens of over- and undernutri- Bull World Health Organ 2021;99:464–472I\| doi: http://dx.doi.org/10.2471/BLT.20.269308 Systematic reviews Diet and physical activity in Africa and the Caribbean Eleanor Turner-Moss et al. Table 2. Factors associated with diet and physical activity in Africa and the Caribbean, scoping review of reviews, 1998–2019 Social model of health categorya Distal factors International health, policy and politics General socioeconomic, cultural and environmental conditions Proximal factors Living and working conditions (including agriculture, food production, education, work environment, unemployment, water, sanitation, health-care services and housing) Social and community networks Age, sex and constitutional factors Factors associated with diet Colonization;17 high-economic-value or cash crops;17,24 humanitarian aid (such as donated cereals);17 development aid and poverty reduction;24 gross domestic product;24 nutritional or epidemiological transition;39 dominance of major international retailers and producers;23 globalized (i.e. western) diet – high energy and low nutritional value;23 infectious diseases (including HIV/AIDS);23 dual burden of under- and overnutrition;24,39 and climate change or variability (e.g. erratic rainfall)23 Access to, and availability of, food;35 price of food;23,24 individual purchasing power;23,35 availability of energy-rich, cheaper foods;23 frequency, quality or size of meals;23 socioeconomic status;20,23,33 parental socioeconomic status;25 mass media;34 cultural beliefs;35 extreme weather (e.g. drought);17,19 food security;17,23 wild food sources;22,23 indigenous vegetable crops;23 infectious disease;23 gendered roles;21–24 institutional exclusion of women (e.g. powerlessness, vulnerability and lack of control over assets);23 deagrarianization;23 urbanization;17,39 habitat loss;23 human– environment interactions;23 lack of desire to engage in agriculture (signal of poverty);23,24 and social grants (particularly for HIV/AIDS)23,24 Poverty;23,24 occupation;33 unemployment;23 distance from markets;23,32 market access for rural development;23 available land and land rights;23,24 geography (e.g. coastal versus inland, highlands versus lowlands, and particular regions or provinces);17 seasonality (particularly of fresh fruit and vegetables);23 locally grown produce;23,24,32 food quality;32 street-food nutritional composition;36 convenience and taste of food;36,39 fortified foods;32 urban versus rural areas;23,24 school meals;24,29,34,35 nutrition education interventions;26,28 cooking demonstrations;25,40 agricultural interventions (e.g. for poverty reduction);24 agricultural expertise and training;23,24 urban agriculture (including food gardens);23 home gardening;25 nutritional advice from health-care workers;34 road improvements;24 personal assets;23,33 education on nutrition and health;33 integration of nutrition education into existing curriculum;26,28 school physical environment;26 school nutrition policies (e.g. availability of healthy snacks);26,28 cognitive, behavioural or psychosocial approaches to nutrition;26,27,40 prompts or rewards for healthy food choices;26,28 parental education;25 water availability;23,24 agricultural inputs;23,24 household size and composition;23,31 household food allocation;35 food preparation techniques;22,32 antiretroviral medication (needs to be taken with food);23 antenatal and postnatal care;25 and multicomponent interventions26,27 Social capital, networks, support and relationships;23 trust, reciprocity and exchange;23 exclusion and power imbalances;23 social meaningmaking;23 church membership;23 collective action and cooperation (such as a savings club);23 self-esteem;23 social interaction and skills acquisition;22 perception of the consumption of healthy food;39 community-based platforms or committees;24,32 social behavioural change interventions;35 key influencers or family members;35 caregiver involvement;26 paternal involvement;25 maternal diet;25 knowledge of quantity of food to eat during pregnancy;35 advice from health-care professionals;25 intervention delivered by community members;26,27 peer support;26–28 counselling and communication skills;35 declining indigenous knowledge;23 strategies to procure food (e.g. selling assets);23 perspectives and experience of food security;23 consumer acceptability and perceptions of processed cereals;23 taboos, beliefs, rules and norms;23,35 and psychosocial determinants39 Sex;21–23,31,34 age;22,32 and infection status24 Factors associated with physical activity Epidemiological transition39 Socioeconomic status;33 cultural heritage and gender disparity;19,20,22 weather (e.g. heavy rain disrupting travel across unbridged river);19 cultural practices and norms (e.g. running to school);19 urbanization;39 indoor leisure activities;39 technology (e.g. television, computer or mobile phone use);39 gendered roles;19 household responsibilities and work burden;19 punishment (including corporal) if late for school;19 fear of attack from people (e.g. violence, rape or harassment);19 dangerous vehicles;19 dangerous animals;19 and topography (such as rivers to cross or difficult terrain)19 Urban versus rural areas;16,18 built environment and perceived access to destinations (e.g. schools, shops and bus stops);19 lack of green space;39 unsafe neighbourhoods;39 multicomponent interventions;26,27 agricultural interventions (e.g. for poverty reduction);24 personal assets;33 education;33 integration of physical activity into existing curriculum;26,28 exercise classes or after-school sports;26 school travel time;19 school type (e.g. public versus private);19 school physical environment;26 physical activity equipment;26,28 and gendered roles19 Cultural practices and norms (e.g. running to school);19 girls afraid of encounters with strangers;19 restriction of girls’ mobility after puberty;19 feeling travel to school is safe;19 insecure neighbourhoods;19 caregiver involvement;28 sport tournaments;26,28 peer training and support;26–28 perceived importance of physical activity;39 and psychosocial determinants39 Sex;16,18–20 ethnicity;16 and age18,19 HIV/AIDS: human immunodeficiency virus and acquired immunodeficiency syndrome. a Factors reported in reviews as associated with diet or physical activity were categorized using Dahlgren and Whitehead’s social model of health.9 Bull World Health Organ 2021;99:464–472I\| doi: http://dx.doi.org/10.2471/BLT.20.269308 467 Systematic reviews Eleanor Turner-Moss et al. Diet and physical activity in Africa and the Caribbean tion; infectious and chronic disease; and the impact of climate change. In addition, associations were described with: socioeconomic, cultural and environmental conditions, including access to food, the availability of food, prices, food security, deagrarianization and urbanization; living and working conditions, including education, poverty, household composition, land rights, skills, assets, rurality, and agricultural and school-based interventions; social and community networks, involving for example social capital, skills acquisition, peer support, key influencers, taboos and norms; and constitutional factors, particularly age and sex. On physical activity, only one review described evidence on determinants in the most distal category of the social model of health (i.e. international health, policy and politics; Table 2). As expected, there were similarities and differences between the associations described for diet and physical activity. For example, both featured urbanization, socioeconomic status and gendered roles. In contrast, certain associations were described only for physical activity: (i) topography and climate; (ii) aspects of the built environment; (iii) dangerous traffic; (iv) fear of violent crime; (v) access to leisure facilities and green spaces; and (vi) restrictions on girls’ mobility after puberty. Many reviews reported the heterogeneity and lack of standardization of the assessment methods used in the primary studies. For example, one review on food insecurity reported that the studies included used 26 distinct indicators of food insecurity and that many studies neither directly measured food insecurity nor adequately reported the measures they used.23 On physical activity, reviews typically reported that the primary studies tended to use selfreport assessments and not objective assessments or measuring tools.18–20,28 Discussion We identified 25 reviews published between 1998 and 2019 that described factors associated with diet and physical activity in Africa and the Caribbean. Although our scoping review considered only evidence from these regions, our findings confirm that evidence is generally lacking from such settings on which to base policy and design interventions for improving diet and 468 physical activity. Moreover, our findings are consistent with those of a previous study,10 which carried out a systematic review of research from low- and lowermiddle-income countries published between 1990 and 2015 on the effect of interventions aligned with WHO’s “best buy” interventions on noncommunicable disease. 41 They identified 36 studies, which covered only nine of the 83 low- and lower-middle-income countries. Only two of the 36, both from Pakistan, concerned diet and physical activity. In our study, we found no review from Africa or the Caribbean that summarized evidence relevant to WHO’s “best buy” interventions. Similarly, none of the literature we identified assessed primary research relevant to WHO’s global action plan targets on noncommunicable diseases or to targets set for the relevant sustainable development goals (SDGs).4,42 Although there may be research from Africa and the Caribbean that has not yet been reviewed, our findings suggest that, to date, policies on diet and physical activity are not informed by summarized research evidence on their determinants from these settings. This conclusion has two clear implications: (i) relevant primary research that has not yet been reviewed should be identified and evaluated; and (ii) new research should be undertaken to fill gaps in the evidence. The policy responses and types of intervention required to improve health outcomes associated with diet and physical inactivity may be quite different in Africa and the Caribbean than in higher-income settings. In the absence of evidence indicating how different they need to be, current international guidance (e.g. WHO’s “best buy” interventions and recommendations in the global action plan on noncommunicable diseases) should be followed, so long as the interventions employed are robustly evaluated and can subsequently contribute to the evidence available from Africa and the Caribbean.43 Research funding bodies could help fill knowledge gaps and encourage the production of evidence summaries to guide policy. It would help if the terminology and definitions used for outcomes and their hypothesized determinants were much more consistent than we found in our study. In addition, international research networks that cover a range of different settings across Africa and the Caribbean could help develop and promote the high-quality, multidisciplinary research needed to address the complexity inherent in understanding how behavioural determinants vary between different contexts.44 In choosing to carry out a broad scoping review of factors associated with diet and physical activity and by adopting the review as the unit of analysis, our intention was to highlight gaps in the summarized literature (rather than in the primary literature) as an aid to policy-making. We did not directly look for primary research on the determinants of diet and physical activity, nor did we summarize policy documents. Consequently, our review does not indicate, for example, whether or not there exists a large number of primary research studies that have not yet been included in systematic appraisals of the evidence. Nor can we evaluate the degree to which existing policies are evidencebased; we can only comment on whether there is sufficient summarized evidence to inform those policies. Our search strategy and the study’s conclusions were limited to factors that had a hypothesized or demonstrated association with behaviours affecting diet or physical activity. It is likely that, in some settings, academic research investigated factors associated with obesity or noncommunicable disease but did not explicitly categorize behaviour. Consequently, given that we were primarily interested in factors associated with behaviour rather than disease, our search strategy – though broad – could have missed some reviews of the determinants of diet and physical activity. Moreover, the cut-off date for inclusion in our review was 2019, which was just 4 years into the period covered by the SDGs. Most of the research included was, therefore, conducted during the era of the millennium development goals, which focused on undernutrition and did not stipulate any targets or indicators for noncommunicable disease. Another limitation was that we did not appraise the quality of the reviews or the robustness of their evidence because our scoping review was intended primarily to map work in this area. Moreover, we identified papers only in English and may have missed reviews in other languages. We did not search the grey literature as our focus was on peerreviewed academic journals. However, having identified gaps in the literature, Bull World Health Organ 2021;99:464–472I\| doi: http://dx.doi.org/10.2471/BLT.20.269308 Systematic reviews Diet and physical activity in Africa and the Caribbean Eleanor Turner-Moss et al. we plan to include both academic and grey literature in a range of languages in future reviews. In conclusion, our scoping review of reviews provides an overview of an important and rapidly evolving area of work. As our search strategy was kept broad by design, we found that a wide range of factors were reported to be associated with diet and physical activity in Africa and the Caribbean. However, evidence on which to base policy or to design interventions was lacking, which highlights the need for further reviews of the primary evidence to inform policy responses where the evidence exists, and to establish whether additional primary research is needed. As the modifiability of determinants of diet and physical activity and the feasibility of modify- ing them vary widely, future research should be aligned with policy targets and should evaluate the effectiveness of policy responses in different contexts. ■ Acknowledgements We thank members of the Global Diet and Activity Research Network: Rosemary Musuva, Charles O Obonyo, Pamela Wadende and Vincent Were (Kenya Medical Research Institute, Kenya); Anna Brugulat, Ebele Mogo, Tolu Oni, Lambed Tatah, Nicholas J Wareham and James Woodcock (University of Cambridge, England); Estelle Victoria Lambert, Feyi Odunitan-Wayas, Kufre Okop, Maylene Shung-King and Amy Weimann (University of Cape Town, South Africa); Nadia Bennett, Ishtar Govia, Nathalie Guthrie-Dixon, Ian Hambleton, Alafia Samuels, Joanne Smith and Marshall Tulloch Reid (University of the West Indies, Jamaica); Agnes Erzse, Karen J Hofman, Kelsey Lebard, Gugulethu Mabena, Lisa K Micklesfield, Molebogeng Motlhalhedi and Shane A Norris (University of Witwatersrand, South Africa); and Felix Assah, Clarisse Mapa, Jean Claude Mbanya and Yves Wasnyo (University of Yaoundé, Cameroon). Funding: This research was funded by the National Institute for Health Research (NIHR; 16/137/64) using UK aid from the UK Government to support global health research. Competing interests: None declared. ‫ملخص‬ ‫أدلة عىل العوامل املرتبطة بالنظام الغذائي والنشاط البدين يف الدول األفريقية ودول البحر الكاريبي‬ ً ‫وشملت ثالثة منها فقط دلي‬ ‫ تضمنت‬.‫ال من منطقة البحر الكاريبي‬ ‫ أدلة أولية تم نرشه بني عامي‬،‫ جنب ًا إىل جنب‬،25 ‫املراجعات الـ‬ ‫ القليل من الدليل املوجز تعلق باالرتباطات‬.2018‫ و‬1926 ‫ والنظام الغذائي أو‬،‫بني العوامل الصحية أو السياسية الدولية‬ ‫االرتباطات بني أي عامل والنشاط البدين عرب كل فئات النموذج‬ .‫االجتامعي للصحة‬ ‫االستنتاج وجدت املراجعة عن كثب جمموعة واسعة من العوامل‬ ‫التي تم اإلبالغ عن ارتباطها بالنظام الغذائي والنشاط البدين يف‬ ‫ إال أن األدلة املوجزة الذي يمكن‬،‫إفريقيا ومنطقة البحر الكاريبي‬ ‫أن يساعد يف توجيه السياسات التي تشجع السلوكيات املرتبطة‬ ‫ كانت‬،‫بالنظم الغذائية الصحية والنشاط البدين يف هذه املناطق‬ ‫ هناك حاجة إىل مزيد من املراجعات لتوجيه السياسة حينام‬.‫مفقودة‬ .‫ وما إذا كانت هناك حاجة إىل بحث أويل إضايف‬،‫يكون هناك دليل‬ ‫الغرض حتديد ووصف الدليل املوجز عىل العوامل املرتبطة بالنظام‬ ‫الغذائي والنشاط البدين يف البلدان منخفضة الدخل ومتوسطة‬ ‫ عن طريق إجراء‬،‫الدخل يف أفريقيا ومنطقة البحر الكاريبي‬ .‫مراجعة عن كثب للمراجعات‬ ،LILACS‫ و‬،®Medline ‫الطريقة قمنا بالبحث يف قواعد بيانات‬ ‫ عن‬،Web of Science‫ و‬،Global Health‫ و‬،Scopus‫و‬ ،‫ملراجعات للعوامل املرتبطة بالنظام الغذائي أو النشاط البدين‬ 25% ‫ كان جيب أن تأيت‬.2019‫ و‬1998 ‫واملنشورة بني عامي‬ ‫عىل األقل من دراسات املراجعات من إفريقيا أو منطقة البحر‬ ‫ تم تصنيف العوامل باستخدام نموذج داجلرين ووايتهيد‬.‫الكاريبي‬ .‫ ومل يكن هناك تقييم للجودة‬،‫االجتامعي للصحة‬ ‫ وأربعة‬،‫ منها عن النظام الغذائي‬13 ،‫ مراجعة‬25 ‫النتائج لقد حددنا‬ ‫ كانت ثامين عرشة مقالة‬.‫معا‬ ً ‫ وثامنية منها عنهام‬،‫عن النشاط البدين‬ 25% ‫ كانت‬،‫ مراجعة‬12 ‫ يف‬.‫عبارة عن مراجعات منهجية كمية‬ .‫ من الدراسات من أفريقيا أو منطقة البحر الكاريبي‬50% ‫إىل‬ 摘要非洲和加勒比国家饮食和身体活动相关因素的证据目的旨在通过对综述文献进行概况性评价，确定和说明非洲和加勒比地区中低收入国家饮食和身体活动相关因素的汇总证据。方法我们搜索了 Medline®、LILACS、Scopus、全球健康和科学网数据库，以查看 1998 年至 2019 年期间发布的饮食或身体活动相关因素综述文献。在这些综述文献中，至少有 25 ％的研究项目来自非洲或加勒比地区。使用 Dahlgren 和 Whitehead 的健康社会模型对因素进行了分类。但并未进行质量鉴定。结果我们确定了 25 份综述文献：其中 13 份与饮食有关，4 份关于身体活动，8 份与两者均有关。十八篇文章采用了定量系统评价方法。在 12 份综述文献中， 25-50% 的研究项目来自非洲或加勒比地区。其中仅三份证据来自加勒比地区。这 25 份综述文献整体涵括了 1926 年至 2018 年期间公布的主要证据。汇总证据很少涉及国际健康或政治因素与饮食之间的关联性，或健康社会模型所有类别中任何因素与身体活动之间的关联性。结论通过概况性评价，我们发现非洲和加勒比地区众多因素均与饮食和身体活动有关，但缺少汇总证据来帮助推行适当政策，以鼓励在这些地区实施与健康饮食和身体活动有关的行为。需要进一步查询综述文献，以基于已有证据推行适当政策并确定是否需要开展其他初步研究工作。 Bull World Health Organ 2021;99:464–472I\| doi: http://dx.doi.org/10.2471/BLT.20.269308 469 Systematic reviews Eleanor Turner-Moss et al. Diet and physical activity in Africa and the Caribbean Résumé Éléments de preuve concernant les facteurs liés au régime alimentaire et à l'activité physique dans les pays d’ Afrique et des Caraïbes Objectif Identifier et décrire les synthèses de données probantes consacrées aux facteurs liés au régime alimentaire et à l'activité physique dans les pays à faible et moyen revenu en Afrique et dans les Caraïbes, en examinant la portée des revues. Méthodes Nous avons examiné les bases de données Medline®, LILACS, Scopus, Global Health et Web of Science en quête de revues publiées entre 1998 et 2019, consacrées aux facteurs liés au régime ou à l'activité physique. Au moins 25% des études citées dans ces revues devaient provenir d'Afrique ou des Caraïbes. Nous avons ensuite classé les facteurs à l'aide du modèle social de santé Dahlgren et Whitehead. Aucune évaluation de la qualité n'a été effectuée. Résultats Nous avons repéré 25 revues: 13 sur le régime, quatre sur l'activité physique et huit réunissant les deux thèmes. Dix-huit articles étaient des revues systématiques quantitatives. Dans 12 revues, 25 à 50% des études avaient été réalisées en Afrique ou dans les Caraïbes. Seulement trois contenaient des éléments de preuve relatifs aux Caraïbes. Prises dans leur ensemble, les 25 revues renfermaient des preuves primaires publiées entre 1926 et 2018. Rares étaient les synthèses de données probantes consacrées aux liens entre la santé internationale ou les facteurs politiques d'une part, et le régime alimentaire de l'autre, ou encore entre n'importe quel facteur et l'activité physique dans toutes les catégories du modèle social de santé. Conclusion L'examen de la portée a permis de découvrir un large éventail de facteurs considérés comme en lien avec le régime alimentaire et l'activité physique en Afrique et dans les Caraïbes. Toutefois, il manquait une synthèse des données probantes, qui aurait pu contribuer à orienter les politiques destinées à encourager les comportements favorisant un régime alimentaire sain et la pratique d'une activité physique dans ces régions. D'autres revues sont nécessaires pour fournir des informations aux politiques lorsqu'il existe une preuve, et pour déterminer si une recherche préliminaire supplémentaire est requise. Резюме Данные о факторах, связанных с питанием и физической активностью, в странах Африки и Карибского бассейна Цель Выявить и описать обобщенные данные о факторах, связанных с питанием и физической активностью, в странах Африки и Карибского бассейна с низким и средним уровнем доходов путем составления обзора проведенных исследований. Методы Авторы выполнили поиск информации в базах данных Medline®, LILACS, Scopus, Global Health и Web of Science, опубликованной в период с 1998 по 2019 год, с целью изучения факторов, связанных с питанием или физической активностью. Минимум 25% исследований в обзорах должны были проводиться в странах Африки или Карибского бассейна. Эти факторы были классифицированы с использованием социальной модели факторов здоровья Дальгрена и Уайтхед. Оценка качества не проводилась. Результаты Было составлено 25 обзоров: 13 по питанию, 4 по физической активности и 8 по обоим факторам. Восемнадцать статей являлись количественными систематическими обзорами. В 12 обзорах от 25 до 50% исследований приходилось на страны Африки и Карибского бассейна. Только три из них содержали данные по странам Карибского бассейна. В общей сложности 25 обзоров содержали данные первичных исследований, опубликованные в период с 1926 по 2018 год. Малый процент обобщенных данных касался взаимосвязей между международным здравоохранением или политическими факторами и питанием либо взаимосвязей между любыми факторами и физической активностью по всем категориям социальной модели здоровья. Вывод Обзор проведенных исследований выявил широкий спектр факторов, связанных с питанием и физической активностью, в странах Африки и Карибского бассейна, однако обобщенные данные, которые могли бы способствовать разработке политики поощрения здорового питания и физической активности, по этим регионам отсутствовали. Дальнейшие обзоры необходимы для подкрепления политических мер в случае наличия таких данных, а также для определения необходимости проведения дополнительных первичных исследований. Resumen Pruebas de los factores asociados a la dieta y la actividad física en los países de África y el Caribe Objetivo Identificar y describir las pruebas resumidas sobre los factores asociados a la dieta y la actividad física en los países de ingresos bajos y medios de África y el Caribe mediante la realización de una revisión del alcance de las revisiones. Métodos Realizamos búsquedas en las bases de datos Medline®, LILACS, Scopus, Global Health y Web of Science de revisiones de factores asociados a la dieta o la actividad física publicadas entre 1998 y 2019. Al menos el 25% de los estudios de las revisiones debían proceder de África o el Caribe. Los factores se clasificaron utilizando el modelo social de salud de Dahlgren y Whitehead. No hubo evaluación de la calidad. Resultados Identificamos 25 revisiones: 13 sobre la dieta, cuatro sobre la actividad física y ocho sobre ambas. Dieciocho artículos eran revisiones sistemáticas cuantitativas. En 12 revisiones, entre el 25 y el 50% de los estudios eran de África o el Caribe. Solo tres incluyeron pruebas del 470 Caribe. En conjunto, las 25 revisiones incluyeron evidencia primaria publicada entre 1926 y 2018. Pocas de las pruebas resumidas se referían a las asociaciones entre los factores políticos o de salud internacionales y la dieta o las asociaciones entre cualquier factor y la actividad física en todas las categorías del modelo social de salud. Conclusión En la revisión del alcance encontramos una gran variedad de factores que, según los informes, se asocian con la dieta y la actividad física en África y el Caribe, pero carecemos de pruebas resumidas que puedan ayudar a informar las políticas que fomentan los comportamientos relacionados con las dietas saludables y la actividad física en estas regiones. Es necesario realizar más revisiones para informar a las políticas sobre los puntos en los que existen pruebas y sobre la necesidad de realizar investigaciones primarias adicionales. Bull World Health Organ 2021;99:464–472I\| doi: http://dx.doi.org/10.2471/BLT.20.269308 Systematic reviews Diet and physical activity in Africa and the Caribbean Eleanor Turner-Moss et al. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. Global status report on noncommunicable diseases 2014. Geneva: World Health Organization; 2014. Available from: https://www.who.int/nmh/ publications/ncd-status-report-2014/en/ [cited 2021 Mar 11]. Report on the status of major health risk factors for noncommunicable diseases: WHO African Region, 2015. Brazzaville: WHO Regional Office for Africa; 2016. Available from: https://www.afro.who.int/publications/report -status-major-health-risk-factors-noncommunicable-diseases-who-african -region-0 [cited 2021 Mar 11]. Forouzanfar MH, Afshin A, Alexander LT, Anderson HR, Bhutta ZA, Biryukov S, et al.; GBD 2015 Risk Factors Collaborators. Global, regional, and national comparative risk assessment of 79 behavioural, environmental and occupational, and metabolic risks or clusters of risks, 1990–2015: a systematic analysis for the Global Burden of Disease Study 2015. Lancet. 2016 Oct 8;388(10053):1659–724. doi: http://dx.doi.org/10.1016/S0140 -6736(16)31679-8 PMID: 27733284 Global action plan for the prevention and control of NCDs 2013–2020. Geneva: World Health Organization; 2013. Available from: https://www.who .int/publications-detail-redirect/9789241506236 [cited 2021 Mar 11]. WHO guidelines on physical activity and sedentary behaviour. Geneva: World Health Organization; 2020. Available from: https://www.who.int/ publications-detail-redirect/9789240015128 [cited 2021 Mar 11]. Global health data exchange: GBD results tool [internet]. Seattle: Institute for Health Metrics and Evaluation; 2019. Available from: http://ghdx .healthdata.org/gbd-results-tool [cited 2021 Jan 25]. Juma PA, Mohamed SF, Matanje Mwagomba BL, Ndinda C, Mapa-Tassou C, Oluwasanu M, et al. Non-communicable disease prevention policy process in five African countries. BMC Public Health. 2018 Aug 15;18(S1) Suppl 1:961. doi: http://dx.doi.org/10.1186/s12889-018-5825-7 PMID: 30168393 Murphy MM, Unwin N, Samuels TA, Hassel TA, Bishop L, Guell C. Evaluating policy responses to noncommunicable diseases in seven Caribbean countries: challenges to addressing unhealthy diets and physical inactivity. Rev Panam Salud Publica. 2018 Dec 17;42:e174. doi: http://dx.doi.org/10 .26633/RPSP.2018.174 PMID: 31093202 Dahlgren G, Whitehead M. Policies and strategies to promote social equity in health. Background document to WHO strategy paper for Europe. September 1991. Arbetsrapport 2007:14. Stockholm: Institute for Futures Studies; 2018. Allen LN, Pullar J, Wickramasinghe KK, Williams J, Roberts N, Mikkelsen B, et al. Evaluation of research on interventions aligned to WHO ‘Best Buys’ for NCDs in low-income and lower-middle-income countries: a systematic review from 1990 to 2015. BMJ Glob Health. 2018 Feb 19;3(1):e000535. doi: http://dx.doi.org/10.1136/bmjgh-2017-000535 PMID: 29527342 Ebrahim S, Pearce N, Smeeth L, Casas JP, Jaffar S, Piot P. Tackling non-communicable diseases in low- and middle-income countries: is the evidence from high-income countries all we need? PLoS Med. 2013;10(1):e1001377. doi: http://dx.doi.org/10.1371/journal.pmed.1001377 PMID: 23382655 Arksey H, O’Malley L. Scoping studies: towards a methodological framework. Int J Soc Res Methodol. 2005;8(1):19–32. doi: http://dx.doi.org/ 10.1080/1364557032000119616 Global Diet and Activity Research Network (GDAR) [internet]. Cambridge: Global Diet and Activity Research Group and Network; 2020. Available from: https://www.gdarnet.org/ [cited 2021 Jan 25]. Tricco AC, Lillie E, Zarin W, O’Brien KK, Colquhoun H, Levac D, et al. PRISMA extension for scoping reviews (PRISMA-ScR): checklist and explanation. Ann Intern Med. 2018 Oct 2;169(7):467–73. doi: http://dx.doi.org/10.7326/M18 -0850 PMID: 30178033 Scoping review protocol. Factors associated with diet and physical activity in Africa and the Caribbean: a scoping review of reviews. Cambridge: Global Diet and Activity Research Group and Network; 2019. Available from: https://www.gdarnet.org/wp-content/uploads/2019/07/Scoping-review -protocol-v1.8.pdf [cited 2021 Jan 25]. Abubakari AR, Bhopal RS. Systematic review on the prevalence of diabetes, overweight/obesity and physical inactivity in Ghanaians and Nigerians. Public Health. 2008 Feb;122(2):173–82. doi: http://dx.doi.org/10.1016/j .puhe.2007.06.012 PMID: 18035383 Raschke V, Oltersdorf U, Elmadfa I, Wahlqvist ML, Kouris-Blazos A, Cheema B. Investigation of the dietary intake and health status in East Africa in the 1960s: a systematic review of the historic Oltersdorf collection. Ecol Food Nutr. 2008;47(1):1–43. doi: http://dx.doi.org/10.1080/03670240701454683 18. Abubakari AR, Lauder W, Jones MC, Kirk A, Agyemang C, Bhopal RS. Prevalence and time trends in diabetes and physical inactivity among adult West African populations: the epidemic has arrived. Public Health. 2009 Sep;123(9):602–14. doi: http://dx.doi.org/10.1016/j.puhe.2009.07.009 PMID: 19748643 19. Larouche R, Oyeyemi AL, Prista A, Onywera V, Akinroye KK, Tremblay MS. A systematic review of active transportation research in Africa and the psychometric properties of measurement tools for children and youth. Int J Behav Nutr Phys Act. 2014 Oct 18;11(1):129. doi: http://dx.doi.org/10.1186/ s12966-014-0129-5 PMID: 25326031 20. Muthuri SK, Wachira LJ, Leblanc AG, Francis CE, Sampson M, Onywera VO, et al. Temporal trends and correlates of physical activity, sedentary behaviour, and physical fitness among school-aged children in sub-Saharan Africa: a systematic review. Int J Environ Res Public Health. 2014 Mar 20;11(3):3327– 59. doi: http://dx.doi.org/10.3390/ijerph110303327 PMID: 24658411 21. Sobers-Grannum N, Murphy MM, Nielsen A, Guell C, Samuels TA, Bishop L, et al. Female gender is a social determinant of diabetes in the Caribbean: a systematic review and meta-analysis. PLoS One. 2015 May 21;10(5):e0126799. doi: http://dx.doi.org/10.1371/journal.pone.0126799 PMID: 25996933 22. Lew-Levy S, Reckin R, Lavi N, Cristóbal-Azkarate J, Ellis-Davies K. How do hunter-gatherer children learn subsistence skills?: a meta-ethnographic review. Hum Nat. 2017 Dec;28(4):367–94. doi: http://dx.doi.org/10.1007/ s12110-017-9302-2 PMID: 28994008 23. Misselhorn A, Hendriks SL. A systematic review of sub-national food insecurity research in South Africa: missed opportunities for policy insights. PLoS One. 2017 Aug 22;12(8):e0182399. doi: http://dx.doi.org/10.1371/ journal.pone.0182399 PMID: 28829787 24. Pullar J, Allen L, Townsend N, Williams J, Foster C, Roberts N, et al. The impact of poverty reduction and development interventions on non-communicable diseases and their behavioural risk factors in low and lower-middle income countries: a systematic review. PLoS One. 2018 Feb 23;13(2):e0193378. doi: http://dx.doi.org/10.1371/journal.pone.0193378 PMID: 29474454 25. Abdurahman AA, Chaka EE, Bule MH, Niaz K. Magnitude and determinants of complementary feeding practices in Ethiopia: a systematic review and meta-analysis. Heliyon. 2019 Jul 2;5(7):e01865. doi: http://dx.doi.org/10 .1016/j.heliyon.2019.e01865 PMID: 31317077 26. Adom T, De Villiers A, Puoane T, Kengne AP. School-based interventions targeting nutrition and physical activity, and body weight status of African children: a systematic review. Nutrients. 2019 Dec 30;12(1):95. doi: http://dx .doi.org/10.3390/nu12010095 PMID: 31905832 27. Gyawali B, Bloch J, Vaidya A, Kallestrup P. Community-based interventions for prevention of type 2 diabetes in low- and middle-income countries: a systematic review. Health Promot Int. 2019 Dec 1;34(6):1218–30. doi: http:// dx.doi.org/10.1093/heapro/day081 PMID: 30329052 28. Klingberg S, Draper CE, Micklesfield LK, Benjamin-Neelon SE, van Sluijs EMF. Childhood obesity prevention in Africa: a systematic review of intervention effectiveness and implementation. Int J Environ Res Public Health. 2019 Apr 4;16(7):1212. doi: http://dx.doi.org/10.3390/ijerph16071212 PMID: 30987335 29. Kristjansson EA, Robinson V, Petticrew M, MacDonald B, Krasevec J, Janzen L, et al. School feeding for improving the physical and psychosocial health of disadvantaged elementary school children. Cochrane Database Syst Rev. 2007 Jan 24; (1):CD004676. PMID: 17253518 30. Lee SE, Talegawkar SA, Merialdi M, Caulfield LE. Dietary intakes of women during pregnancy in low- and middle-income countries. Public Health Nutr. 2013 Aug;16(8):1340–53. doi: http://dx.doi.org/10.1017/ S1368980012004417 PMID: 23046556 31. Johnston D, Stevano S, Malapit HJ, Hull E, Kadiyala S. Agriculture, gendered time use, and nutritional outcomes: a systematic review. IFPRI discussion paper 01456. Washington, DC: International Food Policy Research Institute; 2015. Available from: https://www.ifpri.org/publication/agriculture -gendered-time-use-and-nutritional-outcomes-systematic-review [cited 2021 Mar 11]. 32. Osendarp SJ, Broersen B, van Liere MJ, De-Regil LM, Bahirathan L, Klassen E, et al. Complementary feeding diets made of local foods can be optimized, but additional interventions will be needed to meet iron and zinc requirements in 6- to 23-month-old children in low- and middle-income countries. Food Nutr Bull. 2016 Dec;37(4):544–70. doi: http://dx.doi.org/10 .1177/0379572116655239 PMID: 27334774 Bull World Health Organ 2021;99:464–472I\| doi: http://dx.doi.org/10.2471/BLT.20.269308 471 Systematic reviews Eleanor Turner-Moss et al. Diet and physical activity in Africa and the Caribbean 33. Allen L, Williams J, Townsend N, Mikkelsen B, Roberts N, Foster C, et al. Socioeconomic status and non-communicable disease behavioural risk factors in low-income and lower-middle-income countries: a systematic review. Lancet Glob Health. 2017 Mar;5(3):e277–89. doi: http://dx.doi.org/ 10.1016/S2214-109X(17)30058-X PMID: 28193397 34. Graziose MM, Downs SM, O’Brien Q, Fanzo J. Systematic review of the design, implementation and effectiveness of mass media and nutrition education interventions for infant and young child feeding. Public Health Nutr. 2018 Feb;21(2):273–87. doi: http://dx.doi.org/10.1017/ S1368980017002786 PMID: 29081315 35. Kavle JA, Landry M. Addressing barriers to maternal nutrition in low- and middle-income countries: a review of the evidence and programme implications. Matern Child Nutr. 2018 Jan;14(1):e12508. doi: http://dx.doi .org/10.1111/mcn.12508 PMID: 28836343 36. Abrahale K, Sousa S, Albuquerque G, Padrão P, Lunet N. Street food research worldwide: a scoping review. J Hum Nutr Diet. 2019 Apr;32(2):152–74. doi: http://dx.doi.org/10.1111/jhn.12604 PMID: 30311276 37. Audate PP, Fernandez MA, Cloutier G, Lebel A. Scoping review of the impacts of urban agriculture on the determinants of health. BMC Public Health. 2019 May 31;19(1):672. doi: http://dx.doi.org/10.1186/s12889-019 -6885-z PMID: 31151393 38. Boneya DJ, Ahmed AA, Yalew AW. The effect of gender on food insecurity among HIV-infected people receiving anti-retroviral therapy: a systematic review and meta-analysis. PLoS One. 2019 Jan 7;14(1):e0209903. doi: http:// dx.doi.org/10.1371/journal.pone.0209903 PMID: 30615692 472 39. Leandro CG, Fonseca EVDSD, de Lim CR, Tchamo ME, Ferreira-E-Silva WT. Barriers and enablers that influence overweight/obesity/obesogenic behavior in adolescents from lower-middle income countries: a systematic review. Food Nutr Bull. 2019 Dec;40(4):562–71. doi: http://dx.doi.org/10 .1177/0379572119853926 PMID: 31272220 40. Webb Girard A, Waugh E, Sawyer S, Golding L, Ramakrishnan U. A scoping review of social-behaviour change techniques applied in complementary feeding interventions. Matern Child Nutr. 2020 Jan;16(1):e12882. doi: http:// dx.doi.org/10.1111/mcn.12882 PMID: 31386791 41. Tackling NCDs: 'best buys' and other recommended interventions for the prevention and control of noncommunicable diseases. Geneva: World Health Organization; 2017. Available from: https://apps.who.int/iris/handle/ 10665/259232 [cited 2021 Mar 11]. 42. Resolution A/RES/70/1. Transforming our world: the 2030 agenda for sustainable development. In: Seventieth United Nations General Assembly, New York, 25 September 2015. New York: United Nations; 2015. Available from: http://www.un.org/ga/search/view_doc.asp?symbol=A/RES/70/1& Lang=E [cited 2021 Mar 11]. 43. Ogilvie D, Adams J, Bauman A, Gregg EW, Panter J, Siegel KR, et al. Using natural experimental studies to guide public health action: turning the evidence-based medicine paradigm on its head. J Epidemiol Community Health. 2020 Feb;74(2):203–8. doi: http://dx.doi.org/10.1136/jech-2019 -213085 PMID: 31744848 44. Oni T, Assah F, Erzse A, Foley L, Govia I, Hofman KJ, et al.; GDAR network. The global diet and activity research (GDAR) network: a global public health partnership to address upstream NCD risk factors in urban low and middle-income contexts. Global Health. 2020 Oct 19;16(1):100. doi: http:// dx.doi.org/10.1186/s12992-020-00630-y PMID: 33076935 Bull World Health Organ 2021;99:464–472I\| doi: http://dx.doi.org/10.2471/BLT.20.269308 Systematic reviews Eleanor Turner-Moss et al. Diet and physical activity in Africa and the Caribbean Box 1. Search strategies, scoping review of reviews of factors associated with diet and physical activity in Africa and the Caribbean, 1998–2019 Medline® search strategy 1. diet.mp. OR exp DIET 2. exp NUTRITION DISORDERS/ OR nutrition.mp. 3. food intake.mp. OR exp Eating/ 4. exp Feeding Behavior/ OR eating behavio?r.mp. 5. junk* food.mp. 6. (calori adj2 intake).mp. 7. meat consumption.mp. 8. (high adj2 (fat OR salt* OR sugar)).mp. 9. malnutrition.mp. OR exp MALNUTRITION/ 10. exp Malnutrition/ OR malnourish.mp. 11. (fruit AND veg).mp. 12. exp Energy Intake/ OR energy intake.mp. 13. (physical* adj2 activ).mp. 14. exp Exercise/ 15. exercis.mp. 16. (active adj2 (living OR transport* OR travel)).mp. 17. walk.mp. OR exp Walking/ 18. (bike OR bicycl* OR biking).mp. 19. exp SEDENTARY LIFESTYLE/ OR exp Physical Exertion/ OR sedentary.mp. 20. (physical* adj2 exert).mp. 21. (screen time OR screentime).mp. 22. manual labo?r.mp. 23. subsistence.mp. 24. mobil.mp. 25. or/1–24 26. determinant.mp. OR exp “SOCIAL DETERMINANTS OF HEALTH”/ 27. exp SOCIOECONOMIC FACTORS/ OR socioeconomic.mp. 28. associat.mp. 29. correlat.mp. 30. (policy OR policies).mp. 31. legislat.mp. 32. exp Risk Factors/ OR risk factor.mp. 33. built environment.mp. OR exp Environment Design/ 34. exp SOCIAL ENVIRONMENT/ OR exp ENVIRONMENT/ OR environment.mp. 35. cultur.mp. OR exp Culture/ 36. ethnograph.mp. 37. psychosocial.mp. 38. exp Demography/ OR demograph.mp. OR exp Population Dynamics/ OR exp Population Characteristics/ 39. exp Epidemiology/ OR exp Epidemiologic Studies/ OR exp Epidemiologic Methods/ OR exp Epidemiological Monitoring/ OR epidemiolog.mp. 40. (cohort OR longitudinal* OR observation).mp. 41. or/26–40 42. Developing Countries.sh,kf. 43. ((developing OR less developed OR under developed OR underdeveloped OR middle income OR low* income OR underserved OR under served OR deprived OR poor) adj (countr OR nation? OR population? OR world)).ti,ab. 44. ((developing OR less* developed OR under developed OR underdeveloped OR middle income OR low* income) adj (economy OR economies)). ti,ab. 45. (low* adj (gdp OR gnp OR gross domestic OR gross national)).ti,ab. 46. (low adj3 middle adj3 countr).ti,ab. 47. (lmic OR lmics OR third world OR lami countr).ti,ab. 48. transitional countr.ti,ab. continues ... Bull World Health Organ 2021;99:464–472I\| doi: http://dx.doi.org/10.2471/BLT.20.269308 472A Systematic reviews Diet and physical activity in Africa and the Caribbean Eleanor Turner-Moss et al. ... continued 49. review.pt. 50. review.ab,ti. 51. 49 OR: 50 52. (Africa OR Caribbean OR West Indies).hw,ti,ab,cp. 53. exp AFRICA/ 54. exp Caribbean Region/ 55. (Africa OR Caribbean OR Sub-Sahara* OR “Sub Sahara” OR Algeria OR Angola OR Belize OR Benin OR Botswana OR “Burkina Faso” OR Burundi OR “Cabo Verde” OR “Cape verde” OR Cameroon OR “Central African Republic” OR Chad OR Comoros OR Comores OR Comoro OR Congo OR “Cote d’Ivoire” OR Cuba OR Djibouti OR Dominica OR “Dominican Republic” OR Egypt OR Eritrea OR Ethiopia OR Gabon OR Gambia OR Ghana OR Grenada OR Grenadines OR Guinea OR “Guinea Bisau” OR Guyana OR Haiti OR Jamaica OR Kenya OR Lesotho OR Liberia OR Libya OR Madagascar OR Malawi OR Mali OR Mauritania OR Mauritius OR Morocco OR Mozambique OR Namibia OR Niger OR Nigeria OR Principe OR Rwanda OR Ruanda OR “Sao Tome” OR Senegal OR “Sierra Leone” OR Somalia OR “South Africa” OR “South Sudan” OR “St Lucia” OR “St Vincent” OR Sudan OR Surinam OR Suriname OR Swaziland OR Tanzania OR Togo OR Tunisia OR Uganda OR Zambia OR Zimbabwe).tw. 56. or/52–55 57. (non-infectious OR noncommunicable* OR NCD OR non-communicable).mp. 58. 42 OR: 43 OR: 44 OR: 45 OR: 46 OR: 47 OR: 48 OR: 56 59. 25 OR: 57 60. 59 AND 41 AND 58 AND 51 61. review.m_titl. 62. 59 AND 41 AND 58 AND 61 63. (‘scoping review’ OR ‘systematic review’ OR ‘narrative review’ OR ‘literature review’ OR ‘evidence review’ OR ‘mixed methods review’ OR ‘realist review’ OR ‘realist synthesis’ OR ‘meta-ethnography’ OR ‘meta ethnography’).ab,ti. 64. 59 AND 41 AND 58 AND 63 Search strategy for other databases #1 TS = (diet) #2 TS = nutrition #3 TS = food intake #4 TS = eating behavio?r* #5 TS = (junk* food) #6 TS = (“calori intake”) #7 TS = (meat consumption) #8 TS = (“high fat” OR “high salt” OR “high sugar” #9 TS = malnutrition #10 TS = malnourish* #11 TS = (fruit* AND veg) #12 TS = (energy intake) #13 TS = (“physical* activ”) #14 TS = exercis #15 TS = (“active living” OR “active transport” OR “active travel”) #16 TS = walk* #17 TS = (bike OR bicycl* OR biking) #18 TS = sedentary lifestyle #19 TS = physical* exert* #20 TS = (screentime OR “screen time”) #21 TS = manual labo?r #22 TS = subsistence #23 TS = mobilisa* #24 #23 OR #22 OR #21 OR #20 OR #19 OR #18 OR #17 OR #16 OR #15 OR #14 OR #13 OR #12 OR #11 OR #10 OR #9 OR #8 OR #7 OR #6 OR #5 OR #4 OR #3 OR #2 OR #1 #25 TS = (determinant* OR socioeconomic* OR associat* OR correlat* OR policy OR policies OR legislat* OR risk factor* OR built environment OR environment* OR cultur* OR ethnograph* OR psychosocial* OR demograph* OR epidemiolog* OR cohort* OR longitudinal* OR observation) #26 TS = (“scoping review” OR “systematic review” OR “narrative review” OR “literature review” OR “evidence review” OR “mixed methods review” OR “realist review” OR “realist synthesis” OR “meta-ethnography” OR “meta ethnography”) continues ... 472B Bull World Health Organ 2021;99:464–472I\| doi: http://dx.doi.org/10.2471/BLT.20.269308 Systematic reviews Eleanor Turner-Moss et al. Diet and physical activity in Africa and the Caribbean ... continued #27 TS = (Africa OR Caribbean OR “West Indies”) #28 TS = (Africa OR Caribbean OR Sub-Sahara OR “Sub Sahara” OR Algeria OR Angola OR Belize OR Benin OR Botswana OR “Burkina Faso” OR Burundi OR “Cabo Verde” OR “Cape verde” OR Cameroon OR “Central African Republic” OR Chad OR Comoros OR Comores OR Comoro OR Congo OR “Cote d’Ivoire” OR Cuba OR Djibouti OR Dominica OR “Dominican Republic” OR Egypt OR Eritrea OR Ethiopia OR Gabon OR Gambia OR Ghana OR Grenada OR Grenadines OR Guinea OR “Guinea Bisau” OR Guyana OR Haiti OR Jamaica OR Kenya OR Lesotho OR Liberia OR Libya OR Madagascar OR Malawi OR Mali OR Mauritania OR Mauritius OR Morocco OR Mozambique OR Namibia OR Niger OR Nigeria OR Principe OR Rwanda OR Ruanda OR “Sao Tome” OR Senegal OR “Sierra Leone” OR Somalia OR “South Africa” OR “South Sudan” OR “St Lucia” OR “St Vincent” OR Sudan OR Surinam OR Suriname OR Swaziland OR Tanzania OR Togo OR Tunisia OR Uganda OR Zambia OR Zimbabwe) #29 TS = “developing countries” #30 TS = (“developing countr” OR “less* developed countr” OR “under developed countr” OR “underdeveloped countr” OR “middle income countr” OR “low* income countr” OR “underserved countr” OR “under served countr” OR “deprived countr” OR “poor countr” OR “developing nation” OR “less developed nation” OR “under developed nation” OR “underdeveloped nation” OR “middle income nation” OR “low* income nation” OR “underserved nation” OR “under served nation” OR “deprived nation” OR “poor nation” OR “developing population” OR “less* developed population” OR “under developed population” OR “underdeveloped population” OR “middle income population” OR “low* income population” OR “underserved population” OR “under served population” OR “deprived population” OR “poor population” OR “developing world” OR “less* developed world” OR “under developed world” OR “underdeveloped world” OR “middle income world” OR “low* income world” OR “underserved world” OR “under served world” OR “deprived world” OR “poor world”) #31 TS = (low gdp OR low* GNP OR low* gross domestic OR low* gross national) #32 TS = low middle countr* #33 TS = (lmic OR lmics OR third world OR lami countr) #34 TS = transitional countr #35 #34 OR #33 OR #32 OR #31 OR #30 OR #29 OR #28 OR #27 #36 #35 AND #26 AND #25 AND #24 Note: Each numbered line was run as a separate search. Then, the searches were combined in different ways using Boolean operators and the line numbers for each search. Bull World Health Organ 2021;99:464–472I\| doi: http://dx.doi.org/10.2471/BLT.20.269308 472C 472D Publication years of studies in review Type of review No. studies in review 1964–2003 1982–2013 1967–2013 Abubakari et al. (2009)18 Larouche et al. (2014)19 Muthuri et al. (2014)20 Systematic review of quantitative studies Systematic review and meta-analysis of quantitative studies Systematic review of quantitative studies 71 20a 15 71 20 SubSaharan Africa Africa West Africa East Africa 6 15 Ghana and Nigeria Review setting 16 No. studies in Africa or the Caribbean ≥ 50% of studies in review from Africa or the Caribbean Abubakari and 1964–2003 Systematic 16 Bhopal (2008)16 review of quantitative studies 6 Raschke et al. 1963–1969 Systematic (2008)17 review of historic data (design of included studies unclear) Review author (publication year) Burkina Faso, Cameroon, Côte d'Ivoire, Gambia, Ghana, Mali, Nigeria and Senegal Algeria, Botswana, Djibouti, Egypt, Ghana, Kenya, Libya, Malawi, Mauritius, Morocco, Namibia, Nigeria, Senegal, Seychelles, South Africa, Uganda, United Republic of Tanzania, Zambia and Zimbabwe Botswana, Cameroon, Côte d’Ivoire, Eswatini, Ethiopia, Ghana, Kenya, Mozambique, Namibia, Nigeria, Senegal, Seychelles, South Africa, Uganda, United Republic of Tanzania, Zambia and Zimbabwe Kenya, Uganda and United Republic of Tanzania Ghana and Nigeria African or Caribbean countries in studies reviewed Age, socioeconomic status, sex and urban or rural residence Socioeconomic status and urban or rural residence Children and young people Children and young people Age, sex and urban or rural residence Colonialization, natural environment and urbanization Sex, socioeconomic status and urban or rural residence Factors associated with diet or physical activity Adults Children and adults Adults Population group studied Table 1. Reviews included in scoping review of factors associated with diet and physical activity in Africa and the Caribbean, 1998–2019 NA NA (i) Cash-crop farming and replacement of indigenous crops; (ii) global food systems; (iii) urbanization; and (iv) destruction of natural ecosystems NA NA Notes on factors Physical activity Physical activity Physical activity Diet Physical activity Outcomes Diet and physical activity in Africa and the Caribbean (continues. . .) Additional outcomes of interest were sedentary behaviour and physical fitness Active travel (walking, running or cycling for transport) An additional outcome of interest was diabetes prevalence Additional outcomes of interest were diabetes prevalence and body composition (i) Food shortages; (ii) dependence on introduced or donated cereals; and (iii) loss of dietary diversity Notes on outcomes Systematic reviews Eleanor Turner-Moss et al. Bull World Health Organ 2021;99:464–472I\| doi: http://dx.doi.org/10.2471/BLT.20.269308 Publication years of studies in review 2007–2013 1939–2015 1997–2014 Review author (publication year) SobersGrannum et al. (2015)21 Lew-Levy et al. (2017)22 Misselhorn and Hendriks (2017)23 (. . .continued) Systematic review (design of included studies unclear) Metaethnographic review of quantitative and qualitative studies Systematic review and meta-analysis of quantitative studies Type of review 169 58 50 No. studies in review 169 31 50 No. studies in Africa or the Caribbean South Africa (mainly rural areas) Hunter– gatherer societies Caribbean Review setting Botswana, Cameroon, Central African Republic, Democratic Republic of the Congo, Ethiopia, Madagascar, South Africa and United Republic of Tanzania South Africa Bahamas, Barbados, Cuba, Grenada, Guadeloupe, Jamaica, Puerto Rico, Saba, Suriname, Trinidad and Tobago and Virgin Islands (USA) African or Caribbean countries in studies reviewed Assumed children and adults (unclear from article and appendices) Access to food, food prices, urban or rural residence, socioeconomic status and sex Age, interventions, sex and social environment Ethnicity, socioeconomic status and sex Children and adults Children Factors associated with diet or physical activity Population group studied (i) Food stability (variability over time in supply and access); (ii) access to food (mediating factors of affordability, allocation and power relations); (iii) food utilization (nutritional value in terms of dietary quality, diversity and quantity, social value, food preparation and safety); and (iv) food availability (production, distribution and exchange) Interventions included teaching, imitation and participation (i) Only findings on sex were summarized in the review; and (ii) socioeconomic status was derived from educational level, occupation and income Notes on factors Bull World Health Organ 2021;99:464–472I\| doi: http://dx.doi.org/10.2471/BLT.20.269308 Food insecurity Diet (continues. . .) (i) More studies were on physical activity than on diet; and (ii) additional outcomes of interest were body composition, tobacco smoking, metabolic syndrome and diabetes Self-sufficiency and subsistence skills for hunter–gatherer societies Diet and physical activity Diet and physical activity Notes on outcomes Outcomes Eleanor Turner-Moss et al. Diet and physical activity in Africa and the Caribbean Systematic reviews 472E 472F 2003–2015 2009–2016 Gyawali et al. (2019)27 Klingberg et al. (2019)28 2013–2018 Abdurahman et al. (2019)25 2000–2018 1999–2015 Pullar et al. (2018)24 Adom et al. (2019)26 Publication years of studies in review Review author (publication year) (. . .continued) 17 10 10 26 29 No. studies in review 17 5 10 26 15 No. studies in Africa or the Caribbean Africa Low- and middleincome countries Africa Ethiopia Low- and middleincome countries Review setting South Africa, Tunisia and Uganda Cameroon, South Africa and Uganda South Africa and Tunisia Ethiopia Burkina Faso, Burundi, Democratic Republic of the Congo, Egypt, Ethiopia, Ghana, Kenya, Malawi, Mali, Mozambique, Niger, Nigeria, Rwanda, Senegal, Uganda and United Republic of Tanzania African or Caribbean countries in studies reviewed Children Adults School children Interventions Interventions Antenatal care, age, household composition, interventions, parental socioeconomic status, region and urban or rural residence Interventions Interventions Children and adults Infants and young children Factors associated with diet or physical activity Population group studied Primarily school or after school programmes School-based interventions targeting diet, physical activity or weight Community-based interventions for the prevention of type 2 diabetes (i) Poverty reduction; and (ii) development interventions targeting economic development, social inequalities, community engagement, agriculture, fisheries, water or sanitization, or human rights NA Notes on factors Diet and physical activity Diet and physical activity Diet and physical activity (continues. . .) Additional outcomes of interest were glycated haemoglobin levels, fasting blood glucose levels, blood pressure and weight (i) More studies were on physical activity than on diet; and (ii) additional outcomes of interest were anthropometry, physical fitness and screen time An additional outcome of interest was weight Infant and young child feeding practices More studies were on diet than on physical activity Diet and physical activity Diet Notes on outcomes Outcomes Diet and physical activity in Africa and the Caribbean Systematic review of quantitative intervention studies Systematic review of quantitative intervention studies Systematic review of quantitative intervention studies Systematic review of quantitative studies Systematic review of quantitative intervention studies Type of review Systematic reviews Eleanor Turner-Moss et al. Bull World Health Organ 2021;99:464–472I\| doi: http://dx.doi.org/10.2471/BLT.20.269308 Publication years of studies in review Type of review No. studies in review No. studies in Africa or the Caribbean 1978–2014 2001–2014 1994–2015 Johnston et al. (2015)31 Osendarp et al. (2016)32 Allen et al. (2017)33 Bull World Health Organ 2021;99:464–472I\| doi: http://dx.doi.org/10.2471/BLT.20.269308 Systematic review of quantitative studies Systematic review of quantitative and qualitative studies Systematic review of quantitative studies 75 23 89 35 10 27 25–50% of studies in review from Africa or the Caribbean 18 5 Kristjansson et 1926–2004 Systematic al. (2007)29 review and meta-analysis of quantitative intervention studies Lee et al. 1989–2010 Systematic 62 16 (2013)30 review of quantitative studies Review author (publication year) (. . .continued) Low- and lowermiddleincome countries Low- and middleincome countries (rural areas) Low- and middleincome countries Cambodia, Ethiopia, Malawi, South Africa, United Republic of Tanzania and Zimbabwe Benin, Burkina Faso, Chad, Comoros, Côte d'Ivoire, Democratic Republic of the Congo, Egypt, Eritrea, Eswatini, Ethiopia, Ghana, Kenya, Malawi, Mali, Mauritania, Morocco, Nigeria, Senegal, Togo, United Republic of Tanzania, Zambia and Zimbabwe Children and adults Infants and young children Children and adults Age, sex and socioeconomic status Age, household composition, interventions, sex, social environment and socioeconomic status Age, interventions Socioeconomic status based on household or individual measures of income, wealth, assets, education, caste and occupation The hypothetical optimization of intake of locally available foods Agricultural interventions and practices Region and country NA Pregnant women Burkina Faso, Egypt, Ethiopia, Ghana, Jamaica, Kenya, Malawi, Morocco, Seychelles and South Africa Unspecified countries in sub-Saharan Africa, the Middle East, North Africa and Latin America Notes on factors Interventions were school meal programmes Low- and middleincome countries Factors associated with diet or physical activity Age, interventions, sex and socioeconomic status Population group studied School children (low socioeconomic status) African or Caribbean countries in studies reviewed Worldwide Jamaica and Kenya Review setting (i) More studies were on physical activity than on diet; and (ii) additional outcomes of interest were harmful use of alcohol and tobacco use Diet and physical activity (continues. . .) NA (i) Diet and nutritional outcomes; and (ii) time use related to agriculture Additional outcomes of interest were physical health, psychological health, behavioural variables and adverse events NA Notes on outcomes Diet Diet Diet Diet Outcomes Eleanor Turner-Moss et al. Diet and physical activity in Africa and the Caribbean Systematic reviews 472G Publication years of studies in review 2006–2016 2004–2015 1985–2017 1996–2017 2009–2017 2001–2016 Review author (publication year) Graziose et al. (2018)34 Kavle et al. (2018)35 Abrahale et al. (2019)36 Audate et al. (2019)37 Boneya et al. (2019)38 Leandro et al. (2019)39 (. . .continued) 472H Systematic review and meta-analysis of quantitative studies Systematic review of quantitative studies Systematic review of quantitative and qualitative intervention studies Systematic review of quantitative and qualitative studies Systematic review Systematic review of quantitative intervention studies Type of review 4 Infants and young children Population group studied Lowermiddleincome countries Ghana, Lesotho, Nigeria, Sudan and Uganda Worldwide Ethiopia, Senegal and Uganda HIV-infected adults receiving antiretroviral therapy Adolescents Children and Worldwide Benin, Botswana, adults (in Cameroon, Côte urban areas) d'Ivoire, Eswatini, Ghana, Kenya, Lesotho, Malawi, Mozambique, Namibia, Nigeria, South Africa, Uganda, United Republic of Tanzania, Zambia and Zimbabwe Children and adults Burkina Faso, Egypt, Pregnant Ethiopia, Kenya, Nigeria and lactating and Senegal women Burkina Faso, Kenya, Madagascar and Nigeria African or Caribbean countries in studies reviewed Worldwide Unspecified countries in Africa Low- and middleincome countries Low- and middleincome countries Review setting Barriers to and enablers of obesogenic behaviour Sex Street food availability and consumption Interventions Access to food, food prices, socioeconomic status and social environment Interventions Factors associated with diet or physical activity NA NA Urban agriculture Specific barriers and facilitating factors associated with maternal diet during pregnancy and the postpartum period NA Mass media and nutrition education interventions Notes on factors Diet and physical activity Diet Diet Diet Diet Diet Outcomes (continues. . .) Additional outcomes of interest were overweight and obesity An additional outcome of interest was food safety Additional outcomes of interest were food security, nutrition, social capital, health, sanitation, socioeconomic status, natural or physical environment, cultural connections and lifestyle Food insecurity Infants’ and young children’s feeding practices and related psychosocial factors, including the knowledge, attitudes and beliefs of caregivers NA Notes on outcomes Diet and physical activity in Africa and the Caribbean 11 6 36 101 17 162 8 23 441 5 No. studies in Africa or the Caribbean 18 No. studies in review Systematic reviews Eleanor Turner-Moss et al. Bull World Health Organ 2021;99:464–472I\| doi: http://dx.doi.org/10.2471/BLT.20.269308 2000–2017 Webb Girard et al. (2020)b,40 Systematic review of quantitative intervention studies Type of review 64 No. studies in review 23 No. studies in Africa or the Caribbean Low- and middleincome countries Review setting Egypt and unspecified countries in subSaharan Africa African or Caribbean countries in studies reviewed Infants and young children Population group studied Interventions Factors associated with diet or physical activity HIV: human immunodeficiency virus; NA: not applicable. a An additional 19 studies assessed the psychometric properties of assessment tools. These were not restricted to Africa and predominantly included high-income countries. b First published online in 2019. Publication years of studies in review Review author (publication year) (. . .continued) Interventions to shift complementary feeding behaviours Notes on factors Diet Outcomes Infant and young child feeding practices Notes on outcomes Eleanor Turner-Moss et al. Diet and physical activity in Africa and the Caribbean Systematic reviews Bull World Health Organ 2021;99:464–472I\| doi: http://dx.doi.org/10.2471/BLT.20.269308 472I
https://openalex.org/W3120682132	https://www.researchsquare.com/article/rs-29729/latest.pdf	English	null	Impact of Doxycycline on COVID-19 Patients with Risk Factors: DYNAMIC, a Multi-Centre, Randomised, Placebo-Controlled, Double-Blind Trial	Research Square (Research Square)	2,021	cc-by	8,656	Impact of Doxycycline on Risk Factors: DYNAMIC, a Randomised, Placebo-Con Alexandra Poinas (  alexandra.poinas@chu-nante Centre Hospitalier Universitaire de Nantes https:// David Boutoille Centre Hospitalier Universitaire de Nantes Florence Vrignaud Centre Hospitalier Universitaire de Nantes Jean-Michel Nguyen Centre Hospitalier Universitaire de Nantes Fabrice Bonnet Centre Hospitalier Universitaire de Bordeaux Cédric Rat Universite de Nantes Gilles Garcia Hopital Bicetre Anne Dompmartin Centre Hospitalier Universitaire de Caen Marie-Thérèse Leccia Centre Hospitalier Universitaire Grenoble Alpes Lionel Piroth Centre Hospitalier Universitaire de Dijon Eve Maubec Hopital Avicenne Pierre Gandon dermatologiste Alexandra Jobert Centre Hospitalier Universitaire de Nantes Soizic Boinet Centre Hospitalier Universitaire de Nantes Julie Cassecuel Centre Hospitalier Universitaire de Nantes Laurent Flet Impact of Doxycycline on COVID-19 Patients with Risk Factors: DYNAMIC, a Multi-Centre, Randomised, Placebo-Controlled, Double-Blind Trial Alexandra Poinas (  alexandra.poinas@chu-nantes.fr ) Centre Hospitalier Universitaire de Nantes https://orcid.org/0000-0003-4183-483X Alexandra Poinas (  alexandra.poinas@chu-nantes.fr ) Alexandra Poinas (  alexandra.poinas@chu-nantes.fr ) Centre Hospitalier Universitaire de Nantes https://orcid.org/0000-0003-418 Page 1/22 Page 1/22 Study protocol Keywords: COVID-19, doxycycline, patient with risk factors, outpatient, randomised controlled trial Posted Date: January 25th, 2021 DOI: https://doi.org/10.21203/rs.3.rs-29729/v4 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 2/22 Abstract Background: The DYNAMIC study is based on three properties of tetracyclines. (1) Tetracyclines are known to chelate zinc from matrix metalloproteinases. It is possible their chelating activity may help inhibit COVID-19 infection by limiting its ability to replicate in the host. (2) As seen with dengue virus, tetracyclines may also be able to inhibit the replication of positive polarity single-stranded RNA viruses, such as COVID-19. (3) Tetracyclines are also modulators of innate immunity (anti-inflammatory activity), a property that has been used to treat inflammatory skin diseases for many years. They could therefore participate in limiting the cytokine storm induced by COVID-19. Moreover, the lipophilic nature of tetracyclines and their strong pulmonary penetration could allow them to inhibit viral replication at this level. Among the tetracyclines, doxycycline has three advantages: its long safety history (side effects are uncommon with no notable risks), its short treatment duration and its low cost. Methods: The trial will involve 330 patients who are positive for SARS-CoV-2 infection and have one or more risk factors for worsening the disease. These patients will be included as outpatients for early treatment of illness. For logistical reasons and in order to be able to standardise the study as much as possible, recruitment will take place in 6 hospital departments covering the whole of France. For 14 days they will be given either 200mg of doxycycline a day or placebo. Our hypothesis is a considerable reduction in the number of patients hospitalised due to COVID-19 thanks to the treatment of doxycycline. Discussion: This study could have an impact on the overcrowding of patients with COVID-19 at the hospital which is one of the major world-wide problems of this pandemic. This treatment would therefore contribute to supporting the deconfinement strategy by blocking the viral infection early and reducing the infectious period. Trial Registration: On ClinicalTrials.gov, registration number NCT04371952, first published on 30 April 2020. Background In Wuhan, China, in the last quarter of 2019, a new coronavirus emerged, which is the third documented transmission from animals to humans[1]. Known as SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2) [2], it spread rapidly across China and many other countries [3,4]. On 11 February 2020, the World Health Organization (WHO) announced the name of the epidemic disease caused by SARS- CoV-2: COVID-19 for COronaVIrus Disease 2019 (https://www.who.int/dg/speeches/detail/who-director- general-s-remarks-at-the-media-briefing-on-2019-ncov-on-11-february-2020). The latest Chinese meta-analysis performed on 43 studies involving 3,600 patients provides an overview of the clinical characteristics, laboratory results, chest imaging results, disease severity and case fatality rate of COVID-19 patients [5]. The dominant clinical features of COVID-19, which can be present in a variety of combinations, are: fever, cough, asthenia, rhinorrhoea, headache, dysgeusia, dysosmia and Page 3/22 Page 3/22 diarrhoea [6,7]. The most frequently reported laboratory abnormalities are a decreased lymphocyte count, elevated C-reactive protein (CRP) and D-dimer levels and elevated lactate dehydrogenase [6]. diarrhoea [6,7]. The most frequently reported laboratory abnormalities are a decreased lymphocyte count, elevated C-reactive protein (CRP) and D-dimer levels and elevated lactate dehydrogenase [6]. The vast majority of patients with COVID-19 have a good prognosis, but there are critical situations and even deaths [8]. Most of these critically ill and deceased patients did not develop severe clinical manifestations in the early stages of the disease. Some of the patients only had a mild fever, cough or muscle pain, with their condition suddenly deteriorating in the latter stages of the disease. Acute Respiratory Distress Syndrome (ARDS) and multi-organ failure occur rapidly, resulting in death within a short time [9]. The pandemic has shown that some patients may have one or more risk factors for adverse outcomes: here are the relevant characteristics identified by the French High Council for Public Health (HCSP) : 70 years of age and older, cardiovascular history, chronic respiratory disease that may undergo decompensation due to viral infection, respiratory failure, poorly controlled and/or complicated diabetes, patients with chronic renal failure on dialysis and cancer patients under treatment [10]. We also added body mass index (BMI) higher than 30 (obesity) [11–13]. The massive release of cytokines, known as a cytokine storm, is considered one of the major causes of ARDS and multi-organ failure [14]. It plays an important role in the process of worsening the disease [15]. Background In addition, tetracyclines are modulators of innate immunity (anti-inflammatory activity), a property that has been used to treat inflammatory skin diseases for many years. These modulatory effects are apparent on several targets of innate immunity. They can decrease the expression of NFB and the release of inflammatory cytokines such as TNF-α, IL-1β and IL-6, and inhibit the formation of inflammatory granulomas and the release of free radicals, independently of their antibiotic mechanism [18]. Furthermore, a recent publication has shown that species-independent coronaviruses induce mast cell proliferation in the respiratory submucosa, which in turn produces inflammatory agents such as histamine and protease, in addition to inflammatory cytokines such as IL-1 and IL-33 [27]. Two other studies have shown that chemically modified tetracyclines can induce mast cell apoptosis and activation of protein kinase C, thereby lowering the levels of inflammatory agents [28]. These teams have suggested that tetracyclines can be used to treat inflammatory disorders, including those induced by coronaviruses [27,28]. It should also be noted that, because of their anti-inflammatory capabilities, tetracyclines have also been documented to have in vitro activity on other viral infections such as HIV, West Nile virus and viral encephalitis [29]. Doxycycline may therefore contribute to limiting the cytokine storm induced by SARS-CoV-2. Lastly, the lipophilic nature of tetracyclines and their strong pulmonary penetration could enable them to inhibit viral replication directly at the inflammatory site. The interest of doxycycline also lies in its long safety history (side effects are uncommon with no notable risks), its short treatment duration, which will be 14 days in our study (corresponding to the period during which SARS-CoV-2 can induce serious signs in the patient) and its low cost. As already mentioned, it is often elderly patients with comorbidities who worsen during the second week of the disease and have to be hospitalised for respiratory deterioration or even put on mechanical ventilation. The HCSP has determined the patients with risk factors [10] who have become the target population of our study in France. We have added obesity as a risk factor since it seems to be present in the literature for COVID-19-positive patients in intensive care units [30]. Background Effective suppression of the cytokine storm is therefore an important tool in preventing the deterioration of health and saving the lives of patients with SARS-CoV-2 infection. Controlling the cytokine storm in its early stages using treatments such as immunomodulators and anti-cytokines, as well as reducing infiltration of lung inflammatory cells, is one of the keys to reducing the mortality rate of patients with COVID-19 [16]. Different therapeutics are currently being evaluated in clinical trials, although no therapy has proven its effectiveness to date. Doxycycline is a second-generation semi-synthetic tetracycline that is chemically derived from first- generation tetracyclines, originally found in soil bacteria: actinomycetes [17]. Doxycycline was approved by the Food and Drug Administration (FDA) as an antibiotic in 1967 and to this day remains part of the antibiotic arsenal of most clinicians [17]. Following recent advances in knowledge of the anti- inflammatory effects of doxycycline on the skin, its use has been extended in dermatology where its anti- inflammatory property is now being used more than its antimicrobial property. In addition to rosacea, acne and hidradenitis suppurativa, doxycycline is used for other dermatological diseases, including bullous dermatoses, cutaneous sarcoidosis, Kaposi's sarcoma and neutrophil dermatoses (neutrophil chemotaxis) [18]. Based on these three properties of tetracyclines, we believe, like Sodhit and Etminan [19], Sargiacomo et al. [20] and Farouk and Salman [21], that doxycycline could be an effective treatment for COVID-19. The coronavirus family is known to bind to host matrix metalloproteinases (MMPs), particularly for viral survival. Tetracyclines are known to chelate zinc from MMPs [22]. It is therefore possible that their chelating activity may help inhibit SARS-CoV-2 infection by limiting its ability to replicate in the host [23]. Tetracyclines may also be able to inhibit the replication of positive polarity single-stranded RNA viruses, Page 4/22 Page 4/22 such as SARS-CoV-2. Indeed, the antiviral activity of doxycycline had already been reported against retroviruses 20 years ago, and a significant reduction in retrovirus titre has been observed after incubation of doxycycline-infected cells [24]. Other studies have shown that doxycycline inhibits the formation of dengue virus plaques by disrupting the conformational changes in the viral envelope that are necessary for virus entry [25]. They have also shown that at normal human body temperature and fever conditions, doxycycline significantly inhibited the serine protease of the virus and a concentration-dependent decrease in viral replication was observed [26]. required access to equipment and team facilities dedicated to clinical research. required access to equipment and team facilities dedicated to clinical research. required access to equipment and team facilities dedicated to clinical research. Our study will be a multi-centre, randomised, placebo-controlled study to determine the efficacy of doxycycline in this context, by measuring the decrease in the number of patients hospitalised compared to the control arm. Our study will be a multi-centre, randomised, placebo-controlled study to determine the efficacy of doxycycline in this context, by measuring the decrease in the number of patients hospitalised compared to the control arm. Objectives The primary objective is to evaluate the decrease in the number of patients hospitalised after at least 48 hours of experimental treatment or requiring hospitalisation for COVID-19. The 48-hour delay allows a more objective attribution of the clinical course to advanced treatment. The embedded second primary objective is to evaluate the decrease in the number of patients requiring mechanical ventilator support. The secondary objectives are to evaluate: - The decrease in the number of patients with a SARS-CoV-2-positive PCR test 7 days after the start of the experimental treatment. - The decrease in the number of patients with a SARS-CoV-2-positive PCR test 7 days after the start of the experimental treatment. - The decrease in the total length of hospital stay. - The decrease in the length of hospitalisation in the intensive care unit. - The decrease in the duration of mechanical ventilator support. - The decrease in the rate of deaths related to SARS-CoV-2 infection. - An assessment of the safety of doxycycline. The main outcome is an efficacy outcome corresponding to the percentage of patients hospitalised after at least 48 hours of experimental treatment. The outcome corresponding to the embedded second primary objective corresponds to the percentage of patients requiring mechanical ventilator support. These three outcomes are monitored in the two arms from 48 hours of treatment to 28 days corresponding to the end-of-study visit. For the secondary outcomes: Efficacy: These outcomes correspond to the report of the: Background Our hypothesis is to offer this treatment as soon as a patient with a risk factor is confirmed as being COVID-19-positive by a PCR test, with only a few symptoms, with no serious signs and before the onset of oxygen dependence, to reduce or even stop the cytokine storm, and thus the progression to a serious form of the disease, which will in turn avoid the need for hospitalisation. Page 5/22 The importance of finding medicines to keep patients at home and relieve hospital overcrowding is crucial during this pandemic. However, this outpatient trial will be carried out in hospital due to the The importance of finding medicines to keep patients at home and relieve hospital overcrowding is crucial during this pandemic. However, this outpatient trial will be carried out in hospital due to the Page 5/22 Page 5/22 required access to equipment and team facilities dedicated to clinical research. - Number of positive SARS-CoV-2 PCR tests at the inclusion visit and on D7. - Number of positive SARS-CoV-2 PCR tests at the inclusion visit and on D7 - Number of positive SARS-CoV-2 PCR tests at the inclusion visit and on D7. - Total length of hospital stay. - Total length of hospital stay. - Total length of hospital stay. Page 6/22 Page 6/22 - Total length of hospital stay in the intensive care unit. - Duration of mechanical ventilator support. - Duration of mechanical ventilator support. - Number of deaths related to SARS-CoV-2 infection. Safety: Report of the number of adverse events (AE) and serious adverse eve Safety: Report of the number of adverse events (AE) and serious adverse events (SAE) over 28 days. Apart from the first and the last secondary outcomes, the other outcomes will be recorded at 28 days. If the patient is hospitalised during the study period, this data will be recorded three months after the beginning of the treatment. For all of these secondary outcomes, percentages will be calculated. For all of the outcomes, the data will be reported as percentages with confidence intervals and a p value. Furthermore, the treatment effects, reported in the results and which meets all the outcomes except that of safety, will be estimated by risk ratio (plus confidence intervals and p value). We will focus on the reduction of hospitalisations (main objective), mortality and the duration of respiratory care. Moreover, we will look at the correlation between the number of risk factors and these outcomes in the results. Study design The DYNAMIC study is a superiority study of the efficacy of doxycycline. It is a prospective, phase III, randomised, stratified, double-blind, placebo-controlled, multi-centre national study to evaluate the use of doxycycline in patients with at least one risk factor for worsening COVID-19. As the study is double-blind, the study treatment and its placebo will be managed by the pharmacist of the coordinating centre, CHU Nantes. The oral anti-inflammatory dose of 200 mg of doxycycline or the matching placebo will be taken once a day in the evening one hour before going to bed, for 14 days. It should be noted that the placebo being used is marketed by COOPER; it is made of lactose and is very similar to the doxycycline tablet. The treatment (doxycycline or placebo) will be dispensed at the inclusion/randomisation visit. No dose adjustment is required. The treatment will end if the patient is hospitalised between day 0 (D0) and day 14 (D14). Six French university hospitals: CHU Nantes, AP-HP [Assistance Publique – Hôpitaux de Paris; Hôpital Avicenne], CHU Bordeaux, CHU Caen, CHU Dijon and CHU Grenoble, will include 330 patients to provide at least 280 analysable patients. The inclusion period may be three to six months depending on the recruitment rate. Advertising, approved by the ethics committee, will be displayed to improve the recruitment rate. The treatment period is 14 days per patient and the last patient visit will be at 28 days (+/- 2 days) from the start of treatment. Study population The study population is made up of male or female patients over 45 years of age, suspected of having COVID-19 with one or more risk factors for worsening the disease. They may be included in the study after a positive SARS-CoV-2 PCR test performed at the hospital. We chose 45 years as the minimum age in this study because this is the low end of the range for COVID-19-positive patients likely to be admitted to intensive care units [31]. In order to be eligible, as already mentioned, these patients, older than 45, must have one or more risk factors for worsening the disease as described by HCSP [10]: 70 years of age or older, cardiovascular history (stroke, coronary artery disease, complicated hypertension, cardiac surgery, NYHA class III or IV heart failure), poorly controlled insulin-dependent diabetes or complicated secondary diabetes, respiratory disease likely to undergo decompensation due to viral infection, patients with chronic renal failure on dialysis, and cancer patients under treatment. We have also added BMI (BMI>30) as a risk criterion, based on recent data from the literature [11–13,32]. Exclusion criteria are vulnerable populations and contraindication of the use of doxycycline. Box 1 presents all of the inclusion/exclusion criteria. Furthermore, patients cannot be included in any other interventional research, unless they are hospitalised, and provided there is no drug interaction with any other experimental treatment. Patients who are hospitalised will be considered as a failed patient for the primary objective. These patients will then be followed up only for the management of this SAE. If hospitalisation occurs between D2 and D7, the PCR will not be performed at D7 and the corresponding objective will not be performed for this patient. Randomisation Page 7/22 Randomisation will be stratified by centre and by severity factors (1 versus ≥2). It will be performed according to a 1:1 ratio and balanced by blocks; the block size was random. The random numbers will be generated by computer. The software used to collect the data from the electronic report form allocated the patients automatically. Randomisation was performed according to the Interactive Web Response System. Subjects are randomised into blocks as the allocation progresses, with a block being a sub-group of a predetermined size which contains a random allocation of patients. The software used for the randomisation is R version 9.4. An email alert will notify the investigator’s team and the pharmacist that a patient has been randomised. For the pharmacist, the allocation will be revealed. The randomisation key is known only to the biostatistician and the data managers, to make it impossible for the investigator to assign a particular treatment. It should be noted that the biostatistician who carries out the randomisation is different to the biostatistician who will perform the statistical analysis. Study schedule The plan for the study described in this section is presented in Figure 1 and the flowchart in Figure 2. The screening is performed by COVID-19 emergency facilities or the COVID-19 unit of the investigating centre. Page 8/22 A patient suspected of having COVID-19, with one or more characteristic clinical signs, and having one or more risk factors for worsening the disease is sent to hospital for a SARS-CoV-2 test. In the waiting room of the department specially dedicated to SARS-CoV-2 PCRs, a poster presenting DYNAMIC will be displayed. If the PCR is positive, either the patient who has seen the poster will contact the DYNAMIC team or the investigation team will contact the patient because of the PCR result. The hospital investigator explains the DYNAMIC study to the patient orally, checks the eligibility criteria, provides information on DYNAMIC and obtains the patient’s signed informed consent. If the version of the informed consent changes during the patient's participation in the study, they will be informed of this immediately during the telephone calls held during the trial, and the new consent will be signed at the next consultation. The Supplementary Material file contains the French informed consent form that patients sign prior to their inclusion in the trial (the updated protocol is version 1.8 as of 28 October 2020). Furthermore, blood samples for serology and immunological markers will be collected, stored and kept for Dynamic study if the patient signed in addition the biocollection informed consent. This second informed consent form states that the samples may be used for scientific research. This biocollection and its consent procedure have been registered with French ethics committee CPP (Comité de protection des Personnes) Ouest IV under number DC-2011-1399. After a clinical examination, patients will be randomised either in the doxycycline arm or in the placebo arm. The patients, the investigators and their teams will be blind and will not know the assigned treatment. As Karanicolas et al. pointed out in their article, the biostatisticians will also be blinded until the analyses are performed to reduce the study bias [33]. On D3 and D14, a telephone call will be made by the investigator or their team. A clinical assessment of the patient will be carried out using the questionnaire shown in Figure 3. Study schedule Furthermore, the patient will answer daily this questionnaire and if their answers indicate warning signs they will call the investigators or the emergency department. If necessary, a face-to-face consultation will be conducted immediately after this call to accurately assess the extent to which the disease has worsened. On D7, a face-to-face follow-up visit will be performed. After a clinical examination, a SARS-CoV-2 qualitative PCR test will be carried out. On D28, the face-to-face end-of-study visit will be performed. A blood sample will be kept for serology and other immunological markers. The patient will return any unused therapeutic units and the empty blister packs. An early end-of-study visit will be carried out corresponding to the end-of-study visit (D28 visit), with the exception of serology if the patient leaves the study before 14 days. For included patients hospitalised before 28 days, the experimental treatment is stopped as soon as they are hospitalised and they are considered a failure. Clinical data related to their hospitalisation will be reported in the CRF. For patients hospitalised after 28 days and up to 90 days post-treatment, they will be followed up until discharged. This follow-up will include the collection of vigilance data and various hospitalisation data (date of end Page 9/22 Page 9/22 of hospitalisation, length of hospitalisation in an intensive care unit, duration of mechanical respiratory support, possible death). During the trial, any drug judged necessary for the patient’s well-being, which should not interfere with the evaluation of the test drug, may be administered at the investigator’s discretion. Absorption of doxycycline is impaired by antacids containing aluminium and calcium, as well as magnesium, bismuth subsalicylate and iron- and zinc-containing preparations. The study drug should therefore be taken at a different time to antacids. Other prohibited treatments are those prohibited by the Summary of Product Characteristics of doxycycline and are listed in the exclusion criteria (see Box 1). However, in the absence of a change to the therapeutic management of a patient arriving at hospital according to their treatment arm, a blinding procedure is not required in this protocol. Treatment will be stopped if hospitalisation occurs. Statistical methods The embedded testing strategy will be used to successively test: The embedded testing strategy will be used to successively test: - The decrease in the percentage of patients requiring hospitalisation related to SARS-CoV-2 infection after at least 48 hours of treatment. - The decrease in the percentage of patients requiring hospitalisation related to SARS-CoV-2 infection after at least 48 hours of treatment. - A decrease in the percentage of patients requiring mechanical ventilator support if the first null hypothesis is rejected. - A decrease in the percentage of patients requiring mechanical ventilator support if the first null hypothesis is rejected. An intermediate futility analysis will be carried out after 100 patients have been evaluated, with the objective of stopping the clinical trial if there is no probability that the null hypothesis will be rejected at the end of the full study. The sole response to this analysis is whether or not to stop the trial because of a lack of efficacy. The Peto method will be used with an alpha risk of 1 per 1,000 at the intermediate analysis and of 5% at the final analysis [34]. As already quoted, the biostatistician will be part of the blinded team. They will give the results of this intermediate analysis to the Data and Safety Monitoring Committee (DSMC), which may or may not unblind depending on the results. The analysis will be stratified by centre and number of severity factors (1 versus ≥ 2). The severity factors are ≥ 70 years, BMI > 30, cardiovascular history (stroke, coronary artery disease, complicated hypertension, cardiac surgery, NYHA class III or IV heart failure), poorly controlled insulin-dependent diabetes or complicated secondary diabetes, respiratory disease likely to undergo decompensation due to viral infection, patients with chronic renal failure on dialysis, and cancer patients under treatment at the time of inclusion. A modified intention-to-treat analysis will be applied. It will include all eligible randomised patients who provided their consent to participate in the trial. It will exclude patients who withdrew their consent prior to the assessment of the first primary endpoint, randomised patients who were incorrectly included (inclusion or exclusion criteria not met) and patients who did not receive any dose of treatment. Page 10/22 A per-protocol analysis will also be performed, including all patients evaluated in their treatment group who received more than 75% of the total treatment dose. Sample size It is estimated that 25% of the eligible patients in the study will require hospitalisation. In the first wave, hospitalisation rates ranged from 18.1% [38] to 31.4% [31], we chose the mean. It is hypothesised that with doxycycline treatment, this rate would decrease to 12%. Indeed, the article concerning the effect of doxycycline on dengue patients: "Dengue Patients Treated With Doxycycline Showed Lower Mortality Associated with a Reduction in IL-6 and TNF Levels" [39], shows that the group on doxycycline had a lower mortality rate than those in the untreated group (11.2% [13/116] vs 20.9% [24/115], respectively, p=0.05). In addition, doxycycline administration resulted in a significant drop in IL-6 and TNF. These results therefore confirm our hypothesis of reducing the hospitalisation rate to 12%. An embedded testing strategy is used for the primary objective, and as the report of the European Medicine Agency stated, no adjustment is needed for multiplicity [40]. For the second embedded hypothesis, it is estimated that 23% (https://www.data.gouv.fr/fr/datasets/donnees-hospitalieres-relatives-a-lepidemie-de-covid-19/) of hospitalised patients are admitted to intensive care units. With the recruitment level we have, we will have a power of 80% to show a decrease from 23% to 10%. Under these hypotheses, with a 5% alpha risk in a bilateral situation and a power of 80%, we would need to evaluate a total of 280 patients in both arms. If the first hypothesis is rejected, the power of the test of the second hypothesis would then be 83.5%. Three hundred and thirty (330) patients will be included to compensate for study dropouts and non-assessable patients. Statistical methods For the main outcome (percentage of patients), we will use a Mantel-Haenszel test (stratified on centres and on the number of severity factors) to compare the rate of success (success means no hospitalisation). If the null hypothesis is rejected, we will compare the rate of mechanical ventilator between the two arms. [35,36]. For the quantitative secondary criteria, a linear model stratified on hospital centres and on the number of risk factors will be developed [37] if the minimum number of recruitments per centre allows, otherwise the model will be stratified across centres and adjusted for the number of risk factors. The statistical software used will be SAS and R, version 3.8 or later. The significance thresholds will be set at 5% in a bilateral situation. Adverse event management Page 11/22 For doxycycline, the main AEs expected are skin disorders (photosensitivity reaction, rash), immune system disorders (urticaria, rash, pruritus, angioedema, anaphylactic reaction) or digestive disorders (nausea, epigastralgia, diarrhoea, anorexia, glossitis, enterocolitis, and anal or genital candidiasis). There have been no reports of overdose. AEs reported for other tetracyclines following renal impairment (hepatic toxicity, hyperazotemia, hyperphosphataemia, acidosis), are unlikely to occur with doxycycline Page 11/22 due to the lack of a change in blood levels in relation to the functional value of the kidney. For the placebo, the main expected effects are digestive disorders (excess gas, feeling of abdominal bloating, abdominal cramps and pain, and diarrhoea). As regards the pathology, patients with an uncomplicated viral infection of the upper respiratory tract may have non-specific symptoms such as fever, fatigue, cough (with or without discharge), anorexia, malaise, muscle aches, sore throat, dyspnoea, nasal congestion or headache. Rarely, patients may also experience diarrhoea, nausea and vomiting [6,41], and loss of taste or smell [42]. The disease can worsen to pneumonia, acute respiratory distress syndrome [43], sepsis and septic shock [44] . All SAEs, whether expected or unexpected, require the completion of an SAE report. The investigator must ensure that the information entered in this report is accurate and clear. The SAE should be reported to the sponsor immediately (within 24 hours of being highlighted by the investigator). After receiving an unexpected SAE report, the sponsor will notify the authorities. Furthermore, a DSMC has been set up; it is a consultative committee responsible for reviewing the safety of a study on behalf of the study sponsor and coordinator/principal investigator. Members of the committee who are competent in the field of clinical trials (pathology methodology and pharmacovigilance) are not involved in the study. The DSMC is a referral point for pharmacovigilance if an SUSAR (Suspected Unexpected Serious Adverse Reaction) or SAE poses particular analytical difficulty, or if a doubt arises concerning the risk/benefit of the study. It will also make a decision on the outcome of the futility analysis. If the study is ended early following a decision of the DSMC or the study sponsor, the regulatory authorities and the ethics committee will be informed by post within a maximum of 15 days. In any event, written confirmation will be sent to the coordinating investigator of the study (specifying the reasons for early termination) and to the principal investigator of each centre, if applicable. Adverse event management All patients involved in the study will be informed and will be required to attend their early discharge visit. Ethical, regulatory and dissemination aspects The clinical study will be conducted in accordance with the relevant versions of the French Public Health Code, national and international good clinical practice guidelines, and the Declaration of Helsinki. In accordance with French law, the study protocol was submitted to French regulatory authority the ANSM (Agence Nationale de Sécurité du Médicament et des Produits de Santé). This clinical study was submitted to and approved by the ethics committee of Boulogne-Billancourt (CPP Ile de France VIII) on 13 May 2020. Requests for substantial modifications should be addressed by the sponsor for approval or notification to the ANSM and/or the ethics committee concerned in compliance with Law 2004-806 of 9 August 2004 and its implementing decrees. The amended protocol will be a dated and updated version. If necessary, the information form and consent form will be amended. All of the submissions/declarations were made by the Sponsor Department at CHU Nantes, which manages the quality of the data collected. The data collected during the study will be processed electronically in accordance with the requirements of French Data Protection Authority the CNIL (in Page 12/22 Page 12/22 compliance with French Reference Methodology MR001). Data will only be shared between the investigators. However, the datasets analysed during this study will be available from the corresponding author upon reasonable request. An electronic Case Report Form (eCRF) shall be drawn up for each included patient. Subjects will be identified by the first letter of their family name, the first letter of their first name, the centre number and the inclusion number. This code should be the only information featured on the eCRF enabling a retrospective link to the patient. The investigator, or their team, shall also encode the patient data on any documents that may be in their possession (imaging, biology test reports, etc.) attached to the eCRF. At the end of the study, database reconciliation is carried out between the CRF database and the safety database. This reconciliation is performed before database locking. Similarly, an annual reconciliation is carried out when updating the Annual Safety Report. As required, the sponsor has provided an insurance policy to cover the financial consequences of its civil liability in accordance with the regulations. This protocol was created thanks to a Scientific Committee. The Scientific Committee is coordinated by Professor B Dréno and its members include external and internal experts in COVID-19, experts in clinical trials and a methodologist. Ethical, regulatory and dissemination aspects An inspection or audit may take place as part of this study, performed by the sponsor and/or the regulatory authorities. Inspectors will check the documents, logistics, records and any other resources the authorities consider to be associated with the clinical trial and that may be located at the trial site itself. The trial results will be published in international, medical and scientific journals and presented at national and international conferences. The investigators will follow the rules and guidelines of the International Committee of Medical Journal Editors [45]. In practice, the Scientific Committee will be among the authors of the publication, as will the investigators who included the patients in the trial. Discussion The treatment is inexpensive at around 3 euros per 15 tablets, and we hope to avoid the transfer of patients to other intensive care units, which are very expensive for the community. Recently, in Italy, Bonzano et al. reported the benefit of doxycycline in patients with COVID-19 [46]. Their retrospective study, albeit on only six patients, suggests promising results that our randomised clinical trial will be able to strengthen by removing the bias of a retrospective study on a few cases. Furthermore, Yates et al. have presented a series of four high-risk, symptomatic, COVID-19-positive patients with known pulmonary disease, treated with doxycycline with subsequent rapid clinical improvement. Doxycycline is an attractive candidate. If the results are as positive as we hope, this treatment could be given to the entire population, potentially for prevention of the disease. Trial status The updated protocol is version 1.8 as of 28 October 2020. The first patient inclusion is expected by 15 January 2021. With an inclusion period of six months, the last patient may be included on 15 June 2021 and the study will end after their follow-up visits one month later. Discussion The COVID-19 pandemic is affecting all continents. Saturation of the hospital care system is feared in many countries, and the first and the second lockdown of the French population and practically the whole world was decided in an attempt to avoid hospital overcrowding. Researchers are looking for a life-saving drug. To date, 3,904 clinical studies have been published at https://clinicaltrials.gov/ (viewed on 13 November 2020) since the beginning of the pandemic. Of these studies, 2,217 are intervention research. However, there are only 113 studies that deal with outpatient clinical research. The great strength of our study is that it tests doxycycline on at-risk patients on an outpatient basis before the cytokine wave. In its "Clinical management of severe acute respiratory infection (SARI) when COVID-19 disease is suspected" guidance document, WHO proposes that elderly patients and those with comorbidities such Page 13/22 as cardiovascular disease and type II diabetes should be hospitalised even if they do not show signs of severity. By avoiding hospitalisation, the impact of our treatment could be to relieve congestion by allowing healthcare personnel to focus on serious cases and freeing up resuscitation places. Moreover, this will prevent the need to cancel at least 20% of their usual activity in surgery and medicine, which is currently the case in French hospitals in autumn 2020. as cardiovascular disease and type II diabetes should be hospitalised even if they do not show signs of severity. By avoiding hospitalisation, the impact of our treatment could be to relieve congestion by allowing healthcare personnel to focus on serious cases and freeing up resuscitation places. Moreover, this will prevent the need to cancel at least 20% of their usual activity in surgery and medicine, which is currently the case in French hospitals in autumn 2020. This experimental treatment could support the deconfinement strategy by blocking the viral infection early in cases of infection, by reducing the infectious period, and by making home care possible for infected patients with a treatment whose tolerance has been proven for nearly sixty years, even in elderly patients. The medico-economic interest is also very important. The treatment is inexpensive at around 3 euros per 15 tablets, and we hope to avoid the transfer of patients to other intensive care units, which are very expensive for the community. The medico-economic interest is also very important. List Of Abbreviations Page 14/22 List Of Abbreviations AE Adverse event ANSM Agence Nationale de Sécurité du Médicament et des Produits de Santé [French National Agency for the Safety of Medicines and Health Products] AP-HP Assistance Publique – Hôpitaux de Paris [university hospital trust of Paris and its suburbs] ARDS Acute Respiratory Distress Syndrome Adverse event ANSM Agence Nationale de Sécurité du Médicament et des Produits de Santé [French National Agency for the Safety of Medicines and Health Products] Assistance Publique – Hôpitaux de Paris [university hospital trust of Paris AP-HP Assistance Publique – Hôpitaux de Paris [university hospital trust of Paris and its suburbs] Agence régionale de Santé [regional health authority] Body mass index Commission Nationale de l’Informatique et des Libertés [French data Coronavirus disease year 2019 Comité de protection des personnes (French ethics committee) Case Report Form C-reactive protein Day Data and Safety Monitoring Committee Electronic Case Report Form Food and Drug Administration Haut Conseil de la Santé Publique [French High Council for Public Health] Matrix metalloproteinases Serious adverse event Oxygen saturation Severe Acute Respiratory Syndrome Coronavirus 2 World Health Organisation ent to Participate th t d ill b i l d itt i f ti b t thi t i l d ill ARS Agence régionale de Santé [regional health authority] BMI Body mass index CNIL Commission Nationale de l’Informatique et des Libertés [French data protection authority] COVID-19 Coronavirus disease year 2019 CPP Comité de protection des personnes (French ethics committee) CRF Case Report Form CRP C-reactive protein D Day DSMC Data and Safety Monitoring Committee eCRF Electronic Case Report Form FDA Food and Drug Administration HCSP Haut Conseil de la Santé Publique [French High Council for Public Health] MMP Matrix metalloproteinases SAE Serious adverse event Sa O2 Oxygen saturation SARS-CoV2 Severe Acute Respiratory Syndrome Coronavirus 2 WHO World Health Organisation Declaration Ethics Approval and Consent to Participate All patients participating in the study will be given oral and written information about this trial and will sign the informed consent form. An independent ethics committee, the Comité de Protection des Personnes of Boulogne-Billancourt, CPP Ile de France VIII, issued a favourable opinion for this clinical trial and gave its informed consent on 28 April 2020. Page 15/22 Page 15/22 Consent for publication The Supplementary Material file contains the French informed consent form, and information materials are available from the corresponding author on request. Funding The “Doxycycline Versus Placebo in COVID-19-Positive Patients Without Hospitalisation Criteria: Prospective, Multi-centre, Randomised, Double-blind Study” was supported by Laboratoires Pierre Fabre, Bailleul, the Endowment Fund of CHU Nantes, Nantes Métropole and a Foundation of a French Bank (Fondation Grand Ouest de la Banque Populaire). The pharmaceutical company Laboratoires Pierre Fabre, Bailleul played no part in the study’s design, the collection, management, analysis, or interpretation of data, writing the report, or the decision to submit the report for publication. Availability of data and materials Any data required to support the protocol can be supplied on request. The data from the completed trial will not be shared and will only be transmitted to the sponsor. Data collected during the test may be processed electronically in accordance with the requirements of the CNIL (compliance with French Reference Methodology MR001). The authors declare that they have no competing interests. The authors declare that they have no competing interests. References 1. Lu H, Stratton CW, Tang Y. Outbreak of pneumonia of unknown etiology in Wuhan, China: The mystery and the miracle. J Med Virol. 2020;92:401–2. 2. Coronaviridae Study Group of the International Committee on Taxonomy of Viruses. The species Severe acute respiratory syndrome-related coronavirus: classifying 2019-nCoV and naming it SARS- CoV-2. Nat Microbiol. 2020;5:536–44. 3. Li Q, Guan X, Wu P, Wang X, Zhou L, Tong Y, et al. Early Transmission Dynamics in Wuhan, China, of Novel Coronavirus–Infected Pneumonia. N Engl J Med. 2020;382:1199–207. 4. Wang C, Horby PW, Hayden FG, Gao GF. A novel coronavirus outbreak of global health concern. The Lancet. 2020;395:470–3. 5. Fu L, Wang B, Yuan T, Chen X, Ao Y, Fitzpatrick T, et al. Clinical characteristics of coronavirus disease 2019 (COVID-19) in China: a systematic review and meta-analysis. J Infect. 2020; 80 (6): 656-665 6. Chen N, Zhou M, Dong X, Qu J, Gong F, Han Y, et al. Epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in Wuhan, China: a descriptive study. The Lancet. 2020;395:507–13. 7. Guan W, Ni Z, Hu Y, Liang W, Ou C, He J, et al. Clinical Characteristics of Coronavirus Disease 2019 in China. New England Journal of Medicine. 2020;382:1708–20. 7. Guan W, Ni Z, Hu Y, Liang W, Ou C, He J, et al. Clinical Characteristics of Coronavirus Disease 2019 in China. New England Journal of Medicine. 2020;382:1708–20. 8. Lai C-C, Shih T-P, Ko W-C, Tang H-J, Hsueh P-R. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and coronavirus disease-2019 (COVID-19): The epidemic and the challenges. Int J Antimicrob Agents. 2020;55:105924. 8. Lai C-C, Shih T-P, Ko W-C, Tang H-J, Hsueh P-R. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and coronavirus disease-2019 (COVID-19): The epidemic and the challenges. Int J Antimicrob Agents. 2020;55:105924. 9. Guo Y-R, Cao Q-D, Hong Z-S, Tan Y-Y, Chen S-D, Jin H-J, et al. The origin, transmission and clinical therapies on coronavirus disease 2019 (COVID-19) outbreak - an update on the status. Mil Med Res. 2020;7:11. 9. Guo Y-R, Cao Q-D, Hong Z-S, Tan Y-Y, Chen S-D, Jin H-J, et al. The origin, transmission and clinical therapies on coronavirus disease 2019 (COVID-19) outbreak - an update on the status. Mil Med Res. 2020;7:11. 10. HCSP. Avis provisoire Recommandations relatives à la prévention et à la prise en charge du COVID-19 chez les patients à risque de formes sévères https://www.hcsp.fr/explore.cgi/avisrapportsdomaine? Authors’ contribution AP; FV; AK; BD and JMN wrote the manuscript, DB; FB; CR; GG; AD; MTL; LP; EM; PG; AJ; SB; JC and LF; assisted with drafting the manuscript. BD; DB; FB; GG; AP; AK; FV and JMN designed the trial. BD; AP; AK; FV; DB; CR; SB; LF and AJ wrote the protocol and/or the file for the experimental drug and assisted with drafting the manuscript. SB coordinated the protocol’s submission and follow-up with (1) the ethics committee and (2) the regulatory authorities and coordinated the trial. JMN wrote the methodological/statistical analyses in the protocol. BD; DB; FB; MTL; LP; EM and JC participated in patient enrolment and follow-up. AJ assisted with the trial’s pharmacovigilance. LF wrote the experimental drug file and coordinates the provision of treatment in the centres. All authors have read and approved the final manuscript. Acknowledgement: In March 2020, Pierre Gandon submitted an opinion to the Nouvelle-Aquitaine French regional public health authority (Agence Régionale de Santé - ARS) regarding the potential interest of doxycycline with respect to COVID-19, having observed that acne sufferers on doxycycline escaped seasonal viral infections. Page 16/22 Page 16/22 References clefr=775 (2020) Accessed 19 Apr 2020 11. Kalligeros M, Shehadeh F, Mylona EK, Benitez G, Beckwith CG, Chan PA, et al. Association of Obesity with Disease Severity among Patients with COVID-19. Obesity (Silver Spring). 2020;28 (7):1200–4. 11. Kalligeros M, Shehadeh F, Mylona EK, Benitez G, Beckwith CG, Chan PA, et al. Association of Obesity with Disease Severity among Patients with COVID-19. Obesity (Silver Spring). 2020;28 (7):1200–4. 12. Richardson S, Hirsch JS, Narasimhan M, Crawford JM, McGinn T, Davidson KW, et al. Presenting Characteristics, Comorbidities, and Outcomes Among 5700 Patients Hospitalized With COVID-19 in the New York City Area. JAMA. 2020;323 (20):2052–9. 12. Richardson S, Hirsch JS, Narasimhan M, Crawford JM, McGinn T, Davidson KW, et al. Presenting Characteristics, Comorbidities, and Outcomes Among 5700 Patients Hospitalized With COVID-19 in the New York City Area. JAMA. 2020;323 (20):2052–9. 13. Zheng KI, Gao F, Wang X-B, Sun Q-F, Pan K-H, Wang T-Y, et al. Obesity as a risk factor for greater severity of COVID-19 in patients with metabolic associated fatty liver disease. Metabolism. 2020;108:154244. 13. Zheng KI, Gao F, Wang X-B, Sun Q-F, Pan K-H, Wang T-Y, et al. Obesity as a risk factor for greater severity of COVID-19 in patients with metabolic associated fatty liver disease. Metabolism. 2020;108:154244. 14. Chousterman BG, Swirski FK, Weber GF. Cytokine storm and sepsis disease pathogenesis. Semin Immunopathol. 2017;39:517–28. 14. Chousterman BG, Swirski FK, Weber GF. Cytokine storm and sepsis disease pathogenesis. Semin Immunopathol. 2017;39:517–28. 14. Chousterman BG, Swirski FK, Weber GF. Cytokine storm and sepsis disease pathogenesis. Semin Immunopathol. 2017;39:517–28. Page 17/22 Page 17/22 15. Shimabukuro-Vornhagen A, Gödel P, Subklewe M, Stemmler HJ, Schlößer HA, Schlaak M, et al. Cytokine release syndrome. J Immunother Cancer. 2018;6:56. 16. Ye Q, Wang B, Mao J. The pathogenesis and treatment of the `Cytokine Storm’ in COVID-19. J Infect. 2020;80 (6):607–13. 17. Nelson ML, Levy SB. The history of the tetracyclines: The history of the tetracyclines. Annals of the New York Academy of Sciences. 2011;1241:17–32. 18. Henehan M, Montuno M, De Benedetto A. Doxycycline as an anti-inflammatory agent: updates in dermatology. J Eur Acad Dermatol Venereol. 2017;31:1800–8. 19. Sodhi M, Etminan M. Therapeutic Potential for Tetracyclines in the Treatment of COVID-19. Pharmacotherapy. 2020;40(5):487–8. 20. Sargiacomo C, Sotgia F, Lisanti MP. COVID-19 and chronological aging: senolytics and other anti- aging drugs for the treatment or prevention of corona virus infection? Aging (Albany NY). 2020;12 (8):6511–8. 21. Farouk A, Salman S. References Dapsone and doxycycline could be potential treatment modalities for COVID-19. Medical Hypotheses. 2020;140:109768. 22. Aggarwal HK, Jain D, Talapatra P, Yadav RK, Gupta T, Kathuria KL. Evaluation of role of doxycycline (a matrix metalloproteinase inhibitor) on renal functions in patients of diabetic nephropathy. Renal Failure. 2010;32:941–6. 23. Phillips JM, Gallagher T, Weiss SR. Neurovirulent Murine Coronavirus JHM.SD Uses Cellular Zinc Metalloproteases for Virus Entry and Cell-Cell Fusion. J Virol. 2017;91. 24. Sturtz FG. Antimurine retroviral effect of doxycycline. Methods Find Exp Clin Pharmacol. 1998;20:643–7. 25. Yang J-M, Chen Y-F, Tu Y-Y, Yen K-R, Yang Y-L. Combinatorial computational approaches to identify tetracycline derivatives as flavivirus inhibitors. PLoS ONE. 2007;2:e428. 26. Rothan HA, Mohamed Z, Paydar M, Rahman NA, Yusof R. Inhibitory effect of doxycycline against dengue virus replication in vitro. Arch Virol. 2014;159:711–8. 27. Kristas SK, Ronconi G, Caraffa A, Gallenga C, Ross R, Conti P. Mast cells contribute to coronavirus- induced inflammation: new anti-inflammatory strategy. J Biol Regul Homeost Agents. 2020;34 (1):9– 14. 28. Sandler C, Nurmi K, Lindstedt KA, Sorsa T, Golub LM, Kovanen PT, et al. Chemically modified tetracyclines induce apoptosis in cultured mast cells. Int Immunopharmacol. 2005;5:1611–21. 29. Dutta K, Basu A. Use of minocycline in viral infections. Indian J Med Res. 2011;133 (5):467–70. 29. Dutta K, Basu A. Use of minocycline in viral infections. Indian J Med R 30. Petrakis D, Margină D, Tsarouhas K, Tekos F, Stan M, Nikitovic D, et al. Obesity ‑a risk factor for increased COVID‑19 prevalence, severity and lethality (Review). Mol Med Rep. 2020; 31. CDC COVID-19 Response Team. Severe Outcomes Among Patients with Coronavirus Disease 2019 (COVID-19) — United States, February 12–March 16, 2020. MMWR Morb Mortal Wkly Rep. 2020;69 (12):343–6. Page 18/22 Page 18/22 32. Caussy C, Wallet F, Laville M, Disse E. Obesity is associated with severe forms of COVID-19. Obesity (Silver Spring). 2020; 28 (7):1175 33. Karanicolas PJ, Farrokhyar F, Bhandari M. Practical tips for surgical research: blinding: who, what, when, why, how? Can J Surg. 2010;53 (5) :345–8. 34. Peto R, Pike MC, Armitage P, Breslow NE, Cox DR, Howard SV, et al. Design and analysis of randomized clinical trials requiring prolonged observation of each patient. I. Introduction and design. Br J Cancer. 1976;34:585–612. 35. Mantel N, Haenszel W. Statistical aspects of the analysis of data from retrospective studies of disease. J Natl Cancer Inst. 1959;22 (4):719–48. 36. Mantel N. References Chi-Square Tests with One Degree of Freedom; Extensions of the Mantel-Haenszel Procedure. Journal of the American Statistical Association. 1963;58 (303):690–700. 37. Nelder JA, Wedderburn RWM. Generalized Linear Models. Journal of the Royal Statistical Society Series A (General). 1972;135:370. 38. COVID-19 National Incident Room Surveillance Team. COVID-19, Australia: Epidemiology Report 9 (Reporting week to 23:59 AEDT 29 March 2020). Commun Dis Intell (2018). 2020;44. doi:10.33321/cdi.2020.44.29. 38. COVID-19 National Incident Room Surveillance Team. COVID-19, Australia: Epidemiology Report 9 (Reporting week to 23:59 AEDT 29 March 2020). Commun Dis Intell (2018). 2020;44. doi:10.33321/cdi.2020.44.29. 39. Fredeking T, Zavala-Castro J, Gonzalez-Martinez P, Moguel-Rodríguez W, Sanchez E, Foster M, et al. Dengue Patients Treated with Doxycycline Showed Lower Mortality Associated to a Reduction in IL-6 and TNF Levels. PRI. 2015;10:51–8. 39. Fredeking T, Zavala-Castro J, Gonzalez-Martinez P, Moguel-Rodríguez W, Sanchez E, Foster M, et al. Dengue Patients Treated with Doxycycline Showed Lower Mortality Associated to a Reduction in IL-6 and TNF Levels. PRI. 2015;10:51–8. 40. EMA. COMMITTEE FOR PROPRIETARY MEDICINAL PRODUCTS (CPMP) - POINTS TO CONSIDER ON MULTIPLICITY ISSUES IN CLINICAL TRIALS. Available from: https://www.ema.europa.eu/en/documents/scientific-guideline/points-consider-multiplicity-issues- clinical-trials_en.pdf (2002) Accessed 5 Nov 2020 40. EMA. COMMITTEE FOR PROPRIETARY MEDICINAL PRODUCTS (CPMP) - POINTS TO CONSIDER ON MULTIPLICITY ISSUES IN CLINICAL TRIALS. Available from: https://www.ema.europa.eu/en/documents/scientific-guideline/points-consider-multiplicity-issues- clinical-trials_en.pdf (2002) Accessed 5 Nov 2020 https://www.ema.europa.eu/en/documents/scientific-guideline/points-consider-multiplicity-issues- clinical-trials_en.pdf (2002) Accessed 5 Nov 2020 41. Huang C, Wang Y, Li X, Ren L, Zhao J, Hu Y, et al. Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China. Lancet. 2020;395:497–506. 41. Huang C, Wang Y, Li X, Ren L, Zhao J, Hu Y, et al. Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China. Lancet. 2020;395:497–506. 42. Lechien JR, Chiesa-Estomba CM, De Siati DR, Horoi M, Le Bon SD, Rodriguez A, et al. Olfactory and gustatory dysfunctions as a clinical presentation of mild-to-moderate forms of the coronavirus disease (COVID-19): a multicenter European study. Eur Arch Otorhinolaryngol. 2020; 42. Lechien JR, Chiesa-Estomba CM, De Siati DR, Horoi M, Le Bon SD, Rodriguez A, et al. Olfactory and gustatory dysfunctions as a clinical presentation of mild-to-moderate forms of the coronavirus disease (COVID-19): a multicenter European study. Eur Arch Otorhinolaryngol. 2020; 43. ARDS Definition Task Force, Ranieri VM, Rubenfeld GD, Thompson BT, Ferguson ND, Caldwell E, et al. Acute respiratory distress syndrome: the Berlin Definition. JAMA. 2012;307 (23):2526–33. 43. Figures Figures g References ARDS Definition Task Force, Ranieri VM, Rubenfeld GD, Thompson BT, Ferguson ND, Caldwell E, et al. Acute respiratory distress syndrome: the Berlin Definition. JAMA. 2012;307 (23):2526–33. 44. Rhodes A, Evans LE, Alhazzani W, Levy MM, Antonelli M, Ferrer R, et al. Surviving Sepsis Campaign: International Guidelines for Management of Sepsis and Septic Shock: 2016. Intensive Care Med. 2017;43:304–77. 45. International Committee of Medical Journal Editors (ICMJE). International Committee of Medical Journal Editors (ICMJE): Uniform Requirements for Manuscripts Submitted to Biomedical Journals: writing and editing for biomedical publication. Haematologica. 2004;89:264. Page 19/22 46. Bonzano C, Borroni D, Lancia A, Bonzano E. Doxycycline: From Ocular Rosacea to COVID-19 Anosmia. New Insight Into the Coronavirus Outbreak. Front Med. 2020;7:200. Figures Figure 1 Study Schedule Page 20/22 Figure 3 Patient clinical re-evaluation questionnaire (D3 and D14) and daily patient self-reporting questionnaire S l Fil Patient clinical re-evaluation questionnaire (D3 and D14) and daily patient self-reporting questionnaire Figure 2 Flowchart Study Page 21/22 Supplementary Files This is a list of supplementary files associated with this preprint. Click to d Page 22/22 Box1revised.docx SPIRITDYNAMICrevised.doc s1.docx EthicCommiteeapprobationtranslatedversion.pdf EthicCommiteeapprobationFrenchversion.pdf Informedconsent.pdf Fundingdocumentation.doc Box1revised.docx Box1revised.docx SPIRITDYNAMICrevised.doc s1.docx EthicCommiteeapprobationFrenchversion.pdf Fundingdocumentation.doc
https://openalex.org/W3177825949	https://hrcak.srce.hr/file/377867	Croatian	null	O održivosti semantičkog poimanja presupozicija	Fluminensia	2,021	cc-by	6,299	izvorni znanstveni članak rukopis primljen: 15. travnja 2021; prihvaćen za tisak: 12. svibnja 2021. U radu se problematiziraju presupozicije i njihove temeljne karakteristike te se razmatra još uvijek neriješeno pitanje jesu li one semantički ili pragmalingvistički fenomen. Na temelju povezanosti presupozicija i leksičkih elemenata te sintaktičkih struktura propituje se uvjerenje da je moguće kakvu određenu presupoziciju uvijek vezati uz točno određeni leksičko-sintaktički element, odnosno propituje se mišljenje da postoje takozvani garanti presupozicija. Na posve konkretnom problemu projekcije presupozicija, odnosno njihova prijenosa iz surečenice u složenu rečenicu u koju je ta surečenica uključena, razmatra se njihova kontekstualna ovisnost, pri čemu kontekst predstavlja rečenica unutar koje se presupozicija pojavljuje. Cilj je rada na konkretnim primjerima pokazati da kontekst utječe na stvaranje presupozicija te da nije održivo njihovo poimanje kao inferencija koje nastaju na temelju semantičkih garanata. Ključne riječi: presupozicije; projekcija; semantika; pragmalingvistika * Ovaj je rad financiralo Sveučilište u Rijeci projektom uniri-human-1813 1140. Nikolina Palašić O ODRŽIVOSTI SEMANTIČKOG POIMANJA PRESUPOZICIJA* Nikolina Palašić O ODRŽIVOSTI SEMANTIČKOG POIMANJA PRESUPOZICIJA* dr. sc. Nikolina Palašić, Sveučilište u Rijeci, Filozofski fakultet nikolina.palasic@ffri.uniri.hr orcid.org/0000-0002-0054-1830 Nikolina Palašić, O održivosti semantičkog poimanja presupozicija FLUMINENSIA, god. 33 (2021), br. 1, str. 7–23 Nikolina Palašić, O održivosti semantičkog poimanja presupozicija FLUMINENSIA, god. 33 (2021), br. 1, str. 7–23 Nikolina Palašić, O održivosti semantičkog poimanja presupozicija FLUMINENSIA, god. 33 (2021), br. 1, str. 7–23 7 7 https://doi.org/10.31820/f.33.1.11 1 Ovdje moramo napomenuti da referenciju shvaćamo u širem smislu negoli je primjerice opisana kod Russela (1905) i Austina (2014) – naime i jedan i drugi tvrdili su da ako nema onoga na što se referiramo, onda su takvi iskazi ništavni. Ako referenciju shvatimo na taj način, onda bismo mogli čak reći da je presupozicija rečenice Kepler je umro u bijedi zapravo kvazipresupozicija ili pseudopresupozcija jer tom rečenicom referiramo na nešto što ne postoji. Međutim jasno je da vlastita imena referiraju na osobu koja ih nosi, bila ona živa ili ne, pa ako bismo se složili s tvrdnjom da su iskazi bez (postojećeg) denotata ništavni, onda ne bismo ništa mogli reći ni o čemu što je nekada postojalo ili bismo, u manje ekstremnom slučaju, mogli reći da ništa od onoga što kažemo o onome što više ne postoji ne odgovara uvjetima istinitosti. Prema tome, kada je riječ o pitanju što presupozicija u odnosu na referenciju uop će znači, zadržat ćemo se na Fregeovu poimanju temeljnih karakteristika presupozicija: one sadrže informaciju koja je prethodila onome što mi nekim izrazom tvrdimo. Takav nam pri stup omogućuje da se referiramo i na ono što je nekada postojalo, ali i na primjerice izmišljene denotate (mitologija, književnost i sl.). Strawson se kasnije nadovezao na Fregeo vu ideju te je razradio pojam presupozicije, a referenciju je odredio ne kao karakteristiku kakva izraza, već kao rezultat upotrebe toga izraza. U skladu s tim on više ne govori o istini tosti izraza, već o istinitosti upotrebe izraza (1950: 326). 1. Neka preliminarna razmišljanja o presupozicijama Ideja se o presupozicijama prvi put javila u Fregeovim (1892) promišlja njima o smislu i referenciji, pri čemu je on utvrdio da određenoj izjavi mora Nikolina Palašić, O održivosti semantičkog poimanja presupozicija FLUMINENSIA, god. 33 (2021), br. 1, str. 7–23 8 prethoditi informacija koja tu izjavu uopće čini mogućom. Ta je informacija zapravo presupozicija. Pritom je zaključio dvije stvari: prvo, da bi neki iskaz bio istinit, njegova presupozicija mora biti istinita i drugo, presupozicija „preživljava” negaciju. Fregeov primjer, koji podupire obje navedene tvrdnje, glasi: Kepler je umro u bijedi. Presupozicija pritom glasi: ime Kepler nešto znači, odnosno po stojala je osoba imena Kepler odnosno „Kepler” referira na određeni denotat. Dakle ako je postojala osoba imena Kepler, onda o toj osobi možemo nešto i reći, dakle možemo se referirati1 na nju određenim iskazima, što znači da smo utvrđivanjem istinitosti te presupozicije uspostavili i uvjete istinitosti za iskaze kojima se na tu osobu referiramo. Negiramo li početnu rečenicu, dobit ćemo izjavu Kepler nije umro u bijedi te vidimo da presupozicija ime Kepler ne što znači ostaje i u tom slučaju netaknuta, dakle „preživljava” negaciju. Na temelju navedenoga možemo reći da presupozicija prethodi govor nom događaju i predstavlja nužan uvjet za njegovu realizaciju. Budući da ne predstavlja dio asercije/propozicije (sadržaja, onoga što se iskazom poruču je), opstaje i u situacijama u kojima se propozicija negira. Nakon što je Frege u svojim filozofskim ogledima postavio temelje za razmišljanje o presupozicijama, rasprava je o njima zaživjela i u lingvistici, pa su svoje definicije presupozicija i kriterije za njihovo određivanje iznijeli primjerice Strawson (1950, 1952), Katz i Postal (1964), Horn (1969), Stal naker (1973, 1974), Montague (1973) i drugi. Strawsonov je pristup Nikolina Palašić, O održivosti semantičkog poimanja presupozicija FLUMINENSIA, god. 33 (2021), br. 1, str. 7–23 9 problematici presupozicija logičko-semantički, iako u svojim obrazloženji ma referencije, u okviru kojih dolazi do definicije presupozicije, ima izrazito pragmalingvističkih elemenata. Na njegova se promišljanja nastavljaju sva ostala istraživanja presupozicija u lingvističkoj domeni. U nastavku ćemo ukratko iznijeti najvažnije njegove teze. Strawson (1950) u svom je članku On referring, suprotstavljajući se Russelovu poimanju referencije (s osnovnom tezom da je Russel pomiješao rečenice i upotrebu rečenica) i nastavljajući se na Fregea, uveo sam pojam presupozicije. 1. Neka preliminarna razmišljanja o presupozicijama Pošao je od toga da određeni izrazi naprosto ne moraju imati istinosnu vrijednost, odnosno istinosnovrijednosno značenje, ali to ne znači da ih mi ne možemo upotrebljavati (ne znači da su ništavni, kako je to tvr dio Russel). Za primjer je uzeo rečenicu Francuski je kralj ćelav. Da je ta rečenica izgovorena u nekom davnom stoljeću, njezina bi propozicija bila istinita ako bi odgovarala izvanjezičnim okolnostima, drugim riječima, ako je tadašnji francuski kralj doista bio ćelav. Ta je izvanjezična okolnost prov jerljiva. Međutim ako istu tu rečenicu netko upotrijebi u današnje vrijeme, kada Francuska više nema kralja, onda njezina propozicija nije ni istinita ni neistinita, već takva propozicija naprosto nema istinosnu vrijednost. Strawson to argumentira tvrdnjom da ako kažemo da neka propozicija nije istinita, primjerice nije istinito da je francuski kralj ćelav, onda negacija te propozicije mora biti istinita, tj. onda bi rečenica Francuski kralj nije ćelav imala istinitu propoziciju. Budući da Francuska nema kralja, jasno je da ni propozicija negirane rečenice ne može biti istinita, pa stoga Strawson za ključuje da se tvrdnje čijim se propozicijama izjavljuje nešto o denotatu koji više ne postoji ne mogu promatrati s obzirom na uvjete istinitosti. Da bi se takve tvrdnje mogle razmatrati u kontekstu uvjeta istinitosti, potrebno ih je „aktualizirati”, i to u smislu da im je potrebno dodati određene elemente koji u sebi sadrže informacije o vremenu i mjestu govornoga događaja, od nosno da bismo mogli određivati istinosnu vrijednost vremenske odrednice „sada”, moramo znati na koji se trenutak to „sada” odnosi, odnosno kada je određena rečenica upotrijebljena. U tom smislu možemo reći da rečenica Trenutni francuski kralj sada je ćelav ne zadovoljava uvjete istinitosti (jer Francuska trenutno nema kralja), dok to za rečenicu Francuski je kralj ćelav ne možemo tvrditi. U tom kontekstu Strawson donosi definiciju presupozicija, koju može mo formulirati na sljedeći način: uzmimo da su A i B dvije propozicije; A presuponira B ako i samo ako B predstavlja preduvjet istinitosti ili neistinitosti A. Drugim riječima, ako je A istinit ili neistinit, B je istinit. Nikolina Palašić, O održivosti semantičkog poimanja presupozicija FLUMINENSIA, god. 33 (2021), br. 1, str. 7–23 10 Kako vidimo, njegova definicija sadrži temeljnu karakteristiku pre supozicija, koju je već utvrdio Frege, naime njihovu neosjetljivost na negaciju. 1. Neka preliminarna razmišljanja o presupozicijama Međutim ono što je zanimljivo u tom kontekstu jest činjenica da i sam Strawson (kao i svi koji ga kasnije citiraju) smatra da je njegova de finicija presupozicija isključivo logičko-semantička, ali tu ipak moramo uočiti jedan izrazito pragmalingvistički aspekt – naime on je sadržan u dijelu definicije koji kaže „ako je A istinit ili neistinit”. Strawson je, kako smo gore pokazali, tvrdio da samo aktualizirana rečenica, dakle ona koja ima jasno označene vremensko-prostorne koordinate, može biti osjetljiva na uvjete istinitosti. Budući da je aktualizirana rečenica (iskaz) element govornoga događaja, ne možemo tvrditi da Strawson presupozicije sma tra isključivo semantičkim fenomenom. Iz toga bismo nadalje mogli zaključiti da presupozicije, ako ih vežemo uz uvjete istinitosti, ne može mo tražiti u apstraktnim, već samo u konkretnim rečenicama, dakle iskazima. Pomak prema proučavanju presupozicija kao u potpunosti pragma lingvističkog fenomena nalazimo kod Stalnakera (1973: 2ff), koji u svom članku Pragmatics iznosi tezu da se presupozicije trebaju analizirati u kon tekstu u kojemu su nastale, odnosno da se razlika između asercije i presupozicije ne treba tražiti u sadržaju iskazane propozicije, već u situaciji u kojoj je propozicija upotrijebljena, konkretno, u stavovima i intuiciji go vornika i sugovornika. Presupozicije on smatra uvjerenjima koja čine pozadinu neke asercije: propozicija p pragmatička je presupozicija samo ako govornik pretpostavlja ili vjeruje da p, pretpostavlja ili vjeruje da njegov su govornik pretpostavlja ili vjeruje da p te pretpostavlja ili vjeruje da njegov sugovornik prepoznaje to da on pri iskazivanju propozicije stvara te pretpo stavke (ibid., 5). Prema takvu shvaćanju presupozicija nije vezana uz verbalni dio govornoga čina, odnosno presupoziciju ne stvaraju asercije ni propozicije, već ih stvara onaj tko asercije i propozicije upotrebljava u go vornom činu. Iako se takvo poimanje presupozicije može činiti kao prilično radikalan obrat u odnosu na njezinu definiciju iz semantičke perspektive, potrebno je napomenuti da presupozicija shvaćena na taj način ne gubi ni šta od svojih karakteristika koje joj pripisuje semantičko-logička definicija – ona i dalje ostaje stabilna pri negaciji te mora biti istinita da bi iskazana propozicija uopće mogla biti istinita. Stalnaker polazi od činjenice da se ko munikacija mora odvijati na određenom temelju dijeljenih (zajedničkih) uvjerenja – kada ne bismo neke stvari pritom uzimali zdravo za gotovo, ko munikacija ne bi bila moguća. 1. Neka preliminarna razmišljanja o presupozicijama U skladu s tim on tvrdi da ne izgovaramo ono j j j j p p j gi j – ona i dalje ostaje stabilna pri negaciji te mora biti istinita da bi iskazana propozicija uopće mogla biti istinita. Stalnaker polazi od činjenice da se ko munikacija mora odvijati na određenom temelju dijeljenih (zajedničkih) uvjerenja – kada ne bismo neke stvari pritom uzimali zdravo za gotovo, ko munikacija ne bi bila moguća. U skladu s tim on tvrdi da ne izgovaramo ono – ona i dalje ostaje stabilna pri negaciji te mora biti istinita da bi iskazana propozicija uopće mogla biti istinita. Stalnaker polazi od činjenice da se ko munikacija mora odvijati na određenom temelju dijeljenih (zajedničkih) uvjerenja – kada ne bismo neke stvari pritom uzimali zdravo za gotovo, ko munikacija ne bi bila moguća. U skladu s tim on tvrdi da ne izgovaramo ono Nikolina Palašić, O održivosti semantičkog poimanja presupozicija FLUMINENSIA, god. 33 (2021), br. 1, str. 7–23 11 što uzimamo zdravo za gotovo te da ne govorimo ono što nije kompatibilno s tom pozadinom dijeljenih uvjerenja.2 što uzimamo zdravo za gotovo te da ne govorimo ono što nije kompatibilno s tom pozadinom dijeljenih uvjerenja.2 Stalnaker smatra da promatranje presupozicija iz takve, pragmalingvi stičke perspektive donosi neke prednosti u odnosu na semantičko-logičku perspektivu. Jedna je od tih prednosti primjerice ta što nam takav pristup ukida napor oko stroga određivanja razlika između presupozicija i seman tičkih implikacija3 (npr. rečenica Ivan shvaća da p i presuponira i semantički implicira da p). Pragmalingvistički pristup omogućuje nam tvrdnju da je ponekad ono što je presuponirano istovremeno i semantički implicirano, a ponekad nije. Međutim iako se Stalnakerovo poimanje presupozicija može činiti pri vlačnim jer nam uvelike olakšava njihovo određivanje i omogućuje nam da naprosto kažemo: u ovom kontekstu govornik presuponira ovo, u nekom drugom ono, u literaturi o presupozicijama vlada uvjerenje da na semantič koj razini postoje elementi koji služe kao okidači presupozicija, i to bez obzira na kontekst u kojem su upotrijebljeni. Iako govornikova namjera i njegova uvjerenja, dakako, uvjetuju ono što se nekim iskazom presuponira, i dalje stoji tvrdnja da je u komunikaciji okidač za određenu presupoziciju sadržan u verbalnom dijelu govornoga čina, odnosno propozicija je ta koja navodi sugovornika na iščitavanje onoga što je presuponirano. 2 U literaturi pronalazimo mišljenja da postoje slučajevi nastanka presupozicija koji i ta kav pristup dovode u pitanje jer ne pretpostavljaju dijeljeno znanje, primjerice uzmimo da u firmu dolazi novozaposlena osoba i već prvi dan kasni na uvodni sastanak te kaže: Oprostite što kasnim, morao sam odvesti sina liječniku. Presupozicija koja pritom nastaje glasi: ta osoba ima sina. Međutim nikako ne možemo reći da je ta presupozicija nastala na zajedničkom znanju sudionika komunikacije jer o toj osobi nitko od prisutnih ne mora ništa znati, a opet će svi na temelju rečenoga presuponirati da ima sina. Takvom argumentacijom Stalnakerov pristup u pitanje dovode primjerice Burton-Roberts (1989) i Gauker (1998). Međutim mogli bismo reći da kritičari Stalnakerova poimanja presupozicija zajednički kontekst/dijeljeno znanje možda preusko shvaćaju te da u ovom slučaju to dijeljeno znanje glasi: ako netko govo ri o svom sinu, onda se podrazumijeva da ima sina. 3 O razlici između presupozicija i semantičkih implikacija (entailments) vidi u Palašić (2018: 53 ff). 4 Usp. Fillmore (1969, 1971), Kiparsky i Kiparski (1970), Keenan (1971), Karttunen (1973), Jäger (2010) i dr. 2. Propozicijski elementi kao okidači presupozicija Iako je pojam presupozicija u svojim začecima bio relevantan tek u vezi s referencijom, odnosno referirajućim izrazima, nakon što su se njime Nikolina Palašić, O održivosti semantičkog poimanja presupozicija FLUMINENSIA, god. 33 (2021), br. 1, str. 7–23 12 počeli baviti lingvisti, bitno mu je proširena domena relevantnosti, pa su se u tom smislu istraživali propozicijski elementi koji su odgovorni za nasta nak presupozicija, dakle presupozicijski okidači ili garanti. U kontekstu (još uvijek aktualne) rasprave o tome predstavljaju li presupozicije semantički ili pragmatički fenomen u literaturi se razlikuju semantički i kontekstualni okidači. Semantički su okidači jezična sredstva koja zadovoljavaju sljedeću definiciju: G je garant presupozicije P samo ako za sve rečenice koje sadrže G u svim mogućim kontekstima vrijedi da G izaziva presupoziciju P (Reis 1977: 28). Obrnuto, semantičke su presupozicije neke rečenice samo one koje nastaju na temelju garanta sadržanog u danoj rečenici. Međutim takva definicija semantičkih okidača – i općenito presupozi cija kao semantičkog fenomena – problematična je već iz tog razloga što se presupozicije u određenim kontekstima mogu dokinuti, i to bez obzira na to što su nastale na temelju kakva semantičkog garanta. U lingvističkoj lite raturi o presupozicijama postoji niz takvih elemenata za koje se smatra da uvijek izazivaju određenu presupoziciju4, ali ono što se pritom rijetko dovo di u pitanje jest stabilnost presupozicije bez obzira na jezične i druge okolnosti. Naime, kako smo već rekli, čak i tako nastale presupozicije kon tekstualno su ovisne, što onda dovodi u pitanje njihovu pripadnost semantici. Jedan od takvih semantičkih presupozicijskih okidača predstavljaju primjerice definitni nominalni izrazi, na temelju kojih nastaju referencijal ne presupozicije: a) Ivan dolazi. (p = postoji Ivan) b) Ivan ne dolazi. (p = postoji Ivan) c) Ivan ne dolazi jer ne postoji. (presupozicija p nestaje) 5 R. Lakoff (1971) tvrdi da su dva temeljna obilježja veznika ali suprotnost i koncesiv nost. Pritom veznik ali na suprotnost ukazuje kada se upotrebljava za kontrastiranje dviju semantički povezanih karakteristika (npr. antonima siromašan/bogat: Ivo je siromašan, ali Marko je bogat), a koncesivno mu se značenje pripisuje kada se presuponira kakva suprot c) Ivan ne dolazi jer ne postoji. (presupozicija p nestaje) U navedenom primjeru riječ je o postojanju/nepostojanju referenta te uvjetima istinitosti povezanih s presupozicijom, o čemu smo već govorili na početku ovoga rada. Na takvu je primjeru stvaranja presupozicija Strawson ustvrdio da neke rečenice, pa onda ni presupozicije koje iz njih nastaju, nisu osjetljive na uvjete istinitosti (jer nisu primijenjene u kontekstu). Ako je kon tekst odgovoran za nastanak takva tipa presupozicija, onda ne možemo reći da su definitni nominalni izrazi semantički persupozicijski garanti, što osim Nikolina Palašić, O održivosti semantičkog poimanja presupozicija FLUMINENSIA, god. 33 (2021), br. 1, str. 7–23 13 toga vidimo i iz primjera c), koji predstavlja jezični kontekst u kojemu presu pozicija nastala na temelju definitnog nominalnog izraza ne preživljava. Nadalje se semantičkim garantima smatraju određene čestice i veznici – riječ je pritom o uvjerenju da pojedini leksički elementi, zahvaljujući svo jim semantičkim obilježjima, uvijek stvaraju istu presupoziciju. Kada je riječ o česticama, one po svojoj definiciji sadrže oznaku modalnosti, što ih svrstava više u ilokucijski nego u propozicijski segment iskaza (Reis 1977: 55), dakle vezane su uz govorni čin, pa ih već i ta činjenica isključuje iz po pisa semantičkih presupozicijskih okidača. Osim toga ti elementi ne stvaraju iste presupozicije u različitim kontekstima, čak ni onda kada njiho va modalnost ne dolazi u prvi plan. Uzmimo za primjer česticu još upotrijebljenu u čisto temporalnom značenju: a) Ona je još tu. b) Je li ona još tu? c) Ona još nije došla. d) Može još doći. nost, odnosno kada se pretpostavlja da postoji kakva karakteristika unatoč očekivanju: Ona je siromašna, ali poštena (ta se rečenica može formulirati na način da dopusnost postane ek splicitna: Iako je siromašna, poštena je). O koncesivnosti veznika ali vidi i Silić–Pranjković (2005: 325), Badurina (2008: 104) te Badurina–Palašić (2010: 254). e) Još (samo) ona nije došla. U primjeru a) asercija te rečenice glasi: ona je u nekom vremenskom isječ ku t tu. Presupozicija, čiji je okidač čestica još, glasi: ona je bila tu prije vremenskoga isječka t. U primjeru b) presupozicija ostaje ista, no u primjeru c) čestica još ne stvara istu presupoziciju, već upravo oprečnu: ona nije bila tu prije vremenskoga isječka t. Primjer d) ovdje je naveden kao kontekst koji ne sadrži negaciju (ako bi netko možda pomislio da navođenje negirana konteksta nije valjan odabir za dokazivanje nepostojanosti presupozicije) i u kojem presupozicija nastala u primjerima a) i b) također ne opstaje, već se stvara presupozicija kao u primjeru c). U primjeru e) stvara se presupozicija kao i u primjeru c), ali i presupozicija koja nam govori da su svi ostali došli. Dakle presupozicije koje se stvaraju na temelju čestice još ovisne su i o kon tekstu i o mjestu na kojemu se još nalazi u propoziciji i o intonaciji, odnosno mjestu koje se u propoziciji naglašava. Veznik ali stvara presupozicije koje ukazuju na kakvu suprotnost, ali i dopusnost5. Budući da ovisno o upotrebi Nikolina Palašić, O održivosti semantičkog poimanja presupozicija FLUMINENSIA, god. 33 (2021), br. 1, str. 7–23 14 stvara dva tipa presupozicija, također ga ne možemo smatrati semantičkim garantom, već zapravo elementom koji stvara pragmatičke, kontekstualno ovisne presupozicije. Rečenični je naglasak također vrlo važan element u određivanju presu pozicija, a na koji ćemo rečenični segment staviti naglasak, opet ovisi o govornikovoj intenciji, odnosno o kontekstu u kojemu stvaramo iskaz upo trebljavajući određenu rečenicu. Uzmimo naprimjer rečenicu Ivan je prevario Anu. Presupozicije koje takva rečenica stvara glase: Postoji osoba koja se zove Ivan i Postoji osoba koja se zove Ana. Negiramo li prvotnu rečenicu, dobiva mo: Ivan nije prevario Anu, što nam potvrđuje da su navedene inferencije doista presupozicije jer ostaju nepromijenjene. Međutim što se događa s presupozicijama ako odlučimo naglasiti koju riječ u navedenom primjeru? a) IVAN je prevario Anu. b) Ivan je PREVARIO Anu. c) Ivan je prevario ANU. c) Ivan je prevario ANU. U primjeru a) osim navedenih presupozicija stvara se još jedna: netko je prevario Anu, odnosno Ana je prevarena. Da je ta inferencija doista presu pozicija, možemo provjeriti negacijskim testom: IVAN nije prevario Anu – presupozicija ostaje netaknuta. U primjeru b) stvara se dodatna presupo zicija Ivan je nešto učinio Ani, a u primjeru c) Ivan je prevario nekoga. Kada je riječ o rečeničnim strukturama kao okidačima presupozicija, i tu možemo pronaći primjere u kojima se presupozicije dokidaju u vrlo slič nim kontekstima. Uzmimo naprimjer slučaj koji je naveo Levinson (1983): a) Ona je plakala prije nego što je završila doktorat. b) Ona je umrla prije nego što je završila doktorat. 6 Primjeri su preuzeti iz Reis (1977: 56). Ovdje je potrebno napomenuti da Reis na broj nim primjerima nastoji pokazati kako pojedini leksemi doista ne mogu služiti kao semantički garanti presupozicija, međutim promiče joj jedna vrlo važna stvar: brojni slučaje vi koje ona navodi kao različite presupozicije nastale u različitim kontekstima upotrebe jednog te istog leksičkog elementa zapravo nisu presupozicije, već implikature, što je razvid no iz činjenice da ne preživljavaju negaciju propozicije u kojoj su nastale. 7 To su pitanje u lingvistička razmatranja uveli Langendoen i Savin (1971) tvrdnjom da ako klauza stvara određenu presupoziciju, onda ta presupozicija vrijedi i za cijelu rečenicu. b) Ona je umrla prije nego što je završila doktorat. U primjeru a) presupozicija glasi: Ona je završila doktorat, no u primje ru b) ta se presupozicija dokida, i to na temelju našega općeg znanja, konkretno, na temelju našega znanja o tome da mrtva osoba ne može zavr šiti neki rukopis. Nikolina Palašić, O održivosti semantičkog poimanja presupozicija FLUMINENSIA, god. 33 (2021), br. 1, str. 7–23 15 Osim navedenoga neki propozicijski elementi u različitim konteksti ma uopće ne sudjeluju u stvaranju presupozicija, već implikatura: a) Iskrenost je opet na cijeni. b) Nije točno da je iskrenost opet na cijeni. c) Je li iskrenost opet na cijeni?6 U navedenim trima primjerima upotrebe čestice opet nastaje presupo zicija iskrenost je jednom prije bila na cijeni. Neki drugi primjeri upotrebe čestice opet ne aktiviraju nikakve presupozicije, već zapravo semantičke im plikacije: a) Opet se javio Ivan. b) Opet se nije javio Ivan. Inferencija koju stvaramo na temelju čestice opet u primjeru a) glasi: Ivan se već ranije javio, no kako ta inferencija u primjeru b) ne preživljava, odnosno glasi: Ivan se već ranije nije javio, zaključujemo da je riječ o implika turi, a ne o presupoziciji. Slična je situacija s kvantifikatorima poput svi i neki. Kvantifikator neki znači ne svi, pa ako kažemo primjerice Neki su njezini prijatelji plavokosi, do bit ćemo inferenciju: Nisu svi njezini prijatelji plavokosi, a negiramo li prvu izjavu (da bismo testirali je li dobivena inferencija presupozicija), dobit ćemo propoziciju: Neki njezini prijatelji nisu plavokosi, pri čemu vidimo da nam inferencija ostaje nepromijenjena, što znači da je ona zapravo presu pozicija. Međutim promijenimo li kontekst, pa kažemo: Neki njezini prijatelji nisu plavokosi samo u njezinoj mašti, dobivena nam presupozicija više ne vri jedi. Takav nam rezultat može ukazivati na dvije stvari: ili dobivena presupozicija nije semantička ili uopće nije presupozicija (iako iz negacij skog testa proizlazi suprotno). Iz navedenoga možemo zaključiti da su semantičke presupozicije upitan fenomen, posebice ako ih definiramo kao inferencije koje nastaju na temelju semantičkih garanata, i to neovisno o kontekstu u kojemu se pojavljuju. Nikolina Palašić, O održivosti semantičkog poimanja presupozicija FLUMINENSIA, god. 33 (2021), br. 1, str. 7–23 16 Svaku od tako nastalih presupozicija možemo modificirati ili dokinuti promjenom konteksta, što zapravo više ide u prilog mišljenju da se presupo zicije trebaju analizirati iz pragmalingvističke perspektive, odnosno da predstavljaju pragmalingvistički, komunikacijski fenomen. 3. O problemu projekcije presupozicija Dosad smo pokazali kako stroga semantička definicija presupozicija i njihovih okidača nije održiva u komunikaciji te da ako želimo presupozicije promatrati kao semantički fenomen, moramo u najmanju ruku odustati od ideje presupozicijskih garanata. Pitanje projekcije još je jedan nerješiv pro blem u semantičkom pristupu presupozicijama (usp. npr. Levinson 1983, Gazdar 1979). Problem je projekcije presupozicija također vezan uz njihovu kontekstualnu ovisnost, s tim da je kontekst u tom slučaju shvaćen u užem smislu od komunikacijskog konteksta, naime predstavlja ga sama rečenica unutar koje presupozicija nastaje. Naime riječ je o tome kako složena reče nica, nastala od jednostavne rečenice, „nasljeđuje” njezinu presupoziciju, odnosno o tome prenosi li se presupozicija iz zavisne rečenice na glavnu ili ne.7 U nastavku donosimo nekoliko primjera u kojima ćemo pokazati kako se presupozicije projiciraju na cijelu složenu rečenicu i kako ta projekcija u određenim sintaktičkim strukturama izostaje. Na temelju rečenice a) Ivan je opet napravio grešku javlja se presupozicija a’) Ivan je već ranije napravio grešku/greške. U složenim rečeničnim struktu rama koje slijede ta se presupozicija održava te se projicira na cijelu rečenicu: b) Nije istina da je Ivan opet napravio grešku. c) Ako je Ivan opet napravio grešku, to će biti loše. d) Postoji velika vjerojatnost da će Ivan opet napraviti grešku. e) Ivan je požalio što je opet napravio grešku. Sintaktički konteksti koji su „propusni” za presupozicije u literaturi se nazivaju „rupama” (holes) (Karttunen (1973)). Takve rečenične strukture uključuju primjerice faktivne i finitivne glagole (poput požaliti i prestati). S druge pak strane postoji niz glagola koji tvore takav sintaktički kontekst Nikolina Palašić, O održivosti semantičkog poimanja presupozicija FLUMINENSIA, god. 33 (2021), br. 1, str. 7–23 17 koji ne „propušta” presupozicije, odnosno koji onemogućuje njihovu pro jekciju, a oni se nazivaju „čepovima” (plugs). Sintaktičke okolnosti koje ne dopuštaju projekciju obično se konstituiraju oko glagola govorenja (pitati, govoriti, tvrditi i dr.), uz iznimku rečeničnih struktura koje sadrže takav gla gol u prvom licu aktivnog prezenta ili kada je zavisna rečenica indirektno pitanje (Pitam se je li Ivan opet napravio grešku). Primjerice u rečenici Ivan tvrdi/govori da je opet napravio grešku presupozicija Ivan je već ranije napravio grešku ne pojavljuje se (kao u prethodno navedenim primjerima), odnosno ta inferencija koja se pojavljuje zapravo je implikacija (što vidimo iz toga da ne preživljava negaciju). 8 Primjeri su preuzeti iz Jäger (32010: 416) te minimalno modificirani. 3. O problemu projekcije presupozicija Osim pri upotrebi glagola govorenja projekcija se presupozicija ne ostvaruje ni u nekim pogodbenim rečeničnim strukturama, a takve okolno sti Kartunnen naziva „filtrima” – naime neke se presupozicije „propuštaju”, odnosno selektivno se projiciraju, druge pak ne. Uzmimo za pojašnjenje sljedeće primjere8: f) Opet je Ivan taj koji je upravo napravio grešku. g) Ako je Ivan taj koji je zadnji put napravio grešku, onda je vjerojatno opet Ivan taj koji je upravo napravio grešku. g) Ako je Ivan taj koji je zadnji put napravio grešku, onda je vjerojatno opet Ivan taj koji je upravo napravio grešku. h) Ivan ili nikada ne pravi greške ili je opet Ivan taj koji je upravo napravio grešku. h) Ivan ili nikada ne pravi greške ili je opet Ivan taj koji je upravo napravio grešku. Presupozicije koje se stvaraju na temelju primjera f) glase: Ivan je već ranije napravio grešku i Netko je upravo napravio grešku. Međutim u primjeri ma g) i h) opstaje, odnosno biva projicirana, samo druga presupozicija, dok prva ne prolazi kroz „filtar” navedenih rečeničnih struktura. Štoviše, kod takvih primjera možemo reći da su implikature koje nastaju na temelju ta kvih rečeničnih struktura odgovorne za ukidanje presupozicije: Naime primjeri g) i h) impliciraju to da govornik nije siguran u istinosnu vrije dnost rečenoga: stvara se implikatura da je moguće da je Ivan već napravio grešku, ali i implikatura da je moguće da Ivan nije već napravio grešku. Drugim riječima, nije održivo implicirati da postoje dvije mogućnosti, a presuponirati jednu od njih. Brojni su autori u svojim opisima projekcije presupozicija nastojali konstruirati sintaktičko-semantička pravila prema kojima se projekcija do gađa ili ne, ali su naposljetku došli do zaključka da to ne mogu konzistentno Nikolina Palašić, O održivosti semantičkog poimanja presupozicija FLUMINENSIA, god. 33 (2021), br. 1, str. 7–23 18 izvesti (npr. Liberman (1973), Reis (1974), Wilson (1975), Soames (1983) i dr.). izvesti (npr. Liberman (1973), Reis (1974), Wilson (1975), Soames (1983) i dr.). izvesti (npr. Liberman (1973), Reis (1974), Wilson (1975), Soames (1983) i dr.). Soames (1983: 483) primjerice navodi čak i to da je temeljni kriterij koji se upotrebljava za dokazivanje postojanja presupozicija, naime negaci ja, također samo vrsta projekcije presupozicije iz potvrdne u negiranu rečenicu, odnosno negirana rečenica predstavlja „rupu”, ako se želimo drža ti Karttunenove terminologije. 9 Gazdar (1979) također pokazuje odmak od semantičkog poimanja presupozicija, pa za primjer John je prestao pušiti, koji stvara presupoziciju John je pušio, navodi da takva rečenica uključena u neke kompleksnije rečenične strukture (npr. Ako je John pušio, onda je prestao pušiti) gubi navedenu presupoziciju iz jednostavnog razloga što ona nije konzistentna s kon verzacijskim pretpostavkama koje trebaju biti ispunjene da bi takva kompleksna rečenica predstavljala primjeren iskaz. 3. O problemu projekcije presupozicija U svom nastojanju da iznađe kriterij koji bi jasno ukazivao na to koje će rečenice u cjelini naslijediti sve presupozicije svojih sastavnica, koje će naslijediti samo neke, a koje će u potpunost bloki rati projekciju, Soames je morao odustati od nabrajanja pojedinih rečeničnih struktura i uspostavljanja semantičkih okvira te je došao do zaključka da je u konačnici kontekst u kojemu je pojedina rečenica upotrijebljena odgovoran za ono što se s presupozicijama događa. On navodi, nastavljajući se na Stalnakera i Gazdara9, sljedeće pravilo (njegovim riječima: strategiju), koje je primjenjivo na sve slučajeve projekcije i izostanka projekcije presupozicija, a koje uključuje govornika, sugovorni ka i kontekst (ibid., 490): Iskazivanje S u kakvu (konzistentnu) konverzacijskom kontekstu C presuponira P, osim ako vrijedi sljedeće: Iskazivanje S u kakvu (konzistentnu) konverzacijskom kontekstu C presuponira P, osim ako vrijedi sljedeće: a) P je nekompatibilno s kontekstom C b) iskaz konverzacijski implicira da govornik ne uzima P zdravo za gotovo. b) iskaz konverzacijski implicira da govornik ne uzima P zdravo za gotovo. Drugim riječima, ako se neka presupozicija kakve klauze blokira kada se ta klauza uklopi u složenu rečenicu, razlog je taj što kontekst koji je zah tijevao takvu formulaciju rečenice nije kompatibilan s prethodno nastalom presupozicijom, odnosno kontekst je taj koji je blokira, a ne rečenična struktura. Nadalje presupozicija može nestati naprosto zato što je govornik namjerno upotrijebio kakav iskaz kojim daje do znanja da se ne slaže s po tencijalnim stvaranjem takve presupozicije. Što se tiče kompatibilnosti s kontekstom, konkretnije, sa zajedničkim znanjem, potrebno je dodati da Nikolina Palašić, O održivosti semantičkog poimanja presupozicija FLUMINENSIA, god. 33 (2021), br. 1, str. 7–23 19 nije uvijek presupozicija ta koja se mijenja ili izostaje kada se dogodi da u kontekstu ne postoji zajednička pozadina, već se ponekad mijenja i sam kontekst. Ta se pojavnost u literaturi naziva akomodacija, odnosno prilagod ba (usp. Stalnaker 1974, Karttunen 1974). Do akomodacije dolazi kada govornik iznese neku informaciju koja je sugovorniku posve nova, a u toj je je informaciji sadržana kakva presupozicija. Primjerice čujemo li na vijesti ma Jučer je umro kralj Lesota, nećemo dokinuti presupoziciju Lesoto ima kralja samo zato što vjerojatno nismo znali da je Lesoto kraljevina, nego ćemo promijeniti kontekst, odnosno u svoje ćemo opće znanje dodati tu in formaciju. Na taj način sudionici govornoga događaja mijenjaju kontekst kako bi iskazi imali smisla, odnosno kako bi presupozicije koje takvi iskazi stvaraju opstale. 10 Karttunen (1974: 191) u tom smislu kaže da govornici upotrebljavaju prečace u iznoše nju svojih misli te se tako služe rečenicama čije presupozicije ne zadovoljavaju konverzacijski kontekst, odnosno govornik se može ponašati kao da je određena propozicija dio zajedničkih iskustava. Takvo je ponašanje u komunikaciji više pravilo nego iznimka. Zbog toga sugovor nici često proširuju kontekst izvan postojećih granica u skladu s onime što konkretna situacija zahtijeva kako bi došlo do sporazumijevanja. 3. O problemu projekcije presupozicija Ako neki iskaz stvara presupoziciju koju govornik ne može konzistentno prihvatiti, onda se obično takav iskaz smatra komunikacijski neprimjerenim, tj. neuspjelim.10 4. Zaključak S obzirom na sve gore rečeno semantičko poimanje presupozicija koje se u ovome radu nastojalo preispitati jasno pokazuje nedostatke. Problemi koji se javljaju s tumačenjem presupozicija (njihova vezanost uz pojedine riječi, tj. garanti presupozicija, njihovo ukidanje u različitim rečeničnim strukturama koje sadrže iste garante itd.) teško se mogu u potpunosti uklo niti promatramo li presupozicije iz semantičke perspektive – naime svi dosadašnji pokušaji prezentirani u literaturi u vezi s tom problematikom nisu bili posve uspješni jer je uvijek moguće pronaći primjere (poput pri mjera navedenih u ovom radu) iz kojih jasno proizlazi da nije uvijek moguće slijediti uspostavljeni okvir, tj. koji jasno upućuju na potrebu šireg konte ksta od rečeničnog da bi se u potpunosti semantički razlučili. Iz gore navedenih promišljanja jasno proizlazi da u semantičkom pri stupu presupozicijama jedan od najvećih problema predstavlja problem njihove projekcije jer takav pristup zahtijeva stvaranje pravila koja će odre diti kako istinosna vrijednost ili njezin izostanak u složenoj rečenici Nikolina Palašić, O održivosti semantičkog poimanja presupozicija FLUMINENSIA, god. 33 (2021), br. 1, str. 7–23 20 predstavlja funkciju istinosne vrijednosti klauza koje tvore tu rečenicu (Stalnaker 2002: 703 ff). Kada do projekcije dolazi, odnosno kada i klauza i cijela složena rečenica imaju istinosnu vrijednost, situacija je jednostavna, no moramo se zapitati kako će takva pravila glasiti kada jedna klauza ima pogrešnu semantičku presupoziciju, odnosno kada nema istinosnu vrije dnost i kada do projekcije ne dolazi – u takvim situacijama nastaju problemi za kakve semantički pristup još uvijek nije uspio ponuditi adekvatan odgo vor. Na temelju svega dosad rečeno zaključujemo da je za razliku od se mantičkoga pragmalingvistički pristup u stanju riješiti sve probleme koji se tradicionalno javljaju pri semantičkom poimanju presupozicija iz jedno stavnog razloga što je komunikacija dinamički proces, u kojemu se presupozicije stvaraju i nestaju u skladu s komunikacijskim potrebama. Pragmalingvistički pristup pritom ne negira mogućnost pojedinih leksičko- sintaktičkih elemenata da stvaraju određene presupozicije, već samo polazi od toga da su te presupozicije potencijalne, upravo kao i samo značenje, te se ili aktualiziraju ili ne aktualiziraju tijekom komunikacijskoga procesa, ovisno o sudionicima i smjeru komunikacije. Literatura Austin, John Langshaw (2014) Kako djelovati riječima, prev. Andrea Milanko, Zagreb: Disput. Badurina, Lada (2008) Između redaka: Studije o tekstu i diskursu, Zagreb – Rijeka: Hrvatska sveučilišna naklada – Izdavački centar Rijeka. Badurina, Lada, Palašić, Nikolina (2010) „Pragmatika veznih sredstava”, u: Palić, I. (ur.): Sarajevski filološki susreti I, zbornik radova, Sarajevo: Bosansko filološko društvo, str. 252–265. Burton-Roberts, Noel (1989) The Limits to Debate: A Revised Theory of Semantic Presupposition. Cambridge: Cambridge University Press. Fillmore, Charles J. (1969) „Types of lexical information”, u: Kiefer, F. (ur.): Studies in Syntax and Semantics, Dordrecht: D. Reidel Publishing Company, str. 109–137. Fillmore, Charles J. (1971) „Some problems for case grammar”, u: O’Brien R. J. (ur.): 22nd annual Round Table. Linguistics: developments of the sixties – viewpoints of the seventies, volume 24 of Monograph Series on Language and Linguistics, Washington D.C: Georgetown University Press, str. 35–56. Nikolina Palašić, O održivosti semantičkog poimanja presupozicija FLUMINENSIA, god. 33 (2021), br. 1, str. 7–23 21 Frege, Gottlob (1892) „Über Sinn und Bedeutung”, u: Fichte, I. H, Ulrici, H. (ur.): Zeitschrift für Philosophie und philosophische Kritik, br. 100, Leipzig: Verlag von C. E. M. Pfeffer, str. 25–50. Gauker, Christopher (1998) „What Is a Context of Utterance? Philosophical Studies 91, str. 149–172. Gazdar, Gerald (1979) Pragmatics. Implicature, Presupposition and Logical Form, London: Academic Press. Horn, Laurence (1969) „A presuppositional analysis of only and even”, u: Papers from the fifth regional meeting of the Chicago Linguistics Society, Chicago, 98–107. Jäger, Gerhard (32010) „Implikaturen und Präsuppositionen”, u: Carsensten, Kai‑Uwe/Ebert, Cornelia/Ebert, Christian et al. (ur.): Computerlinguistik und Sprachtechnologie. Eine Einführung, Heidelberg: Spektrum Akademischer Verlag. Kartunnen, Lauri (1973) „On defining ‘presupposition’”, u: Linguistic Inquiry, br. 4, str. 256–260. Karttunnen, Lauri (1974) „Presuppositions and linguistic context”, u Krifka, M. (ur.): Theoretical linguistics 1, Berlin: De Gruyter Mouton, str. 181–194. Katz, Jerrold R. (1979) „A Solution to the Projection Problem for Presupposition”, u: Oh. C-K, Dineen, D. A. (ur.): Syntax and Semantics, New York–London: Academic Press, str. 91–126. Katz, Jerrold J., Postal, Paul M. (1964) An Intergrated Theory of Linguistic Description, Cambridge, MA: The MIT Press. Keenan, Edward L. (1971) „Two kinds of presupposition in natural language” u: Fillmore, C. J. i Langendoen, D. T. (ur.): Studies in Linguistic Semantics, Austin: Rinehart & Winston, str. 45–54. Kiparsky, Paul i Kiparsky, Carol (1970) „Fact”, u: Bierwisch, M. i Heidelpf, K. (ur.) Progress in Linguistics, Berlin: de Gruyter Mouton, str. 143–173. Literatura Lakoff, Robin (1971) „If’s, And’s and But’s about conjunction”, u: Fillmore, C. J., Langendoen, D. T. (ur.): Studies in Linguistic Semantics, Austin: Rinehart & Winston, str. 114–149. Langendoen, Donald T.; Savin, Harris B. (1971): „The projection problem for presuppositions”. In: Petöfi, J.; Franck, D. (Hrsg.); Präsuppositionen in Philosophie und Linguistik, Frankfurt a. Main. Levinson (1983) Pragmatik, Tübingen: Max Niemeyer Verlag. Nikolina Palašić, O održivosti semantičkog poimanja presupozicija FLUMINENSIA, god. 33 (2021), br. 1, str. 7–23 22 Liberman, M. (1973) „Alternatives”, u: Corum, C., Smith-Stark, T. C., Weiser, A. (ur.): Papers from the Ninth Regional Meeting of the Chicago Linguistic Society, Chicago, Illinois: Unversity of Chicago, 346–355. Montague, Richard (1973) „Presupposing” u: Petöfi, J., Franck, D. (ur.): Präsuppositionen in Philosophie und Linguistik, Frankfurt a. Main: Athenäum Verlag. Palašić, Nikolina (2018) „Presupozicije vs. implikature – na razmeđu semantike i pragmatike”, u: Badurina, L., Palašić, N. Riječki filološki dani, Zbornik radova, Rijeka: Filozofski fakultet, str. 51–62. Reis, Marga (1977) Präsupposition und Syntax, Max Niemeyer Verlag, Tübingen. Russel, Bertrand (1905) „On Denoting”, u Stout, G. F. (ur.): Mind, New Series, vol. 14, br. 56, Oxford: Oxford University Press, str. 479–493. Soames, Scott (1983) „How Presuppositions Are Inherited: A Solution to the Projection Problem”, u: Linguistic Inquiry, vol. 13, br 3, Cambridge, Massachusetts: The MIT Press, str. 483–545. Sperber, Dan, Wilson, Deirdre (1986) Relevance: Communication and Cognition, Oxford: Blackwell. Stalnaker, Robert C. (1973) „Pragmatics”, u: Petöfi, J.; Franck, D. (ur.): Präsuppositionen in Philosophie und Linguistik, Frankfurt a. Main. Stalnaker, Robert C. (1974) „Pragmatic Presuppositions”, u: Munitz M . K., Unger, P. K. Semantics and philosophy, New York: New York University Press, str. 197–213. Stalnaker, Robert C. (2002) „Common ground”, u: Linguistics and Philosophy, Nr. 25, 701–721. Strawson, Peter Frederick (1950) „On Referring”, u: Rile, Gilbert (ur.) Mind, A Quarterly Rewiev of Psychology and Philosophy, vol. 59, br. 235, Oxford: University Press, str. 320–344. Strawson, Peter Frederick (1952) Introduction to Logical Theory, London: Methuen. Wilson, Deirdre (1975) Presuppositions and Non-Truth-Conditional Semantics, London/New York/San Francisco: Academic Press. Nikolina Palašić, O održivosti semantičkog poimanja presupozicija FLUMINENSIA, god. 33 (2021), br. 1, str. 7–23 Nikolina Palašić, O održivosti semantičkog poimanja presupozicija FLUMINENSIA, god. 33 (2021), br. 1, str. 7–23 23 SUMMARY Nikolina Palašić ON THE SUSTAINABILITY OF THE SEMANTIC UNDERSTANDING OF PRESUPPOSITIONS The paper deals with presuppositions and their basic characteristics, as well as the still unresolved question of whether they are a semantic or pragmalinguistic phenomenon. Special emphasis is placed on the connection between presuppositions and individual lexical elements and syntactic structures, questioning the belief that it is possible to always link a particular presupposition to a specific lexical-syntactic element, i.e., the opinion that there are so-called guarantors of presuppositions. In addition, the paper discusses a very specific problem of projection of presuppositions, i.e., their transfer from a clause to a complex sentence in which that clause is included. Key words: presuppositions; projection; semantics; pragmalinguistics
https://openalex.org/W4391255635	https://www.arkat-usa.org/get-file/81542/	English	null	Silver nitrate promoted synthesis of tetraphenyl derivatives and diaryl ketones	ARKIVOC	2,024	cc-by	5,947	Free to Authors and Readers Free to Authors and Readers Silver nitrate promoted synthesis of tetraphenyl derivatives and diaryl ketones Nian-Qi Chen, a Alageswaran Jayaram, a Karthick Govindan, a Yen Jung Lee, b and Wei-Yu Lin a, c,d * aDepartment of Medicinal and Applied Chemistry, Kaohsiung Medical University, Kaohsiung, Taiwan, ROC bCenter for Research Resources and Development, Kaohsiung Medical University, Kaohsiung, Taiwan, ROC cDepartment of Medical Research, Kaohsiung Medical University Hospital, Kaohsiung 80708, Taiwan, ROC dDrug Development and Value Creation Research Centre, Kaohsiung Medical University, Taiwan, ROC Email: wylin@kmu.edu.tw Dedicated to Prof. Tien-Yau Luh on the occasion of his 76th anniversary Received 05-01-2023 Accepted Manuscript 01-26-2024 Published on line 02-12-2024 Abstract Here in we report a novel silver nitrate-promoted oxidative pathway for the efficient synthesis of tetra substituted aryl derivatives (homodimers) and diaryl ketones. The oxidation of sp3 C-H bond has been extensively studied by tuning the amount of the silver nitrate to afford the tetraphenyl or diaryl ketone, respectively with good selectivity under mild and simple conditions. This developed protocol offers a facile and general route to access a variety of value of tetraphenyl and diaryl ketones derivatives with moderate to good yield. Homocoupling Abstract A Platinum Open Access Journal for Organic Chemistry Abstract Here in we report a novel silver nitrate-promoted oxidative pathway for the efficient synthesis of tetra substituted aryl derivatives (homodimers) and diaryl ketones. The oxidation of sp3 C-H bond has been extensively studied by tuning the amount of the silver nitrate to afford the tetraphenyl or diaryl ketone, respectively with good selectivity under mild and simple conditions. This developed protocol offers a facile and general route to access a variety of value of tetraphenyl and diaryl ketones derivatives with moderate to good yield. Abstract Cite as Arkivoc 2023 (ii) 202312012 DOI: https://doi.org/10.24820/ark.5550190.p012.012 Page 1 of 10 ©AUTHOR(S) R1 R1/R2 R1 R1/R2 O R1 R1 K2S2O8 MeCN/H2O, 60 °C Homocoupling Oxidation 20 mol % AgNO3 2.0 equiv. AgNO3 Keywords: C(sp3)-H bond, Oxidation, Silver nitrate, Homocoupling, Aryl ketones Keywords: C(sp3)-H bond, Oxidation, Silver nitrate, Homocoupling, Aryl ketones Cite as Arkivoc 2023 (ii) 202312012 DOI: https://doi.org/10.24820/ark.5550190.p012.012 Page 1 of 10 ©AUT ©AUTHOR(S) Nian-Qi Chen, a Alageswaran Jayaram, a Karthick Govindan, a Yen Jung Lee, b and Wei-Yu Lin a, c,d * aDepartment of Medicinal and Applied Chemistry, Kaohsiung Medical University, Kaohsiung, Taiwan, ROC bCenter for Research Resources and Development, Kaohsiung Medical University, Kaohsiung, Taiwan, ROC cDepartment of Medical Research, Kaohsiung Medical University Hospital, Kaohsiung 80708, Taiwan, ROC dDrug Development and Value Creation Research Centre, Kaohsiung Medical University, Taiwan, ROC Email: wylin@kmu.edu.tw Dedicated to Prof. Tien-Yau Luh on the occasion of his 76th anniversary Received 05-01-2023 Introduction The selection of carbon-carbon (C-C) bond formation is indispensable in organic synthetic chemistry, being extensively utilized for the synthesis of numerous derivatives in various applications. Despite the array of methods available for C-C bond formations, C(sp3)-H bond activation is still a rare occurrence in synthetic transformations. The di-benzyl core structure is a highly versatile scaffold, offering the potential for the synthesis of a range of compounds in natural products and pharmaceuticals. 1-4 The most conventional method for obtaining di-benzyl derivatives is reducing carbon-carbon multiple bonds. 5, 6 The oxidative homo coupling reaction imposes valuable tools, and the use of oxidizing reagents triggered the transformation of toluene derivatives into dibenzylic compounds. 7 Given their significance, various metal-catalyzed methods (Rh, Cu, Pt, Pd ) have been developed utilizing benzyl magnesium halide, benzyl boronic acid, phenyl acetic acid, benzyl zinc bromide through a homocoupling approach (Scheme 1a). 8-11 In recent years, the rapid advancement of Ag-catalyzed transformations in organic synthesis has overcome the early idea that silver impeded catalytic activity. Silver catalysts combined with K2S2O8 allow for various oxidative C-H bond functionalizations to generate distinct C-C, C-O, and C-N bonds. 12-14 Herein, a novel silver-mediated oxidative homocoupling strategy is presented, facilitating the coupling of two molecules of the same substrate. In contrast, diarylketones are well known compounds that are widely employed in the synthesis of various applications. 15, 16 The oxidation of sp3 C-H bonds has been extensively studied as an effective way to convert alkylarenes into their respective ketones, yet it remains largely underexplored. However, several methods have been known for the synthesis of aromatic ketones such as Friedel Crafts acylation, 17 transition-metal catalyzed coupling reactions, 18 or by oxidation of alcohols into ketones. 19 Tremendous progress has been made through the use of catalytic amounts of transition metal complexes, and various oxidants (Scheme 1b). 20-23 In addition, the Xu group developed potassium tert-butoxide promoted oxidative process by using molecular oxygen. 24 Based on the literature mentioned above, there are certain drawbacks, such as toxic, expansive catalysts, explosive oxidants, and hostile environments. We have successfully overcome and uncovered an effective way to achieve oxidation of benzylic C(sp3)-H via a silver-catalyzed reaction. Based on the above literature precedent, we show that the oxidation of benzylic C(sp3)-H can be achieved through a silver-catalyzed reaction. ©AUTHOR(S) Arkivoc 2023 (ii) 202312012 Chen, N-Q. et al. Introduction By controlling the equivalence of silver nitrate in the oxidative process, two selective products were obtained under mild conditions (Scheme 1c). Page 2 of 10 Arkivoc 2023 (ii) 202312012 Chen, N-Q. et al. Scheme 1. Previous and this work. Results and Discussion At the outset, we assessed the viability of the oxidative reaction of diphenylmethane (1a, 0.2mmol) and AgNO3 ( 0.04 mmol), K2S2O8 (0.2 mmol) in acetonitrile and water (1:1) ratio at 60 ֯ C for 12 h which afforded a homocoupling dimer of tetaphenyl product 2a in 38%. With this result in hand, we prepared a number of derivatives of benzylic substrates (Table 1). Bis (4-fluorophenyl) methane was tolerated under this condition to get the dimer product 2b. Next, cyclic benzylic substrates were examined. The reaction of fluorene was a useful substrate and yielded the bisfluorene 2c in 40% yield. In addition, 9H – xanthene could also be employed to give the desired bixanthene 2d in 45% yield. Arkivoc 2023 (ii) 202312012 Chen, N-Q. et al. Scheme 1. Previous and this work. Results and Discussion At the outset, we assessed the viability of the oxidative reaction of diphenylmethane (1a, 0.2mmol) and AgNO3 ( 0.04 mmol), K2S2O8 (0.2 mmol) in acetonitrile and water (1:1) ratio at 60 ֯ C for 12 h which afforded a homocoupling dimer of tetaphenyl product 2a in 38%. With this result in hand, we prepared a number of derivatives of benzylic substrates (Table 1). Bis (4-fluorophenyl) methane was tolerated under this condition to get the dimer product 2b. Next, cyclic benzylic substrates were examined. The reaction of fluorene was a useful substrate and yielded the bisfluorene 2c in 40% yield. In addition, 9H – xanthene could also be employed to give the desired bixanthene 2d in 45% yield. At the outset, we assessed the viability of the oxidative reaction of diphenylmethane (1a, 0.2mmol) and AgNO3 ( 0.04 mmol), K2S2O8 (0.2 mmol) in acetonitrile and water (1:1) ratio at 60 ֯ C for 12 h which afforded a homocoupling dimer of tetaphenyl product 2a in 38%. With this result in hand, we prepared a number of derivatives of benzylic substrates (Table 1). Bis (4-fluorophenyl) methane was tolerated under this condition to get the dimer product 2b. Next, cyclic benzylic substrates were examined. The reaction of fluorene was a useful substrate and yielded the bisfluorene 2c in 40% yield. In addition, 9H – xanthene could also be employed to give the desired bixanthene 2d in 45% yield. Conclusions We have developed a selective and efficient single protocol for the synthesis of symmetrical tetraphenyl and diaryl ketone derivatives by controlling the equivalence of silver nitrate. The oxidative silver-catalyzed homocoupling was successfully implemented with both acyclic and cyclic substrates, thus extending the scope of the oxidation reaction for the effective production of diaryl ketones. The versatility of synthetic transformations has been made evident through their practical applications. We have developed a selective and efficient single protocol for the synthesis of symmetrical tetraphenyl and diaryl ketone derivatives by controlling the equivalence of silver nitrate. The oxidative silver-catalyzed homocoupling was successfully implemented with both acyclic and cyclic substrates, thus extending the scope of the oxidation reaction for the effective production of diaryl ketones. The versatility of synthetic transformations has been made evident through their practical applications. Page 3 of 10 Arkivoc 2023 (ii) 202312012 Chen, N-Q. et al. Table 1. Tetraphenyl derivatives 2 Table 1. Tetraphenyl derivatives 2 Table 2. Diaryl ketones 3 Page 4 of 10 ©AUTHOR( e 2. Diaryl ketones 3 Next, we embarked on the study of oxidative processes to extend our strategy by selecting silver nitrat valence and observing selective oxidation to give diaryl ketones (Table 2). Diphenylmethane 1a (0 ol), silver nitrate (0.4 mmol), potassium persulfate (0.2 mmol) in acetonitrile and water (1:1) ratio at 60 12-24 h reaction was performed. The yield of product 3a was found to be 70% yield. We assessed th ad applicability of the oxidative transformation by utilizing a range of diaryl methane compounds. Th oduction of fluoro group 3b was tolerated. Electron-deficient diaryl methane derivative was employed a ting substrate, resulting of 3c in a 48% yield. Moreover, the incorporation of a dimethylamine moiety on yl methane substrate was also evaluated, yielding 3d with moderate results. The reaction progresse othly for the cyclic derivatives of fluorene, and 9H-xanthene substrates to give the corresponding keton vatives (3e-f). The oxidation of 9, 10-dihydroanthracene to anthracne-9, 10-dione 3g was observed upo able 2. Diaryl ketones 3 Table 2. Diaryl ketones 3 Table 2. Diaryl ketones 3 Table 2. Diaryl ketones 3 Next, we embarked on the study of oxidative processes to extend our strategy by selecting silver nitrate equivalence and observing selective oxidation to give diaryl ketones (Table 2). Diphenylmethane 1a (0.2 mmol), silver nitrate (0.4 mmol), potassium persulfate (0.2 mmol) in acetonitrile and water (1:1) ratio at 60 ֯ C for 12-24 h reaction was performed. The yield of product 3a was found to be 70% yield. We assessed the broad applicability of the oxidative transformation by utilizing a range of diaryl methane compounds. The introduction of fluoro group 3b was tolerated. Electron-deficient diaryl methane derivative was employed as starting substrate, resulting of 3c in a 48% yield. Moreover, the incorporation of a dimethylamine moiety on a diaryl methane substrate was also evaluated, yielding 3d with moderate results. The reaction progressed smoothly for the cyclic derivatives of fluorene, and 9H-xanthene substrates to give the corresponding ketone derivatives (3e-f). The oxidation of 9, 10-dihydroanthracene to anthracne-9, 10-dione 3g was observed upon Page 4 of 10 Page 4 of 10 Arkivoc 2023 (ii) 202312012 Chen, N-Q. et al. Chen, N-Q. et al. reaction. Page 3 of 10 The process was further demonstrated through its application to the synthesis of a hydrazine derivative from benzophenone, thus illustrating its synthetic utility (Scheme 2). Scheme 2 Synthetic transformations Scheme 2. Synthetic transformations. Scheme 2. Synthetic transformations. Experimental Section General. All chemicals were purchased from commercial vendors (Sigma Aldrich, Alfa Aesar, TCI, and matrix scientific) and used directly without further purification unless otherwise noted. Well-cleaned and oven-dried glassware was used for the experiments. The reaction was monitored by thin layer chromatography (TLC), purchased as pre-coated with silica gel 60 F254 from Merck. Column chromatography was performed using the MACHEREY-NAGEL silica gel 60 mit with a mixture of ethyl acetate/hexane or hexane as an eluent. 1H NMR spectra were recorded on 400 MHz & 600 MHz, 13C-NMR spectra were recorded on 100 MHz & 151 MHz, and Varian Mercury spectrometer using CDCl3 as solvent. The spectra were recorded and presented as chemical shifts (ppm). The multipliers were provided in s (singlet), d (doublet), t (triplet), q (quartet), br (broad singlet), m (multiplet), and dd (doublet of doublet). The coupling constants (J) were reported in Hz. General procedure for synthesis of tetraphenyl derivatives. A 15 mL vial was charged with 1a-d (0.2 mmol), silver nitrate (0.04 mmol), potassium persulfate (0.2 mmol) in acetonitrile and water (1:1) ratio were added. The resulting mixture was stirred under nitrogen atmosphere at 60 °C for 12-24 h and the reaction was carried out. After completion of the reaction extracted with ethyl acetate (3 mL x 5), the organic layer was combined and washed with brine solution (5 mL). The organic layer was dried over anhydrous MgSO4, filtered and concentrated under vacuum. The crude product was purified by column chromatography (Hexane, silica gel) and obtained as solid 2a-2d. Furthermore, the obtained desired products were characterized by NMR, the data are shown given below and the 1H-NMR and 13C-NMR spectra of the products were matched with the literature data. 25, 26 Page 5 of 10 ©AUTHOR(S) ©AUTHOR(S) Page 5 of 10 ©AUTHOR(S) ©AUTHOR(S) Page 5 of 10 Arkivoc 2023 (ii) 202312012 Chen, N-Q. et al. General procedure for synthesis of aryl ketones derivatives. A 15 mL vial was charged with 1a-g (0.2 mmol), silver nitrate (0.4 mmol), potassium persulfate (0.2 mmol) in the acetonitrile and water (1:1) ratio were added. The resulting mixture was stirred under nitrogen atmosphere at 60 °C for 12-24 h and the reaction was carried out. After completion of the reaction extracted with ethyl acetate (3 mL x 5), organic layer combined and washed with brine solution (5 mL). The organic layer was dried over anhydrous MgSO4, filtered and concentrated under vacuum. The crude product was purified by column chromatography (Hexane, silica gel) and obtained as solid 3a-3g. Furthermore, the obtained desired products were characterized by NMR, the data are shown below and the 1H-NMR & 13C-NMR spectra of the products were matched with the previous literatures. 24, 27-29 Arkivoc 2023 (ii) 202312012 Chen, N-Q. et al. Chen, N-Q. et al. General procedure for synthesis of aryl ketones derivatives. A 15 mL vial was charged with 1a-g (0.2 mmol), silver nitrate (0.4 mmol), potassium persulfate (0.2 mmol) in the acetonitrile and water (1:1) ratio were added. The resulting mixture was stirred under nitrogen atmosphere at 60 °C for 12-24 h and the reaction was carried out. After completion of the reaction extracted with ethyl acetate (3 mL x 5), organic layer combined and washed with brine solution (5 mL). The organic layer was dried over anhydrous MgSO4, filtered and concentrated under vacuum. The crude product was purified by column chromatography (Hexane, silica gel) and obtained as solid 3a-3g. Furthermore, the obtained desired products were characterized by NMR, the data are shown below and the 1H-NMR & 13C-NMR spectra of the products were matched with the previous literatures. 24, 27-29 1,1,2,2-Tetraphenylethane (2a).30 After the reaction based on the general procedure: Diphenyl methane (0.2 mmol), silver nitrate (0.04 mmol), potassium persulfate (0.2 mmol) in acetonitrile and water (1:1) ratio were added. The resulting mixture was stirred under nitrogen atmosphere at 60 °C for 12 h. The crude mixture was purified by column chromatography (silica gel) using hexane as solvent and obtained as white solid (26 mg, 38%); mp 225-226 °C ; 1H-NMR (400 MHz, CDCl3) δ 7.18-7.16 (m, 8H), 7.13-7.09 (m, 8H), 7.03-7.00 (m, 4H), 4.78 (s, 2H); 13C-NMR (100 MHz, CDCl3) δ143.6, 128.7, 128.3, 126.0, 56.5. ©AUTHOR(S) The crude mixture was purified by column chromatography (silica gel) using hexane as solvent and obtained as white solid (33 mg, 45%); mp 201-202 °C ; 1H-NMR (400 MHz, CDCl3) δ 7.24-7.20 (m, 4H), 6.96-6.88 (m, 8H), 6.68 (dd, J = 8.0 Hz, 4.0 Hz, 4H), 4.21(s, 2H); 13C-NMR (100 MHz, CDCl3) δ 153.0, 129.1, 128.1, 122.6, 121.8, 115.8, 49.5 9H,9'H-9,9'-Bixanthene (2d).31 After the reaction according to the general procedure: 9H-xanthene (0.2 mmol), silver nitrate (0.04 mmol), potassium persulfate (0.2 mmol) in the ratio of acetonitrile and water (1:1). The resulting mixture was stirred under nitrogen atmosphere at 60 °C for 24 h. The crude mixture was purified by column chromatography (silica gel) using hexane as solvent and obtained as white solid (33 mg, 45%); mp 201-202 °C ; 1H-NMR (400 MHz, CDCl3) δ 7.24-7.20 (m, 4H), 6.96-6.88 (m, 8H), 6.68 (dd, J = 8.0 Hz, 4.0 Hz, 4H), 4.21(s, 2H); 13C-NMR (100 MHz, CDCl3) δ 153.0, 129.1, 128.1, 122.6, 121.8, 115.8, 49.5 9H,9'H-9,9'-Bixanthene (2d).31 After the reaction according to the general procedure: 9H-xanthene (0.2 mmol), silver nitrate (0.04 mmol), potassium persulfate (0.2 mmol) in the ratio of acetonitrile and water (1:1). The resulting mixture was stirred under nitrogen atmosphere at 60 °C for 24 h. The crude mixture was purified by column chromatography (silica gel) using hexane as solvent and obtained as white solid (33 mg, 45%); mp 201-202 °C ; 1H-NMR (400 MHz, CDCl3) δ 7.24-7.20 (m, 4H), 6.96-6.88 (m, 8H), 6.68 (dd, J = 8.0 Hz, 4.0 Hz, 4H), 4.21(s, 2H); 13C-NMR (100 MHz, CDCl3) δ 153.0, 129.1, 128.1, 122.6, 121.8, 115.8, 49.5 Benzophenone (3a).32 After the reaction based on the general procedure: Diphenyl methane (0.2 mmol), silver nitrate (0.4 mmol), potassium persulfate (0.2 mmol) was added in acetonitrile and water (1: 1) ratio. The resulting mixture was stirred under nitrogen atmosphere at 60 °C for 24 h. The crude mixture was purified by column chromatography (silica gel) using hexane as solvent and obtained as white solid (25 mg, 70%); mp 48- 49 °C ; 1H-NMR (400 MHz, CDCl3) δ 7.82-7.80 (m, 4H), 7.61-7.58 (m, 2H), 7.51-7.47 (m,4H); 13C-NMR (100 MHz, CDCl3) δ 196.9, 137.7, 132.6, 130.2, 128.4. Benzophenone (3a).32 After the reaction based on the general procedure: Diphenyl methane (0.2 mmol), silver nitrate (0.4 mmol), potassium persulfate (0.2 mmol) was added in acetonitrile and water (1: 1) ratio. ©AUTHOR(S) ( ) ( ) 1,1,2,2-Tetrakis(4-fluorophenyl)ethane (2b).30 After the reaction according to the general procedure: bis(4- fluorophenyl)methane (0.2 mmol), silver nitrate (0.04 mmol), potassium persulfate (0.2 mmol) in acetonitrile and water (1:1) ratio were added. The resulting mixture was stirred under nitrogen atmosphere at 60 °C for 12 h. The crude mixture was purified by column chromatography (silica gel) using hexane as solvent and obtained as white solid (25 mg, 30%); mp 312-313 °C ;1H-NMR (600 MHz, CDCl3) δ 7.05-7.03 (m, 8H),6.84-6.81 (m, 8H), 4.62 (s, 2H); 13C-NMR (151 MHz, CDCl3) δ 161.3 (d, JC-F = 246.5 Hz), 138.7 (d, JC-F = 3 Hz), 129.8 (d, JC-F = 8.0 Hz), 115.5 (d, JC-F = 21 Hz), 55.3. 1,1,2,2-Tetrakis(4-fluorophenyl)ethane (2b).30 After the reaction according to the general procedure: bis(4- fluorophenyl)methane (0.2 mmol), silver nitrate (0.04 mmol), potassium persulfate (0.2 mmol) in acetonitrile and water (1:1) ratio were added. The resulting mixture was stirred under nitrogen atmosphere at 60 °C for 12 h. The crude mixture was purified by column chromatography (silica gel) using hexane as solvent and obtained as white solid (25 mg, 30%); mp 312-313 °C ;1H-NMR (600 MHz, CDCl3) δ 7.05-7.03 (m, 8H),6.84-6.81 (m, 8H), 4.62 (s, 2H); 13C-NMR (151 MHz, CDCl3) δ 161.3 (d, JC-F = 246.5 Hz), 138.7 (d, JC-F = 3 Hz), 129.8 (d, JC-F = 8.0 Hz), 115.5 (d, JC-F = 21 Hz), 55.3. 9H,9'H-9,9'-Bifluorene (2c).7 After the reaction according to the general procedure: 9H-fluorene (0.2 mmol), silver nitrate (0.04 mmol), potassium persulfate (0.2 mmol) in the acetonitrile and water (1:1) ratio were added. The resulting mixture was stirred under nitrogen atmosphere at 60 °C for 24 h. The crude mixture was purified by column chromatography (silica gel) using hexane as solvent and obtained as white solid (26.5 mg, 40%); mp 241-242 °C ; 1H-NMR (400 MHz, CDCl3) δ 7.65 (d, J = 8.0 Hz, 4H), 7.29 (d, J = 8.0 Hz, 4H),7.10 (t, J = 8.0 Hz, 4H), 6.96 (d, J = 8.0 Hz, 4H), 4.85(s,2H); 13C-NMR (100 MHz, CDCl3) δ 144.6, 141.5, 127.3, 126.7, 124.0, 119.6, 49.8 9H,9'H-9,9'-Bixanthene (2d).31 After the reaction according to the general procedure: 9H-xanthene (0.2 mmol), silver nitrate (0.04 mmol), potassium persulfate (0.2 mmol) in the ratio of acetonitrile and water (1:1). The resulting mixture was stirred under nitrogen atmosphere at 60 °C for 24 h. ©AUTHOR(S) The resulting mixture was stirred under nitrogen atmosphere at 60 °C for 24 h. The crude mixture was purified by column chromatography (silica gel) using hexane as solvent and obtained as white solid (25 mg, 70%); mp 48- 49 °C ; 1H-NMR (400 MHz, CDCl3) δ 7.82-7.80 (m, 4H), 7.61-7.58 (m, 2H), 7.51-7.47 (m,4H); 13C-NMR (100 MHz, CDCl3) δ 196.9, 137.7, 132.6, 130.2, 128.4. Bis(4-fluorophenyl)methanone (3b).33 After the reaction according to the general procedure: bis(4- fluorophenyl)methane (0.2 mmol), silver nitrate (0.4 mmol), potassium persulfate (0.2 mmol) in the acetonitrile and water (1:1) ratio were added. The resulting mixture was stirred under nitrogen atmosphere at ©AUTHOR(S) Page 6 of 10 Page 6 of 10 Arkivoc 2023 (ii) 202312012 Chen, N-Q. et al. Chen, N-Q. et al. 60 °C for 24 h. The crude mixture was purified by column chromatography (silica gel) using hexane as solvent and obtained as white solid (22 mg, 50%); mp 101-102 °C ; 1H-NMR (400 MHz, CDCl3) δ 7.84-7.80 (m, 4H), 7.19-7.15 (m, 4H); 13C-NMR (100 MHz, CDCl3) δ 193.8, 165.3 (d, JC-F 254 Hz), 133.7 (d, JC-F 3 Hz), 132.5 (d, JC-F 8 Hz), 115.5 (d, JC-F 22 Hz) 60 °C for 24 h. The crude mixture was purified by column chromatography (silica gel) using hexane as solvent and obtained as white solid (22 mg, 50%); mp 101-102 °C ; 1H-NMR (400 MHz, CDCl3) δ 7.84-7.80 (m, 4H), 7.19-7.15 (m, 4H); 13C-NMR (100 MHz, CDCl3) δ 193.8, 165.3 (d, JC-F 254 Hz), 133.7 (d, JC-F 3 Hz), 132.5 (d, JC-F 8 Hz), 115.5 (d, JC-F 22 Hz) Phenyl(2-(trifluoromethyl)phenyl)methanone (3c).34After the reaction according to the general procedure: 1- benzyl-2-(trifluoromethyl)benzene (0.2 mmol), silver nitrate (0.4 mmol), potassium persulfate (0.2 mmol) in acetonitrile and water (1:1) ratio were added. The resulting mixture was stirred under nitrogen atmosphere at 60 °C for 24 h. The crude mixture was purified by column chromatography (silica gel) using hexane as solvent and obtained as white solid (24 mg, 48%); mp 60-61 °C ;1H-NMR (400 MHz, CDCl3) δ 7.79-7.77 (m, 3H), 7.64- 7.58 (m, 3H), 7.47-7.43 (m, 2H), 7.40-7.37 (m, 1H); 13C-NMR (100 MHz, CDCl3) δ 198.9, 138.4, 136.5, 134.0, 131.5, 130.3, 129.9, 128.6, 128.2, 126.8 (q, JC-F =4.6 Hz), 123.7 (d, JC-F 272.2Hz) (4-(Dimethylamino)phenyl)(phenyl)methanone (3d).32 After the reaction according to the general procedure: 4-benzyl-N,N-dimethylaniline (0.2 mmol), silver nitrate (0.4 mmol), potassium persulfate (0.2 mmol) in acetonitrile and water (1:1) ratio were added. ©AUTHOR(S) Anthracene-9,10-dione (3g).37 After the reaction according to the general procedure: 9,10-dihydroanthracene (0.2 mmol), silver nitrate (0.4 mmol), potassium persulfate (0.2 mmol) in acetonitrile and water (1:1) ratio were added. The resulting mixture was stirred under nitrogen atmosphere at 60 °C for 24 h. The crude mixture was purified by column chromatography (silica gel) using hexane as solvent and obtained as white solid (22 mg, 52%); mp 283-285 °C ; 1H-NMR (600 MHz, CDCl3) δ 8.33 (q, J 6.0 Hz, 4H), 7.82 (q, J 6.0 Hz, 4H); 13C-NMR (151 MHz, CDCl3) δ 183.3, 134.3, 133.7, 127.4. (Diphenylmethylene)hydrazine (4a).38 A mixture of benzophenone (0.2 mmol), hydrazine monohydrate (2.0 equiv.), and acetic acid (0.06 mmol) was stirred at 100 °C for 21 h. After cooled to room temperature, solvent and volatile materials were removed under reduced pressure to afford corresponding product as a white solid (35 mg, 90%) ; mp 94-95 °C ; 1H-NMR (400 MHz, CDCl3) δ 7.52-7.44 (m, 5H), 7.29-7.26 (m, 5H), 5.42 (bs, 2H); 13C-NMR (100 MHz, CDCl3) δ 149.2, 138.5, 133.1, 129.5, 128.2, 126.6. Page 7 of 10 ©AUTHOR(S) The resulting mixture was stirred under nitrogen atmosphere at 60 °C for 12 h. The crude mixture was purified by column chromatography (silica gel) using hexane as solvent and obtained as white solid (21 mg, 46%); mp 89-90 °C ;1H-NMR (400 MHz, CDCl3) δ 7.80 (d, J 8.0 Hz, 2H), 7.73 (d, J 4.0 Hz, 2H), 7.53-7.473 (m, 3H), 6.68 (d, J 8.0 Hz, 2H); 13C-NMR (100 MHz, CDCl3) δ 195.3, 153.4, 139.4, 132.9, 131.2, 129.6, 128.1, 124.9, 110.7, 40.2. 9H-Fluoren-9-one (3e).35 After the reaction according to the general procedure: 9H-fluorene (0.2 mmol), silver nitrate (0.4 mmol), potassium persulfate (0.2 mmol) in acetonitrile and water (1:1) ratio were added. The resulting mixture was stirred under nitrogen atmosphere at 60 °C for 12 h. The crude mixture was purified by column chromatography (silica gel) using hexane as solvent and obtained as white solid (21 mg, 58%); mp 82- 83 °C ;1H-NMR (400 MHz, CDCl3) δ 7.76 (d, J 7.4 Hz, 2H), 7.54-7.47 (m, 4H), 7.30 (td, J 7.36 Hz, J 1.24Hz, 2H); 13C-NMR (100 MHz, CDCl3) δ 192.4, 144.6, 134.8, 134.3, 129.2, 124.5, 120.4. 9H-Xanthen-9-one (3f).36 After the reaction according to the general procedure: 9H-xanthene (0.2 mmol), silver nitrate (0.4 mmol), potassium persulfate (0.2 mmol) was added in acetonitrile and water (1: 1) ratio. The resulting mixture was stirred under nitrogen atmosphere at 60 °C for 24 h. The crude mixture was purified by column chromatography (silica gel) using hexane as solvent and obtained as white solid (22 mg, 56%); mp 171- 172 °C ; 1H-NMR (600 MHz, CDCl3) δ 8.36-8.35 (m, 2H), 7.75-7.72 (m, 2H), 7.52-7.50 (m, 2H), 7.41-7.38 (m, 2H); 13C-NMR (151 MHz, CDCl3) δ177.4, 156.4, 135.0, 126.9, 124.1, 118.1, 116.7. Anthracene-9,10-dione (3g).37 After the reaction according to the general procedure: 9,10-dihydroanthracene (0.2 mmol), silver nitrate (0.4 mmol), potassium persulfate (0.2 mmol) in acetonitrile and water (1:1) ratio were added. The resulting mixture was stirred under nitrogen atmosphere at 60 °C for 24 h. The crude mixture was purified by column chromatography (silica gel) using hexane as solvent and obtained as white solid (22 mg, 52%); mp 283-285 °C ; 1H-NMR (600 MHz, CDCl3) δ 8.33 (q, J 6.0 Hz, 4H), 7.82 (q, J 6.0 Hz, 4H); 13C-NMR (151 MHz, CDCl3) δ 183.3, 134.3, 133.7, 127.4. ©AUTHOR(S) Page 7 of 10 ©AUTHOR(S) Page 7 of 10 Page 7 of 10 Arkivoc 2023 (ii) 202312012 Chen, N-Q. et al. Acknowledgements The authors gratefully acknowledge funding from the National Science and Technology Council (MOST 111- 2113-M-037-016, Taiwan) and Kaohsiung Medical University “NSYSU-KMU JOINT RESEARCH PROJECT” (112- NK112P20) The authors also wish to thank the Centre for Research Resources and Development of Kaohsiung Medical University for Mass and 400 MHz NMR analyses. Arkivoc 2023 (ii) 202312012 Chen, N-Q. et al. Acknowledgements The authors gratefully acknowledge funding from the National Science and Technology Council (MOST 111- 2113-M-037-016, Taiwan) and Kaohsiung Medical University “NSYSU-KMU JOINT RESEARCH PROJECT” (112- NK112P20) The authors also wish to thank the Centre for Research Resources and Development of Kaohsiung Medical University for Mass and 400 MHz NMR analyses. The authors gratefully acknowledge funding from the National Science and Technology Council (MOST 111- 2113-M-037-016, Taiwan) and Kaohsiung Medical University “NSYSU-KMU JOINT RESEARCH PROJECT” (112- NK112P20) The authors also wish to thank the Centre for Research Resources and Development of Kaohsiung Medical University for Mass and 400 MHz NMR analyses. Supplementary Material For NMR spectra see the Supplementary Material. For NMR spectra see the Supplementary Material. https://doi.org/10.1021/ol501619w 11. Zhu, Y.; Xiong, T.; Han, W.; Shi, Y. Org. Lett. 2014, 16, 6144-6147. https://doi.org/10.1021/ol5030103 11. Zhu, Y.; Xiong, T.; Han, W.; Shi, Y. Org. Lett. 2014, 16, 6144 6147. https://doi.org/10.1021/ol5030103 12. Mandal, S.; Bera, T.; Dubey, G.; Saha, J.; Laha, J. K. ACS Catalysis 2018, 8, 5085-5144. https://doi.org/10.1021/acscatal.8b00743 https://doi.org/10.1002/anie.201612548 6. Kim, Y. J.; Kim, S. M.; Cho, E. J.; Hosono, H.; Yang, J. W.; Kim, S. W. Chem. Sci. 2015, 6, 3577-3581. https://doi.org/10.1039/C5SC00933B 6. Kim, Y. J.; Kim, S. M.; Cho, E. J.; Hosono, H.; Yang, J. W.; Kim, S. W. Chem. Sci. 2015, 6, 3577-3581. h //d i /10 1039/C5SC00933B 7. Sahoo, S. K. Tetrahedron Lett. 2016, 57, 3476-3480. h //d i /10 1016/j l 2016 06 092 7. Sahoo, S. K. Tetrahedron Lett. 2016, 57, 3476-3480. https://doi.org/10.1016/j.tetlet.2016.06.092 7. Sahoo, S. K. Tetrahedron Lett. 2016, 57, 3476-3480. https://doi.org/10.1016/j.tetlet.2016.06.092 8. Lei, A.; Zhang, X. Org. Lett. 2002, 4, 2285-2288. https://doi.org/10.1021/ol0258536 9. Manley, D. W.; Walton, J. C. Org. Lett. 2014, 16, 5394-5397. https://doi.org/10.1021/ol502625w 9. Manley, D. W.; Walton, J. C. Org. Lett. 2014, 16, 5394 5397. https://doi.org/10.1021/ol502625w 10. Sato, K.; Inoue, Y.; Mori, T.; Sakaue, A.; Tarui, A.; Omote, M.; Kumadaki, I.; Ando, A. Org. Lett. 2014, 16, 3756-3759. References 1. Liu, C.; Jin, L.; Lei, A. Synlett 2010, 2010, 2527-2536. https://doi.org/10.1055/s-0030-1258802 p // g/ / 2. Sheldon, R. A.; Kochi, J. K. Metal-catalyzed oxidations of organic compounds in the liquid phase: A mechanistic approach. In Advances in catalysis, Vol. 25; Elsevier, 1976; pp 272-413. https://doi.org/10.1016/S0360-0564(08)60316-8 3 W C Y S R J Xi Y X Li J H S th i 2016 223 230 2. Sheldon, R. A.; Kochi, J. K. Metal-catalyzed oxidations of organic compounds in the liquid phase: A mechanistic approach. In Advances in catalysis, Vol. 25; Elsevier, 1976; pp 272-413. https://doi.org/10.1016/S0360-0564(08)60316-8 3. Wang, C.-Y.; Song, R.-J.; Xie, Y.-X.; Li, J.-H. Synthesis 2016, 223-230. https://www.thieme-connect.com/products/ejournals/abstract/10.1055/s-0035-156037 4. Wang, Q.; Zhang, W.-W.; Song, H.; Wang, J.; Zheng, C.; Gu, Q.; You, S.-L. J. Am. Chem. Soc. 2020, 142, 15678- 15685. htt //d i /10 1021/j 0 08205 4. Wang, Q.; Zhang, W.-W.; Song, H.; Wang, J.; Zheng, C.; Gu, Q.; You, S.-L. J. Am. Chem. Soc. 2020, 142, 15678- 15685. // / / 5. Gieshoff, T. N.; Chakraborty, U.; Villa, M.; Jacobi von Wangelin, A. Angew. Chem. Int. Ed. 2017, 56, 3585- 3589. htt //d i /10 1002/ i 201612548 5. Gieshoff, T. N.; Chakraborty, U.; Villa, M.; Jacobi von Wangelin, A. Angew. Chem. Int. Ed. 2017, 56, 3585- 3589. https://doi.org/10.1002/anie.201612548 For NMR spectra see the Supplementary Material. https://doi.org/10.1021/ja710349j Zhou, Q.; Wei, S.; Han, W. J. Org. Chem. 2014, 79, 1454 1460. https://doi.org/10.1021/jo402366p 30. Pan, F.-F.; Guo, P.; Huang, X.; Shu, X.-Z. Synthesis 2021, 53, 3094-3100. 30. Pan, F.-F.; Guo, P.; Huang, X.; Shu, X.-Z. Synthesis 2021, 53, 3094-3100. https://doi.org/10.1055/a-1467-2432 31. Schönberg, A.; Singer, E.; Stephan, W.; Sheldrick, W. S. Tetrahedron 1983, 39, 2429-2437. https://doi.org/10.1016/S0040-4020(01)91970-2 31. Schönberg, A.; Singer, E.; Stephan, W.; Sheldrick, W. S. Tetrahedron 1983, 39, 2429-2437. htt //d i /10 1016/S0040 4020(01)91970 2 32. Mkrtchyan, S.; Purohit, V. B.; Khutsishvili, S.; Nociarová, J.; Yar, M.; Mahmood, T.; Ayub, K.; Budzák, Š.; Skoršepa, M.; Iaroshenko, V. O. Adv. Synth. Catal. 2023, 365, 2026-2035. https://doi.org/10.1002/adsc.202300260 32. Mkrtchyan, S.; Purohit, V. B.; Khutsishvili, S.; Nociarová, J.; Yar, M.; Mahmood, T.; Ayub, K.; Budzák, Š.; Skoršepa, M.; Iaroshenko, V. O. Adv. Synth. Catal. 2023, 365, 2026-2035. 33. Wang, L.; Yu, J.; Duan, Z.; Jin, J.; Zhang, Y. Org. Biomol. Chem. 2022, 20, 6554-6557. https://doi.org/10.1039/D2OB01275H Page 8 of 10 Arkivoc 2023 (ii) 202312012 Chen, N-Q. et al. Chen, N-Q. et al. 13. Mir, B. A.; Rajamanickam, S. Potassium Persulfate as an Eco-Friendly Oxidant for Oxidative Transformations. In Green Chemistry-New Perspectives, IntechOpen, 2022. 14. Syper, L. Tetrahedron Lett. 1967, 8, 4193-4198. 14. Syper, L. Tetrahedron Lett. 1967, 8, 4193-4198. https://doi.org/10.1016/S0040-4039(01)89720-3 14. Syper, L. Tetrahedron Lett. 1967, 8, 4193-4198. https://doi.org/10.1016/S0040-4039(01)89720-3 15. Luque-Ortega, J. R.; Reuther, P.; Rivas, L.; Dardonville, C. J. Med. Chem. 2010, 53, 1788-1798. https://doi.org/10.1021/jm901677h 15. Luque-Ortega, J. R.; Reuther, P.; Rivas, L.; Dardonville, C. J. Med. Chem. 2010, 53, 1788-1798. h //d i /10 1021/j 901677h p // g/ /j 16. Yang, H.-B.; Ghosh, K.; Zhao, Y.; Northrop, B. H.; Lyndon, M. M.; Muddiman, D. C.; White, H. S.; Stang, P. J. J. Am. Chem. Soc. 2008, 130, 839-841. 16. Yang, H.-B.; Ghosh, K.; Zhao, Y.; Northrop, B. H.; Lyndon, M. M.; Muddiman, D. C.; White, H. S.; Stang, P. J. J. Am. Chem. Soc. 2008, 130, 839-841. https://doi.org/10.1021/ja710349j https://doi.org/10.1021/ja710349j 17. Chebolu, R.; Bahuguna, A.; Sharma, R.; Mishra, V. K.; Ravikumar, P. Chem. Commun. 2015, 51, 15438-15441. https://doi.org/10.1039/C5CC05713B 18. Li, M.; Wang, C.; Ge, H. Org. Lett. 2011, 13, 2062-2064. https://doi.org/10.1021/ol200459v 18. Li, M.; Wang, C.; Ge, H. Org. Lett. 2011, 13, 2062 2064. https://doi.org/10.1021/ol200459v https://doi.org/10.1021/ol200459v 19. Kamijo, S.; Tao, K.; Takao, G.; Tonoda, H.; Murafuji, T. Org. Lett. 2015, 17, 3326-3329. https://doi.org/10.1021/acs.orglett.5b01550 20. Catino, A. J.; Nichols, J. M.; Choi, H.; Gottipamula, S.; Doyle, M. P. Org. Lett. 2005, 7, 5167-5170. https://doi.org/10.1021/ol0520020 20. Catino, A. J.; Nichols, J. M.; Choi, H.; Gottipamula, S.; Doyle, M. P. Org. Lett. 2005, 7, 5167-5170. 21. Murahashi, S.-I.; Komiya, N.; Oda, Y.; Kuwabara, T.; Naota, T. J. Org. Chem. 2000, 65, 9186-9193. h //d i /10 1021/j 001348f 21. Murahashi, S.-I.; Komiya, N.; Oda, Y.; Kuwabara, T.; Naota, T. J. Org. Chem. 2000, 65, 9186-9193. https://doi.org/10.1021/jo001348f 22. Wu, X.-F. Tetrahedron Lett. 2012, 53, 6123-6126. https://doi.org/10.1016/j.tetlet.2012.08.149 23. Xie, Y.; Yang, Y.; Huang, L.; Zhang, X.; Zhang, Y. Org. Lett. 2012, 14, 1238-1241. https://doi.org/10.1021/ol300037p , ; g, ; g, ; g, ; g, g , , https://doi.org/10.1021/ol300037p 24 W H W Z H H T J X K O L 2016 18 5680 5683 24. Wang, H.; Wang, Z.; Huang, H.; Tan, J.; Xu, K. Org. Lett. 2016, 18, 5680-5683. https://doi.org/10.1021/acs.orglett.6b02914 24. Wang, H.; Wang, Z.; Huang, H.; Tan, J.; Xu, K. Org. Lett. 2016, 18, 5680 5683. https://doi.org/10.1021/acs.orglett.6b02914 25. Li, P.-C.; Wang, T.-S.; Lee, G.-H.; Liu, Y.-H.; Wang, Y.; Chen, C.-T.; Chao, I. J. Org. Chem. 2002, 67, 8002-8009. https://doi.org/10.1021/jo020196g 25. Li, P.-C.; Wang, T.-S.; Lee, G.-H.; Liu, Y.-H.; Wang, Y.; Chen, C.-T.; Chao, I. J. Org. Chem. 2002, 67, 8002-8009. p g j g 26. Okajima, M.; Soga, K.; Nokami, T.; Suga, S.; Yoshida, J.-i. Org. Lett. 2006, 8, 5005-5007. // / / 26. Okajima, M.; Soga, K.; Nokami, T.; Suga, S.; Yoshida, J.-i. Org. Lett. 2006, 8, 5005-5007. https://doi.org/10.1021/ol061647c 27. Natarajan, P.; Vagicherla, V. D.; Vijayan, M. T. Tetrahedron Lett. 2014, 55, 5817-5821. 27. Natarajan, P.; Vagicherla, V. D.; Vijayan, M. T. Tetrahedron Lett. 2014, 55, 5817-5821. https://doi.org/10.1016/j.tetlet.2014.08.121 28. Zhang, J.; Du, J.; Zhang, C.; Liu, K.; Yu, F.; Yuan, Y.; Duan, B.; Liu, R. Org. Lett. 2022, 24, 1152-1157. https://doi.org/10.1021/acs.orglett.1c04154 https://doi.org/10.1021/acs.orglett.1c04154 29 Zhou Q Wei S Han W J Org Chem 2014 79 1454 1460 29. Zhou, Q.; Wei, S.; Han, W. J. Org. Chem. 2014, 79, 1454-1460. https://doi.org/10.1021/jo402366p 29. ©AUTHOR(S) Page 9 of 10 Arkivoc 2023 (ii) 202312012 Chen, N-Q. et al. 34. Buu‐Hoï, N.; Nam, N.; Xuong, N. Recl. Trav. Chim. Pays-Bas 1966, 85, 367-372. https://doi.org/10.1002/recl.19660850407 34. Buu‐Hoï, N.; Nam, N.; Xuong, N. Recl. Trav. Chim. Pays-Bas 1966, 85, 367-372. https://doi.org/10.1002/recl.19660850407 35. Liu, C.; Yu, J.; Bao, L.; Zhang, G.; Zou, X.; Zheng, B.; Li, Y.; Zhang, Y. J. Org. Chem. 2023, 88, 3794-3801. 35. Liu, C.; Yu, J.; Bao, L.; Zhang, G.; Zou, X.; Zheng, B.; Li, Y.; Zhang, Y. J. Org. Chem. 2023, 88, 3794-3801. htt //d i /10 1021/ j 2 03071 35. Liu, C.; Yu, J.; Bao, L.; Zhang, G.; Zou, X.; Zheng, B.; Li, Y.; Zhang, Y. J. Org. Chem. 2023, 88, 3794-3801. https://doi.org/10.1021/acs.joc.2c03071 35. Liu, C.; Yu, J.; Bao, L.; Zhang, G.; Zou, X.; Zheng, B.; Li, Y.; Zhang, Y. J. Org. Chem. 2023, 88, 3794-3801. https://doi.org/10.1021/acs.joc.2c03071 36. Pradhan, S.; Sharma, V.; Chatterjee, I. Org. Lett. 2021, 23, 6148-6152. https://doi.org/10.1021/acs.orglett.1c02272 36. Pradhan, S.; Sharma, V.; Chatterjee, I. Org. Lett. 2021, 23, 6148-6152. https://doi.org/10.1021/acs.orglett.1c02272 36. Pradhan, S.; Sharma, V.; Chatterjee, I. Org. Lett. 2021, 23, 6148-6152. h //d i /10 1021/ l 1 02272 37. Yahuaca-Juárez, B.; González, G.; Ramírez-Morales, M. A.; Alba-Betancourt, C.; Deveze-Álvarez, M. A.; Mendoza-Macías, C. L.; Ortiz-Alvarado, R.; Juárez-Ornelas, K. A.; Solorio-Alvarado, C. R.; Maruoka, K. Synth Commun 2020, 50, 539-548. https://doi.org/10.1080/00397911.2019.1707225 37. Yahuaca-Juárez, B.; González, G.; Ramírez-Morales, M. A.; Alba-Betancourt, C.; Deveze-Álvarez, M. A.; Mendoza-Macías, C. L.; Ortiz-Alvarado, R.; Juárez-Ornelas, K. A.; Solorio-Alvarado, C. R.; Maruoka, K. Synth Commun 2020, 50, 539-548. https://doi org/10 1080/00397911 2019 1707225 38. Bhosle, A. A.; Banerjee, M.; Hiremath, S. D.; Bhasikuttan, A. C.; Chatterjee, A Chem Asian J. 2023, 18, e202300048 https://onlinelibrary.wiley.com/doi/full/10.1002/asia.202300048 38. Bhosle, A. A.; Banerjee, M.; Hiremath, S. D.; Bhasikuttan, A. C.; Chatterjee, A Chem Asian J. 2023, 18, e202300048 https://onlinelibrary.wiley.com/doi/full/10.1002/asia.202300048 This paper is an open access article distributed under the terms of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/) This paper is an open access article distributed under the terms of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/) This paper is an open access article distributed under the terms of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/) Page 10 of 10 Page 10 of 10
https://openalex.org/W2833068522	https://www.nature.com/articles/s41598-018-29062-w.pdf	English	null	Buparlisib is a brain penetrable pan-PI3K inhibitor	Scientific reports	2,018	cc-by	6,848	Buparlisib is a brain penetrable pan- PI3K inhibitor Mark C. de Gooijer 1,2, Ping Zhang1,2,3, Levi C. M. Buil1,2, Ceren H. Çitirikkaya1,2, Nishita Thota1,2, Jos H. Beijnen1,4,5 & Olaf van Tellingen 1,2 Mark C. de Gooijer 1,2, Ping Zhang1,2,3, Levi C. M. Buil1,2, Ceren H. Çitirikkaya1,2, Nishita Thota1,2, Jos H. Beijnen1,4,5 & Olaf van Tellingen 1,2 Received: 30 January 2018 Accepted: 4 July 2018 Published: xx xx xxxx Characterization of the genomic landscapes of intracranial tumours has revealed a clear role for the PI3K-AKT-mTOR pathway in tumorigenesis and tumour maintenance of these malignancies, making phosphatidylinositol 3-kinase (PI3K) inhibition a promising therapeutic strategy for these tumours. Buparlisib is a novel pan-PI3K inhibitor that is currently in clinical development for various cancers, including primary and secondary brain tumours. Importantly however, earlier studies have revealed that sufficient brain penetration is a prerequisite for antitumor efficacy against intracranial tumours. We therefore investigated the brain penetration of buparlisib using a comprehensive set of in vitro and in vivo mouse models. We demonstrate that buparlisib has an excellent brain penetration that is unaffected by efflux transporters at the blood-brain barrier, complete oral bioavailability and efficient intracranial target inhibition at clinically achievable plasma concentrations. Together, these characteristics make buparlisib the ideal candidate for intracranially-targeted therapeutic strategies that involve PI3K inhibition. Phosphatidylinositol 3-kinase (PI3K) is a key component of the PI3K-AKT-mTOR pathway and as such impor- tant for cell proliferation and survival1. The pivotal role of PI3K in this signalling makes it an attractive anticancer target, especially in tumours harbouring an overactivated PI3K pathway2. Overactivation of the PI3K pathway is seen in many cancers, including primary intracranial cancers such as glioblastoma (GBM)3, diffuse intrinsic pontine glioma4 and paediatric high-grade glioma4, and cancers that frequently metastasize to the brain such as cutaneous melanoma5 and breast cancer6. PI3K inhibition has thus been proposed as a promising treatment strategy for various intracranial tumours7–9.fi gy We and others have previously demonstrated that modest efficacy of PI3K inhibition can be achieved in pre- clinical mouse models of glioblastoma and brain metastases, but only if the compound used to inhibit PI3K exhibits sufficient brain penetration10–14. Therefore, it is important to assess whether a PI3K inhibitor has suffi- cient brain penetration prior to starting its development for treatment of intracranial cancers.h The brain penetration of a small molecular compound is generally restricted by the blood-brain barrier (BBB), which is composed of the brain endothelial cells (BECs) that are being supported by astrocytes and pericytes15. 1Division of Pharmacology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX, Amsterdam, The Netherlands. 2Mouse Cancer Clinic, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX, Amsterdam, The Netherlands. 3Department of Neurosurgery, Qilu Hospital, Shandong University, Wenhua Xi Road 107, 250012, Jinan, P.R. China. 4Department of Pharmacy and Pharmacology, The Netherlands Cancer Institute / MC Slotervaart Hospital, Louwesweg 6, 1066 EC, Amsterdam, The Netherlands. 5Division of Pharmacoepidemiology and Clinical Pharmacology, Department of Pharmaceutical Sciences, Faculty of Science, Utrecht University, Universiteitsweg 99, 3584 CG, Utrecht, The Netherlands. Correspondence and requests for materials should be addressed to O.v.T. (email: o.v.tellingen@nki.nl) www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Results Remarkably, the brain concentration of buparlisib was even slightly higher than the concentrations in other well-perfused organs that contain fenestrated endothelium such as the liver, kidney and spleen (Fig. 2D–F). Buparlisib exhibits excellent oral bioavailability and achieves intracranial target inhibi- tion. Buparlisib is given orally in both preclinical19 and clinical studies21, since it is claimed to have excellent oral bioavailability17,18,21. However, no data supporting this claim are available in the public domain. We therefore set out to determine the systemic exposure and brain concentrations after oral administration of buparlisib in mice.it First, we could confirm that buparlisib also achieves excellent brain penetration after oral administration, since the observed brain-plasma ratio in WT FVB mice was in line with those obtained following i.v. administra- tion (Figs 2C, 3A). Interestingly, a dose-proportional increase in plasma and brain concentrations was observed, yielding similar brain-plasma ratios at three different dose levels. Although we only measured one time point, these data suggest that buparlisib displays linear pharmacokinetics between the 1 mg/kg and 5 mg/kg dose levels in mice. Next, we studied the intracranial pharmacodynamics of buparlisib in WT FVB mice as measured by target inhibition. Immunoblotting of brain lysates revealed that buparlisib dose-dependently decreased the phosphoryl- ation of AKT, the main target of PI3K, without affecting signalling through ERK (Fig. 3B). Importantly, efficient intracranial target inhibition could be reached in mice at clinically relevant plasma concentrations, since 5 mg/kg oral buparlisib resulted in plasma levels of approximately 1200 ng/mL (Fig. 3A)21. p p pp y g g Finally, we studied the pharmacokinetics of buparlisib in male and female mice and assessed the oral bioavail- ability in both genders by comparing the area-under-the-plasma concentration-time-curves (AUCs) following intravenous and oral administration. Both after oral and i.v. administration there was no difference in phar- macokinetics between male and female mice (Fig. 3C, Table 1). Strikingly, buparlisib displayed complete oral bioavailability since no difference in dose-corrected AUC could be observed between i.v. and oral administration in both genders. Interestingly, buparlisib exhibits a relatively small volume of distribution being less than 1 L/kg (Table 1), which is unusual for a small-molecule kinase inhibitor27. We therefore also assessed the buparlisib concentrations in a range of other tissues and indeed found that the levels were equal (liver) or even lower (kid- ney, spleen) than concurrent plasma levels (Fig. 2D–F). Results Buparlisib is only very weakly transported by murine BCRP in vitro. We first sought to investigate whether buparlisib (Fig. 1A) is transported by P-gp or BCRP in vitro using concentration equilibrium trans- port assays (CETAs). CETAs are sensitive assays to determine translocation over a cellular monolayer and are frequently used to determine substrate affinity for ABC transporters by comparing cell lines that overexpress ABC transporters with their parental counterparts24–26. Since P-gp and BCRP are apically located transporters, a prerequisite for detecting substrate affinity for these transporters in a CETA is the capacity of a molecule to pen- etrate cell membranes. We therefore first confirmed that buparlisib could efficiently diffuse over a cellular mon- olayer using a conventional transwell set-up. In this set-up, buparlisib plateaued to almost complete equilibrium between the apical and basal compartments within 4 hours, independent of the direction of diffusion (Fig. 1B). This diffusion was studied in the parental LLC-PK1 cell line and in presence of the P-gp inhibitor zosuquidar to avoid any confounding effects of porcine P-gp on buparlisib diffusion. y gf p gp pf Next, we studied buparlisib translocation in CETAs using cell lines that overexpress murine P-gp (Abcb1a/ Mdr1a), murine BCRP (Abcg2/Bcrp1) or their human orthologues (ABCB1/MDR1, ABCG2). Only a minimal buparlisib translocation was observed in the MDCK-Bcrp1 CETA, whereas no translocation was found in any of the other CETAs, indicating that buparlisib is not a substrate of human P-gp, murine P-gp and human BCRP and a very weak substrate of murine BCRP (Fig. 1C). Note that functionality of the cell lines was confirmed as these assays were also performed in parallel using other compounds that did show basolateral to apical translocation. Buparlisib brain penetration is not restricted by P-gp and BCRP in vivo. To study whether P-gp and BCRP attenuate buparlisib brain penetration in vivo, we measured the brain and plasma concentration in wildtype (WT), Abcg2−/−, Abcb1a/b−/− and Abcb1a/b; Abcg2−/− mice 1 hour after i.v. administration of 2 mg/kg buparlisib. No differences could be observed among the mouse strains in both the plasma and brain concentra- tions of buparlisib (Fig. 2A,B). These results demonstrate that P-gp and BCRP do not restrict the brain penetra- tion of buparlisib in vivo. Interestingly, the brain-plasma ratio was between 1.5 and 2 in all strains, indicating that buparlisib exhibits excellent brain penetration (Fig. 2C). Results Thus, the brain contains the highest concentration of all tissues included in this study, suggesting that buparlisib may be a very suitable PI3K inhibitor candidate for treatment of intracranial tumours. Buparlisib is a brain penetrable pan- PI3K inhibitor Mark C. de Gooijer 1,2, Ping Zhang1,2,3, Levi C. M. Buil1,2, Ceren H. Çitirikkaya1,2, Nishita Thota1,2, Jos H. Beijnen1,4,5 & Olaf van Tellingen 1,2 BECs abundantly express ATP-binding cassette (ABC) efflux transporters on their apical membranes that very efficiently pump xenobiotics back into the bloodstream, thereby protecting the brain parenchyma from poten- tially harmful substances. Among these transporters, P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) are the most dominant. Together, these efflux transporters are responsible for restricting the brain pen- etration of many anticancer drugs, be it classical chemotherapeutics or novel targeted agents16. In line with this, almost all PI3K inhibitors that have been analysed to date were demonstrated to be transported, ZSTK474 and GNE-317 appearing to be exceptions12,13. pp g p Buparlisib is a novel pan-PI3K inhibitor that has been developed to inhibit all class I PI3K isoforms17. It has shown preclinical efficacy in various PI3K pathway overactivated cancer models, including GBM18–20. Buparlisib has thus far proceeded through phase I and phase II clinical trials in various extracranial solid tumours21,22 1Division of Pharmacology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX, Amsterdam, The Netherlands. 2Mouse Cancer Clinic, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX, Amsterdam, The Netherlands. 3Department of Neurosurgery, Qilu Hospital, Shandong University, Wenhua Xi Road 107, 250012, Jinan, P.R. China. 4Department of Pharmacy and Pharmacology, The Netherlands Cancer Institute / MC Slotervaart Hospital, Louwesweg 6, 1066 EC, Amsterdam, The Netherlands. 5Division of Pharmacoepidemiology and Clinical Pharmacology, Department of Pharmaceutical Sciences, Faculty of Science, Utrecht University, Universiteitsweg 99, 3584 CG, Utrecht, The Netherlands. Correspondence and requests for materials should be addressed to O.v.T. (email: o.v.tellingen@nki.nl) SCIeNTIFIC REPOrts \| (2018) 8:10784 \| DOI:10.1038/s41598-018-29062-w 1 www.nature.com/scientificreports/ and is now also being tested in primary and secondary intracranial cancers (e.g., ClinicalTrials.gov Identifiers NCT02000882, NCT02452294, NCT01339052). Despite some reports mentioning that buparlisib is BBB penetrable17,18,21,23, there are no pharmacokinetic data in the public domain supporting this claim. We here report a detailed analysis of the BBB penetration and oral bioavailability of buparlisib and demonstrate that it is a blood-brain barrier penetrable PI3K inhibitor with excellent oral bioavailability and intracranial target engagement. Discussionh This study demonstrates that buparlisib has pharmacokinetic properties that make it an attractive candidate for treatment of intracranial malignancies in patients. The accumulation of buparlisib in the brain is higher than in most other organs and buparlisib inhibits PI3K in the brain at clinically achievable plasma concentrations. SCIeNTIFIC REPOrts \| (2018) 8:10784 \| DOI:10.1038/s41598-018-29062-w 2 www.nature.com/scientificreports/ ure.com/scientificreports/ Figure 1. Analysis of buparlisib substrate affinity for ABC transporters using in vitro transport assays. (A) The chemical structure of buparlisib. (B) A conventional transport assay (CTA) using LLC-PK1 cells in presence of zosuquidar to block endogenous (porcine) P-gp activity. Buparlisib efficiently diffuses over a cellular monolayer irrespective of direction, plateauing to near-equilibrium in 4 hours. (C) Concentration equilibrium transport assays (CETAs) using MDCK or LLC cells that overexpress murine BCRP, (Bcrp1), human BCRP, murine P-gp (Mdr1a) or human P-gp (MDR1). No substrate affinity of buparlisib for BCRP, Mdr1a or MDR1 could be observed, whereas very minimal buparlisib transport was found in the MDCK-Bcrp1 cell line. The P-gp inhibitor zosuquidar was used in all MDCK cell lines to inhibit endogenous P-gp activity and in LLC cell lines to validate possible P-gp-mediated translocations. The dual BCRP/P-gp inhibitor elacridar was used to confirm possible BCRP-mediated translocations by abolishing buparlisib translocation in presence of elacridar. Data are represented as mean ± SD (n ≥ 4); p < 0.01; p < 0.001. Figure 1. Analysis of buparlisib substrate affinity for ABC transporters using in vitro transport assays. (A) The chemical structure of buparlisib. (B) A conventional transport assay (CTA) using LLC-PK1 cells in presence of zosuquidar to block endogenous (porcine) P-gp activity. Buparlisib efficiently diffuses over a cellular monolayer irrespective of direction, plateauing to near-equilibrium in 4 hours. (C) Concentration equilibrium transport assays (CETAs) using MDCK or LLC cells that overexpress murine BCRP, (Bcrp1), human BCRP, murine P-gp (Mdr1a) or human P-gp (MDR1). No substrate affinity of buparlisib for BCRP, Mdr1a or MDR1 could be observed, whereas very minimal buparlisib transport was found in the MDCK-Bcrp1 cell line. The P-gp inhibitor zosuquidar was used in all MDCK cell lines to inhibit endogenous P-gp activity and in LLC cell lines to validate possible P-gp-mediated translocations. The dual BCRP/P-gp inhibitor elacridar was used to confirm bl d d l b b l h b l b l f l d Figure 1. Analysis of buparlisib substrate affinity for ABC transporters using in vitro transport assays. Discussionh All data are represented as mean ± SD (n = 4). The ability of a compound to access the brain is largely determined by two factors, namely the ability to diffuse over cellular membranes and the affinity for the drug efflux transporters P-gp and BCRP that are expressed at the BBB. Of all PI3K inhibitors that have thus far been reported, buparlisib shows by far the best brain penetration. The brain-plasma ratio ranges between 1.5 and 2 (Figs 2C, 3A), whereas the brain-plasma ratio of several other PI3K inhibitors that are considered brain penetrable (NVP-BEZ235, ZSTK474 and GNE-317) do not exceed 112,13. Importantly, the buparlisib brain concentration is also higher than in most other tissues, which is in line with the relatively low distribution volume of this drug (<1 L/kg; Table 1)27. The reason why buparlisib distributes more to the brain than to other tissues is unclear, but could be related to more preferential protein binding in the brain or substrate affinity for uptake transporters that are present at the BBB. Regardless of the cause, this char- acteristic makes buparlisib a clear frontrunner among all PI3K inhibitors for development against intracranial tumours provided that the mouse mimics the human in this respect.hifh p p The plasma concentration-time profiles of buparlisib in mice and humans are very different. The peak plasma levels in humans receiving the maximum tolerated dose of buparlisib of 100 mg per day is about 1700 ng/ml (4.15 μM) and remains above 800 ng/ml (1.95 μM) for the remaining period of 24 h until the next dose21. By contrast, the peak plasma level in mice receiving 5 mg/kg is in the same range as in humans, but steeply declines to below 1 ng/ml (2.4 nM) after 24 h. Notably, a plasma concentration between 500 and 1000 nM was needed for inhibition of PI3K in the brain of WT mice (Fig. 3A). Single agent antitumor efficacy in preclinical models has already been shown, but only at 30 to 50 mg/kg dose levels17,18. The lack of efficacy at lower doses is likely due to the short duration of pharmacologically active plasma levels. Discussionh (A) The chemical structure of buparlisib. (B) A conventional transport assay (CTA) using LLC-PK1 cells in presence of zosuquidar to block endogenous (porcine) P-gp activity. Buparlisib efficiently diffuses over a cellular monolayer irrespective of direction, plateauing to near-equilibrium in 4 hours. (C) Concentration equilibrium transport assays (CETAs) using MDCK or LLC cells that overexpress murine BCRP, (Bcrp1), human BCRP, murine P-gp (Mdr1a) or human P-gp (MDR1). No substrate affinity of buparlisib for BCRP, Mdr1a or MDR1 could be observed, whereas very minimal buparlisib transport was found in the MDCK-Bcrp1 cell line. The P-gp inhibitor zosuquidar was used in all MDCK cell lines to inhibit endogenous P-gp activity and in LLC cell lines to validate possible P-gp-mediated translocations. The dual BCRP/P-gp inhibitor elacridar was used to confirm possible BCRP-mediated translocations by abolishing buparlisib translocation in presence of elacridar. Data are represented as mean ± SD (n ≥ 4); p < 0.01; **p < 0.001. Characterization of the genomic landscapes of intracranial tumours has revealed a clear role for the PI3K-AKT-mTOR pathway in tumorigenesis and tumour maintenance of these malignancies3–6. Further work also supports that PI3K pathway hyperactivated tumours are vulnerable to PI3K inhition2,18–20. Thus, targeting this pathway is expected to be a promising therapeutic strategy for these tumours. Importantly however, suf- ficient brain penetration of any therapeutic agent is a prerequisite for antitumor efficacy against intracranial tumours10–14,28. SCIeNTIFIC REPOrts \| (2018) 8:10784 \| DOI:10.1038/s41598-018-29062-w 3 www.nature.com/scientificreports/ p Figure 2. The impact of P-gp and BCRP on the brain and tissue penetration of buparlisib. Buparlisib was administered intravenously to wildtype, Abcg2−/−, Abcb1a/b−/− and Abcb1a/b;Abcg2−/− mice at a dose of 2 mg/kg. One hour after injection, blood and tissues were collected for LC-MS/MS analysis. No difference in buparlisib (A) plasma concentration, (B) brain concentration, (C) brain-plasma ratio, (D) liver concentration, (E) kidney concentration and (F) spleen concentration could be observed among the different mouse strains. All data are represented as mean ± SD (n = 4). Figure 2. The impact of P-gp and BCRP on the brain and tissue penetration of buparlisib. Buparlisib was administered intravenously to wildtype, Abcg2−/−, Abcb1a/b−/− and Abcb1a/b;Abcg2−/− mice at a dose of 2 mg/kg. One hour after injection, blood and tissues were collected for LC-MS/MS analysis. No difference in buparlisib (A) plasma concentration, (B) brain concentration, (C) brain-plasma ratio, (D) liver concentration, (E) kidney concentration and (F) spleen concentration could be observed among the different mouse strains. Discussionh (2 mg/kg) Plasma AUC (ng/ml.h) 0-∞ 5600 ± 860 6300 ± 1500 Cmax (ng/ml) 1900 ± 410 2100 ± 190 t1/2 (h) 1.60 ± 0.07 1.67 ± 0.07 Vz (L/kg) 0.86 ± 0.14 0.80 ± 0.23 CL (L/kg.h) 0.37 ± 0.06 0.33 ± 0.09 p.o. (5 mg/kg) Plasma AUC (ng/ml.h) 0-∞ 16000 ± 1700 16000 ± 1200 Cmax (ng/ml) 3000 ± 490 3300 ± 1300 tmax (h) 0.67 ± 0.39 1.00 ± 0.00 t1/2 (h) 1.40 ± 0.04 1.44 ± 0.06 F (%) 112 ± 21.1 103 ± 26.1 Vz/F (L/kg) 0.63 ± 0.08 0.64 ± 0.03 CL/F (L/kg.h) 0.32 ± 0.04 0.31 ± 0.02 Table 1. Pharmacokinetic parameters of buparlisib after oral and i.v. administration to male and female FVB mice. AUC, area under the curve; Cmax, maximum concentration in plasma; tmax, time to reach maximum plasma concentration; t1/2, elimination half-life; Vz, apparent volume of distribution; CL, apparent clearance, F, oral bioavailability; Vz/F, apparent volume of distribution after oral administration; CL/F, apparent clearance after oral administration. Data are represented as mean ± SD (n = 4). Administration route Parameter Time (h) Gender female male i.v. (2 mg/kg) Plasma AUC (ng/ml.h) 0-∞ 5600 ± 860 6300 ± 1500 Cmax (ng/ml) 1900 ± 410 2100 ± 190 t1/2 (h) 1.60 ± 0.07 1.67 ± 0.07 Vz (L/kg) 0.86 ± 0.14 0.80 ± 0.23 CL (L/kg.h) 0.37 ± 0.06 0.33 ± 0.09 p.o. (5 mg/kg) Plasma AUC (ng/ml.h) 0-∞ 16000 ± 1700 16000 ± 1200 Cmax (ng/ml) 3000 ± 490 3300 ± 1300 tmax (h) 0.67 ± 0.39 1.00 ± 0.00 t1/2 (h) 1.40 ± 0.04 1.44 ± 0.06 F (%) 112 ± 21.1 103 ± 26.1 Vz/F (L/kg) 0.63 ± 0.08 0.64 ± 0.03 CL/F (L/kg.h) 0.32 ± 0.04 0.31 ± 0.02 Table 1. Pharmacokinetic parameters of buparlisib after oral and i.v. administration to male and female FVB mice. AUC, area under the curve; Cmax, maximum concentration in plasma; tmax, time to reach maximum plasma concentration; t1/2, elimination half-life; Vz, apparent volume of distribution; CL, apparent clearance, F, oral bioavailability; Vz/F, apparent volume of distribution after oral administration; CL/F, apparent clearance after oral administration. Data are represented as mean ± SD (n = 4). Table 1. Pharmacokinetic parameters of buparlisib after oral and i.v. administration to male and female FVB mice. Discussionh AUC, area under the curve; Cmax, maximum concentration in plasma; tmax, time to reach maximum plasma concentration; t1/2, elimination half-life; Vz, apparent volume of distribution; CL, apparent clearance, F, oral bioavailability; Vz/F, apparent volume of distribution after oral administration; CL/F, apparent clearance after oral administration. Data are represented as mean ± SD (n = 4). Figure 3. Buparlisib has excellent intracranial target engagement and oral bioavailability. (A) Buparlisib was orally administered to wildtype mice at a dose of 1 mg/kg, 2 mg/kg or 5 mg/kg. One hour after injection, blood and tissues were collected for LC-MS/MS analysis. Buparlisib plasma and brain levels increase dose-dependently, yielding similar and excellent brain-plasma ratios at all dose levels that were tested. (B) Immunoblotting of brain tissue lysates from (A). Buparlisib efficiently inhibited phosphorylation of AktS473 in the brains of wildtype mice following oral administration at a dose of 5 mg/kg, without affecting signalling through ERK. (C) Plasma-time curves of male and female wildtype mice following intravenous (2 mg/kg) or oral (5 mg/kg) administration. No differences could be observed in buparlisib plasma pharmacokinetics among both genders. The oral bioavailability of buparlisib is excellent, since oral and intravenous administration yielded similar dose-adjusted AUCs (see Table 1). All data are represented as mean ± SD (n = 4). Figure 3. Buparlisib has excellent intracranial target engagement and oral bioavailability. (A) Buparlisib was orally administered to wildtype mice at a dose of 1 mg/kg, 2 mg/kg or 5 mg/kg. One hour after injection, blood and tissues were collected for LC-MS/MS analysis. Buparlisib plasma and brain levels increase dose-dependently, yielding similar and excellent brain-plasma ratios at all dose levels that were tested. (B) Immunoblotting of brain tissue lysates from (A). Buparlisib efficiently inhibited phosphorylation of AktS473 in the brains of wildtype mice following oral administration at a dose of 5 mg/kg, without affecting signalling through ERK. (C) Plasma-time curves of male and female wildtype mice following intravenous (2 mg/kg) or oral (5 mg/kg) administration. No differences could be observed in buparlisib plasma pharmacokinetics among both genders. The oral bioavailability of buparlisib is excellent, since oral and intravenous administration yielded similar dose-adjusted AUCs (see Table 1). All data are represented as mean ± SD (n = 4). There is a growing body of clinical and preclinical evidence that PI3K inhibitors used in combination with other drugs may even be more promising for broader antitumor efficacy33. Discussionh However, high dose levels will cause high and non-clinically relevant plasma levels during the first hours and create the risk that some of the profound single agent antitumor activities observed in mice are in fact due to off-target effects29.h gf gf Thus far, only one PI3K inhibitor has received FDA approval. Idelalisib is a class I PI3Kδ inhibitor and has been approved as monotherapy for treatment of follicular lymphoma and small lymphocytic lymphoma30. Buparlisib inhibits all class I PI3K isoforms. Next to the canonical function of class I PI3Kα in regulating tumour cell pro- liferation and survival, roles for other class I PI3K isoforms in the immune system, including tumour-induced immune suppression, are emerging31,32. Thus, pan-PI3K inhibition may be advantageous for antitumor efficacy. Buparlisib is at an advanced stage of development, with several phase III trials underway in extracranial malig- nancies (e.g., NCT01633060, NCT02756247) and phase II trials ongoing for primary and secondary intracranial tumours (e.g., NCT02000882, NCT01339052). SCIeNTIFIC REPOrts \| (2018) 8:10784 \| DOI:10.1038/s41598-018-29062-w 4 www.nature.com/scientificreports/ Administration route Parameter Time (h) Gender female male i.v. (2 mg/kg) Plasma AUC (ng/ml.h) 0-∞ 5600 ± 860 6300 ± 1500 Cmax (ng/ml) 1900 ± 410 2100 ± 190 t1/2 (h) 1.60 ± 0.07 1.67 ± 0.07 Vz (L/kg) 0.86 ± 0.14 0.80 ± 0.23 CL (L/kg.h) 0.37 ± 0.06 0.33 ± 0.09 p.o. (5 mg/kg) Plasma AUC (ng/ml.h) 0-∞ 16000 ± 1700 16000 ± 1200 Cmax (ng/ml) 3000 ± 490 3300 ± 1300 tmax (h) 0.67 ± 0.39 1.00 ± 0.00 t1/2 (h) 1.40 ± 0.04 1.44 ± 0.06 F (%) 112 ± 21.1 103 ± 26.1 Vz/F (L/kg) 0.63 ± 0.08 0.64 ± 0.03 CL/F (L/kg.h) 0.32 ± 0.04 0.31 ± 0.02 Table 1. Pharmacokinetic parameters of buparlisib after oral and i.v. administration to male and female FVB mice. AUC, area under the curve; Cmax, maximum concentration in plasma; tmax, time to reach maximum plasma concentration; t1/2, elimination half-life; Vz, apparent volume of distribution; CL, apparent clearance, F, oral bioavailability; Vz/F, apparent volume of distribution after oral administration; CL/F, apparent clearance after oral administration. Data are represented as mean ± SD (n = 4). Administration route Parameter Time (h) Gender female male i.v. Methods Methods Drugs. Buparlisib (NVP-BKM120) and AZD8055 were purchased from Selleck Chemicals (Houston, TX) and zosuquidar from Eli Lilly (Indianapolis, IN). Elacridar was generously made available by GlaxoSmithKline (Research Triangle Park, NC). Cell culture. All cell lines used in this study were previously generated in our institute and generously pro- vided by Dr. A.H. Schinkel38–40. All cells were cultured in MEM supplemented with 10% FBS, 1% L-glutamine, 1% sodium pyruvate, 1% MEM vitamins, 1% non-essential amino acids and 1% penicillin/streptomycin (all from Life Technologies, Carlsbad, CA) under 37 °C and 5% CO2 conditions. Concentration equilibrium transport assays. Conventional bidirectional transport assays (CTAs) and concentration equilibrium transport assays (CETAs) were performed as described previously25. Buparlisib was used at a concentration of 100 nM and, when appropriate, specific transport was inhibited using the P-gp inhibi- tor zosuquidar (5 μM) or the dual P-gp/BCRP inhibitor elacridar (5 μM). Transwell leakiness was determined as Carboxyl-[14C]-inulin translocation exceeding 1.5% per hour and these wells were excluded from the analysis. y g y To prepare buparlisib transport assay samples for LC-MS/MS analysis, 10 μL medium samples were mixed with 30 μL of acetonitrile:formic acid (100:1 v/v). After centrifugation, the supernatant was 5-fold diluted in water and the buparlisib concentration was measured using an LC-MS/MS system as described below. Animals. All animal housing and studies were approved by the Animal Experimental Committee of the Netherlands Cancer Institute and conducted according to national law and institutional guidelines. Mice were housed at 20.9 °C on a 12 hour light/dark cycle with food and water ad libitum. Pharmacokinetic studies. The pharmacokinetics of buparlisib were analysed in WT, Abcg2−/−, Abcb1a/b−/− and Abcb1a/b; Abcg2−/− FVB mice. All knockout mice strains have been developed within our institute41–43. For i.v. administration, buparlisib was dissolved in DMSO and injected at a dose of 2 mg/kg. For oral administration, bupar- lisib was formulated in DMSO:Cremophor EL:water (1:1:8 v/v/v) and administered at 1, 2 or 5 mg/kg as indicated. Tail vein bleeding was used to collect blood at intermediate time points, whereas at the last time point blood was drawn by cardiac puncture and various tissue were collected. Plasma was obtained from whole blood by centrifu- gation (5 min, 5,000 rpm, 4 °C). After weighing, tissues were homogenized using a FastPrep®-24 (MP-Biomedicals, NY) in 1% (w/v) bovine serum albumin in water. Methods Buparlisib was extracted from plasma and tissue homogenate by liquid-liquid extraction with ethyl acetate using AZD8055 as an internal standard. LC-MS/MS analysis. Buparlisib was measured in samples from in vitro transport assays and in vivo pharma- cokinetic studies using an LC-MS/MS system comprised of an UltiMate 3000 LC Systems (Dionex, Sunnyvale, CA) and an API 4000 mass spectrometer (Sciex, Framingham, MA). Samples were run through a Securityguard C18 pre-column (Phenomenex, Utrecht, The Netherlands) prior to separation on a ZORBAX Extend-C18 column (Agilent, Santa Clara, CA). Elution was done using a in a 5 minute gradient from 20% to 95% B (mobile phase A was 0.1% HCOOH in water (v/v) and mobile phase B was methanol). 95% B was maintained for 3 min followed by re-equilibration at 20% B. Multiple reaction monitoring was performed at 411.3/367.2 (buparlisib) and 418.2/138.4 (AZD8055). Analyst® 1.6.2 software (AB Sciex; Foster City, CA) was used for system control and data analysis. Western blotting. Mouse brains were homogenized in RIPA buffer supplemented with sodium fluoride (2 mM), sodium orthovanadate (1 mM), sodium pyrophosphate (1 mM), β-glycerophosphate (2.5 mM), PMSF (1 mM), DTT (1 mM) and protein inhibitor cocktail (Roche; Basel, Switzerland). The primary antibodies that were used in this study are phospho-AktS473 (1:1000; D9E, #4060; Cell Signaling Technology; Danvers, MA), total Akt (1:1000; #9272; Cell Signaling Technology), phospho-Erk1T202/204/Erk2T183/Y185 (1:1000; sc-16982; Santa Cruz Biotechnology, Dallas, TX), total Erk1/2 (1:1000; C16, sc-93; Santa Cruz Biotechnology) and β–tubulin (1:1000; T3952; Sigma-Aldrich, St. Louis, MO). Goat-anti-rabbit-HRP (1:2000; DAKO, Santa Clara, CA) was used a secondary antibody. Pharmacokinetic and statistical analysis. As described in detail before, CETA results were analysed with the General linear model repeated measures procedure of SPSS (v22; SPSS Inc, Chicago, IL)25. PK solver was used to determine pharmacokinetic parameters44. The standard error of the oral bioavailability was calculated using the formula below: =       +       . . . . . . . . SE F SE AUC SE AUC F AUC p o AUC i v 2 2 p o i v Buparlisib concentrations in WT and transporter knockout mice in vivo were compared using one-way analy- sis of variance followed by post hoc Bonferroni tests. Statistical differences between the buparlisib PK parame- ters in male and female mice were calculated as Bonferroni corrected p-values using multiple Student’s t-tests. www.nature.com/scientificreports/ www.nature.com/scientificreports/ instance combination with a MEK inhibitor34, Bcl-2 inhibitor35 and CSF-1R inhibitor36 in GBM or a Smo antag- onist in medulloblastoma37. The finding that pharmacologically relevant plasma levels of buparlisib are achieved in patients warrants further clinical investigation of such combination therapies. instance combination with a MEK inhibitor34, Bcl-2 inhibitor35 and CSF-1R inhibitor36 in GBM or a Smo antag- onist in medulloblastoma37. The finding that pharmacologically relevant plasma levels of buparlisib are achieved in patients warrants further clinical investigation of such combination therapies. p g p In summary, buparlisib is a pan-PI3K inhibitor with excellent brain penetration, complete oral bioavailability and efficient intracranial target inhibition at clinically achievable plasma concentrations, making it the attractive candidate for intracranially-targeted therapeutic strategies involving PI3K inhibition. Discussionh For instance in orthotopic mouse models of glioblastoma, the efficacy of PI3K inhibition as monotherapy has previously been shown to be modest at best, even when sufficient brain penetration and intracranial target inhibition could be reached12,13. Several pre- clinical studies have already pinpointed interesting strategies for combination treatment involving buparlisib, for There is a growing body of clinical and preclinical evidence that PI3K inhibitors used in combination with other drugs may even be more promising for broader antitumor efficacy33. For instance in orthotopic mouse models of glioblastoma, the efficacy of PI3K inhibition as monotherapy has previously been shown to be modest at best, even when sufficient brain penetration and intracranial target inhibition could be reached12,13. Several pre- clinical studies have already pinpointed interesting strategies for combination treatment involving buparlisib, for SCIeNTIFIC REPOrts \| (2018) 8:10784 \| DOI:10.1038/s41598-018-29062-w 5 Methods Differences were considered statistically significant when p < 0.05. 6 SCIeNTIFIC REPOrts \| (2018) 8:10784 \| DOI:10.1038/s41598-018-29062-w www.nature.com/scientificreports/ Referencesh Decreased affinity for efflux transporters increases brain penetrance and molecular targeting of a PI3K/mTOR inhibitor in a mouse model of glioblastoma. Neuro Oncol. 17, 1210–1219 (2015).h 4. Osswald, M. et al. Impact of Blood-Brain Barrier Integrity on Tumor Growth and Therapy Response in Brain Metastases. Clin Cancer Res. 22, 6078–6087 (2016).f 5. van Tellingen, O. et al. Overcoming the blood-brain tumor barrier for effective glioblastoma treatment. Drug Resist. Updat. 19, 1–12 (2015) f 6. Durmus, S., Hendrikx, J. J. M. A. & Schinkel, A. H. Apical ABC Transporters and Cancer Chemotherapeutic Drug Disposition. Adv Cancer Res. 125, 1–41 (2015).i 7. Maira, S. M. et al. Identification and characterization of NVP-BKM120, an orally available pan-class I PI3-kinase inhibitor. Mol Cancer Ther. 11, 317–328 (2012).i h 8. Burger, M. T. et al. Identification of NVP-BKM120 as a Potent, Selective, Orally Bioavailable Class I PI3 Kinase Inhibitor for Treating Cancer. ACS Med. Chem. Lett. 2, 774–779 (2011).f 9. Koul, D. et al. Antitumor activity of NVP-BKM120–a selective pan class I PI3 kinase inhibitor showed differential forms of cell death based on p53 status of glioma cells. Clin. Cancer Res. 18, 184–195 (2012). 20. Speranza, M. C. et al. BKM-120 (Buparlisib): A Phosphatidyl-Inositol-3 Kinase Inhibitor with Anti-Invasive Properties in Glioblastoma. Sci. Rep. 6, 20189 (2016). p 1. Bendell, J. C. et al. Phase I, Dose-Escalation Study of BKM120, an Oral Pan-Class I PI3K Inhibitor, in Patients With Advanced Solid Tumors. J. Clin. Oncol. 30, 282–290 (2011). 22. Soulières, D. et al. Buparlisib and paclitaxel in patients with platinum-pretreated recurrent or metastatic squamous cell carcinoma of the head and neck (BERIL-1): a randomised, double-blind, placebo-controlled phase 2 trial. Lancet Oncol. 18, 323–335 (2017). 3. Cho, C.-F. et al. Blood-brain-barrier spheroids as an in vitro screening platform for brain-penetrating agents. Nat. Comm. 8, 15623 (2017) 23. Cho, C.-F. et al. Blood-brain-barrier spheroids as an in vitro screening platform for brain-penetrating agents. Nat. Comm. 8, 15623 (2017). 24. Luna-Tortós, C., Fedrowitz, M. & Löscher, W. Several major antiepileptic drugs are substrates for human P-glycoprotein. Neuropharmacology 55, 1364–1375 (2008). 4. Luna-Tortós, C., Fedrowitz, M. & Löscher, W. Several major antiepileptic drugs are substrates for human P-glycoprotein Neuropharmacology 55, 1364–1375 (2008). p gy 5. Zhang, P. et al. ABCB1 and ABCG2 restrict the brain penetration of a panel of novel EZH2-Inhibitors. Int. J. Cancer 137, 2007–2018 (2015).i 26. Zhang, C., Zuo, Z., Kwan, P. & Baum, L. Referencesh In vitro transport profile of carbamazepine, oxcarbazepine, eslicarbazepine acetate, an active metabolites by human P-glycoprotein. Epilepsia 52, 1894–1904 (2011). 27. Di Gion, P. et al. Clinical Pharmacokinetics of Tyrosine Kinase Inhibitors. Clin. Pharmacokinet. 50, 551–603 (2011) 28. Sarkaria, J. N. et al. Is the blood–brain barrier really disrupted in all glioblastomas? A critical assessment of existing clini Neuro Oncol. 20, 184–191 (2018). 9. Brachmann, S. M. et al. Characterization of the Mechanism of Action of the Pan Class I PI3K Inhibitor NVP-BKM120 across a Broad Range of Concentrations. Mol. Cancer Ther. 11, 1747–1757 (2012). h 30. Miller, B. W. et al. FDA Approval: Idelalisib Monotherapy for the Treatment of Patients with Follicular Lymphoma and Lymphocytic Lymphoma. Clin. Cancer Res. 21, 1525–1529 (2015). y p y y p 31. Kaneda, M. M. et al. PI3Kγ is a molecular switch that controls immune suppression. Nature 539, 437–442 (2016). Regulation of T cell alloimmunity by PI3Kγ and PI3Kδ. Nat. Com 32. Uehara, M. et al. Regulation of T cell alloimmunity by PI3Kγ an g y y 33. Fruman, D. A. & Rommel, C. PI3K and cancer: lessons, challenges and opportunities. Nat. Rev. Drug Discov. 13, 140–156 (201 33. Fruman, D. A. & Rommel, C. PI3K and cancer: lessons, challenges and opportunities. Nat. Rev 33. Fruman, D. A. & Rommel, C. PI3K and cancer: lessons, challenges and opportunities. Nat. Rev. Drug Discov. 13, 140 156 (2014). 34. El Meskini, R. et al. A preclinical orthotopic model for glioblastoma recapitulates key features of human tumors and demonstrates , , , g pp g , ( ) 34. El Meskini, R. et al. A preclinical orthotopic model for glioblastoma recapitulates key features of human tumors and demonstrates sensitivity to a combination of MEK and PI3K pathway inhibitors Dis Model Mech 8 45 56 (2014) 34. El Meskini, R. et al. A preclinical orthotopic model for glioblastoma recapitulates key features of human tumors and demonst sensitivity to a combination of MEK and PI3K pathway inhibitors. Dis. Model. Mech. 8, 45–56 (2014). 35. Jane, E. P., Premkumar, D. R., Morales, A., Foster, K. A. & Pollack, I. F. Inhibition of phosphatidylinositol 3-kinase/AKT signaling by NVP-BKM120 promotes ABT-737-induced toxicity in a caspase-dependent manner through mitochondrial dysfunction and DNA damage response in established and primary cultured glioblastoma cells. J. Pharm. Exp. Ther. 350, 22–35 (2014).h g p p y g ph 36. Quail, D. F. et al. Referencesh 1. Thorpe, L. M., Yuzugullu, H. & Zhao, J. J. PI3K in cancer: divergent roles of isoforms, modes of activation and therapeutic targeting. Nat. Rev. Cancer 15, 7–24 (2015). 2. Engelman, J. A. Targeting PI3K signalling in cancer: opportunities, challenges and limitations. Nat. Rev. Cancer 9, 550–562 (2009). 3. Brennan, C. W. et al. The somatic genomic landscape of glioblastoma. Cell 155, 462–477 (2013).f g , J g g g g pp , g , ( ) 3. Brennan, C. W. et al. The somatic genomic landscape of glioblastoma. Cell 155, 462–477 (2013).f h 4. Mackay, A. et al. Integrated Molecular Meta-Analysis of 1,000 Pediatric High-Grade and Diffuse Intrinsic Pontine Glioma. Cance Cell 32, 520–537.e525 (2017).i 5. Cancer Genome Atlas, N. Genomic Classification of Cutaneous Melanoma. Cell 161, 1681–1696 (2015). i 6. Network, T. C. G. A. Comprehensive molecular portraits of human breast tumours. Nature 490, 61–70 (2012). 7. Lin, F. et al. Targeting core (mutated) pathways of high-grade gliomas: challenges of intrinsic resistance and drug efflux. CNS Oncol 2, 271–288 (2013). 2, 271 288 (2013). 8. Niessner, H. et al. PI3K Pathway Inhibition Achieves Potent Antitumor Activity in Melanoma Brain Metastases In Vitro and In Vivo. Clin Cancer Res 22 5818 5828 (2016) 8. Niessner, H. et al. PI3K Pathway Inhibition Achieves Potent Antitumor Activity in Melanoma Brain Metastases In Vitro and In Vivo Clin. Cancer Res. 22, 5818–5828 (2016). 9. Ni, J. et al. Combination inhibition of PI3K and mTORC1 yields durable remissions in mice bearing orthotopic patient-derived xenografts of HER2-positive breast cancer brain metastases. Nat. Med. 22, 723–726 (2016). ff l f l l l d d h b f d t 0. Heffron, T. P. et al. Discovery of Clinical Development Candidate GDC-0084, a Brain Penetrant Inhibitor of PI3K and mTOR. ACS Med. Chem. Lett. 7, 351–356 (2016).fi 11. Salphati, L. et al. Targeting the PI3K Pathway in the Brain–Efficacy of a PI3K Inhibitor Optimized to Cross the Blood-Brain Barrier. Clin. Cancer Res. 18, 6239–6248 (2012).fi 2. Lin, F. et al. PI3K–mTOR Pathway Inhibition Exhibits Efficacy Against High-grade Glioma in Clinically Relevant Mouse Models Clin. Cancer Res. 23, 1286–1298 (2017).fifl ( ) 3. Becker, C. M. et al. Decreased affinity for efflux transporters increases brain penetrance and molecular targeting of a PI3K/mTOR i hibit i d l f li bl t N O l 17 1210 1219 (2015) 3. Becker, C. M. et al. Acknowledgementsh g This work was supported by a research grant from the foundation Stophersentumoren.nl (O.v.T.). M.C.d.G., J.H.B. and O.v.T. conceived the study, designed the experiments and wrote the manuscript. J.H.B. and O.v.T. supervised the study. M.C.d.G., P.Z., L.C.M.B., C.H.Ç. and N.T. performed and analysed the experiments. SCIeNTIFIC REPOrts \| (2018) 8:10784 \| DOI:10.1038/s41598-018-29062-w Referencesh The tumor microenvironment underlies acquired resistance to CSF-1R inhibition in gliomas. Science 352, aad3018 (2016). 7. Buonamici, S. et al. Interfering with resistance to smoothened antagonists by inhibition of the PI3K pathway in medulloblastoma Sci. Transl. Med. 2, 51ra70 (2010).f ( ) 38. Schinkel, A. H., Wagenaar, E., van Deemter, L., Mol, C. A. & Borst, P. Absence of the mdr1a P-Glycoprotein in mice affects tissue distribution and pharmacokinetics of dexamethasone, digoxin, and cyclosporin A. J. Clin. Invest. 96, 1698–1705 (1995). 39. Jonker, J. W. et al. Role of Breast Cancer Resistance Protein in the Bioavailability and Fetal Penetration of Topotecan. J. Natl. Canc. Inst. 92, 1651–1656 (2000). 0. Pavek, P. et al. Human Breast Cancer Resistance Protein: Interactions with Steroid Drugs, Hormones, the Dietary Carcinogen 2-Amino-1-methyl-6-phenylimidazo(4,5-b)pyridine, and Transport of Cimetidine. J. Pharm. Exp. Ther. 312, 144–152 (2005).i h 1. Schinkel, A. H. et al. Disruption of the mouse mdr1a P-glycoprotein gene leads to a deficiency in the blood-brain barrier and to increased sensitivity to drugs. Cell 77, 491–502 (1994). 42. Jonker, J. W. et al. The breast cancer resistance protein protects against a major chlorophyll-derived dietary phototoxin and protoporphyria. Proc. Natl. Acad. Sci. USA 99, 15649–15654 (2002). protoporphyria. Proc. Natl. Acad. Sci. USA 99, 15649–15654 (2002) p p p y 3. Jonker, J. W. et al. Contribution of the ABC Transporters Bcrp1 and Mdr1a/1b to the Side Population Phenotype in Mammary Gland and Bone Marrow of Mice. Stem Cells 23, 1059–1065 (2005). 44. Zhang, Y., Huo, M., Zhou, J. & Xie, S. PKSolver: An add-in program for pharmacokinetic and pharmacodynamic data analysis in Microsoft Excel. Comput. Methods Programs Biomed. 99, 306–314 (2010). SCIeNTIFIC REPOrts \| (2018) 8:10784 \| DOI:10.1038/s41598-018-29062-w 7 www.nature.com/scientificreports/ Additional Informationh Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 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https://openalex.org/W3082297889	https://scholarlypublications.universiteitleiden.nl/access/item%3A3134949/view	English	null	Intention to imitate: Top-down effects on 4-year-olds’ neural processing of others’ actions	Developmental cognitive neuroscience	2,020	cc-by	11,382	A R T I C L E I N F O Keywords: Top-down attention Action observation Young children EEG Motor activity Imitation Keywords: Top-down attention Action observation Young children EEG Motor activity Imitation From early in life, we activate our neural motor system when observing others’ actions. In adults, this so-called mirroring is modulated not only by the saliency of an action but also by top-down processes, like the intention to imitate it. Yet, it remains unknown whether neural processing of others’ actions can be modulated by top-down processes in young children who heavily rely on learning from observing and imitating others but also still develop top-down control skills. Using EEG, we examined whether the intention to imitate increases 4-year-olds’ motor activation while observing others’ actions. In a within-subjects design, children observed identical actions preceded by distinct instructions, namely to either imitate the action or to name the toy’s color. As motor activation index, children’s alpha (7−12 Hz) and beta (16−20 Hz) power over motor cortices was analyzed. The results revealed more motor activity reflected by significantly lower beta power for the Imitation compared to the Color-naming Task. The same conditional difference, although differently located, was detected for alpha power. Together, our results show that children’s neural processing of others’ actions was amplified by their intention to imitate the action. Thus, already at age 4 top-down attention to others’ actions can modulate neural action processing. Intention to imitate: Top-down effects on 4-year-olds’ neural processing of others’ actions☆ Marlene Meyer a,b,, Hinke M. Endedijk c, Sabine Hunnius a Donders Institute for Brain, Cognition and Behaviour, Radboud University, the Netherlands b Department of Psychology, University of Chicago, USA c Education Science, Leiden University, Leiden, The Netherlands a Donders Institute for Brain, Cognition and Behaviour, Radboud University, the Netherlands b Department of Psychology, University of Chicago, USA c Education Science, Leiden University, Leiden, The Netherlands a Donders Institute for Brain, Cognition and Behaviour, Radboud University, the Netherlands b Department of Psychology, University of Chicago, USA c Education Science, Leiden University, Leiden, The Netherlands ☆The data that support the findings of this study are available from the corresponding author (Marlene Meyer) upon reasonable request. Corresponding author. Present Address: Montessorilaan 3, 6525 HR, Nijmegen, the Netherlands. E-mail address: m.meyer@donders.ru.nl (M. Meyer). Contents lists available at ScienceDirect Contents lists available at ScienceDirect Available online 27 August 2020 1878-9293/© 2020 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). https://doi.org/10.1016/j.dcn.2020.100851 Received 6 November 2019; Received in revised form 28 July 2020; Accepted 25 August 2020 Developmental Cognitive Neuroscience 45 (2020) 100851 Developmental Cognitive Neuroscience 45 (2020) 100851 Available online 27 August 2020 1878-9293/© 2020 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). g 1878-9293/© 2020 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). 1. Introduction Paying attention to other people’s actions allows us to better un derstand, predict, and learn from what they do. Especially in early childhood, others’ actions provide rich information and form the basis for learning a diverse range of new skills. A substantial body of research in developmental psychology has contributed to our understanding of how children process others’ actions and how they imitate and learn from them (Hunnius and Bekkering, 2014; Marshall and Meltzoff, 2014; Meltzoff and Marshall, 2018). Given the plethora of actions happening around children every day, children face the challenge to focus on ac tions that allow them to extract useful information for their own behavior and to learn novel actions. Unravelling the neural un derpinnings of how children process others’ actions, and in particular how to focus on action-related information, will inform ongoing in vestigations of action understanding and learning (Marshall and Meltzoff, 2014). Decades of cognitive neuroscience research have advanced our un derstanding of how actions are processed. Research on primates, human adults and children demonstrated that the neural activation found during observation of others’ actions closely resembles the neural pat terns of performing the same action. Electrophysiological studies with adults and developmental populations, for instance, show that the alpha and beta rhythms overlaying sensorimotor regions are suppressed both during observation of another person’s actions as well as during action execution, with less power indicating more activation (e.g. Fox et al., 2016; Hari et al., 1998). This neural overlap, often called mirror mechanism or mirroring (Rizzolatti and Fogassi, 2014) is suggested to be the neural basis for understanding of and learning from others’ actions (Hunnius and Bekkering, 2014; Woodward and Gerson, 2014). Although the term ‘mirroring’ and different interpretations of this mechanism are under discussion (Csibra, 2008; Hickok, 2014) the finding itself is established based on more than a decade of research (e.g. Rizzolatti and ☆The data that support the findings of this study are available from the corresponding author (Marlene Meyer) upon reasonable request. * Corresponding author. Present Address: Montessorilaan 3, 6525 HR, Nijmegen, the Netherlands. E-mail address: m.meyer@donders.ru.nl (M. Meyer). Developmental Cognitive Neuroscience 45 (2020) 100851 M. Meyer et al. phase (Southgate and Begus, 2013) and action observation phase (Langeloh et al., 2018; Patzwald et al., 2020). Furthermore, as in adult research (e.g. 2.1. Participants Together these findings provide ample evidence for top-down mod ulations of action perception in adults and, specifically, for enhanced neural motor activity when actions are relevant to the observer. It is particularly in early childhood that observing others’ actions is valuable for learning and improving own skills. However, top-down control as reflected in cognitive flexibility and attentional control are still under going significant development in children’s first years of life (Rueda et al., 2004; Zelazo and Carlson, 2012). Thus, it is currently unknown whether neural processing of others’ actions in young children is modulated by top-down processes. Is young children’s neural motor activation increased for actions they intend to imitate? To our knowl edge no previous developmental study has manipulated top-down pro cesses on children’s neural processing of others’ actions employing manipulations in task demands like adult research has done (e.g. Frey and Gerry, 2006; Muthukumaraswamy and Singh, 2008). While relevant developmental EEG research has contributed to our understanding of how bottom-up differences in actions might affect children’s neural mirroring (Woodward and Gerson, 2014), little is known about top-down modulations. Recent findings suggest for instance that neural motor activation during action observation can differ depending on in fants’ subsequent imitation responses (Filippi et al., 2016). This effect is likely reflecting bottom-up mechanisms, such that certain actions appear more salient to the infants thereby leading to both, enhanced neural processing and increased likelihood of subsequent imitation. Recent studies have also demonstrated that contextual information might effect infants’ neural motor activation during an anticipation Twenty-nine 4-year-old children (19 girls) with a mean age of 52 months (SD = 1.94 months) participated in this study. Three partici pants were excluded from the final sample due to an insufficient number of artifact-free trials (see EEG data analysis for details). Participants were recruited from a database with families who had indicated interest in participating in developmental studies living in the region of Nijmegen, a middle-sized city in the Netherlands. These participants belong to a subset of participants who took part in a longitudinal experiment (Endedijk et al., 2017). For none of the children atypical development was reported. Children were accompanied by their caregiver to the testing session. At the testing session, informed consent was obtained from the child’s legal guardian. After participation, children received a gift (book or 20 euros). 1.2. Top-down effects on processing others’ actions Top-down processes like paying attention to actions relevant to one’s own behavior, for instance in order to reproduce an action or to coor dinate with another person, amplify neural responses to the observed action. Studies with adults suggest that brain regions involved in action execution are activated more strongly during action perception when the observed action is relevant to the observer (Campbell and Cun nington, 2017). This evidence comes from studies in which adults watched actions after the explicit instruction to later reproduce the ac tion rather than to passively view the same action or to perform a non-action related task (Grezes et al., 1998; Frey and Gerry, 2006; Muthukumaraswamy and Singh, 2008). For instance, the MEG study by Muthukumaraswamy and Singh (2008) found more motor activity as reflected by less beta power over motor cortices when adults observed an action in an imitation condition compared to a passive viewing condition. An EEG study with adults (Schuch et al., 2010) showed that observing a scene with action-related instructions led to increased motor activation as indexed by less sensorimotor alpha power compared to observing the same scene with instructions directed at color features. More indirectly, top-down effects on neural processing of others’ actions are observed in studies comparing different social contexts, for instance showing enhanced motor activity during action observation when adults were engaged in a social interaction with the observed person (Kilner et al., 2006; Kourtis et al., 2010; M´enoret et al., 2014). 1. Introduction Kourtis et al., 2010), top-down effects on neural pro cessing of others’ actions are observed more indirectly in a study comparing different social contexts. Meyer et al. (2011) demonstrated that 3-year-old children showed more motor activation (as indexed by less beta power) when observing an action partner perform an action than when observing the same action while not being engaged in the joint action. While these findings might suggest that young children’s neural motor system is sensitive to actions relevant for coordinating with others, it remains an indirect and thereby limited manipulation of top-down effects on processing others’ actions. Fogassi, 2014). The precise functionality of the mirror mechanism, however, is still matter of debate. In particular, the question whether the mirror mechanism is activated automatically during action observation or whether it is sensitive to top-down modulations like the relevance of actions to the observer has received attention recently (see Campbell and Cunnington, 2017 for a review). In contrast to bottom-up attention which relies on the properties of a stimulus, top-down attention to the environment is not driven by the features of a stimulus itself but rather by one’s prior experience, knowledge and internal goals (Katsuki and Constantinidis, 2014). 1.4. The current study Here, we investigated the effect of top-down processes on children’s neural action processing. Based on previous adult work (Muthukumar aswamy and Singh, 2008; Schuch et al., 2010), we hypothesized that children’s neural motor activity during action observation would be increased when the action is relevant to their own actions (here: for imitating that action) compared to a control condition. In our within-subjects design, we manipulated top-down attention to an observed action by contrasting two action observation conditions in 4-year-old children. We tested children at the age of 4 because at that age children are able to follow explicit task instructions (pivotal for isolating top-down factors) but their attentional and cognitive control skills are still developing. Before watching a short video clip of an action, the 4-year-olds were asked to either subsequently imitate the action they saw (Imitation Task) or label the color of the toy that was acted on (Color-naming Task). The actions children observed in both conditions were identical. To assess children’s neural motor activation during ac tion observation, we measured their EEG throughout the task. In our analysis, we then focused on the alpha (also called mu) and beta fre quency bands since less power in these frequency bands over motor-related brain regions is associated with neural action processing (Fox et al., 2016; Hari et al., 1998). 2.1. Participants This study was approved by the local ethics committee of Radboud University Nijmegen and consent was obtained according to the Declaration of Helsinki (1991; p. 1194). 2. Methods 1.3. Top-down effects on processing others’ actions in the developing brain? 2.2. Procedure For this purpose, we inspected normalized power values of the grand mean, averaged over central electrodes C3 and C4 between 3 Hz and 30 Hz. By determining frequency ranges with less power during action execution compared to baseline we identified the sample-specific frequency band of alpha at 7−12 Hz and of beta at 16−20 Hz (see Fig. 2). To compare children’s neural motor activity between the two action observation conditions (Imitation Task vs. Color-naming Task), we then extracted the normalized power values for these sample-specific bands averaged across central electrodes (C3/C4) for each condition. We hypothesized to find less alpha and beta power for the Imitation Task compared to the Color-naming Task. In our main, hypothesis-driven part of the analysis, we statistically compared the normalized power values between condi tions using a paired samples t-test per frequency band. To further examine the topographic specificity of the effects we provide topo graphic plots of normalized power in the alpha and beta frequency range. Additionally, in a data-driven part of the analysis we then con ducted a cluster based permutation test per frequency band across all counter-balanced across participants. Before watching, in one block children were instructed to imitate the action they observed (Imitation Task) and in another block to later label the color of the toy that was acted on (Color-naming Task). Then children saw the action clip three times, each preceded by a fixation cross (1 s) before they got to execute the action (Imitation Task) or label the toy’s color (Color-Naming). Consistent with Endedijk et al. (2017) the fixation cross was used as baseline. As highlighted in Cuevas et al., 2014, it is favorable to have a baseline temporally close to the test events rather than using a baseline in a separate block. Children thus saw each unique action three times per condition, six times in total. Throughout the experimental session EEG was recorded to assess top-down effects on children’s neural response to action observation and to use their neural response during action execution to identify sample-specific frequency bands associated with action processing. Moreover, we video recorded children throughout the EEG recording for offline coding of children’s task performance and movements during action observation. 2.2. Procedure Recordings from this experiment were also used as part of a longitudinal study on peer cooperation and neural mirroring (Endedijk et al., 2017) and to investigate the role of theta oscillations in task processing (Meyer et al., 2019). During the same recording session in a separate block the children also watched abstract movement clips (i.e. short screensaver clips) without any task. The abstract movement clips were played after every two unique ac tions. This was the same for both conditions. This was subject to a comparison in the study by Endedijk et al. (2017). For transparency, the findings for these stimuli can be found in Endedijk et al. (2017). 2.3. Behavioral analysis Children’s behavior was coded offline to determine their task per formance in the Imitation and Color-naming task (also see Endedijk et al., 2017). To score children’s imitation performance, each action was divided into three parts. The three parts were specified as follows: Movie Clip 1 – stack all cups, stack from left to right (or vice versa), pick up cups using both hands (at least 3 out of 4 times); Movie Clip 2 – shake the rattle, hold it up to the left side of the head, hold it up to the right side of the head; Movie Clip 3 – pick up cup, pick up towel, make sweeping movement with towel inside cup; Movie Clip 4 – take apart all blocks, alternately place blocks left and right (at least 3 times), arrange blocks in one line; Movie Clip 5 – move toy car into the box with one hand, change hands, move toy car out of box with other hand; Movie Clip 6 – activate light, use two hands to touch lamp, push the lamp twice. For each part of an action children imitated correctly, they received one point. Thus, for each action children could score a maximum of three points. To evaluate children’s performance in the Color-naming Task, each correct label of the toy color was scored with one point. 2.2. Procedure Note that analysis of alpha power was also part of supplementary analyses in Meyer et al. (2019). However, this article examined a different research question and the analyses involved different EEG processing steps (e.g. no baseline-correction). As frequency bands shift throughout early development, we used normalized power values from the action execution period to identify our sample-specific alpha and beta fre quency ranges associated with action processing (see Fig. 2). For this purpose, we inspected normalized power values of the grand mean, averaged over central electrodes C3 and C4 between 3 Hz and 30 Hz. By determining frequency ranges with less power during action execution compared to baseline we identified the sample-specific frequency band of alpha at 7−12 Hz and of beta at 16−20 Hz (see Fig. 2). To compare children’s neural motor activity between the two action observation conditions (Imitation Task vs. Color-naming Task), we then extracted the normalized power values for these sample-specific bands averaged across central electrodes (C3/C4) for each condition. We hypothesized to find less alpha and beta power for the Imitation Task compared to the Color-naming Task. In our main, hypothesis-driven part of the analysis, we statistically compared the normalized power values between condi tions using a paired samples t-test per frequency band. To further examine the topographic specificity of the effects we provide topo graphic plots of normalized power in the alpha and beta frequency range. Additionally, in a data-driven part of the analysis we then con ducted a cluster-based permutation test per frequency band across all electrodes, to further explore differences between conditions across all sites. Comparable studies with children of the same age are rare and thus details such as electrode sites of the planned analyses were mostly based ith i f t d lt lit t electrodes. Subsequently, all 1 s segments, experimental and baseline segments, in which children moved or looked away from the stimulus were discarded. Moving was defined as any fine or gross motor move ment children performed during each 1 s segment. The remaining seg ments were visually inspected and EEG artifacts (e.g. due to noisy channels or eye blinks) were rejected. For three children no baseline trials for the Color-naming condition remained. Therefore, they were removed from the final analysis. 2.2. Procedure For the remaining sample of 26 chil dren, per child 65 segments remained on average for action observation in the Imitation condition (SD = 30), 59 segments for action observation in the Color-naming condition (SD = 33), with a minimum of 13 seg ments for the Imitation condition and a minimum of 20 segments for the Color-naming condition. On average, 7 segments for the baseline pre ceding action observation in the Imitation condition (SD = 3), and 6 segments for the baseline preceding action observation in the Color-naming condition (SD = 4) remained. Comparing the number of segments for experimental and baseline periods between conditions did not yield any evidence for a difference between conditions (Experi mental: t(25) = 1.022, p = .316, d = .20; Baseline: t(25) = -1.113, p = .276, d = -.21). We used a DFT filter to remove line noise from the data, and we subtracted the mean signal of each segment from each time point of the segment to take out potential offset differences. To estimate spectral power we used Fast Fourier transform with a Hanning taper and finally calculated the average power for each child over all segments separately for each condition and corresponding baseline. As suggested by Cuevas et al. (2014) the neural signal recorded during action obser vation was normalized using a baseline which did not contain any action (here: fixation cross period). More specifically, we calculated the ratio of average power during action observation for each condition relative to the average baseline of that condition. Taking into account that ratios are not normally distributed, we then applied a log transform to the ratio data. This resulted in normalized values for all frequency bands. We focused on alpha and beta frequency ranges of electrodes overlaying sensorimotor regions (C3, C4) in line with previous adult and child research (Pfurtscheller and Da Silva, 1999; Endedijk et al., 2017). Note that analysis of alpha power was also part of supplementary analyses in Meyer et al. (2019). However, this article examined a different research question and the analyses involved different EEG processing steps (e.g. no baseline-correction). As frequency bands shift throughout early development, we used normalized power values from the action execution period to identify our sample-specific alpha and beta fre quency ranges associated with action processing (see Fig. 2). 2.2. Procedure Children and their caregivers were invited to an EEG testing session of approximately an hour. Upon arrival, caregivers and children were informed about the procedure and written consent was obtained from the child’s legal guardian. Then the child was fitted with a child-sized EEG cap containing 32 active electrodes (actiCap) arranged in a stan dard 10–20 system layout. The online reference was placed at electrode position FCz. For the EEG recording we used a BrainAmp DC EEG 2 Developmental Cognitive Neuroscience 45 (2020) 100851 M. Meyer et al. amplifier to digitize the signal at 500 Hz and band-pass filter the signal between 0.1 and 125 Hz. To reduce environmental noise on the EEG signal, parent and child were accompanied to a shielded room for the experimental part of the session. In the shielded room, children sat at a table with about 60 cm distance to a screen. The experimenter sat next to them so as to provide prompt instructions and parents observed the session from a chair in the back of the room. Children were presented with short movie clips (~7−8 s). Action movie clips displayed a person performing one of six unique goal-directed actions using a toy. These actions were 1) stacking four upside-down cups to a tower, 2) shaking a rattle, 3) wiping the inside of a cup with a towel, 4) disassembling a stack of blocks, 5) moving a toy car into a box and out again and 6) turn on a lamp with two hands (see Fig. 1 for an illustration). Children watched the same action movie clips in two within-subjects conditions implemented by two distinct task instructions (Imitation Task, Color- naming Task). The two task instructions were presented in blocks, 3 ips (~7−8 s). Action movie clips displayed a person naming Task). The two task instructions were p all six actions demonstrated in the movie clips. The pictures represent snapshots of the movie clips with the first picture he following two pictures showing snapshots of the subsequent action unfolding. Fig. 1. Illustration of all six actions demonstrated in the movie clips. The pictures represent snapshots of the movie clips with the first picture in each row showing the start frame and the following two pictures showing snapshots of the subsequent action unfolding. 3 M. Meyer et al. Developmental Cognitive Neuroscience 45 (2020) 100851 electrodes. 2.2. Procedure Subsequently, all 1 s segments, experimental and baseline segments, in which children moved or looked away from the stimulus were discarded. Moving was defined as any fine or gross motor move ment children performed during each 1 s segment. The remaining seg ments were visually inspected and EEG artifacts (e.g. due to noisy channels or eye blinks) were rejected. For three children no baseline trials for the Color-naming condition remained. Therefore, they were removed from the final analysis. For the remaining sample of 26 chil dren, per child 65 segments remained on average for action observation in the Imitation condition (SD = 30), 59 segments for action observation in the Color-naming condition (SD = 33), with a minimum of 13 seg ments for the Imitation condition and a minimum of 20 segments for the Color-naming condition. On average, 7 segments for the baseline pre ceding action observation in the Imitation condition (SD = 3), and 6 segments for the baseline preceding action observation in the Color-naming condition (SD = 4) remained. Comparing the number of segments for experimental and baseline periods between conditions did not yield any evidence for a difference between conditions (Experi mental: t(25) = 1.022, p = .316, d = .20; Baseline: t(25) = -1.113, p = .276, d = -.21). We used a DFT filter to remove line noise from the data, and we subtracted the mean signal of each segment from each time point of the segment to take out potential offset differences. To estimate spectral power we used Fast Fourier transform with a Hanning taper and finally calculated the average power for each child over all segments separately for each condition and corresponding baseline. As suggested by Cuevas et al. (2014) the neural signal recorded during action obser vation was normalized using a baseline which did not contain any action (here: fixation cross period). More specifically, we calculated the ratio of average power during action observation for each condition relative to the average baseline of that condition. Taking into account that ratios are not normally distributed, we then applied a log transform to the ratio data. This resulted in normalized values for all frequency bands. We focused on alpha and beta frequency ranges of electrodes overlaying sensorimotor regions (C3, C4) in line with previous adult and child research (Pfurtscheller and Da Silva, 1999; Endedijk et al., 2017). 2.4. EEG data analysis Bottom row: Topographic distribution of the negative peak of normalized alpha power at 9 Hz (left) and beta power at 18 Hz (right) overlaid with circles visualizing the a priori defined electrode locations for sample-specific frequency selection and analyses (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.). Fig. 2. Top row: Normalized power at electrode sites C3 and C4 (averaged) shown as a function of frequency (Hz). Shaded areas around the mean difference line illustrate the standard error. The left green shaded area indicating the sample-specific frequency range identified for alpha (7–12 Hz), and the right green shaded area indicating the frequency range for beta (16–20 Hz). Negative normalized power values represent suppression during action execution with respect to baseline. Bottom row: Topographic distribution of the negative peak of normalized alpha power at 9 Hz (left) and beta power at 18 Hz (right) overlaid with circles visualizing the a priori defined electrode locations for sample-specific frequency selection and analyses (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.). 2.4. EEG data analysis In accordance with previous studies, we focused in our analysis on two specific frequency bands: the alpha and beta frequency bands (Fox et al., 2016; Marshall and Meltzoff, 2011; Meyer et al., 2011, 2016). While power changes in both frequency bands are associated with pro cessing of own and others’ actions, their functional role may differ. For instance, while alpha activity was proposed to link sensory and motor processes (Pineda, 2005), beta activity was suggested to be involved in monitoring and updating (Engel and Fries, 2010) as well as top-down predictive signaling (van Pelt et al., 2016). As their precise similarity and distinction remains underspecified, we examine these two fre quency bands separately. We used the open source Matlab toolbox FieldTrip (Oostenveld et al., 2011) to conduct EEG data processing in line with Endedijk et al. (2017). More specifically, we first segmented the data in 1 s segments, separate for fixation cross (with 18 possible segments in total per condition) and action movie clip periods (with 144 possible segments in total per condition), eight segments per action movie clip. We then re-referenced the data to the average of all 4 M. Meyer et al. Developmental Cognitive Neuroscience 45 (2020) 100851 2. Top row: Normalized power at electrode sites C3 and C4 (averaged) shown as a function of frequency (Hz). Shaded areas around the mean differen trate the standard error. The left green shaded area indicating the sample-specific frequency range identified for alpha (7–12 Hz), and the right green shade cating the frequency range for beta (16–20 Hz). Negative normalized power values represent suppression during action execution with respect to baseline. Topographic distribution of the negative peak of normalized alpha power at 9 Hz (left) and beta power at 18 Hz (right) overlaid with circles visualizin ri defined electrode locations for sample-specific frequency selection and analyses (For interpretation of the references to colour in this figure legend, the ferred to the web version of this article.). Fig. 2. Top row: Normalized power at electrode sites C3 and C4 (averaged) shown as a function of frequency (Hz). Shaded areas around the mean difference line illustrate the standard error. The left green shaded area indicating the sample-specific frequency range identified for alpha (7–12 Hz), and the right green shaded area indicating the frequency range for beta (16–20 Hz). Negative normalized power values represent suppression during action execution with respect to baseline. 3.2.3. Topography of conditional differences 3.2.3. Topography of conditional differences 3.246, p < .05, d = .63; Color-naming condition, t(25) = 5.471, p < .05, d = 1.07; beta: Imitation condition, t(25) = 2.364, p < .05, d = .46; Color-naming condition, t(25) = 5.215, p < .05, d = 1.02). For more information on the neural response to action observation (averaged across conditions), a topographic plot and spectral distribution across the C3/C4 channels is provided in the Supplementary Material (Sup plementary Fig. S1). Although the lack of overall suppression is unex pected, it is in line with a number of previous developmental studies (e. g. Marshall et al., 2013; Ruysschaert et al., 2013). To exclude the pos sibility that differences in the baseline are driving any effects, we ran a paired samples t-test for each frequency band comparing power values at C3/C4 across conditions in the baseline. Neither in the alpha (t(25) = -.103, p = .919, d = -.02), nor the beta range (t(25) = -.153, p = .138, d = -.03) did we find evidence for differences between the baselines. 3.246, p < .05, d = .63; Color-naming condition, t(25) = 5.471, p < .05, d = 1.07; beta: Imitation condition, t(25) = 2.364, p < .05, d = .46; Color-naming condition, t(25) = 5.215, p < .05, d = 1.02). For more information on the neural response to action observation (averaged across conditions), a topographic plot and spectral distribution across the C3/C4 channels is provided in the Supplementary Material (Sup plementary Fig. S1). Although the lack of overall suppression is unex pected, it is in line with a number of previous developmental studies (e. g. Marshall et al., 2013; Ruysschaert et al., 2013). To exclude the pos sibility that differences in the baseline are driving any effects, we ran a paired samples t-test for each frequency band comparing power values at C3/C4 across conditions in the baseline. Neither in the alpha (t(25) = -.103, p = .919, d = -.02), nor the beta range (t(25) = -.153, p = .138, d = -.03) did we find evidence for differences between the baselines. Fig. 4 (top row) displays the topography of differences between the two action observation conditions (Imitation Task vs. Color-naming Task) in the alpha and beta frequency bands. 3.2.1. Alpha frequency band (7−12 Hz) Based on our a priori hypothesis we tested for condition differences in children’s motor activity by comparing normalized power in the alpha range at central electrode sites (C3/C4). The paired samples t-test did not reveal any significant differences between the mean normalized power in alpha at electrode sites (C3/C4) between the Color-naming condition (M = .35, SE = .06) and the Imitation condition (M = .30, SE = .09), t(25) = .589, p = .561, d = .11. The results are illustrated in Fig. 3 (left). Thus, results of the hypothesis-driven analysis of the alpha frequency range at electrode sites (C3/C4) does not provide any evi dence for increased motor activation during observation of an action with the intention to later imitate that action. 3.2.3. Topography of conditional differences As illustrated by cooler colors over left and right fronto-central sites, children’s beta power was less over the motor regions of the brain for the Imitation compared to the Color-naming task. This distribution of the effect is consistent with the a priori selected electrode sites (C3/C4). A similar data pattern is visible at lateral frontal and parietal-occipital sites. Interestingly, also in the alpha frequency range normalized power appears to be less for the Imitation compared to the Color-naming task over motor regions. While the a priori defined sites of interest (C3/C4) did not reveal evidence for any conditional differences, the topographic distribution of this alpha contrast suggests an effect in the same hy pothesized direction at more centrally located sites as a priori assumed. To examine condition differences across all electrode sites more rigor ously, we conducted a data-driven cluster-based permutation test (Maris and Oostenveld, 2007) for both frequency ranges. 3.2.4. Data-driven comparison of conditional differences across all electrodes We explored conditional differences across all electrode sites by means of a cluster-based permutation test. As a result, the contrast for alpha reveals a negative cluster over fronto-central (F7, Fz, FC5, FC1, Cz) and right parietal (P4, P8) electrode sites. At this cluster, the dif ference between conditions was significant (p < .05). While we found no evidence for a difference in the a priori defined electrodes C3/C4, the negative cluster over slightly more frontal and midline central elec trodes suggests that also alpha range activity was modulated by task instructions. Consistent with our beta findings from the hypothesis- driven analysis, these alpha findings indicate stronger neural motor activation when children watched an action with the intention to imitate it rather than to perform a non-action related task. In contrast to the beta effect, the alpha effect was located more along the midline in central regions rather than over left and right sensorimotor cortices. Also, there were no occipital clusters with a significant difference between condi tions for alpha. Comparing beta power between conditions in a data- driven manner also yielded a negative cluster. Overlapping with the a priori defined electrodes (C3/C4) the cluster that was detected spreads over left fronto-central sites (F7, F3, C3). This cluster-based statistic 3.1. Behavioral results Fig. 3 illustrates normalized power in alpha (left) and beta (right) frequency bands for the two conditions, Imitation and Color-naming. Besides highlighting condition differences, the figure shows that across conditions there is no indication for power suppression (i.e. represented in negative values) in the alpha or beta band with respect to baseline. One-sample t-tests against zero rather suggest an increase in power with respect to baseline (alpha: Imitation condition, t(25) = In the Imitation Task, all children performed all actions. On average, children had an imitation score of 2.59 (range 1–3) out of a maximum of 3. In the Color-naming Task, one child labelled 3 out of 6, two children labelled 5 out of 6, and all remaining children labeled all colors correctly. Together, children performed at ceiling level on both tasks. 5 Developmental Cognitive Neuroscience 45 (2020) 100851 Fig. 3. Boxplots displaying normalized alpha power (left) and normalized beta power (right) averaged over a priori defined electrodes C3 and C4 dependent on the within-subjects condition. Negative values reflect less power and positive more power with respect to baseline. The asterisk indicates a significant difference between the Color-naming and Imitation Task. M. Meyer et al. Developmental Cognitive Neuroscience 45 (2020) 100851 M. Meyer et al. M. Meyer et al. Fig. 3. Boxplots displaying normalized alpha power (left) and normalized beta power (right) averaged over a priori defined electrodes C3 and C4 dependent on the within-subjects condition. Negative values reflect less power and positive more power with respect to baseline. The asterisk indicates a significant difference between the Color-naming and Imitation Task. 3.2.5. Exploration of spectral distribution and topography of the negative peak in beta 3.2.5. Exploration of spectral distribution and topography of the negative peak in beta 3.2.5. Exploration of spectral distribution and topography of the negative peak in beta Besides the data-driven comparison, we further provide a depiction of the spectral specificity of the conditional difference across frequencies 3–30 Hz at the pre-defined electrode sites (C3/C4) and the topography of the negative peak in the beta power at 18 Hz in Fig. 5. Note that this topographic map of the beta effect is only for descriptive purposes and Fig. 4. Top row: Topographic distribution of differences in normalized alpha power (left) and normalized beta power (right) between the Imitation condition and Color-naming condition. Cooler colors represent less power for the Imitation compared to the Color-naming condition. Warmer colors represent more power for the Imitation compared to the Color-naming condition. Less power in these frequency bands over sensorimotor regions is associated with more motor activity. Bottom row: Topographic distribution of conditional differences in normalized alpha power (left) and normalized beta power (right) overlaid with circles visualizing the electrode locations identified as part of a cluster based on the cluster-based permutation test. Fig. 4. Top row: Topographic distribution of differences in normalized alpha power (left) and normalized beta power (right) between the Imitation condition and Color-naming condition. Cooler colors represent less power for the Imitation compared to the Color-naming condition. Warmer colors represent more power for the Imitation compared to the Color-naming condition. Less power in these frequency bands over sensorimotor regions is associated with more motor activity. Bottom row: Topographic distribution of conditional differences in normalized alpha power (left) and normalized beta power (right) overlaid with circles visualizing the electrode locations identified as part of a cluster based on the cluster-based permutation test. comparing the conditions was marginally significant (p < .1). Thus, for beta, hypothesis-driven and data-driven results converge. Fig. 4 (bottom row) illustrates the distribution of all electrode locations identified by the cluster statistics. Additionally, Supplementary Fig. S2 displays the conditional difference across all frequencies (3−30 Hz) for the electrode clusters identified for the alpha and beta frequency band. no statistical tests were conducted to avoid multiple testing of a previ ously identified effect. To further explore potential influences of the baseline, we provide the same illustration as direct contrast without baseline-correction between the two conditions in the Supplementary Material (Supplementary Fig. S3). The spectral and topographic distri butions show the same pattern as our main effect, i.e. less power for the Imitation compared to the Color-naming Task in the beta frequency range with a peak distributed over fronto-central brain regions. This demonstrates the robustness of the condition difference we observe. 3.2.2. Beta frequency band (16−20 Hz) Analogously to alpha, we contrasted the two action observation conditions in the beta band. The paired samples t-test revealed a sig nificant difference of the mean normalized power in beta at central electrode sites (C3/C4) between the Color-naming condition (M = .33, SE = .06) and the Imitation condition (M = .16, SE = .07), t(25) = 2.407, p = .024, d = .47. For an illustration of the results see Fig. 3 (right). These findings indicate significantly less beta power, thus stronger motor activation, when 4-year-olds watched another person’s actions with the intention to imitate that action compared to watching the same action with the intention to report visual aspects of the action (i.e. toy color). 6 Developmental Cognitive Neuroscience 45 (2020) 10 4. Top row: Topographic distribution of differences in normalized alpha power (left) and normalized beta power (right) between the Imitation condition r-naming condition. Cooler colors represent less power for the Imitation compared to the Color-naming condition. Warmer colors represent more power for ation compared to the Color-naming condition. Less power in these frequency bands over sensorimotor regions is associated with more motor activity. Bo Topographic distribution of conditional differences in normalized alpha power (left) and normalized beta power (right) overlaid with circles visualizing rode locations identified as part of a cluster based on the cluster-based permutation test. eyer et al. M. Meyer et al. Developmental Cognitive Neuroscience 45 (2020) 100851 4 Top row: Topographic distribution of differences in normalized alpha power (left) and normalized beta power (right) between the Imitation condition 4. Discussion In this study, we investigated whether 4-year-olds’ neural motor activation when observing others’ actions is modulated by top-down 7 M. Meyer et al. Developmental Cognitive Neuroscience 45 (2020) 100851 Fig. 5. Top row: Normalized power difference between conditions at electrode sites C3 and C4 (averaged) shown as a function of frequency (Hz). Positive values represent less power for the Color-naming Task and negative values represent less power for the Imitation Task. Shaded areas around the mean difference line illustrate the standard error. The light blue shaded areas indicates the sample-specific frequency range for alpha (7-12 Hz), and the dark blue shaded area indicates the beta frequency range (16-20 Hz). Bottom row: Topographic distribution of the negative peak of the normalized difference in beta power identified at 18 Hz (right) (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.). Fig. 5. Top row: Normalized power difference between conditions at electrode sites C3 and C4 (averaged) shown as a function of frequency (Hz). Positive values represent less power for the Color-naming Task and negative values represent less power for the Imitation Task. Shaded areas around the mean difference line illustrate the standard error. The light blue shaded areas indicates the sample-specific frequency range for alpha (7-12 Hz), and the dark blue shaded area indicates the beta frequency range (16-20 Hz). Bottom row: Topographic distribution of the negative peak of the normalized difference in beta power identified at 18 Hz (right) (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.). processes. Based on cognitive neuroscience research with adults (Muthukumaraswamy and Singh, 2008; Schuch et al., 2010), we hy pothesized that children’s neural motor activity would be increased when watching an action with the intention to imitate it (Imitation Task) compared to when watching the same action with a non-action related task (Color-naming Task). Our planned and data-driven analyses pro vide converging evidence that in contrast to the Color-naming condition, children’s neural motor activation was increased when observing an action with the intention to imitate it. Our findings suggest that children processed the same action with more engagement of their neural motor system when it was relevant for their own subsequent behavior. with the intention to reproduce it (Muthukumaraswamy and Singh, 2008). 4. Discussion It is further in line with beta effects induced by different social contexts in children and adults (Kourtis et al., 2010; M´enoret et al., 2014; Meyer et al., 2011). Although only marginally significant, out comes of the data-driven analysis of beta power across all electrodes overlap and converge with results from the planned analyses. The frontal topography reported in the data-driven analysis also fits with the frontal topography during children’s action execution (see Fig. 2) and is in line with evidence of top-down connections of signals in the beta frequency range between prefrontal and parietal regions (van Pelt et al., 2016). In contrast, there was no initial evidence for condition differences in the alpha frequency band. However, data-driven cluster-based tests revealed that rather than over left and right sensorimotor cortices (as used in the planned comparison) there were condition differences in the alpha band over central-midline areas, left frontal and right parietal sites. Like in the beta effect, baseline-corrected power in the alpha range (although at more midline-central sites) was less for the Imitation Task compared to the Color-naming Task. The topography of the alpha effect is comparable to that found by Schuch et al. (2010) when contrasting alpha range activity of adults solving action-related and color-related tasks. Moreover, the fronto-parietal results fit with the notion that fronto-parietal networks reflect action processing (Pineda, 2005). The topographic distribution of alpha and beta power, in particular with respect to their frontal activation sites might further suggest links be tween movement learning and planning with premotor cortices. In accordance with that, Suchan et al. (2008) found that adults’ premotor cortex is active when they were asked to observe actions to later imitate 4.2. Limitations of the current study Contrary to what was expected (see e.g., Fox et al., 2016), our study did not yield evidence for overall suppression with respect to baseline in alpha or beta frequency ranges (see Fig. 3). However, several other developmental EEG studies report similar results to ours (K¨oster et al., 2020; Marshall et al., 2013; Ruysschaert et al., 2013; also cf. Endedijk et al., 2017, for a detailed discussion). A potential explanation might be that observing televised compared to live actions elicits overall less motor activity (Shimada and Hiraki, 2006). Also, one might speculate that children actively inhibited their motor system because they were asked to sit still during the action videos, which might have resulted in an increase in power with respect to baseline. This fits with research showing an increase in sensorimotor alpha when adults are asked to inhibit an action, an effect stronger in younger compared to older adults (e.g. B¨onstrup et al., 2015). Another possibility is that the task instruc tion which preceded the action observation might have led children to activate their motor system already during the baseline phase, which in turn might have led to a lack of suppression with respect to the action clips. This hypothesis, however, is not supported by our data (see Sup plementary Material). Despite this unexpected outcome, all effects of conditional differences in our within-subjects contrast are in the hy pothesized direction, with less power in the Imitation Task compared to the Color-naming Task. We are confident that irrespective of the overall motor activation with respect to baseline, the relative difference in motor activation between conditions, validly reflects differences in children’s neural processing of others’ actions. Another potential limi tation of the current study is the loss of temporal information in the analysis. Since addressing the current research question did not require a time-resolved analysis, data of each stimulus movie were epoched in 1 s segments. This allowed us to include more data by not having to discard entire 7−8 second data epochs when only a small fraction was artifacted. While that allowed for more robust results, this analysis de cision also limits the current findings because temporal information is lost. As we suggest below, future research could take this into account by using shorter movie clips such that temporal information is retained while keeping data loss at a minimum. 4.1. Intention to imitate increases 4-year-olds’ neural motor activation We analyzed the power in two frequency bands, alpha and beta, which are well-established indices of neural motor activation (e.g. Fox et al., 2016; Hari et al., 1998; Pfurtscheller and Da Silva, 1999). Our planned analyses of these frequency bands over left and right sensori motor cortices revealed more motor activity indexed by significantly lower baseline-corrected beta power for the Imitation Task compared to the Color-naming Task. The topography of the negative peak of this beta effect illustrates that the modulation is strongest at electrode sites overlaying motor cortices and spreads out to lateral frontal sites over laying premotor regions as well as parietal sites overlaying sensorimotor cortices. This suggests top-down effects in brain regions involved in action processing such as motor and premotor cortices. Moreover, this is consistent with previous findings of top-down effects on beta oscillations reflecting modulations of motor activity when adults observe an action 8 8 Developmental Cognitive Neuroscience 45 (2020) 100851 M. Meyer et al. them (see also Frey and Gerry, 2006). Similarly, this fronto-central and partially parietal distribution also fits with other adult neuroimaging studies on action observation and observational learning (e.g. Buccino et al., 2001; Cross et al., 2009; Molenberghs et al., 2012). Besides this, the cluster-based tests did not provide any evidence for condition dif ferences in occipital channels (i.e. no condition differences at occipital clusters reached significance) suggesting that sensorimotor alpha rather than occipital alpha was affected by children’s intention to imitate. All in all, the results provide evidence for top-down attentional effects on neural processing of others’ actions already in early childhood. properties of a given stimulus, such as its visual appearance (Katsuki and Constantinidis, 2014). While previous findings cannot dissociate be tween these alternative interpretations, the current results can exclude stimulus saliency as a driver of enhanced neural activation because the observed actions were identical across conditions. This allowed us to study the neural effect of having the prior intention to imitate an observed action in isolation. Since children’s performance in both tasks was at ceiling level, we could not assess potential beneficial effects of selective neural enhancement for imitation performance on an individ ual differences basis. 4.2. Limitations of the current study Although the focus of the current study was on the effect of top-down attention on the processing of others’ actions, when those were impor tant for children, one might speculate on how top-down attention might have influenced children’s processing in the Color-Naming task. For instance, children might have paid particular attention to the color of the toy instead of the actions, leading to enhanced visual processing. Modulation of alpha power over occipital sites is thought to reflect enhanced visual attention to a stimulus (Herring et al., 2015). Therefore, one might have expected occipital alpha power to be suppressed in the Color-naming compared to Imitation condition. However, as apparent from the results of our data-driven comparison in the alpha range and Fig. 4, our data do not seem to support this speculation. For discussions on potential language-related effects on top-down processing in the theta frequency range see Meyer et al. (2019). 4.4. Top-down attention and the role of motor-related brain areas Do top-down attentional effects in this context originate from and are they confined to motor-related brain regions such as (pre-)motor and sensorimotor areas or is the modulation we observe in the 4-year-olds’ neural motor activity a downstream result stemming from a more wide- spread attentional network? Although the current data cannot provide an ultimate answer to this question, previous theoretical and empirical work in cognitive neuroscience suggests a network of cortical oscilla tions spanning different brain regions involved in top-down processes. As proposed by Clayton et al. (2015) and Cavanagh and Frank (2014), supramodal theta oscillations from frontomedial brain regions might modulate modality-specific activity for instance in alpha and beta os cillations downstream. In line with this, van Ede et al. (2017) found top-down control effects in an MEG study in which adults had to either solve a visual or tactile working memory task. Their results show that medial prefrontal theta synchronization predicts subsequent modality-specific alpha and beta suppression (in visual and somato sensory regions, respectively). Consistent with this idea, results of post-hoc analyses of the current dataset showing that frontomedial theta power was modulated by task engagement and task demands (Meyer 4.1. Intention to imitate increases 4-year-olds’ neural motor activation Although beyond the scope of the current study, one might expect amplified neural motor activity to predict better imitation performance given prior work linking attentional effects re flected in alpha and beta oscillations and overt performance in adults (Haegens et al., 2011; van Ede et al., 2012) and recent infant EEG work (Filippi et al., 2016). Relatedly, in adults more precise imitation per formance is related to alpha power suppression when later observing the previously imitated action again, further supporting a tight link between neural motor activity during action observation and imitation perfor mance (Marshall et al., 2009). Together this might imply that instruction prior to demonstrating an action could not only enhance children’s neural response during action perception but also has the potential to affect children’s action learning. In infancy, at an age at which children cannot be instructed explicitly, using other forms of highlighting the relevance of the action and engaging the child might have similar effects on children’s neural processing of others’ actions. Strategies such as social engagement through gaze cues (Michel et al., 2015), turn-taking (Meyer et al., 2011) or infant-directed behaviors like infant-directed speech (Zhang et al., 2011) and infant-directed actions (van Schaik et al., 2019) might help to underline the relevance of an action for children too young to be explicitly instructed. 5. Conclusion Hunnius, S., Bekkering, H., 2014. What are you doing? How active and observational experience shape infants’ action understanding. Philos. Trans. Biol. Sci. 369 (1644), 20130490. The current findings with 4-year-old children provide the first evi dence that top-down attention to another person’s action modulates young children’s neural processing of that action. In particular, we found higher motor activation as indexed by alpha and beta rhythm activity when children observed an action with the intention to imitate it rather than solving a non-action related task (labeling the color of a toy). This suggests that already young children flexibly process others’ ac tions depending on the relevance of the observed actions for their own behavior. Katsuki, F., Constantinidis, C., 2014. Bottom-up and top-down attention: different processes and overlapping neural systems. Neuroscientist 20 (5), 509–521. Katsuki, F., Constantinidis, C., 2014. Bottom-up and top-down attention: different processes and overlapping neural systems. Neuroscientist 20 (5), 509–521. Kilner, J.M., Marchant, J.L., Frith, C.D., 2006. Modulation of the mirror system by social relevance. Soc. Cogn. Affect. Neurosci. 1 (2), 143–148. p pp g y , Kilner, J.M., Marchant, J.L., Frith, C.D., 2006. Modulation of the mirror system by social relevance. Soc. Cogn. Affect. Neurosci. 1 (2), 143–148. ¨ l h li h i hl K¨oster, M., Langeloh, M., Kliesch, C., Kanngiesser, P., Hoehl, S., 2020. Motor cortex activity during action observation predicts subsequent action imitation in human infants. NeuroImage, 116958. Kourtis, D., Sebanz, N., Knoblich, G., 2010. Favouritism in the motor system: social interaction modulates action simulation. Biol. Lett. 6 (6), 758–761. Langeloh, M., Buttelmann, D., Matthes, D., Grassmann, S., Pauen, S., Hoehl, S., 2018. Reduced mu power in response to unusual actions is context-dependent in 1-year- olds. Front. Psychol. 9, 36. Maris, E., Oostenveld, R., 2007. Nonparametric statistical testing of EEG-and MEG-data. J. Neurosci. Methods 164 (1), 177–190. References Modulation of neural activity during observational learning of actions and their sequential orders. J. Neurosci. 26 (51), 13194–13201. G J C t N D t J 1998 T d ff t f th t t t i it t th Grezes, J., Costes, N., Decety, J., 1998. Top down effect of the strategy to imitate on the brain areas engaged in perception of biological motion: a PET study. Cogn. Neuropsychol. 15 (6), 7. p y Haegens, S., H¨andel, B.F., Jensen, O., 2011. Top-down controlled alpha band activity in somatosensory areas determines behavioral performance in a discrimination task. J. Neurosci. 31 (14), 5197–5204. Hari, R., Forss, N., Avikainen, S., Kirveskari, E., Salenius, S., Rizzolatti, G., 1998. Activation of human primary motor cortex during action observation: a neuromagnetic study. Proc. Natl. Acad. Sci. 95 (25), 15061–15065. Herring, J.D., Thut, G., Jensen, O., Bergmann, T.O., 2015. Attention modulates TMS- locked alpha oscillations in the visual cortex. J. Neurosci. 35 (43), 14435–14447. Hickok, G., 2014. The Myth of Mirror Neurons: the Real Neuroscience of Communication Hickok, G., 2014. The Myth of Mirror Neurons: the Real Neuroscience of Communication and Cognition. WW Norton & Company. , , y and Cognition. WW Norton & Company. 4.3. Top-down attention to action and imitation in development The current findings not only add to adult literature but also extend the growing body of developmental literature on the role of mirroring for children’s imitation and learning from others (Hunnius and Bek kering, 2014; Marshall and Meltzoff, 2014; Meltzoff and Marshall, 2018; Woodward and Gerson, 2014). As summarized by Marshall and Meltzoff (2014), converging evidence suggests a tight link between imitating others’ actions and the neural processing of others’ actions from early in life. In line with this, recent results from an EEG study with 7-month-old infants show that neural motor activation to another person’s actions is stronger when it preceded infants’ subsequent imitation of the observed action (Filippi et al., 2016). This might be due to infants preferring one action over another (bottom-up influence) or it might result from in fants’ prior intention to imitate what they are about to see (top-down influence). Important in the distinction between bottom-up and top-down attention is that bottom-up attention is solely caused by the 9 Developmental Cognitive Neuroscience 45 (2020) 100851 M. Meyer et al. final version of the manuscript for submission. et al., 2019) hints at similar networks being at play in young children. Our design and limited number of trials did not allow us to conduct the same trial-wise correlational analysis as in van Ede et al. (2017) to systematically investigate the potential relation between theta syn chronization in frontomedial sites and alpha and beta power over pre motor and sensorimotor regions in the current data. Although optimal for addressing the main question of this study, the current presentation of three subsequent movie clips and the segmenting of each movie clip in 1 s segments resulted in the loss of time-locking critical for a trial-wise analysis as in van Ede et al. (2017). Moreover, van Ede et al. (2017) Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.dcn.2020.100851. presented close to 500 trials to their adult participants which is far beyond the number of trials in the current study with 4-year-olds. While trial-level analyses were not possible with the current data we did conduct a first exploratory analysis on the subject-level. More specif ically, we correlated frontal theta power (3−6 Hz) at Fz prior and during action observation (averaged across conditions) with the normalized alpha and beta power difference between conditions (Imitation – Color-naming Task) at C3/C4 (see Supplementary Analyses and Sup plementary Figs. S4-S7). This exploratory analysis yielded a negative correlation between frontal theta and the central beta power effect, which provides a first indication that frontal theta power predicts sub sequent top-down effects reflected in central beta oscillations. Rather than conclusive evidence, these exploratory results provide more leverage to pursuing a systematic investigation of this question. Future investigations are needed to test the idea that top-down effects on action processing involve a cortical oscillation network in which frontomedial theta synchronization predicts alpha/beta power effects in premotor and sensorimotor regions. For instance, by having very short movie clips (of about 2–3 seconds) each immediately followed by the response of the child, more time-locked data could be obtained. Moreover, testing a large number participants would further help to compensate for the lower number of trials inevitable when testing 4-year-old children. Addressing this type of oscillatory framework is particularly interesting in developmental populations given the drastic changes, functionally and structurally, of frontomedial brain regions such as medial frontal and anterior cingulate cortex which are thought to generate theta os cillations. This opens up an avenue with potential to inform questions from both, the field of developmental psychology and cognitive neuro science. For instance, does the emergence of top-down attentional ef fects on action processing change as a function of structural development in frontal brain regions or rather motor cortical regions which mature significantly earlier in development? How does this pre dict children’s imitation behavior? Moreover, research on the precise functionality of oscillatory models of top-down attention might benefit from isolating the role of frontomedial theta and pre-motor and senso rimotor alpha/beta by harnessing the differences in developmental trajectories of frontal and sensorimotor cortices. References B¨onstrup, M., Hagemann, J., Gerloff, C., Sauseng, P., Hummel, F.C., 2015. Alpha oscillatory correlates of motor inhibition in the aged brain. Front. Aging Neurosci. 7, 193. Buccino, G., Binkofski, F., Fink, G.R., Fadiga, L., Fogassi, L., Gallese, V., et al., 2001. Action observation activates premotor and parietal areas in a somatotopic manner: an fMRI study. Eur. J. Neurosci. 13 (2), 400–404. Campbell, M.E., Cunnington, R., 2017. More than an imitation game: top-down modulation of the human mirror system. Neurosci. Biobehav. Rev. 75, 195–202. Cavanagh, J.F., Frank, M.J., 2014. Frontal theta as a mechanism for cognitive control. Trends Cogn. Sci. 18 (8), 414–421. Clayton, M.S., Yeung, N., Kadosh, R.C., 2015. The roles of cortical oscillations in sustained attention. Trends Cogn. Sci. 19 (4), 188–195. Cross, E.S., Kraemer, D.J., Hamilton, A.F.D.C., Kelley, W.M., Grafton, S.T., 2009. Sensitivity of the action observation network to physical and observational learning. Cereb. Cortex 19 (2), 315–326. Csibra, G., 2008. Action Mirroring and Action Understanding: an Alternative Account. Sensorymotor foundations of higher cognition. Attention and performance XXII, pp. 435–459. Cuevas, K., Cannon, E.N., Yoo, K., Fox, N.A., 2014. The infant EEG mu rhythm: methodological considerations and best practices. Dev. Rev. 34 (1), 26–43. Endedijk, H.M., Meyer, M., Bekkering, H., Cillessen, A.H.N., Hunnius, S., 2017. Neural mirroring and social interaction: motor system involvement during action observation relates to early peer cooperation. Dev. Cogn. Neurosci. 24, 33–41. Engel, A.K., Fries, P., 2010. Beta-band oscillations—signalling the status quo? Curr. Opin. Neurobiol. 20 (2), 156–165. Filippi, C.A., Cannon, E.N., Fox, N.A., Thorpe, S.G., Ferrari, P.F., Woodward, A.L., 2016. Motor system activation predicts goal imitation in 7-month-old infants. Psychol. Sci. 27 (5), 675–684. Fox, N.A., Bakermans-Kranenburg, M.J., Yoo, K.H., Bowman, L.C., Cannon, E.N., Vanderwert, R.E., et al., 2016. Assessing human mirror activity with EEG mu rhythm: a meta-analysis. Psychol. Bull. 142 (3), 291. Fox, N.A., Bakermans-Kranenburg, M.J., Yoo, K.H., Bowman, L.C., Cannon, E.N., Vanderwert, R.E., et al., 2016. Assessing human mirror activity with EEG mu rhythm: a meta-analysis. Psychol. Bull. 142 (3), 291. Frey, S.H., Gerry, V.E., 2006. Modulation of neural activity during observational learning of actions and their sequential orders. J. Neurosci. 26 (51), 13194–13201. rhythm: a meta-analysis. Psychol. Bull. 142 (3), 291. Frey, S.H., Gerry, V.E., 2006. Modulation of neural activity during observational learning of actions and their sequential orders. J. Neurosci. 26 (51), 13194–13201. rhythm: a meta analysis. Psychol. Bull. 142 (3), 291. Frey, S.H., Gerry, V.E., 2006. Declaration of Competing Interest The authors report no declarations of interest. Author contributions Marshall, P.J., Meltzoff, A.N., 2011. Neural mirroring systems: exploring the EEG mu rhythm in human infancy. Dev. Cogn. Neurosci. 1 (2), 110–123. rhythm in human infancy. Dev. Cogn. Neurosci. 1 (2), 110–123. Marshall, P.J., Meltzoff, A.N., 2014. Neural mirroring mechanisms and imitation in h i f t Phil T Bi l S i 369 (1644) 20130620 MM, HME and SH jointly developed the study concept and design. MM and HME collected the data. MM and HME performed data analyses and all authors interpreted the data. MM drafted the manuscript, and HME and SH contributed critical revisions. All authors approved the Marshall, P.J., Meltzoff, A.N., 2014. Neural mirroring mechanisms and imitation in human infants. Philos. Trans. Biol. Sci. 369 (1644), 20130620. Marshall, P.J., Meltzoff, A.N., 2014. Neural mirroring mechanisms and imitation in human infants. Philos. Trans. Biol. Sci. 369 (1644), 20130620. 10 M. Meyer et al. Developmental Cognitive Neuroscience 45 (2020) 100851 Marshall, P.J., Bouquet, C.A., Shipley, T.F., Young, T., 2009. Effects of brief imitative experience on EEG desynchronization during action observation. Neuropsychologia 47 (10), 2100–2106. Rizzolatti, G., Fogassi, L., 2014. The mirror mechanism: recent findings and perspectives. Philos. Trans. Biol. Sci. 369 (1644), 20130420. Rueda, M.R., Posner, M.I., Rothbart, M.K., 2004. Attentional control and self-regulation. Handbook of Self-Regulation: Research, Theory, and Applications, pp. 284–299, 2. Marshall, P.J., Saby, J.N., Meltzoff, A.N., 2013. Infant brain responses to object weight: exploring goal-directed actions and self-experience. Infancy 18 (6), 942–960. Ruysschaert, L., Warreyn, P., Wiersema, J.R., Metin, B., Roeyers, H., 2013. Neural mirroring during the observation of live and video actions in infants. Clin. Neurophysiol. 124 (9), 1765–1770. mirroring during the observation of live and video actions in infants. Clin. Neurophysiol. 124 (9), 1765–1770. Meltzoff, A.N., Marshall, P.J., 2018. Human infant imitation as a social survival circuit. Curr. Opin. Behav. Sci. 24, 130–136. M´enoret, M., Varnet, L., Fargier, R., Cheylus, A., Curie, A., Des Portes, V., et al., 2014. Neural correlates of non-verbal social interactions: a dual-EEG study. Neuropsychologia 55, 85–97. Schuch, S., Bayliss, A.P., Klein, C., Tipper, S.P., 2010. Attention modulates motor system activation during action observation: evidence for inhibitory rebound. Exp. Brain Res. 205 (2), 235–249. Meyer, M., Hunnius, S., van Elk, M., van Ede, F., Bekkering, H., 2011. Joint action modulates motor system involvement during action observation in 3-year-olds. Exp. Brain Res. 211 (3–4), 581–592. Shimada, S., Hiraki, K., 2006. Infant’s brain responses to live and televised action. Author contributions Neuroimage 32 (2), 930–939. Southgate, V., Begus, K., 2013. Motor activation during the prediction of nonexecutable actions in infants. Psychol. Sci. 24 (6), 828–835. Meyer, M., Braukmann, R., Stapel, J.C., Bekkering, H., Hunnius, S., 2016. Monitoring others’ errors: the role of the motor system in early childhood and adulthood. Br. J. Dev. Psychol. 34 (1), 66–85. Suchan, B., Melde, C., Herzog, H., H¨omberg, V., Seitz, R.J., 2008. Activation differences in observation of hand movements for imitation or velocity judgement. Behav. Brain Res. 188 (1), 78–83. Meyer, M., Endedijk, H.M., van Ede, F., Hunnius, S., 2019. Theta oscillations in 4-year- olds are sensitive to task engagement and task demands. Sci. Rep. 9 (1), 6049. van Ede, F., de Lange, F.P., Maris, E., 2012. Attentional cues affect accuracy and reaction time via different cognitive and neural processes. J. Neurosci. 32 (30), 10408–10412. i Michel, C., Stets, M., Parise, E., Reid, V.M., Striano, T., Hoehl, S., 2015. Theta-and alpha- band EEG activity in response to eye gaze cues in early infancy. Neuroimage 118, 576–583. van Ede, F., Jensen, O., Maris, E., 2017. Supramodal theta, gamma, and sustained fields predict modality-specific modulations of alpha and beta oscillations during visual and tactile working memory. J. Cogn. Neurosci. 29 (8), 1455–1472. P lt S H il L K i th t J O d b k S R ij I B kk i H 2016 B t Molenberghs, P., Cunnington, R., Mattingley, J.B., 2012. Brain regions with mirror properties: a meta-analysis of 125 human fMRI studies. Neurosci. Biobehav. Rev. 36 (1), 341–349. van Pelt, S., Heil, L., Kwisthout, J., Ondobaka, S., van Rooij, I., Bekkering, H., 2016. Beta- and gamma-band activity reflect predictive coding in the processing of causal events. Soc. Cogn. Affect. Neurosci. 11 (6), 973–980. Muthukumaraswamy, S.D., Singh, K.D., 2008. Modulation of the human mirror neuron system during cognitive activity. Psychophysiology 45 (6), 896–905. ld i i h ff l i ld i and gamma-band activity reflect predictive coding in the processing of causal events. Soc. Cogn. Affect. Neurosci. 11 (6), 973–980. van Schaik, J.E., Meyer, M., van Ham, C.R., Hunnius, S., 2019. Motion tracking of parents’ infant-versus adult-directed actions reveals general and action-specific modulations. Dev. Sci., e12869 Oostenveld, R., Fries, P., Maris, E., Schoffelen, J.M., 2011. FieldTrip: open source software for advanced analysis of MEG, EEG, and invasive electrophysiological data. Comput. Intell. Neurosci. 2011, 1. Woodward, A.L., Gerson, S.A., 2014. Author contributions Mirroring and the development of action understanding. Philos. Trans. Biol. Sci. 369 (1644), 20130181. ld d l l f l k d l h Patzwald, C., Matthes, D., Elsner, B., 2020. Eighteen-month-olds integrate verbal cues into their action processing: evidence from ERPs and mu power. Infant Behav. Dev. 58, 101414. World Medical Association, 1991. Declaration of Helsinki. Law Med. Health Care 19 (3–4), 264–265. Pfurtscheller, G., Da Silva, F.L., 1999. Event-related EEG/MEG synchronization and desynchronization: basic principles. Clin. Neurophysiol. 110 (11), 1842–1857. Pineda J A 2005 The functional significance of mu rhythms: translating “seeing” and Pfurtscheller, G., Da Silva, F.L., 1999. Event-related EEG/MEG synchronization and desynchronization: basic principles. Clin. Neurophysiol. 110 (11), 1842–1857. Pineda, J.A., 2005. The functional significance of mu rhythms: translating “seeing” and “hearing” into “doing”. Brain Res. Rev. 50 (1), 57–68. Zelazo, P.D., Carlson, S.M., 2012. Hot and cool executive function in childhood and adolescence: development and plasticity. Child Dev. Perspect. 6 (4), 354–360. desynchronization: basic principles. Clin. Neurophysiol. 110 (11), 1842 1857. Pineda, J.A., 2005. The functional significance of mu rhythms: translating “seeing” and “hearing” into “doing”. Brain Res. Rev. 50 (1), 57–68. Pineda, J.A., 2005. The functional significance of mu rhyth “hearing” into “doing”. Brain Res. Rev. 50 (1), 57–68. Pineda, J.A., 2005. The functional significance of mu rhythms: translating “seeing” and “hearing” into “doing”. Brain Res. Rev. 50 (1), 57–68. i “hearing” into “doing”. Brain Res. Rev. 50 (1), 57–68. Zhang, Y., Koerner, T., Miller, S., Grice-Patil, Z., Svec, A., Akbari, D., et al., 2011. Neural coding of formant-exaggerated speech in the infant brain. Dev. Sci. 14 (3), 566–581. 11 11
https://openalex.org/W4392639484	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0297331&type=printable	English	null	Integrating image and gene-data with a semi-supervised attention model for prediction of KRAS gene mutation status in non-small cell lung cancer	PloS one	2,024	cc-by	12,989	OPEN ACCESS KRAS is a pathogenic gene frequently implicated in non-small cell lung cancer (NSCLC). However, biopsy as a diagnostic method has practical limitations. Therefore, it is important to accurately determine the mutation status of the KRAS gene non-invasively by combining NSCLC CT images and genetic data for early diagnosis and subsequent targeted therapy of patients. This paper proposes a Semi-supervised Multimodal Multiscale Attention Model (S2MMAM). S2MMAM comprises a Supervised Multilevel Fusion Segmentation Network (SMF-SN) and a Semi-supervised Multimodal Fusion Classification Network (S2MF-CN). S2MMAM facilitates the execution of the classification task by transferring the useful infor- mation captured in SMF-SN to the S2MF-CN to improve the model prediction accuracy. In SMF-SN, we propose a Triple Attention-guided Feature Aggregation module for obtaining segmentation features that incorporate high-level semantic abstract features and low-level semantic detail features. Segmentation features provide pre-guidance and key information expansion for S2MF-CN. S2MF-CN shares the encoder and decoder parameters of SMF- SN, which enables S2MF-CN to obtain rich classification features. S2MF-CN uses the pro- posed Intra and Inter Mutual Guidance Attention Fusion (I2MGAF) module to first guide seg- mentation and classification feature fusion to extract hidden multi-scale contextual information. I2MGAF then guides the multidimensional fusion of genetic data and CT image data to compensate for the lack of information in single modality data. S2MMAM achieved 83.27% AUC and 81.67% accuracy in predicting KRAS gene mutation status in NSCLC. This method uses medical image CT and genetic data to effectively improve the accuracy of predicting KRAS gene mutation status in NSCLC. Citation: Xue Y, Zhang D, Jia L, Yang W, Zhao J, Qiang Y, et al. (2024) Integrating image and gene- data with a semi-supervised attention model for prediction of KRAS gene mutation status in non- small cell lung cancer. PLoS ONE 19(3): e0297331. https://doi.org/10.1371/journal.pone.0297331 Editor: Jeonghwan Gwak, Korea National University of Transportation, REPUBLIC OF KOREA Received: June 5, 2023 Accepted: January 3, 2024 Published: March 11, 2024 Peer Review History: PLOS recognizes the benefits of transparency in the peer review process; therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. The editorial history of this article is available here: https://doi.org/10.1371/journal.pone.0297331 Copyright: © 2024 Xue et al. PLOS ONE PLOS ONE RESEARCH ARTICLE Integrating image and gene-data with a semi- supervised attention model for prediction of KRAS gene mutation status in non-small cell lung cancer Yuting Xue1, Dongxu Zhang1, Liye Jia1☯, Wanting Yang1☯, Juanjuan ZhaoID2,3, Yan Qiang1, Long Wang3, Ying Qiao4, Huajie Yue4 1 College of Information and Computer, Taiyuan University of Technology, Taiyuan, Shanxi, China, 2 School of Software, Taiyuan University of Technology, Taiyuan, Shanxi, China, 3 College of Information, Jinzhong College of Information, Taiyuan, Shanxi, China, 4 First Hospital of Shanxi Medical University, Taiyuan, Shanxi, China a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 ☯These authors contributed equally to this work. zhaojuanjuan@tyut.edu.cn OPEN ACCESS This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Introduction available on a GitHub repository at https://github. com/xyttttboom/SSMMAM. available on a GitHub repository at https://github. com/xyttttboom/SSMMAM. Lung cancer is specifically divided into non-small cell lung cancer (NSCLC) and small cell lung cancer. NSCLC accounts for approximately 85% of newly diagnosed lung cancers yearly [1]. The emergence of targeted therapy has substantially increased the survival rate of NSCLC patients. Prior to targeted therapy, it should be determined whether important disease-causing genes are mutated. KRAS is a common causative gene in NSCLC, and approximately one- third of patients with NSCLC have KRAS mutations. The usual diagnostic tool is a puncture biopsy. However, this invasive method has many limitations, such as it is unsuitable for all body types and has unpredictable consequences such as increased risk of cancer metastasis [2]. Therefore, there is an urgent need for a non-invasive diagnostic method that can accurately predict KRAS mutations in lung cancer patients. This method will not only improve the treat- ment outcome of patients but also guide prognosis. Funding: This work was supported by the National Natural Science Foundation of China (Grant No. U21A20469); the National Natural Science Foundation of China (Grant No. 61972274); the Central Government Guides Local Science and Technology Development Fund Project (Grant No. YDZJSX2022C004); the Natural Science Foundation of Shanxi Province (Grant No. 202103021224066); and NHC Key Laboratory of Pneumoconiosis Shanxi China Project, (Grant No.2020-PT320-005), the Non-profit Central Research Institute Fund of Chinese Academy of Medical Science. The funders had a role in decision to publish and preparation of the manuscript. In recent years, researchers have used CT images to predict gene mutations based on tradi- tional radiomics and machine learning. Song et al. [3] propose a machine-learning model for predicting EGFR and KRAS mutation status. They used the model to extract statistical, shape, pathological, and deep learning features from 144 CT scans of tumor regions. Shiri et al. [4] used minimum redundancy, maximum correlation feature selection, and random forest classi- fier to build a multivariate model. The model analyzed radiological features extracted from images of tumors and successfully predicted EGFR and KRAS mutation status in cancer patients. Competing interests: The authors have declared that no competing interests exist. The radiomics and machine learning methods mentioned above have successfully predicted gene mutations. However, most of these methods rely on hand-crafted features. Data Availability Statement: The data are available from the website https://wiki. Data Availability Statement: The data are available from the website https://wiki. cancerimagingarchive.net/display/Public/NSCLC +Radiogenomics. The code for S2MMAM is +Radiogenomics. The code for S2MMAM is 1 / 25 PLOS ONE \| https://doi.org/10.1371/journal.pone.0297331 March 11, 2024 PLOS ONE Integrating image and gene-data for prediction of KRAS gene mutation status in non-small cell lung cancer PLOS ONE \| https://doi.org/10.1371/journal.pone.0297331 March 11, 2024 Introduction In recent years, deep learning based on convolutional neural networks has attracted much attention in the field of medical image computing. This data-driven approach can automatically extract complex image features [5–7]. In addition, imaging genomics is more expected to develop in the field of deep learning than single modality data for analytical studies. It integrates disease imaging data and genomic data. Imaging genomics is a high-throughput research method cor- relating imaging features with genomic data. In recent imaging genomics studies, researchers have proposed a series of deep learning algorithms and theoretical models based on image or genetic data. Dong et al. [8] proposed a multichannel and multitasking deep learning (MMDL) model. They used the fusion of radiological features of CT images and clinical infor- mation of patients to improve the accuracy of the model to predict KRAS gene mutations. Hou et al. [9] proposed a multimodal information fusion module based on attention that suc- cessfully predicted lymph node metastasis using deep learning features of CT images fused with genetic data. Therefore, machine learning and deep learning-based imaging genomics approaches have great potential and application in predicting KRAS gene mutation status in NSCLC. Although the above model achieved considerable performance, there are still some chal- lenges in the study of deep learning methods based on image and genetic data for predicting KRAS mutation status in NSCLC: 1) Majority of deep learning methods [8, 9] that study classification tasks focus only on classification methods. However, these studies did not use the segmentation features generated by the segmentation task to facilitate the classification task to improve the performance and effectiveness of the classification task. Lesion segmen- tation and classification are two highly related tasks. The segmentation can help remove dis- tractions from CT images and thus is highly beneficial for improving the accuracy of lesion classification. 2) Most of the studied fusion methods used simple fusion means of direct concatenation. However, they ignore the correlation and difference between medical images and genetic data. It not only leads to ineffective mining of useful semantic features between PLOS ONE \| https://doi.org/10.1371/journal.pone.0297331 March 11, 2024 2 / 25 PLOS ONE Integrating image and gene-data for prediction of KRAS gene mutation status in non-small cell lung cancer multi-scale image features and gene features but also fails to make full use of the comple- mentarity of multimodal information. Introduction 3) Many studies used models that overemphasized the deep features of lesion abstraction. Nonetheless, they did not pay sufficient attention to the importance of detailed shallow features in prediction results. This leads to limitations in improving accuracy. To overcome these difficulties and achieve non-invasive and accurate prediction of KRAS gene mutations in NSCLC. We propose a Semi-supervised Multimodal Multiscale Attention Model (S2MMAM) for predicting KRAS gene mutation status in NSCLC. The model uses the Mean Teacher [10] framework as the main structure of the network. Mean Teacher can make full use of labeled images to achieve analytical prediction of unlabeled images in order to diminish the dependence of the network on manual annotation. In order to compensate for the information loss of single-modal unlabeled image data to the net- work, the model not only uses Semi-supervised Multimodal Fusion Classification Networks (S2MF-CN) to share the parameter strategy of the Supervised Multilevel Fusion Segmenta- tion Network (SMF-SN) to enrich the key information of the lesion. S2MMAM also multi- modally fuses the patient’s genetic data with the image data to expand the mutation knowledge. Specifically, SMF-SN designs a new Triple Attention-guided Feature Aggrega- tion (TAFA) module. It aims to adaptively fuse high-level semantic features with low-level semantic features using an attention-guided mechanism. TAFA can ignore background noise and localize the extraction of lesion key features. In S2MF-CN, we propose an Intra and Inter Mutual Guidance Attention Fusion (I2MGAF) module to guide the fusion between inter-information and between intra-information in a staged manner. I2MGAF can effectively extract complementary information from different modalities at different scales to facilitate classification efficiency improvement. In contrast to conventional radiomics and machine learning [3, 4], we used a convolutional neural network technique for CT image feature extraction as compared to previous studies for KRAS mutation prediction. This technique is more efficient and reduces the cost of manual annotation. Moreover, it can realize the prospect of end-to-end applications. Studies [5–9] that have made predictions for other diseases in multimodal-based classification tasks have used simple multimodal fusion methods. In contrast, our proposed method focuses more on extracting different dimensions of information from different modal data to achieve comple- mentary fusion. PLOS ONE \| https://doi.org/10.1371/journal.pone.0297331 March 11, 2024 Mean Teacher in semi-supervised learning Semi-supervised learning has been studied in the medical imaging community for a long time [11, 12]. It can reduce the human workload on labeled data. Current research has shown the potential to improve network performance when labels are scarce. There are three semi-super- vised models based on the principle of consistency: the P-Model [13], Temporal Ensembling (TE) [13], and the Mean Teacher model. In order to show the advantages and disadvantages of three consistency-based semi-supervised methods more succinctly, we summarize Table 1, which allows a more precise comparison of the three approaches. In recent years, Mean Teacher has achieved good results as a basic framework in semi- supervised classification tasks. Wang et al. [14] successfully identified diabetic macular edema based on the Mean Teacher model using a small amount of roughly labeled data and a large amount of unlabeled data. Liu et al. [15] used the Mean Teacher-based framework of the net- work model to successfully achieve skin lesion diagnosis with ISIC 2018 challenge and thorax disease classification with ChestX-ray14. Wang et al. [16] proposed a model that unifies diverse knowledge into a generic knowledge distillation framework for skin disease classification. It enables the student model to acquire richer knowledge from the faculty model. The above model demonstrates that Mean Teacher achieves excellent results in semi-supervised classifi- cation tasks, so we use it as the basic framework for Our S2MMAM. Introduction The contributions of this paper are as follows: The contributions of this paper are as follows: • A Semi-supervised Multimodal Multiscale Attention Model (S2MMAM) based on imaging genomics is proposed, which effectively solves the problem of difficult intermediate fusion of multimodal heterogeneous data. S2MMAM exploits the facilitation of supervised segmenta- tion features for semi-supervised classification tasks to improve the model performance for predicting KRAS gene mutation status in NSCLC. • A new Triple Attention-guided Feature Aggregation (TAFA) module is designed. It is based on the attention module to adaptively fuse high-level semantic features with low-level semantic features. TAFA can suppress low-level background noise and retain detailed local semantic information. • We use the Intra and Inter Mutual Guidance Attention Fusion (I2MGAF) module to guide segmentation and classification feature fusion, as well as CT image and genetic data fusion, respectively. It can achieve multi-scale multimodal information fusion and improve classifi- cation performance. 3 / 25 PLOS ONE \| https://doi.org/10.1371/journal.pone.0297331 March 11, 2024 PLOS ONE Integrating image and gene-data for prediction of KRAS gene mutation status in non-small cell lung cancer https://doi.org/10.1371/journal.pone.0297331.t001 PLOS ONE \| https://doi.org/10.1371/journal.pone.0297331 March 11, 2024 Segmentation facilitates classification Using segmentation tasks to facilitate classification network tasks is a basic form of multitask learning [17]. In multitask learning, the segmentation task associated with the classification task can assist the learning of the target by the classification task, thus improving the perfor- mance of the classification task [18]. Similarly, in a single-task classification model, this idea is borrowed from above. The information captured by the segmentation branch of the model can be transferred to the classification model to expand the foci information. The supervised segmentation task is trained using masked labeled data. The aim is to obtain the most compre- hensive high-level semantic features of the target region and reduce the learning of noisy back- grounds. Rich segmentation features can support the classification task to learn more and richer semantic information. Thus, a supervised segmentation network can assist the classifica- tion task by suppressing the background noise introduced by missing physician labeling infor- mation in semi-supervised classification networks and improving the classification accuracy. According to Table 2 the above works demonstrate that segmentation has a facilitating g y According to Table 2, the above works demonstrate that segmentation has a facilitating effect on classification. However, there is a common problem: they are all studied for super- vised models. Supervised models have high requirements for data labeling costs. We believe Table 1. Comparison of three commonly used consistency-based semi-supervised methods. Methods Purpose Limitations P-Model Based on the consistency principle and perturbs the input data High complexity and nosiy-prone results Temporal Ensembling (TE) Employs an exponential moving average (EMA) prediction for each unlabeled data as the consistency target. Maintain a huge prediction matrix during the prediction process, and the training time complexity is high for large data sets. Mean Teacher Improves the problem of high time complexity caused by the TE method. Constructs a teacher model using the EMA weights of the student model. https://doi.org/10.1371/journal.pone.0297331.t001 Table 1. Comparison of three commonly used consistency-based semi-supervised methods. 4 / 25 PLOS ONE \| https://doi.org/10.1371/journal.pone.0297331 March 11, 2024 PLOS ONE Integrating image and gene-data for prediction of KRAS gene mutation status in non-small cell lung cancer Table 2. Comparison of three commonly used consistency-based semi-supervised methods. Methods Contributions Limitations Xie et al. [18] Proposed the Mutual Bootstrapping Deep Convolutional Neural Networks (MB-DCNN) model for simultaneous segmentation and classification of skin lesions. Segmentation facilitates classification The rough lesion masks generated by the segmentation network in MB-DCNN help the classification network for training. The segmentation and classification networks transfer knowledge to each other in a bootstrap manner and facilitate each other. 1. Non-end-to-end model 2. Professional doctors are needed to manually label each image Zhao et al. [19] Proposed a Segmentation-based Sequence Residual Attention Model (SSRAM) for the dual task of colorectal cancer lesion segmentation and KRAS gene mutation status prediction. The SSRAM utilizes the information provided by the segmentation network and the mask to successfully improve the accuracy of the classification task. 1. Data pre-processing is more complex 2. Professional doctors are needed to manually label each image Song et al. [20] Utilized the lung nodule segmentation task to assist the lung nodule malignant development prediction task. Professional doctors are needed to manually label each image https://doi.org/10.1371/journal.pone.0297331.t002 https://doi.org/10.1371/journal.pone.0297331.t002 that the combination of segmentation and classification tasks can make the network more informative. Therefore, our research aims to combine the idea of segmentation facilitating classification with semi-supervised models. We combined two related tasks of NSCLC lesion segmentation and KRAS gene mutation status prediction. S2MMAM allows S2MF-CN to obtain the key features of lesions upon initialization through the strategy of sharing network parameters between SMF-SN and S2MF-CN. In S2MF-CN, the segmentation features are guided to merge with the classification features to obtain the extracted key features. This strat- egy can enrich the lesion information and improve the network model classification performance. PLOS ONE \| https://doi.org/10.1371/journal.pone.0297331 March 11, 2024 Multiscale features and attention learning Traditional convolution operations mostly focus on extracting local features. However, due to the limited information contained in local features, the model cannot learn the full range of region of interest contents well. Multi-scale features contain local features of multiple regions of interest. The extracted local features are fused with other operations to obtain comprehen- sive information about the target, which helps the network model to learn. To extract multi- scale features, The Atrous Spatial Pyramid Pooling (ASPP) module [21] captures contextual information by multi-step convolution of the target region using different expansion rates. In the medical image domain, the PSE [22] module uses a patch-level pyramid design to extend SE operations to multiple scales, allowing the network to adaptively focus on vessels of variable width. The scale-aware Feature Aggregation (SFA) module [23] effectively extracts hidden multi-scale background information and aggregates multi-scale features to improve the mod- el’s ability to handle complex vasculature. The Convolutional Block Attention Module (CBAM) [24] introduces channel and spatial attention. It extracts multiple key feature information from both dimensions to enrich the net- work content. In the medical image application domain, Context-assisted full Attention Net- work (CAN) [25] combines Non-Local Attention (NLA), Channel Attention (CA), and Dual- pathway Spatial Attention (DSA) to extract lesion information in multiple directions. Currently, it is widely believed that both multi-scale features and attention mechanisms can help models enhance the recognition of feature maps from different dimensions. However, the above papers have a common problem: they do not combine the ideas of multi-scale and atten- tion mechanism. Therefore, we combine these two techniques and design the TAFA module. PLOS ONE \| https://doi.org/10.1371/journal.pone.0297331 March 11, 2024 5 / 25 PLOS ONE Integrating image and gene-data for prediction of KRAS gene mutation status in non-small cell lung cancer On the one hand, fuse high and low dimensional segmentation features to obtain abstract and detailed information. On the other hand, we fuse segmentation and classification features of different levels to guide the features to learn key factors adaptively and enhance the ability of the network to capture lesions. Thus, the predictive capability of the model is improved. https://doi.org/10.1371/journal.pone.0297331.g001 Overview In this paper, we propose a Semi-supervised Multimodal Multiscale Attention Model (S2MMAM). The overall architecture of the model is divided into two parts: Supervised Multi- level Fusion Segmentation Network (SMF-SN) and Semi-supervised Multimodal Fusion Clas- sification Network (S2MF-CN), as shown in Fig 1. In this model, the useful information of CT images is captured by SMF-SN and transferred to S2MF-CN to facilitate the execution of image prediction tasks. The S2MMAM utilizes the fusion of CT images and genetic data to accurately predict whether KRAS is mutated in NSCLC. Fig 1. Overview of our S2MMAM, including: (a) Supervised Multilevel Fusion Segmentation Network (SMF-SN). The inputs are CT images and pixel-level mask images, and the outputs are segmented lesion images, (b) Semi-supervised Multimodal Fusion Classification Network (S2MF-CN), and (c) processing of gene data. In the S2MMAM, the useful information of CT images is captured by SMF-SN and transferred to S2MF-CN to facilitate the execution of image prediction tasks. The S2MMAM utilizes the fusion of CT images and genetic data to accurately predict whether KRAS is mutated in NSCLC. Fig 1. Overview of our S2MMAM, including: (a) Supervised Multilevel Fusion Segmentation Network (SMF-SN). The inputs are CT images and pixel-level mask images, and the outputs are segmented lesion images, (b) Semi-supervised Multimodal Fusion Classification Network (S2MF-CN), and (c) processing of gene data. In the S2MMAM, the useful information of CT images is captured by SMF-SN and transferred to S2MF-CN to facilitate the execution of image prediction tasks. The S2MMAM utilizes the fusion of CT images and genetic data to accurately predict whether KRAS is mutated in NSCLC. Fig 1. Overview of our S2MMAM, including: (a) Supervised Multilevel Fusion Segmentation Network (SMF-SN). The inputs are CT images and pixel-level mask images, and the outputs are segmented lesion images, (b) Semi-supervised Multimodal Fusion Classification Network (S2MF-CN), and (c) processing of gene data. In the S2MMAM, the useful information of CT images is captured by SMF-SN and transferred to S2MF-CN to facilitate the execution of image prediction tasks. The S2MMAM utilizes the fusion of CT images and genetic data to accurately predict whether KRAS is mutated in NSCLC. Overview https://doi.org/10.1371/journal.pone.0297331.g001 https://doi.org/10.1371/journal.pone.0297331.g001 6 / 25 PLOS ONE \| https://doi.org/10.1371/journal.pone.0297331 March 11, 2024 PLOS ONE Integrating image and gene-data for prediction of KRAS gene mutation status in non-small cell lung cancer In the NSCLC dataset, each patient corresponds to a set of CT images and gene data (Sec- tion Dataset). Specifically, in our problem setting, we are given a training set containing N labeled data and M unlabeled data where N<<M. Let the labeled training dataset be denoted by SL ¼ fXi L; Yi Lg N i¼1 and CL ¼ fXi L; Yi L; Zi Lg N i¼1, where SL represents dataset for segmentation, CL represents dataset for classification, Xi L represents i-th labeled CT image, Yi L represents the pixel-level annotation of Xi L and Zi L represents the results of whether the KRAS gene is mutated. Zi L 2 f0; 1g where 0 means negative and 1 means positive. Let the unlabeled training dataset be denoted by CU ¼ fXi Ug M i¼1, where Xi U represents i-th unlabeled image. The entire model pipline can be summarized as follows: First, we pre-train SMF-SN, which is initialized on SL, to train the network’s ability to capture focal regions. It can eliminate problems such as large noise from CT and promote the ability of classification—meanwhile, the network body of S2MF-CN shares encoder and decoder parameters with SMF-SN. Therefore, the encoder and decoder of S2MF-CN are also initialized in this step, and practical segmentation features for different levels of lesions are obtained. The classification network in S2MF-CN can capture the key classification features of lesions using these segmentation features. Finally, after S2MF-CN fuses segmentation, classification, and genetic data features, the semi-supervised Student Model is trained to determine patients’ KRAS gene mutation status accurately. PLOS ONE \| https://doi.org/10.1371/journal.pone.0297331 March 11, 2024 PLOS ONE \| https://doi.org/10.1371/journal.pone.0297331 March 11, 2024 Supervised multilevel fusion segmentation network The architecture of SMF-SN. This section introduces a supervised segmentation network based on multidimensional feature fusion. SMF-SN can precisely localize lesion edges and internal regions and greatly reduce the impact of image background noise on network perfor- mance. SMF-SN mainly utilizes our proposed SE-ResNeXt and TAFA modules. We use the enhanced segmentation training dataset SL to train SMF-SN to obtain rich seg- mentation features. The obtained segmentation features can provide the semi-supervised clas- sification network with a priori information about the lesion location. This improves the classification network’s ability to localize and identify lesions. As shown in Fig 2, SMF-SN includes a stem block, three encoder blocks, three TAFA blocks, a bridge block, three decoder blocks, and an output block. g In the encoder, each encoder is composed of a SE-ResNeXt and a max-pooling layer with step size 2. As shown in Fig 3, SE-ResNeXt is improved from ResNeXt with SENet. ResNext achieves aggregating a set of transitions with the same topology by repeating multiple blocks. SENet can perform feature learning on the aggregated features in the channel dimension to form the impor- tance of each channel. SE-ResNeXt can enhance the network in both the channel and spatial dimensions to capture richer segmentation features. Applying the MaxPooling layer can reduce the spatial dimension of the feature map by half to reduce the computational cost. The output of the encoder is passed through a bridge consisting of SE-ResNeXt and Atrous Spatial Pyramid Pooling (ASPP). It provides the largest receptive domain for TAFA to include a wider range of contextual information, facilitating more efficient integration between multiple levels. Between high-level and low-level semantics, we use the proposed TAFA module. This module utilizes multi-scale and attention fusion mechanisms. The module both suppresses low-level irrelevant background noise and complements each other with contextual difference information, preserv- ing more detailed local semantic information and better learning of focal information. TAFA module is depicted in detail in Section Triple Attention-guided Feature Aggregation. Triple attention-guided feature aggregation. Since CT images of lung nodules may con- tain a large amount of noise, for example, there are problems of grayscale overlap between lung tissues, blurred boundaries, and challenging to distinguish. High-level features of the decoder and low-level features of the encoder are crucial for capturing lesion features. PLOS ONE \| https://doi.org/10.1371/journal.pone.0297331 March 11, 2024 Supervised multilevel fusion segmentation network PLOS ONE \| https://doi.org/10.1371/journal.pone.0297331 March 11, 2024 7 / 25 PLOS ONE E Integrating image and gene-data for prediction of KRAS gene mutation status in non-small cell lung cancer Integrating image and gene-data for prediction of KRAS gene mutation status in non-small cell lung cancer Fig 2. Block diagram of the proposed SMF-SN architecture. We adjust the dilation rates in ASPP in the bridge from 6,12,18 to 3,6,9 to better adapt SMF-SN to our segmentation task. https://doi.org/10.1371/journal.pone.0297331.g002 Fig 2. Block diagram of the proposed SMF-SN architecture. We adjust the dilation rates in ASPP in the bridge from 6,12,18 to 3,6,9 to better adapt SMF-SN to our segmentation task. Fig 2. Block diagram of the proposed SMF-SN architecture. We adjust the dilation rates in ASPP in the bridge from 6,12,18 to 3,6,9 to better adapt SMF-SN to our segmentation task. https://doi.org/10.1371/journal.pone.0297331.g002 https://doi.org/10.1371/journal.pone.0297331.g002 8 / 25 PLOS ONE \| https://doi.org/10.1371/journal.pone.0297331 March 11, 2024 PLOS ONE Integrating image and gene-data for prediction of KRAS gene mutation status in non-small cell lung cancer Fig 3. The architecture of SE-ResNext. SE-ResNeXt is improved from ResNeXt with SENet. https://doi.org/10.1371/journal.pone.0297331.g003 Fig 3. The architecture of SE-ResNext. SE-ResNeXt is improved from ResNeXt with SENet. https://doi.org/10.1371/journal.pone.0297331.g003 https://doi.org/10.1371/journal.pone.0297331.g003 PLOS ONE \| https://doi.org/10.1371/journal.pone.0297331 March 11, 2024 9 / 25 PLOS ONE Integrating image and gene-data for prediction of KRAS gene mutation status in non-small cell lung cancer Fig 4. Framework diagram of the proposed TAFA module. https://doi.org/10.1371/journal.pone.0297331.g004 Fig 4. Framework diagram of the proposed TAFA module. https://doi.org/10.1371/journal.pone.0297331.g004 However, most of the existing UNet-based connection methods directly connect shallow and deep semantic features of different scales. This behavior ignores that high-level features con- tain rich semantic information that can help low-level features identify semantically important locations. Likewise, low-level features contain rich spatial information that can help high-level features reconstruct accurate details. Considering the above factors, we design a Triple attention-guided feature aggregation (TAFA) module to guide the fusion between high and low-dimensional features. TAFA can guide different layers to extract key feature information individually and then fuse after retain- ing the domain invariant key information, as shown in Fig 4. In the TAFA module, we first upsample the high-dimensional feature Fiþ1 highto have the same size as the low-dimensional fea- ture Fi lowði 2 f1; 2; 3gÞ. After that, we perform the high and low-dimensional feature concatenating based on channels to obtain Fi C. PLOS ONE \| https://doi.org/10.1371/journal.pone.0297331 March 11, 2024 Supervised multilevel fusion segmentation network Fi ¼ ConvðWglobal ðConcatðFi low att; Fiþ1 high attÞÞÞ ð5Þ ð5Þ Where Conv represents 1×1 convolution operation, represents element-wise sum and represents element-wise multiplication. Our proposed TAFA transfers features from shallower convolutional layers to deeper con- volutional layers. Performing the shallow features in the deeper convolutional layers prevents the shallow features from being forgotten. It makes the obtained features have more vital char- acterization ability. By gradually guiding the fusion between high and low features, SMF-SN can be guided to adaptively combine high and low-dimensional semantic information to reas- sign feature weights and better capture critical domain invariant information. Thus, lung nod- ules can be separated from the noise. Supervised multilevel fusion segmentation network Fi C ¼ ConcatðFi low; fupðFiþ1 highÞÞ ð1Þ ð1Þ Where Concat represents the concatenation operation, fup represents up-sampling opera- tions. Then, to better mine the most useful feature channels between different levels. We intro- duce a scale channel attention-aware mechanism to automatically select the appropriate receptive domain for the feature map and suppress the interference of irrelevant background noise. We feed the concatenate feature Fi C of high and low dimensional features into global average pooling (GAP) and global max pooling (GMP) respectively. TAFA uses the GAP mod- ule to excite the feature channel information and the GMP layer to retain the semantic PLOS ONE \| https://doi.org/10.1371/journal.pone.0297331 March 11, 2024 10 / 25 PLOS ONE Integrating image and gene-data for prediction of KRAS gene mutation status in non-small cell lung cancer maximum information. Afterward, the corresponding feature maps Fi AM and Fi MM are obtained using a multi-layer perceptron (MLP) sharing the same parameters. The feature maps Fi AM and Fi MM are summed. Then the sum feature passes through a sigmoid function to generate a global bootstrap feature coefficient Wglobal. Wglobal ¼ fsffmlpðfgmpðFi CÞÞ fmlpðfgapðFi CÞÞg ð2Þ Wglobal ¼ fsffmlpðfgmpðFi CÞÞ fmlpðfgapðFi CÞÞg ð2Þ Where fσ represents sigmoid activation, fmlp represents the MLP operator, fgap represents the global average pooling, fgmp represents the global max pooling. In addition, using the high and low level semantic binding information Fi AM and Fi MM as guidance, they are combined with high and low dimensional features, respectively, and the high level guidance semantic features Fiþ1 high att and low level guidance semantic features Fi low att are obtained after the attention opera- tion, respectively. Whigh ¼ fsðFiþ1 high Fi AMÞ; Wlow ¼ fsðFi low Fi MMÞ ð3Þ Fiþ1 high att ¼ Fiþ1 high Whigh Fiþ1 high; Fi low att ¼ Fi low Wlow Fi low ð4Þ Whigh ¼ fsðFiþ1 high Fi AMÞ; Wlow ¼ fsðFi low Fi MMÞ ð3Þ ð3Þ Fiþ1 high att ¼ Fiþ1 high Whigh Fiþ1 high; Fi low att ¼ Fi low Wlow Fi low ð4Þ ð4Þ Finally, the weighted features are concatenated. The concatenated feature maps are multi- plied with Wglobal. Then domain-invariant information is captured while reducing the dimensionality through 1x1 convolutional layers to obtain the final fusion module Fi. PLOS ONE \| https://doi.org/10.1371/journal.pone.0297331 March 11, 2024 PLOS ONE \| https://doi.org/10.1371/journal.pone.0297331 March 11, 2024 Semi-supervised multimodal fusion classification networks The architecture of S2MF-CN. The proposed S2MF-CN structure is shown in Fig 1(B), which adopts the Mean Teacher model structure as the main framework of the classification network. In Mean Teacher, the Teacher network has the same structure as the Student net- work. The Student model is the target model to be trained. It assigns the exponential moving average (EMA) of its weights to the Teacher model at each step of training. The predictions of the Teacher model will be considered as additional supervision of the learning of the Student model. Our model uses the final Student model to make predictions. The specific training Stu- dent model is shown in Fig 5(A) and consists of three parts: encoder, decoder, and Intra and Inter Mutual Guidance Attention Fusion (I2MGAF) Module. The encoder and decoder have the same structure and parameters as the SMF-SN. This allows focusing on the lesion region and capturing the necessary segmentation features through the encoder and decoder. I2MGAF performs feature purification using mutual guidance attention modules. It is able to extract multi-scale lung CT image features and genetic features fully. It also performs an adaptive fusion of features through an attentional fusion mechanism for KRAS gene mutation PLOS ONE \| https://doi.org/10.1371/journal.pone.0297331 March 11, 2024 11 / 25 PLOS ONE Integrating image and gene-data for prediction of KRAS gene mutation status in non-small cell lung cancer Fig 5. The overview of the Student Module, including (a) the specific implementation details of the Student Model, (b) Intra fusion component (IntraFC) aims to fuse classification and segmentation features at different levels, and (c) Inter fusion component (InterFC) aims to fuse CT image features and genetic features. Fig 5. The overview of the Student Module, including (a) the specific implementation details of the Student Model, (b) Intra fusion component (IntraFC) aims to fuse classification and segmentation features at different levels, and (c) Inter fusion component (InterFC) aims to fuse CT image features and genetic features https://doi.org/10.1371/journal.pone.0297331.g005 https://doi.org/10.1371/journal.pone.0297331.g005 prediction in NSCLC. I2MGAF is described in detail in Section Intra and Inter Mutual Guid- ance Attention Fusion Module. Intra and inter mutual guidance attention fusion module. In the S2MF-CN network, we propose an I2MGAF module. I2MGAF fully fuses multi-scale image segmentation, classifi- cation features, and genetic features by using the IntraFC component and InterFC component with a dual attention fusion mechanism. Its aim is to improve the classification capability of the classification network. Semi-supervised multimodal fusion classification networks 1. Intra Fusion Component (IntraFC) 1. Intra Fusion Component (IntraFC) We propose the IntraFC based on the MultiRes Block, which can capture multi-scale infor- mation [26]. We adopted a strategy of fusing classification features with segmentation fea- tures at each level. The information favoring the prediction of KRAS gene mutation status is jointly retained. The specific structure of the IntraFC component is shown in Fig 5(B). The final level seg- mentation features F3 S are subjected to convolutional operations to obtain the initial classifi- cation features FC. Due to the problem of induction bias inherent in the convolution mechanism, it is easy to lose the key features of the lesion after multiple convolutions. Therefore, it is necessary for us to fuse the previous segmentation features with the existing classification features to compensate for the bias problem due to the deep network. First we p We propose the IntraFC based on the MultiRes Block, which can capture multi-scale infor- mation [26]. We adopted a strategy of fusing classification features with segmentation fea- tures at each level. The information favoring the prediction of KRAS gene mutation status is jointly retained. We propose the IntraFC based on the MultiRes Block, which can capture multi-scale infor- mation [26]. We adopted a strategy of fusing classification features with segmentation fea- tures at each level. The information favoring the prediction of KRAS gene mutation status is jointly retained. The specific structure of the IntraFC component is shown in Fig 5(B). The final level seg- mentation features F3 S are subjected to convolutional operations to obtain the initial classifi- cation features FC. Due to the problem of induction bias inherent in the convolution mechanism, it is easy to lose the key features of the lesion after multiple convolutions. Therefore, it is necessary for us to fuse the previous segmentation features with the existing classification features to compensate for the bias problem due to the deep network. First we reshape the segmented feature Fi Sfi 2 ð1; 2; 3Þg through dimensionality until C×W×H is Therefore, it is necessary for us to fuse the previous segmentation features with the existing classification features to compensate for the bias problem due to the deep network. 2. Inter Fusion Component (InterFC) We propose the InterFC to find the bidirectional mapping relationship between lung cancer image features and causative genes from the sagittal view (x-axis), coronal view (y-axis), and axial view (z-axis), respectively. InterFC can adaptively enhance the necessary informa- tion in different modal features, allowing a more adequate fusion of multimodal features. The specific structure of the InterFC component is shown in Fig 5(C). The initial classifica- tion feature FC, the fusion result Fi Intrafi 2 ð1; 2; 3Þg output by IntraFC, and the processed genetic data G are firstly subjected to a splicing operation to obtain the multimodal fusion feature MC. After that, MC is delivered to InterFC to further model the importance of each modal data. MC ¼ ConcatðF1 Intra; F2 Intra; F3 Intra; FC; GÞ ð6Þ ð6Þ Where Concat denotes the concatenation operation. Then the concatenated multimodal data features are fed to three convolutional layers with BN and ReLU. The size of the convo- lution kernel is 1×3×1, 3×1×1 and 1×1×3 respectively, to produce three feature maps Quer- y2RC×H×W, Key2RC×H×W and Value2RC×H×W (where C,H,W indicate the channel, height, width of the input features F respectively). We first transpose the Query feature. Then, we perform a softmax layer on the matrix multiplication of QueryT and Key to encode the fea- ture relationships in sagittal and coronal views. Finally, matrix multiplication is multiplied with Value to obtain the voxel-level attention enhanced fusion features FInter, which are then reshaped to be in RC×H×W. FInter ¼ MC softmaxðQueryT KeyÞ Value ð7Þ ð7Þ Where denotes element-wise sum, denotes element-wise multiplication. Semi-supervised multimodal fusion classification networks First we reshape the segmented feature Fi Sfi 2 ð1; 2; 3Þg through dimensionality until C×W×H is 12 / 25 PLOS ONE \| https://doi.org/10.1371/journal.pone.0297331 March 11, 2024 PLOS ONE Integrating image and gene-data for prediction of KRAS gene mutation status in non-small cell lung cancer the same size as the classified feature FC. Then, after the segmentation features and classifi- cation features are each applied 3×3 convolution. We will introduce the convolutional fea- tures from the previous stage and the initial fusion feature Fi SC before the subsequent convolution. This can effectively model the correlation between segmentation and classifi- cation features. It ensures that the features from the shallow convolutional layer of segmen- tation and classification are better transferred to the deeper layers. The final fused result Fi Intrafi 2 ð1; 2; 3Þg is obtained after several feature fusions. 2. Inter Fusion Component (InterFC) PLOS ONE \| https://doi.org/10.1371/journal.pone.0297331 March 11, 2024 Implementation details Our model S2MMAM is divided into SMF-SN and S2MF-CN. The labeled image data applied to SMF-SN is 30% of the total dataset, about 2100 images. The training dataset applied to S2MF-CN consists of 30% labeled data and 70% unlabeled data. Our experiments are mainly done on 2 NVIDIA RTX A5000 GPUs and 64 GB of memory. All models in the experiments are trained using 10-fold cross-validation. The specific initialization network configurations are shown in Table 4. PLOS ONE Table 3. Patients’ medical record information in the dataset. Category Total Mutation Wildtype Amount 124 30 94 Gender Male 93 24 69 Female 31 6 25 Smoking History Smoking 107 30 77 Non-smoking 17 0 17 Pathological type Adenocarcinoma 105 29 76 Squamous Carcinoma 17 0 17 Other 2 1 1 https://doi.org/10.1371/journal.pone.0297331.t003 and 30 were of the mutation type. The clinical information of these patients is shown in Table 3. All data were randomly divided into training and test datasets in a 4:1 ratio. and 30 were of the mutation type. The clinical information of these patients is shown in Table 3. All data were randomly divided into training and test datasets in a 4:1 ratio. Data preprocessing CT image. In our experiments, for 124 sets of CT images inspired by Cubuk et al. [28], we use the simple procedure of AutoAugment to automatically search for improved data enhance- ment strategies. By designing a search space in which a strategy consists of many sub-strate- gies, one sub-strategy is randomly selected for each image in each small batch. The sub- strategies contain two operations, each of which is an image processing function, such as clip- ping or applying the probability and magnitude of that function. Thus, we obtained 6696 images with a fixed size of 512×512. Genes selection. The gene expression data used in this study is RNA-seq data. Since the vast gene dataset contains more than 20,000 gene expression data per patient, the huge amount of gene expression data can significantly increase the computational cost and decrease the pre- diction accuracy. Therefore, before training the model, we screened the gene expression data from RNA-seq sequencing by the feature selection algorithm [29] to retain the most relevant genes with KRAS mutations. A total of 115 relevant genes were finally screened. The obtained correlated genes were fed into MLP to obtain effective gene features, which achieved mapping high-dimensional gene data to low-dimensional space. Dataset In this study, we applied NSCLC-Radiogenomics [27], directly accessible on the Cancer Imag- ing Archive (TCIA) website. NSCLC-Radiogenomics is part of a public dataset. The patients involved in the dataset have been ethically approved. Users can download the relevant data for research and publication free of charge. Our study is based on open-source data and is there- fore free from ethical issues and other conflicts of interest. NSCLC-Radiogenomics has devel- oped a unique radiogenomic dataset from the NSCLC dataset of 211 subjects. The imaging data include mainly CT, semantic annotation of tumors observed on CT images using con- trolled vocabulary, and segmentation maps of tumor lesions (lung nodules) on CT scans; the genetic data include mainly RNA sequencing (RNA-seq) data. In the training and testing data- sets, patients would be excluded for 1) lack of RNA-seq data, 2) lack of CT images, and 3) lack of physician-annotated segmentation maps of CT lesions. After screening, the number of cases with complete images and genetic data was 124. Of the 124 patients, 94 were of the wildtype, PLOS ONE \| https://doi.org/10.1371/journal.pone.0297331 March 11, 2024 13 / 25 Integrating image and gene-data for prediction of KRAS gene mutation status in non-small cell lung cancer PLOS ONE \| https://doi.org/10.1371/journal.pone.0297331 March 11, 2024 Evaluation metrics To quantitatively analyze the experimental results, we used six performance metrics to evaluate the classification results obtained, including Accuracy (AC), Recall, Precision, Specificity (SP), Area Under the receiver operating Curve (AUC) and F1 score (F1). They are defined as 14 / 25 PLOS ONE \| https://doi.org/10.1371/journal.pone.0297331 March 11, 2024 PLOS ONE Integrating image and gene-data for prediction of KRAS gene mutation status in non-small cell lung cancer follows: AC ¼ TP þ TN TP þ TN þ FN þ FP ð8Þ Recall ¼ TP TP þ FN ð9Þ Precision ¼ TP TP þ FP ð10Þ SP ¼ TN TN þ FP ð11Þ AUC ¼ Z1 0 tprðfprÞdfpr ¼ PðX1 > X0Þ ð12Þ R ll P i i Table 4. The initialization network configurations of model. Network Configurations Setting Epochs per fold 20 Optimizer Adams Initial learning rate 0.001 Batch size 16 https://doi.org/10.1371/journal.pone.0297331.t004 follows: AC ¼ TP þ TN TP þ TN þ FN þ FP ð8Þ Recall ¼ TP TP þ FN ð9Þ Precision ¼ TP TP þ FP ð10Þ SP ¼ TN TN þ FP ð11Þ AUC ¼ Z1 0 tprðfprÞdfpr ¼ PðX1 > X0Þ ð12Þ Table 4. The initialization network configurations of model. Network Configurations Setting Epochs per fold 20 Optimizer Adams Initial learning rate 0.001 Batch size 16 https://doi.org/10.1371/journal.pone.0297331.t004 follows: Table 4. The initialization network configurations of model. Network Configurations Setting Epochs per fold 20 Optimizer Adams Initial learning rate 0.001 Batch size 16 https://doi.org/10.1371/journal.pone.0297331.t004 AC ¼ TP þ TN TP þ TN þ FN þ FP ð8Þ Recall ¼ TP TP þ FN ð9Þ Precision ¼ TP TP þ FP ð10Þ SP ¼ TN TN þ FP ð11Þ ð8Þ ð9Þ ð10Þ SP ¼ TN TN þ FP ð11Þ ð11Þ AUC ¼ Z1 0 tprðfprÞdfpr ¼ PðX1 > X0Þ ð12Þ ð12Þ F1 ¼ 2 Recall Precision Recall þ Precision ð13Þ ð13Þ Where TP is true positive, TN is true negative, FP is false positive, FN is false negative, tpr is the true positive rate, fpr is the false positive rate, X1 and X0 are the confidence scores for nega- tive instances of sexual instances, respectively. PLOS ONE SE-ResNeXt(Ours) achieved the best results in all six comparative metrics. Methods AC(%) Recall(%) Precision(%) SP(%) AUC(%) F1(%) UNet [30] 71.29±0.53 71.35±0.11 73.24±0.64 70.09±0.36 70.33±0.38 72.28±0.37 ResNet [31] 73.95±1.05 74.01±2.19 74.15±0.41 76.32±2.46 76.48±0.47 74.07±1.29 ResNeXt [32] 77.99±3.16 76.92±1.37 78.14±3.21 75.28±3.14 77.31±2.22 77.53±2.27 Inception-v3 [33] 75.29±2.86 77.39±3.15 75.14±2.21 77.20±2.18 76.84±1.43 76.25±2.66 SE-ResNeXt(Ours) 81.67±2.67 82.31±2.51 83.15±1.21 82.66±2.07 83.27±1.49 82.73±1.86 https://doi.org/10.1371/journal.pone.0297331.t005 between features by utilizing multi-scale and attention mechanisms. This enables SE-ResNeXt to outperform other traditional networks trained on the data and helps the model to better localize the lesion area. Ablation study of TAFA module. Using TAFA as the basic module to build S2MMAM can better capture the key and complementary information of high-level semantic features and Table 5. Comparison of classification performance of UNet, ResNet, ResNeXt, Inception-v3 and SE-ResNeXt on S2MMAM. SE-ResNeXt(Ours) achieved the best results in all six comparative metrics. Methods AC(%) Recall(%) Precision(%) SP(%) AUC(%) F1(%) UNet [30] 71.29±0.53 71.35±0.11 73.24±0.64 70.09±0.36 70.33±0.38 72.28±0.37 ResNet [31] 73.95±1.05 74.01±2.19 74.15±0.41 76.32±2.46 76.48±0.47 74.07±1.29 ResNeXt [32] 77.99±3.16 76.92±1.37 78.14±3.21 75.28±3.14 77.31±2.22 77.53±2.27 Inception-v3 [33] 75.29±2.86 77.39±3.15 75.14±2.21 77.20±2.18 76.84±1.43 76.25±2.66 SE-ResNeXt(Ours) 81.67±2.67 82.31±2.51 83.15±1.21 82.66±2.07 83.27±1.49 82.73±1.86 https://doi.org/10.1371/journal.pone.0297331.t005 Table 5. Comparison of classification performance of UNet, ResNet, ResNeXt, Inception-v3 and SE-ResNeXt on S2MMAM. SE-ResNeXt(Ours) achieved the best results in all six comparative metrics. Methods AC(%) Recall(%) Precision(%) SP(%) AUC(%) F1(%) UNet [30] 71.29±0.53 71.35±0.11 73.24±0.64 70.09±0.36 70.33±0.38 72.28±0.37 ResNet [31] 73.95±1.05 74.01±2.19 74.15±0.41 76.32±2.46 76.48±0.47 74.07±1.29 ResNeXt [32] 77.99±3.16 76.92±1.37 78.14±3.21 75.28±3.14 77.31±2.22 77.53±2.27 Inception-v3 [33] 75.29±2.86 77.39±3.15 75.14±2.21 77.20±2.18 76.84±1.43 76.25±2.66 SE-ResNeXt(Ours) 81.67±2.67 82.31±2.51 83.15±1.21 82.66±2.07 83.27±1.49 82.73±1.86 https://doi.org/10.1371/journal.pone.0297331.t005 ssification performance of UNet, ResNet, ResNeXt, Inception-v3 and SE-ResNeXt on S2MMAM. SE-ResNeXt(Ours) achie t i of UNet, ResNet, ResNeXt, Inception-v3 and SE-ResNeXt on S2MMAM. SE-ResNeXt(Ours) achieved the best Table 5. Comparison of classification performance of UNet, ResNet, ResNeXt, Inception-v3 and SE-ResNeXt on S2M results in all six comparative metrics. between features by utilizing multi-scale and attention mechanisms. This enables SE-ResNeXt to outperform other traditional networks trained on the data and helps the model to better localize the lesion area. between features by utilizing multi-scale and attention mechanisms. This enables SE-ResNeXt to outperform other traditional networks trained on the data and helps the model to better localize the lesion area. Ablation study of TAFA module. Using TAFA as the basic module to build S2MMAM can better capture the key and complementary information of high-level semantic features and low-level semantic features. PLOS ONE It further enhances the feature representation capability, improves the model to extract segmented feature quality and promotes classification performance. To val- idate the performance of our proposed TAFA, we compare our proposed S2MMAM (Ours) with Addition, Concatenation, Adaptive Enhanced Attention Fusion (AEAF) [34], and Adap- tive Spatiotemporal Semantic Calibration Module (ASSCM) [35] on the test dataset, respec- tively. The results are shown in Table 6. The results show that the highest performance metrics were achieved on the classification task using our proposed S2MMAM constructed from TAFA. TAFA (Ours) not only obtained the highest AUC value of 83.27% compared to the other four models. It also achieved the best results on the other five classification performance metrics, with a maximum AC of 81.67% and a maximum SP of 82.66%. The AUC is 4.39% higher compared to the second place AEAF, proving that TAFA can effectively fuse multi-scale information. It proves that our model S2MMAM can better detect more patients and effectively reduce the underdiagnosis rate. TAFA achieved 82.73% in F1 score, which is higher than the AEAF at 4.46% and the ASSCM at 4.3%. It is demonstrated that our TAFA has a more stable classification performance and better classification ability. Ablation study of I2MGAF module. The I2MGAF module was implemented to guide the fusion of features in segmentation and classification tasks, as well as the fusion of image fea- tures with genetic data. To demonstrate that the I2MGAF module can better guide the fusion of multimodal and multiscale features in the model. We replaced the IntraFC module in I2MGAF with Addition, Concatenation, and Adaptive Feature Fusion (AFF Block) [23], respectively. The InterFC module was replaced with Group Feature Learning (GFL Block) [36] and Non-Local Attention (NLA Block) [25], respectively. The five obtained models are com- pared with the performance of I2MGAF on the classification test dataset. The results are shown in Figs 6 and 7. Table 6. Comparison of classification performance of TAFA on S2MMAM and four models with different fusion blocks. TAFA(Ours) achieved the best results in all six comparative metrics. PLOS ONE PLOS ONE between features by utilizing multi-scale and attention mechanisms. This enables SE-ResNeXt to outperform other traditional networks trained on the data and helps the model to better localize the lesion area. Ablation study of TAFA module. Using TAFA as the basic module to build S2MMAM can better capture the key and complementary information of high-level semantic features and low-level semantic features. It further enhances the feature representation capability, improves the model to extract segmented feature quality and promotes classification performance. To val- idate the performance of our proposed TAFA, we compare our proposed S2MMAM (Ours) with Addition, Concatenation, Adaptive Enhanced Attention Fusion (AEAF) [34], and Adap- tive Spatiotemporal Semantic Calibration Module (ASSCM) [35] on the test dataset, respec- tively. The results are shown in Table 6. The results show that the highest performance metrics were achieved on the classification task using our proposed S2MMAM constructed from TAFA. TAFA (Ours) not only obtained the highest AUC value of 83.27% compared to the other four models. It also achieved the best results on the other five classification performance metrics, with a maximum AC of 81.67% and a maximum SP of 82.66%. The AUC is 4.39% higher compared to the second place AEAF, proving that TAFA can effectively fuse multi-scale information. It proves that our model S2MMAM can better detect more patients and effectively reduce the underdiagnosis rate. TAFA achieved 82.73% in F1 score, which is higher than the AEAF at 4.46% and the ASSCM at 4.3%. It is demonstrated that our TAFA has a more stable classification performance and better classification ability. Ablation study of I2MGAF module. The I2MGAF module was implemented to guide the fusion of features in segmentation and classification tasks, as well as the fusion of image fea- tures with genetic data. To demonstrate that the I2MGAF module can better guide the fusion of multimodal and multiscale features in the model. We replaced the IntraFC module in I2MGAF with Addition, Concatenation, and Adaptive Feature Fusion (AFF Block) [23], respectively. The InterFC module was replaced with Group Feature Learning (GFL Block) [36] and Non-Local Attention (NLA Block) [25], respectively. The five obtained models are com- pared with the performance of I2MGAF on the classification test dataset. The results are shown in Figs 6 and 7. Table 5. Comparison of classification performance of UNet, ResNet, ResNeXt, Inception-v3 and SE-ResNeXt on S2MMAM. Ablation studies In this section, we evaluate the impact of the SE-ResNeXt, TAFA module, and the I2MGAF module on our S2MMAM respectively. Ablation study of SE-ResNeXt. Using SE-ResNeXt as the backbone of the network can not only enhance the network to extract focal features. It can also take advantage of the light- weight feature of ResNeXt to reduce the computational burden of the network and improve the network’s efficiency. To verify the performance of our proposed SE-ResNeXt, we replace the backbone network with S2MMAM(UNet), S2MMAM(ResNet), S2MMAM(ResNeXt) and S2MMAM(Inception-V3), respectively. These methods compare with our proposed SE-Res- NeXt on the same dataset. The results are shown in Table 5. As shown in Table 5, it is evident from the results that our S2MMAM(Ours) performed the best in KRAS gene mutation prediction among the five models. S2MMAM(Ours) achieved the best results in all six comparative metrics. The AUC was 83.27%, 5.96% higher than the sec- ond-place S2MMAM(ResNeXt). Compared to the more popular S2MMAM (Inception-V3), the AUC was 6.43% higher. SE-ResNeXt has a simpler architecture and lower computational complexity than Inception-v3. SE-ResNeXt effectively eliminates the semantic differences PLOS ONE \| https://doi.org/10.1371/journal.pone.0297331 March 11, 2024 15 / 25 Integrating image and gene-data for prediction of KRAS gene mutation status in non-small cell lung cancer Fig 6. Comparison of the classification performance of IntraFC and three models using other fusion methods. https://doi.org/10.1371/journal.pone.0297331.g006 Fig 6. Comparison of the classification performance of IntraFC and three models using other fusion methods. https://doi.org/10.1371/journal.pone.0297331.g006 Fig 6 shows a visual comparison of the six classification performance metrics after replacing the IntraFC module in I2MGAF with addition, concatenation, and AFF Block, respectively. From Fig 6, we find that the Concatenation fusion method achieves the lowest AUC value, so the [5–9] method cannot fully take advantage of the multimodal information. AFF Block is 5.9% lower than our IntraFC in AUC. This is due to the fact that AFF Block only focuses on inter-channel fusion of features at different levels, ignoring the potential loss of information PLOS ONE Methods AC(%) Recall(%) Precision(%) SP(%) AUC(%) F1(%) Addition 72.56±1.02 72.28±1.69 71.43±2.14 72.47±1.79 72.49±1.23 71.85±1.97 Concatenation 73.03±1.52 73.63±1.46 75.24±1.22 73.13±1.86 72.15±0.87 74.43±1.34 AEAF [34] 77.25±1.67 78.22±1.62 77.73±2.44 76.56±2.77 78.88±2.45 78.27±2.72 ASSCM [35] 78.39±1.42 78.37±1.19 78.49±0.76 78.57±2.06 77.89±1.06 78.43±0.97 TAFA(Ours) 81.67±2.67 82.31±2.51 83.15±1.21 82.66±2.07 83.27±1.49 82.73±1.86 https://doi org/10 1371/journal pone 0297331 t006 nce of TAFA on S2MMAM and four models with different fusion blocks. TAFA(Ours) achieved the best results in all on of classification performance of TAFA on S2MMAM and four models with different fusion blocks. TAFA(Ours) achiev rics Table 6. Comparison of classification performance of TAFA on S2MMAM and four models with different fusion blocks. TAFA(Ours) achieved the best results in all six comparative metrics. Methods AC(%) Recall(%) Precision(%) SP(%) AUC(%) F1(%) Addition 72.56±1.02 72.28±1.69 71.43±2.14 72.47±1.79 72.49±1.23 71.85±1.97 Concatenation 73.03±1.52 73.63±1.46 75.24±1.22 73.13±1.86 72.15±0.87 74.43±1.34 AEAF [34] 77.25±1.67 78.22±1.62 77.73±2.44 76.56±2.77 78.88±2.45 78.27±2.72 ASSCM [35] 78.39±1.42 78.37±1.19 78.49±0.76 78.57±2.06 77.89±1.06 78.43±0.97 TAFA(Ours) 81.67±2.67 82.31±2.51 83.15±1.21 82.66±2.07 83.27±1.49 82.73±1.86 https://doi.org/10.1371/journal.pone.0297331.t006 PLOS ONE \| https://doi.org/10.1371/journal.pone.0297331 March 11, 2024 16 / 25 Comparison of classification performance of TAFA on S2MMAM and four models with different fusion blocks. TAFA( arative metrics. 16 / 25 PLOS ONE Integrating image and gene-data for prediction of KRAS gene mutation status in non-small cell lung cancer PLOS ONE \| https://doi.org/10.1371/journal.pone.0297331 March 11, 2024 Comparison experiment We compare the proposed S2MMAM with the classical Semi-supervised Learning (SSL), and the recently published SSL image classification models with better results, trained on data with 100% and 30% of labeled data, respectively. Among the classical SSL methods include P- Model [13] and Mean Teacher. The competing methods include Relation-driven Self-ensem- bling Model (RSM) [15], SS-TBN [37], and DAB [38]. Note that we reproduce the above meth- ods on the same testset for the sake of fairness. Table 7 shows that the key evaluation metrics of S2MMAM outperform the other models on both 100% and 30% of the data with labeled data. This means that our S2MMAM can be used not only for supervised training but also for semi-supervised applications. We use the fully supervised model with 100% labeled data as the upper bound. And the SSL model trained on 30% labeled data as the target model. As can be seen from the Table 7, S2MMAM(Ours) achieved an AUC of 83.27% on the 30% labeled dataset. Mean Teacher only obtains an AUC result of 80.04% on the 100% labeled dataset. This shows the superiority of our S2MMAM for the classification task and even achieves accurate prediction with less cost. Compared with other models, our S2MMAM has the smallest gap of AUC, which is only 4.65% between 30% of the labeled dataset and the upper bound. This result indicates that our TAFA module and I2MGAF module effectively fuse the key features of multi-scale multi-modality. They can solve the problem of feature disappearance due to deep convolution and re-establish the fusion of high and low dimensional semantic key features. Compared with other SSL models that use only CT images for classification, our model has an AUC 6.9% higher than the second best Table 7. Comparison of the classification performance of S2MMAM and five other semi-supervised medical image classification models. Fig 7. Comparison of the classification performance of InterFC and two models using other fusion methods. https://doi.org/10.1371/journal.pone.0297331.g007 Fig 7. Comparison of the classification performance of InterFC and two models using other fusion methods. https://doi.org/10.1371/journal.pone.0297331.g007 Fig 7. Comparison of the classification performance of InterFC and two models using other fusion methods. Fig 7. Comparison of the classification performance of InterFC and two models using other fusion methods. htt //d i /10 1371/j l 0297331 007 PLOS ONE \| https://doi.org/10.1371/journal.pone.0297331 March 11, 2024 17 / 25 PLOS ONE Integrating image and gene-data for prediction of KRAS gene mutation status in non-small cell lung cancer due to network depth. Our IntraFC module not only focuses on channel fusion of segmenta- tion and classification features but also solves the problem of information loss caused by multi- ple fusions. Fig 7 shows the comparison of the six classification performance metrics after replacing the InterFC module in I2MGAF with the GFL Block and NLA Block, respectively. Our InterFC outperforms the second-place NLA Block by 4.11% and 3.6% in AUC and F1 scores, respec- tively. Our InterFC solves the limitation that NLA Block only focuses on the fusion of informa- tion in a single dimension. InterFC can fully combine the information in three dimensions to fuse the data of different modalities and improve the model sensitivity, thus obtaining a better prediction of KARS mutation. Comparison experiment Methods Labeled Unlabeled Data Result(%) CT Gene AC Recall Precision SP AUC F1 P-Model [13] 100% 0 p 76.35±2.32 78.21±2.36 79.32±2.68 77.32±2.65 76.23±2.31 78.76±2.51 Mean Teacher [10] p 81.24±2.18 80.15±1.81 82.34±1.79 81.86±2.43 80.04±2.78 81.23±1.8 RSM [15] p 84.21±1.26 81.93±2.15 84.21±2.03 84.72±2.07 83.41±2.65 83.05±2.09 SS-TBN [37] p 80.25±1.71 79.38±2.16 79.88±2.71 78.62±3.89 81.23±2.44 80.35±2.66 DAB [38] p 81.79±2.3 80.42±1.51 82.11±2.23 82.8±2.43 83.56±2.36 82.37±1.97 S2MMAM(Ours) p p 86.94±3.12 85.97±2.19 84.28±1.73 86.11±2.54 87.92±1.69 85.12±1.96 P-Model [13] 30% 70% p 71.23±2.49 72.11±1.65 72.56±1.24 71.16±2.77 70.15±2.17 72.33±1.45 Mean Teacher [10] p 74.28±2.53 74.16±2.73 75.29±2.94 74.62±2.82 74.21±3.48 74.72±2.83 RSM [15] p 75.91±2.37 75.13±3.21 76.37±2.86 75.49±3.53 75.94±2.34 75.74±3.04 SS-TBN [37] p 76.01±1.54 75.17±1.89 77.22±1.47 77.01±2.15 76.37±2.22 77.74±1.98 DAB [38] p 75.73±2.46 77.22±2.49 76.16±2.73 76.49±2.87 76.06±1.87 76.58±2.12 S2MMAM(Ours) p p 81.67±2.67 82.31±2.51 83.15±1.21 82.66±2.07 83.27±1.49 82.73±1.86 https://doi org/10 1371/journal pone 0297331 t007 cation performance of S2MMAM and five other semi-supervised medical image classification models. Comparison of the classification performance of S2MMAM and five other semi-supervised medical image classification Table 7. Comparison of the classification performance of S2MMAM and five other semi-supervised medical image classification models. PLOS ONE \| https://doi.org/10.1371/journal.pone.0297331 March 11, 2024 18 / 25 PLOS ONE Integrating image and gene-data for prediction of KRAS gene mutation status in non-small cell lung cancer Fig 8. AUC of our S2MMAM and five other medical image classification models on 30% labeled image dataset. https://doi.org/10.1371/journal.pone.0297331.g008 Fig 8. AUC of our S2MMAM and five other medical image classification models on 30% labeled image dataset. https://doi.org/10.1371/journal.pone.0297331.g008 SS-TBN model and 7.21% higher than the DBA model. This is due to our design of a new mul- timodal fusion module, I2MGAF. I2MGAF guides the fusion of features for multiple tasks and the fusion of multimodal data. It utilizes segmentation features to facilitate the classification task and efficiently extract important features from different modalities. I2MGAF has the abil- ity to compensate for the specificity information that can be easily overlooked by a single data modality and achieve the complementary effects of multi-modal data. As well as to find the pathogenic features of lesions based on multi-dimensionality, thus enhancing the classification ability. We also plot the AUC curves of our S2MMAM with the other five models in Fig 8 to demonstrate the classification performance of our S2MMAM more visually. PLOS ONE PLOS ONE Table 8. Six metrics were achieved on the test set by Baseline, Baseline+SMF-SN, Baseline+Gene, and our S2MMAM when using 30%, 40%, and 100% labeled train- ing images. Methods Labeled Unlabeled Data Result(%) CT Gene AC Recall Precision SP AUC F1 Baseline 100% 0 p 76.29±5.22 75.39±2.31 74.92±2.64 77.57±2.84 78.26±3.14 75.15±2.39 Baseline+SMF-SN p 83.19±2.46 79.38±1.37 81.31±1.67 82.47±3.64 84.29±4.36 80.33±1.52 Baseline+Gene p p 82.37±1.67 80.06±3.49 78.15±2.17 81.66±3.58 82.2±2.61 79.09±2.81 S2MMAM(Ours) p p 86.94±3.12 85.97±2.19 84.28±1.73 86.11±2.54 87.92±1.69 85.12±1.96 Baseline 40% 60% p 74.04±1.21 73.72±3.11 74.3±2.16 73.74±2.94 75.61±2.47 73.28+2.76 Baseline+SMF-SN p 78.37±2.14 77.49±2.76 78.06±1.98 78.34±2.48 79.23±3.16 79.35+2.54 Baseline+Gene p p 78.41±1.43 78.16±1.32 79.64±2.34 78.14±1.79 78.02±1.99 79.87+1.29 S2MMAM(Ours) p p 82.35±1.72 83.14±1.48 83.78±1.77 81.87±1.76 83.98±1.01 83.62+1.52 Baseline 30% 70% p 73.65±2.18 72.91±1.03 72.11±2.36 73.67±2.72 73.33±2.31 72.51±1.7 Baseline+SMF-SN p 77.29±5.22 76.43±4.21 77.24±3.21 77.17±1.2 77.44±5.32 78.8±3.69 Baseline+Gene p p 75.11±2.3 78.81±2.92 79.35±2.16 75.39±3.44 75.14±3.26 79.08±2.54 S2MMAM(Ours) p p 81.67±2.67 82.31±2.51 83.15±1.21 82.66±2.07 83.27±1.49 82.73±1.86 https://doi.org/10.1371/journal.pone.0297331.t008 Baseline, Baseline+SMF-SN, Baseline+Gene, and our S2MMAM when using 30%, 40%, and 100% labeled train- Table 8. Six metrics were achieved on the test set by Baseline, Baseline+SMF-SN, Baseline+Gene, and our S2MMAM ing images. features for the classification task, 2) the superiority of multimodal data over single modal data, and 3) the selection of the proportion of labeled images within the training dataset. We designed three sets of experiments and empirically used data with the proportion of labeled data of 100%, 40%, and 30% as the training dataset. Baseline is used as our base archi- tecture, where Baseline is only constructed by S2MF-CN using CT image data for the classifica- tion task. Based on this, we conducted a comparative study by gradually adding SMF-SN, genetic data, and both SMF-SN and genetic data. The experimental results are shown in Table 8. PLOS ONE \| https://doi.org/10.1371/journal.pone.0297331 March 11, 2024 Superiority of the model Although ablation studies and comparison experiments have demonstrated the merits of our proposed method, further discussions are needed on 1) the positive effects of segmentation PLOS ONE \| https://doi.org/10.1371/journal.pone.0297331 March 11, 2024 19 / 25 Integrating image and gene-data for prediction of KRAS gene mutation status in non-small cell lung cancer 1) The positive effects of segmentation features for the classification task As shown in Table 8, better classification results are obtained when the model utilizes the idea of segmentation to facilitate classification. Compared to Baseline, Baseline+SMF-SN improves the AUC values by 6.03%, 3.62%, and 4.11% in 30%, 40%, and 100% labeled datasets, respectively. We also visualize some of our Baseline and Baseline+SMF-SN segmentation results in Fig 9. The results are output in the form of a segmentation graph, which visualizes the ability of the network to localize the lesion area. As can be seen from Fig 9, the model with segmentation task can better localize the lesion area. It can avoid mixing impurities that can easily interfere with the judgment to improve the accuracy of diagnosis. 2) The superiority of multimodal data over single modal data 2) The superiority of multimodal data over single modal data As shown in Table 8, when we used genetic data, the AUC improved by 3.94%, 2.41%, and 2.81%, respectively, compared with Baseline. This indicates that image data can also extract genotypic features from biological data that can express individual differences and reflect dis- ease characteristics at the micro level. Further, enhances the network information richness and promotes the classification performance. 3) The selection of the proportion of labeled images within the training dataset As shown in Table 8, when the proportion of labeled data was 30% and 40%, respectively, the difference in the values of the four metrics was small, with a 0.71% difference in AUC and a 0.83% difference in Recall. Compared with the cost of physician labeling, this result indicates that the guidance information contained in 30% labeled training images is sufficient for the PLOS ONE \| https://doi.org/10.1371/journal.pone.0297331 March 11, 2024 20 / 25 PLOS ONE Integrating image and gene-data for prediction of KRAS gene mutation status in non-small cell lung cancer Fig 9. Comparison of the segmentation results obtained after training on Baseline strategy and Baseline+SMF-SN strategy: Baseline: Only classification task. Baseline+SMF-SN: classification task and segmentation task. (a) and (b) are the wild type of NSCLC. (c) and (d) are the mutation of NSCLC. The region surrounded by the red line is the ground truth, and the region surrounded by the green line is the segmentation results. https://doi org/10 1371/journal pone 0297331 g009 Fig 9. Comparison of the segmentation results obtained after training on Baseline strategy and Baseline+SMF-SN strategy: Baseline: Only classification task. Baseline+SMF-SN: classification task and segmentation task. (a) and (b) are the wild type of NSCLC. (c) and (d) are the mutation of NSCLC. The region surrounded by the red line is the ground truth, and the region surrounded by the green line is the segmentation results. https://doi.org/10.1371/journal.pone.0297331.g009 https://doi.org/10.1371/journal.pone.0297331.g009 https://doi.org/10.1371/journal.pone.0297331.g009 network to learn the key information of the lesion. Therefore, we used 30% labeled images and 70% unlabeled images as the training ratio of the model. To show the classification performance of our S2MMAM more visually, we also plotted the 3D comparison histograms of AUC and F1 score, as shown in Figs 10 and 11. To show the classification performance of our S2MMAM more visually, we also plotted the 3D comparison histograms of AUC and F1 score, as shown in Figs 10 and 11. 2) The superiority of multimodal data over single modal data In summary, the strategy of sharing segmentation network parameters by the classification network can assist the network to better localize the lesion region. The complementary nature of multimodal data allows the network to learn more abstract features besides addressing the challenge of less information in semi-supervised strategies. Therefore, our S2MMAM is better able to preserve the pathogenic regions, ignore irrelevant information, and improve model sensitivity. This leads to better KRAS mutation prediction results for NSCLC. Performance in supervised learning In order to demonstrate the scalability of our model, our application scenarios will not be lim- ited to semi-supervised learning but will be extended to supervised learning. We compare our S2MMAM with current multimodal classification models that have better results. The compet- ing methods include Multimodal Feature Fusion Diagnostic Model (MFFDM) [39], PLNM [9]. Note that we reproduce the above methods on the same test set for the sake of fairness. As shown in Table 9, our S2MMAM achieved the best AC, SP, and AUC values. This shows that our model has excellent classification performance even in supervised learning applica- tions. The AUC is 1.6% more than the second place PLNM and 3.75% more than the MFFDM. The fusion method of the MFFDM employs a simple splicing fusion, which we believe is the reason for the poor classification performance. Our S2MMAM employs a multidimensional fusion, which means that it is better able to adaptively fuse complementary information. Our S2MMAM and PLNM are similar in classification performance, but our method achieves bet- ter AUC values. We believe that SSL models can achieve the purpose of utilizing limited PLOS ONE \| https://doi.org/10.1371/journal.pone.0297331 March 11, 2024 21 / 25 PLOS ONE Integrating image and gene-data for prediction of KRAS gene mutation status in non-small cell lung cancer Fig 10. AUC were achieved on the test set by Baseline, Baseline+SMF-SN, Baseline+Gene and our S2MMAM, when using 30%, 40% and 100% labeled training images. Fig 10. AUC were achieved on the test set by Baseline, Baseline+SMF-SN, Baseline+Gene and our S2MMAM, when using 30%, 40% and 100% labeled training images. https://doi.org/10.1371/journal.pone.0297331.g010 information to achieve accurate prediction. When we train with more labeled data, our information to achieve accurate prediction. When we train with more labeled data, our S2MMAM can have a better ability to extract information and integrate information. In sum- mary, as described, our S2MMAM can be used not only in SSL but also in supervised learning. Fig 11. F1 score were achieved on the test set by Baseline, Baseline+SMF-SN, Baseline+Gene and our S2MMAM, when using 30%, 40% and 100% labeled training images. https://doi.org/10.1371/journal.pone.0297331.g011 Fig 11. F1 score were achieved on the test set by Baseline, Baseline+SMF-SN, Baseline+Gene and our S2MMAM, when using 30% 40% and 100% labeled training images Fig 11. Conclusion In this paper, we propose an integrating Image and Gene Data with a Semi-Supervised Atten- tion Model for the Prediction of KRAS Gene Mutation Status in Non-Small Cell Lung. The model consists of two components: supervised multilevel fusion segmentation network (SMF-SN) and semi-supervised multimodal fusion classification network (S2MF-CN) fusion. The results on the NSCLC-Radiogenomics dataset demonstrate that S2MMAM can achieve a more accurate prediction of KRAS gene mutation status. However, our S2MMAM still has some limitations. First, the model tested in this study used a single dataset and was not tested on multiple different datasets. Second, although CT images have been shown to aid in the prediction of KRAS gene mutations. However, in the clinical set- ting, histopathology images are the gold standard. We will try to combine CT images, histopa- thology images, and genetic data to further improve the accuracy of KRAS gene mutation status prediction in non-small cell lung cancer. Performance in supervised learning F1 score were achieved on the test set by Baseline, Baseline+SMF-SN, Baseline+Gene and our S2MMAM, when using 30%, 40% and 100% labeled training images. https://doi org/10 1371/journal pone 0297331 g011 Fig 11. F1 score were achieved on the test set by Baseline, Baseline+SMF-SN, Baseline+Gene and our S2MMAM, when using 30%, 40% and 100% labeled training images. Fig 11. F1 score were achieved on the test set by Baseline, Baseline+SMF-SN, Baseline+Gene and our S2MMAM, when using 30%, 40% and 100% labeled training images. https://doi.org/10.1371/journal.pone.0297331.g011 Fig 11. F1 score were achieved on the test set by Baseline, Baseline+SMF-SN, Baseline+Gene and our S2MMAM, when using 30%, 40% and 100% labeled training images. https://doi.org/10.1371/journal.pone.0297331.g011 https://doi.org/10.1371/journal.pone.0297331.g011 22 / 25 PLOS ONE \| https://doi.org/10.1371/journal.pone.0297331 March 11, 2024 Integrating image and gene-data for prediction of KRAS gene mutation status in non-small cell lung cancer PLOS ONE Table 9. Comparison of the classification performance of S2MMAM and two other supervised medical image classification models. Methods AC(%) Recall(%) Precision(%) SP(%) AUC(%) F1(%) MFFDM [39] 84.15±1.45 84.22±2.04 83.98±2.77 84.02±1.97 84.17±1.03 84.16±1.17 PLNM [9] 86.34±2.11 86.21±2.61 85.24±1.69 85.73±2.65 86.32±1.87 85.23±2.31 S2MMAM(Ours) 86.94±3.12 85.97±2.19 84.28±1.73 86.11±2.54 87.92±1.69 85.12±1.96 https://doi org/10 1371/journal pone 0297331 t009 arison of the classification performance of S2MMAM and two other supervised medical image classification models. Table 9. Comparison of the classification performance of S2MMAM and two other supervised medical image classifi It is a non-invasive method to determine whether the KRAS gene is mutated or not, to deter- mine the treatment for patients early, and to improve the survival rate of patients. It is a non-invasive method to determine whether the KRAS gene is mutated or not, to deter- mine the treatment for patients early, and to improve the survival rate of patients. Author Contributions Conceptualization: Yuting Xue, Dongxu Zhang, Liye Jia, Ying Qiao, Huajie Yue. Software: Yuting Xue, Dongxu Zhang. Validation: Liye Jia, Wanting Yang, Long Wang. Writing – original draft: Yuting Xue. Writing – review & editing: Dongxu Zhang, Wanting Yang, Juanjuan Zhao, Ying Qiao, Hua- jie Yue. References 1. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2019. CA Cancer J Clin. 2019 Jan; 69(1):7–34. https:// doi.org/10.3322/caac.21551 PMID: 30620402 23 / 25 PLOS ONE \| https://doi.org/10.1371/journal.pone.0297331 March 11, 2024 PLOS ONE \| https://doi.org/10.1371/journal.pone.0297331 March 11, 2024 PLOS ONE Integrating image and gene-data for prediction of KRAS gene mutation status in non-small cell lung cancer 2. Johannet P, Coudray N, Donnelly DM, Jour G, Illa-Bochaca I, Xia Y, et al. Using Machine Learning Algorithms to Predict Immunotherapy Response in Patients with Advanced Melanoma. Clin Cancer Res. 2021 Jan 1; 27(1):131–140. https://doi.org/10.1158/1078-0432.CCR-20-2415 PMID: 33208341 3. Song Y. CT Radio Genomics of Non-Small Cell Lung Cancer Using Machine and Deep Learning. ICCECE.2021, January.128–139. 4. Shiri I, Amini M, Nazari M, Hajianfar G, Haddadi Avval A, Abdollahi H, et al. Impact of feature harmoni- zation on radiogenomics analysis: Prediction of EGFR and KRAS mutations from non-small cell lung cancer PET/CT images. Comput Biol Med. 2022 Mar; 142:105230. https://doi.org/10.1016/j. compbiomed.2022.105230 PMID: 35051856 5. Ma Y, Wang J, Song K, Qiang Y, Jiao X, Zhao J. Spatial-Frequency dual-branch attention model for determining KRAS mutation status in colorectal cancer with T2-weighted MRI. Comput Methods Pro- grams Biomed. 2021 Sep; 209:106311. https://doi.org/10.1016/j.cmpb.2021.106311 PMID: 34352652 6. Yang W, Dong Y, Du Q, Qiang Y, Wu K, Zhao J, et al. Integrate domain knowledge in training multi-task cascade deep learning model for benign–malignant thyroid nodule classification on ultrasound images. Eng Appl Artif Intell.2021; 98:104064. https://doi.org/10.1016/j.engappai.2020.104064 7. Zhao Z, Zhao J, Song K, Hussain A, Du Q, Dong Y, et al. Joint DBN and Fuzzy C-Means unsupervised deep clustering for lung cancer patient stratification. Eng Appl Artif Intell. 2020; 91: 103571. https://doi. org/10.1016/j.engappai.2020.103571 8. Dong Y, Hou L, Yang W, Han J, Wang J, Qiang Y, et al. Multi-channel multi-task deep learning for pre- dicting EGFR and KRAS mutations of non-small cell lung cancer on CT images. Quant Imaging Med Surg. 2021 Jun; 11(6):2354–2375. https://doi.org/10.21037/qims-20-600 PMID: 34079707 9. Hou G, Jia L, Zhang Y, Wu W, Zhao L, Zhao J, et al. Deep learning approach for predicting lymph node metastasis in non-small cell lung cancer by fusing image–gene data. Eng Appl Artif Intell. 2023; 122:106140. https://doi.org/10.1016/j.engappai.2023.106140 10. Tarvainen A, Valpola H. Mean teachers are better role models: Weight-averaged consistency targets improve semi-supervised deep learning results. Adv Neural Inf Process Syst. 2017;30. https://doi.org/ 10.5555/3294771.3294885 11. Zhu F, Zhao S, Wang P, Wang H, Yan H, Liu S. PLOS ONE \| https://doi.org/10.1371/journal.pone.0297331 March 11, 2024 References 2021; 70: 102025. https://doi.org/10.1016/j.media.2021.102025 PMID: 33721692 24. Woo S, Park J, Lee J. Y, Kweon I. S. Cbam: Convolutional block attention module. ECCV. 2018;3–19. https://doi.org/10.1007/978-3-030-01234-2_1 25. Li Z, Zhang C, Zhang Y, Wang X, Ma X, Zhang H, et al. CAN: Context-assisted full Attention Network for brain tissue segmentation. Med Image Anal. 2023; 85: 102710. https://doi.org/10.1016/j.media. 2022.102710 PMID: 36586394 26. Ibtehaz N, Rahman MS. MultiResUNet: Rethinking the U-Net architecture for multimodal biomedical image segmentation. Neural Netw. 2020 Jan; 121:74–87. https://doi.org/10.1016/j.neunet.2019.08.025 PMID: 31536901 27. Bakr S, Gevaert O, Echegaray S, Ayers K, Zhou M, Shafiq M, et al. A radiogenomic dataset of non- small cell lung cancer. Sci Data. 2018 Oct 16; 5:180202. https://doi.org/10.1038/sdata.2018.202 PMID: 30325352 28. Cubuk E.D, Zoph B, Mane D, Vasudevan V, Le Q.V. Autoaugment: Learning augmentation strategies from data. CVPR.2019;113–123. https://doi.org/10.48550/arXiv.1805.09501 29. Jia L, Wu W, Hou G, Zhang Y, Zhao J, Qiang Y, et al. DADFN: dynamic adaptive deep fusion network based on imaging genomics for prediction recurrence of lung cancer. Phys Med Biol. 2023 Mar 23; 68 (7). https://doi.org/10.1088/1361-6560/acc168 PMID: 36867882 30. Ronneberger O, Fischer P, Brox T. U-net: Convolutional networks for biomedical image segmentation. MICCAI.2015;Part III:234–241. https://doi.org/10.1007/978-3-319-24574-4_28 31. He K, Zhang X, Ren S, Sun J. Deep residual learning for image recognition. CVPR.2016; 770–778. https://doi.org/10.1109/CVPR.2016.90 32. Xie S, Girshick R, Dolla´r P, Tu Z, He K. Aggregated residual transformations for deep neural networks. CVPR.2017;1492–1500. https://doi.org/10.1109/CVPR.2017.634 33. Szegedy C, Vanhoucke V, Ioffe S, Shlens J, Wojna Z. Rethinking the inception architecture for com- puter vision. CVPR.2016; 2818–2826. https://doi.org/10.1109/CVPR.2016.308 34. Cai M, Zhao L, Zhang Y, Wu W, Jia L, Zhao J, et al. A progressive phased attention model fused histo- pathology image features and gene features for lung cancer staging prediction. Int J Comput Assist Radiol Surg. 2023 Oct; 18(10):1857–1865 https://doi.org/10.1007/s11548-023-02844-y PMID: 36943546 35. Wu H, Liu J, Xiao F, Wen Z, Cheng L, Qin J. Semi-supervised segmentation of echocardiography vid- eos via noise-resilient spatiotemporal semantic calibration and fusion. Med Image Anal. 2022; 78: 102397. https://doi.org/10.1016/j.media.2022.102397 PMID: 35259635 36. Zhao C, Chen W, Qin J, Yang P, et al. IFT-Net: Interactive Fusion Transformer Network for Quantitative Analysis of Pediatric Echocardiography. Med Image Anal. 2022; 82: 102648. https://doi.org/10.1016/j. media.2022.102648 PMID: 36242933 37. Zeng LL, Gao K, Hu D, Feng Z, Hou C, Rong P, et al. SS-TBN: A Semi-Supervised Tri-Branch Network for COVID-19 Screening and Lesion Segmentation. IEEE Trans Pattern Anal Mach Intell. References Semi-supervised wide-angle portraits correction by multi-scale transformer. IEEE Conf. Comput. Vis. Pattern Recognit. 2022;19689–19698. https://doi.org/ 10.48550/arXiv.2109.08024 12. Kwon D, Kwak S. Semi-supervised semantic segmentation with error localization network. CVPR. 2022;9957–9967. https://doi.org/10.48550/arXiv.2204.02078 13. Laine S, Aila T. Temporal ensembling for semi-supervised learning. arXiv preprint arXiv:1610. 02242, 2016. https://doi.org/10.48550/arXiv.1610.02242 14. Wang X, Tang F, Chen H, Cheung C. Y, Heng P. A. Deep semi-supervised multiple instance learning with self-correction for DME classification from OCT images. Med Image Anal. 2023; 83: 102673. https://doi.org/10.1016/j.media.2022.102673 PMID: 36403310 15. Liu Q, Yu L, Luo L, Dou Q, Heng PA. Semi-Supervised Medical Image Classification With Relation- Driven Self-Ensembling Model. IEEE Trans Med Imaging. 2020 Nov; 39(11):3429–3440. https://doi. org/10.1109/TMI.2020.2995518 PMID: 32746096 16. Wang Y, Wang Y, Cai J, Lee TK, Miao C, Wang ZJ. Ssd-kd: A self-supervised diverse knowledge distil- lation method for lightweight skin lesion classification using dermoscopic images. Med Image Anal. 2023; 84: 102693. https://doi.org/10.1016/j.media.2022.102693 PMID: 36462373 17. Ruder S. An overview of multi-task learning in deep neural networks. arXiv preprint arXiv:1706. 05098, 2017. Preprint at https://doi.org/10.48550/arXiv.1706.05098. 18. Xie Y, Zhang J, Xia Y, Shen C. A Mutual Bootstrapping Model for Automated Skin Lesion Segmentation and Classification. IEEE Trans Med Imaging. 2020 Jul; 39(7):2482–2493. https://doi.org/10.1109/TMI. 2020.2972964 PMID: 32070946 19. Zhao L, Song K, Ma Y, Cai M, Qiang Y, Sun J, et al. A segmentation-based sequence residual attention model for KRAS gene mutation status prediction in colorectal cancer. Appl Intell, 2022; 53:10232– 10254. https://doi.org/10.1007/s10489-022-04011-3 20. Song P, Hou J, Xiao N, Zhao J, Zhao J, Qiang Y, et al. MSTS-Net: malignancy evolution prediction of pulmonary nodules from longitudinal CT images via multi-task spatial-temporal self-attention network. Int J Comput Assist Radiol Surg. 2023 Apr; 18(4):685–693. https://doi.org/10.1007/s11548-022-02744- 7 PMID: 36447076 21. Papandreou G, Kokkinos I, Savalle P. A. Modeling local and global deformations in deep learning: Epi- tomic convolution, multiple instance learning, and sliding window detection. CVPR. 2015;390–399. https://doi.org/10.1109/CVPR.2015.7298636 24 / 25 PLOS ONE \| https://doi.org/10.1371/journal.pone.0297331 March 11, 2024 PLOS ONE Integrating image and gene-data for prediction of KRAS gene mutation status in non-small cell lung cancer 22. Ye Y, Pan C, Wu Y, Wang S, Xia Y. MFI-Net: Multiscale Feature Interaction Network for Retinal Vessel Segmentation. IEEE J Biomed Health Inform. 2022 Sep; 26(9):4551–4562. https://doi.org/10.1109/ JBHI.2022.3182471 PMID: 35696471 23. Wu H, Wang W, Zhong J, Lei B, Wen Z, Qin J. Scs-net: A scale and context sensitive network for retinal vessel segmentation. Med Image Anal. PLOS ONE \| https://doi.org/10.1371/journal.pone.0297331 March 11, 2024 References 2023 Aug; 45 (8):10427–10442 https://doi.org/10.1109/TPAMI.2023.3240886 PMID: 37022260 38. Chen X, Bai Y, Wang P, Luo J. Data augmentation based semi-supervised method to improve COVID- 19 CT classification. Math Biosci Eng. 2023 Feb 6; 20(4):6838–6852. https://doi.org/10.3934/mbe. 2023294 PMID: 37161130 39. Tu Y, Lin S, Qiao J, Zhuang Y, Zhang P. Alzheimer’s disease diagnosis via multimodal feature fusion. Computers in Biology and Medicine, 2022, 148: 105901 https://doi.org/10.1016/j.compbiomed.2022. 105901 PMID: 35908497 25 / 25
W2963434388.txt	https://www.aclweb.org/anthology/N16-1041.pdf	en		Leverage Financial News to Predict Stock Price Movements Using Word Embeddings and Deep Neural Networks	null	2,016	cc-by	3,881	Leverage Financial News to Predict Stock Price Movements Using Word Embeddings and Deep Neural Networks Yangtuo Peng and Hui Jiang Department of Electrical Engineering and Computer Science, York University, 4700 Keele Street, Toronto, Ontario, M3J 1P3, Canada emails: tim@cse.yorku.ca, hj@cse.yorku.ca Abstract Financial news contains useful information on public companies and the market. In this paper we apply the popular word embedding methods and deep neural networks to leverage financial news to predict stock price movements in the market. Experimental results have shown that our proposed methods are simple but very effective, which can significantly improve the stock prediction accuracy on a standard financial database over the baseline system using only the historical price information. 1 Introduction In the past few years, deep neural networks (DNNs) have achieved huge successes in many data modelling and prediction tasks, ranging from speech recognition, computer vision to natural language processing. In this paper, we are interested in applying the powerful deep learning methods to financial data modelling to predict stock price movements. Traditionally neural networks have been used to model stock prices as time series for the forecasting purpose, such as in (Kaastra and Boyd, 1991; Adya and Collopy, 1991; Chan et al., 2000; Skabar and Cloete, 2002; Zhu et al., 2008). In these earlier work, due to the limited training data and computing power available back then, normally shallow neural networks were used to model various types of features extracted from stock price data sets, such as historical prices, trading volumes, etc, in order to predict future stock yields and market returns. More recently, in the community of natural language processing, many methods have been proposed to explore additional information (mainly online text data) for stock forecasting, such as financial news (Xie et al., 2013; Ding et al., 2014), twitter sentiments (Si et al., 2013; Si et al., 2014), financial reports (Lee et al., 2014). For example, (Xie et al., 2013) has proposed to use semantic frame parsers to generalize from sentences to scenarios to detect the roles of specific companies (positive or negative), where support vector machines with tree kernels are used as predictive models. On the other hand, (Ding et al., 2014) has proposed to use various lexical and syntactic constraints to extract event features for stock forecasting, where they have investigated both linear classifiers and deep neural networks as predictive models. In this paper, we propose to use the recent word embedding methods (Mikolov et al., 2013b; Liu et al., 2015; Chen et al., 2015) to select features from on-line financial news corpora, and employ deep neural networks (DNNs) to predict the future stock movements based on the extracted features. Experimental results have shown that the features derived from financial news are very useful and they can significantly improve the prediction accuracy over the baseline system that only relies on the historical price information. 2 Our Approach In this paper, we use deep neural networks (DNNs) as our predictive models, which provide us with the advantage of easily combining various types of features from different domains with the minimum 374 Proceedings of NAACL-HLT 2016, pages 374–379, San Diego, California, June 12-17, 2016. c 2016 Association for Computational Linguistics pre-processing and normalization effort. The DNN model takes as input the features extracted from both historical price information and on-line financial news to predict the stock movements in the future (either up or down) (Peng and Jiang, 2015). 2.1 Deep Neural Networks The structure of DNNs used in this paper is a conventional multi-layer perceptron with many hidden layers. An L-layer DNN consisting of L − 1 hidden nonlinear layers and one output layer. The output layer is used to model the posterior probability of each output target. In this paper, we use the rectified linear activation function, i.e., f (x) = max(0, x), to compute from activations to outputs in each hidden layer, which are in turn fed to the next layer as inputs. For the output layer, we use the softmax function to compute posterior probabilities between two nodes, standing for stock-up and stock-down. 2.2 Features from historical price data In this paper, for each target stock on a target date, we choose the previous five days’ closing prices and concatenate them to form an input feature vector for DNNs: P = (pt−5 , pt−4 , pt−3 , pt−2 , pt−1 ), where t denotes the target date, and pm denotes the closing price on the date m. We then normalize all prices by the mean and variance calculated from all closing prices of this stock in the training set. In addition, we also compute first and second order differences among the five days’ closing prices, which are appended as extra feature vectors. For example, we compute the first order difference as follows: ∆P = (pt−4 , pt−3 , pt−2 , pt−1 ) − (pt−5 , pt−4 , pt−3 , pn−2 ). In the same way, the second order difference is calculated by taking the difference between two adjacent values in each ∆P : ∆∆P = (∆Pt−3 , ∆Pt−2 , ∆Pt−1 ) − (∆Pt−4 , ∆Pt−3 , ∆Pt−2 ). Finally, for each target stock on a particular date, the feature vector representing the historical price information consists of P , ∆P and ∆∆P . 2.3 Financial news features In order to extract fixed-size features suitable to DNNs from financial news corpora, we need to preprocess the text data. For all financial articles, we first split them into sentences. We only keep those 375 sentences that mention at least a stock name or a public company. Each sentence is labelled by the publication date of the original article and the mentioned stock name. It is possible that multiple stocks are mentioned in one sentence. In this case, this sentence is labeled several times for each mentioned stock. We then group these sentences by the publication dates and the underlying stock names to form the samples. Each sample contains a list of sentences that were published on the same date and mention the same stock or company. Moreover, each sample is labelled as positive (“price-up”) or negative (“price-down”) based on its next day’s closing price consulted from the CRSP financial database (Booth, 2012). In the following, we introduce our method to extract three types of features from each sample. (1) Bag of keywords (BoK): We first select the keywords based on the recent word embedding methods in (Mikolov et al., 2013a; Mikolov et al., 2013b). Using the popular word2vec method 1 , we first compute the vector representations for all words occurring in the training set. Secondly, we manually select a small set of seed words, namely, nine seed words of {surge, rise, shrink, jump, drop, fall, plunge, gain, slump} in this work, which are believed to have a strong indication to the stock price movements. Next, we will repeat an iterative searching process to collect other useful keywords. In each iteration, we compute the cosine distance between other words occurring in the training set and each seed word. The cosine distance represents the similarity between two words in the word vector space. For example, based on the pre-calculated word vectors, we have found other words, such as rebound, decline, tumble, slowdown, climb, which are very close to at least one of the above seed words. The top 10 most similar words are chosen and added back into the set of seed words at the end of each iteration. The updated seed words will be used to repeat the searching process again to find another top 10 most similar words, the size of the seed words will increase as we repeat the procedure. In this way, we have searched all words occurring in training set and finally selected 1,000 words (including the nine initial seed words) as the keywords for our prediction 1 https://code.google.com/p/word2vec/ task. In this iterative process to collect keywords, we have found that the final set of the derived keywords is usually very similar as long as we start from a small set of seed words that all strongly indicate the stock price movements. Finally, a 1000-dimension feature vector, called bag-of-keywords or BoK, is generated for each sample. Each dimension of the BoK vector is the TFIDF score computed for each selected keyword from the whole training corpus. (2) Polarity score (PS): We further compute socalled polarity scores (Turney and Littman, 2003; Turney and Pantel, 2010) to measure how each keyword is related to stock movements and how each keyword applies to a target stock in each sentence. To do this, we first compute the point-wise mutual information for each keyword w: PMI(w, pos) = log freq(w, pos) × N , freq(w) × freq(pos) where freq(w, pos) denotes the frequency of the keyword w occurring in all positive samples, N denotes the total number of samples in the training set, freq(w) denotes the total number of keyword w occurring in the whole training set and freq(pos) denotes the total number of positive samples in the training set. Furthermore, we calculate the polarity score for each keyword w as: PS(w) = PMI(w, pos) − PMI(w, neg). Obviously, the above polarity score PS(w) measures how each keyword is related to stock movements (either positively or negatively) and by how much. Next, for each sentence in all samples, we need to detect how each keyword is related to the mentioned stock. To do this, we use the Stanford parser (Marneffe et al., 2006) to detect whether the target stock is a subject of the keyword or not. If the target stock is the direct object of the keyword, we assume the keyword is oppositely related to the underlying stock. As a result, we need to flip the sign of the polarity score. Otherwise, if the target stock is the subject of the keyword, we keep the keyword’s polarity score as it is. For example, in a sentence like “Apple slipped behind Samsung and Microsoft in a 2013 customer experience survey from Forrester Research”, which contains an identified keyword slip. 376 Based on the parsing result, we know Apple is the subject of slip while Samsung and Microsoft are the object of slip. Therefore, if this sentence is used as a sample for Apple, the above polarity score of slip is directly used. However, if this sentence is used as a sample for Samsung or Microsoft, the polarity score of slipped is flipped by multiplying −1. Finally, the resultant polarity scores are multiplied to the TFIDF scores to generate another 1000-dimension feature vector for each sample. (3) Category tag (CT): During the preprocessing of the financial news data, we have discovered that certain type of events are frequently described in the financial news, and the stock price will change significantly after the publication of such financial news. In order to discover the impact of these specific events on the stock price, we further define a list of categories that may indicate the specific events or activities of a public company, which we call as category tags. In this paper, the defined category tags include: new-product, acquisition, pricerise, price-drop, law-suit, fiscal-report, investment, bankrupt, government, analyst-highlights. Each category is first manually assigned with a few words that are closely related to the category. For example, we have chosen released, publish, presented, unveil as a list of seed words for the category newproduct, which indicates the company’s announcement of new products. Similarly, we use the above word embedding model to automatically expand the word list by searching for more words that have closer cosine distances with the selected seed words. At last, we choose the top 100 words to assign to each category tag. After we have collected all key words for each category, for each sample, we count the total number of occurrences of all words under each category, and then we take the logarithm to obtain a feature vector as V = (log N1 , log N2 , log N3 , ..., log Nc ), where Nc denotes the total number of times the words in category c appear in the sample. In the case where Nc is zero, it is replaced by a large negative number, for example -99.0 in this work. 2.4 Predicting Unseen Stocks via Correlation Graph There are a large number of stocks trading in the market. However, we normally can only find a Figure 1: Illustration of a part of correlation graph fraction of them mentioned in daily financial news. Hence, for each date, the above method can only predict those stocks mentioned in the news. In this section, we propose a new method to extend to predict more stocks that may not be directly mentioned in the financial news. Here we propose to use a stock correlation graph, shown in Figure 1, to predict those unseen stocks. The stock correlation graph is an undirected graph, where each node represents a stock and the arc between two nodes represents the correlation between these two stocks. In this way, if some stocks in the graph are mentioned in the news on a particular day, we first use the above method to predict these mentioned stocks. Afterwards, the predictions are propagated along the arcs in the graph to generate predictions for those unseen stocks. (1) Build the graph: We choose the top 5,000 stocks from the CRSP database (Booth, 2012) to construct the correlation graph. At each time, any two stocks in the collection are selected to align their closing prices based on the related dates (between 2006/01/01 - 2012/12/31), and we only keep the stock pairs that have an overlapped trading period of at least 252 days (number of trading days in one year). Then we calculate the correlation coefficients between the closing prices of these two stocks. The computed correlation coefficients (between −1 and 1) are attached to the arc connecting these two stocks in the graph, indicating their price correlation. The correlation coefficients are calculated for all stock pairs from the collection of 5,000 stocks. In this paper, we only keep the arcs with an absolute correlation value greater than 0.8, all other edges are considered to be unreliable and pruned from the graph. (2) Predict unseen stocks: In order to predict 377 price movements of unseen stocks, we first take the prediction results of those mentioned stocks from the DNN outputs, by which we construct a 5000dimension vector x. The value of each dimension in this vector is set to indicate the probability of price moving up or down. For the dimensions corresponding to the stocks that are mentioned in the financial news, we set the values using the prediction outputs of the DNN. Since the DNN has two outputs, each of which represents the probabilities of two categories, i.e. stock price moving up or down. If a sample is recognized as price-up, we set this dimension as its probability. Otherwise, if it is recognized as price-down, we set this dimension as its probability multiplied by −1.0. Next, we set zeros for all other dimensions corresponding to unseen stocks. The graph propagation process can be mathematically represented as a matrix multiplication: x0 = Ax where A is a symmetric 5000-by-5000 matrix denoting all arc correlation weights in the graph. Of course, this graph propagation may be repeated for several times until the prediction x0 converges. 3 Dataset The financial news data we used in this paper are provided by (Ding et al., 2014), which contains 106,521 articles from Reuters and 447,145 from Bloomberg. The news articles were published in the time period from October 2006 to December 2013. The historical stock security data are obtained from the Centre for Research in Security Prices (CRSP) database (Booth, 2012), which is published by the Chicago Business School and is widely used in the financial modelling. The CRSP database is properly adjusted for all special price events such as stock splits as well as dividend yields. We only use the security data from 2006 to 2013 to match the time period of the financial news. Base on the samples’ publication dates, we split the dataset into three sets: a training set (all samples between 2006-10-01 and 2012-12-31), a validation set (2013-01-01 and 201306-15) and a test set (2013-06-16 to 2013-12-31). The training set contains 65,646 samples, the validation set contains 10,941 samples, and the test set contains 9,911 samples. 4 Experiments 4.1 Stock Prediction using DNNs In the first set of experiments, we use DNNs to predict stock price movements based on a variety of features, namely producing a polar prediction of the price movement on next day (either price-up or price-down). Here we have trained a set of DNNs using different combinations of feature vectors and found that the DNN structure of 4 hidden layers (with 1024 hidden nodes in each layer) yields the best performance in the validation set. We use the historical price feature alone to create the baseline and various features derived from the financial news are added on top of it. We measure the final performance by calculating the error rate on the test set. As shown in Table 1, the features derived from financial news can significantly improve the prediction accuracy and we have obtained the best performance (an error rate of 43.13%) by using all the features discussed in Sections 2.2 and 2.3. We have also applied the structured event features proposed in (Ding et al., 2014) to our samples and the result is also listed in Table 1, which shows that our proposed features produce better performance in predicting a pool of individual stock prices. feature combination price price + BoK price + BoK + PS price + BoK + CT price + PS price + CT price + PS +CT price + BoK + PS + CT structured events (Ding et al., 2014) error rate 48.12% 46.02% 43.96% 45.86% 45.00% 46.10% 46.03% 43.13% 44.79% Table 1: Stock prediction error rates on the test set. 4.2 Predict Unseen Stocks via Correlation Here we group all outputs from DNNs based on the dates of all samples on the test set. For each date, we create a vector x based on the DNN prediction results for all observed stocks and zeros for all unseen stocks, as described in section 2.4. Then, the vector is propagated through the correlation graph to generate another set of stock movement prediction. Dur378 Figure 2: Predict unseen stocks via correlation ing the propagation, we compute the results from different iterations by multiplying the vector with the correlation matrix (x0 = Ax). Our experimental results show that the prediction accuracy stops to increase after the 4th iteration. After the propagation converges, we may apply a threshold on the propagated vector to prune all low-confidence predictions. The remaining ones may be used to predict some stocks unseen on the test set. The prediction of all unseen stocks is compared with the actual stock movement on next day. Experimental results are shown in Figure 2, where the left y-axis denotes the prediction accuracy and the right y-axis denotes the percentage of stocks predicted out of all 5000 per day under various pruning thresholds. For example, using a large threshold (0.9), we may predict with an accuracy of 52.44% on 354 extra unseen stocks per day, in addition to predicting only 110 stocks per day on the test set. 5 Conclusion In this paper, we have proposed a simple method to leverage financial news to predict stock movements based on the popular word embedding and deep learning techniques. Our experiments have shown that the financial news is very useful in stock prediction and the proposed methods can improve the prediction accuracy on a standard financial data set. Acknowledgement This work is partially supported by a Discovery Grant and an Engage Grant from Natural Sciences and Engineering Research Council (NSERC) of Canada. References Monica Adya and Fred Collopy. 1991. How effective are neural networks at forecasting and prediction? a review and evaluation. Journal of Forecasting, 17:481– 495. Chicago Booth. 2012. CRSP Data Description Guide for the CRSP US Stock Database and CRSP US Indices Database. Center for Research in Security Prices, The University of Chicago Graduate School of Business. Man-Chung Chan, Chi-Cheong Wong, and Chi-Chung Lam. 2000. Financial time series forecasting by neural network using conjugate gradient learning algorithm and multiple linear regression weight initialization. Computing in Economics and Finance, 61. Zhigang Chen, Wei Lin, Qian Chen, Xiaoping Chen, Si Wei, Xiaodan Zhu, and Hui Jiang. 2015. Revisiting word embedding for contrasting meaning. In Proceedings of the 53th Annual Meeting of the Association for Computational Linguistics (ACL). Association for Computational Linguistics. Xiao Ding, Yue Zhang, Ting Liu, and Junwen Duan. 2014. Using structured events to predict stock price movement: An empirical investigation. In Proceedings of the 2014 Conference on Empirical Methods in Natural Language Processing (EMNLP), pages 1415– 1425. Association for Computational Linguistics. Iebeling Kaastra and Milton Boyd. 1991. Designing a neural network for forecasting financial and economic time series. Neurocomputing, 10:215–236. Heeyoung Lee, Mihai Surdeanu, Bill Maccartney, and Dan Jurafsky. 2014. On the importance of text analysis for stock price prediction. In Proceedings of the Ninth International Conference on Language Resources and Evaluation (LREC). Quan Liu, Hui Jiang, Si Wei, Zhenhua Ling, and Yu Hu. 2015. Learning semantic word embeddings based on ordinal knowledge constraints. In Proceedings of the 53th Annual Meeting of the Association for Computational Linguistics (ACL). Association for Computational Linguistics. Marie-Catherine Marneffe, Bill MacCartney, and Christopher D. Manning. 2006. Generating typed dependency parses from phrase structure parses. In Proceedings of LREC. Tomas Mikolov, Kai Chen, Greg Corrado, and Jeffrey Dean. 2013a. Efficient estimation of word representations in vector space. In Proceedings of ICLR Workshop. Tomas Mikolov, Ilya Sutskever, Kai Chen, Greg S Corrado, and Jeff Dean. 2013b. Distributed representations of words and phrases and their compositionality. In Proceedings of NIPS, pages 3111–3119. 379 Yangtuo Peng and Hui Jiang. 2015. Leverage financial news to predict stock price movements using word embeddings and deep neural networks. In arXiv:1506.07220. Jianfeng Si, Arjun Mukherjee, Bing Liu, Qing Li, Huayi Li, and Xiaotie Deng. 2013. Exploiting topic based twitter sentiment for stock prediction. In Proceedings of the 51st Annual Meeting of the Association for Computational Linguistics (ACL), pages 24–29. Association for Computational Linguistics. Jianfeng Si, Arjun Mukherjee, Bing Liu, Sinno Jialin Pan, Qing Li, and Huayi Li. 2014. Exploiting social relations and sentiment for stock prediction. In Proceedings of the 2014 Conference on Empirical Methods in Natural Language Processing (EMNLP), pages 1139– 1145. Association for Computational Linguistics. Andrew Skabar and Ian Cloete. 2002. Neural networks, financial trading and the efficient markets hypothesis. In Proc. of the Twenty-Fifth Australasian Computer Science Conference (ACSC). Peter D. Turney and Michael L. Littman. 2003. Measuring praise and criticism: Inference of semantic orientation from association. ACM Trans. Inf. Syst., 21(4):315–346. Peter D. Turney and Patrick Pantel. 2010. From frequency to meaning: Vector space models of semantics. Journal of Artificial Intelligence Research, 37(1):141– 188. Boyi Xie, Rebecca Passonneau, Leon Wu, and Germán G. Creamer. 2013. Semantic frames to predict stock price movement. In Proceedings of the 51st Annual Meeting of the Association for Computational Linguistics (ACL), pages 873–883. Association for Computational Linguistics. Xiaotian Zhu, Hong Wang, Li Xu, and Huaizu Li. 2008. Predicting stock index increments by neural networks: The role of trading volume under different horizons. Expert Systems with Applications, 34:3043–3054.
https://openalex.org/W3154513371	https://www.e3s-conferences.org/articles/e3sconf/pdf/2021/24/e3sconf_caes2021_01070.pdf	English	null	Numerical Simulation of Wind Turbine Aerodynamic Characteristics under Wind Shear Based on Lattice-Boltzmann Method	E3S web of conferences	2,021	cc-by	2,621	1 Introduction Under the trend of large-scale development of wind turbines, it becomes particularly important to accurately analyze the aerodynamic performance of large wind turbines. According to the aerodynamic loads of wind turbines obtained from the analysis, the aerodynamic layout optimization design of wind turbines is carried out, which ensures the structural strength and at the same time obtains a higher wind energy utilization coefficient [1].At present, experimental and numerical simulation methods are mainly used to study the aerodynamic performance of wind turbines. The numerical simulation methods mainly include Blade-element Theory (BEM), eddy current theory and computational fluid dynamics method (CFD) [2]. With the improvement of computer performance, large commercial CFD software has been more and more widely used and has become a sharp tool for wind turbine aerodynamic performance research [3]. This paper takes T bl 1 M i Numerical Simulation of Wind Turbine Aerodynamic Characteristics under Wind Shear Based on Lattice-Boltzmann Method Chengdong Yuan1, Jinlong Wang2, Yueyue Pan2, , Hui Chen1 and Xiaqi Zhang1 Abstract: In order to study the influence of wind shear on the aerodynamic characteristics of large wind turbines, taking the 5MW wind turbine blade model published by NREL as the research object, large eddy simulation (LES) of wind turbines was carried out by using XFlow fluid simulation software based on Lattice- Boltzmann method (LBM). WALE turbulence model was used to study wind shear at 3, 11.2 and 25m/s wind speeds. The effect of factors on the axial thrust and torque of wind turbines is compared with the data published by NREL. The results show that the XFlow software based on LBM and LES method has good capturing ability for the eddy wake of wind turbine; wind shear causes the airfoil section of each section of blade to deviate from the best designed attack angle in theory and results in a decrease in torque applied to the wind turbine. 5MW wind turbine of National Renewable Energy Laboratory (NREL) as the research object, large eddy simulation (LES) of wind turbine was carried out by XFlow fluid simulation software, and studies the influence of wind shear factor on axial thrust and torque of wind turbine at 3, 11.2 and 25 m/s wind speeds, and verifies the reliability of simulation software by comparing it with published data of NREL. 2.1 Three-dimensional model of wind turbine This paper takes NREL 5 MW wind turbine as the research object. The wind turbine is a horizontal axis, three-blade and windward wind power unit [4]. Its main parameters are shown in Table 1. [ ] p p Table 1. Main parameters of NREL5 MW wind turbine © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). https://doi.org/10.1051/e3sconf/202124801070 https://doi.org/10.1051/e3sconf/202124801070 E3S Web of Conferences 248, 01070 (2021) CAES 2021 Numerical Simulation of Wind Turbine Aerodynamic Characteristics under Wind Shear Based on Lattice-B Method Chengdong Yuan1, Jinlong Wang2, Yueyue Pan2, , Hui Chen1 and Xiaqi Zhang1 1 Shandong university of science and technology, Shandong, China, 266590 2 Weifang university, Shandong, China, 261061 * Corresponding author e-mail: panyueyue1022@wfu.edu.cn; panyueyue_1022@163.com y EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 icenses/by/4.0/). 2.2 Wind field modeling and meshing After verification of irrelevance of wind field size, the selected wind field size is 600 m x 290 m x 400 m. Figure 2. calculation domain of wind field Figure 2. calculation domain of wind field 3.2 LBM Method The LBM method is to discrete the Boltzmann-BGK equation in space, time and velocity [5]. The Boltzmann transport equation is as follows: [ ] p p Table 1. Main parameters of NREL5 MW wind turbine y p [ ] p p Table 1. Main parameters of NREL5 MW wind turbine [ ] p p Table 1. Main parameters of NREL5 MW wind turbine * Corresponding author e-mail: panyueyue1022@wfu.edu.cn; panyueyue_1022@163.com Parameters Numerical value Parameters Numerical value Rating 5 MW Cut-in wind speed 3 m/s The number of blades 3 Cut-out wind speed 25 m/s Wind turbine diameter 123 m Rated wind speed 11.4 m/s Hub diameter 3 m Cut-in rotor speed 6.9 r/min Hub height 90 m Rated rotor speed 12.1 r/min Shaft tilt 5 ° Cone angle 2.5 ° he Authors published by EDP Sciences This is an open access article distributed under the terms of the Creative Commons Attribution Licens © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). E3S Web of Conferences 248, 01070 (2021) CAES 2021 https://doi.org/10.1051/e3sconf/202124801070 tower. The wind turbine has elevation and cone angle. The wind turbine model is shown in Figure 1. In this paper, the numerical simulation calculation model only considers the wind turbine part, without considering the influence of engine compartment and Figure 1. Geometric model of wind turbine Figure 1. Geometric model of wind turbine Considering the influence of ground, the height of wind wheel center from ground is 90 m, and the distance from inlet of inflow is 200 m. Both left and right boundary and upper boundary of calculation area are 200 m, as shown in Figure 2. Considering the influence of ground, the height of wind wheel center from ground is 90 m, and the distance from inlet of inflow is 200 m. Both left and right boundary and upper boundary of calculation area are 200 m, as shown in Figure 2. 3.3 large eddy simulation Method Large eddy simulation method (LES) filters continuity equation and N-S equation, filters subgrid vortices smaller than filter scale, decomposes control equation describing the motion of large eddy, and introduces subgrid stress term into the equation to consider the influence of momentum and energy of subgrid vortices on large eddy [7]. The subgrid turbulence model used in XFlow software is a wall-adaptive local eddy viscosity model (WALE), which has good characteristics in both laminar and turbulent flows, whether close to the wall or far from the wall. The model restores the asymmetric characteristics of the turbulent boundary layer without adding artificial turbulent viscosity to the wake shear zone, which can be directly solved. The WALE model [8] is represented as follows: 3.1 Introduction to XFlow software XFlow is a new generation of fluid dynamics simulation analysis software based on Lattice-Boltzmann method (LBM). It uses particle-based and complete Lagrange function method, has advanced large eddy simulation (LES) model and uses uniform non-equilibrium wall function to simulate boundary layer.       1 , , ,..., B i i i i b f r c t t t f r t f f     (1) Where if is the distribution function in the direction; B i  is the collision operator; t is the discrete time, s; ic is the velocity in the i direction, m/s; r is the 2 E3S Web of Conferences 248, 01070 (2021) CAES 2021 https://doi.org/10.1051/e3sconf/202124801070   2 2 1 2 d ij ij iji S g g   (6) position on the lattice. position on the lattice. (6) p The lattice Boltzmann method (LBM) defines a simplified operator under the Bhatnagar-Gross-Krook (BGK) [6] approximation as follows: Where WALE constant w C is usually 0.2; d ij S is Where WALE constant w C is usually 0.2; d ij S is the strain rate tensor, which reflects not only the function of the symmetric part of the velocity gradient tensor, but also the effect of the asymmetric part.   1 BGK eq i i i f f     (2) (2) Where eq if is a local equilibrium function;  is a relaxation characteristic time. 3.4 Wind shear model Typically, the balanced distribution function expression is as follows: The NREL 5 MW large wind turbine is 123 m in diameter and the effects of wind shear must be taken into account. The NREL 5 MW large wind turbine is 123 m in diameter and the effects of wind shear must be taken into account. Changes in wind speed perpendicular to the wind plane are called wind shear or wind speed profiles, where an exponential law distribution is used, as shown in the Equation (8):   2 2 2 , 1 2 i i eq ia a i i s s s v v c c c v f r t t c c c                          (3) Changes in wind speed perpendicular to the wind plane are called wind shear or wind speed profiles, where an exponential law distribution is used, as shown in the Equation (8): (3) Where sc is the sound velocity, m/s; v and v are the macro-viscosities, Pa.s;   is the Croneker function; it is the parameter to ensure spatial isotropy;  is the macro-density, kg/m3. ref ref y U U y         (7) (7) In the formula, U is the average wind speed at y above ground level, m/s; ref y is the reference height, taking the hub center height of 90 m; ref U is the average wind speed at the hub center height, m/s;  is the wind speed profile index, and here take 1/7. 4 Numerical simulation results and analysis Under three wind speeds of 3 m/s, 11.2 m/s and 25 m/s, the wind turbine is simulated under two conditions of wind shear and no wind shear, and the axial thrust and torque changes of the wind turbine are monitored. The lattice analytical scale of calculation domain is 4 m for far-field solution, 0.25 m for near-wall solution and 0.25 m for eddy wake solution. Dynamic adaptive tracking encryption control is used for eddy wake. At this time, the number of grids is about 19.32 million and the calculation time is about 15 hours. The calculation accuracy and cost of eddy wake simulation are relatively balanced. The lattice analytical scale is shown in Figure 3 and the three- dimensional volume field cloud of vorticity is shown in Figure 4.       3/2 2 t s 5/2 5/4 d d ij ij d d d d ij ij ij ij S S L S S S S        (4)   1/3 min , s w L d C V   (5) (4) (5) 3 https://doi.org/10.1051/e3sconf/202124801070 E3S Web of Conferences 248, 01070 (2021) CAES 2021 E3S Web of Conferences 248, 01070 (2021) Figure 3. The lattice analytical scale Figure 4. The 3D volume field cloud of vorticity Figure 4. The 3D volume field cloud of vorticity Figure 3. The lattice analytical scale Figure 4. The 3D volume field cloud of vorticity Figure 3. The lattice analytical scale Figure 4. The 3D volume field cloud of vorticity It can be seen from Figure 3 that obvious vortex wake is formed behind the blade, and the grid analytical scale in the wake area is automatically adjusted. From Figure 4, it can be seen that the spiral vortex wake is formed in the wind field due to the rotation of the wind turbine, and vortices of different sizes are generated in the wake, which can well simulate the flow state of subgrid vortex. In other words, XFlow software can automatically detect and capture the large and sub-grid vortices in the wake area, update and encrypt the grid analytical scale of the wake area in real time, accurately simulate the vortex wake of wind turbine, and effectively save computing resources. 4 Numerical simulation results and analysis The data of numerical simulation results select the average load value within 5 rotation cycles after stabilization and compare with NREL published data, as shown in Table 2, where FXB is the axial thrust of the wind turbine, and MXB is the torque of the wind turbine. Operating conditions Wind turbine load Wind speed (m/s) Wind shear FXB (kN) FXB of NREL (kN) Error (%) MXB (kN·m) MXB of NREL (kN·m) Error (%) 3 Yes 149 168 11% 58 56 4% No 155 168 8% 61 56 9% 11.4 Yes 760 794 4% 4318 4181 3% No 766 794 4% 4362 4181 4% 25 Yes 345 373 8% 4320 4181 3% No 350 373 6% 4370 4181 5% According to the data comparison in Table 2, under all working conditions, the axial thrust of the wind turbine is slightly less than the NREL open value, and the wind turbine torque is slightly greater than the NREL open value, and the error at cut-in wind speed is relatively large, and the error at rated wind speed is relatively small. The results show that the relative errors between the numerical simulation results and NREL open results of the axial force and the torque of the wind turbine are within a reasonable range, which shows that the XFlow software based on Lattice-Boltzmann method and large eddy simulation has high accuracy for the simulation of wind turbine vortex wake. In addition, compared with the effect of wind shear, it is found that the thrust and torque of the rotor shaft under the action of wind shear are relatively less than the value of no wind shear under the corresponding wind speed. This is because the wind shear causes the wind speed of the wind turbine blade to be larger when it is at the high position in the rotation cycle, and the wind speed is small when it is at the low position, and the incoming wind speed of the blade presents periodic fluctuations, which deviates from the theoretical design wind speed. The results show that the angle of attack of each airfoil periodically deviates from the optimal design angle of attack, resulting in the reduction of wind turbine torque, which is less than the value without wind shear. 5 Conclusion (1) The XFlow software based on LBM and LES method has good capture ability and high simulation accuracy for large eddy and subgrid vortices in the wake area of wind turbine. (2) Wind shear makes the attack angle of each section of the blade periodically deviate from the theoretical optimal design angle of attack, which leads to the decrease of wind turbine torque. 4 https://doi.org/10.1051/e3sconf/202124801070 E3S Web of Conferences 248, 01070 (2021) CAES 2021 Acknowledgments The authors thank financial support from the Youth Program of National Natural Science Foundation of China (NSFC) under Grant No. 51908430. Reference 1. Jia Y.Y., Chen J.J., Wang H.J. (2018) Research on Influence of Wind Wheel Elevation Angle on Wind Turbine Aerodynamic Performance. Acta Energiae Solaris Sinica, 39(04): 1135-1141. 2. Yu H.N., Zhu F.L., Liu Y. (2008) Overview of Aeroelastic Stability of Wind Turbines. Journal of Machine Design, (06):1-3. 3. Hansen M.O.L., SΦRensen J.N., Voutsinas S., et al. (2006) State of the art in wind turbine aerodynamics and aeroelasticity. Progress in Aerospace Sciences, 42(4): 285-330. 4. Jonkman J., Butterfield S., Musial W., and Scott G. (2009) Definition of a 5-MW reference wind turbine for offshore system development. Office of Scientific & Technical Information, Golden, Colorado. 5. He Y.L., Wang Y., Li Q. (2009) Lattice Boltzmann method: theory and applications. Beijing Science Press, Beijing. 6. Qian Y.H., D'Humières D., Lallemand P. (1992) Lattice BGK models for Navier-Stokes equation. Europhysics Letters, 17(6): 479-484. 7. Xu C., Huang H.Q., Shi C., et al. (2020) Numerical Simulation of Wind Turbine Wake Based on LBM- LES Method under Typical Complex Terrain. Proceedings of the CSEE, (13). 8. Nicoud F., Ducros F. (1999) Subgrid-scale stress modelling based on the square of the velocity gradient tensor. Flow, Turbulence and Combustion, 62(3): 183-200. 5
https://openalex.org/W4393316479	https://www.explorationpub.com/uploads/Article/A1001212/1001212.pdf	English	null	Association of TGFBR2 gene polymorphisms (rs6785358 and rs764522) with congenital heart disease susceptibility in Egyptians	Exploration of medicine	2,024	cc-by	5,476	Nahed Dawood1, El-Shaimaa Shabana2, Ashraf A.H. El-Midany3, Faten R. Abdelghaffar1, Islam El-Garawani1* , Rizk Elbaz2 1Department of Zoology, Faculty of Science, Menoufia University, Shebin El-Kom 32512, Egypt enetics Unit, Children Hospital, Faculty of Medicine, Mansoura University, Mansoura 35516, Egypt 2Genetics Unit, Children Hospital, Faculty of Medicine, Mansoura University, Mansoura 35516, Egypt 3Department of Cardiothoracic Surgery, Faculty of Medicine, Ain Shams University, Cairo 11331, Egyp 3Department of Cardiothoracic Surgery, Faculty of Medicine, Ain Shams University, Cairo 11331, Egypt Correspondence: Islam El-Garawani, Department of Zoology, Faculty of Science, Menoufia University, Shebin El-Kom 32512, Egypt. dr.garawani@science.menofia.edu.eg orrespondence: Islam El-Garawani, Department of Zoology, Faculty of Science, Menoufia University, Shebin El 512, Egypt. dr.garawani@science.menofia.edu.eg Academic Editor: Guo-Chang Fan, University of Cincinnati College of Medicine, USA Received: November 17, 2023 Accepted: January 30, 2024 Published: March 29, 2024 Cite this article: Dawood N, Shabana ES, El-Midany AAH, Abdelghaffar FR, El-Garawani I, Elbaz R. Association of TGFBR2 gene polymorphisms (rs6785358 and rs764522) with congenital heart disease susceptibility in Egyptians. Explor Med. 2024;5:148–57. https://doi.org/10.37349/emed.2024.00212 Exploration of Medicine Exploration of Medicine Exploration of Medicine Open Access Original Article © The Author(s) 2024. This is an Open Access article licensed under a Creative Commons Attribution 4.0 International License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, sharing, adaptation, distribution and reproduction in any medium or format, for any purpose, even commercially, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Explor Med. 2024;5:148–57 \| https://doi.org/10.37349/emed.2024.00212 Introduction Congenital heart defects (CHD) are structural issues with the heart that exist at birth [1]. These abnormalities can affect the heart’s chambers, valves, or blood vessels that transport blood to and from the heart. The defects are frequently found alone or in combination with other defects [2, 3]. Physiologically, they are classified as cyanotic and acyanotic heart defects [4]. Cardiac abnormalities can range in complexity from simple that have no symptoms to complex lesions that have significant, life-threatening symptoms [5]. Congenital birth malformations can arise in children for a variety of reasons [6]. According to previous studies, genetic factors may be the primary cause of CHD [7]. Transforming growth factor beta (TGF-β) receptor II (TGFBR2), a transmembrane protein, is a member of the serine/threonine protein kinase family and the TGFBR subfamily with a molecular weight of 70/80 kDa [8]. The TGFBR2 gene, which is found on chromosome 3p22 and has seven exons and a 567 codon open reading frame, is responsible for encoding the TGFBR2 protein [9]. The TGF-β ligands bind to the TGFBR2 for the initiation of TGF-β signalling leading to the activation of the TGFBR1, which then causes the translocation to the nucleus following a series of processes, including phosphorylation of mothers against decapentaplegic homolog 2 (SMAD2) and SMAD3 with the association of SMAD4. SMAD proteins are involved in managing transcription of target genes which can be affected by TGFBR2 inactivation [10]. The TGFBR2 gene mutation in aortic pathology has been reported, and during cardiac development, the endothelial cells might be affected by TGFB signalling through TGFBR2 [11]. The valvuloseptal heart defect may occur due to disruptions in the signal transformation of endocardial cushions [12]. TGFBR2 is expressed during heart development in embryonic myocytes, endothelial cells, and endocardial cushion of mice [13]. It is known that genetic polymorphisms may have an impact on the susceptibility or resistance to certain infections [14] or have a key role in the contribution of the susceptibility to some diseases [15, 16] and personalized nutrition [17]. The relation between TGFBR2 gene polymorphisms (rs6785358 and rs764522), the 5’ upstream were also named -3779A/G and -1444C/G, respectively, and some disorders, such as Marfan syndrome and cardiac arrest, were reported [18, 19]. This study aims to explore the link between TGFBR2 gene polymorphisms (rs6785358 and rs764522) and CHD in Egyptians. To our knowledge, among the studied population, this is the first report focusing on this issue. Abstract Aim: Transforming growth factor beta (TGF-β) receptor II (TGFBR2) is a basic constituent of TGF-β signalling pathway and is important in heart development. This study investigates the relationship between TGFBR2 gene variance and congenital heart defects (CHD) among Egyptians. Methods: The study involved 75 CHD-affected subjects and 100 healthy controls. Genotyping of two selected tag single nucleotide polymorphisms (tagSNPs, rs6785358, rs764522) within the TGFBR2 gene was conducted using polymerase chain reaction-restriction fragment length polymorphism method (PCR- RFLP) assays. Results: Significant genotype differences were found for rs764522 and rs6785358 (P < 0.05). In the case of rs6785358, the G/G genotype was more prevalent in cases than controls (18.7% vs. 4.0%). This significance was observed in both the codominant model [A/A vs. A/G vs. G/G; odds ratio (OR) = 0.20, 95% confidence interval (CI) = 0.06–0.66, P = 0.0073] and the recessive model (A/A + A/G vs. G/G; OR = 0.19, 95% CI = 0.06–0.60, P = 0.0018). For rs764522, the G/G genotype was more prevalent in cases than controls (21.3% vs. 0.0%). Significant associations were observed in the codominant model (C/C vs. C/G vs. G/G; OR = 0.43, 95% CI = 0.02–0.90, P < 0.0001), as well as in the dominant model (C/C vs. C/G + G/G) and recessive model (C/C + C/G vs. G/G; P < 0.0001). Gender-specific analysis indicated that the C/G genotype was less common in male cases compared to females and controls (OR = 0.24, 95% CI = 0.07–0.84). For rs6785358, the G/G genotype frequency was higher in male cases compared to females and controls (OR = 0.10, 95% CI = 0.01–0.88 and OR = 0.22, 95% CI = 0.05–0.94, respectively). Conclusions: These findings indicate that TGFBR2 gene SNPs (rs6785358 and rs764522) may be risk factors for CHD in Egyptians. Explor Med. 2024;5:148–57 \| https://doi.org/10.37349/emed.2024.00212 Page 148 © The Author(s) 2024. This is an Open Access article licensed under a Creative Commons Attribution 4.0 International License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, sharing, adaptation, distribution and reproduction in any medium or format, for any purpose, even commercially, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Page 148 Page 148 Keywords Transforming growth factor beta receptor II (TGFBR2), single nucleotide polymorphisms (SNP), congenital heart disease, case-control study Transforming growth factor beta receptor II (TGFBR2), single nucleotide polymorphisms (SNP), congenital heart disease, case-control study Samples collection Peripheral blood samples were taken in sterile EDTA tubes (Kemico Vacutainer, Egypt) for genotyping and hematological evaluations. Additionally, blood aliquots were taken in serum tubes for other laboratory tests. Sera were stored at –20°C until use. All samples were identified and given matched numbers that corresponded to all investigations. Materials and methods This study involved 75 cases affected by CHD, 21 had atrial septal defect (ASD), 43 had ventricular septal defect (VSD), and 11 had tetralogy of Fallot (TOF), from the Department of Cardiology, Internal Medicine Specialized Hospital, Mansoura University, Egypt. While normal controls were enrolled via invitations. Isolation of total genomic DNA In an EDTA-containing tube, approximately 4 mL of venous blood samples were collected. Genomic DNA extraction was done from samples using the phenol-chloroform method in the presence of proteinase K digestion (Sigma-Aldrich, Germany). Using a UV spectrophotometer (Spectronic 1201, Milton Roy, USA), the absorbance at 260 nm was used for DNA concentration, however, the A260/A280 nm was used for purity determination. Demographic and clinical characteristics The characteristics of the cases (n = 75), and control (n = 100) groups are listed in Table 1. Regarding age or sex, the demographic results revealed a non-significant difference between the studied groups (P > 0.05). However, non-significant differences among studied groups regarding all laboratory investigations (P > 0.05) were noticed, except AST, hemoglobin, and platelets (P < 0.05). Statistical analysis Fisher’s exact χ2 test was used for Hardy-Weinberg equilibrium assessment (HWE 14 program in SPSS). The SPSS for Windows version 13.0 (SPSS Inc, USA) was used for performing statistical analyses. The Chi-square (χ2) test was done for the analysis of allelic variation among studied subject groups. The relation was illustrated as odds ratio (OR) with 95% confidence interval (95% CI) of the risk. The level of statistical significance was set at 0.05 (two tails). Also, Binary logistic regression was done for the association between different variables and the disease status. Laboratory investigations Physical inspections and routine questionnaires were done for all patients. Hematological parameters, such as hemoglobin, white blood cells (WBCs), and platelets, were investigated using Sysmex KX21 Hematology Physical inspections and routine questionnaires were done for all patients. Hematological parameters, such as hemoglobin, white blood cells (WBCs), and platelets, were investigated using Sysmex KX21 Hematology Explor Med. 2024;5:148–57 \| https://doi.org/10.37349/emed.2024.00212 Page 149 Analyzer (Sysmex Corporation, Japan). Other standard biochemical analyses, such as fasting blood sugar, aspartate transaminase (AST), alanine transaminase (ALT), urea, and creatinine (Biosystem, Spain), were performed. Single nucleotide polymorphisms selection Single nucleotide polymorphisms (SNPs) under investigation were selected according to PubMed published data (SNP database). To see if the promoter region harboured any genetic variants susceptible to CHD, we chose two tagSNPs, 5’ upstream, of the TGFBR2 gene (rs6785358 and rs764522). Isolation of total genomic DNA Isolation of total genomic DNA Genotype assessment To examine the allelic variance, the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay was done. The primer sequences of the rs6785358 were 5’- GAACTGCAAACAAGAGAATGGAT-3’ (forward) and 5’-TTAGAATTCTACCCTAATGATTGTAAGG-3’ (reverse), however, the rs764522 primers were 5’-GAGTGAAAGAGCCCAGAACG-3’ (forward) and 5’- GGGCTAGGCATCTTCTTTCC-3’ (reverse) [13]. A total volume of 10 μL of PCR reaction containing 10 ng of DNA, 0.5 pmol of each primer, and 1 × PCR master mix was prepared. Using an GeneAmpTM PCR System 9700 (Thermo Fisher, USA), the amplifications were accomplished. The reaction program proceeded as follows: one cycle for 5 min at 95°C, 30 cycles at 95°C for 30 s, 30 s for annealing at 61°C (rs6785358)/63°C (rs764522), and 30 s at 72°C. A final extension cycle at 72°C for 10 min. The PCR products were digested using the restriction enzymes BsuRI and MvaI (Thermo Fisher, USA), for rs6785358 and rs764522, respectively [13]. Results Demographic and clinical characteristics Genotyping and agarose gel electrophoresis for rs764522 The MvaI-digested PCR fragments were separated using the agarose gel electrophoresis method. The PCR amplicon was seen at 192 bp. For rs764522, the C allele was not digested and appeared as a single band at 192 bp. However, the G allele strand was digested into 41 bp and 151 bp fragments. Explor Med. 2024;5:148–57 \| https://doi.org/10.37349/emed.2024.00212 Page 150 Table 1. Demographic and laboratory data of the studied groups Table 1. Demographic and laboratory data of the studied groups Table 1. Demographic and laboratory data of the studied groups Parameter Controls (n = 100) CHD (n = 75) P-value Age (years) 4.59 ± 2.34 4.56 ± 2.30 0.934 (t-test) Sex 0.300 (χ 2) Female Male 55 (55%) 45 (45%) 35 (47%) 40 (53%) Hb (mg/dL) 11.91 ± 0.60 86.01 ± 5.00 0.001 WBCs × 10 3 6.28 ± 0.62 6.40 ± 0.62 0.212 Platelets × 10 3 246.70 ± 31.96 261.38 ± 45.98 0.014* FBS (mg/dL) 86.07 ± 5.66 86.01 ± 5.00 0.945 ALT (U/L) 23.03 ± 2.90 23.92 ± 3.32 0.061 AST (U/L) 25.27 ± 2.91 27.82 ± 6.72 0.001* Urea (mg/dL) 24.59 ± 3.47 24.09 ± 3.61 0.359 Creatinine (mg/dL) 0.53 ± 0.09 0.50 ± 0.11 0.050 FBS: fasting blood sugar; Hb: hemoglobin; : P-value significant < 0.05. Data were illustrated as mean ± standard deviation (SD) In Table 2, there are different models for testing the association of TGFBR2 rs764522 gene polymorphism with CHD. Herein, the G/G genotype frequency is much higher among cases compared to controls (21.3% vs. 0.0%). This might imply that the G/G genotype is a predisposing factor to the occurrence of CHD. A positive significance in the codominant model (C/C vs. C/G vs. G/G; OR = 0.43, 95% CI = 0.02–0.90, P < 0.0001) was noticed, as well as, in the dominant model (C/C vs. C/G + G/G) and recessive one (C/C + C/G vs. G/G; P < 0.0001). Table 2. Genotyping and agarose gel electrophoresis for rs764522 Testing genetic association of TGFBR2 (rs764522) gene polymorphism with CHD (n = 175, adjusted by sex) Genotypic states Genotype Cases Controls OR (95% CI) P-value C/C 14 (18.7%) 40 (40.0%) 1.00 C/G 45 (60%) 60 (60%) 0.43 (0.20–0.90) Codominant G/G 16 (21.3%) 0 (0.0%) 0.00 (0.00–NA) < 0.0001 C/C 14 (18.7%) 40 (40.0%) 1.00 Dominant C/G + G/G 61 (81.3%) 60 (60.0%) 0.31 (0.15–0.65) 0.001* C/C + C/G 59 (78.7%) 100 (100.0%) 1.00 Recessive G/G 16 (21.3%) 0 (0.0%) 0.00 (0.00–NA) < 0.0001* C/C + G/G 30 (40%) 40 (40%) 1.00 Overdominant C/G 45 (60%) 60 (60%) 0.94 (0.51–1.76) 0.86 NA: not applicable; : P-value significant < 0.05 Table 2. Testing genetic association of TGFBR2 (rs764522) gene polymorphism with CHD (n = 175, adjust The TGFBR2 (rs764522) gene polymorphism cross interaction with gender of cases and controls is displayed in Table 3. The genotype C/G frequency was noticed to be lower in male cases than in females and controls (OR = 0.24, 95% CI = 0.07–0.84). Table 3. TGFBR2 (rs764522) gene polymorphism cross interaction with gender of cases and controls (n = 175, crude analysis) Female Male TGFBR2 (rs764522) genotype Cases Controls OR (95% CI) Cases Controls OR (95% CI) C/C 4 18 1.00 10 22 0.49 (0.13–1.82) C/G 24 37 0.34 (0.10–1.14) 21 23 0.24 (0.07–0.84) G/G 7 0 0.00 9 0 0.00 P-value P < 0.0001 P = 0.0013* : P-value significant < 0.05 Table 3. TGFBR2 (rs764522) gene polymorphism cross interaction with gender of cases and controls (n = 175, 2 (rs764522) gene polymorphism cross interaction with gender of cases and controls (n = 175, crude analysis) Explor Med. 2024;5:148–57 \| https://doi.org/10.37349/emed.2024.00212 Page 151 Page 151 In Table 4, the TGFBR2 (rs764522) gene polymorphic genotypes are revealed among cases of different types of CHDs compared to controls. The C/C genotype frequency in the VSD cases was much lower compared to controls (14% vs. 40%). On the other hand, the G/G genotype frequency was higher in ASD cases compared to controls (20.9% vs. 0.0%) with a significant statistical difference (P < 0.0001). Table 4. Genotyping and agarose gel electrophoresis for rs6785358 Genotyping and agarose gel electrophoresis for rs6785358 Genotyping and agarose gel electrophoresis for rs6785358 For rs6785358, the BsuRI-digested PCR products were resolved on agarose electrophoresis. The results revealed that the A allele remained intact as a single 176 bp band. However, the G allele strand was digested into 147 bp and 29 bp fragments. The different models for testing the association of TGFBR2 (rs6785358) gene polymorphism with CHD are given in Table 5. The results revealed that the G/G genotype frequency is much higher among cases compared to controls (18.7% vs. 4.0%, respectively). This might imply that this genotype is a predisposing factor to the occurrence of CHDs. Moreover, a positive significance in the codominant model (A/A vs. A/G vs. G/G; OR = 0.20, 95% CI = 0.06–0.66, P = 0.0073) was noticed, as well as in the recessive model (A/A + A/G vs. G/G; OR = 0.19, 95% CI = 0.06–0.60, P = 0.0018). Table 5. Testing genetic association of TGFBR2 (rs6785358) gene polymorphism with CHD (n = 175, adjusted by sex) Genotypic states Genotype Cases Controls OR (95% CI) P-value A/A 31 (41.3%) 46 (46.0%) 1.00 A/G 30 (40%) 50 (50%) 1.13 (0.59–2.14) Codominant G/G 14 (18.7%) 4 (4.0%) 0.20 (0.06–0.66) 0.0073 A/A 31 (41.3%) 46 (46.0%) 1.00 Dominant A/G + G/G 44 (58.7%) 54 (54.0%) 0.83 (0.46–1.53) 0.56 A/A + A/G 61 (81.3%) 96 (96.0%) 1.00 Recessive G/G 14 (18.7%) 4 (4.0%) 0.19 (0.06–0.60) 0.0018* A/A + G/G 45 (60%) 50 (50%) 1.00 Overdominant A/G 30 (40%) 50 (50%) 1.49 (0.81–2.74) 0.19 : P-value significant < 0.05 Table 5. Testing genetic association of TGFBR2 (rs6785358) gene polymorphism with CHD (n = 175, adjus The TGFBR2 (rs6785358) gene polymorphism cross interaction with gender of subjects is shown in Table 6. The results indicated that the genotype G/G frequency was noticed to be higher in male than female cases and controls (OR = 0.10, 95% CI = 0.01–0.88 and OR = 0.22, 95% CI = 0.05–0.94, respectively). The TGFBR2 (rs6785358) gene polymorphic genotypes among cases of different types of CHD compared to controls revealed that the G/G allelic frequency in the VSD cases was much higher in cases compared to controls (25.6% vs. 4.0%) with a significant statistical difference (P = 0.002, Table 7). Genotyping and agarose gel electrophoresis for rs764522 TGFBR2 (rs764522) gene polymorphic genotypes among different types of CHD cases compared to controls TGFBR2 (rs764522) genotype Controls VSD ASD TOF Total 40 6 4 4 54 C/C 40.0% 14.0% 19.0% 36.4% 30.9% 0 9 5 2 16 G/G 0.0% 20.9% 23.8% 18.2% 9.1% 60 28 12 5 105 C/G 60.0% 65.1% 57.1% 45.5% 60.0% Fisher’s exact test – P-value 34.143 – P < 0.0001 : P-value significant < 0.05 522) gene polymorphic genotypes among different types of CHD cases compared to controls Genotyping and agarose gel electrophoresis for rs6785358 TGFBR2 (rs6785358) gene polymorphism cross interaction with gender of cases and controls (n = 175, crude analysis) Female Male TGFBR2 (rs6785358) genotype Cases Controls OR (95% CI) Cases Controls OR (95% CI) A/A 15 26 1.00 16 20 0.72 (0.29–1.80) A/G 14 28 1.15 (0.47–2.85) 16 22 0.79 (0.32–1.96) G/G 6 1 0.10 (0.01–0.88) 8 3 0.22 (0.05–0.94) P-value P = 0.0287 P = 0.1845 : P value significant < 0 05 2 (rs6785358) gene polymorphism cross interaction with gender of cases and controls (n = 175, crude analysis) Table 7. TGFBR2 (rs6785358) gene polymorphic genotypes among cases of different types of CHD compared to control TGFBR2 (rs6785358) genotype Controls VSD ASD TOF Total 46 18 9 4 77 A/A 46.0% 41.9% 42.9% 36.4% 44.0% 4 11 0 3 18 G/G 4.0% 25.6% 0.0% 27.3% 10.3% 50 14 12 4 80 A/G 50.0% 32.6% 57.1% 36.4% 45.7% Fisher’s exact test – P-value 19.690 – P = 0.002 : P-value significant < 0.05 BR2 (rs6785358) gene polymorphic genotypes among cases of different types of CHD compared to control Regarding another gene variant (rs6785358) within TGFBR2, the analysis revealed a positive equilibrium in both the studied patients and controls, with P-values greater than or equal to 0.05, indicating no significant deviation from the equilibrium. Regression modeling is a statistical method employed to examine the connections between variables. In the context of assessing the correlation between genetic markers and disease status, logistic regression is commonly used. The analysis involved evaluating the association of each SNP. In cases of binary responses, the outcomes of logistic regression analysis are succinctly presented in Table 8, encompassing significance levels, OR, and 95% CI. The findings indicate a noteworthy association between the GG genotype of rs6785358, as opposed to the CC genotype, and cardiovascular disease in children. Additionally, the CC genotype of rs764522 exhibited a significant association with the disease. Conversely, the variable of sex displayed a lack of significant association with the disease. Table 8. Genotyping and agarose gel electrophoresis for rs6785358 Binary logistic regression between each SNP and the incidence of CHD 95% CI Variable B Chi-square P-value OR Lower Upper Sex 0.423 1.332 0.248 1.526 0.745 3.127 rs6785358 (total) 8.528 0.014 rs6785358 (AA) –0.022 0.003 0.955 0.979 0.460 2.083 rs6785358 (GG) 1.836 7.802 0.005 6.272 1.729 22.747 rs764522 (total) 4.951 0.084 rs764522 (CC) –0.934 4.951 0.026 0.393 0.172 0.895 rs764522 (GG) 21.342 0.000 0.998 0.000 Constant –0.718 3.294 0.070 0.488 B: binary logistic regression coefficient. Blank cells represent “not detected”. rs6785358 (AG), rs764522 (CG) are used as reference for each related SNP Genotyping and agarose gel electrophoresis for rs6785358 The TGFBR2 (rs6785358) gene polymorphic genotypes among cases of different types of CHD compared to controls revealed that the G/G allelic frequency in the VSD cases was much higher in cases compared to controls (25.6% vs. 4.0%) with a significant statistical difference (P = 0.002, Table 7). The Hardy-Weinberg genetic equilibrium analysis for the TGFBR2 gene variant (rs764522) showed a negative equilibrium in both the studied subjects and control samples, with significant P-values of 0.0008 and 0.0001, respectively. This deviation is likely due to the very low frequency of the G/G genotype among the controls. However, in the cases group, a positive Hardy-Weinberg equilibrium (P = 0.11) was observed. The Hardy-Weinberg genetic equilibrium analysis for the TGFBR2 gene variant (rs764522) showed a negative equilibrium in both the studied subjects and control samples, with significant P-values of 0.0008 and 0.0001, respectively. This deviation is likely due to the very low frequency of the G/G genotype among the controls. However, in the cases group, a positive Hardy-Weinberg equilibrium (P = 0.11) was observed. Explor Med. 2024;5:148–57 \| https://doi.org/10.37349/emed.2024.00212 Page 152 Table 6. TGFBR2 (rs6785358) gene polymorphism cross interaction with gender of cases and controls (n = 175, crude analysis) Female Male TGFBR2 (rs6785358) genotype Cases Controls OR (95% CI) Cases Controls OR (95% CI) A/A 15 26 1.00 16 20 0.72 (0.29–1.80) A/G 14 28 1.15 (0.47–2.85) 16 22 0.79 (0.32–1.96) G/G 6 1 0.10 (0.01–0.88) 8 3 0.22 (0.05–0.94) P-value P = 0.0287 P = 0.1845 : P-value significant < 0.05 Table 7. TGFBR2 (rs6785358) gene polymorphic genotypes among cases of different types of CHD compared to control TGFBR2 (rs6785358) genotype Controls VSD ASD TOF Total 46 18 9 4 77 A/A 46.0% 41.9% 42.9% 36.4% 44.0% 4 11 0 3 18 G/G 4.0% 25.6% 0.0% 27.3% 10.3% 50 14 12 4 80 A/G 50.0% 32.6% 57.1% 36.4% 45.7% Fisher’s exact test – P-value 19.690 – P = 0.002 *: P-value significant < 0.05 Table 6. Discussion A set of structural and functional deficiencies called CHD occurs during the development of the heart. Furthermore, CHD is the most common cause of child death which is linked to birth defects, and it accounts for one-third of all serious congenital malformations [20]. Since 2000, the prevalence of CHD has increased by more than 50% worldwide. There are notable geographic variances, with Asia reporting the greatest CHD birth frequency while Europe was much higher than North America [21–23]. Explor Med. 2024;5:148–57 \| https://doi.org/10.37349/emed.2024.00212 Page 153 The TGFB signalling pathway plays an important role in the generation of the heart. The signalling of TGF-β regulates a variety of biological roles, including cell growth, differentiation, matrix production, and apoptosis in a wide range of cell types [10, 24]. The lethal aortic and cardiac defects were reported due to the inactivation of TGFBR2 in smooth muscle cells and epicardium [25]. Moreover, the TGFB is required during in vivo cardiac development [26]. Numerous human congenital illnesses, such as Marfan syndrome, Loeys-Dietz syndrome, neoplasms, aortic aneurysms and dissections, nonsegmental vitiligo, intracerebral hemorrhage, and sudden cardiac arrest in coronary artery disease patients are associated with genetic changes of the TGFBR2 gene [18, 19], which can be detected during the generation of the heart. Several studies propped up to determine the role of TGFBR2 in heart development based on mouse models of depleting TGFBR2 in special-cells. The endocardial depletion of TGFBR2 caused defects in the ventricular septal and double-inlet left ventricle [26]. The conditional deletion of TGFBR2 gene in smooth muscle cell- specific protein-expressing mice cells caused death during the last third of gestation, heart defects such as hypoplasia of the compact zone of the myocardium, ventricular, and atrial abnormalities were noticed in about half of mice [26]. TGFBR2 was essential for the development of the heart’s endothelial cells, and when its expression was inhibited, the ventricular septum was not properly formed [11]. The substantial reduction in transcriptional activities and loss of TGFBR2 gene expression may result in promoter mutation [27]. In the present study, it is revealed that the G/G genotype frequencies of TGFBR2 (rs764522) were much higher among cases compared to controls (21.3% vs. 0.0%, respectively). This might imply that this genotype could be a predisposing factor to the occurrence of CHD. Moreover, a positive significance within the codominant and dominant model (P ≤ 0.001) was evidenced. Discussion Our results were consistent with the previous study on TGFBR2 (rs6785358) which revealed a significant association between the carrier of the A/G + G/G genotype and the risk of congenital heart defects compared to A/A genotype in the Chinese population [13]. Similarly, in Han Chinese population, the study of Li et al. [28] proved the association of TGFBR2 (rs6785358) SNP, A/G + G/G variant, with the susceptibility to congenital ventricular septal heart defects. An increased risk of CHD in males was noticed among G allele-carrying individuals (A/G + G/G genotypes, rs6785358) but not in females [13]. Moreover, the allelic variants in rs6785358 were significantly different between the male and female subgroups in cases and controls [28]. In the current study, the cross interaction with gender revealed that the C/G frequency (rs764522) was lower in male than female cases and controls. However, the genotype G/G frequency (rs6785358) was noticed to be higher in male than female cases and controls. Previously, gender variation was confirmed to be significant in specific CHD subgroups [29]. Hormone modulation controls the activation of the TGF-signalling pathway [30], and this may help to explain why sex influences the relationship between the TGFBR2 gene and the risk of congenital cardiac abnormalities. Additionally, mutations in sex and autosomal chromosomes may have an impact on the risk for CHD [31]. We should draw attention to the possible limitations in the present study, including its small sample size and pilot-study design among just two tagSNPs in the TGFBR2 gene. In order to determine whether tagSNPs or functional SNPs covering the TGFBR2 gene possess any mutations that are sensitive to congenital cardiac abnormalities in Egyptian patients, we advise doing further, larger, and multicenter investigations with more inclusion of other CHD medical manifestations. Funding Not applicable. Copyright Copyright © The Author(s) 2024. Conclusions This study showed that SNP rs6785358 and rs764522 of TGFBR2 gene were associated with an elevated risk of CHD in the Egyptian population. In the future, the results offer an opportunity for the development of a novel early genetic detection of CHD risk. Abbreviations ASD: atrial septal defect AST: aspartate transaminase CHD: congenital heart defects Explor Med. 2024;5:148–57 \| https://doi.org/10.37349/emed.2024.00212 Explor Med. 2024;5:148–57 \| https://doi.org/10.37349/emed.2024.00212 Page 154 Page 154 CI: confidence interval OR: odds ratio PCR: polymerase chain reaction SMAD2: mothers against decapentaplegic homolog 2 SNPs: single nucleotide polymorphisms tagSNPs: tag single nucleotide polymorphisms TGFBR2: transforming growth factor beta receptor II TGF-β: transforming growth factor beta TOF: tetralogy of Fallot VSD: ventricular septal defect VSD: ventricular septal defect Availability of data and materials The datasets that support the findings of this study are available from the corresponding author upon reasonable request. Ethical approval The Ethical Committee at the Faculty of Medicine, Menoufia University approved the study (No. 11.2/2020 INTM2). The study is in accordance with the ethical standards of institutional research committee and with the 1964 Helsinki Declaration and its later amendments. Consent to participate An informed consent was obtained from the parents of all participating subjects of this study. Consent to publication Consent to publication Not applicable. Availability of data and materials Author contributions ND: Formal analysis, Investigation, Writing—original draft. ESS and AAHE: Formal analysis, Investigation, Writing—original draft, Writing—review & editing. FRA, IE, and RE: Conceptualization, Writing—original draft, Writing—review & editing. All authors read and approved the submitted version. Conflicts of interest Conflicts of interest All authors declare that they have no conflicts of interest. All authors declare that they have no conflicts of interest. Ethical approval References Rahim F, Younas M, Gandapur AJ, Talat A. Pattern of congenital heart diseases in children at tertiary care center in Peshawar. Pakistan J Med Sci. 2003;19:19–22. 1. Brown KL, Ridout DA, Hoskote A, Verhulst L, Ricci M, Bull C. Delayed diagnosis of congenital heart disease worsens preoperative condition and outcome of surgery in neonates. Heart. 2006;92: 1298–302. 2. Explor Med. 2024;5:148–57 \| https://doi.org/10.37349/emed.2024.00212 Page 155 Peterson C, Dawson A, Grosse SD, Riehle-Colarusso T, Olney RS, Tanner JP, et al. Hospitalizations, costs, and mortality among infants with critical congenital heart disease: How important is timely detection? Birth Defects Res A Clin Mol Teratol. 2013;97:664–72. 3. Rashid U, Qureshi AU, Hyder SN, Sadiq M. Pattern of congenital heart disease in a developing country tertiary care center: factors associated with delayed diagnosis. Ann Pediatr Cardiol. 2016;9:210–5. 4. Sadiq M, Roshan B, Khan A, Latif F, Bashir I, Sheikh SA. Pattern of pediatric heart diseases in Pakistan. J Coll Physicians Surg Pakistan. 2002;12:149–53. 5. Diab NS, Barish S, Dong W, Zhao S, Allington G, Yu X, et al. Molecular genetics and complex inheritance of congenital heart disease. Genes (Basel). 2021;12:1020. 6. Pierpont ME, Brueckner M, Chung WK, Garg V, Lacro RV, McGuire AL, et al.; American Heart Association Council on Cardiovascular Disease in the Young; Council on Cardiovascular and Stroke Nursing; Council on Genomic and Precision Medicine. Genetic basis for congenital heart disease: revisited: a scientific statement from the American Heart Association. Circulation. 2018;138: e653–711. 7. Hart PJ, Deep S, Taylor AB, Shu Z, Hinck CS, Hinck AP. Crystal structure of the human TβR2 ectodomain–TGF-β3 complex. Nat Struct Biol. 2002;9:203–8. 8. Takenoshita S, Hagiwara K, Nagashima M, Gemma A, Bennett WP, Harris CC. The genomic structure of the gene encoding the human transforming growth factor β type II receptor (TGF-β RII). Genomics. 1996;36:341–4. 9. Arthur HM, Bamforth SD. TGFβ signaling and congenital heart disease: insights from mouse studies. Birth Defects Res A Clin Mol Teratol. 2011;91:423–34. 10. Robson A, Allinson KR, Anderson RH, Henderson DJ, Arthur HM. The TGFβ type II receptor plays a critical role in the endothelial cells during cardiac development. Dev Dyn. 2010;239:2435–42. 11. Markwald RR, Fitzharris TP, Manasek FJ. Structural development of endocardial cushions. Am J Anat. 1977;148:85–119. 12. Huang F, Li L, Shen C, Wang H, Chen J, Chen W, et al. Association between TGFBR2 gene polymorphisms and congenital heart defects in Han Chinese population. Nutr Hosp. 2015;31:710–5. 13. References El-Nabi SH, Sayed S, Abd-Elhafez MA, Elfiky M, Abdel Moneim AE, El-Garawani I. Arg753Gln polymorphisms in the Toll-like receptor 2 gene are associated with cytomegalovirus infection in Egyptian bone marrow recipients. Endocr Metab Immune Disord Drug Targets. 2020;20:619–24. 14. El-Garawani I, Hassab El-Nabi S, Gadallah M, Abdelsameea E. Association between IFN-λ 3 gene polymorphisms and outcome of treatment with direct acting antivirals in chronic HCV-infected Egyptian patients. Immunol Invest. 2021;50:12–22. 15. El-Garawani IM, Shaheen EM, El-Seedi HR, Khalifa SMA, Mersal GAM, Emara MM, et al. Angiotensinogen gene missense polymorphisms (rs699 and rs4762): the association of end-stage renal failure risk with type 2 diabetes and hypertension in Egyptians. Genes (Basel). 2021;12:339. 16. Vesnina A, Prosekov A, Kozlova O, Atuchin V. Genes and eating preferences, their roles in personalized nutrition. Genes (Basel). 2020;11:357. 17. Mizuguchi T, Collod-Beroud G, Akiyama T, Abifadel M, Harada N, Morisaki T, et al. Heterozygous TGFBR2 mutations in Marfan syndrome. Nat Genet. 2004;36:855–60. 18. Tseng ZH, Vittinghoff E, Musone SL, Lin F, Whiteman D, Pawlikowska L, et al. Association of TGFBR2 polymorphism with risk of sudden cardiac arrest in patients with coronary artery disease. Heart Rhythm. 2009;6:1745–50. 19. Greutmann M, Tobler D. Changing epidemiology and mortality in adult congenital heart disease: looking into the future. Future Cardiol. 2012;8:171–7. 20. Dolk H, Loane M, Garne E; European Surveillance of Congenital Anomalies (EUROCAT) Working Group. Congenital heart defects in Europe: prevalence and perinatal mortality, 2000 to 2005. Circulation. 2011;123:841–9. 21. Explor Med. 2024;5:148–57 \| https://doi.org/10.37349/emed.2024.00212 Page 156 Hoffman JI. The global burden of congenital heart disease. Cardiovasc J Afr. 2013;24:141–5. 2. Lozano R, Naghavi M, Foreman K, Lim S, Shibuya K, Aboyans V, et al. Global and regional mortality from 235 causes of death for 20 age groups in 1990 and 2010: a systematic analysis for the Global Burden of Disease Study 2010. Lancet. 2012;380:2095–128. 23. Heldin CH, Miyazono K, ten Dijke P. TGF-β signalling from cell membrane to nucleus through SMAD proteins. Nature. 1997;390:465–71. 24. Langlois D, Hneino M, Bouazza L, Parlakian A, Sasaki T, Bricca G, et al. Conditional inactivation of TGF- β type II receptor in smooth muscle cells and epicardium causes lethal aortic and cardiac defects. Transgenic Res. 2010;19:1069–82. 25. Jiao K, Langworthy M, Batts L, Brown CB, Moses HL, Baldwin HS. Tgfβ signaling is required for atrioventricular cushion mesenchyme remodeling during in vivo cardiac development. Development. 2006;133:4585–93. 26. Hoffman JI, Kaplan S. Explor Med. 2024;5:148–57 \| https://doi.org/10.37349/emed.2024.00212 References The incidence of congenital heart disease. J Am Coll Cardiol. 2002;39:1890–900. 27. Li XT, Shen CQ, Zhang R, Shi JK, Li ZH, Liu HY, et al. Association of TGFBR2 rs6785358 polymorphism with increased risk of congenital ventricular septal defect in a Chinese population. Pediatr Cardiol. 2015;36:1476–82. 28. Pugnaloni F, Felici A, Corno AF, Marino B, Versacci P, Putotto C. Gender differences in congenital heart defects: a narrative review. Transl Pediatr. 2023;12:1753–64. 29. Buck MB, Knabbe C. TGF-beta signaling in breast cancer. Ann N Y Acad Sci. 2006;1089:1 30. Rossouw JE. Hormones, genetic factors, and gender differences in cardiovascular disease. Cardiovasc Res. 2002;53:550–7. 31. Explor Med. 2024;5:148–57 \| https://doi.org/10.37349/emed.2024.00212 Page 157
https://openalex.org/W3013417724	https://www.nature.com/articles/s41598-020-72574-7.pdf	English	null	Myoelectric digit action decoding with multi-label, multi-class classification: an offline analysis	bioRxiv (Cold Spring Harbor Laboratory)	2,020	cc-by	8,758	OPEN Agamemnon Krasoulis1* & Kianoush Nazarpour1,2 The ultimate goal of machine learning-based myoelectric control is simultaneous and independent control of multiple degrees of freedom (DOFs), including wrist and digit artificial joints. For prosthetic finger control, regression-based methods are typically used to reconstruct position/ velocity trajectories from surface electromyogram (EMG) signals. Unfortunately, such methods have thus far met with limited success. In this work, we propose action decoding, a paradigm-shifting approach for independent, multi-digit movement intent prediction based on multi-output, multi-class classification. At each moment in time, our algorithm decodes movement intent for each available DOF into one of three classes: open, close, or stall (i.e., no movement). Despite using a classifier as the decoder, arbitrary hand postures are possible with our approach. We analyse a public dataset previously recorded and published by us, comprising measurements from 10 able-bodied and two transradial amputee participants. We demonstrate the feasibility of using our proposed action decoding paradigm to predict movement action for all five digits as well as rotation of the thumb. We perform a systematic offline analysis by investigating the effect of various algorithmic parameters on decoding performance, such as feature selection and choice of classification algorithm and multi- output strategy. The outcomes of the offline analysis presented in this study will be used to inform the real-time implementation of our algorithm. In the future, we will further evaluate its efficacy with real-time control experiments involving upper-limb amputees. Upper-limb loss can negatively impact an affected individual’s ability to perform activities of daily living. To mitigate this effect, prosthetic devices have historically aimed at restoring the appearance and basic functionality of a missing limb using artificial components. The advancement of robotics research in the last decades has led to the advent of upper-limb prostheses with highly-sophisticated mechanical capabilities. Sensing technologies and control algorithms, however, have not kept pace; as a result, they currently impose a bottleneck on the control dexterity enjoyed by prosthesis users1–3. y j y y p Prosthetic hands are typically controlled using muscle activity signals, called electromyograms (EMGs), recorded on the skin surface. Traditionally, clinical solutions have deployed simple, amplitude-based control algorithms which rely on monitoring the activity of a pair of antagonist remnant muscles. The amplitude of each recorded muscle is usually linked to the activation of a specific function, for example, wrist rotation or hand opening/closing. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports 1School of Engineering, Newcastle University, Newcastle upon Tyne NE1 7RU, UK. 2School of Informatics, University of Edinburgh, Edinburgh EH8 9AB, UK. *email: Agamemnon.Krasoulis@newcastle.ac.uk OPEN i.e., multi-output) classification has been proposed as a means of decoding multiple hand and wrist functions ogether, hence resulting in greater flexibility and dexterity5–10.i l Intuitive selection and activation of grasp and wrist functions can be greatly beneficial for the user in per- forming activities of daily living. Yet, this paradigm results in severe prosthesis under-actuation and can thus offer much less functionality and dexterity than a natural hand. From a technical perspective, the ultimate goal of the myoelectric control field is to approximate this dexterity via simultaneous and independent control of multiple degrees of freedom (DOFs) in a continuous space. To that end, several groups, including us, have used regression-based methods to reconstruct wrist kinematic trajectories11–15, finger positions16–22 and velocities23, 24, as well as fingertip forces25–28. Only a few studies have, however, thus far demonstrated the feasibility of real-time prosthetic finger control in amputee users17, 21, 22. This indicates that independent prosthetic digit control is indeed a challenging problem, which calls for new and more efficient methods for tackling it. fi In this work, we propose a novel approach for simultaneous and independent control of prosthetic digits. Our algorithm is based on multi-output, multi-class classification. This is in contrast with previous work in this area, which has focused on the use of multi-output regression algorithms to achieve the same goal. At each time step, our algorithm uses surface EMG features to decode movement intent for each available DOF into one of three classes: open, close, or stall (i.e., no movement). Our motivation is to replace the multi-output regressor with a multi-output classifier with the aim of simplifying the decoding part. This is achieved by using discrete as opposed to real-valued (i.e., continuous) targets (i.e., outputs). We term our approach action decoding, since it is based on predicting digit actions rather than positions and/or velocities. A schematic of the approach is shown in Fig. 1. h l l d h d ll b h d l k h We have previously evaluated the proposed action controller in a robotic hand tele-operation task with a data glove and found that it can achieve comparable performance to digit position (i.e., joint angle) control29. Here, we provide a first implementation of the method in the context of myoelectric decoding. We demonstrate the feasibility of using surface EMG measurements to decode digit actions. OPEN We also perform a systematic offline analysis investigating several aspects of the method, including feature selection and choice of classifier and multi-output strategy. With regard to the latter, we evaluate the efficacy of a state-of-the-art method for multi- output classification, namely, classifier chains (CC), which takes output dependencies into account when making predictions. The outcomes of our analysis are used to inform the real-time implementation of the algorithm, which we present in a separate study30. OPEN To access a different function, the user has to switch between the available modes by using a trigger signal, such as muscle co-contraction2. Although this algorithm has proven robust, it results in limited control and can also be non-intuitive and cumbersome for the end-user. Unfortunately, this leads to an increased prosthesis rejection rate4. p j To improve control dexterity, machine learning algorithms can be used to infer movement intent from EMG recordings. Typically, a classifier is used to map features extracted from multiple EMG channels onto a discrete output variable encoding grasp type and/or other prosthesis functions. This paradigm has been highly-successful and, in the last decade, has found its way towards commercial adoption3. One caveat of this approach is that it can only support a single function activation at a time. That is, to achieve outcomes that require activating more than one prosthesis functions, for example, wrist rotation and hand closing. the user has to trigger a sequence of commands. This results in sub-optimal and unnatural control. To address this issue, simultaneous Scientific Reports \| (2020) 10:16872 \| https://doi.org/10.1038/s41598-020-72574-7 www.nature.com/scientificreports/ Figure 1. Action decoding paradigm. Multi-channel raw EMG measurements are pre-processed and fed as inputs into a multi-output classifier. The classifier has six outputs corresponding to the following DOFs: thumb rotation and flexion/extension of the thumb, index, middle, ring, and little digits. For each DOF, the algorithm classifies movement intent into one of three actions: open, close, or stall (i.e., no movement). Predictions can then be used to control the digits of a prosthesis using discrete actions. Any type of multi-output, multi-class classifier can be used as the decoder. Figure 1. Action decoding paradigm. Multi-channel raw EMG measurements are pre-processed and fed as inputs into a multi-output classifier. The classifier has six outputs corresponding to the following DOFs: thumb rotation and flexion/extension of the thumb, index, middle, ring, and little digits. For each DOF, the algorithm classifies movement intent into one of three actions: open, close, or stall (i.e., no movement). Predictions can then be used to control the digits of a prosthesis using discrete actions. Any type of multi-output, multi-class classifier can be used as the decoder. (i.e., multi-output) classification has been proposed as a means of decoding multiple hand and wrist functions together, hence resulting in greater flexibility and dexterity5–10. Results l We analysed data from 10 able-bodied and two transradial amputee subjects. We extracted features from 16 surface EMG channels and used them to decode digit movement (i.e., actions) for the following DOFs: thumb rotation and flexion/extension of the thumb, index, middle, ring, and little digits.i l g g As a first step, we performed an exhaustive feature analysis including a large number of commonly used time-domain EMG features. For each subject, features were ranked using a forward feature selection algorithm and we computed mean ranks for all features. The results of this analysis are presented in Fig. 2a. The highest- performing feature was Wilson amplitude (WAmp), followed by log-variance (LogVar) and Hjorth (Hjorth) parameters. Fig. 2b shows average classification performance by means of F1-score for an increasing number of added features. We observed a plateau in performance after including six to eight features. Based on this obser- vation, we selected the seven most highly-ranked features for the rest of our analysis: WAmp, LogVar, Hjorth, kurtosis (Kurt), auto-regressive (AR) coefficients, waveform length (WL), and skewness (Skew).i gfi g Next, we investigated the potential of using CC and ensembles of CC to improve classification performance by exploiting output dependencies. The results of this analysis are presented in Fig. 3a. For each participant, we report the performance of the best- and worst-performing CC, as well as average performance of ensemble CC consisting of 10 chains with random label orders. We compare the performance of CCs and ensemble CC to that of multiple independent classifiers. In all cases, we used linear discriminant analysis (LDA) models as the base classifiers for the CC. We use F1-score (see Methods section) throughout as our main performance measure, unless noted otherwise. Scientific Reports \| (2020) 10:16872 \| https://doi.org/10.1038/s41598-020-72574-7 www.nature.com/scientificreports/ Figure 2. Feature analysis. (a) Average ranking for individual features using the sequential forward selection method. The procedure was run independently for each participant and average rankings were computed ( n = 12 ). Lower ranks indicate larger feature importance. (b) Performance as a function of the number of features used for decoding. Higher F1-scores indicate better performance. Points, means; error bars, standard errors estimated via bootstrapping (1000 iterations). Figure 2. Feature analysis. (a) Average ranking for individual features using the sequential forward selection method. The procedure was run independently for each participant and average rankings were computed ( n = 12 ). Lower ranks indicate larger feature importance. Results l (b) Performance as a function of the number of features used for decoding. Higher F1-scores indicate better performance. Points, means; error bars, standard errors estimated via bootstrapping (1000 iterations). Figure 3. Performance comparisons. (a) Comparison of multi-output classification strategies using an LDA base classifier. CC best and CC worst correspond to best- and worst-performing single CC models, respectively. For each participant, 100 random chains were generated and evaluated. Ensemble CC corresponds to a model with 10 random chains. Straight lines, medians; solid boxes, interquartile ranges; whiskers, overall ranges of non-outlier data; dots, individual data points ( n = 12 ); asterisk, p < 0.05 ; n.s., p > 0.05 . (b) Comparison of classification algorithms using F1-score and the independent multi-output strategy. Classifiers are presented in order of decreasing median performance and statistical comparisons are performed only against the highest- performing algorithm. CC classifier chain, RDA regularised discriminant analysis, LDA linear discriminant analysis, QDA quadratic discriminant analysis, RF random forest, GNB Gaussian naive Bayes, KNN k-nearest neighbours, LR logistic regression, ET extra trees, BL baseline. Figure 3. Performance comparisons. (a) Comparison of multi-output classification strategies using an LDA b l fi CC b d CC d b d f l CC d l Figure 3. Performance comparisons. (a) Comparison of multi-output classification strategies using an LDA base classifier. CC best and CC worst correspond to best- and worst-performing single CC models, respectively. For each participant, 100 random chains were generated and evaluated. Ensemble CC corresponds to a model with 10 random chains. Straight lines, medians; solid boxes, interquartile ranges; whiskers, overall ranges of non-outlier data; dots, individual data points ( n = 12 ); asterisk, p < 0.05 ; n.s., p > 0.05 . (b) Comparison of classification algorithms using F1-score and the independent multi-output strategy. Classifiers are presented in order of decreasing median performance and statistical comparisons are performed only against the highest- performing algorithm. CC classifier chain, RDA regularised discriminant analysis, LDA linear discriminant analysis, QDA quadratic discriminant analysis, RF random forest, GNB Gaussian naive Bayes, KNN k-nearest neighbours, LR logistic regression, ET extra trees, BL baseline. Figure 3. Performance comparisons. (a) Comparison of multi-output classification strategies using an LDA base classifier. CC best and CC worst correspond to best- and worst-performing single CC models, respectively. For each participant, 100 random chains were generated and evaluated. Ensemble CC corresponds to a model with 10 random chains. Results l Straight lines, medians; solid boxes, interquartile ranges; whiskers, overall ranges of non-outlier data; dots, individual data points ( n = 12 ); asterisk, p < 0.05 ; n.s., p > 0.05 . (b) Comparison of classification algorithms using F1-score and the independent multi-output strategy. Classifiers are presented in order of decreasing median performance and statistical comparisons are performed only against the highest- performing algorithm. CC classifier chain, RDA regularised discriminant analysis, LDA linear discriminant analysis, QDA quadratic discriminant analysis, RF random forest, GNB Gaussian naive Bayes, KNN k-nearest neighbours, LR logistic regression, ET extra trees, BL baseline. We did not observe a difference in F1-score between the best-performing CC (CC-best) and independent output classifiers ( p = 0.51 ). Moreover, performance was not statistically different when we used ensembles of 10 CC ( p = 0.61 ). F1-scores for the worst-performing CC (CC-worst) were statistically lower than independent ( p = 0.02 ), CC-best ( p = 0.03 ), and ensemble CC ( p = 0.02 ). This finding indicates that a poor label ordering can in fact decrease performance, as compared to independent classifiers. A performance summary with all evaluation metrics is presented in Table 1 and detailed results are provided in Supplementary Figure S1. Scientific Reports \| (2020) 10:16872 \| https://doi.org/10.1038/s41598-020-72574-7 www.nature.com/scientificreports/ Table 1. Multi-output strategy benchmark. Median scores and overall ranges are reported for each strategy using the LDA classifier. Bold values indicate highest average performance for each evaluation metric. Multi-output strategy F1-score (macro- average) Exact match ratio Hamming score Recall (macro- average) Precision (macro- average) Independent 0.63 (0.54, 0.68) 0.52 (0.43, 0.59) 0.80 (0.71, 0.83) 0.61 (0.54, 0.68) 0.67 (0.53, 0.72) CC (best) 0.63 (0.53, 0.66) 0.53 (0.44, 0.60) 0.80 (0.70, 0.83) 0.61 (0.54, 0.67) 0.66 (0.51, 0.71) CC (worst) 0.61 (0.53, 0.66) 0.53 (0.44, 0.60) 0.80 (0.70, 0.83) 0.60 (0.54, 0.67) 0.65 (0.52, 0.71) Ensemble CC 0.63 (0.55, 0.67) 0.53 (0.44, 0.60) 0.80 (0.71, 0.83) 0.61 (0.55, 0.68) 0.66 (0.53, 0.72) Table 1. Multi-output strategy benchmark. Median scores and overall ranges are reported for each strategy using the LDA classifier. Bold values indicate highest average performance for each evaluation metric. Table 2. Classifier benchmark. Median scores and overall ranges are reported for each classifier using the independent multi-output strategy. Bold values indicate highest average performance for each evaluation metric. Results l A performance summary with all evaluation metrics is presented in Table 2.i Figure 4 shows average confusion matrices obtained with the independent multi-output RDA classifier for individual DOFs. The best performance was achieved for the ring digit, followed by the middle, index, and thumb digits. The lowest performance was achieved for the thumb rotation and little digit flexion/extension DOFs. gh p gl Additional results from the benchmark analysis are provided in the supplementary material: Figure S2 shows algorithmic comparisons for both independent classifiers and CC for all evaluation metrics; Figure S3 shows performance of the two highest-performing algorithms (i.e., LDA and RDA) for individual subjects; and Figure S4 summarises performance of all classifiers for individual DOFs using independent classifiers for each output. Results l Classifier F1-score (macro-average) Exact match ratio Hamming score Recall (macro-average) Precision (macro- average) RDA 0.64 (0.56, 0.69) 0.58 (0.52, 0.62) 0.82 (0.79, 0.84) 0.63 (0.54, 0.70) 0.67 (0.59, 0.70) LDA 0.64 (0.55, 0.70) 0.59 (0.53, 0.62) 0.83 (0.79, 0.85) 0.62 (0.54, 0.69) 0.70 (0.58, 0.73) QDA 0.63 (0.39, 0.67) 0.53 (0.03, 0.60) 0.78 (0.40, 0.81) 0.66 (0.58, 0.73) 0.61 (0.51, 0.65) RF 0.61 (0.50, 0.68) 0.62 (0.57, 0.64) 0.84 (0.81, 0.85) 0.57 (0.47, 0.66) 0.77 (0.65, 0.81) GNB 0.57 (0.49, 0.63) 0.54 (0.10, 0.57) 0.76 (0.61, 0.78) 0.59 (0.54, 0.69) 0.58 (0.47, 0.61) KNN 0.56 (0.46, 0.63) 0.58 (0.51, 0.62) 0.82 (0.77, 0.83) 0.52 (0.44, 0.61) 0.65 (0.50, 0.70) LR 0.56 (0.45, 0.66) 0.58 (0.55, 0.60) 0.82 (0.80, 0.84) 0.53 (0.43, 0.65) 0.69 (0.62, 0.72) ET 0.51 (0.43, 0.62) 0.59 (0.56, 0.62) 0.83 (0.80, 0.84) 0.47 (0.41, 0.60) 0.76 (0.68, 0.83) BL 0.33 (0.33, 0.33) 0.10 (0.08, 0.15) 0.61 (0.58, 0.66) 0.33 (0.33, 0.33) 0.33 (0.33, 0.33) Table 2. Classifier benchmark. Median scores and overall ranges are reported for each classifier using the independent multi-output strategy. Bold values indicate highest average performance for each evaluation metric. Figure 4. Confusion matrices. For each DOF, the average confusion matrices obtained with the independent multi-output RDA classifier are shown. Colour bar and annotated scores indicate normalised prediction rates. Figure 4. Confusion matrices. For each DOF, the average confusion matrices obtained with the independen multi-output RDA classifier are shown. Colour bar and annotated scores indicate normalised prediction rate Figure 3b summarises the results of the classifier benchmark analysis using independent output classifiers. The highest median performance was achieved by regularised discriminant analysis (RDA) ( F1med = 0.64 ), closely followed by LDA. We performed statistical comparisons between the highest-performing algorithm (i.e., RDA) and all other classifiers and found that RDA significantly outperformed all classifiers except LDA. All classifiers performed higher than chance. A performance summary with all evaluation metrics is presented in Table 2.i Figure 3b summarises the results of the classifier benchmark analysis using independent output classifiers. The highest median performance was achieved by regularised discriminant analysis (RDA) ( F1med = 0.64 ), closely followed by LDA. We performed statistical comparisons between the highest-performing algorithm (i.e., RDA) and all other classifiers and found that RDA significantly outperformed all classifiers except LDA. All classifiers performed higher than chance. www.nature.com/scientificreports/ www.nature.com/scientificreports/ decode digit actions; and (2) to carry-out a systematic offline investigation prior to implementing the algorithm in real-time and testing it with upper-limb amputees.h We have shown that it is feasible, in principle, to decode digit actions from surface EMG signals. The median F1-score of the best-performing configuration (i.e., independent multi-output RDA classifiers) was F1med = 0.64 . The median Hamming score, exact match ratio, and macro-average precision and recall scores were all signifi- cantly and substantially higher than chance (supplementary material). We observed the highest performance for the ring and middle digits (Fig. 4). This is in agreement with previous work on regression-based reconstruction of digit position/velocity trajectories18, 21, 23. This finding is expected from a physiological perspective, given that these two digits are controlled by extrinsic superficial muscles, as opposed to the thumb, for example, which is controlled by intrinsic and deep extrinsic muscles, both of which are not easily accessible from the surface of the forearm.fl Our offline investigation scrutinised several important aspects of the method, including feature analysis, choice of decoding algorithm, and evaluation of two multi-output strategies. It has been previously demonstrated that both feature selection and choice of classifier can substantially influence the performance of myoelectric classification systems31, 32. In line with previous reports, which were mainly concerned with upper-limb motion/ grip classification9, 31–33, we found that discriminant analysis-based classifiers, such as LDA and RDA, can achieve the highest level of performance. The results of our feature selection/ranking analysis (Fig. 2a) are also largely in agreement with previous reports from the motion classification literature32, 34.it gi In multi-output classification settings, it is often desirable to exploit output dependencies to improve decoding performance. In this regard, we investigated the potential of using the state-of-the-art method of CC to improve classification. The exact match ratio was marginally improved with both CC and ensemble CC (see supplemen- tary material). Nevertheless, we did not observe an increase in F1-score using either method. The improvement in exact match ratio is expected, since it has been theoretically shown that CC maximise this metric exactly by equivalently minimising 0/1 loss35. On the other hand, when labels are evaluated independently, as with macro- average F1-score, there is no guarantee that CC will outperform independent classifiers, although this may often happen in practice36. www.nature.com/scientificreports/ We attribute the ineffectiveness of CC in our case to the fact that the dataset comprised both single-digit exercises as well as full-hand grips (five exercises of each type). Including a large number of single-digit motions results in outputs becoming largely independent and, thus, there is less structure in the output domain that CC can exploit to leverage performance.i p p g p In myoelectric control, multi-output classification has been previously used only to decode simultaneous wrist and hand motions5–10. For control of prosthetic digits, however, previous efforts have focused on using multi-output regression to reconstruct position16–19, 21, velocity23, 24 or fingertip force trajectories26–28. It is worth noting that despite using a discrete decoder in our approach, intermediate positions are possible. We have previ- ously shown in a hand tele-operation task with a data glove29 that by using a small action step (i.e., digit position increase/decrease step), control becomes approximately continuous. In other words, despite using a classifier as the decoder, our approach allows for arbitrary hand configurations. From a control theory perspective, our action-based paradigm can be viewed as an extreme, discretised case of velocity decoding; velocity can either be zero, or take a constant value, which is only parametrised by its sign/direction.fi y p y g One study has previously adopted a similar approach to ours37, but with four main differences: firstly, the labels corresponded to isometric muscle contractions while the hand was kept fixed, as opposed to digit actions during unconstrained finger movement in our case; secondly, labels were binary (i.e., stimuli corresponded to fully open or closed digits), whereas with our approach actions can take three values (i.e., open, close, or stall); thirdly, our approach requires less computational resources for both training and inference, due to using a set of linear classifiers as opposed to a convolutional neural network; and finally, our recording setup was simpler. That is, we used sparse EMG electrodes as opposed to a two-dimensional electrode grid, and we did not constrain the participants’ hand or forearm. In comparison with regression-based methods, our approach only requires discrete ground truth labels for training the decoders. This offers a theoretical advantage over regression, since it can potentially remove the need for using data gloves or motion tracking systems, which are typically required for acquiring real-valued (i.e., continuous) ground truth labels16–19, 21, 23, 24. Discussion h k In this work, we introduced a novel paradigm for upper-limb prosthetic digit control using surface EMG signals. In the proposed approach, EMG features are used to decode discrete digit actions via multi-output classification. At each time step, the algorithm classifies movement intent for each DOF into one of three categories: open, close, or stall (i.e., no movement). We have previously evaluated this type of controller in a robotic hand tele-operation task with a data glove and have found that it can achieve comparable performance to digit position control29. The aim of this study was twofold: (1) to demonstrate the feasibility of using surface EMG signals from the forearm to Scientific Reports \| (2020) 10:16872 \| https://doi.org/10.1038/s41598-020-72574-7 Methods et ods Dataset. The dataset used in the study was previously collected and made publicly available by us21. For completeness, we briefly describe here the experimental protocol and refer the reader to the original study for more details. Ten able-bodied and two right-arm transradial (i.e., below-elbow) amputee participants were included in the study. All able-bodied participants were right-hand dominant. All experiments were performed in accordance with the Declaration of Helsinki and were approved by the local Ethics Committees of the School of Informatics, University of Edinburgh (#201507160854) and School of Engineering, Newcastle University (#14-NAZ-056). Prior to the experiments, subjects read a participant information sheet and gave written informed consent. We placed 16 Delsys Trigno surface EMG sensors (Delsys, Inc.) on the participants’ skin below the right elbow arranged in two rows of eight equidistant electrodes and without targeting specific muscles. Prior to electrode placement, we cleansed participants’ skin using 70% isopropyl alcohol. We used adhesive elastic bandage to secure the locations of the electrodes throughout the sessions. The sampling rate of EMG data acquisition was fixed at 1111 Hz. i In addition, we recorded hand kinematic data using an 18-DOF Cyberglove II data glove (CyberGlove Sys- tems, LLC), which we placed on the participants’ left hand. Data glove measurements were calibrated for each participant using a quick calibration routine provided by the manufacturer. The sampling rate of glove data acquisition was fixed at 25 Hz.fih i Participants sat comfortably on an office chair and rested both arms on a table. They were asked to per- form a series of bilateral mirrored hand exercises whilst these were instructed on a computer display placed approximately 1 m in front of them. The selection of movements was done such that both single-finger as well as full-hand exercises were included. The following nine unique movements were selected: thumb abduction/ adduction; thumb, index, middle, and combined ring and little fingers flexion/extension; cylindrical, lateral, and tripod grips; and index pointer. Three blocks of exercises were recorded for each participant: datasets A and B comprised 10 repetitions of each exercise and dataset C only two. Consecutive trials were interleaved with 3 s of rest. The experimental protocol and apparatus used are shown in Fig. 5. Pre‑processing. Myoelectric and glove data were upsampled to 2 kHz and synchronised using linear inter- polation. We processed myoelectric data using an overlapping window approach. www.nature.com/scientificreports/ In this study, we analysed a previously collected dataset comprising data glove measurements21, thus we obtained the discrete labels by thresholding the respective velocity profiles. Alternatively, discrete ground truth labels can be acquired by prompting the user to perform imaginary finger movements with specified direction30, in a similar fashion that machine learning-based commercial systems are typically calibrated. This feature renders our approach more suitable than regression in a clinical setting, and also makes it suitable for people with bilateral limb deficiency or amputation.fl p pi y p Our study has three limitations. Firstly, it was limited to offline analyses. It is well-accepted in the myoe- lectric control community that offline performance measures are not always a good proxy of real-time control performance10, 14, 21, 38, 39. Therefore, it is imperative to evaluate control algorithms with real-time implementations and user-in-the-loop experiments. Given that we have introduced a completely novel paradigm for prosthetic digit control, the main purpose of this work was to systematically explore different parameters of the method and lay the groundwork for the subsequent real-time implementation. We report our implementation and evaluation of the proposed algorithm with amputee participants in a separate study30.hi The second limitation of the study was that we did not consider neural networks in our classifier benchmarks. Neural networks can naturally handle multi-output, multi-class classification problems via appropriate design of the output layers. For our application, it is likely that parameter sharing in the early layers of a network may benefit overall performance by optimising a combination of ouput loss functions, one for each DOF (i.e., multi- task learning). This is currently seen as a future research direction.h h The third limitation of the study is that we did not investigate aspects of decoding robustness under n stationary conditions and/or generalisation to novel finger configurations. It is well-known that mach Scientific Reports \| (2020) 10:16872 \| https://doi.org/10.1038/s41598-020-72574-7 www.nature.com/scientificreports/ Figure 5. Data collection. (a) Sixteen wireless EMG sensors were placed on the surface of the skin and were arranged in two rows of eight equidistant sensors below the elbow. (b–c) Hand kinematic data were collected using an instrumented data glove placed on the contralateral side. Participants were instructed to perform bilateral mirrored movements. Parts of the figure were previously published by us under a Creative Commons Attribute License (CC BY 4.0)21. Figure 5. Data collection. www.nature.com/scientificreports/ (a) Sixteen wireless EMG sensors were placed on the surface of the skin and were arranged in two rows of eight equidistant sensors below the elbow. (b–c) Hand kinematic data were collected using an instrumented data glove placed on the contralateral side. Participants were instructed to perform bilateral mirrored movements. Parts of the figure were previously published by us under a Creative Commons Attribute License (CC BY 4.0)21. learning-based myoelectric control algorithms typically suffer from poor generalisation under different limb positions and/or muscle contraction levels40, 41. Moreover, generalisation to novel postures is a much desired feature, as it allows to extrapolate to movements not present in the training set. In principle, this feature is sup- ported by our framework, given that the motion of each digit is controlled independently. It will be invaluable in the future to systematically investigate all the above aspects of decoding generalisation, ideally outside a lab- controlled environment. In conclusion, we have proposed a new paradigm for prosthetic digit control based on multi-output, multi- class classification. We have demonstrated the feasibility of decoding actions for all five digits and rotation of the thumb using surface EMG measurements recorded on the forearm in both able-bodied and transradial amputee participants. Our algorithm warrants further investigation with real-time, user-in-the-loop experiments with upper-limb amputees. Methods We set the window length to 128 ms and the increment to 50 ms (i.e., approximately 60% overlap). We filtered the EMG signals using a 4th-order band-pass Butterworth digital filter with lower and upper cutoff frequencies of 10 and 500 Hz, respec- tively. y We used a linear mapping21 to transform the calibrated glove measurements into joint angles for the follow- ing six DOFs: thumb rotation, flexion/extension of the thumb, index, middle, ring, and little digits. Joint angles were then normalised in the range 0–1, with 0 corresponding to a DOF being fully open (or thumb rotator fully reposed) and 1 to fully closed (thumb rotator fully opposed). Finally, we smoothed the calibrated glove measure- ments with a 1st-order low-pass Butterworth filter with cutoff frequency of 1 Hz. Scientific Reports \| (2020) 10:16872 \| https://doi.org/10.1038/s41598-020-72574-7 www.nature.com/scientificreports/ Figure 6. Finger action estimation from glove data. The mapping from position to action space is demonstrated using data from one participant. The position trajectory for each DOF (grey traces, left y-axes) is normalised between 0 (i.e., fully open) and 1 (i.e., fully closed). The first-order discrete position difference is computed and transformed into action via thresholding (black traces, right y-axes). The shown excerpt corresponds to two repetitions of the cylindrical grasp exercise. Figure 6. Finger action estimation from glove data. The mapping from position to action space is demonstrated using data from one participant. The position trajectory for each DOF (grey traces, left y-axes) is normalised between 0 (i.e., fully open) and 1 (i.e., fully closed). The first-order discrete position difference is computed and transformed into action via thresholding (black traces, right y-axes). The shown excerpt corresponds to two repetitions of the cylindrical grasp exercise. Digit action estimation from joint angle trajectories. To extract digit action labels from calibrated glove data we used the following procedure. Firstly, we estimated joint velocities by computing the first-order difference of normalised joint angle trajectories. We then thresholded the computed differences using a tolerance ǫ = 0.004 , such that joint velocities larger than ǫ were assigned the “close” label and velocities smaller than −ǫ were assigned the “open” label. Velocity values in the range [−ǫ, ǫ] were assigned the “stall” label, corresponding to no movement. Finally, for joint angles less than 7.5% away from either boundary (0 or 1), we assumed that the respective actions were “open” and “close”, respectively, regardless of the joint velocity. Methods The digit action estima- tion procedure was performed independently for each DOF. An illustration is provided in Fig. 6 using data from one participant as an example.h p p p The digit movement (i.e., target) variables were highly-imbalanced: approximately 75% of the samples cor- responded to the “stall” class, whereas the remaining 25% was equally split between the “open” and “close” classes. Performance evaluation and metrics. We considered a range of performance metrics to characterise classification performance. In multi-label classification, which is a special case of multi-output classification whereby the outputs are binary, the following metrics are commonly used42–44: (1) exact match ratio or accuracy is the percentage of samples that have all their labels correctly classified; (2) hamming score is the fraction of correctly classified labels to the total number of labels; (3) precision, recall and F1-score (i.e., harmonic mean of precision and recall) can be used in a similar way to multi-class classification, that is, either on a per-label basis or by using an appropriate method to average across labels. Macro- and micro-averaging are common choices: macro-averaging computes the metric of interest for each label independently and averages across labels; whereas micro-averaging aggregates contributions from each label to compute the average metric.hi g g gg g p g The exact match ratio is a strict measure, since it requires all labels to be correctly classified for a sample to be considered correct. Given that the number of labels in our case was relatively large (i.e., six), we did not consider this measure as our main evaluation metric. On the other hand, training/testing datasets for individual participants were highly-imbalanced, due to domination of the “stall” class over the “open” and “close” classes. Therefore, hamming loss was not an appropriate evaluation metric either. Taking the above into consideration, we selected F1-score as our main performance measure and used macro-averaging to account both for multiple labels as well as multiple classes within each label. We additionally considered the following metrics: exact match ratio (i.e., accuracy), hamming score, recall (macro-average) and precision (macro-average). EMG feature extraction and selection. We experimented with a large group of time-domain EMG fea- tures. www.nature.com/scientificreports/ yielded the highest performance was added to the pool and the procedure was repeated until all features were included. In that way, each EMG feature was assigned a rank, which was equal to the order that it was added to the pool. The forward selection procedure was performed independently for each participant and respective feature ranks were averaged. For each participant, models were fitted using independent multi-output LDA classifiers on dataset A and were evaluated on dataset B. yielded the highest performance was added to the pool and the procedure was repeated until all features were included. In that way, each EMG feature was assigned a rank, which was equal to the order that it was added to the pool. The forward selection procedure was performed independently for each participant and respective feature ranks were averaged. For each participant, models were fitted using independent multi-output LDA classifiers on dataset A and were evaluated on dataset B. Based upon the results of the feature selection analysis (Fig. 2), we used the following features in the rest of the study: WAmp, LogVar, Hjorth, Kurt, AR, WL, and Skew. Classifier chain analysis. Classifier chains (CC) is a popular machine learning tool for multi-label clas- sification (i.e., a special case of multi-output classification whereby outputs are binary) that takes into account label dependencies. We briefly describe the method here and refer the interested reader to the original paper for more details36.i Given a set of labels L , a CC model learns \|L\| classifiers, which are linked in a chain. Firstly, the label chain (i.e., order of labels) needs to be defined: {L1, L2, . . . , L\|L\|} . The first classifier in the chain L1 is then fitted using input features only. The ground truth data from L1 are then included as an additional input feature for training the second classifier in the chain L2 . This process is repeated for all remaining labels in the chain by including for each label Ln ground truth data from previous labels in the chain {L1, L2, . . . Ln−1} . For inference, the same procedure is followed, except predictions from previous labels in the chain are used at each step. A popular vari- ant of the method is the ensemble of CC, whereby several CC models are trained with random orders of labels and their predictions are aggregated using a voting scheme. www.nature.com/scientificreports/ In our application, the number of labels (i.e., outputs) was \|L\| = 6 , that is, the number of DOFs: thumb opposition/reposition and flexion/extension of thumb, index, middle, ring, and little digits. For each label, the set of classes was C = {open, close, stall} and thus the number of classes was \|C \| = 3 . In our CC analysis, we tested all possible orders of labels, that is, a total of 6! = 720 chains. For each participant, we report performance for the best- and worst-performing chains in terms of F1-score. In addition, we report best performance from a set of 100 ensemble CC models, each trained with a random set of 10 label orders. We implemented the ensemble CC using a soft voting scheme, which predicts class labels based on the predicted probabilities from each chain in the ensemble. We compared the performance of CC and ensemble CC to that of independent multi-output classification, whereby an independent classifier is trained for each output. This method is often referred to as binary relevance42, 43 when dealing with binary multi-output classification problems (i.e., multi-label). Note, however, that this term is not appropriate for our problem, which is multi-output and multi-class. Therefore, we refer to this strategy as independent multi-output classification. Classifier training and hyper‑parameter optimisation. We considered a wide range of classifiers in our classification benchmark analysis. With few exceptions (e.g., LDA, quadratic discriminant analysis (QDA) and Gaussian naive Bayes (GNB)), most algorithms have hyper-parameters which were systematically tuned with hold-out validation. For each participant, we fitted models on dataset A and tuned hyper-parameters using randomised search with 50 iterations on dataset B. We finally report performance on dataset C. There were approximately 25 × 103 samples in datasets A and B (training and validation, respectively) and 5 × 103 samples in dataset C (testing). We performed 10 independent runs for each participant/classifier experiment and report average performance results across runs. Baseline performance was assessed using a dummy classifier always predicting the “stall” class for each label, which was the dominating class in the training dataset. www.nature.com/scientificreports/ The list of algo- rithms used in the benchmark along with hyper-parameters and respective search ranges for each classifier are provided in Supplementary Table S1.i p pp y Using 16 EMG electrodes and the optimal feature set identified as part of the feature selection analysis (i.e., WAmp, LogVar, Hjorth, Kurt, 4th-order AR coefficients, WL, and Skew), the input dimensionality was D = 192 . For the k-nearest neighbours (KNN) and logistic regression (LR) classifiers we reduced the input dimensionality to D = 50 using principal component analysis to speed-up training. We performed model training and testing in Python 3.7 (https://www.python.org/) using the scikit-learn library v. 0.22 (https://scikit-learn.org/ stable/)51 and custom-written code. Statistical analysis. We used two-sided Wilcoxon signed-rank tests to compare performance between pairs of classifiers. All comparisons were performed at the population level using participant-average scores, thus the number of samples was n = 12 . To account for multiple comparisons, we used the Holm-Bonferroni correction method. Statistical analysis was performed in Python 3.7 using the Pingouin library v. 0.3.1 (https://pingouin-stats.org/)52. Methods In the feature analysis investigation, we included the following time-domain features: mean absolute value (MAV)45, waveform length (WL)32, rate of zero-crossings (ZC)46, slope sign changes (SSC)32, Wilson amplitude (WAmp)46, root mean square (RMS)32, integrated EMG (IEMG)46, variance (VAR)46, log-variance (LogVar)12, kurtosis (Kurt)47, skewness (Skew)48, 4th-order auto-regressive (AR) coefficients46, histogram (Hist) counts46 ( nbins = 5 ), and Hjorth (Hjorth) parameters (i.e., activity, mobility, and complexity)49.i j j p y y p y For the feature analysis investigation, we used a modified version of the sequential forward feature selection algorithm50. The algorithm was initialised with an empty feature set. Within each iteration, all available features were tentatively added to the pool, one at a time, and the respective F1-scores were estimated. The feature that Scientific Reports \| (2020) 10:16872 \| https://doi.org/10.1038/s41598-020-72574-7 www.nature.com/scientificreports/ Data availabilityh The dataset used in the study is available as part of the NINAPRO database (http://ninapro.hevs.ch/DB8) and the Newcastle University data repository (https://doi.org/10.25405/data.ncl.9577598.v1). Meta-data are available from the authors upon reasonable request. Received: 4 April 2020; Accepted: 2 September 2020 References References 1. Farina, D. et al. The extraction of neural information from the surface EMG for the control of upper-limb prostheses: emerging avenues and challenges. IEEE Trans. Neural Syst. Rehabilit. Eng. 22, 797–809 (2014). Scientific Reports \| (2020) 10:16872 \| https://doi.org/10.1038/s41598-020-72574-7 www.nature.com/scientificreports/ 3. Roche, A. D. et al. Clinical perspectives in upper limb prostheses: an update. Curr. Surg. Rep. 7, 5 (2019). 4. Biddiss, E. A. & Chau, T. T. Upper limb prosthesis use and abandonment: a survey of the last 25 years. Prosthet. Orthot. Int. 31 236–257 (2007).i 5. Young, A. J., Smith, L. H., Rouse, E. J. & Hargrove, L. J. Classification of simultaneous movements using surface EMG pattern recognition. IEEE Trans. Biomed. Eng. 60, 1250–1258 (2013). g g 6. Young, A. J., Smith, L. H., Rouse, E. J. & Hargrove, L. J. A comparison of the real-time controllability of pattern recognition to conventional myoelectric control for discrete and simultaneous movements. J. NeuroEng. Rehabil. 11, 5 (2014). 7. Fougner, A. L., Stavdahl, Ø. & Kyberd, P. J. System training and assessment in simultaneous proportional myoelectric prosthesis control. J. NeuroEng. Rehabil. 11, 75 (2014). g 8. Wurth, S. M. & Hargrove, L. J. A real-time comparison between direct control, sequential pattern recognition control and simul- taneous pattern recognition control using a Fitts’ law style assessment procedure. J. NeuroEng. Rehabil. 11, 91 (2014).i 9. Ortiz-Catalan, M., Håkansson, B. & Brånemark, R. Real-time and simultaneous control of arti tion algorithms. IEEE Trans. Neural Syst. Rehabil. Eng. 22, 756–764 (2014).fl 9. Ortiz-Catalan, M., Håkansson, B. & Brånemark, R. Real-time and simultaneous control of artificial limbs based on pattern recogni- tion algorithms. IEEE Trans. Neural Syst. Rehabil. Eng. 22, 756–764 (2014).fl 10. Ortiz-Catalan, M., Rouhani, F., Brånemark, R. & Håkansson, B. Offline accuracy: a potentially misleading metric in myoelectric pattern recognition for prosthetic control. In 37th Annual International Conference of the IEEE Engineering in Medicine and Biology Society (EMBC), 1140–1143 (2015). y 11. Muceli, S., Jiang, N. & Farina, D. Extracting signals robust to electrode number and shift for online simultaneous and proportional myoelectric control by factorization algorithms. IEEE Trans. Neural Syst. Rehabil. Eng. 22, 623–633 (2014). y y g y g 12. Hahne, J. M. et al. Linear and nonlinear regression techniques for simultaneous and proportional myoelectric control. IEEE Trans. Neural Syst. Rehabil. Eng. 22, 269–279 (2014). y g 3. Smith, L. H., Kuiken, T. A. & Hargrove, L. J. References 24 Xiloyannis M Gavriel C Thomik A A C & Faisal A A Gaussian process autoregression for simultaneous proportional multi- 4. Xiloyannis, M., Gavriel, C., Thomik, A. A. C. & Faisal, A. A. Gaussian process autoregression for simultaneous proportional multi modal prosthetic control with natural hand kinematics. IEEE Trans. Neural Syst. Rehabil. Eng. 25, 1785–1801 (2017). 5 C lli i C & d S P S f EMG i d d h d h i Bi l C b 100 35 47 (2009) p y g 25. Castellini, C. & van der Smagt, P. Surface EMG in advanced hand prosthetics. Biol. Cybern. 100, 35–47 (2009). 25. Castellini, C. & van der Smagt, P. Surface EMG in advanced hand prosthetics. Biol. Cybern. 100, 35–47 (2009). 26 Gij b A l S bl l i l f h d h i i li i l l i F N b 8 g y 6. Gijsberts, A. et al. Stable myoelectric control of a hand prosthesis using non-linear incremental learning. Front. Neurorobot. 8, 8 (2014). 27. Gailey, A., Artemiadis, P. & Santello, M. Proof of concept of an online EMG-based decoding of hand postures and individual digit forces for prosthetic hand control. Front. Neurol. 8, 7 (2017).i 8. Barsotti, M. et al. Online finger control using high-density EMG and minimal training data for robotic applications. IEEE Robot Autom. Lett. 4, 217–223 (2018).i 9. Krasoulis, A., Vijayakumar, S. & Nazarpour, K. Continuous versus discrete simultaneous control of prosthetic fingers. In 40th Annual International Conference of the IEEE Engineering in Medicine and Biology Society (EMBC), 3774–3777 (2018). 30. Krasoulis, A. & Nazarpour, K. Discrete action control for prosthetic digits. bioRxiv. https://doi.org/10.1101/2020.03.25.007203 (2020). 1. Scheme, E. J. & Englehart, K. B. Electromyogram pattern recognition for control of powered upper-limb prostheses: state of the art and challenges for clinical use. J. Rehabil. Res. Dev. 48, 643 (2011).i 31. Scheme, E. J. & Englehart, K. B. Electromyogram pattern recognition for control of pow art and challenges for clinical use. J. Rehabil. Res. Dev. 48, 643 (2011). g 32. Phinyomark, A., Phukpattaranont, P. & Limsakul, C. Feature reduction and selection for EMG signal classification. Expert Syst. Appl. 39, 7420–7431 (2012). pp 3. Krasoulis, A., Nazarpour, K. & Vijayakumar, S. Use of regularized discriminant analysis improves myoelectric hand movement classification. In 2017 8th International IEEE/EMBS Conference on Neural Engineering (NER), 395–398 (2017). i 4. Boostani, R. & Moradi, M. H. References Real-time simultaneous and proportional myoelectric control using intramuscular EMG. J. Neural Eng. 11, 066013 (2014). g ( ) 4. Jiang, N., Vujaklija, I., Rehbaum, H., Graimann, B. & Farina, D. Is accurate mapping of EMG signals on kinematics needed for precise online myoelectric control?. IEEE Trans. Neural Syst. Rehabil. Eng. 22, 549–558 (2014). p y y g 5. Hahne, J. M., Schweisfurth, M. A., Koppe, M. & Farina, D. Simultaneous control of multiple functions of bionic hand prostheses performance and robustness in end users. Sci. Robot. 3, eaat3630 (2018).hi 16. Smith, R. J., Tenore, F., Huberdeau, D., Etienne-Cummings, R. & Thakor, N. V. Continuous decoding of finger position from surface EMG signals for the control of powered prostheses. In 30th Annual International Conference of the IEEE Engineering in Medicine and Biology Society (EMBC), 197–200 (2008). gy y 7. Cipriani, C. et al. Online myoelectric control of a dexterous hand prosthesis by transradial amputees. IEEE Trans. Neural Syst Rehabil. Eng. 19, 260–270 (2011).i g 18. Ngeo, J. G., Tamei, T. & Shibata, T. Continuous and simultaneous estimation of finger kinematics using inputs from an EMG-to- muscle activation model. J. NeuroEng. Rehabil. 11, 122 (2014).i 19. Krasoulis, A., Vijayakumar, S. & Nazarpour, K. Evaluation of regression methods for the continuous decoding of finger movement from surface EMG and accelerometry. In 7th International IEEE/EMBS Conference on Neural Engineering (NER), 631–634 (2015). y f g g 0. Krasoulis, A., Nazarpour, K. & Vijayakumar, S. Towards low-dimensionsal proportional myoelectric control. In 37th Annua International Conference of the IEEE Engineering in Medicine and Biology Society (EMBC), 7155–7158 (2015).fi 21. Krasoulis, A., Vijayakumar, S. & Nazarpour, K. Effect of user practice on prosthetic finger control with an intuitive myoele decoder. Front. Neurosci. 13, 585703 (2019). 22. Zhuang, K. Z. et al. Shared human–robot proportional control of a dexterous myoelectric prosthesis. Nat. Mach. Intell. 1, 400–411 (2019).h 3. Xiloyannis, M., Gavriel, C., Thomik, A. A. & Faisa, A. A. Gaussian process regression for accurate prediction of prosthetic limb movements from the natural kinematics of intact limbs. In 2015 7th International IEEE/EMBS Conference on Neural Engineering (NER), 659–662 (2015).h 23. Xiloyannis, M., Gavriel, C., Thomik, A. A. & Faisa, A. A. Gaussian process regression for accurate prediction of prosthetic limb movements from the natural kinematics of intact limbs. In 2015 7th International IEEE/EMBS Conference on Neural Engineering (NER), 659–662 (2015). References & Al-Jumaily, A. Orthogonal fuzzy neighborhood discriminant analysis for multifunction myoelectric hand control. IEEE Trans. Biomed. Eng. 57, 1410–1419 (2010). i g ( ) 48. Khushaba, R. N., Al-Ani, A. & Al-Jumaily, A. Orthogonal fuzzy neighborhood discriminant analysis for multifunction myoelectric hand control. IEEE Trans. Biomed. Eng. 57, 1410–1419 (2010). 9. Hjorth, B. EEG analysis based on time domain properties. Electroencephalogr. Clin. Neurophysiol. 29, 306–310 (1970). 0. Pudil, P., Novovičová, J. & Kittler, J. Floating search methods in feature selection. Pattern Recogn. Lett. 15, 1119–1125 (1994). , , , J , J g g , ( ) 1. Pedregosa, F. et al. Scikit-learn: machine learning in Python. J. Mach. Learn. Res. 12, 2825–2830 (2011).t Acknowledgements g AK and KN are supported by the Engineering and Physical Sciences Research Council (EPSRC) under Grant EP/R004242/1. g AK and KN are supported by the Engineering and Physical Sciences Research Council (EPSRC) under Grant EP/R004242/1. Competing interest h p g The authors declare no competing interests. Author contributions A.K. designed the study. A.K. analysed the data and prepared figures. A.K. wrote the manuscript. A.K. and K.N. read and approved the final manuscript. A.K. designed the study. A.K. analysed the data and prepared figures. A.K. wrote the manuscript. A.K. and K.N. read and approved the final manuscript. References Evaluation of the forearm EMG signal features for the control of a prosthetic hand. Physiol. Meas 24, 309–319 (2003).i 35. Dembczynski, K., Waegeman, W. & Hüllermeier, E. An analysis of chaining in multi-label classification. ECAI 294–299 (2012). 36 Read J Pfahringer B Holmes G & Frank E Classifier chains for multi-label classification Mach Learn 85 333 (2011) gii 37. Olsson, A. E. et al. Extraction of multi-labelled movement information from the raw HD-sEMG image with time-domain depth. Sci. Rep. 9, 1–10 (2019).i p 8. Jiang, N., Dosen, S., Müller, K.-R. & Farina, D. Myoelectric control of artificial limbs: is there a need to change focus? [In the spotlight]. IEEE Signal Proc. Mag. 29, 150–152 (2012). 39. Vujaklija, I. et al. Translating research on myoelectric control into clinics: are the performance assessment methods adequate?. Front. Neurorobot. https://doi.org/10.3389/fnbot.2017.00007 (2017).f 40. Fougner, A., Scheme, E. J., Chan, A. D. C., Englehart, K. B. & Stavdahl, Ø. Resolving the limb position effect in myoelectric pattern recognition. IEEE Trans. Neural Syst. Rehabil. Eng. 19, 644–651 (2011).l . Fougner, A., Scheme, E. J., Chan, A. D. C., Englehart, K. B. & Sta recognition. IEEE Trans. Neural Syst. Rehabil. Eng. 19, 644–651 (2 g y g 1. Khushaba, R. N., Al-Timemy, A., Kodagoda, S. & Nazarpour, K. Combined influence of forearm orientation and muscular contrac tion on EMG pattern recognition. Expert Syst. Appl. 61, 154–161 (2016).ii 42. Godbole, S. & Sarawagi, S. Discriminative methods for multi-labeled classification. In Pacific-Asia Conference on Knowledge Discovery and Data Mining, 22–30 (Springer, 2004). k k l l b l l fi h ( ) y g p g 43. Tsoumakas, G. & Katakis, I. Multi-label classification: an overview. Int. J. Data Warehous. Min. (IJDWM) 3, 1–13 (2007). https://doi.org/10.1038/s41598-020-72574-7 Scientific Reports \| (2020) 10:16872 \| www.nature.com/scientificreports/ 45. Hudgins, B. S., Parker, P. A. & Scott, R. N. A new strategy for multifunction myoelectric control. IEEE Trans. Biomed. Eng. 40, 82–94 (1993). 6. Zardoshti-Kermani, M., Wheeler, B., Badie, K. & Hashemi, R. EMG feature evaluation for movement control of upper extremity prostheses. IEEE Trans. Rehabil. Eng. 3, 324–333 (1995). p g ( ) 7. Nazarpour, K., Sharafat, A. R. & Firoozabadi, S. M. P. Application of higher order statistics to surface electromyogram signal clas- sification. IEEE Trans. Biomed. Eng. 54, 1762–1769 (2007). sification. IEEE Trans. Biomed. Eng. 54, 1762–1769 (2007). i g , ( ) 8. Khushaba, R. N., Al-Ani, A. © The Author(s) 2020 Additional information Supplementary information is available for this paper at https://doi.org/10.1038/s41598-020-72574-7. Supplementary information is available for this paper at https://doi.org/10.1038/s41598-020-72574-7. Supplementary information is available for this paper at https://doi.org/10.1038/s41598-020-72574-7. Correspondence and requests for materials should be addressed to A.K. Correspondence and requests for materials should be addressed to A.K. Reprints and permissions information is available at www.nature.com/reprints. Reprints and permissions information is available at www.nature.com/reprints. Reprints and permissions information is available at www.nature.com/reprints. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. 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https://openalex.org/W2795084742	https://eprints.whiterose.ac.uk/129082/8/Dhankher_et_al-2018-Plant%252C_Cell_%2526_Environment.pdf	English	null	Climate resilient crops for improving global food security and safety	Plant, cell & environment/Plant, cell and environment	2,018	cc-by	7,072	1 \| INTRODUCTION cropping systems by low diversity and the high intensity of inputs, climate‐associated yield instabilities being higher in grain legumes such as soybean (Figure 1) and broad leaved crops than in autumn‐sown cereals (Reckling et al., 2018). The predicted increased frequency of drought and intense precipitation events, elevated temperatures, as well as increased salt and heavy metals contamination of soils, will often be accompanied by increased infestation by pests, and patho- gens are expected to take a major toll on crop yields (Figure 2) leading to enhanced risks of famine (Long, Marshall‐Colon, & Zhu, 2015). For example, the frequency and intensity of extreme temperature events in the tropics are increasing rapidly as a result of climate change. Trop- ical biomes are currently experiencing temperatures that may already exceed physiological thresholds. The ability of tropical species to with- stand such “heat peaks” is poorly understood, particularly with regard to how plants prevent precocious senescence and retain photosynthe- sis in the leaves during these high temperature (HT) conditions. Such environmental stresses are among the main causes for declining crop productivity worldwide leading to billions of dollars of annual losses. Throughout history, farmers have adopted new crop varieties and adjusted their practices in accordance with changes in the environ- ment. But with the global temperatures rising, the pace of environ- mental change will likely be unprecedented. Furthermore, with the expansion of crop cultivation to nonoptimal environments and nonar- able lands, development of climate‐resilient crops is becoming increas- ingly important for ensuring food security (Kathuria, Giri, Tyagi, & Tyagi, 2007). The United Nations Sustainable Development Goals (SDGs) present an urgent and formidable challenge to scientists and society alike, highlighting the urgent requirement to transform agriculture and the food sector to achieve food and nutrition security, ecosystem sustain- ability, economic growth, and social equity over the coming decades. Global food demand is predicted to grow by 70–85% as the popula- tion increases to over 9 billion people by 2050 (FAO, 2017; Ray, Mueller, West, & Foley, 2013). A “next generation Green Revolution” is required to achieve future food security. Radical new concepts and approaches are needed to achieve a more sustainable develop- ment of agriculture. The next Green Revolution requires a much broader and systems‐based approach including environment, econ- omy, and society, across all levels of organization (Nüsslein & Dhankher, 2016). Abstract Food security and the protection of the environment are urgent issues for global society, particularly with the uncertainties of climate change. Changing climate is predicted to have a wide range of negative impacts on plant physiology metabolism, soil fertility and carbon sequestration, microbial activity and diversity that will limit plant growth and productivity, and ultimately food production. Ensuring global food security and food safety will require an intensive research effort across the food chain, starting with crop production and the nutritional quality of the food products. Much uncertainty remains concerning the resil- ience of plants, soils, and associated microbes to climate change. Intensive efforts are currently underway to improve crop yields with lower input requirements and enhance the sustainability of yield through improved biotic and abiotic stress tolerance traits. In addition, significant efforts are focused on gaining a better understanding of the root/soil interface and associated microbiomes, as well as enhancing soil properties. Received: 23 March 2018 Accepted: 28 March 2018 DOI: 10.1111/pce.13207 Received: 23 March 2018 Accepted: 28 March 2018 DOI: 10.1111/pce.13207 Accepted: 28 March 2018 This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2018 The Authors Plant, Cell & Environment Published by John Wiley & Sons Ltd e terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided Plant Cell Environ. 2018;41:877–884. © 2018 The Authors Plant, Cell & Environment Published by John Wiley & Sons Ltd 1 \| INTRODUCTION Transformative science across the agri‐food sector is required if a major crisis in food production to meet the needs of a growing world population is to be avoided. Future agriculture requires tailored solutions that not only incorporate fundamental step‐changes in current knowledge and enabling technologies but also take into account of the need to protect the earth and respect societal demands. Climate change has far‐reaching implications for global food secu- rity and has already substantially impacts agricultural production worldwide through effects on soil fertility and carbon sequestration, microbial activity and diversity, as well as on plant growth and produc- tivity. Negative environmental impacts are exacerbated in current wileyonlinelibrary.com/journal/pce 877 Plant Cell Environ. 2018;41:877–884. EDITORIAL EDITORIAL 878 FIGURE 1 Pollinators such as bees are very attracted to legumes because of the high nutrient content of their pollen and nectar. Bumblebee feeding on soybean (variety Sultana) in a field experiment in 2015 in Müncheberg, Germany important constraints to crop yields. Drought stress alone is expected to limit the productivity of more than half of the earth's arable land in the next 50 years, competition for water between urban and agricul- tural areas compounding the problem. Several papers in this volume (Herzog, Konnerup, Pedersen, Winkel, & Colmer, 2017; Kerr et al., 2017; Pérez‐Jiménez, Hernández‐Munuera, Piñero, López‐Ortega, & Amor, 2017) describe the physiological, molecular, and biochemical responses of plants to drought and flooding. Although the use of brackish and saline water could help alleviate the world's water prob- lems, this option is only possible with the development of salt‐tolerant crops (Figure 3) or management practices that alleviate salt stress. A number of manuscripts in this volume (Herzog et al., 2017; Joshi et al., 2017; Lakra, Kaur, Anwar, Pareek, & Pareek, 2017; Oyiga et al., 2017; Patishtan, Hartley, Fonseca de Carvalho, & Maathuis, 2017) describe how plants tolerate high levels of salt. Soil phytoremediation and tolerance to heavy metals are also highlighted (Fasani, Manara, Martini, Furini, & DalCorso, 2017). A number of papers describe the mechanisms that enable plants to withstand extremes of temperature (Bredow, Tomalty, Smith, & Walker, 2017; D'Amelia et al., 2017; Djanaguiraman et al., 2017; Djanaguiraman, Perumal, Ciampitti, Gupta, & Prasad, 2017; Charrier, Isabelle, Marc, & Thierry, 2017; Izydorczyk et al., 2017; Xia et al., 2017). 1 \| INTRODUCTION Taken together, the new information provided in these manuscripts increases our current understanding of the biochemical and molecular basis of crop adaptation to abiotic stresses, highlighting promising candidate genes/enzymes that are targets for manipulation to improve the ability of plants to produce better yields under changing climate conditions. The acclimation mechanisms that facilitate optimization of photosyn- thesis and associated processes to changing irradiance are considered in two papers (Karpinska et al., 2017; Townsend, Ware, & Ruban, 2017). Within this context, the stress‐induced accumulation of reac- tive oxygen species (ROS) controls numerous growth and develop- mental processes by modifying enzyme activity and protein–protein interactions. Several papers describe the roles of ROS, redox signalling and antioxidants in the plant stress signalling network, and in the interactions with phytohormone signalling cascades that govern plant responses to biotic and abiotic stresses (Ahammed et al., 2017; Karpinska et al., 2017; Song et al., 2017; Xia et al., 2017; Zhou et al., 2017). Cell proliferation and fate can also be regulated by control of FIGURE 1 Pollinators such as bees are very attracted to legumes because of the high nutrient content of their pollen and nectar. Bumblebee feeding on soybean (variety Sultana) in a field experiment in 2015 in Müncheberg, Germany FIGURE 2 Xerohalophytes growing in soil impacted by severe salinity and drought FIGURE 2 Xerohalophytes growing in soil impacted by severe salinity and drought Sustainable innovation of the agricultural sector within SDG constraints is urgently required to improve the way that food and animal feed are produced. Current scientific advances offer considerable potential tomeet the challenges of increasing agricultural production with conservation of the environment and the earth's ecosystems, compliant to the SDGs. The papers published in this special issue cover basic and applied research focused on enabling crops to grow under over a wider range of environmental conditions with sustainable and reliable crop yields. Particular emphasis is placed on the development of climate‐resil- ient crops that are able to adapt rapidly to changing climatic conditions and on how climate change impacts on the resilience of plant/soil inter- face and soil microbiomes. The manuscripts that comprise this volume address the challenges imposed by the increased frequency of abiotic and biotic stresses with a view developing strategies to minimize the impact of changing climate on agriculture and the environment. FIGURE 3 Cultivation of salt‐tolerant crops such as Agave sp. on marginal lands in India The use of phytoremediation to improve contaminated soils and/or water is proposed as a cost‐effective and environmental friendly “green‐clean” technology. The study described in the paper by Dixit et al. (2017) highlights the role of novel stress‐associated proteins (SAPs) in providing toler- ance to the multiple abiotic stresses experienced by plants. The Arabidopsis and rice genomes were found to contain 14 and 18 genes encoding SAP‐related proteins, respectively. Most of the SAP genes in plants are differentially regulated in response to multiple environmen- tal stresses such as low temperatures (LTs), salinity, drought, heavy metals, wounding, and submergence. Transgenic Arabidopsis lines overexpressing AtSAP13 were found to show improved tolerance to drought and salt stresses, and also toxic metals including arsenic (As), cadmium (Cd), and zinc (Zn) (Dixit et al., 2017). The mode of action of AtSAP13 proteins and their roles in tolerance to multiple abiotic stresses was analysed using DNA‐protein interaction assays (Dixit et al., 2017). Several transcription factors related to abiotic stress toler- ance were shown to bind to the AtSAP13 promoter. AtSAP13 and its homologs could therefore be used to develop climate resilient crops. oxygen availability. The role of physiological hypoxia in the control of the bud burst is also discussed (Meitha et al., 2017). branching with more grains filled per plant under unstressed and salin- ity stress conditions (Joshi et al., 2017). These findings shed new light on the complex crosstalk between cytokinin metabolism, abiotic stress tolerance, and grain yield. Two manuscripts (Dixit et al., 2017; Gupta et al., 2017) describe the development of strategies for improving crop resiliency against multiple stresses for producing better yields with limited agronomic inputs. In particular, the paper by Gupta et al. (2017) reports the generation of rice plants with improved adaptation towards multiple abiotic and biotic stresses with reduced yield penalty through manip- ulation of the glyoxalase pathway. Methylglyoxal (MG) is a cytotoxic metabolite that is accumulated as a consequence of many abiotic and biotic stresses. MG accumulation may therefore be a linking factor in plant responses to diverse stresses. This paper reports that genetic manipulation of the two‐step glyoxalase pathway that removes MG led to improved tolerance of rice to multiple abiotic and biotic stresses. The enhanced stress tolerance observed in the glyoxalase‐ overexpressing rice plants was attributed to improved MG detoxifica- tion, reduced levels of ROS accumulation, and better protection of photosynthesis (Gupta et al., 2017). Hence, prevention of MG accu- mulation is a promising strategy to develop improved crops with enhanced tolerance to a range of abiotic and biotic stresses. , g y Increasing soil contamination as a result of industrial activities and agricultural practices, such as use of recycled wastewater and under- ground water contaminated with heavy metals such as arsenic (Figure 4), has not only caused a decline in crop productivity but has also led to serious food safety concerns. Phytoremediation approaches are therefore crucial in the removal of toxic pollutants from soil and water so that crop production can be increased on (nonarable) contam- inated soils. This topic is described in a comprehensive review by Fasani et al. (2017), which describes the problems associated with heavy metals toxicity in soil, as well as the potential of genetic engineering approaches to improve plant phytoremediation capacity in contami- nated soils. The mechanisms that plants employ for uptake, transloca- tion, detoxification, and accumulation of toxic metals are highlighted in this review, which also provides a comprehensive list of the recent studies undertaken in this field (Fasani et al., 2017). 1.1 \| Abiotic stress tolerance Of the multitude of diverse abiotic and biotic stresses faced by plants in the field, water availability is widely accepted to be one of the most FIGURE 3 Cultivation of salt‐tolerant crops such as Agave sp. on marginal lands in India EDITORIAL 879 number of hot days over the past 50–60 years with mean temperature increase by 0.9 °C (Deo, McAlpine, Syktus, McGowan, & Phinn, 2007). Warming over the Indian subcontinent (both land and ocean) has been recorded over first decade of this century (Roxy et al., 2015), and recent studies have warned the increased occurrences of heatwaves over the land (Rohini, Rajeevan, & Srivastava, 2016). Temperatures are projected to rise faster in Africa than in the rest of the world, with increases exceeding 2 °C by mid‐21st century and 4 °C by the end of 21st century (Niang et al., 2014). the underlying physiological, molecular, and biochemical mechanisms and identify related genes and gene networks. Patishtan et al. (2017) report the results of a genome‐wide association studies of salt‐related traits in 306 rice cultivars. An important region on chromosome 8 was identified that contains a number of genes related to the ubiquitination pathway (Patishtan et al., 2017). The process of protein degradation is therefore proposed as a major target for improving salt tolerance in rice. Several hundred nonsynonymous single nucleotide polymorphisms were found in coding regions, specific genomic regions with increased numbers of nonsynonymous single nucleotide poly- morphisms were identified. A mechanistic understanding of plant responses to HT, particu- larly when the stress is imposed at flowering, is crucial for the devel- opment of stress tolerant genotypes because plant reproductive organs are very sensitive to HT stress, (Farooq, Bramley, Palta, & Siddique, 2011; Prasad, Bheemanahalli, & Jagadish, 2017). HT reduce pollen viability and shorten the grain‐filling period, temperature increases of 3–4 °C are likely to cause crop yields to fall by 15–35% in Africa and Asia and by 25–35% in the Middle East (Ortiz et al., 2008). Pearl millet (Pennisetum glaucum) has a higher HT tolerance than many other cereals and is hence considered to be an important climate resilient crop. Hence, like sorghum (Sorghum biclor), pearl millet is an important cereal crop in the agriculture of arid and semiarid regions. The impacts of HT stress in pearl millet are reported in the paper by Djanaguiraman, Perumal, Ciampitti, et al. (2017), who identi- fied sensitive stages and determined parameters such as temperature thresholds, genetic variability, and the fertility of pollen and the pistil. Exposure to HT stress was found to decrease pollen germination and seed yield per panicle (Djanaguiraman, Perumal, Ciampitti, et al., 2017), the periods of gametogenesis and anthesis being the most sensitive to HT stress in terms of effects on seed yield. Negative impacts of HT stress on the fertility of both pollen and pistil tissues were observed, the pistil being more sensitive than pollen (Djanaguiraman, Perumal, Ciampitti, et al., 2017). The screening of pearl millet germplasm and identification of HT tolerant lines in this paper will be extremely useful in future breeding programs designed to develop parental lines or hybrids with HT tolerance. Two rice genotypes (the salt‐sensitive IR64 and the salt tolerant Pokkali) with contrasting responses to salinity stress were used to investigate the temporal differences in proteome profiles in the study reported by Lakra et al. (2017). This paper not only demonstrates the usefulness of the proteomics (2D‐DIGE: two‐dimensional differential in‐gel electrophoresis) approaches to unravel the proteins involved in salt stress tolerance in rice genotypes but also highlights the finding that tolerant genotypes were “ready in anticipation” of stress, that is, the stress responsive machinery remained active and responsive to the stress signals (Lakra et al., 2017). The proteins identified in these studies will be helpful in improving salinity tolerance in crop plants. Genetic variations in salt tolerance were also reported in the paper by Oyiga et al. (2017), which reports a comprehensive evaluation and identification of quantitative trait loci conferring salt tolerance in 150 winter wheat cultivars, using a genome‐wide association study approach. A large number of SNPs were reported in 37 quantitative trait loci associated with the salt tolerance traits. Candidate genes linked to these polymorphisms were identified and results confirmed by transcriptomics and qRT‐PCR on samples harvested from plants grown under salt stress and control conditions (Oyiga et al., 2017). The polymorphisms identified in these two papers have biological rel- evance that can be exploited in future breeding programs directed at enhancing salt tolerance in wheat and rice. The flooding of paddy fields is a common practice that could adversely affect global rice production because complete submergence can lead to severe damage and death of rice seedlings. Hence, the anal- ysis of the role of gas films on leaves as a tolerance mechanisms presented in the paper by Herzog et al. (2017) has relevance for rice crop survival. The gas films on leaves of rice plants submerged in saline water were shown to delay the entry of salt. Moreover, the natural loss or removal of the leaf gas films resulted in a severe decline in photosyn- thesis and the growth of the rice plants (Herzog et al., 2017). Understanding of mechanisms that afford tolerance will assist in the development of HT stress tolerant lines and hybrids. Similarly, deployment and adoption of HT tolerant genotypes and/or hybrids will increase the resilience of millet‐based cropping systems to future climate changes. Both the pollen and pistil functions were found to decrease in response to HT stress in grain sorghum in the study reported by Djanaguiraman, Perumal, Jagadish, et al. (2017). In this case, an analysis of direct and reciprocal crosses showed that sorghum pollen was more sensitive to HT stress than the pistil, with greater decreases in seed‐set (Djanaguiraman, Perumal, Jagadish, et al., 2017). Loss of sorghum gamete functions under HT stress were asso- ciated with changes in anatomy, and phospholipid composition and level of saturation, as well as ROS levels and antioxidant enzyme activities. 1.2 \| Genomics and proteomics approaches to improve salt tolerance Salinity is an ever‐increasing menace to agriculture worldwide. This is particularly important for the cultivation of salt‐sensitive crops such as rice and wheat. Rice is the second largest crop in the world and is planted on about one tenth of the earth's arable land and is the single largest source of food for half of humanity (FAO, 2015). Of the 130 million hectares of land used for rice cultivation, approximately 30% contain levels of salt high enough to affect rice yield (Vinocur & Altman, 2005; Wang, Vinocur, & Altman, 2003). The degree of suscep- tibility to salinity varies widely between rice cultivars, pointing to extensive genetic diversity that can be exploited to identify genes and their corresponding proteins that are important for rice salt toler- ance. To develop crops tolerant to salinity, it is essential to understand The role of abscisic acid‐responsive transcription factors (ABFs) in the regulation of drought tolerance in cotton is described in detail in the paper by Kerr et al. (2017). A functional analysis of two genes that encode representative ABFs from Arabidopsis and cotton was under- taken. Expression of the Arabidopsis or cotton ABFs in transgenic cotton plants led to increased drought stress tolerance both under controlled greenhouse conditions and in the field (Kerr et al., 2017). Some of the transgenic lines analysed were better able to maintain yields during dry conditions in the field than the wild‐type or nonexpressing controls. Hence, the increased expression of ABFs could provide a realistic mechanism to improve the performance of cotton in the field and develop more drought tolerant cotton varieties (Kerr et al., 2017). FIGURE 4 Field trials of rice germplasms grown on arsenic contaminated sites in West Bengal as a collaborative research project of CSIR‐NBRI, Lucknow and RRS, Chinsurah, West Bengal, India. Enhanced rice grain yields, achieved through manipulation of cytokinin homeostasis in the inflorescence meristem, are reported in the paper by Joshi et al. (2017). Cytokinin is degraded by the enzyme cytokinin oxidase (CKX) in the rice inflorescence. Knockdown of the inflorescence meristem‐specific CKX, OsCKX2, resulted in elevated cellular cytokinin levels, which in turn, lead to enhanced panicle FIGURE 4 Field trials of rice germplasms grown on arsenic contaminated sites in West Bengal as a collaborative research project of CSIR‐NBRI, Lucknow and RRS, Chinsurah, West Bengal, India. 880 EDITORIAL EDITORIAL 1.4 \| Phytohormones signalling and stress tolerance in plants ROS accumulation is controlled by a complex antioxidant scav- enging system that includes enzymes such as superoxide dismutase, ascorbate peroxidase, catalase, glutathione peroxidase, glutathione reductase, and peroxiredoxins (Foyer & Shigeoka, 2011). However, enzymes that are considered to play important antioxidative roles such as peroxidases can also promote ROS production or ROS‐depen- dent processes and different ROS forms (such as superoxide and H2O2) may antagonize each other in terms of the regulation of gene expression and low molecular weight antioxidants such as glutathione may play an integral role in transmitting oxidative signals as well as controlling ROS accumulation (Foyer et al., 2017). Photosynthesis is the major source of ROS in plants. H2O2 is generated in chloroplasts via the action of superoxide dismutase during photosynthesis, and this oxidant is also produced in the peroxisomes during photorespiration (Foyer & Noctor, 2005). ROS production, signalling, and removal by the antioxidant systems associated with photosynthesis provide flexi- bility and control in the management of high light and other stresses (Foyer et al., 2017). For example, using transgenic tobacco lines with low and high ascorbate oxidase activity, Karpinska et al. (2017) demonstrate how the redox state of the apoplast influences the acclimation of photosynthesis and leaf metabolism to the changing irradiance. High light‐dependent changes in photosynthesis rates were significantly higher in high‐light grown leaves when the apoplast was less oxidized, demonstrating that the redox state of the apoplast influ- ences the extent of susceptibility of photosynthesis to high light‐ induced inhibition (Karpinska et al., 2017). Seasonal shifts in temperature induced by climate change are likely to affect seed germination and increase the risk of crop failure, particu- larly in economically important cereals such as wheat. Understanding the temperature‐dependent mechanisms that influence seed germina- tion is important in considerations of the resilience of wheat to chang- ing environmental conditions. The molecular mechanisms underlying LT‐regulation of abscisic acid (ABA) and gibbrelic acid (GA) metabolism and signalling during wheat seed germination are reported in the paper by Izydorczyk et al. (2017). LT modulation of the spatiotemporal balance between ABA and GA levels and tissue sensitivity was reported to occur via altered expression of genes involved in the metabolic and signalling pathways of these phytohormones (Izydorczyk et al., 2017). Like ABA and GA, brassinosteroids (BR) play important roles in developmental processes as well as abiotic and biotic stresses tolerance (Vriet, Russinova, & Reuzeau, 2012; Zhou et al., 2014). 1.5 \| Oxidative signalling, photosynthesis and biotic stress responses ROS have multifaceted roles in plant biology. Despite the compelling evidence that ROS mainly act as beneficial signalling agents that pro- tect plants against stress, the concept that oxidative damage is a major cause of stress‐induced loss of cellular functions remains in the litera- ture (Foyer, Ruban, & Noctor, 2017). This is particularly evident in the field of photosynthesis where the idea persists that light‐induced damage to photosystem (PS)II causes photoinhibition requiring subse- quent repair of the PSII D1 protein (Foyer et al., 2017). However, the interplay between photodamage and photoprotection in PSII is shown to be much more complex in the paper by Townsend et al. (2017). Light‐induced photoinhibition is prevented by thermal energy dissipa- tion processes in the thylakoid membrane that are together called nonphotochemical quenching (NPQ). Using a new pulse amplitude modulation fluorescence methodology, the relative contributions of NPQ and D1 repair to photoprotection were determined under short periods of illumination using photoinhibitors and mutant Arabidopsis thaliana lines. These studies show that NPQ makes a much greater contribution to PSII yield than D1 repair under short periods of illumi- nation (Townsend et al., 2017). and ice recrystallization inhibition proteins, which play important roles in freezing tolerance via antinucleating activities that inhibit nucleation and formation of ice crystals were reported in the model grass Brachypodium distachyon (Bredow et al., 2017). Frost damage to flower buds is a particularly important stress for perennial crops, such as fruit trees, causing severe economic losses. The importance of frost damage at the developmental stage to walnut trees was highlighted using dynamic simulation modelling of temperature and photoperiod interactions (Charrier et al., 2017). Frost hardiness monitoring of three walnut genotypes at low and high elevation locations over 5 years revealed contrasting phenologies and maximum hardiness. Frost damage was shown to be controlled primality by frost exposure and not the vulnerability of the walnut genotypes to frost damage during the dormant periods (Charrier et al., 2017). Such studies emphasize the importance of the mechanisms of perception of frost signals in order to minimize the damage. Phenolic compounds such as anthocyanins have been implicated in LT tolerance. Allelic diversity and the evolutionary significance of gene duplication on anthocyanin metabolism is reported in the paper by D'Amelia et al. (2017). This study highlights the role of the R2R3 MYB transcription factor called AN2, and its paralog AN1, in cold tolerant and cold susceptible potato (Solanum tuberosum) varieties. The duplication of MYB genes appears to have resulted in divergent functions, with AN1 specializing in anthocyanin production whereas AN2 serves to activate cold stress responses, inducing the synthesis of hydroxycinnamic acid derivatives (D'Amelia et al., 2017). paper by Song et al. (2017). Parasitic nematodes cause more than $150 billion losses annually to susceptible crops worldwide (Hassan, Pham, Shi, & Zheng, 2013). BR were shown to be positive regulators acting to prevent infestation by root‐knot nematodes via RESPIRA- TORY BURST OXIDASE HOMOLOG‐dependent increases in mitogen‐activated protein kinases (Song et al., 2017). 1.3 \| Adaptation of extreme temperature stress Exposure to extreme temperatures (chilling, freezing, or HT) causes detrimental effects on plant productivity and crop yields. The semiarid regions of the world are particularly vulnerable to the weather vari- ability associated with climate change (Arab Water Council, 2009). Simulation studies have predicted extreme hot summers will occur twice a decade in the future in contrast to twice a century during 2000s (Christidis, Jones, & Scott, 2014). Russia recorded the worst ever heatwave in three decades in 2012 leading to about 55,000 casu- alties (Russo et al., 2014). Australia recorded doubling of the annual Like HT, LT stresses such as chilling and freezing also severely impair seedling survival and lower crop yields worldwide. Several stud- ies in this volume provide new insights into the mechanisms by which plants perceive cold stress and how they transduce the LT signal to activate adaptive responses (Mantri, Patade, Penna, Ford, & Pang, 2012). For example, the in planta functions of an ice‐binding protein EDITORIAL 881 KEYWORDS abiotic stresses, climate change, food safety, food security, oxidative stress Om Parkash Dhankher1 Christine H. Foyer2 1Stockbridge School of Agriculture, University of Massachusetts Amherst MA, Amherst, MA 01003, USA 2Centre for Plant Sciences, School of Biology, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, UK Correspondence Christine H. Foyer, Centre for Plant Sciences, School of Biology, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, UK. Email: c.foyer@leeds.ac.uk Correspondence Christine H. Foyer, Centre for Plant Sciences, School of Biology, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, UK. Email: c.foyer@leeds.ac.uk ACKNOWLEDGMENTS We thank the Worldwide University Network (WUN) for financial support for the Climate Resiliency Open Partnership for Food Security (CROP‐FS) consortium. We thank Mrs. Gunhild Rosner, ZALF, Germany, for Figure 1, Prof. Ashwani Pareek, School of Life Sciences, Jawaharlal Nehru University, New Delhi, India, for Figures 2 and 3, and Dr. R. D. Tripathi, National Botanical Research Institute Lucknow, India for Figure 4. A role for photorespiration in activating tomato leaf defenses against Pseudomonas syringae was reported in the paper by Ahammed et al. (2017). Climate change‐associated increase in atmo- spheric CO2 levels will shift the balance between photosynthetic carbon assimilation and photorespiration in C3 plants (Walker, VanLooke, Bernacchi, & Ort, 2016). Evidence is presented showing that photorespiration contributes to the basal defense against P. syringae via glycolate‐oxidase‐derived H2O2 production and hence climate changes associated decreases in photorespiration may impair such defense response (Ahammed et al., 2017). This report supports the concept that biotic defense pathways are promoted by photores- piration, which is inhibited at high CO2. However, apparently contra- dictory observations show that growth under elevated CO2 can induce salicylic acid‐dependent defenses and thus increase plant resistance to pathogens (Noctor & Mhamdi, 2017). Moreover, ele- vated atmospheric CO2 concentrations were shown to protect sweet cherry plants from the damaging effects of waterlogging (Pérez‐ Jiménez et al., 2017). Waterlogging often leads to hypoxia and decreases plant survival via inhibition of processes such as photosyn- thesis. However, high CO2 was able to offset some of negative effects of hypoxia in sweet cherry (Pérez‐Jiménez et al. 2017). Resolution of such apparently contradictory observations requires a much greater understanding of climate change‐related changes in the relationships between primary metabolism, inducible defenses, and resistance to abiotic and biotic stresses (Noctor & Mhamdi, 2017). 1.4 \| Phytohormones signalling and stress tolerance in plants The BR‐mediated regulation of chilling stress in tomato plants was studied in the paper by Xia et al. (2017). BR is shown to positively regulate chilling tolerance through a signalling cascade involving glutaredoxin genes and the redox status of 2‐Cys peroxiredoxin, as well as antioxidant enzymes activities (Xia et al., 2017). The BR‐induced increases in antioxidant capacity that underpin enhanced chilling toler- ance were found to be largely dependent on the activation of RESPIRA- TORY BURST OXIDASE HOMOLOG and associated increases in apoplastic ROS. Direct evidence of a crosstalk between BR and ROS in the resistance of tomato to root‐knot nematodes is reported in the 882 EDITORIAL ORCID Om Parkash Dhankher http://orcid.org/0000-0003-0737-6783 Christine H. Foyer http://orcid.org/0000-0001-5989-6989 Om Parkash Dhankher http://orcid.org/0000-0003-0737-6783 Christine H. Foyer http://orcid.org/0000-0001-5989-6989 Stress‐induced oxidation of the cellular environment leads to posttranslational modifications in protein structure and function that provide a high degree of plasticity and control in response to environ- mental stimuli. The influence of oxidative stress on two poorly charac- terized plant posttranslational modifications, protein succinylation and acetylation, is reported in the paper of Zhou et al. (2017). Using a pro- teomics approach to study the oxidative stress‐induced interactions between the succinyl‐ and acetyl‐proteomes, these authors provide evidence of the presence of H2O2‐triggered interactions between the lysine succinylome and acetylome in rice leaves (Zhou et al., 2017). Large numbers of acetylated and succinylated proteins were identified in rice leaves. However, exposure to oxidative stress did not cause large global changes in the rice acetylome or succinylome profiles but rather led to modifications on a specific subset of the identified sites. Moreover, succinylation exerted a strong influence on the activities of catalase and glutathione S‐transferase 6 recombi- nant proteins (Zhou et al., 2017). These studies presented in the different manuscripts that com- prise this special issue not only increase current knowledge of the genes, processes, and underlying mechanisms of stress tolerance/ resistance and associated crop resilience but they also identify poten- tial new strategies for developing climate‐resilient crops that will have enhanced and sustained productivity traits that will help to ensure global food security. These papers also contribute essential new ideas that are required to transform food production and so address the SDGs, with all their complex interactions and trade‐offs. D'Amelia, V., Aversano, R., Ruggiero, A., Batelli, G., Appelhagen, I., Dinacci, C., … Carputo, D. (2017). Subfunctionalization of duplicate MYB genes in Solanum commersonii generated the cold‐induced ScAN2 and the anthocyanin regulator ScAN1. Plant, Cell & Environment, 41. 1038–1051. Kerr, T. C., Abdel‐Mageed, H., Aleman, L., Lee, J., Payton, P., Cryer, D., & Allen, R. D. (2017). Ectopic expression of two AREB/ABF orthologs increases drought tolerance in cotton (Gossypium hirsutum). Plant, Cell & Environment, 41, 898–907. Lakra, N., Kaur, C., Anwar, K., Pareek, S., & Pareek, A. (2017). Proteomics of contrasting rice genotypes: Identification of potential targets for raising crops for saline environment. Plant, Cell & Environment, 41, 947–969. Deo R.C., McAlpine C.A., Syktus J., McGowan H.A. & Phinn S. (2007) On Australian teat Waves: Time series analysis of extreme temperature events in Australia, 1950–2005. Modsim 2007: International Congress on Modelling and Simulation. 626–635. Long, S. P., Marshall‐Colon, A., & Zhu, X.‐G. (2015). Meeting the global food demand of the future by engineering crop photosynthesis and yield potential. Cell, 161, 56–66. Dixit, A., Tomar, P., Vaine, E., Abdullah, H., Hazen, S., & Dhankher, O. P. (2017). A stress‐associated protein, AtSAP13, from Arabidopsis thaliana provides tolerance to multiple abiotic stresses. Plant, Cell & Environment, 41. 1171–1185. Mantri, N., Patade, V., Penna, S., Ford, R., & Pang, E. (2012). Abiotic stress responses in plants: Present and future. In P. Ahmad, & M. N. V. Prasad (Eds.), Abiotic stress responses in plants: Metabolism, productivity and sustainability (pp. 1–19). New York, NY: Springer New York. Djanaguiraman, M., Perumal, R., Ciampitti, I. A., Gupta, S. K., & Prasad, P. V. V. (2017). Quantifying pearl millet response to high temperature stress: thresholds, sensitive stages, genetic variability and relative sensitivity of pollen and pistil. Plant, Cell & Environment, 41. 993–1007. Meitha, K., Agudelo‐Romero, P., Signorelli, S., Gibbs, D., Considine, J., Foyer, C. H., & Considine, M. (2017). Developmental control of hypoxia during bud burst in grapevine. Plant, Cell & Environment, 41, 1154–1170. Djanaguiraman, M., Perumal, R., Jagadish, S. V. K., Ciampitti, I. A., Welti, R., & Prasad, P. V. V. (2017). Sensitivity of sorghum pollen and pistil to high‐temperature stress. Plant, Cell & Environment, 41. 1065–1082. Niang, I., Ruppel, O. C., Abdrabo, M. A., Essel, A., Lennard, C., Padgham, J., & Leary, N. A. (2014). Africa in climate change: Impacts, adaptation, and vulnerability. Part B: Regional aspects. In V. R. Barros, C. B. Field, D. J. Dokken, M. D. Mastrandrea, K. J. Mach, T. E. Bilir, et al. (Eds.), Contribu- tion of working group II to the fifth assessment report of the intergovernmental panel on climate change (pp. 1199–1265). Cambridge, United Kingdom and New York, NY: Cambridge University Press. Farooq, M., Bramley, H., Palta, J. A., & Siddique, K. H. M. (2011). Heat stress in wheat during reproductive and grain‐filling phases. Critical Reviews in Plant Sciences, 30, 1–17. Fasani, E., Manara, A., Martini, F., Furini, A., & DalCorso, G. (2017). The potential of genetic engineering of plants for the remediation of soils con- taminated with heavy metals. Plant, Cell & Environment, 41. 1201–1232. Noctor, G., & Mhamdi, A. (2017). Climate change, CO2, and defense: The metabolic, redox, and signaling perspectives. Trends in Plant Science, 22, 857–870. Food and Agriculture Organization of the United Nations (2015). FAOSTAT. Rome: Food and Agriculture Organization of the United Nations. Nüsslein, K., & Dhankher, O. P. (2016). Project management: Food security needs social‐science input. Nature, 535, 37–37. Food and Agriculture Organization of the United Nations (2017). The future of food and agriculture—Trends and challenges. Rome: Food and Agriculture Organization of the United Nations. Ortiz, R., Braun, H. J., Crossa, J., Crouch, J. H., Davenport, G., Dixon, J., … Iwanaga, M. (2008). Wheat genetic resources enhancement by the International Maize and Wheat Improvement Center (CIMMYT). Genetic Resources and Crop Evolution, 55, 1095–1140. Foyer, C. H., & Noctor, G. (2005). Redox homeostasis and antioxidant sig- nalling: A metabolic interface between stress perception and physiological responses. Plant Cell, 17, 1866–1875. Oyiga, B., Sharma, R., Baum, M., Ogbonnaya, F., Léon, J., & Ballvora, A. (2017). Allelic variations and differential expressions detected at QTL loci for salt stress tolerance in wheat. Plant, Cell & Environment, 41, 919–935. Foyer, C. H., Ruban, A. V., & Noctor, G. (2017). Viewing oxidative stress through the lens of oxidative signalling rather than damage. Biochemical Journal, 474, 877–883. Patishtan, J., Hartley, T., Fonseca de Carvalho, R., & Maathuis, F. J. M. (2017). Genome wide association studies to identify rice salt tolerance markers. Plant, Cell & Environment, 41, 970–982. Foyer, C. H., & Shigeoka, S. (2011). Understanding oxidative stress and antioxidant functions to enhance photosynthesis. Plant Physiology, 155, 93–100. Pérez‐Jiménez, M., Hernández‐Munuera, M., Piñero, M. C., López‐Ortega, G., & del Amor, F. (2017). Are commercial sweet cherry rootstocks adapted to climate change? Short‐term waterlogging and CO2 effects on sweet cherry cv. ‘Burlat’. Plant, Cell & Environment, 41, 908–918. Gupta, B., Sahoo, K., Ghosh, A., Tripathi, A., Anwar, K., Das, P., … Pareek, S. (2017). Manipulation of glyoxalase pathway confers tolerance to multi- ple stresses in rice. Plant, Cell & Environment, 41, 1186–1200. Hassan, A. W., Pham, T. H., Shi, H., & Zheng, J. (2013). Nematodes threats to global food security. Acta Agriculturae Scandinavica, Section B‐ Soil & Plant Science, 63, 420–425. Prasad, P. V. V., Bheemanahalli, R., & Jagadish, S. V. K. (2017). Field crops and the fear of heat stress‐ Opportunities, challenges and future direc- tions. Field Crops Research, 200, 114–121. Herzog, M., Konnerup, D., Pedersen, O., Winkel, A., & Colmer, T. (2017). Leaf gas films contribute to rice (Oryza sativa) submergence tolerance during saline floods. Plant, Cell & Environment, 41, 885–897. Ray, D. K., Mueller, N. D., West, P. C., & Foley, J. A. (2013). Yield trends are insufficient to double global crop production by 2050. PLoS one, 8. e66428 Izydorczyk, C., Nguyen, T.‐N., Jo, S. H., Son, S. H. T., Anh, P., & Ayele, B. (2017). Spatiotemporal modulation of abscisic acid and gibberellin metabolism and signalling mediates the effect of suboptimal and supraoptimal temperatures on seed germination in wheat (Triticum aestivum L.). Plant, Cell & Environment, 41, 1022–1037. Reckling, M., Doring, T. F., Berkvist, G., Chmielewski, F.‐M., Stoddard, F. L., Watson, C. A., … Bachinger, J. (2018). Grain legume yield instability has increased over 60 years in long‐term field experiments as measured by a scale‐adjusted coefficient of variation. Aspects of Applied Biology, 138, 15–20. Joshi, R., Sahoo, K., Tripathi, A., Kumar, R., Gupta, B., Pareek, A., & Pareek, S. (2017). Knockdown of an inflorescence meristem‐specific cytokinin oxidase—OsCKX2 in rice reduces yield penalty under salinity stress condition. Plant, Cell & Environment, 41, 936–946. Rohini, P., Rajeevan, M., & Srivastava, A. K. (2016). On the variability and increasing trends of heat waves over India. Science Reports, 6, 26153. Roxy, M. K., Kapoor, R., Terray, P., Murtugudde, R., Ashok, K., & Goswami, B. N. (2015). Drying of Indian subcontinent by rapid Indian Ocean warming and a weakening land‐sea thermal gradient. Nature Communi- cations, 6, 7423. Karpinska, B., Zhang, K., Rasool, B., Pastok, D., Morris, J., Verrall, S., … Foyer, C. H. (2017). The redox state of the apoplast influences the acclimation of photosynthesis and leaf metabolism to changing irradi- ance. Plant, Cell & Environment, 41, 1083–1097. Russo, S., Dosio, A., Graversen, R. REFERENCES Ahammed, G. J., Li, X., Zhang, G., Zhang, H., Shi, J., Pan, C., … Shi, K. (2017). Tomato photorespiratory glycolate oxidase‐derived H2O2 production contributes to basal defense against Pseudomonas syringae. Plant, Cell & Environment, 41. 1126–1138. The developmental control of hypoxia during bud burst in grape- vine (Vitis vinifera L.) is reported in the paper by Meitha et al. (2017). The uniform control of bud burst is important in woody perennial spe- cies because it helps to synchronizing flowering, fruit set, and harvest. Evidence is presented in support of a role for oxygen‐dependent signalling in the coordination of the resumption of transcriptional and metabolic processes during bud burst in grapevine after a period of dormancy (Meitha et al., 2017). The release from physiological hypoxia and associated transcript profiles are documented over the first 24 hr period during the resumption of growth in postdormant buds. Evidence is provided in support of oxygen‐dependent signalling via the grapevine VII ethylene response factors during the transition from quiescence to bud burst (Meitha et al., 2017). Arab Water Council (2009). Vulnerability of arid and semi‐arid regions to climate change—Impacts and adaptive strategies. Perspectives on Water and Climate Change Adaptation, 9, 1–16. Bredow, M., Tomalty, H. E., Smith, L., & Walker, V. K. (2017). Ice and anti‐ nucleating activities of an ice‐binding protein from the annual grass, Brachypodium distachyon. Plant, Cell & Environment, 41. 983–992. Charrier, G., Chuine, I., Bonhomme, M. & Améglio, T. (2017). Assessing frost damages using dynamic models in walnut trees: Exposure rather than vulnerability controls frost risks. Plant, Cell & Environment, 41, 1008–1021. Christidis, N., Jones, G. S., & Scott, P. A. (2014). Dramatically increasing chance of extremely hot summers since the 2003 European heatwave. Nature Climate Change, 5, 46–50. 883 EDITORIAL G., Sillmann, J., Carrao, H., Dunbar, M. B., … Vogt, J. V. (2014). Magnitude of extreme heat waves in present cli- mate and their projection in a warming world. Journal of Geophysical Research: Atmospheres, 119, 12500–12512. Kathuria, H., Giri, J., Tyagi, H., & Tyagi, A. K. (2007). Advances in transgenic rice biotechnology. Critical Reviews in Plant Sciences, 26, 65–103. 884 EDITORIAL Wang, W., Vinocur, B., & Altman, A. (2003). Plant response to drought, salinity, and extreme temperatures: Towards genetic engineering for stress tolerance. Planta, 218, 1–14. Song, L.‐X., Xu, X.‐C., Wang, F.‐N., Wang, Y., Xia, X. J., Shi, K., … Yu, J. Q. (2017). Brassinosteroids act as a positive regulator for resistance against root knot nematode involving RESPIRATORY BURST OXIDASE HOMOLOG‐dependent activation of MAPKs in tomato. Plant, Cell & Environment, 41, 1113–1125. Xia, X.‐J., Fang, P.‐P., Guo, X., Qian, X.‐J., Zhou, J., Shi, K., … Yu, J.‐Q. (2017). Brassinosteroid‐mediated apoplastic H2O2‐glutaredoxin 12/14 cascade regulates antioxidant capacity in response to chilling in tomato. Plant, Cell & Environment, 41, 1052–1064. Townsend, A., Ware, M., & Ruban, A. (2017). Dynamic interplay between photodamage and photoprotection in photosystem II. Plant, Cell & Environment, 41, 1098–1112. Zhou, H., Finkemeier, I., Guan, W., Tossounian, M., Wei, B., Young, D., … Foyer, C. H. (2017). Oxidative stress‐triggered interactions between the succinyl‐ and acetyl‐proteomes of rice leaves. Plant, Cell & Environment, 41, 1139–1153. Vinocur, B., & Altman, A. (2005). Recent advances in engineering plant tolerance to abiotic stress: Achievements and limitations. Current Opin- ion in Plant Science, 16, 1–10. Vriet, C., Russinova, E., & Reuzeau, C. (2012). Boosting crop yields with plant steroids. The Plant Cell, 24, 842–857. Zhou, J., Xia, X. J., Zhou, Y. H., Shi, K., Chen, Z., & Yu, J. Q. (2014). RBOH1‐ dependent H2O2 production and subsequent activation of MPK1/2 play an important role in acclimation‐induced cross‐tolerance in tomato. Journal of Experimental Botany, 65, 595–607. Walker, B.J., VanLoocke, A., Bernacchi, C.J., &, Ort, D.R. (2016). The costs of photorespiration to food production now and in the future. Annual Review of Plant Biology, 67, 107–129.
https://openalex.org/W2981556946	https://www.zora.uzh.ch/id/eprint/175966/1/PhysRevD.100.084043.pdf	English	null	Gravitational-wave amplitudes for compact binaries in eccentric orbits at the third post-Newtonian order: Memory contributions	Physical review. D/Physical review. D.	2,019	cc-by	19,003	Zurich Open Repository and Archive Zurich Open Repository and Archive University of Zurich University Library Strickhofstrasse 39 CH-8057 Zurich www.zora.uzh.ch University of Zurich University Library Strickhofstrasse 39 CH-8057 Zurich www.zora.uzh.ch Year: 2019 I. INTRODUCTION object populations in globular clusters and galactic nuclei [25]. The observation of the first gravitational-wave (GW) signal by LIGO and Virgo opened up the new field of gravitational-wave astronomy [1–4]. So far, ten confirmed binary black hole mergers and one binary neutron star coalescence have been reported [5–10]. KAGRA [11] is expected to join the global network of detectors later this year, followed by LIGO-India in 2025 [12], leading to improved parameter estimation and source localization. These ground-based detectors are sensitive to the deca- hertz–kilohertz frequency of the GW spectrum. In the future, the space-based detector LISA [13] will probe lower frequencies (around the millihertz range), and pulsar timing arrays (PTAs) may measure ultralow (nanohertz) frequency GWs [14]. As soon as LISA is operational, it will be able to observe compact binaries in our galaxy emitting GWs of much lower frequency. At this point they still are expected to have moderate eccentricities [26,27]. On the other hand, LISA should be able to detect supermassive black hole binaries forming in the aftermath of galaxy mergers. Notably, triple-induced coalescences are expected to have large eccentricities that remain significant until merger [28–32]. The above anticipated prospects of future GW observa- tions have motivated the development of eccentric wave- form models. In the inspiraling phase one usually uses the post-Newtonian (PN) formalism to model the dynamics of the binary. This introduces three distinct time scales. The first two, the orbital and periastron precession time scales, are associated with the conservative dynamics and commonly described by the quasi-Keplerian parametriza- tion [33,34]. The third time scale appears when the dissipative radiation-reaction effects are taken into account [35,36]. Several waveform models have been built using this description of a binary [18,37–46]. In general, the far- zone gravitational radiation field receives instantaneous and hereditary contributions. The instantaneous part is deter- mined by the state of its source at a given retarded time, while the hereditary part depends on the entire dynamical history of the source. In particular, the latter contains tail and memory pieces. Currently (and this will most probably not change in the future), compact binaries are the most important sources of observable GW signals. The events detected so far have all been found using circular templates. However, we know that binaries with substantial eccentricity exist, e.g., the Hulse-Taylor binary with an eccentricity of e ∼0.6 [15]. Gravitational-wave amplitudes for compact binaries in eccentric orbits at the third post-Newtonian order: Memory contributions Gravitational-wave amplitudes for compact binaries in eccentric orbits at the third post-Newtonian order: Memory contributions rsold, Michael ; Boetzel, Yannick ; Faye, Guillaume ; Mishra, Chandra Kant ; Iyer, Bala R ; Jetzer, Philippe Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-175966 Journal Article Published Version The following work is licensed under a Creative Commons: Attribution 4.0 International (CC BY 4.0) License. Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-175966 Journal Article Published Version The following work is licensed under a Creative Commons: Attribution 4.0 I Originally published at: Ebersold, Michael; Boetzel, Yannick; Faye, Guillaume; Mishra, Chandra Kant; Iyer, Bala R; Jetzer, Philippe (2019). Gravitational-wave amplitudes for compact binaries in eccentric orbits at the third post-Newtonian order: Mem- ory contributions. Physical review D, 100(8):084043. DOI: https://doi.org/10.1103/physrevd.100.084043 PHYSICAL REVIEW D 100, 084043 (2019) (Received 14 June 2019; published 21 October 2019) We compute the nonlinear memory contributions to the gravitational-wave amplitudes for compact binaries in eccentric orbits at the third post-Newtonian (3PN) order in general relativity. These contributions are hereditary in nature as they are sourced by gravitational waves emitted during the binary’s entire dynamical past. Combining these with already available instantaneous and tail contributions, we get the complete 3PN accurate gravitational waveform. Gravitational-wave amplitudes for compact binaries in eccentric orbits at the third post-Newtonian order: Memory contributions Michael Ebersold ,1 Yannick Boetzel,1 Guillaume Faye,2 Chandra Kant Mishra,3 Bala R. Iyer,4 and Philippe Jetzer1 1Physik-Institut, Universität Zürich, Winterthurerstrasse 190, 8057 Zürich 2GℝεℂO, Institut d’Astrophysique de Paris, UMR 7095, CNRS, Sorbonne Universit´e, 98bis boulevard Arago, 75014 Paris, France 3Department of Physics, Indian Institute of Technology, Madras, Chennai 600036, India 4International Centre for Theoretical Sciences, Tata Institute of Fundamental Research, Bangalore 560089, India g , , 3Department of Physics, Indian Institute of Technology, Madras, Chennai 600036, India 4International Centre for Theoretical Sciences, Tata Institute of Fundamental Research, Bangalore 560089, India DOI: 10.1103/PhysRevD.100.084043 A. Memory contribution to the mass multipole moments Here we briefly state the essentials of the memory calculation. The conventions and notations used are the same as those outlined in Sec. II of Paper I. The gravitational waveform polarizations can be uniquely decomposed into the spherical harmonic modes hlm via hþ −ih× ¼ X ∞ l¼2 X l m¼−l hlmYlm −2 ðΘ; ΦÞ; ð2Þ ð2Þ where the basis is formed by the spin-weighted spherical harmonics Ylm −2 ðΘ; ΦÞ and the amplitude modes hlm ¼ − G ﬃﬃﬃ 2 p Rclþ2 Ulm −i c Vlm ð3Þ For circular binaries, the nonlinear, nonoscillatory memory contributions to the waveform were computed at the 3PN order in Ref. [62]. Regarding eccentric binaries, the leading-order zero-frequency or the so-called direct current (DC) memory terms were obtained in Ref. [63]. In this paper, we extend these computations to the 3PN level by computing all terms coming from the memory contri- bution to the radiative mass multipoles. Note that this yields not only the “genuine” DC memory, but also oscillatory contributions. In the circular limit, the latter have been computed in Ref. [64]. Due to complicated hereditary integrals, we calculate the memory contributions within a small-eccentricity expansion. We present all of our results in modified harmonic (MH) gauge in terms of the post- Newtonian parameter x ¼ ðGm ¯ω=c3Þ2=3 and the eccentric- ity e ¼ ¯et, with ¯ω ¼ ð1 þ ¯kÞ¯n being the orbital frequency and ¯n ¼ 2π=P the mean motion. With the instantaneous contributions already available [65], and the tail and post- adiabatic contributions computed in a companion paper [66]—hereafter called Paper I—this work aims to complete the knowledge of the 3PN waveform valid during the early inspiral of eccentric binary systems. ð3Þ are given in terms of radiative mass and current multipoles, Ulm and Vlm. These contain both instantaneous and hereditary parts. In the latter, we can further distinguish between tail and memory contributions (some of which may actually be tail induced) at the 3PN order by schematically writing Ulm ¼ Ulm inst þ Ulm tail þ Ulm mem þ δUlm; ð4aÞ Vlm ¼ Vlm inst þ Vlm tail þ δVlm; ð4bÞ ð4aÞ ð4bÞ where δUlm and δVlm represent possible higher-order hereditary terms. Note that there is no memory contribution to the radiative current-type moments [51]. Now, employ- ing the multipolar post-Minkowskian post-Newtonian (PN) formalism, the radiative moments can be written in terms of the source moments. I. INTRODUCTION It arises from GWs sourced by previously emitted GWs. Since the nonlinear memory is not produced directly by the source but rather by its radiation, it is present in all sources of GWs. From a more theoretical perspective, the memory effect and its variants can be interpreted in terms of conserved charges at null infinity and “soft theorems” [53,54]. Several methods to look for the memory effect have been devised. PTAs would observe a sudden change in the pulse frequency of a pulsar [55–58] and ground-based detectors like LIGO—although not sensitive enough to the memory of a single event—could allow for a detection from the accumulation of several events. [59–61]. In an ideal, freely falling GW detector the GW memory causes a permanent displacement after the GW has passed. There are two main types of GW memory. The linear memory [47] originates from a net change in the time derivatives of the source multipole moments between early and late times, present mainly in unbound (e.g., hyperbolic binary) systems. For bound systems the linear memory is negligible, as long as the components were formed, captured, or underwent mass loss long before the GW- driven regime. The nonlinear memory, also called the “Christodoulou memory” [48–52], is a phenomenon directly related to the nonlinearity of general relativity. It arises from GWs sourced by previously emitted GWs. Since the nonlinear memory is not produced directly by the source but rather by its radiation, it is present in all sources of GWs. From a more theoretical perspective, the memory effect and its variants can be interpreted in terms of conserved charges at null infinity and “soft theorems” [53,54]. Several methods to look for the memory effect have been devised. PTAs would observe a sudden change in the pulse frequency of a pulsar [55–58] and ground-based detectors like LIGO—although not sensitive enough to the memory of a single event—could allow for a detection from the accumulation of several events. [59–61]. I. INTRODUCTION Nonetheless, at the time this binary enters the detection band of ground-based GW detectors, it will have circular- ized to a negligible e ∼10−5 and not be distinguishable from a circular binary with current detector sensitivity [16– 18]. In particular, in globular clusters and galactic nuclei there are expected to be binaries with non-negligible eccentricity (e > 0.1) emitting detectable GWs [19–24]. Hence, the detection of GWs from eccentric compact binaries could provide important information on compact In this work, we concentrate on the memory contribu- tions to the waveform from eccentric binaries. Normally we think of gravitational waves as oscillatory perturbations 2470-0010=2019=100(8)=084043(23) 084043-1 © 2019 American Physical Society PHYS. REV. D 100, 084043 (2019) MICHAEL EBERSOLD et al. propagating on the background metric at the speed of light. However, all GW sources are subject to the so-called gravitational-wave memory effect, which manifests in a difference of the observed GW amplitudes at late and early times: This paper is structured as follows. In Sec. II we discuss how the nonlinear memory arises from the gravitational- wave energy flux and how it can be computed by integrating this flux over the binary’s past history. In Sec. III we explicitly evaluate the past-history integrals, which lead to two types of memory terms—DC memory and oscillatory memory—that are discussed separately in Secs. III B and III C. We next combine our results with the already available instantaneous and tail contributions and discuss the full 3PN waveform in Sec. IV. In Sec. V we give a brief summary and conclude our work. Most expressions in this paper are presented only to leading order in eccentricity for convenience, though we provide the complete results to Oðe6Þ in a supplemental Mathematica notebook [67]. Δhmem ¼ lim t→þ∞hðtÞ −lim t→−∞hðtÞ: ð1Þ ð1Þ In an ideal, freely falling GW detector the GW memory causes a permanent displacement after the GW has passed. There are two main types of GW memory. The linear memory [47] originates from a net change in the time derivatives of the source multipole moments between early and late times, present mainly in unbound (e.g., hyperbolic binary) systems. For bound systems the linear memory is negligible, as long as the components were formed, captured, or underwent mass loss long before the GW- driven regime. The nonlinear memory, also called the “Christodoulou memory” [48–52], is a phenomenon directly related to the nonlinearity of general relativity. A. Memory contribution to the mass multipole moments These relations can be found in Sec. III A of Ref. [65] for the instantaneous parts, which only require the knowledge of the source motion at a given 084043-2 PHYS. REV. D 100, 084043 (2019) GRAVITATIONAL-WAVE AMPLITUDES FOR COMPACT … the memory contribution before integration over past history, may thus be expressed as moment in retarded time TR, and in Sec. II B of Paper I for the hereditary parts, which involve integrals over the entire dynamical past of the source. Ulmð1Þ mem ¼ clþ1R2 G ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ 2ðl −2Þ! ðl þ 2Þ! s X ∞ l0¼2 X ∞ l00¼2 X l0 m0¼−l0 X l00 m00¼−l00 × Gll0l00 mm0m00 _hl0m0 _¯h l00m00 ; ð8Þ The nonlinear memory may be expressed in terms of the time derivative of the gravitational waveform by solving one component of Einstein’s equations near future null infinity in Bondi coordinates [53,68]. In this approach, the complex wave amplitude hþ −ih× is decomposed into even-parity and odd-parity pieces, the former being parametrized by a scalar function of the retarded time TR and the angles ðΘ; ΦÞ, namely, ΦeðTR; Θ; ΦÞ ¼ P l≥0;jmj≤l Φlm e ðTRÞYlmðΘ; ΦÞ, where the Φlm e ðTRÞ turn out to be equal to ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ 2ðl −2Þ!=ðl þ 2Þ! p with our con- ventions. The memory then manifests itself as a low- frequency shift of those modes. Since this effect is sourced by GWs, the consequent change for Ulm mem is a functional of the gravitational-wave “flux”1 ð8Þ where Gll0l00 mm0m00 is the angular integral of a product of three spin-weighted spherical harmonics, Gll0l00 mm0m00 ¼ Z dΩ ¯YlmYl0m0 −2 ¯Yl00m00 −2 : ð9Þ ð9Þ Reference [70] provides an explicit formula for this integral: Gll0l00 mm0m00 ¼ ð−1Þmþm0 ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ ð2l þ 1Þð2l0 þ 1Þð2l00 þ 1Þ 4π r × l l0 l00 0 −2 2 l l0 l00 −m m0 −m00 : ð10Þ dEGW dtdΩ ≡c3R2 16πG ð_h2 þ þ _h2 ×Þ: ð5Þ ð5Þ ð10Þ More precisely, the nonlinear memory contribution to the radiative mass moment UlmðTRÞ is given by [53] The brackets denote the Wigner 3-j symbols. The brackets denote the Wigner 3-j symbols. Ulm mem ¼ 32π c2−l ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ ðl −2Þ! 2ðl þ 2Þ! s Z TR −∞ dt Z dΩ dEGW dtdΩ ¯YlmðΩÞ: ð6 1In Eq. (5), at future null infinity the product of R2 with the term between brackets reduces to NABNAB=2 in the notation of Ref. [68]. B. Instantaneous and tail parts of the spherical harmonic modes ð6Þ Remembering that the dominant modes correspond to the quadrupolar case l ¼ 2, with h2m ¼ Oðc−4Þ, we see from Eq. (8) that the memory integrands are of 2.5PN order. However, as discussed below, in addition to oscillatory complex exponentials the Ul0ð1Þ mem also contain nonoscilla- tory terms. Due to the integration over the past history, their contributions at times t ≤TR accumulate and enhance the result by a net factor c5. It follows that the leading memory effect in the polarizations actually arises at the relative Newtonian order. Thus, Eq. (8) implies that, as an input for the computation of the 3PN-accurate Ulmð1Þ mem , we need a priori all nonmemory hlm modes to 3PN order. It is in fact not surprising that part of the waveform is required to calculate the full waveform since the nonlinear memory originates from gravitational waves sourced by the energy flux of gravitational waves emitted in the past, as shown by Eq. (6). Note that the contribution from the memory to the memory itself turns out not to enter the waveform up to the 3PN order. In Ref. [62] it was argued that for circular binaries these contributions would appear at the 5PN level, though for eccentric binaries we find, by explicit calcu- lation, that these appear already at the 4PN order. This is due to additional oscillatory memory contributions that will be discussed in Sec. III C below. However, for the present work, these memory-of-memory terms can be safely ignored. This formula was first shown to hold at quadratic order in G [51] before its validity was extended to the general case (see also Ref. [69], which indicates how to perturbatively construct a radiative-type gauge in which the derivation proposed in Ref. [51] can be adapted in principle to arbitrarily high orders). We will start from Eq. (6) to compute the memory contributions to the GW amplitude to the 3PN order. Inserting the mode decomposition defined in Eq. (2) into Eq. III. COMPUTATION OF THE NONLINEAR MEMORY The instantaneous parts of the 3PN-accurate hlm modes describing inspiraling eccentric binaries have been com- puted in Ref. [65]. The tail contributions were derived in Paper I, as well as the post-adiabatic corrections to the instantaneous contributions. The instantaneous mode amplitudes from Ref. [65] are written in terms of the post-Newtonian parameter x and time eccentricity et, and are parametrized by the eccentric anomaly u. They are valid for arbitrary eccentricities, while the tail contributions in Paper I are given in a small-eccentricity expansion, para- metrized by the mean anomaly l. The same will hold for the memory parts. Inverting the 3PN-accurate Kepler equation by means of the solution developed in Ref. [71], the instantaneous terms can be parametrized by the mean anomaly as well. Instead of restating the quasi-Keplerian parametrization and the phasing formalism describing the dynamics of the binary, we refer the reader to Secs. II C and II D of Paper I where those aspects are summarized with the same conventions and notations. MICHAEL EBERSOLD et al. PHYS. REV. D 100, 084043 (2019) MICHAEL EBERSOLD et al. B. Instantaneous and tail parts of the spherical harmonic modes (5), we find the GW energy flux in terms of the time derivatives of the hlm modes: dEGW dtdΩ ¼ c3R2 16πG X ∞ l0¼2 X ∞ l00¼2 X l0 m0¼−l0 X l00 m00¼−l00 _hl0m0 _¯h l00m00 × Yl0m0 −2 ðθ; ϕÞ ¯Yl00m00 −2 ðθ; ϕÞ: ð7Þ dEGW dtdΩ ¼ c3R2 16πG X ∞ l0¼2 X ∞ l00¼2 X l0 m0¼−l0 X l00 m00¼−l00 _hl0m0 _¯h l00m00 × Yl0m0 −2 ðθ; ϕÞ ¯Yl00m00 −2 ðθ; ϕÞ: ð7Þ ð7Þ We insert this expression in turn into Eq. (6). The time derivative of the memory contribution to the mass multi- pole moment, Ulmð1Þ mem ¼ dUlm mem=dTR, which is nothing but 084043-3 084043-3 A. Memory contributions to the time derivative of the radiative moments The computation of the memory contributions to the radiative mass multipole using Eq. (8) involves products of the time derivatives of the hlm modes given in Eq. (11). These are obtained by expressing ψ in terms of ξ and λξ and applying the following time derivative operator: d dt ¼ n d dξ þ ð1 þ kÞ d dλξ þ dx dt d dx þ de dt d de ; ð14Þ ð14Þ where we have used the facts that dξ=dt ¼ dl=dt ¼ n and dλξ=dt ¼ dλ=dt ¼ ð1 þ kÞn to the required PN order. The secular time evolution of x and e is given, at leading order, by the formulas of Peters and Mathews [16,74] The hlm modes including instantaneous, tail, and post- adiabatic contributions are given in the following form: dx dt ¼ c3ν Gm x5 ð1 −e2Þ7=2 64 5 þ 584 15 e2 þ 74 15 e4 ; ð15aÞ de dt ¼ −c3ν Gm ex4 ð1 −e2Þ5=2 304 15 þ 121 15 e2 : ð15bÞ ð15aÞ hlm ¼ 8Gmν c2R x ﬃﬃﬃπ 5 r e−imψHlm; ð11Þ ð11Þ ð15bÞ where the Hlm are written in terms of the adiabatic post- Newtonian parameter x ≡¯x and time eccentricity e ≡¯et, and are parametrized by the angles ξ and ψ. See, for instance, Eq. (76) of Paper I for the dominant mode (h22) expression. The phase angles ξ and ψ arise naturally when applying a certain shift to the time coordinate aimed at eliminating the arbitrary constant x0 appearing in both the instantaneous and tail parts [72,73] through the redefinitions Note that they cause a 2.5PN correction, and thus the leading order is sufficient here. When computing the time derivatives of the amplitude modes hlm ∼xl=2=c2, we have the following leading-order PN scaling: _hlm ∼ðω=c2Þxl=2 ∼cxl=2þ3=2: ð16Þ ð16Þ As the dominant mode l ¼ 2 is of order cx5=2, the knowledge of the waveform to 3PN order requires modes up to l ¼ 8. According to this argument, the sums in As the dominant mode l ¼ 2 is of order cx5=2, the knowledge of the waveform to 3PN order requires modes up to l ¼ 8. GRAVITATIONAL-WAVE AMPLITUDES FOR COMPACT … PHYS. REV. D 100, 084043 (2019) As an example, we show the leading-order part of the 20-mode up to Oðe2Þ, which will represent the dominant memory contribution: separation and the orbital energy decreases solely due to the emission of gravitational waves. The explicit integrals appearing in Eq. (20) are of two different types. The first one consists of a product of x and e, each with some power p and q, respectively: U20ð1Þ mem ¼ − ﬃﬃﬃﬃﬃπ 15 r c5ν2 G x5 256 7 þ 5008e2 21 þ 768 7 ee−iξ þ 768 7 eeiξ þ 5176 21 e2e−2iξ þ 5176 21 e2e2iξ : ð18Þ Ul0 DC ∼ Z TR −∞ dtxpðtÞeqðtÞ: ð21Þ ð21Þ ð18Þ The leading Newtonian order corresponds to p ¼ 5. The possible values of the integer q range from 0—the quasicircular limit—to the order of the eccentricity expan- sion. These integrals give the nonoscillatory contributions to the waveform, i.e., the DC memory. Note that, as argued above, these terms are only present in the m ¼ 0 modes. The second type of integrals will lead to oscillatory terms appearing at 1.5PN, 2.5PN, and 3PN order in the wave- form. We will discuss these in Sec. III C. The leading Newtonian order corresponds to p ¼ 5. The possible values of the integer q range from 0—the quasicircular limit—to the order of the eccentricity expan- sion. These integrals give the nonoscillatory contributions to the waveform, i.e., the DC memory. Note that, as argued above, these terms are only present in the m ¼ 0 modes. The second type of integrals will lead to oscillatory terms appearing at 1.5PN, 2.5PN, and 3PN order in the wave- form. We will discuss these in Sec. III C. We observe two different type of terms: oscillatory terms proportional to e−im0ξ, and nonoscillatory ones, which give rise to the well-known leading-order DC memory. As argued above, the m ≠0 modes only contain oscil- latory terms since they are proportional to e−imλξ. For instance, the leading order of the 22-mode explicitly is U22ð1Þ mem ¼ − ﬃﬃﬃﬃﬃﬃ 2π 5 r c5ν2 G x5e−2iλξ 40 7 e2 −32 21 ee−iξ þ 32 21 eeiξ −172 21 e2e−2iξ þ 52 21 e2e2iξ : ð19Þ The strategy to evaluate the integral in Eq. GRAVITATIONAL-WAVE AMPLITUDES FOR COMPACT … (21) is to express the PN parameter x in terms of the eccentricity e and change the integration variable from time t to e, so that the integral runs from some initial eccentricity ei at early times to eðTRÞ at the current retarded time: ð19Þ However, in Eq. (8) (used for the calculation of the Ulmð1Þ mem ) _ l00 00 However, in Eq. (8) (used for the calculation of the U ð Þ mem ) there is no need to average the mode products _hl0m0 _¯h l00m00 over several wavelengths. Other derivations of the memory effect, making use of the Isaacson gravitational-wave stress-energy tensor [77], resort to such a procedure. In Ref. [63], which follows this approach, the orbital average entering the calculation of the leading-order eccentric memory effectively removes the terms proportional to eim0ξ in the Ul0ð1Þ mem , so that only the terms yielding the DC memory are left over, while the discarded pieces do not affect the amplitude of the DC memory. In the absence of the orbital average, these pieces lead to small-amplitude oscillatory contributions to the waveform, which here we will call oscillatory memory contributions. It would actually be difficult to introduce an orbital average in the m ≠0 modes because these terms oscillate not only on the orbital time scale, but also on the much longer precession time scale. Ul0 DC ∼ Z eðTRÞ ei de de dt −1 xpðeÞeq: ð22Þ ð22Þ The time evolution of x and e due to radiation reaction is stated to leading order in Eqs. (15a)–(15b). Here, we need the evolution equations up to 3PN order, which are provided in Appendix B. We form the ratio of the two equations, thereby canceling the time dependence, and expand the right-hand side in x and e. This yields a differential equation with the following structure: dx de ¼ fNðeÞx þ f1ðeÞx2 þ f1.5ðeÞx5=2 þ f2ðeÞx3 þ f2.5ðeÞx7=2 þ f3ðe; ln xÞx4: ð23Þ ð23Þ Here, the fiðeÞ terms represent the coefficients of xiþ1 in the expansion of dx=de, with fN ¼ f0. To solve this differential equation, we search for the unknown function xðeÞ in the form of a perturbative expansion, according to A. Memory contributions to the time derivative of the radiative moments According to this argument, the sums in ξ ¼ l −3GM c3 n ln x x0 0 ; ð12aÞ λξ ¼ λ −3GM c3 ð1 þ kÞn ln x x0 0 ; ð12bÞ ð12aÞ Eq. (8) consisting of products _hl0m0 _¯h l00m00 may be truncated at l0 ¼ l00 ¼ 8. Moreover, the appearance of the 3-j symbols in Eq. (10) imply some selection rules: the three lower entries have to add up to zero, i.e., m ¼ m0 −m00. Since the mode products appearing in Eq. (8) scale like λξ ¼ λ −3GM c3 ð1 þ kÞn ln x x0 0 ; ð12bÞ ð12bÞ _hl0m0 _¯h l00m00 ∼xn=2e−iðm0−m00Þλξ ; ð17Þ where M ¼ mð1 −νx=2Þ denotes the Arnowitt-Deser- Misner (ADM) mass, m ¼ m1 þ m2 is the total mass, ν ¼ m1m2=m2 is the symmetric mass ratio, and x0 0 is related to x0 by ð17Þ for some integer n, only memory modes with m ¼ 0 will contain DC terms, as was previously found for circular orbits [62]. The scaling being the same as in that case, ln x0 0 ¼ 11 18 −2 3 γE −4 3 ln 2 þ 2 3 ln x0; ð13Þ we have to compute the Ul0ð1Þ mem up to l ¼ 10. On the other hand, a mode separation property holds for planar orbits [75,76]: the hlm only depend on the mass (current) radiative moments if l þ m is even (odd). Thus, as there is no memory effect in the current radiative moment, there is no memory effect when l þ m is odd. ð13Þ with γE being Euler’s constant. We refer to the Appendix B of Paper I for the relations between the orbital elements (l, λ, ϕ) and their redefined counterparts (ξ, λξ, ψ). 084043-4 B. DC memory The next step consists in evaluating the hereditary time integral x ¼ xN þ ϵx1 þ ϵ3=2x1.5 þ ϵ2x2 þ ϵ5=2x2.5 þ ϵ3x3; ð24Þ ð24Þ Ulm mem ¼ Z TR −∞ dtUlmð1Þ mem : ð20Þ where ϵ is a formal parameter that allows one to keep track of the PN order. Inserting this expansion into Eq. (23) and identifying the coefficients of ϵi on the left- and right-hand sides of the resulting equation, we find the set of differential relations satisfied by the post-Newtonian orders of x. This system can be straightforwardly solved by quadrature. Putting the pieces together yields the PN parameter x as a function of eccentricity. At leading order in the PN and eccentricity expansion, we recover [35] ð20Þ To do so, we need a model for the secular evolution of the binary undergoing gravitational radiation-reaction forces. To do so, we need a model for the secular evolution of the binary undergoing gravitational radiation-reaction forces. The secular 3PN-order evolution equations of the orbital elements for a quasielliptical, inspiraling binary were obtained in Refs. [78–80]. This model is an idealization since it assumes that the two components start at infinite 084043-5 MICHAEL EBERSOLD et al. PHYS. REV. D 100, 084043 (2019) xðeÞ ¼ x0 e0 e 12=19 ; ð25Þ terms. A final Taylor expansion then yields the DC memory pieces of the mass multipole moments. ð25Þ We present the memory contributions to the spherical harmonic modes in the following form: where x0 is the value of x at some reference eccentricity e0. The full 3PN result to leading order in eccentricity is provided in Appendix B. Note that for the expansion in eccentricity to be valid, the eccentricity has to be small at all times, and hence e0 has to be small as well. hlm mem ¼ − G ﬃﬃﬃ 2 p clþ2R Ulm mem ¼ 8Gmν c2R x ﬃﬃﬃπ 5 r e−imψHlm mem: ð26Þ ð26Þ We are now in the position to insert the evolution equation for e and the solution for xðeÞ into Eq. (22). Expanding again in x and e yields elementary integrals, which must be calculated. We then reexpress this result in terms of the time- dependent quantities x and e by solving their relation [Eqs. B. DC memory (25) and (B8)] for x0 and reinsert the expression of this quantity in terms of x and e into the calculated memory With this convention, the memory pieces directly add to the waveform modes stated in Eq. (11). As the expressions are quite long, we present here only the H20 DC mode to 3PN and leading order in eccentricity: With this convention, the memory pieces directly add to the waveform modes stated in Eq. (11). C. Oscillatory memory ð31Þ Before considering the oscillatory integrals, let us recall some properties of the nonlinear memory. As mentioned at the beginning of Sec. II B, the memory contribution to the radiative mass multipole is formally of 2.5PN order. But due to the hereditary nature, the non- oscillatory terms are raised by 2.5PN orders to appear already at the Newtonian level. From the oscillatory terms we cannot expect the same behavior, due to the fact that the oscillations in the remote past effectively cancel each other out. Thus, we expect that only the recent past will contribute. Since p ¼ 5 at Newtonian order and the leading terms in the waveform are of order x, these integrals lead to 2.5PN contributions to the waveform. As we have expected, these kinds of terms oscillating on the orbital time scale keep their formal PN order, and we call them the fast oscillatory memory. Since p ¼ 5 at Newtonian order and the leading terms in the waveform are of order x, these integrals lead to 2.5PN contributions to the waveform. As we have expected, these kinds of terms oscillating on the orbital time scale keep their formal PN order, and we call them the fast oscillatory memory. On the other hand, for r ¼ −s we find Ulm osc ∼−i 3s xp−5=2 þ xp−3=2 −3 2 þ 7ν 3 × eqeisðλξ−ξÞ þ Oðeqþ2Þ: ð32Þ Examining the remaining oscillatory integrals, we notice that they are of the following form: ð32Þ Ulm osc ∼ Z TR −∞ dtxpðtÞeqðtÞeiðsλξþrξÞ: ð29Þ This corresponds to terms that oscillate solely on the periastron precession time scale, and we therefore call these terms the slow oscillatory memory. Because of the much slower oscillations, they are enhanced by 1PN order (corresponding to the PN order of precession) and enter the waveform at 1.5PN. Note also that in Eq. (32) eccentricity corrections of Oðeqþ2Þ appear, whereas Eq. (31) would This corresponds to terms that oscillate solely on the periastron precession time scale, and we therefore call these terms the slow oscillatory memory. Because of the much slower oscillations, they are enhanced by 1PN order (corresponding to the PN order of precession) and enter the waveform at 1.5PN. Note also that in Eq. (32) eccentricity corrections of Oðeqþ2Þ appear, whereas Eq. (31) would ð29Þ Note that we have s ¼ −m. B. DC memory REV. D 100, 084043 (2019) þ e ei 36=19 −142763304914707 25758100279296 þ 48901891428821ν 919932152832 −400181473249ν2 3650524416 þ 2295879173ν3 43458624 þ e ei 48=19385621605844415513 5740376633671680 −157405π2 25992 −3317γE 399 þ −49590995147570629 478364719472640 þ 1271π2 1216 ν þ 3194536246463ν2 34514049024 −1672948713ν3 45653504 −12091 ln 2 5985 −78003 ln 3 5320 −3317 ln x 798 −6634 2527 ln e ei : ð27gÞ ð27gÞ All nonzero DC memory modes are presented to leading order in eccentricity in Appendix D and to Oðe6Þ in the Supplemental Material [67]. All nonzero DC memory modes are presented to leading order in eccentricity in Appendix D and to Oðe6Þ in the Supplemental Material [67]. An important check is to take the circular limit of our calculated memory modes and compare to the circular 3PN memory modes computed in Ref. [62]. To illustrate this fact, we take the circular limit of the 20-mode stated in Eqs. (27) by setting e ¼ 0 and find p e ¼ 0 and find H20 DC ¼ − 5 14 ﬃﬃﬃ 6 p 1 þ x −4075 4032 þ 67ν 48 þ x2 −151877213 67060224 −123815ν 44352 þ 205ν2 352 þ x5=2 −253π 336 þ 253πν 84 þ x3 −4397711103307 532580106240 þ 700464542023 13948526592 −205π2 96 ν þ 69527951ν2 166053888 þ 1321981ν3 5930496 ; ð28Þ ð28Þ in perfect agreement with Eq. (4.3a) of Ref. [62]. The higher DC modes up to l ¼ 10 in the circular limit are consistent with Eq. (4.3) of Ref. [62] as well. Moreover, we can check the leading eccentricity part at Newtonian order against Eq. (2.35) in Ref. [63]. They are found to be equal. Note that at Newtonian order the computation of the DC memory is in principle possible for arbitrary eccentricities [see Eq. (2.34) in Ref. [63] ]; however, this becomes difficult at higher PN orders, especially when tail terms come into play. integral is essentially given by the contributions at the current time, we find Ulm osc ∼− i nðr þ sð1 þ kÞÞ xpeqeiðsλξþrξÞ; ð30Þ ð30Þ where the time dependence on TR is not written explicitly. Expanding the denominator, we have to distinguish between two different cases. The first applies if r ≠−s; we then find Ulm osc ∼− i r þ s xp−3=2eqeiðsλξþrξÞ: ð31Þ B. DC memory As the expressions are quite long, we present here only the H20 DC mode to 3PN and leading order in eccentricity: H20 DC ¼ − 5 14 ﬃﬃﬃ 6 p ðH20 Newt þ xH20 1PN þ x3=2H20 1.5PN þ x2H20 2PN þ x5=2H20 2.5PN þ x3H20 3PNÞ; ð27aÞ H20 Newt ¼ 1 − e ei 12=19 ; ð27bÞ H20 DC ¼ − 5 14 ﬃﬃﬃ 6 p ðH20 Newt þ xH20 1PN þ x3=2H20 1.5PN þ x2H20 2PN þ x5=2H20 2.5PN þ x3H20 3PNÞ; ð27aÞ ð27bÞ H20 1PN ¼ −4075 4032 þ 67ν 48 þ e ei 12=19 −2833 3192 þ 197ν 114 þ e ei 24=19145417 76608 −2849ν 912 ; ð27cÞ H20 1.5PN ¼ −377π 228 e ei 12=19 þ 377π 228 e ei 30=19 ; ð27dÞ 12=19 −2833 3192 þ 197ν 114 þ e ei 24=19145417 76608 −2849ν 912 ; ð27cÞ ð27cÞ H20 1.5PN ¼ −377π 228 e ei 12=19 þ 377π 228 e ei 30=19 ; ð27dÞ ð27dÞ H20 2PN ¼ −151877213 67060224 −123815ν 44352 þ 205ν2 352 þ e ei 12=19358353209 366799104 −738407ν 727776 −20597ν2 17328 þ e ei 24=19411966361 122266368 −825950ν 68229 þ 561253ν2 51984 þ e ei 36=19 −50392977379 24208740864 þ 764295307ν 48033216 −11654209ν2 1143648 ; ð27eÞ ð27eÞ H20 2.5PN ¼ −253π 336 þ 253πν 84 þ e ei 12=193763903π 7277760 þ 12788779πν 1819440 þ e ei 24=1954822209π 8733312 −1074073πν 103968 þ e ei 30=195340205π 1455552 −371345πν 51984 þ e ei 42=19 −424020733π 43666560 þ 27049187πν 3638880 ; ð27fÞ ð27fÞ H20 3PN ¼ −4397711103307 532580106240 þ 700464542023 13948526592 −205π2 96 ν þ 69527951ν2 166053888 þ 1321981ν3 5930496 þ e ei 12=19 −4942027570449143 96592876047360 −81025π2 103968 þ 3317γE 399 þ −10309531979 7466981760 þ 3977π2 3648 ν þ 267351733ν2 82966464 þ 772583ν3 2222316 þ 12091 ln 2 5985 þ 78003 ln 3 5320 þ 3317 ln x 798 þ 710645π2 103968 e ei 30=19 þ e ei 24=19 −31102835980319 14049872879616 þ 279737759653ν 167260391424 þ 26730466283ν2 1991195136 −397176241ν3 23704704 H20 3PN ¼ −4397711103307 532580106240 þ 700464542023 13948526592 −205π2 96 ν þ 69527951ν2 166053888 þ 1321981ν3 5930496 þ e ei 12=19 −4942027570449143 96592876047360 −81025π2 103968 þ 3317γE 399 þ −10309531979 7466981760 þ 3977π2 3648 ν þ 267351733ν2 82966464 þ 772583ν3 2222316 þ 12091 ln 2 5985 þ 78003 ln 3 5320 þ 3317 ln x 798 þ 710645π2 103968 e ei 30=19 þ e ei 24=19 −31102835980319 14049872879616 þ 279737759653ν 167260391424 þ 26730466283ν2 1991195136 −397176241ν3 23704704 084043-6 GRAVITATIONAL-WAVE AMPLITUDES FOR COMPACT … PHYS. C. Oscillatory memory Here we provide a formula to evaluate these integrals, its derivation is presented in Appendix C. Using the fact that λξ ¼ ð1 þ kÞξ and ξ ¼ nt to the required PN order as well as the notion that the 084043-7 PHYS. REV. D 100, 084043 (2019) MICHAEL EBERSOLD et al. only be affected by eccentricity corrections starting at 3.5PN order. only be affected by eccentricity corrections starting at 3.5PN order. We present the waveform in terms of the secular evolving PN parameter ¯x and the time eccentricity ¯e, parametrized by the angles ξ and ψ. We refer to Sec. V C of Paper I for their definition, and to Appendix B therein for various relations between the orbital elements (l, λ, ϕ) and (ξ, λξ, ψ). The secular evolution of the parameters ¯x and ¯e is given in Appendix B. The spherical harmonic modes describing the waveform are then written in the following form: We provide the oscillatory memory contributions to the spherical harmonic modes in the same form as for the DC memory, according to Eq. (26). Besides the DC memory contribution, the 20-mode also contains fast oscillatory memory at 2.5PN: H20 osc ¼ i16 ﬃﬃﬃ 6 p 7 νex5=2 −e−iξ þeiξ −647 576ee−2iξ þ647 576ee2iξ : ð33Þ H20 osc ¼ i16 ﬃﬃﬃ 6 p 7 νex5=2 −e−iξ þeiξ −647 576ee−2iξ þ647 576ee2iξ hlm ¼ 8Gmν c2R ¯x ﬃﬃﬃπ 5 r e−imψHlm: ð35Þ ð35Þ ð33Þ Modes with m < 0 can be calculated from Note that while the DC memory is purely real and therefore only affects the plus polarization (with the usual conven- tions on the polarization triad), the oscillatory contributions influence both polarizations. hl−m ¼ ð−1Þl ¯hlm: ð36Þ ð36Þ In general, the individual modes can be split into three types of contributions: In the m ≠0 modes, only the oscillatory memory is present. C. Oscillatory memory For the dominant 22-mode we find Hlm ¼ Hlm inst þ Hlm hered þ Hlm post-ad: ð37Þ ð37Þ H22 osc ¼ ie2νe2iξ −13 252x3=2 þ 697 336 −865ν 216 x5=2 −29π 126 x3 þ 21ix5=2eν 19 6 e þ 4 3e−iξ −4eiξ þ 65 24ee−2iξ : ð34aÞ The instantaneous terms depend only on the instantaneous state of the source at a given retarded time, with contri- butions at different orders relative to the leading order for each mode given as Hlm inst ¼ ðHlm instÞLead þ ðHlm instÞ1PN þ ðHlm instÞ1.5PN þ ðHlm instÞ2PN þ ðHlm instÞ2.5PN þ ðHlm instÞ3PN: ð38Þ ð38Þ Here the slow oscillatory part in the first and second lines is proportional to e2iξ, as we factored out e−2iψ according to Eq. (26). Three different PN orders of slow oscillatory memory terms appear in this mode. The first one at 1.5PN arises from the leading-order memory contribution to the radiative mass multipole at 2.5PN, so as expected it is enhanced by one post-Newtonian order. At 2.5PN, there is the 1PN correction to the first term as well as a part coming from the 1PN correction to the multipole. Finally, at 3PN there is a term originating from the 1.5PN correction to the memory part of the multipole; this corresponds to the memory of the gravitational-wave tail. The terms in the second line correspond to fast oscillatory memory entering at the 2.5PN level. These are given in terms of x, e, and u in Eqs. (5.09)–(5.11) and Eq. (A1) of Ref. [65]. The parametrization in terms of u has to be transformed to ξ using Eq. (B2b) in Paper I. These are given in terms of x, e, and u in Eqs. (5.09)–(5.11) and Eq. (A1) of Ref. [65]. The parametrization in terms of u The post-adiabatic contributions are introduced by radiation-reaction corrections to the quasi-Keplerian para- metrization, at relative 2.5PN order: Hlm post-ad ¼ ðHlm post-adÞ2.5PN: ð39Þ ð39Þ They are given in Eqs. (66)–(67) of Paper I. They are given in Eqs. (66)–(67) of Paper I. The hereditary contributions, on the other hand, depend on the entire dynamical past of the binary system. They can be further split into tail and memory parts: Hlm hered ¼ Hlm tail þ Hlm mem: ð40Þ GRAVITATIONAL-WAVE AMPLITUDES FOR COMPACT … (40) and Eq. (76) of Paper I, due to additional memory terms not yet considered in Paper I. Complete expressions for all modes to Oðe6Þ are given in the Supplemental Material [67]. ki h i i l li i f d d ib d i S f h i il Note here the difference at 2.5PN order between Eq. (40) and Eq. (76) of Paper I, due to additional memory terms not yet considered in Paper I. Complete expressions for all modes to Oðe6Þ are given in the Supplemental Material [67]. By taking the quasicircular limit of our modes as described in Sec. V E of Paper I, we can compare the instantaneous, tail, and (fast) oscillatory memory contributions of our waveform modes with Ref. [64] and the DC memory terms with Ref. [62]. In all of them we find perfect agreement. By taking the quasicircular limit of our modes as described in Sec. V E of Paper I, we can compare the instantaneous, tail, and (fast) oscillatory memory contributions of our waveform modes with Ref. [64] and the DC memory terms with Ref. [62]. In all of them we find perfect agreement. GRAVITATIONAL-WAVE AMPLITUDES FOR COMPACT … Hlm osc ¼ ðHlm slow oscÞ1.5PN þ ðHlm slow oscÞ2.5PN þ ðHlm slow oscÞ3PN þ ðHlm fast oscÞ2.5PN: ð44Þ DC memory enters the waveform in the m ¼ 0 modes at all relative orders, ð44Þ Slow oscillatory memory is due to the double-periodic nature of eccentric motion and is not present in quasicir- cular binary systems. All memory modes are computed in this paper and are listed in Appendixes D and E. IV. FULL 3PN ECCENTRIC WAVEFORM ð40Þ In this section we summarize the results necessary to construct the full waveform for eccentric binaries at third post-Newtonian order, including all instantaneous, hereditary, and post-adiabatic contributions, as described in Sec. V of Paper I. Rather than listing the lengthy expressions, we give an overview at which PN order the individual terms enter the waveform and where they can be found. Explicit expressions for all spherical harmonic modes are given in a supplemental Mathematica notebook [67]. For the tails we find contributions at different orders relative to the leading order for each mode as Hlm tail ¼ ðHlm tailÞ1.5PN þ ðHlm tailÞ2.5PN þ ðHlm tailÞ3PN: ð41Þ ð41Þ These are given in Eqs. (47)–(48) of Paper I. These are given in Eqs. (47)–(48) of Paper I. These are given in Eqs. (47) (48) of Paper I. There is both DC memory and oscillatory memory: There is both DC memory and oscillatory memory: Hlm mem ¼ Hl0 DC þ Hlm osc: ð42Þ ð42Þ 084043-8 PHYS. REV. D 100, 084043 (2019) GRAVITATIONAL-WAVE AMPLITUDES FOR COMPACT … Hl0 DC ¼ ðHl0 DCÞLead þ ðHl0 DCÞ1PN þ ðHl0 DCÞ1.5PN þ ðHl0 DCÞ2PN þ ðHl0 DCÞ2.5PN þ ðHl0 DCÞ3PN; ð43Þ ð43Þ As an example, we present here the dominant H22 mode including all contributions to OðeÞ: while slow and fast oscillatory memory enter as while slow and fast oscillatory memory enter as ð45aÞ H22 Newt ¼ 1 þ e 4 e−iξ þ 4 eiξ ; ð45aÞ H22 1PN ¼ ¯x −107 42 þ 55ν 42 þ ¯e e−iξ −257 168 þ 169ν 168 þ eiξ −31 24 þ 35ν 24 ; ð45bÞ H22 1.5PN ¼ ¯x3=2 2π þ ¯e e−iξ 11π 4 þ 27i 2 ln 3 2 þ eiξ 13π 4 þ 3i 2 lnð2Þ ; ð45cÞ 257 168 þ 169ν 168 þ eiξ −31 24 þ 35ν 24 ; ð45bÞ 7i 2 ln 3 2 þ eiξ 13π 4 þ 3i 2 lnð2Þ ; ð45cÞ H22 1PN ¼ ¯x −107 42 þ 55ν 42 þ ¯e e−iξ −257 168 þ 169ν 168 þ eiξ −31 24 þ 35ν 24 ; ð45bÞ H22 1.5PN ¼ ¯x3=2 2π þ ¯e e−iξ 11π 4 þ 27i 2 ln 3 2 þ eiξ 13π 4 þ 3i 2 lnð2Þ ; ð45cÞ ð45bÞ ð45cÞ H22 2PN ¼ ¯x2 −2173 1512 −1069ν 216 þ 2047ν2 1512 þ ¯e eiξ −2155 252 −1655ν 672 þ 371ν2 288 þ e−iξ −4271 756 −35131ν 6048 þ 421ν2 864 ; ð45dÞ ð45dÞ ð45dÞ H22 2.5PN ¼ ¯x5=2 −107π 21 þ −24i þ 34π 21 ν þ ¯e eiξ −9i 2 þ 229π 168 þ −14579i 140 þ 61π 42 ν þ 473i 28 −3iν 7 lnð2Þ þ e−iξ −27i 2 −1081π 168 þ −1291i 180 þ 137π 42 ν þ 27i 4 þ 9iν ln 3 2 ; ð45eÞ ð45eÞ H22 3PN ¼ ¯x3 27027409 646800 þ 428iπ 105 þ 2π2 3 −856γE 105 þ −278185 33264 þ 41π2 96 ν −20261ν2 2772 þ 114635ν3 99792 −1712 lnð2Þ 105 −428 lnð¯xÞ 105 þ ¯e e−iξ 219775769 1663200 þ 749iπ 60 þ 49π2 24 −749γE 30 þ −121717 20790 −41π2 192 ν −86531ν2 8316 −33331ν3 399168 þ −2889 70 þ 81iπ 2 ln 3 2 −81 2 ln2 3 2 −749 lnð2Þ 15 −749 lnð¯xÞ 60 þ eiξ 55608313 1058400 þ 3103iπ 420 þ 29π2 24 −3103γE 210 þ −199855 3024 þ 41π2 48 ν −9967ν2 1008 þ 35579ν3 36288 þ −6527 210 þ 3iπ 2 lnð2Þ þ 3ln2ð2Þ 2 −3103 lnð¯xÞ 420 : ð45fÞ ð45fÞ Note here the difference at 2.5PN order between Eq. V. BRIEF SUMMARY In this paper we computed the memory contribution to the gravitational waveform from nonspinning compact binaries in eccentric orbits at the third post-Newtonian order. Our results complete the previous work on the instantaneous parts [65] 084043-9 084043-9 PHYS. REV. D 100, 084043 (2019) MICHAEL EBERSOLD et al. ACKNOWLEDGMENTS We thank Marc Favata for an early review and useful comments. We also thank Maria Haney and Achamveedu Gopakumar for insightful discussions and comments, as well as Luc Blanchet for stimulating discussions. M. E. and Y. B. are supported by the Swiss National Science Foundation. Y. B. is supported by a Forschungskredit of the University of Zurich, Grant No. FK-18-084. Umem ij ðTRÞ ¼ G c5 Z TR −∞ dτ −2 7 Mð3Þ ahiðτÞMð3Þ jiaðτÞ þ G c7 Z TR −∞ dτ −5 756 Mð4Þ ab ðτÞMð4Þ ijabðτÞ −32 63 Sð3Þ ahiðτÞSð3Þ jiaðτÞ þ εabhi 5 42 Sð4Þ jibcðτÞMð3Þ ac ðτÞ −20 189 Mð4Þ jibcðτÞSð3Þ ac ðτÞ ; ðA1aÞ ðA1aÞ Umem ijk ðTRÞ ¼ G c5 Z TR −∞ dτ −1 3 Mð3Þ ahiðτÞMð4Þ jkiaðτÞ −4 5 εabhiMð3Þ ja ðτÞSð3Þ kibðτÞ ; ðA1bÞ Umem ijk ðTRÞ ¼ G c5 Z TR −∞ dτ −1 3 Mð3Þ ahiðτÞMð4Þ jkiaðτÞ −4 5 εabhiMð3Þ ja ðτÞSð3Þ kibðτÞ ; ðA1bÞ ðτÞMð4Þ jkiaðτÞ −4 5 εabhiMð3Þ ja ðτÞSð3Þ kibðτÞ ; ðA1bÞ ðA1bÞ Umem ijkl ðTRÞ ¼ G c3 Z TR −∞ dτ 2 5 Mð3Þ hij ðτÞMð3Þ kliðτÞ þ G c5 Z TR −∞ dτ 12 55 Mð4Þ ahiðτÞMð4Þ jkliaðτÞ −14 99 Mð4Þ ahijðτÞMð4Þ kliaðτÞ þ 32 45 Sð3Þ hijðτÞSð3Þ kliðτÞ þ εabhi −4 5 Mð3Þ ja ðτÞSð4Þ klibðτÞ þ 32 45 Sð3Þ ja ðτÞMð4Þ klibðτÞ ; ðA1cÞ Umem ijklmðTRÞ ¼ G c3 Z TR −∞ dτ 20 21 Mð3Þ hijðτÞMð4Þ klmiðτÞ ; ðA1dÞ Umem ijklmnðTRÞ ¼ G c3 Z TR −∞ dτ 5 7 Mð4Þ hijkðτÞMð4Þ lmniðτÞ −15 14 Mð3Þ hijðτÞMð4Þ klmniðτÞ : ðA1eÞ Umem ijklmðTRÞ ¼ G c3 Z TR −∞ dτ 20 21 Mð3Þ hijðτÞMð4Þ klmiðτÞ ; ðA1dÞ Umem ijklmnðTRÞ ¼ G c3 Z TR −∞ dτ 5 7 Mð4Þ hijkðτÞMð4Þ lmniðτÞ −15 14 Mð3Þ hijðτÞMð4Þ klmniðτÞ : ðA1eÞ ðA1eÞ Note that the symmetric trace-free (STF) projection h…i only applies to the free indices ijk…. The integrand in those equations consists of products of canonical mass and current moments, MðnÞ L ðτÞ and SðnÞ L ðτÞ, and the superscript in brackets stands for the nth derivative with respect to τ. APPENDIX A: COMPUTATION OF THE MEMORY VIA THE RADIATIVE MASS MULTIPOLES and on the tail and post-adiabatic contributions [66]. These waveforms form the basis for the construction of increas- ingly accurate GW templates from binary systems in eccentric orbits. The computation of the nonlinear memory in the paper is done effectively via the GW energy flux with the formula given in Eq. (8). An alternative way is to directly compute the required moments of the memory contribution to the radiative mass multipole. The leading-order memory piece of the mass quadrupole moment contributes at 2.5PN; however, due to the hereditary integral the DC terms are raised by 2.5PN orders such that they contribute at leading order in the waveform polarization. Reference [81] lists the memory contributions up to 3.5PN. From this we are able to compute the DC memory to 1PN accuracy. The hereditary integral enhances the slow oscillatory memory terms by 1PN; therefore, by knowing the 3.5PN contribu- tion to the mass moments we find the leading-order 2.5PN terms contributing at 1.5PN and 2.5PN in the waveform, and that the 3PN terms appear at 2PN and 3PN and the 3.5PN terms at 2.5PN. However, what we miss are the 4PN terms that appear in the waveform at 3PN level. On the other hand, the fast oscillatory memory is not affected by the hereditary integral in its PN order, and we recover it at 2.5PN and 3PN. The required memory contributions at 3.5PN to the radiative mass moments are There are two fundamentally different types of memory. DC memory is a slowly increasing, nonoscillatory con- tribution to the gravitational-wave amplitude, entering at Newtonian order, leading to a difference in the amplitude between early and late times. Oscillatory memory, on the other hand, enters at higher PN orders as a normal periodic contribution. Due to the double-periodic nature of the eccentric motion, slow oscillatory memory contributions on the periastron precession time scale are enhanced by a factor of 1PN, and thus already enter the waveform at 1.5PN order. This is unlike the quasicircular case, where oscillatory memory only enters at 2.5PN order. ACKNOWLEDGMENTS The canonical moments are related by a gauge transformation to the source moments IL and JL along with some more gauge moments that enter at 2.5PN in the δIL; δJL terms, Note that the symmetric trace-free (STF) projection h…i only applies to the free indices ijk…. The integrand in those equations consists of products of canonical mass and current moments, MðnÞ L ðτÞ and SðnÞ L ðτÞ, and the superscript in brackets stands for the nth derivative with respect to τ. The canonical moments are related by a gauge transformation to the source moments IL and JL along with some more gauge moments that enter at 2.5PN in the δIL; δJL terms, ML ¼ IL þ GδIL þ OðG2Þ; ðA2aÞ ðA2aÞ ML ¼ IL þ GδIL þ OðG2Þ; 084043-10 GRAVITATIONAL-WAVE AMPLITUDES FOR COMPACT … The 1PN mass octupole is The 1PN mass octupole is Iijk ¼ −νmΔ B1xhijki þ B2 r_r c2 xhijvki þ B3 r2 c2 xhivjki ; ðA5Þ Iijk ¼ −νmΔ B1xhijki þ B2 r_r c2 xhijvki þ B3 r2 c2 xhivjki ; 084043-10 GRAVITATIONAL-WAVE AMPLITUDES FOR COMPACT … PHYS. REV. D 100, 084043 (2019) GRAVITATIONAL-WAVE AMPLITUDES FOR COMPACT … C1 ¼ 1 þ 1 c2 v2 13 28 −17ν 7 þ Gm r 27 14 þ 15ν 7 ; ðA9aÞ C2 ¼ 5 28 ð1 −2νÞ; ðA9bÞ SL ¼ JL þ GδJL þ OðG2Þ: ðA2bÞ SL ¼ JL þ GδJL þ OðG2Þ: ðA2bÞ For our purpose of calculating the memory contribution to next-to-leading order, we only need the 1PN part of the source moments. Here we list the relevant source moments at 1PN for two nonspinning compact objects in general orbits [65]. The source moments are written in terms of xi and vi, which denote the binary’s relative separation and relative velocity. Moreover, r is the distance between the two objects, and thus r ¼ jxj and _r is the radial velocity. For the mass quadrupole moment we have C2 ¼ 5 28 ð1 −2νÞ; ðA9bÞ ðA9bÞ and finally the leading order of the current octupole is Jijk ¼ νmεabhixjkiavbð1 −3νÞ: ðA10Þ ðA10Þ Having the source moments in hand (and thus in our case also the canonical moments), we can calculate the products of time derivatives of the canonical moments occurring in the integrands of Eq. (A1a)–(A1e). Before treating the hereditary integral, we transform from the STF moments Umem L computed here to the scalar version of the radiative mass moments using Eq. (4) of Paper I. These are the same moments that we find when computing the memory with Eq. (8). The hereditary integral is evaluated in the same way as described in Secs. III B and III C. Using this method, we find the 1PN DC memory and the 1PN oscillatory memory. Be aware that the DC memory appears in the waveform at leading Newtonian order, while the first slow oscillatory memory terms appear at 1.5PN and the fast oscillatory memory at 2.5PN. Iij ¼ νm A1xhixji þ A2 r_r c2 xhixji þ A3 r2 c2 vhivji ; ðA3Þ where where A1 ¼ 1 þ 1 c2 v2 29 42 −29ν 14 þ Gm r −5 7 þ 8ν 7 ; ðA4aÞ A2 ¼ −4 7 þ 12ν 7 ; ðA4bÞ A3 ¼ 11 21 −11ν 7 : ðA4cÞ ðA4bÞ ðA4cÞ This method of computing the memory contribution serves as a check. We can compare the relative 1PN pieces of the DC and oscillatory memory calculated before and here, and they are found to be in perfect agreement. APPENDIX B: RADIATION-REACTION EVOLUTION EQUATIONS ðA5Þ where In this Appendix we provide the secular 3PN-accurate evolution equations for x and e [78–80] in MH gauge. The instantaneous terms are exact, whereas the eccentricity enhancement functions appearing in the hereditary con- tributions are given in an eccentricity expansion. We begin by listing the pieces needed for the evolution of x: B1 ¼1þ 1 c2 v2 5 6−19ν 6 þGm r −5 6þ13ν 6 ; ðA6aÞ ðA6aÞ B2 ¼ −ð1 −2νÞ; ðA6bÞ B3 ¼ 1 −2ν; ðA6cÞ B2 ¼ −ð1 −2νÞ; ðA6bÞ ðA6bÞ B2 ¼ −ð1 −2νÞ; dx dt ¼2c3νx5 3Gm ðXNewt þxX1PN þx2X2PN þx3X3PN þX heredÞ; ðB1Þ where dx dt ¼2c3νx5 3Gm ðXNewt þxX1PN þx2X2PN þx3X3PN þX heredÞ; ðB1Þ and Δ ¼ ðm1 −m2Þ=m is the mass difference ratio. APPENDIX B: RADIATION-REACTION EVOLUTION EQUATIONS X2PN ¼ 1 ð1 −e2Þ11=2 −11257 945 þ 15677ν 105 þ 944ν2 15 þ e2 −2960801 945 −2781ν 5 þ 182387ν2 90 þ e4 −68647 1260 −1150631ν 140 þ 396443ν2 72 þ e6 925073 336 −199939ν 48 þ 192943ν2 90 þ e8 391457 3360 −6037ν 56 þ 2923ν2 45 þ ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ 1 −e2 p 48 −96ν 5 þ e2 2134 −4268ν 5 þ e4 2193 −4386ν 5 þ e6 175 2 −35ν ; ðB2cÞ ðB2cÞ X3PN ¼ 1 ð1 −e2Þ13=2 614389219 148500 þ −57265081 11340 þ 369π2 2 ν −16073ν2 140 −1121ν3 27 þ e2 19769277811 693000 þ 66358561 3240 þ 42571π2 80 ν −3161701ν2 840 −1287385ν3 324 þ e4 −3983966927 8316000 þ 6451690597 90720 −12403π2 64 ν þ 34877019ν2 1120 −33769597ν3 1296 þ e6 −4548320963 5544000 þ −59823689 4032 −242563π2 640 ν þ 411401857ν2 6720 −3200965ν3 108 þ e8 19593451667 2464000 þ −6614711 480 −12177π2 640 ν þ 92762ν2 7 −982645ν3 162 2464000 480 640 7 162 þ e10 33332681 197120 −1874543ν 10080 þ 109733ν2 840 −8288ν3 81 þ ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ 1 −e2 p −1425319 1125 þ 9874 105 −41π2 10 ν þ 632ν2 5 þ e2 933454 375 þ −2257181 63 þ 45961π2 240 ν þ 125278ν2 15 þ e4 840635951 21000 þ −4927789 60 þ 6191π2 32 ν þ 317273ν2 15 þ e6 702667207 31500 þ −6830419 252 þ 287π2 960 ν þ 232177ν2 30 þ e8 56403 112 −427733ν 840 þ 4739ν2 30 þ log xð1 þ ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ 1 −e2 p Þ x0ð2ð1 −e2ÞÞ 54784 175 þ 465664e2 105 þ 4426376e4 525 þ 1498856e6 525 þ 31779e8 350 ; ðB2dÞ Xhered ¼ 96 5 4πx3=2φðeÞ þ πx5=2 −4159 6 2 ψωðeÞ −189 8 νζωðeÞ ðB2dÞ Xhered ¼ 96 5 4πx3=2φðeÞ þ πx5=2 −4159 672 ψωðeÞ −189 8 νζωðeÞ þ x3 −116761 3675 κðeÞ þ 16π2 3 −1712γE 105 −1712 105 log 4x3=2 x0 FðeÞ : ðB2eÞ ðB2eÞ The helper functions appearing in the hereditary contribution are given by pearing in the hereditary contribution are given by The helper functions appearing in the hereditary contribution are given by The helper functions appearing in the hereditary contribution are given by ψωðeÞ ¼ 1344 4159 1 ð1 −e2Þ3=2 h ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ 1 −e2 p ð1 −5e2ÞφðeÞ −4˜φðeÞ i þ 8191 4159 ψðeÞ; ð ψωðeÞ ¼ 1344 4159 1 ð1 −e2Þ3=2 h ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ 1 −e2 p ð1 −5e2ÞφðeÞ −4˜φðeÞ i þ 8191 4159 ψðeÞ; ðB3aÞ ζωðeÞ ¼ 583 567 ζðeÞ −16 567 φðeÞ: ðB3bÞ ðB3aÞ ζωðeÞ ¼ 583 567 ζðeÞ −16 567 φðeÞ: ðB3bÞ ðB3bÞ The various enhancement functions appearing in these equations are listed below. APPENDIX B: RADIATION-REACTION EVOLUTION EQUATIONS PHYS. REV. D 100, 084043 (2019) PHYS. REV. D 100, 084043 (2019) MICHAEL EBERSOLD et al. APPENDIX B: RADIATION-REACTION EVOLUTION EQUATIONS Moreover, we need also the leading-Newtonian-order part of the mass hexadecapole, where where of the mass hexadecapole, Iijkl ¼ νmxhijklið1 −3νÞ: ðA7Þ From the current source moments we need the quadrupole, which is Jij ¼ −νmΔ C1εabhixjiavb þ C2 r_r c2 εabhivjibxa ; ðA8Þ where XNewt ¼ 1 ð1 −e2Þ7=2 96 5 þ 292e2 5 þ 37e4 5 ; ðB2aÞ X1PN ¼ 1 ð1 −e2Þ9=2 −1486 35 −264ν 5 þ e2 2193 7 −570ν þ e4 12217 20 −5061ν 10 þ e6 11717 280 −148ν 5 ; ðB2bÞ XNewt ¼ 1 ð1 −e2Þ7=2 96 5 þ 292e2 5 þ 37e4 5 ; ðB2aÞ X1PN ¼ 1 ð1 −e2Þ9=2 −1486 35 −264ν 5 þ e2 2193 7 −570ν þ e4 12217 20 −5061ν 10 þ e6 11717 280 −148ν 5 ; ðB2bÞ p Iijkl ¼ νmxhijklið1 −3νÞ: ðA7Þ urrent source moments we need the quadrupole, XNewt ¼ 1 ð1 −e2Þ7=2 96 5 þ 292e2 5 þ 37e4 5 ; ðB2aÞ X1PN ¼ 1 ð1 −e2Þ9=2 −1486 35 −264ν 5 þ e2 2193 7 −570ν Iijkl ¼ νmxhijklið1 −3νÞ: ðA7Þ XNewt ¼ 1 ð1 −e2Þ7=2 96 5 þ 292e2 5 þ 37e4 5 ; ðB2aÞ Iijkl ¼ νmxhijklið1 −3νÞ: ðA7Þ ðA7Þ ðB2aÞ From the current source moments we need the quadrupole, which is X1PN ¼ 1 ð1 −e2Þ9=2 −1486 35 −264ν 5 þ e2 2193 7 −570ν From the current source moments we need the quadrupole, which is Jij ¼ −νmΔ C1εabhixjiavb þ C2 r_r c2 εabhivjibxa ; ðA8Þ where ð1 e Þ 35 5 7 þ e4 12217 20 −5061ν 10 þ e6 11717 280 −148ν 5 ; ðB2bÞ Jij ¼ −νmΔ C1εabhixjiavb þ C2 r_r c2 εabhivjibxa ; ðA8Þ where 084043-11 084043-11 084043-11 084043-11 X2PN ¼ 1 ð1 −e2Þ11=2 −11257 945 þ 15677ν 105 þ 944ν2 15 þ e2 −2960801 945 −2781ν 5 þ 182387ν2 90 þ e4 −68647 1260 −1150631ν 140 þ 396443ν2 72 þ e6 925073 336 −199939ν 48 þ 192943ν2 90 þ e8 391457 3360 −6037ν 56 þ 2923ν2 45 þ ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ 1 −e2 p 48 −96ν 5 þ e2 2134 −4268ν 5 þ e4 2193 −4386ν 5 þ e6 175 2 −35ν ; ðB2cÞ X3PN ¼ 1 ð1 −e2Þ13=2 614389219 148500 þ −57265081 11340 þ 369π2 2 ν −16073ν2 140 −1121ν3 27 þ e2 19769277811 693000 þ 66358561 3240 þ 42571π2 80 ν −3161701ν2 840 −1287385ν3 324 þ e4 −3983966927 8316000 þ 6451690597 90720 −12403π2 64 ν þ 34877019ν2 1120 −33769597ν3 1296 þ e6 −4548320963 5544000 þ −59823689 4032 −242563π2 640 ν þ 411401857ν2 6720 −3200965ν3 108 þ e8 19593451667 2464000 þ −6614711 480 −12177π2 640 ν þ 92762ν2 7 −982645ν3 162 þ e10 33332681 197120 −1874543ν 10080 þ 109733ν2 840 −8288ν3 81 þ ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ 1 −e2 p −1425319 1125 þ 9874 105 −41π2 10 ν þ 632ν2 5 þ e2 933454 375 þ −2257181 63 þ 45961π2 240 ν þ 125278ν2 15 þ e4 840635951 21000 þ −4927789 60 þ 6191π2 32 ν þ 317273ν2 15 þ e6 702667207 31500 þ −6830419 252 þ 287π2 960 ν þ 232177ν2 30 þ e8 56403 112 −427733ν 840 þ 4739ν2 30 þ log xð1 þ ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ 1 −e2 p Þ x0ð2ð1 −e2ÞÞ 54784 175 þ 465664e2 105 þ 4426376e4 525 þ 1498856e6 525 þ 31779e8 350 ; ðB2dÞ Xhered ¼ 96 5 4πx3=2φðeÞ þ πx5=2 −4159 672 ψωðeÞ −189 8 νζωðeÞ þ x3 −116761 3675 κðeÞ þ 16π2 3 −1712γE 105 −1712 105 log 4x3=2 x0 FðeÞ : ðB2eÞ MICHAEL EBERSOLD et al. APPENDIX B: RADIATION-REACTION EVOLUTION EQUATIONS The various enhancement functions appearing in these equations are listed below. The various enhancement functions appearing in these equations are listed below. Next we state the evolution equation for the eccentricity. Note that we observed errors in the 2PN- and 3PN-order expressions in Eqs (C10) and (C11) of Ref [80] These are likely due to the fact that only the relation between eMH and pp g q Next we state the evolution equation for the eccentricity. Note that we observed errors in the 2PN- and 3PN-order expressions in Eqs. (C10) and (C11) of Ref. [80]. These are likely due to the fact that only the relation between eMH and eADM was inserted, but one also has to transform deADM=dt to deMH=dt, PHYS. REV. APPENDIX B: RADIATION-REACTION EVOLUTION EQUATIONS D 100, 084043 (2019) GRAVITATIONAL-WAVE AMPLITUDES FOR COMPACT … de dt ¼ −c3νex4 Gm ðEN þ xE1PN þ x2E2PN þ x3E3PN þ EheredÞ; ðB4Þ where ENewt ¼ 1 ð1 −e2Þ5=2 304 15 þ 121e2 15 ; ðB5aÞ E1PN ¼ 1 ð1 −e2Þ7=2 −939 35 −4084ν 45 þ e2 29917 105 −7753 30 ν þ e4 13929 280 −1664ν 45 ; ðB5bÞ E2PN ¼ 1 ð1 −e2Þ9=2 −949877 1890 þ 18763ν 42 þ 752ν2 5 þ e2 −3082783 2520 −988423ν 840 þ 64433ν2 40 þ e4 23289859 15120 −13018711ν 5040 þ 127411ν2 90 þ e6 420727 3360 −362071ν 2520 þ 821ν2 9 þ ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ 1 −e2 p 1336 3 −2672ν 15 þ e2 2321 2 −2321ν 5 þ e4 565 6 −113ν 3 ; ðB5cÞ E3PN ¼ 1 ð1 −e2Þ11=2 54208557619 6237000 þ 50099023 113400 þ 779π2 10 ν −4088921ν2 2520 −61001ν3 486 þ e2 46226320013 6237000 þ 28141879 900 −139031π2 960 ν −21283907ν2 3024 −86910509ν3 19440 þ e4 −116987170177 16632000 þ 11499615139 907200 −271871π2 1920 ν þ 61093675ν2 4032 −2223241ν3 180 þ e6 5891934893 1232000 þ −5028323 560 −6519π2 640 ν þ 24757667ν2 2520 −11792069ν3 2430 þ e8 302322169 1774080 −1921387ν 10080 þ 41179ν2 216 −193396ν3 1215 þ ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ 1 −e2 p −22713049 15750 þ −5526991 945 þ 8323π2 180 ν þ 54332ν2 45 þ e2 89395687 7875 þ −38295557 1260 þ 94177π2 960 ν þ 681989ν2 90 þ e4 5321445613 378000 þ −26478311 1512 þ 2501π2 2880 ν þ 225106ν2 45 þ e6 186961 336 −289691ν 504 þ 3197ν2 18 þ 730168 23625ð1 þ ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ 1 −e2 p Þ þ 304 15 82283 1995 þ 297674 1995 e2 þ 1147147 15960 e4 þ 61311 21280 e6 ln xð1 þ ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ 1 −e2 p Þ 2x0ð1 −e2Þ ; ðB5dÞ , ( ) de dt ¼ −c3νex4 Gm ðEN þ xE1PN þ x2E2PN þ x3E3PN þ EheredÞ; de dt ¼ −c3νex4 Gm ðEN þ xE1PN þ x2E2PN þ x3E3PN þ EheredÞ; ðB4Þ ðB4Þ where ENewt ¼ 1 ð1 −e2Þ5=2 304 15 þ 121e2 15 ; ðB5aÞ E1PN ¼ 1 ð1 −e2Þ7=2 −939 35 −4084ν 45 þ e2 29917 105 −7753 30 ν þ e4 13929 280 −1664ν 45 ; ðB5bÞ ðB5aÞ E1PN ¼ 1 ð1 −e2Þ7=2 −939 35 −4084ν 45 þ e2 29917 105 −7753 30 ν þ e4 13929 280 −1664ν 45 ; ðB5bÞ ðB5bÞ E2PN ¼ 1 ð1 −e2Þ9=2 −949877 1890 þ 18763ν 42 þ 752ν2 5 þ e2 −3082783 2520 −988423ν 840 þ 64433ν2 40 þ e4 23289859 15120 −13018711ν 5040 þ 127411ν2 90 þ e6 420727 3360 −362071ν 2520 þ 821ν2 9 þ ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ 1 −e2 p 1336 3 −2672ν 15 þ e2 2321 2 −2321ν 5 þ e4 565 6 −113ν 3 ; ðB5cÞ þ 15120 5040 þ 90 þ 3360 2520 þ 9 þ ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ 1 −e2 p 1336 3 −2672ν 15 þ e2 2321 2 −2321ν 5 þ e4 565 6 −113ν 3 ; ðB5cÞ E3PN ¼ 1 ð1 −e2Þ11=2 54208557619 6237000 þ 50099023 113400 þ 779π2 10 ν −4088921ν2 2520 −61001ν3 486 þ e2 46226320013 6237000 þ 28141879 900 −139031π2 960 ν −21283907ν2 3024 −86910509ν3 19440 þ e4 −116987170177 16632000 þ 11499615139 907200 −271871π2 1920 ν þ 61093675ν2 4032 −2223241ν3 180 þ e6 5891934893 1232000 þ −5028323 560 −6519π2 640 ν þ 24757667ν2 2520 −11792069ν3 2430 þ e8 302322169 1774080 −1921387ν 10080 þ 41179ν2 216 −193396ν3 1215 þ ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ 1 −e2 p −22713049 15750 þ −5526991 945 þ 8323π2 180 ν þ 54332ν2 45 þ e2 89395687 7875 þ −38295557 1260 þ 94177π2 960 ν þ 681989ν2 90 þ e4 5321445613 378000 þ −26478311 1512 þ 2501π2 2880 ν þ 225106ν2 45 þ e6 186961 336 −289691ν 504 þ 3197ν2 18 þ 730168 23625ð1 þ ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ 1 −e2 p Þ þ 304 15 82283 1995 þ 297674 1995 e2 þ 1147147 15960 e4 þ 61311 21280 e6 ln xð1 þ ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ 1 −e2 p Þ 2x0ð1 −e2Þ ; ðB5dÞ Ehered ¼ −32 5 −985 48 πx3=2φeðeÞ þ πx5=2 55691 1344 ψeðeÞ þ 19067 126 νζeðeÞ þ x3 89789209 352800 −87419 ln 2 630 þ 78003 ln 3 560 κeðeÞ −769 96 16π2 3 −1712γE 105 −1712 105 ln 4x3=2 x0 FeðeÞ : ðB5eÞ ðB5cÞ E3PN ¼ 1 ð1 −e2Þ11=2 54208557619 6237000 þ 50099023 113400 þ 779π2 10 ν −4088921ν2 2520 −61001ν3 486 þ e2 46226320013 6237000 þ 28141879 900 −139031π2 960 ν −21283907ν2 3024 −86910509ν3 19440 þ e4 −116987170177 16632000 þ 11499615139 907200 −271871π2 1920 ν þ 61093675ν2 4032 −2223241ν3 180 þ e6 5891934893 1232000 þ −5028323 560 −6519π2 640 ν þ 24757667ν2 2520 −11792069ν3 2430 þ e8 302322169 1774080 −1921387ν 10080 þ 41179ν2 216 −193396ν3 1215 þ ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ 1 −e2 p −22713049 15750 þ −5526991 945 þ 8323π2 180 ν þ 54332ν2 45 þ e2 89395687 7875 þ −38295557 1260 þ 94177π2 960 ν þ 681989ν2 90 þ e4 5321445613 378000 þ −26478311 1512 þ 2501π2 2880 ν þ 225106ν2 45 þ e6 186961 336 −289691ν 504 þ 3197ν2 18 þ 730168 23625ð1 þ ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ 1 −e2 p Þ þ 304 15 82283 1995 þ 297674 1995 e2 þ 1147147 15960 e4 þ 61311 21280 e6 ln xð1 þ ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ 1 −e2 p Þ 2x0ð1 −e2Þ ; ðB5dÞ Ehered ¼ −32 5 −985 48 πx3=2φeðeÞ þ πx5=2 55691 1344 ψeðeÞ þ 19067 126 νζeðeÞ þ x3 89789209 352800 −87419 ln 2 630 þ 78003 ln 3 560 κeðeÞ ðB5dÞ −32 5 −985 48 πx3=2φeðeÞ þ πx5=2 55691 1344 ψeðeÞ þ 19067 126 νζeðeÞ þ x3 89789209 352800 −87419 ln 2 630 þ 78003 ln 3 560 κeðeÞ −769 96 16π2 3 −1712γE 105 −1712 105 ln 4x3=2 x0 FeðeÞ : ðB5eÞ 084043-13 084043-13 084043-13 MICHAEL EBERSOLD et al. APPENDIX B: RADIATION-REACTION EVOLUTION EQUATIONS PHYS. REV. D 100, 084043 (2019) The additional functions in the hereditary contribution are The additional functions in the hereditary contribution are The additional functions in the hereditary contribution are φeðeÞ ¼ 192 985 ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ 1 −e2 p e2 h ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ 1 −e2 p φðeÞ −˜φðeÞ i ; ðB6aÞ ψeðeÞ ¼ 18816 55691 1 e2 ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ 1 −e2 p ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ 1 −e2 p 1 −11e2 7 φðeÞ − 1 −3 7 e2 ˜φðeÞ þ 16382 55691 ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ 1 −e2 p e2 h ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ 1 −e2 p ψðeÞ −˜ψðeÞ i ; ð Þ φðeÞ −˜φðeÞ i ; ðB6aÞ ðB6aÞ ðB6bÞ ζeðeÞ ¼ 924 19067 1 e2 ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ 1 −e2 p −ð1 −e2Þ3=2φðeÞ þ 1 −5 11 e2 ˜φðeÞ þ 12243 76268 ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ 1 −e2 p e2 h ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ 1 −e2 p ζðeÞ −˜ζðeÞ i ; ðB6cÞ ζeðeÞ ¼ 924 19067 1 e2 ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ 1 −e2 p −ð1 −e2Þ3=2φðeÞ þ 1 −5 11 e2 ˜φðeÞ þ 12243 76268 ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ 1 −e2 p e2 h ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ 1 −e2 p ζðeÞ −˜ζðeÞ i ; ðB6cÞ κeðeÞ ¼ ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ 1 −e2 p e2 h ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ 1 −e2 p κðeÞ −˜κðeÞ i769 96 −3059665 700566 ln 2 þ 8190315 1868176 ln 3 −1 ; ðB6dÞ FeðeÞ ¼ 96 769 ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ 1 −e2 p e2 h ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ 1 −e2 p FðeÞ −˜FðeÞ i : ðB6eÞ κeðeÞ ¼ ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ 1 −e2 p e2 h ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ 1 −e2 p κðeÞ −˜κðeÞ i769 96 −3059665 700566 ln 2 þ 8190315 1868176 ln 3 −1 ; ðB6dÞ ðB6dÞ FeðeÞ ¼ 96 769 ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ 1 −e2 p e2 h ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ 1 −e2 p FðeÞ −˜FðeÞ i : ðB6eÞ FðeÞ −˜FðeÞ i : ðB6eÞ ðB6eÞ The eccentricity enhancement functions arise from hereditary contributions to the energy flux (nontilde) and the angular momentum flux (tilde). Most of them do not admit closed forms and have to be computed numerically or in a small-eccentricity expansion. GRAVITATIONAL-WAVE AMPLITUDES FOR COMPACT … Here we provide xðeÞ at 3PN and to leading order in eccentricity: ðB8Þ xðeÞ ¼ xNewt þ x1PN þ x1.5PN þ x2PN þ x2.5PN þ x3PN; ðB8Þ xðeÞ ¼ xNewt þ x1PN þ x1.5PN þ x2PN þ x2.5PN þ x3PN; ðB8Þ where where xNewt ¼ x0 e0 e 12=19 ; ðB9aÞ x1PN ¼ x2 0 e0 e 24=19 −2833 3192 þ 197ν 114 þ e0 e 12=192833 3192 −197ν 114 ; ðB9bÞ ðB9aÞ 3 2 þ 197ν 114 þ e0 e 12=192833 3192 −197ν 114 ; ðB9bÞ x1PN ¼ x2 0 e0 e 24=19 −2833 3192 þ 197ν 114 þ e0 e 12=192833 3192 −197ν 114 ; x1PN ¼ x2 0 e0 e 24=19 −2833 3192 þ 197ν 114 þ e0 e 12=192833 3192 −197ν 114 ; ðB9bÞ ðB9bÞ x1.5PN ¼ x5=2 0 377π 228 e0 e 12=19 −377π 228 e0 e 30=19 ; x1.5PN ¼ x5=2 0 377π 228 e0 e 12=19 −377π 228 e0 e 30=19 ; ðB9cÞ ðB9cÞ x2PN ¼ x3 0 e0 e 12=19 −358353209 366799104 þ 738407ν 727776 þ 20597ν2 17328 þ e0 e 24=19 −8025889 5094432 þ 558101ν 90972 −38809ν2 6498 þ e0 e 36=19936217217 366799104 −578135ν 80864 þ 248681ν2 51984 ; ðB9dÞ ðB9dÞ x2.5PN ¼ x7=2 0 e0 e 12=19 −3763903π 7277760 −12788779πν 1819440 þ e0 e 24=19 −1068041π 363888 þ 74269πν 12996 þ e0 e 30=19 −5340205π 1455552 þ 371345πν 51984 þ e0 e 42=1912956437π 1819440 −2651489πν 454860 ; ðB9eÞ x2.5PN ¼ x7=2 0 e0 e 12=19 −3763903π 7277760 −12788779πν 1819440 þ e0 e 24=19 −1068041π 363888 þ 74269πν 12996 þ e0 e 30=19 −5340205π 1455552 þ 371345πν 51984 þ e0 e 42=1912956437π 1819440 −2651489πν 454860 ; ðB9eÞ ðB9eÞ x3PN ¼ x4 0 e0 e 12=194942027570449143 96592876047360 þ 81025π2 103968 −3317γE 399 −12091 ln 2 5985 −78003 ln 3 5320 −3317 ln x0 798 þ 10309531979 7466981760 −3977π2 3648 ν −267351733ν2 82966464 −772583ν3 2222316 þ e0 e 30=19 −710645π2 103968 þ e0 e 24=19605942457431 585411369984 −3267214507ν 2986792704 −543796927ν2 82966464 þ 27463573ν3 2963088 þ e0 e 36=192652303375761 390274246656 −449767537459ν 13938365952 þ 2754579983ν2 55310976 −48990157ν3 1975392 þ e0 e 48=19 −1628129474693173 27597964584960 þ 157405π2 25992 þ 3317γE 399 þ 12091 ln 2 5985 þ 78003 ln 3 5320 þ 6634 2527 ln e0 e þ 3317 ln x0 798 þ 6686551181963 209075489280 þ 3977π2 3648 ν −6641442629ν2 165932928 þ 282310639ν3 17778528 : ðB9fÞ ðB9fÞ APPENDIX B: RADIATION-REACTION EVOLUTION EQUATIONS REV. D 100, 084043 (2019) GRAVITATIONAL-WAVE AMPLITUDES FOR COMPACT … GRAVITATIONAL-WAVE AMPLITUDES FOR COMPACT … PHYS. REV. D 100, 084043 (2019) ˜FðeÞ ¼ 1 þ 389 32 e2 þ 3577 64 e4 þ 43049 256 e6: ðB7jÞ ðB7jÞ By dividing the evolution equations for x and e and expanding in these variables, we can find a solution for the evolution of x in terms of e at each order as described in Sec. III B. Here we provide xðeÞ at 3PN and to leading order in eccentricity: By dividing the evolution equations for x and e and expanding in these variables, we can find a solution for the evolution of x in terms of e at each order as described in Sec. III B. APPENDIX B: RADIATION-REACTION EVOLUTION EQUATIONS Here we list them in an eccentricity expansion to Oðe6Þ: φðeÞ ¼ 1 þ 2335 192 e2 þ 42955 768 e4 þ 6204647 36864 e6; ðB7aÞ ˜φðeÞ ¼ 1 þ 209 32 e2 þ 2415 128 e4 þ 730751 18432 e6; ðB7bÞ ψðeÞ ¼ 1 −22988 8191 e2 −36508643 524224 e4 −1741390565 4718016 e6; ðB7cÞ ˜ψðeÞ ¼ 1 −17416 8191 e2 −14199197 524224 e4 −467169215 4718016 e6; ðB7dÞ κðeÞ ¼ 1 þ e2 62 3 −4613840 350283 ln 2 þ 24570945 1868176 ln 3 þ e4 9177 64 þ 271636085 1401132 ln 2 −466847955 7472704 ln 3 þ e6 76615 128 −4553279605 2802264 ln 2 þ 14144674005 119563264 ln 3 þ 914306640625 1076069376 ln 5 ; ðB7eÞ ˜κðeÞ ¼ 1 þ e2 389 32 −2056005 233522 ln 2 þ 8190315 934088 ln 3 þ e4 3577 64 þ 50149295 467044 ln 2 −155615985 3736352 ln 3 þ e6 43049 256 −12561332945 16813584 ln 2 þ 4709431125 59781632 ln 3 þ 182861328125 538034688 ln 5 ; ðB7fÞ ζðeÞ ¼ 1 þ 1011565 48972 e2 þ 106573021 783552 e4 þ 456977827 854784 e6; ðB7gÞ ˜ζðeÞ ¼ 1 þ 102371 8162 e2 þ 14250725 261184 e4 þ 722230667 4701312 e6; ðB7hÞ FðeÞ ¼ 1 þ 62 3 e2 þ 9177 64 e4 þ 76615 128 e6; ðB7iÞ φðeÞ ¼ 1 þ 2335 192 e2 þ 42955 768 e4 þ 6204647 36864 e6; ˜φðeÞ ¼ 1 þ 209 32 e2 þ 2415 128 e4 þ 730751 18432 e6; φðeÞ ¼ 1 þ 2335 192 e2 þ 42955 768 e4 þ 6204647 36864 e6; ðB7aÞ ˜φðeÞ ¼ 1 þ 209 32 e2 þ 2415 128 e4 þ 730751 18432 e6; ðB7bÞ ψðeÞ ¼ 1 −22988 8191 e2 −36508643 524224 e4 −1741390565 4718016 e6; ðB7cÞ ˜ψðeÞ ¼ 1 −17416 8191 e2 −14199197 524224 e4 −467169215 4718016 e6; ðB7dÞ κðeÞ ¼ 1 þ e2 62 3 −4613840 350283 ln 2 þ 24570945 1868176 ln 3 þ e4 9177 64 þ 271636085 1401132 ln 2 −466847955 7472704 ln 3 þ e6 76615 128 −4553279605 2802264 ln 2 þ 14144674005 119563264 ln 3 þ 914306640625 1076069376 ln 5 ; ðB7eÞ ˜κðeÞ ¼ 1 þ e2 389 32 −2056005 233522 ln 2 þ 8190315 934088 ln 3 þ e4 3577 64 þ 50149295 467044 ln 2 −155615985 3736352 ln 3 þ e6 43049 256 −12561332945 16813584 ln 2 þ 4709431125 59781632 ln 3 þ 182861328125 538034688 ln 5 ; ðB7fÞ ζðeÞ ¼ 1 þ 1011565 48972 e2 þ 106573021 783552 e4 þ 456977827 854784 e6; ðB7gÞ ˜ζðeÞ ¼ 1 þ 102371 8162 e2 þ 14250725 261184 e4 þ 722230667 4701312 e6; ðB7hÞ FðeÞ ¼ 1 þ 62 3 e2 þ 9177 64 e4 þ 76615 128 e6; ðB7iÞ φðeÞ ¼ 1 þ 2335 192 e2 þ 42955 768 e4 þ 6204647 36864 e6; ðB7aÞ ˜φðeÞ ¼ 1 þ 209 32 e2 þ 2415 128 e4 þ 730751 18432 e6; ðB7bÞ ψðeÞ ¼ 1 −22988 8191 e2 −36508643 524224 e4 −1741390565 4718016 e6; ðB7cÞ ˜ψðeÞ ¼ 1 −17416 8191 e2 −14199197 524224 e4 −467169215 4718016 e6; ðB7dÞ κðeÞ ¼ 1 þ e2 62 3 −4613840 350283 ln 2 þ 24570945 1868176 ln 3 þ e4 9177 64 þ 271636085 1401132 ln 2 −466847955 7472704 ln 3 þ e6 76615 128 −4553279605 2802264 ln 2 þ 14144674005 119563264 ln 3 þ 914306640625 1076069376 ln 5 ; ðB7eÞ ˜κðeÞ ¼ 1 þ e2 389 32 −2056005 233522 ln 2 þ 8190315 934088 ln 3 þ e4 3577 64 þ 50149295 467044 ln 2 −155615985 3736352 ln 3 þ e6 43049 256 −12561332945 16813584 ln 2 þ 4709431125 59781632 ln 3 þ 182861328125 538034688 ln 5 ; ðB7fÞ ζðeÞ ¼ 1 þ 1011565 48972 e2 þ 106573021 783552 e4 þ 456977827 854784 e6; ðB7gÞ ˜ζðeÞ ¼ 1 þ 102371 8162 e2 þ 14250725 261184 e4 þ 722230667 4701312 e6; ðB7hÞ FðeÞ ¼ 1 þ 62 3 e2 þ 9177 64 e4 þ 76615 128 e6; ðB7iÞ φðeÞ ¼ 1 þ 2335 192 e2 þ 42955 768 e4 þ 6204647 36864 e6; ðB7aÞ ˜φðeÞ ¼ 1 þ 209 32 e2 þ 2415 128 e4 þ 730751 18432 e6; ðB7bÞ ψðeÞ ¼ 1 −22988 8191 e2 −36508643 524224 e4 −1741390565 4718016 e6; ðB7cÞ ˜ψðeÞ ¼ 1 −17416 8191 e2 −14199197 524224 e4 −467169215 4718016 e6; ðB7dÞ κðeÞ ¼ 1 þ e2 62 3 −4613840 350283 ln 2 þ 24570945 1868176 ln 3 þ e4 9177 64 þ 271636085 1401132 ln 2 −466847955 7472704 ln 3 þ e6 76615 128 −4553279605 2802264 ln 2 þ 14144674005 119563264 ln 3 þ 914306640625 1076069376 ln 5 ; ðB7eÞ ˜κðeÞ ¼ 1 þ e2 389 32 −2056005 233522 ln 2 þ 8190315 934088 ln 3 þ e4 3577 64 þ 50149295 467044 ln 2 −155615985 3736352 ln 3 þ e6 43049 256 −12561332945 16813584 ln 2 þ 4709431125 59781632 ln 3 þ 182861328125 538034688 ln 5 ; ðB7fÞ ζðeÞ ¼ 1 þ 1011565 48972 e2 þ 106573021 783552 e4 þ 456977827 854784 e6; ðB7gÞ ˜ζðeÞ ¼ 1 þ 102371 8162 e2 þ 14250725 261184 e4 þ 722230667 4701312 e6; ðB7hÞ FðeÞ ¼ 1 þ 62 3 e2 þ 9177 64 e4 þ 76615 128 e6; ðB7iÞ ðB7aÞ ðB7bÞ ðB7cÞ ðB7dÞ ðB7eÞ ðB7fÞ ðB7hÞ ðB7iÞ 084043-14 084043-14 AMPLITUDES FOR COMPACT … PHYS. APPENDIX C: OSCILLATORY MEMORY INTEGRAL Here we derive the formula to evaluate the oscillatory memory integrals in Eq. (30). For convenience we set G ¼ c ¼ 1 in this Appendix. We define the integral that has to be computed as Jmem ¼ Z TR −∞ dtxpðtÞeqðtÞeiðsλξþrξÞ: ðC1Þ ðC1Þ 084043-15 MICHAEL EBERSOLD et al. PHYS. REV. D 100, 084043 (2019) Next, we express the time-dependent quantities in the integral in terms of y and their values at the current time TR. For x we find We follow the approach of Ref. [73], where this integral was evaluated in the case of circular orbits (q ¼ 0). The eccentric orbit is assumed to evolve only with the secular radiation-reaction equations given in Eqs. (15a)–(15b) starting from x ¼ 0 and e ¼ 1 in the remote past. Every astrophysical process like capture or mass loss possibly happening to the binary is ignored. We start by restating the evolution equation for x at leading order in x and e, xðyÞ¼xðTRÞð1þyÞ−1=4 1−157 172e2ðTRÞðð1þyÞ19=24 −1Þ ; ðC8Þ ðC8Þ dxðtÞ dt ¼ 64νx5ðtÞ 5m 1 þ 157 24 e2ðtÞ ; ðC2Þ and for the eccentricity we find and for the eccentricity we find ðC2Þ eðyÞ ¼ eðTRÞð1 þ yÞ19=48: ðC9Þ ðC9Þ and integrate it over a time interval up to some coalescence time TC, where the orbital frequency and therefore x tends to infinity: Note that while going back in time, with increasing y, we only let the eccentricity evolve until e ¼ 1 is reached. Furthermore, we need the redefined mean anomaly ξðtÞ in terms of y and its value at the current time. Because ξ is defined in terms of _ξ ¼ n, we have to calculate the integral Z TC t dt ¼ Z ∞ xðtÞ dxðtÞ ðdx=dtÞ : ðC3Þ ðC3Þ Thereby, we find an explicit relation between the orbital frequency (related to x) and time t: ξðtÞ ¼ ξðTCÞ − Z t TC dt0nðt0Þ ¼ ξðTCÞ −1 m Z t TC dt0x3=2ðt0Þ ¼ ξðTCÞ −ðTC −TRÞ m Z y −1 dy0x3=2ðy0Þ: ðC10Þ TC −t ¼ 5m 256ν 1 x4ðtÞ 1 −157 43 e2ðtÞ : ðC4Þ ðC4Þ We can now invert the xðeÞ relation derived in Eq. (25) to find e as a function of x. Considering only the leading order, we find ðC10Þ We can now evaluate the latter integral by inserting the expression for xðyÞ given in Eq. (C8). APPENDIX C: OSCILLATORY MEMORY INTEGRAL This leads to We can now evaluate the latter integral by inserting the expression for xðyÞ given in Eq. (C8). This leads to eðtÞ ¼ eðTRÞ xðTRÞ xðtÞ 19=12 : ðC5Þ ðC5Þ ξðtÞ ¼ ξðTCÞ −8ðTC −TRÞx3=2ðTRÞ 5m ð1 þ yÞ5=8 × 1 −471 11696 e2ðTRÞð15ð1 þ yÞ19=24 −34Þ ; ðC11Þ ξðtÞ ¼ ξðTCÞ −8ðTC −TRÞx3=2ðTRÞ 5m ð1 þ yÞ5=8 × 1 −471 11696 e2ðTRÞð15ð1 þ yÞ19=24 −34Þ ; Using Eqs. (C4) and (C5), we get x as an explicit function of t: xðtÞ¼1 4 5m νðTC −tÞ 1=4 1−157 172e2ðTRÞ TC −t TC −TR 19=24 : ðC6Þ ðC11Þ ðC6Þ where ξðTCÞ is the value of ξ at the moment of coalescence. Thus, at the current time TR the mean anomaly is given by where ξðTCÞ is the value of ξ at the moment of coalescence. Thus, at the current time TR the mean anomaly is given by A quick check reveals that this expression indeed solves the differential equation in Eq. (C2). Since the memory integral runs up to the current time TR, we introduce a new integration variable y which is better suited to the integra- tion limits we have: ξðTRÞ¼ξðTCÞ−8ðTC −TRÞx3=2ðTRÞ 5m 1þ 8949 11696e2ðTRÞ : ðC12Þ ðC12Þ y ¼ TR −t TC −TR : ðC7Þ ðC12Þ Now we are able to express ξðtÞ in terms of ξðTRÞ and y, Þ ξðT Þ 8ðTC −TRÞx3=2ðTRÞ ½ð1 þ yÞ5=8 1 1 471 e2ðT Þ 19 −34ð1 þ yÞ5=8 þ 15ð1 þ yÞ17=12 ðC13Þ y ¼ TR −t TC −TR : ðC7Þ ðC7Þ Now we are able to express ξðtÞ in terms of ξðTRÞ and y, ξðtÞ ¼ ξðTRÞ −8ðTC −TRÞx3=2ðTRÞ 5m ½ð1 þ yÞ5=8 −1 1 −471 11696 e2ðTRÞ 19 −34ð1 þ yÞ5=8 þ 15ð1 þ yÞ17=12 ð1 þ yÞ5=8 −1 ; ðC13Þ ðC13Þ where xðTRÞ and eðTRÞ stand for the respective current values of x and e. 084043-16 GRAVITATIONAL-WAVE AMPLITUDES FOR COMPACT … GRAVITATIONAL-WAVE AMPLITUDES FOR COMPACT … PHYS. REV. D 100, 084043 (2019) At this point, we introduce a dimensionless “adiabatic parameter” χðTRÞ, which is connected with the inspiral rate at the current retarded time TR. We define it as the ratio between the current period and the time left until coalescence, χðTRÞ ¼ 1 nðTRÞðTC −TRÞ ; ðC14Þ ðC14Þ here nðTRÞ ¼ x3=2ðTRÞ=m at leading order. Explicitly in terms of xðTRÞ and eðTRÞ, it reads χðTRÞ ¼ 256ν 5 x5=2ðTRÞ 1 þ 157 43 e2ðTRÞ : ðC15Þ ðC15Þ Inserting χðTRÞ into Eq. (C13), we find ξðtÞ ¼ ξðTRÞ − 8 5χðTRÞ ½ð1 þ yÞ5=8 −1 1 −471 11696 e2ðTRÞ 19 −34ð1 þ yÞ5=8 þ 15ð1 þ yÞ17=12 ð1 þ yÞ5=8 −1 : ðC16Þ ðC16Þ Now we put Eqs. (C8), (C9), and (C16) into the oscillatory integral and write it as an integral over y: Jmem ¼ ðTC −TRÞ Z ∞ 0 dyxpðyÞeqðyÞeiðsλξðyÞþrξðyÞÞ ¼ ðTC −TRÞeiðrþsð1þkÞÞξðTRÞ Z ∞ 0 dyxpðyÞeqðyÞ exp −8iðr þ sð1 þ kÞÞ 5χðTRÞ ½ð1 þ yÞ5=8 −1 × 1 −471 11696 e2ðTRÞ 19 −34ð1 þ yÞ5=8 þ 15ð1 þ yÞ17=12 ð1 þ yÞ5=8 −1 : ðC17Þ Z 0 ¼ ðTC −TRÞeiðrþsð1þkÞÞξðTRÞ Z ∞ 0 dyxpðyÞeqðyÞ exp −8iðr þ sð1 þ kÞÞ 5χðTRÞ ½ð1 þ yÞ5=8 −1 × 1 −471 11696 e2ðTRÞ 19 −34ð1 þ yÞ5=8 þ 15ð1 þ yÞ17=12 ð1 þ yÞ5=8 −1 : ðC17Þ Let us look at the form of this integral: Let us look at the form of this integral: Jmem ∼ Z ∞ 0 dyfðyÞ exp i χðTRÞ gðyÞ : ðC18Þ Jmem ∼ Z ∞ 0 dyfðyÞ exp i χðTRÞ gðyÞ : ðC18Þ ðC18Þ The strategy is to integrate by parts, and therefore we need to know the following type of integral: The strategy is to integrate by parts, and therefore we need to know the following type of integral: Z dyeiσgðyÞ ¼ − i σg0ðyÞ eiσgðyÞ þ Oðg0ðyÞ−2Þ: ðC19Þ Z dyeiσgðyÞ ¼ − i σg0ðyÞ eiσgðyÞ þ Oðg0ðyÞ−2Þ: ðC19Þ ðC19Þ This formula is valid as long as g0ðyÞ is sufficiently large. Integrating Eq. (C18) by parts, we get This formula is valid as long as g0ðyÞ is sufficiently large. Integrating Eq. GRAVITATIONAL-WAVE AMPLITUDES FOR COMPACT … (C18) by parts, we get Jmem ∼fðyÞ −iχðTRÞ g0ðyÞ exp i χðTRÞ gðyÞ ∞ 0 þ iχðTRÞ Z ∞ 0 dy f0ðyÞ g0ðyÞ exp i χðTRÞ gðyÞ : ðC20Þ ðC20Þ As y approaches infinity in the remote past, we notice that fðyÞ ¼ xpðyÞeqðyÞ goes to zero. This is because at early times the frequency reaches zero and the eccentricity cannot grow past e ¼ 1 in our model. Evaluating the first term at y ¼ 0, we recover x and e at the current time and the exponential factor is just 1 since gð0Þ ¼ 0. The derivative g0ðyÞ in the denominator evaluated at y ¼ 0 is effectively 1 multiplied by some constants. What remains in the first term of Eq. (C20) is therefore of order χðTRÞ. Looking at the second term, we find the same integral form as in Eq. (C18). Successively integrating by parts would yield another factor of χðTRÞ each time. Since this parameter is already of order 2.5PN, the higher-order χðTRÞ contributions can be safely ignored. Including everything of order χðTRÞ, we find the formula Jmem ¼ −ðTc −TRÞxpeqeiðsλξþrξÞ iχðTRÞ ðr þ sð1 þ kÞÞ ¼ − i nðr þ sð1 þ kÞÞ xpeqeiðsλξþrξÞ; ðC21Þ Jmem ¼ −ðTc −TRÞxpeqeiðsλξþrξÞ iχðTRÞ ðr þ sð1 þ kÞÞ Jmem ¼ −ðTc −TRÞxpeqeiðsλξþrξÞ iχðTRÞ ðr þ sð1 þ kÞÞ ¼ − i nðr þ sð1 þ kÞÞ xpeqeiðsλξþrξÞ; ðC21Þ which allows us to compute the oscillatory hereditary integrals in Sec. III C. which allows us to compute the oscillatory hereditary integrals in Sec. III C. 084043-17 MICHAEL EBERSOLD et al. MICHAEL EBERSOLD et al. PHYS. REV. D 100, 084043 (2019) APPENDIX D: LIST OF DC MEMORY MODES APPENDIX D: LIST OF DC MEMORY MODES Here we list the 3PN-accurate DC memory contributions to the hlm modes at leading order in eccentricity in the following form: hlm DC ¼ 8Gmν c2R x ﬃﬃﬃπ 5 r Hlm DC; ðD1Þ ðD1Þ where Hlm DC is a function of x and e. The nonzero modes read where Hlm DC is a function of x and e. The nonzero modes read and e. APPENDIX D: LIST OF DC MEMORY MODES The nonzero modes read H20 DC ¼ − 5 14 ﬃﬃﬃ 6 p ðH20 Newt þ xH20 1PN þ x3=2H20 1.5PN þ x2H20 2PN þ x5=2H20 2.5PN þ x3H20 3PNÞ; ðD2aÞ H20 Newt ¼ 1 − e ei 12=19 ; ðD2bÞ H20 DC ¼ − 5 14 ﬃﬃﬃ 6 p ðH20 Newt þ xH20 1PN þ x3=2H20 1.5PN þ x2H20 2PN þ x5=2H20 2.5PN þ x3H20 3PNÞ; ðD2aÞ ðD2aÞ H20 1PN ¼ −4075 4032 þ 67ν 48 þ e ei 12=19 −2833 3192 þ 197ν 114 þ e ei 24=19145417 76608 −2849ν 912 ; ðD2cÞ ðD2cÞ i i H20 1.5PN ¼ −377π 228 e ei 12=19 þ 377π 228 e ei 30=19 ; ðD2dÞ H20 1.5PN ¼ −377π 228 e ei 12=19 þ 377π 228 e ei 30=19 ; ðD2dÞ H20 1.5PN ¼ −377π 228 e ei 12=19 þ 377π 228 e ei 30=19 ; ðD2dÞ H20 2PN ¼ −151877213 67060224 −123815ν 44352 þ 205ν2 352 þ e ei 12=19358353209 366799104 −738407ν 727776 −20597ν2 17328 þ e ei 24=19411966361 122266368 −825950ν 68229 þ 561253ν2 51984 þ e ei 36=19 −50392977379 24208740864 þ 764295307ν 48033216 −11654209ν2 1143648 ; ðD2eÞ H20 2PN ¼ −151877213 67060224 −123815ν 44352 þ 205ν2 352 þ e ei 12=19358353209 366799104 −738407ν 727776 −20597ν2 17328 þ e ei 24=19411966361 122266368 −825950ν 68229 þ 561253ν2 51984 þ e ei 36=19 −50392977379 24208740864 þ 764295307ν 48033216 −11654209ν2 1143648 ; ðD2eÞ ðD2eÞ H20 2.5PN ¼ −253π 336 þ 253πν 84 þ e ei 12=193763903π 7277760 þ 12788779πν 1819440 þ e ei 24=1954822209π 8733312 −1074073πν 103968 þ e ei 30=195340205π 1455552 −371345πν 51984 þ e ei 42=19 −424020733π 43666560 þ 27049187πν 3638880 ; ðD2fÞ ðD2fÞ H20 3PN ¼ −4397711103307 532580106240 þ 700464542023 13948526592 −205π2 96 ν þ 69527951ν2 166053888 þ 1321981ν3 5930496 þ e ei 12=19 −4942027570449143 96592876047360 −81025π2 103968 þ 3317γE 399 þ −10309531979 7466981760 þ 3977π2 3648 ν þ 267351733ν2 82966464 þ 772583ν3 2222316 þ 12091 ln 2 5985 þ 78003 ln 3 5320 þ 3317 ln x 798 þ 710645π2 103968 e ei 30=19 þ e ei 24=19 −31102835980319 14049872879616 þ 279737759653ν 167260391424 þ 26730466283ν2 1991195136 −397176241ν3 23704704 þ e ei 36=19 −142763304914707 25758100279296 þ 48901891428821ν 919932152832 −400181473249ν2 3650524416 þ 2295879173ν3 43458624 þ e ei 48=19385621605844415513 5740376633671680 −157405π2 25992 −3317γE 399 þ −49590995147570629 478364719472640 þ 1271π2 1216 ν þ 3194536246463ν2 34514049024 −1672948713ν3 45653504 −12091 ln 2 5985 −78003 ln 3 5320 −3317 ln x 798 −6634 2527 ln e ei ; ðD2gÞ þ 3194536246463ν2 34514049024 −1672948713ν3 45653504 −12091 ln 2 5985 −78003 ln 3 5320 −3317 ln x 798 −6634 2527 ln e ei ; ðD2gÞ ðD2gÞ 084043-18 PHYS. APPENDIX D: LIST OF DC MEMORY MODES REV. D 100, 084043 (2019) GRAVITATIONAL-WAVE AMPLITUDES FOR COMPACT … D 100, 084043 (2019) H60 2PN ¼ −45661561 6342840 þ 101414ν 2517 −48118ν2 839 þ e ei 24=19 −2833 1596 þ 530740ν 47823 −237188ν2 15941 þ e ei 36=191081489489 120513960 −819202ν 15941 þ 1151430ν2 15941 ; ðD4cÞ 24=19 42=19 H60 2PN ¼ −45661561 6342840 þ 101414ν 2517 −48118ν2 839 þ e ei 24=19 −2833 1596 þ 530740ν 47823 −237188ν2 15941 þ e ei 36=191081489489 120513960 −819202ν 15941 þ 1151430ν2 15941 ; ðD4cÞ H60 2.5PN ¼ 1248π 839 −4992πν 839 þ e ei 24=19 −377π 114 þ 226954πν 15941 þ e ei 42=19174031π 95646 −132106πν 15941 ; ðD4dÞ ðD4cÞ þ ei 120513960 −15941 þ 15941 ; ðD4cÞ H60 2.5PN ¼ 1248π 839 −4992πν 839 þ e ei 24=19 −377π 114 þ 226954πν 15941 þ e ei 42=19174031π 95646 −132106πν 15941 ; ðD4dÞ H60 2.5PN ¼ 1248π 839 −4992πν 839 þ e ei 24=19 −377π 114 þ 226954πν 15941 þ e ei 42=19174031π 95646 −132106πν 15941 ; ðD4dÞ H60 2.5PN ¼ 1248π 839 −4992πν 839 þ e ei 24=19 −377π 114 þ 226954πν 15941 þ e ei 42=19174031π 95646 −132106πν 15941 ; ðD4dÞ H60 3PN ¼ 3012132889099 144921208320 −27653500031ν 191694720 þ 1317967427ν2 4107744 −24793657ν3 342312 þ e ei 24=19213887207 183399552 −7295329871ν 1831812192 −214435261ν2 21807288 þ 41962109ν3 1817274 þ e ei 36=193063859722337 128226853440 −839669231153ν 4579530480 þ 555765673ν2 1211516 −113415855ν3 302879 þ e ei 48=19 −9789584507539 213536964096 þ 206521649193667ν 622816145280 −380487275717ν2 494298528 þ 17456918535ν3 41191544 ; ðD4eÞ H80 DC ¼ − 75601 213497856 ﬃﬃﬃﬃﬃﬃﬃﬃ 119 p ðx2H80 2PN þ x3H80 3PNÞ; ðD5aÞ H80 2PN ¼ 1 −452070ν 75601 þ 733320ν2 75601 þ e ei 36=19 −1 þ 452070ν 75601 −733320ν2 75601 ; ðD5bÞ ðD5aÞ H80 3PN ¼ −265361599 33869248 þ 18177898147ν 321757856 −722521125ν2 5745676 þ 261283995ν3 2872838 þ e ei 36=19 −2833 1064 þ 848864713ν 40219732 −81627030ν2 1436419 þ 72232020ν3 1436419 þ e ei 48=1950791665 4838464 −3566973693ν 45965408 þ 1049029245ν2 5745676 −405748035ν3 2872838 ; ðD5cÞ H80 3PN ¼ −265361599 33869248 þ 18177898147ν 321757856 −722521125ν2 5745676 þ 261283995ν3 2872838 þ e ei 36=19 −2833 1064 þ 848864713ν 40219732 −81627030ν2 1436419 þ 72232020ν3 1436419 þ e ei 48=1950791665 4838464 −3566973693ν 45965408 þ 1049029245ν2 5745676 −405748035ν3 2872838 ; ðD5cÞ ðD5cÞ H100 DC ¼ 525221 6452379648 ﬃﬃﬃﬃﬃﬃﬃﬃ 154 p x3 1 −79841784ν 9979199 þ 198570240ν2 9979199 −172307520ν3 9979199 þ e ei 48=19 −1 þ 79841784ν 9979199 −198570240ν2 9979199 þ 172307520ν3 9979199 : ðD6aÞ ðD6aÞ GRAVITATIONAL-WAVE AMPLITUDES FOR COMPACT … GRAVITATIONAL-WAVE AMPLITUDES FOR COMPACT … H40 DC ¼ − 1 504 ﬃﬃﬃ 2 p ðH40 Newt þ xH40 1PN þ x3=2H40 1.5PN þ x2H40 2PN þ x5=2H40 2.5PN þ x3H40 3PNÞ; ðD3aÞ H40 Newt ¼ 1 − e ei 12=19 ; ðD3bÞ H40 1PN ¼ −180101 29568 þ 27227ν 1056 þ e ei 12=19 −2833 3192 þ 197ν 114 þ e ei 24=193920527 561792 −183995ν 6688 ; ðD3cÞ H40 1.5PN ¼ −377π 228 e ei 12=19 þ 377π 228 e ei 30=19 ; ðD3dÞ ðD3cÞ H40 2PN ¼ 2201411267 158505984 −34829479ν 432432 þ 844951ν2 27456 þ e ei 12=19358353209 366799104 −738407ν 727776 −20597ν2 17328 þ e ei 24=1911106852991 896620032 −584029331ν 8005536 þ 36247015ν2 381216 þ e ei 36=19 −17153749047583 629427262464 þ 24120402175ν 156107952 −1235668217ν2 9911616 ; ðD3eÞ ðD3eÞ H40 2.5PN ¼ −13565π 1232 þ 13565πν 308 þ e ei 12=193763903π 7277760 þ 12788779πν 1819440 þ e ei 24=191478038679π 64044288 −69366115πν 762432 þ e ei 30=195340205π 1455552 −371345πν 51984 þ e ei 42=19 −473166857π 29111040 þ 1255597433πν 26685120 ; ðD3fÞ ðD3fÞ H40 3PN ¼ 15240463356751 781117489152 þ −1029744557245 27897053184 −205π2 96 ν −4174614175ν2 36900864 þ 221405645ν3 11860992 þ e ei 12=19 −4942027570449143 96592876047360 −81025π2 103968 þ 3317γE 399 þ −10309531979 7466981760 þ 3977π2 3648 ν þ 267351733ν2 82966464 þ 772583ν3 2222316 þ 12091 ln 2 5985 þ 78003 ln 3 5320 þ 3317 ln x 798 þ 710645π2 103968 e ei 30=19 þ e ei 24=19 −838550569998089 103032401117184 þ 30467243664175ν 1226576203776 þ 963631094693ν2 14602097664 −25650558955ν3 173834496 þ e ei 36=19 −48596571051802639 669710607261696 þ 13219254870469451ν 23918235973632 −107533340184449ν2 94913634816 þ 243426638749ν3 376641408 þ e ei 48=191289915690995598063 11480753267343360 −157405π2 25992 −3317γE 399 þ −515898615572711953 956729438945280 þ 1271π2 1216 ν þ 297870712456705ν2 253103026176 −520032054523ν3 1004377088 −12091 ln 2 5985 −78003 ln 3 5320 −3317 ln x 798 −6634 2527 ln e ei ; ðD3gÞ H60 DC ¼ 4195 1419264 ﬃﬃﬃﬃﬃﬃﬃﬃ 273 p ðxH60 1PN þ x2H60 2PN þ x5=2H60 2.5PN þ x3H60 3PNÞ; ðD4aÞ H60 1PN ¼ 1 −3612ν 839 ; ðD4bÞ 084043-19 MICHAEL EBERSOLD et al. PHYS. REV. APPENDIX E: LIST OF OSCILLATORY MEMORY MODES Here we list the nonzero oscillatory memory contributions to the hlm modes at 3PN order and to quadratic order in eccentricity in the following way: hlm osc ¼ 8Gmν c2R x ﬃﬃﬃπ 5 r e−imψHlm osc; ðE1Þ ðE1Þ where Hlm osc is a function of x, e, and the modified mean anomaly ξ. To improve readability in the odd-m expressions, we define Δ ¼ ðm1 −m2Þ=m ¼ ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ 1 −4ν p : 084043-20 LITUDES FOR COMPACT … PHYS. REV. D 100, 084043 (2019) RAVITATIONAL-WAVE AMPLITUDES FOR COMPACT … PHYS. REV [1] J. Aasi et al., Classical Quantum Gravity 32, 115012 (2015). [6] B. P. Abbott et al., Phys. Rev. Lett. 118, 221101 (2017). [2] T. Accadia et al., J. Instrum. 7, P03012 (2012). [7] B. P. Abbott et al., Phys. Rev. Lett. 119, 141101 (2017). [6] B. P. Abbott et al., Phys. Rev. Lett. 118, 221101 (2017). [7] B. P. Abbott et al., Phys. Rev. Lett. 119, 141101 (2017). [8] B. P. Abbott et al., Phys. Rev. Lett. 119, 161101 (2017). [9] B. P. Abbott et al., Astrophys. J. 851, L35 (2017). [10] B. P. Abbott et al. (LIGO Scientific and Virgo Collabora- tions), Phys. Rev. X 9, 031040 (2019). [3] H. Grote (LIGO Scientific Collaboration), Classical Quan- tum Gravity 27, 084003 (2010). [5] B. P. Abbott et al., Phys. Rev. Lett. 116, 241103 (2016). y [4] B. P. Abbott et al., Phys. Rev. Lett. 116, 061102 (2016). [10] B. P. Abbott et al. (LIGO Scientific and Virgo Collabora- tions), Phys. Rev. X 9, 031040 (2019). GRAVITATIONAL-WAVE AMPLITUDES FOR COMPACT … GRAVITATIONAL-WAVE AMPLITUDES FOR COMPACT … PHYS. REV. GRAVITATIONAL-WAVE AMPLITUDES FOR COMPACT … D 100, 084043 (2019) H20 osc ¼ ﬃﬃﬃ 6 p ix5=2eν 7 −16e−iξ þ 16eiξ −647 36 ee−2iξ þ 647 36 ee2iξ ; ðE2aÞ H22 osc ¼ ix3=2e2νe2iξ −13 252 þ 697 336 −865ν 216 x −29π 126 x3=2 þ ix5=2eν 21 19 6 e þ 4 3 e−iξ −4eiξ þ 65 24 ee−2iξ ; ðE2bÞ H31 osc ¼ ﬃﬃﬃﬃﬃ 14 p x2νΔ 90 44 3 e2 −44 3 eeiξ −44 3 e2e2iξ þ x −121 7 −43 2 ee−iξ −2987 84 e2e−2iξ þ eeiξ 19801 264 −2521ν 231 þ e2e2iξ 7957 88 −827ν 231 þ e2 −111821 616 þ 827ν 231 ; ðE2cÞ H33 osc ¼ x3νΔ 6 ﬃﬃﬃﬃﬃﬃﬃﬃ 210 p 22 9 þ 19e2 þ 11ee−iξ þ 1 3 eeiξ þ 713 30 e2e−2iξ −119 6 e2e2iξ ; ðE2dÞ H40 osc ¼ ﬃﬃﬃ 2 p ix5=2eν 210 −8e−iξ þ 8eiξ −143 16 ee−2iξ þ 143 16 ee2iξ ; ðE2eÞ H42 osc ¼ ix3=2e2νe2iξ 216 ﬃﬃﬃ 5 p −13 14 þ x 7943 56 −25393ν 66 −29 7 πx3=2 þ ix5=2eν 126 ﬃﬃﬃ 5 p 19 12 e þ 2 3 e−iξ −2eiξ þ 65 48 ee−2iξ ; ðE2fÞ H44 osc ¼ ix5=2ν 6 ﬃﬃﬃﬃﬃ 35 p 2 3 þ 331e2 240 þ 14 15 ee−iξ þ 2eeiξ þ 1037 720 e2e−2iξ þ 217 48 e2e2iξ ; ðE2gÞ H51 osc ¼ x2νΔ 18 ﬃﬃﬃﬃﬃﬃﬃﬃ 385 p 43e2 12 −43 12 eeiξ −43 12 e2e2iξ þ x −26 7 −251 56 ee−iξ −1199 168 e2e−2iξ þ eeiξ 8627 156 −41807ν 312 þ e2e2iξ 785 13 −5156ν 39 þ e2 −8321 104 þ 5156ν 39 ; ðE2hÞ H53 osc ¼ x3νΔ 2 ﬃﬃﬃﬃﬃﬃﬃﬃ 330 p −2 189 þ 27e2 112 þ 41 336 ee−iξ −67 504 eeiξ þ 1531e2e−2iξ 5040 −47 72 e2e2iξ ; ðE2iÞ H55 osc ¼ x3νΔ 14 ﬃﬃﬃﬃﬃ 66 p 18 5 þ 8909e2 720 þ 457 72 ee−iξ þ 197 16 eeiξ þ 787 72 e2e−2iξ þ 4369 144 e2e2iξ ; ðE2jÞ H62 osc ¼ ix5=2e2νe2iξ 352 ﬃﬃﬃﬃﬃ 65 p 2783 168 −53ν ; ðE2kÞ H71 osc ¼ 5x3νeΔﬃﬃﬃ p eiξ 5023 168 −97ν þ ee2iξ 5023 168 −97ν þ e −5023 168 þ 97ν : ðE2lÞ RAVITATIONAL-WAVE AMPLITUDES FOR COMPACT … PHYS. REV. [1] J. Aasi et al., Classical Quantum Gravity 32, 115012 (2015). [2] T. Accadia et al., J. Instrum. 7, P03012 (2012). [3] H. Grote (LIGO Scientific Collaboration), Classical Quan- tum Gravity 27, 084003 (2010). [4] B. P. Abbott et al., Phys. Rev. Lett. 116, 061102 (2016). [5] B. P. Abbott et al., Phys. Rev. Lett. 116, 241103 (2016). [9] B. P. Abbott et al., Astrophys. J. 851, L35 (2017). GRAVITATIONAL-WAVE AMPLITUDES FOR COMPACT … [51] L. Blanchet and T. Damour, Phys. Rev. D 46, 4304 (1992). [52] P. N. Payne, Phys. Rev. D 28, 1894 (1983). [23] D. Park, C. Kim, H. M. Lee, Y.-B. Bae, and K. Belczynski, Mon. Not. R. Astron. Soc. 469, 4665 (2017). [53] A. Strominger and A. Zhiboedov, J. High Energy Phys. 16 (2016) 86. [24] R. M. O’Leary, B. Kocsis, and A. Loeb, Mon. Not. R. Astron. Soc. 395, 2127 (2009). [54] S. Pasterski, A. Strominger, and A. Zhiboedov, J. High Energy Phys. 16 (2016) 53. [25] F. Antonini, S. Chatterjee, C. L. Rodriguez, M. Morscher, B. Pattabiraman, V. Kalogera, and F. A. Rasio, Astrophys. J. 816, 65 (2016). [55] N. Seto, Mon. Not. R. Astron. Soc. 400, L38 (2009). [56] R. van Haasteren and Y. Levin, Mon. Not. R. Astron. Soc. 401, 2372 (2010). [26] G. Nelemans, in The Astrophysics of Gravitational Wave Sources, American Institute of Physics Conference Series Vol. 686, edited by J. M. Centrella (AIP, New York, 2003), pp. 263–272. [57] J. M. Cordes and F. A. Jenet, Astrophys. J. 752, 54 (2012). [58] J. B. Wang, G. Hobbs, W. Coles, R. M. Shannon, X. J. Zhu et al., Mon. Not. R. Astron. Soc. 446, 1657 (2015). [59] M. Favata, Astrophys. J. 696, L159 (2009). [27] A. Sesana, J. Phys. Conf. Ser. 840, 012018 (2017). [60] P. D. Lasky, E. Thrane, Y. Levin, J. Blackman, and Y. Chen, Phys. Rev. Lett. 117, 061102 (2016). [28] O. Blaes, M. H. Lee, and A. Socrates, Astrophys. J. 578, 775 (2002). [61] L. O. McNeill, E. Thrane, and P. D. Lasky, Phys. Rev. Lett. 118, 181103 (2017). [29] L. Hoffman and A. Loeb, Mon. Not. R. Astron. Soc. 377, 957 (2007). [62] M. Favata, Phys. Rev. D 80, 024002 (2009). [30] P. Amaro-Seoane, A. Sesana, L. Hoffman, M. Benacquista, C. Eichhorn, J. Makino, and R. Spurzem, Mon. Not. R. Astron. Soc. 402, 2308 (2010). [63] M. Favata, Phys. Rev. D 84, 124013 (2011). [64] L. Blanchet, G. Faye, B. R. Iyer, and S. Sinha, Classical Quantum Gravity 25, 165003 (2008). [31] M. Bonetti, F. Haardt, A. Sesana, and E. Barausse, Mon. Not. R. Astron. Soc. 477, 3910 (2018). [65] C. K. Mishra, K. G. Arun, and B. R. Iyer, Phys. Rev. D 91, 084040 (2015). [32] M. Bonetti, A. Sesana, F. Haardt, E. Barausse, and M. Colpi, Mon. Not. R. Astron. Soc. 486, 4044 (2019). [66] Y. GRAVITATIONAL-WAVE AMPLITUDES FOR COMPACT … PHYS. REV. D 100, 084043 (2019) [11] T. Akutsu et al., Nat. Astron. 3, 35 (2019). [40] E. A. Huerta, P. Kumar, S. T. McWilliams, R. O’Shaughnessy, and N. Yunes, Phys. Rev. D 90, 084016 (2014). [12] B. R. Iyer et al., LIGO-India Technical Report No. LIGO- M1100296, 2011, https://dcc.ligo.org/LIGO-M1100296/ public/main. [41] E. A. Huerta, P. Kumar, B. Agarwal, D. George, H.-Y. Schive, H. P. Pfeiffer, R. Haas, W. Ren, T. Chu, M. Boyle, D. A. Hemberger, L. E. Kidder, M. A. Scheel, and B. Szilagyi, Phys. Rev. D 95, 024038 (2017). [13] P. Amaro-Seoane, H. Audley, S. Babak, J. Baker, E. Barausse, P. Bender, E. Berti et al., arXiv:1702.00786. [42] A. Gopakumar and G. Schäfer, Phys. Rev. D 84, 124007 (2011). [14] R. N. Manchester and IPTA, Classical Quantum Gravity 30, 224010 (2013). [15] J. M. Weisberg and Y. Huang, Astrophys. J. 829, 55 (2016). [43] S. Tanay, M. Haney, and A. Gopakumar, Phys. Rev. D 93, 064031 (2016). [15] J. M. Weisberg and Y. Huang, Astrophys. J. 829, 55 (2016). [16] P. C. Peters and J. Mathews, Phys. Rev. 131, 435 (1963). [16] P. C. Peters and J. Mathews, Phys. Rev. 131, 435 (1963). [17] B. Sun, Z. Cao, Y. Wang, and H.-C. Yeh, Phys. Rev. D 92, 044034 (2015). [44] I. Hinder, L. E. Kidder, and H. P. Pfeiffer, Phys. Rev. D 98, 044015 (2018). [18] E. A. Huerta, C. J. Moore, P. Kumar, D. George, A. J. K. Chua, R. Haas, E. Wessel, D. Johnson, D. Glennon, A. Rebei, A. M. Holgado, J. R. Gair, and H. P. Pfeiffer, Phys. Rev. D 97, 024031 (2018). [45] Z. Cao and W.-B. Han, Phys. Rev. D 96, 044028 (2017). [46] A. Klein, Y. Boetzel, A. Gopakumar, P. Jetzer, and L. de Vittori, Phys. Rev. D 98, 104043 (2018). [47] Y. B. Zel’dovich and A. G. Polnarev, Sov. Astron. 18, 17 (1974). [19] J. Samsing, Phys. Rev. D 97, 103014 (2018). [48] D. Christodoulou, Phys. Rev. Lett. 67, 1486 (1991). [20] J. Samsing, M. MacLeod, and E. Ramirez-Ruiz, Astrophys. J. 784, 71 (2014). [49] A. G. Wiseman and C. M. Will, Phys. Rev. D 44, R2945 (1991). [21] R. M. O’Leary, R. O’Shaughnessy, and F. A. Rasio, Phys. Rev. D 76, 061504 (2007). [50] K. S. Thorne, Phys. Rev. D 45, 520 (1992). [22] S. Naoz, B. Kocsis, A. Loeb, and N. Yunes, Astrophys. J. 773, 187 (2013). GRAVITATIONAL-WAVE AMPLITUDES FOR COMPACT … D 100, 084043 (2019) H20 osc ¼ ﬃﬃﬃ 6 p ix5=2eν 7 −16e−iξ þ 16eiξ −647 36 ee−2iξ þ 647 36 ee2iξ ; ðE2aÞ H22 osc ¼ ix3=2e2νe2iξ −13 252 þ 697 336 −865ν 216 x −29π 126 x3=2 þ ix5=2eν 21 19 6 e þ 4 3 e−iξ −4eiξ þ 65 24 ee−2iξ ; ðE2bÞ H31 osc ¼ ﬃﬃﬃﬃﬃ 14 p x2νΔ 90 44 3 e2 −44 3 eeiξ −44 3 e2e2iξ þ x −121 7 −43 2 ee−iξ −2987 84 e2e−2iξ þ eeiξ 19801 264 −2521ν 231 þ e2e2iξ 7957 88 −827ν 231 þ e2 −111821 616 þ 827ν 231 ; ðE2cÞ H33 osc ¼ x3νΔ 6 ﬃﬃﬃﬃﬃﬃﬃﬃ 210 p 22 9 þ 19e2 þ 11ee−iξ þ 1 3 eeiξ þ 713 30 e2e−2iξ −119 6 e2e2iξ ; ðE2dÞ ðE2aÞ −13 252 þ 697 336 −865ν 216 x −29π 126 x3=2 þ ix5=2eν 21 19 6 e þ 4 3 e−iξ −4eiξ þ 65 24 ee−2iξ ; ðE2 ðE2cÞ þ eeiξ 264 −231 þ e2e2iξ 88 −231 þ e2 − 616 þ 231 ; ðE2cÞ H33 osc ¼ x3νΔ 6 ﬃﬃﬃﬃﬃﬃﬃﬃ 210 p 22 9 þ 19e2 þ 11ee−iξ þ 1 3 eeiξ þ 713 30 e2e−2iξ −119 6 e2e2iξ ; ðE2dÞ H40 osc ¼ ﬃﬃﬃ 2 p ix5=2eν 210 −8e−iξ þ 8eiξ −143 16 ee−2iξ þ 143 16 ee2iξ ; ðE2eÞ H42 osc ¼ ix3=2e2νe2iξ 216 ﬃﬃﬃ 5 p −13 14 þ x 7943 56 −25393ν 66 −29 7 πx3=2 þ ix5=2eν 126 ﬃﬃﬃ 5 p 19 12 e þ 2 3 e−iξ −2eiξ þ 65 48 ee−2iξ ; ðE2fÞ H44 osc ¼ ix5=2ν 6 ﬃﬃﬃﬃﬃ 35 p 2 3 þ 331e2 240 þ 14 15 ee−iξ þ 2eeiξ þ 1037 720 e2e−2iξ þ 217 48 e2e2iξ ; ðE2gÞ H51 osc ¼ x2νΔ 18 ﬃﬃﬃﬃﬃﬃﬃﬃ 385 p 43e2 12 −43 12 eeiξ −43 12 e2e2iξ þ x −26 7 −251 56 ee−iξ −1199 168 e2e−2iξ þ eeiξ 8627 156 −41807ν 312 þ e2e2iξ 785 13 −5156ν 39 þ e2 −8321 104 þ 5156ν 39 ; ðE2hÞ H53 osc ¼ x3νΔ 2 ﬃﬃﬃﬃﬃﬃﬃﬃ 330 p −2 189 þ 27e2 112 þ 41 336 ee−iξ −67 504 eeiξ þ 1531e2e−2iξ 5040 −47 72 e2e2iξ ; ðE2iÞ H55 osc ¼ x3νΔ 14 ﬃﬃﬃﬃﬃ 66 p 18 5 þ 8909e2 720 þ 457 72 ee−iξ þ 197 16 eeiξ þ 787 72 e2e−2iξ þ 4369 144 e2e2iξ ; ðE2jÞ H62 osc ¼ ix5=2e2νe2iξ 352 ﬃﬃﬃﬃﬃ 65 p 2783 168 −53ν ; ðE2kÞ H71 osc ¼ 5x3νeΔ 30888 ﬃﬃﬃ 2 p eiξ 5023 168 −97ν þ ee2iξ 5023 168 −97ν þ e −5023 168 þ 97ν : ðE2lÞ H40 osc ¼ ﬃﬃﬃ 2 p ix5=2eν 210 −8e−iξ þ 8eiξ −143 16 ee−2iξ þ 143 16 ee2iξ ; ðE2eÞ H42 osc ¼ ix3=2e2νe2iξ 216 ﬃﬃﬃ 5 p −13 14 þ x 7943 56 −25393ν 66 −29 7 πx3=2 þ ix5=2eν 126 ﬃﬃﬃ 5 p 19 12 e þ 2 3 e−iξ −2eiξ þ 65 48 ee−2iξ ; ðE2fÞ H44 osc ¼ ix5=2ν 6 ﬃﬃﬃﬃﬃ 35 p 2 3 þ 331e2 240 þ 14 15 ee−iξ þ 2eeiξ þ 1037 720 e2e−2iξ þ 217 48 e2e2iξ ; ðE2gÞ H51 osc ¼ x2νΔ 18 ﬃﬃﬃﬃﬃﬃﬃﬃ 385 p 43e2 12 −43 12 eeiξ −43 12 e2e2iξ þ x −26 7 −251 56 ee−iξ −1199 168 e2e−2iξ þ eeiξ 8627 156 −41807ν 312 þ e2e2iξ 785 13 −5156ν 39 þ e2 −8321 104 þ 5156ν 39 ; ðE2hÞ H53 osc ¼ x3νΔ 2 ﬃﬃﬃﬃﬃﬃﬃﬃ 330 p −2 189 þ 27e2 112 þ 41 336 ee−iξ −67 504 eeiξ þ 1531e2e−2iξ 5040 −47 72 e2e2iξ ; ðE2iÞ H55 osc ¼ x3νΔ 14 ﬃﬃﬃﬃﬃ 66 p 18 5 þ 8909e2 720 þ 457 72 ee−iξ þ 197 16 eeiξ þ 787 72 e2e−2iξ þ 4369 144 e2e2iξ ; ðE2jÞ H42 osc ¼ ix3=2e2νe2iξ 216 ﬃﬃﬃ 5 p −13 14 þ x 7943 56 −25393ν 66 −29 7 πx3=2 þ ix5=2eν 126 ﬃﬃﬃ 5 p 19 12 e þ 2 3 e−iξ −2eiξ þ 65 48 ee−2iξ ; ðE2fÞ H44 osc ¼ ix5=2ν 6 ﬃﬃﬃﬃﬃ 35 p 2 3 þ 331e2 240 þ 14 15 ee−iξ þ 2eeiξ þ 1037 720 e2e−2iξ þ 217 48 e2e2iξ ; ðE2gÞ H51 osc ¼ x2νΔ 18 ﬃﬃﬃﬃﬃﬃﬃﬃ 385 p 43e2 12 −43 12 eeiξ −43 12 e2e2iξ þ x −26 7 −251 56 ee−iξ −1199 168 e2e−2iξ þ eeiξ 8627 156 −41807ν 312 þ e2e2iξ 785 13 −5156ν 39 þ e2 −8321 104 þ 5156ν 39 ; ðE2hÞ H53 osc ¼ x3νΔ 2 ﬃﬃﬃﬃﬃﬃﬃﬃ 330 p −2 189 þ 27e2 112 þ 41 336 ee−iξ −67 504 eeiξ þ 1531e2e−2iξ 5040 −47 72 e2e2iξ ; ðE2iÞ H55 osc ¼ x3νΔ 14 ﬃﬃﬃﬃﬃ 66 p 18 5 þ 8909e2 720 þ 457 72 ee−iξ þ 197 16 eeiξ þ 787 72 e2e−2iξ þ 4369 144 e2e2iξ ; ðE2jÞ H62 osc ¼ ix5=2e2νe2iξ 352 ﬃﬃﬃﬃﬃ 65 p 2783 168 −53ν ; ðE2kÞ H71 osc ¼ 5x3νeΔ 30888 ﬃﬃﬃ 2 p eiξ 5023 168 −97ν þ ee2iξ 5023 168 −97ν þ e −5023 168 þ 97ν : ðE2lÞ ðE2hÞ ðE2iÞ ðE2jÞ H71 osc ¼ 5x3νeΔ 30888 ﬃﬃﬃ 2 p eiξ 5023 168 −97ν þ ee2iξ 5023 168 −97ν þ e −5023 168 þ 97ν : ðE2lÞ ðE2lÞ 084043-21 MICHAEL EBERSOLD et al. GRAVITATIONAL-WAVE AMPLITUDES FOR COMPACT … Boetzel, C. K. Mishra, G. Faye, A. Gopakumar, and B. R. Iyer, Phys. Rev. D 100, 044018 (2019). [33] T. Damour and N. Deruelle, Ann. Inst. Henri Poincar´e Phys. Th´eor. 43, 107 (1985). [67] See Supplemental Material at http://link.aps.org/ supplemental/10.1103/PhysRevD.100.084043 for a Mathe- matica Notebook containing all memory modes presented in this manuscript. It also lists full expressions for all modes including instantaneous, hereditary and post- adiabatic contributions. [34] R.-M. Memmesheimer, A. Gopakumar, and G. Schäfer, Phys. Rev. D 70, 104011 (2004). [35] T. Damour, A. Gopakumar, and B. R. Iyer, Phys. Rev. D 70, 064028 (2004). [36] C. Königsdörffer and A. Gopakumar, Phys. Rev. D 73, 124012 (2006). [68] D. A. Nichols, Phys. Rev. D 95, 084048 (2017). [69] L. Blanchet, Proc. R. Soc. Ser. A 409, 383 (1987). [37] N. Yunes, K. G. Arun, E. Berti, and C. M. Will, Phys. Rev. D 80, 084001 (2009). [70] DLMF, NIST Digital Library of Mathematical Functions, edited by F. W. J. Olver, A. B. O. Daalhuis, D. W. Lozier, B. I. Schneider, R. F. Boisvert, C. W. Clark, B. R. Miller, and B. V. Saunders (2019), Release 1.0.22 of 2019-03-15, http://dlmf.nist.gov/. [38] N. J. Cornish and J. S. Key, Phys. Rev. D 82, 044028 (2010). [39] J. S. Key and N. J. Cornish, Phys. Rev. D 83, 083001 (2011). 084043-22 PHYS. REV. D 100, 084043 (2019) [77] R. A. Isaacson, Phys. Rev. 166, 1272 (1968). [71] Y. Boetzel, A. Susobhanan, A. Gopakumar, A. Klein, and P. Jetzer, Phys. Rev. D 96, 044011 (2017). [78] K. G. Arun, L. Blanchet, B. R. Iyer, and M. S. S. Qusailah, Phys. Rev. D 77, 064034 (2008). y ( ) [72] L. Blanchet, B. R. Iyer, C. M. Will, and A. G. Wiseman, Classical Quantum Gravity 13, 575 (1996). [79] K. G. Arun, L. Blanchet, B. R. Iyer, and M. S. S. Qusailah, Phys. Rev. D 77, 064035 (2008). [73] K. G. Arun, L. Blanchet, B. R. Iyer, and M. S. S. Qusailah, Classical Quantum Gravity 21, 3771 (2004). [80] K. G. Arun, L. Blanchet, B. R. Iyer, and S. Sinha, Phys. Rev. D 80, 124018 (2009). [74] P. C. Peters, Phys. Rev. 136, B1224 (1964). 75] L. E. Kidder, Phys. Rev. D 77, 044016 (2008). [81] G. Faye, L. Blanchet, and B. R. Iyer, Classical Quantum Gravity 32, 045016 (2015). [81] G. Faye, L. Blanchet, and B. R. Iyer, Classical Quantum Gravity 32, 045016 (2015). [76] G. Faye, S. Marsat, L. Blanchet, and B. R. Iyer, Classical Quantum Gravity 29, 175004 (2012). [76] G. Faye, S. Marsat, L. Blanchet, and B. R. Iyer, Classical [76] G. Faye, S. Marsat, L. Blanchet, and B. R Quantum Gravity 29, 175004 (2012). 084043-23
https://openalex.org/W3013320394	https://www.mdpi.com/2071-1050/12/7/2723/pdf?version=1586764094	English	null	Risk Evaluation of “Not-In-My-Back-Yard” Conflict Potential in Facilities Group: A Case Study of Chemical Park in Xuwei New District, China	Sustainability	2,020	cc-by	13,459	Received: 12 December 2019; Accepted: 23 March 2020; Published: 30 March 2020 Received: 12 December 2019; Accepted: 23 March 2020; Published: 30 March 2020 Abstract: The social risk of chemical industry park projects attracts much attention, as they are perceived to yield strong environmental risks. This paper systematically evaluates the social risk of Xuwei Chemical Park in China, which was investigated as an example to guide the risk control strategy of conﬂict in industrial facilities for developing countries. The results show that residents and government departments have a resistance to the risks of the project as a stronger sense of group risk perception (the value is 7 × 10−6) compared with the basic value of 7 × 10−5. By contrast, the low value of group risk perception was evaluated in an enterprise group (7 × 10−4), indicating that the risks of petrochemical projects are often accepted. The expert group’s risk perception regarding petrochemical projects is consistent with the basic value. This is a very interesting ﬁnding indicating that the greater the experience, the more the support for petrochemical projects. The knowledge and information from education or experience improve the judgment of the risk of the facility, which increases the individual’s rational assessment comprehension of risk. Moreover, factors that are signiﬁcantly related to residents’ attitudes are information cognitive factors (trust in information publicity and petrochemical project understanding), and project inﬂuencing factors (project planning rationality, quality of life improvement, and economic development satisfaction). Among them, the degree of trust in information disclosure has the highest degree of inﬂuence, followed by the level of education, while the satisfaction with economic development has the lowest degree of inﬂuence. Therefore, improving the trust of residents in the information disclosure of petrochemical projects should be the core of the government’s risk control policy. Keywords: environmental risk; chemical park project; social risk; fuzzy comprehensive evaluation method; probit model sustainability sustainability sustainability sustainability Risk Evaluation of “Not-In-My-Back-Yard” Con Potential in Facilities Group: A Case Study of Chemical Park in Xuwei New District, China Yongyou Nie, Jinbu Zhao, Yiyi Zhang and Jizhi Zhou * School of Economics, Shanghai University, Shanghai 200444, China; nyy2000@shu.edu.cn (Y.N.); zhaojin302@shu.edu.cn (J.Z.); zoeyzhang_1222@163.com (Y.Z.) * Correspondence: Jizhi.zhou@t.shu.edu.cn; Tel.: +86-21-66137746 Yongyou Nie, Jinbu Zhao, Yiyi Zhang and Jizhi Zhou * School of Economics, Shanghai University, Shanghai 200444, China; nyy2000@shu.edu.cn (Y.N.); zhaojin302@shu.edu.cn (J.Z.); zoeyzhang_1222@163.com (Y.Z.) * Correspondence: Jizhi.zhou@t.shu.edu.cn; Tel.: +86-21-66137746 Sustainability 2020, 12, 2723; doi:10.3390/su12072723 1. Introduction In the growth of global economy, the petrochemical industry plays an important role. Despite this, the inﬂuence of the petrochemical industry on environmental and human health has attracted much attention for decades, as many pollutants were discharged from the process of petrochemical production. For instance, oil production increases CO2 emissions signiﬁcantly from the ﬁrst to sixth quantiles, with a greater eﬀect at the lowest quantile and a weaker eﬀect at the highest quantile [1]. Chen reported CO2 and NOx emission from ﬂue gas in a petrochemical plant and indicated the essential requirement of pollution reduction [2]. For human health risk, Aghadadashi studied the spatial structure of sedimentary total Polycyclic Aromatic Hydrocarbons(PAHs) and revealed its potential cancer risks [3]. Shaygan examined the prevalence of chronic pain among workers of several petrochemical and petroleum refinery plants for its predictive role of psycho-familial variables (depression, Sustainability 2020, 12, 2723; doi:10.3390/su12072723 www.mdpi.com/journal/sustainability www.mdpi.com/journal/sustainability 2 of 18 Sustainability 2020, 12, 2723 work-family conflict, and job stress) in causing chronic pain when controlling for demographic and occupational factors [4]. This led to a great concern regarding the risk of pollution and eco-system damage from the petrochemical industry. Based on the risk of the petrochemical industry, a public opposition to its construction within a certain range of its own residence occurs. This phenomenon is deﬁned as a “not in my back yard” (NIMBY) conﬂict. It is proposed that this conﬂict is usually activated when a high environmental risk project is recognized by the public. For instance, Signorino indicated that proximity to industrial pollution sources inﬂuences risk perception and assimilates risk perception proﬁles of populations in the risk perception proﬁles of populations residing in the neighborhood of two petrochemical enterprises [5]. Tortosa-Edo showed the corroboration of the direct and indirect eﬀects of personal environmental values on the variables that make up the trust in companies–heuristic-systematic theory (HSM) of information processing–risk perception sequence [6]. Therefore, much eﬀort has been made to reduce the NIMBY conﬂict. Zhiqiang Geng et al. proposed a novel data envelopment analysis (DEA) model based on the aﬃnity propagation (AP) clustering algorithm (AP-DEA), which is eﬃcient in terms of energy saving and carbon emission reduction of a petrochemical industry [7]. Nicolletti studied how petroleum companies can adapt to climate change using social learning approaches [8]. 1. Introduction In 2019, Choi found that the petrochemical industry exhibits 63.5% emission trading scheme (ETS) performance, on average, showing a huge potential for improvement in the sustainable governance of the Korean petrochemical industry [9]. Thus, it is proposed that decreasing the environmental and health risks, combined with increasing the public beneﬁts, are usually the common risk reduction strategies. Recently, the increase in petrochemical production with the continuous increase in the demand for chemicals led to the construction of chemical parks that usually include a group of petrochemical industries [10]. This resulted in a strong NIMBY conﬂict as a result of the increase in risk perception of environmental pollution and health hazards [11]. The conﬂict led to social risk, which had a negative eﬀect on social stability. Therefore, it is necessary to evaluate the risk of chemical parks to the public and its corresponding control strategy. Herein, this paper aims to study the social risks caused by chemical park projects with high environmental risks by taking Xuwei New District as an example. On the basis of identifying the environmental risks existing in chemical projects, from the perspective of risk perception, the social population is assessed based on the social risk perception caused by the project’s environmental risks, and the social risks of the projects are analyzed. Finally, according to the conclusion of the social risk control research, advice and guidance are provided for the government’s risk control policy. 2.1. Social Risk Assessment Risk perception theory is the basic theory of social risk assessment. The World Health Organization (WHO) has provided a guide for risk assessment, which demonstrates that the assessment of risk perception should be considered as a fundamental instrument for creating proper risk communication plans that sustain the implementation of risk-management and territorial-remediation strategies [12]. The theory reveals that risk conﬂict mainly stems from the diﬀerence of risk perceptions between diﬀerent subjects. This diﬀerence is an important driving factor for the formation of group events. Zhang studied the risk of lane-change behaviors in multilane urban expressway oﬀ-ramp areas [13]. Yu et al. explored the impact of risk programs on risk perceptions of nearby residents in 2018 [14]. The results showed that residents’ age, gender, education level, and environmental awareness were signiﬁcantly correlated with their risk perception. This reasoning could be followed by adverse environmental, social, and economic eﬀects that could threaten the sustainable development of urban spaces. Based on risk perception and project environmental risk perspectives, Leﬂey studied the relationship between risk occurrence probability and the possible impact on risk management when an accident does occur [15]. By analyzing the changes in residents’ risk perceptions in risk-disaster accidents, Chiang suggested 3 of 18 Sustainability 2020, 12, 2723 that residents’ risk perceptions should be incorporated into risk communication to promote residents’ adaptive actions in accidents [16]. Zhu et al. studied the key factors of people’s anti-nuclear behavior intentions [17]. The results indicate that people’s behavior intentions are driven by risk perception, which cannot be stimulated by systematic processing. This usually results in an inverse U-relationship between perceived knowledge and behavior intention against facility construction. p g g y The quantitative risk assessment method is based on the quantitative assessment of the risk of high-risk environmental projects to evaluate the social acceptability of the risks associated with accidents. Thus, risk assessment is a concrete method for measuring risk perception and a tool to reveal the potential values of risk in real situations. The assessment method is widely used in the quantitative assessment of risks in various chemical industries and other areas [11,12]. Wang et al. proposed a new dynamic quantitative risk assessment method to analyze the operational performance of chemical processes, and to estimate the probability of the occurrence of risk events by monitoring multiple key variables [18]. Recently, Gholipour et al. 2.1. Social Risk Assessment used quantitative microbial risk assessment (QMRA) to analyze Listeria infections for workers and farmers [19]. Alileche et al. studied the domino-eﬀect quantitative analysis method applied to the chemical park scenario, which provided an overview, comparison, and discussion of the current regulations of the domino eﬀect [20]. Based on the domino eﬀect, Cozzani et al. reported a quantitative risk assessment of accidents caused by process equipment ﬁres and explosion damage in the chemical industry [21]. Markowski and Kotynia incorporated the “bowtie” model into the analytic hierarchy analysis method to achieve the quantitation of the risk, and optimized the safety measures for speciﬁc accident scenarios through optimization of the model’s construction [22]. Fabbrocino used quantitative analysis to study the chemical plant accident hazard caused by seismic risk and gave countermeasures [23]. In 2016, Valencia-Barragán et al. quantitatively analyzed the potential impacts of chemical park accidents on people inside and outside the industry, and conducted a risk analysis of insiders and personnel leaving the factory [24]. In the evaluation of the social risk of the petrochemical project in Xujing New District, this paper establishes a risk perception evaluation index system by using the analytic hierarchy process, and uses the fuzzy comprehensive evaluation method to measure the risk perception value of diﬀerent groups and society to analyze the social risks of the project. 2.2. Social Risk Control The social ampliﬁcation theory of risk is an important theory of social risk research. The theory holds that the relationship between risk and risk events is linked by risk signals, and that the risk signals control the scope and role of risks through reinforcement and weakening [25]. In 2018, Fellenor examined the social ampliﬁcation of risk on Twitter [26]. Jagiello and Hills explored the eﬀects of message dissemination on risk ampliﬁcation and risk communication, which indicated that the more widely messages spread, the more negative statements were contained in the message [27]. The increase in perceived risk and the generation of negative information are closely related to the amount of information received. Moreover, re-exposure to the initial information is ineﬀective for reducing prejudice. Wirz et al. used the risk framework to study the role of social media responsibility and risk perception in risk ampliﬁcation, which pointed out the importance of multilingual approaches in risk communication [28]. In 2002, using the risk ampliﬁcation eﬀect model, Frewer et al. analyzed the collection of attitude data before, during, and after the increased reporting of the risks of genetically modiﬁed food in the United Kingdom (spring 1999). It has been demonstrated that people’s risk perceptions increase and decrease in line with what might be expected upon examination of the ampliﬁcation and attenuation mechanisms integral to the framework [29]. It was concluded that the social ampliﬁcation of risk frameworks is a useful framework for beginning to explain the potential impact of a risk event on risk perceptions, particularly if that risk events are presented to the public as a new hazard occurring in a crisis context. By studying the uncertainty and instability of risk message propagation through the diﬀusion chain, Mehdi et al. provided the quantitative analysis of risk-aware 4 of 18 Sustainability 2020, 12, 2723 social ampliﬁcation to help policymakers with the better prediction and management of public insights into emerging threats [30]. Risk control is an attempt to establish a risk control mechanism as a way to prevent risks and is based on the analysis of risk triggers. An open information environment, as well as high variability and uncertainty are necessary parts of the vacancy of an active distributed network (ADN) risk control system. In 2018, Wang and Ieee applied a cyber physical system (CPS) into ADN risk control. 2.2. Social Risk Control In the formulation of the risk prevention and control system, the main risk control measures are enterprise risk control, personnel risk control, park risk control, and government risk control [31]. For instance, the risk prevention and control system, anti-control system, and supervision system of chemical park enterprises can provide corresponding suggestions and improve the safe production capacity of enterprises. In order to improve the ability of emergency management for storage areas of dangerous chemicals, a framework for a risk management technical system for ﬂammable and explosive dangerous chemicals was proposed [32]. Using the dynamic risk management system, it can eﬀectively achieve the goal of dynamic supervision, risk identiﬁcation, and real-time monitoring, as well as assisting the emergency decision-making of dangerous chemicals in the whole life cycle. Under this circumstance, the government should guide its own branches, the media, experts, and enterprises to conduct risk supervision and risk information judgment to build an eﬃcient chemical park from the perspectives of risk participants. In the formulation of government risk control policies, it is recommended that residents’ risk attitudes be inﬂuenced through the control of diﬀerent risk signals, such as media and experts, to improve residents’ support for petrochemical projects. 2.3. Literature Review of Model Application 2.3. Literature Review of Model Application 2.3.1. Fuzzy Comprehensive Evaluation Method The fuzzy comprehensive evaluation method is based on the fuzzy set theory and the principle of maximum membership degree. The fuzzy set is used to eﬀectively quantify the evaluation target. This method is widely used to solve the complex problem of multi-factor decision-making [33]. The analysis is used to determine the membership degree of each layer of indicators in the indicator set based on the set of indicators [34]. In 2014, Shi et al. obtained the best emergency treatment technical solution immediately after a chemical pollution accident occurred in the planning database [35]. Based on the group decision-making improved fuzzy comprehensive evaluation method, the technical evaluation index system was established. An example analysis was carried out with an aniline pollution accident. The sustained casing pressure (SCP) threatened the wellbore safety signiﬁcantly. Considering the serious SCP situation in the gas ﬁeld in the southwest of China, Dezhi established a SCP risk evaluation model based on the fuzzy comprehensive evaluation method [36]. The social risk assessment of the chemical park project studied in this paper is a multi-index level problem. In the social risk assessment, the fuzzy comprehensive evaluation method is used to study the social risk perception of diﬀerent social groups, and the risk perception value of diﬀerent groups is calculated comprehensively. The social total risk perception value is used for determining the social risk of the project. 3.1. Xuwei New District Xuwei New District (ND) is located in the southeast of Lianyungang city. The petrochemical industry is one of the key port development industries in Xuwei. The construction of a large petrochemical industrial base in Xuwei is an important part of Jiangsu’s petrochemical industry layout adjustment. The development of the petrochemical industry is based on the integration of refining, ethylene, and aromatics, supplemented by diversified raw materials processing, featuring clean energy, organic raw materials, and synthetic materials that feature new chemical materials and fine chemicals. Xuwei New District (ND) is located in the southeast of Lianyungang city. The petrochemical industry is one of the key port development industries in Xuwei. The construction of a large petrochemical industrial base in Xuwei is an important part of Jiangsu’s petrochemical industry layout adjustment. The development of the petrochemical industry is based on the integration of refining, ethylene, and aromatics, supplemented by diversified raw materials processing, featuring clean energy, organic raw materials, and synthetic materials that feature new chemical materials and fine chemicals. A large-scale refining and chemical integration form a multi-product chain and multi-product cluster. The construction of the petrochemical industry has shifted the petrochemical industry along the Yangtze River, promoted industrial adjustment and upgrading, and played a major role in meeting the demand for petrochemical products and raw materials in the Yangtze River Delta region, as well as in the central and western regions A number of companies have officially entered the production and operation Xuwei New District (ND) is located in the southeast of Lianyungang city. The petrochemical industry is one of the key port development industries in Xuwei. The construction of a large petrochemical industrial base in Xuwei is an important part of Jiangsu’s petrochemical industry layout adjustment. The development of the petrochemical industry is based on the integration of refining, ethylene, and aromatics, supplemented by diversified raw materials processing, featuring clean energy, organic raw materials, and synthetic materials that feature new chemical materials and fine chemicals. A large-scale refining and chemical integration form a multi-product chain and multi-product cluster. The construction of the petrochemical industry has shifted the petrochemical industry along the Yangtze River, promoted industrial adjustment and upgrading, and played a major role in meeting the demand for petrochemical products and raw materials in the Yangtze River Delta region, as well as in the central and western regions. 3.1. Xuwei New District A number of companies have officially entered the production and operation stage, including Jiangsu Honggang Petrochemical Co, Ltd. (Lianyungang, China), and Lianyungang Hongyang Thermal Power Co, Ltd. (Lianyungang, China) Xuwei ND includes three administrative villages (Zhangtiao, Dongbanshan, and Xianghe) and four communities (Yuejin, Chengwei, Banqiao, and Gaowei) with a population of 16,000. 3.2. Construction of Social Risk Assessment Model The social risk assessment of the petrochemical project is based on the risk perception perspective, examining the social risks caused by the project’s environmental risks, and establishing the social risk assessment model to analyze the social risks of the project. According to the Figure 1 showed in the following text, the social risk assessment model consists of three parts: (1) the basic theory—risk perception theory, and the setting of the risk perception base value; (2) the social risk perception evaluation index system; and (3) the social risk perception assessment. The analysis process divides the entire social population into residents. Groups, expert groups, government departments, and enterprise groups are the subjects in the fuzzy comprehensive evaluation method; the risk perception value of each group is calculated, along with the total social risk perception value. 2.3.2. Probit Model In the research of social risks, many scholars have chosen to apply econometric models, statistical analysis models, and other methods to both assess risks and prevent accidents [37]. The Probit model is also used as a discrete selection model for the analysis and prediction of risk accidents [38]. In view of the potential accident risks brought by technical operations to the process industry, Crăciu et al. studied the impact of thermal radiation on the population and used diﬀerent probit functions to carry out personal risk calculations. By comparing the case ﬁndings, diﬀerent uses were applied in estimating the consequences. Based on an ordered probit model, the risk degree model of 5 of 18 Sustainability 2020, 12, 2723 bridge damage caused by the collision of disabled ships was established and applied to analyze the risk degree of bridge damage [39]. Ma and Jie aimed to understand four types of vehicle ownership within a household, including the automobile, motorcycle, electric bicycle, and human-powered bicycle [40]. The study presented a cross-sectional multivariate ordered probit model with a composite marginal likelihood estimation approach, which accommodated the eﬀects of explanatory variables and captured the dependence among the propensity of households for vehicle ownership. To ensure that people can safely evacuate during chemical release, James determined the maximum safe shelter time by using the probit model and provided a link between expected response probability and group exposure to speciﬁc risk events [41]. In order to eﬀectively control the risk of petrochemical projects in chemical parks, this paper takes the residents’ group participating in the group event as the research subject of risk control, so as to analyze the factors aﬀecting residents’ risk attitudes using the probit model. We also select key factors to formulate risk control policies. 3.2.1. Risk Perception Base Value Project environmental risk characteristics Personal influence factor Social influence factor Social risk perception impact H e a l t h r i s k A c c i d e n t r i s k P o l l u t i o n r i s k S e c u r i t y R i s k C a r e e r a g e E d u c a t i o n A c c e p t t h e r i s k R i s k e x p e r i e n c e R i s k e d u c a t i o n G o v e r n m e n t s u p e r v i s i o n l e v e l G o v e r n m e n t a s s i s t a n c e C h e m i c a l p a r k i n f o r m a t i o n d i s c l o s u r e C h e m i c a l p a r k a c c i d e n t e m e r g e n c y r e s c u e c a p a b i l i t y M e d i a c r e d i b i l i t y A B C Figure 2. Social Risk Perception Assessment Index System. ded in each influencing factor. h i l i k h i i f h Figure 2 Social Risk Perception Assessment Index System. Figure 2. Social Risk Perception Assessment Index System. y , p j risks, accident risks, pollution risks, and safety risks. The personal influence factors were selected including basic personal characteristics, such as occupation, age, and education, as well as risk-aware factors, such as risk willingness, risk experience, and risk education. Social influence factors include government supervision level, government assistance, chemical park information disclosure, chemical park accident emergency rescue capability, and media credibility. 3 2 3 G Ri k P ti C l l ti 3.2.2. 3.2.1. Risk Perception Base Value The third-level indicator layer (layer C) is the evaluation index included in each influencing factor Project environmental risk characteristics Personal influence factor Social influence factor Social risk perception impact H e a l t h r i s k A c c i d e n t r i s k P o l l u t i o n r i s k S e c u r i t y R i s k C a r e e r a g e E d u c a t i o n A c c e p t t h e r i s k R i s k e x p e r i e n c e R i s k e d u c a t i o n G o v e r n m e n t s u p e r v i s i o n l e v e l G o v e r n m e n t a s s i s t a n c e C h e m i c a l p a r k i n f o r m a t i o n d i s c l o s u r e C h e m i c a l p a r k a c c i d e n t e m e r g e n c y r e s c u e c a p a b i l i t y M e d i a c r e d i b i l i t y A B C Figure 2. Social Risk Perception Assessment Index System. In the assessment system, the environmental risk characteristics of the project include health risks, accident risks, pollution risks, and safety risks. The personal inﬂuence factors were selected including basic personal characteristics, such as occupation, age, and education, as well as risk-aware factors, such as risk willingness, risk experience, and risk education. Social inﬂuence factors include government supervision level, government assistance, chemical park information disclosure, chemical park accident emergency rescue capability, and media credibility. Figure 2 Social Risk Perception Assessment Index System. 3.2.2. 3.2.1. Risk Perception Base Value Risk Perception Assessment Indicator System It can been seen from figure2, in the social risk assessment model, the social risk perception assessment index system consists of three layers, the first of which is the overall goal (level one ndicator layer A), which is the degree of social risk perception. The second level indicator layer (layer B) includes three effects: the factors affecting the environmental risk characteristics of the project personal influence factors, and social influence factors. The third-level indicator layer (layer C) is the evaluation index included in each influencing factor. In the assessment system the environmental risk characteristics of the project include health l t h r i s k d e n t r i s k t i o n r i s k r i t y R i s k a r e e r a g e u c a t i o n t t h e r i s k e x p e r i e n c e e d u c a t i o n e r n m e n t i s i o n l e v e l e r n m e n t i s t a n c e i c a l p a r k o r m a t i o n c l o s u r e i c a l p a r k t e m e r g e n c y c a p a b i l i t y c r e d i b i l i t y C Figure 2 Social Risk Perception Assessment Index System. 3.2.1. Risk Perception Base Value Chemical accident management requires studies estimating the potential scale of chemical accidents’ eﬀects, their unpredictability, and the uncertainties of their consequences for environmental risk assessment [42–44]. The risk value of chemical accidents was evaluated by the natural mortality rate, which served as the benchmark for risk assessment [45]. At the end of 2017, China’s total population was 139.08 million and the population mortality rate was 0.711%. The conservative estimate Sustainability 2020, 12, 2723 6 of 18 of the natural mortality rate of the population was 0.7%. Since the death risk of residents is increased through the potential emissions of the chemical industries, an annual mortality rate of 1% was taken as the basic value. Therefore, the perceived risk base value of the project was 7 × 10−5. Sustainability 2020, 12, 2723 6 of 18 Figure 1 Social Risk Assessment Model. Figure 1. Social Risk Assessment Model. Sustainability 2020, 12, 2723 6 of 18 Figure 1 Social Risk Assessment Model. Figure 1. Social Risk Assessment Model. S i l i k ti i 3.2.2. Risk Perception Assessment Indicator System Project environmental risk characteristics Personal influence factor Social influence factor Social risk perception impact H e A c c P o l S e c E A c c e R i s k R i s k G o s u p e r G oa C h ei nd C h e a c c i d e r e s c u M e d i a B It can been seen from Figure 2, in the social risk assessment model, the social risk perception assessment index system consists of three layers, the ﬁrst of which is the overall goal (level one indicator layer A), which is the degree of social risk perception. The second level indicator layer (layer B) includes three eﬀects: the factors aﬀecting the environmental risk characteristics of the project, personal inﬂuence factors, and social inﬂuence factors. The third-level indicator layer (layer C) is the evaluation index included in each inﬂuencing factor. Figure 1 Social Risk Assessment Model. Figure 2 Social Risk Perception Assessment Index System. 3.2.2. Risk Perception Assessment Indicator System It can been seen from figure2, in the social risk assessment model, the social risk perception assessment index system consists of three layers, the first of which is the overall goal (level one indicator layer A), which is the degree of social risk perception. 3.2.1. Risk Perception Base Value The second level indicator layer (layer B) includes three effects: the factors affecting the environmental risk characteristics of the project, personal influence factors, and social influence factors. The third-level indicator layer (layer C) is the evaluation index included in each influencing factor. In the assessment system, the environmental risk characteristics of the project include health risks, accident risks, pollution risks, and safety risks. The personal influence factors were selected including basic personal characteristics, such as occupation, age, and education, as well as risk-aware factors, such as risk willingness, risk experience, and risk education. Social influence factors include government supervision level, government assistance, chemical park information disclosure, chemical park accident emergency rescue capability, and media credibility. 3.2.3. Group Risk Perception Calculation a l t h r i s k i d e n t r i s k u t i o n r i s k u r i t y R i s k C a r e e r a g e d u c a t i o n p t t h e r i s k e x p e r i e n c e e d u c a t i o n v e r n m e n t v i s i o n l e v e l v e r n m e n t s i s t a n c e m i c a l p a r k f o r m a t i o n s c l o s u r e m i c a l p a r k n t e m e r g e n c y e c a p a b i l i t y c r e d i b i l i t y C Figure 2 Social Risk Perception Assessment Index System. 3.2.2. Risk Perception Assessment Indicator System It can been seen from figure2, in the social risk assessment model, the social risk perception assessment index system consists of three layers, the first of which is the overall goal (level one indicator layer A), which is the degree of social risk perception. The second level indicator layer (layer B) includes three effects: the factors affecting the environmental risk characteristics of the project, personal influence factors, and social influence factors. 3.2.1. Risk Perception Base Value Risk Perception Assessment Indicator System It can been seen from figure2, in the social risk assessment model, the social risk perception assessment index system consists of three layers, the first of which is the overall goal (level one indicator layer A), which is the degree of social risk perception. The second level indicator layer (layer B) includes three effects: the factors affecting the environmental risk characteristics of the project, personal influence factors, and social influence factors. The third-level indicator layer (layer C) is the In the assessment system, the environmental risk characteristics of the project include health risks, accident risks, pollution risks, and safety risks. The personal inﬂuence factors were selected including basic personal characteristics, such as occupation, age, and education, as well as risk-aware factors, such as risk willingness, risk experience, and risk education. Social inﬂuence factors include government supervision level, government assistance, chemical park information disclosure, chemical park accident emergency rescue capability, and media credibility. 7 of 18 Sustainability 2020, 12, 2723 7 of 18 (3) Single factor membership. The rating of the individual factors of the three-level indicator evaluation set UC was analyzed by collecting data from the risk perception questionnaire of the respondents. The evaluation result for the risk perception degree for each sample was combined with the normalization method to calculate the proportion of each index being evaluated at diﬀerent levels. The membership value of the index for the ﬁve evaluation levels was able to be determined, and the maximum membership degree principle could be inferred for a single risk perception level of each diﬀerent factor of the sample. Moreover, according to the membership value of the three-level indicator, the membership degree matrix of each level factor included in the second layer B can be further obtained: VBi =   vc11 · · · vcm1 ... ... ... vc1n · · · vcmn   , i = 1, 2, 3. (2) Determining the weights of indicators at all levels. (2) Determining the weights of indicators at all levels. Assuming that the weight value of each factor of the second-level indicator UB is ωi, the weight set is WB1 = (ωC1, . . . , ωC4), WB2 = (ωC5, . . . , ωC10), WB3 = (ωC11, . . . , ωC15). The weight set of the target layer A layer is WA = (ω1, ω2, ω3). The weight determination of the risk-aware inﬂuence factor was determined by the collected data according to the weight calculation principle of the analytic hierarchy process [49]. According to the opinions of experts who participated in the environmental impact assessment hearing of the overall development plan of the Lianyungang Petrochemical Industrial Base and the weight calculation principle of the above analytic hierarchy process, the weight values of risk-aware factors were obtained. It can be seen that the second-level indicator weight sets are: WB1 = (0.467, 0.278, 0.160, 0.095) WB2 = (0.094, 0.093, 0.238, 0.112, 0.200, 0.263) WB3 = (0.187, 0.230, 0.167, 0.322, 0.094) The weight set of the target layer A is: WA = (0.509, 0.179, 0.312). WB1 = (0.467, 0.278, 0.160, 0.095) WB3 = (0.187, 0.230, 0.167, 0.322, 0.094) The weight set of the target layer A is: WA = (0.509, 0.179, 0.312). (3) Single factor membership. 3.2.3. Group Risk Perception Calculation As the inﬂuence degree of various factors on diﬀerent groups’ risk perception is complex and uncertain, the fuzzy comprehensive evaluation method is an eﬀective method to solve these uncertainties [46]. This method was used to quantitatively evaluate the risk perception of diﬀerent groups. The assessment for the level of risk perception was set to ﬁve levels, namely V1 (strong) to V5 (weak). According to the weight calculation step of the fuzzy comprehensive evaluation method, the evaluation of the bottom layer index was carried out, followed by the calculation of the target layer weight [47]. The speciﬁc calculation was as follows [48]. (1) Building a multi-level evaluation set. (1) Building a multi-level evaluation set. (1) Building a multi-level evaluation set. Suppose A contains the set of all factors of the first level indicator (target layer), UA = (UB1, UB2, UB3), and the single factor set corresponding to layer B is: UB1 = (C1, . . . , C4), UB2 = (C5, . . . , C10), UB3 = (C11, . . . , C15). The evaluation set of risk influencing factors C is a collection of all possible evaluation results of the evaluation object. The evaluation set is defined as UC = VC = (vc1, vc2, . . . , vcn), where vn represents a possible risk-aware evaluation result. Suppose A contains the set of all factors of the first level indicator (target layer), UA = (UB1, UB2, UB3), and the single factor set corresponding to layer B is: UB1 = (C1, . . . , C4), UB2 = (C5, . . . , C10), UB3 = (C11, . . . , C15). The evaluation set of risk influencing factors C is a collection of all possible evaluation results of the evaluation object. The evaluation set is defined as UC = VC = (vc1, vc2, . . . , vcn), where vn represents a possible risk-aware evaluation result. (2) Determining the weights of indicators at all levels. (4) Fuzzy evaluation set of each indicator. The fuzzy comprehensive evaluation set of each evaluation index layer can be calculated in the following way: First, the membership degree matrix of the various factors included in B is Sustainability 2020, 12, 2723 8 of 18 VB1, VB2, VB3, and in the second-level fuzzy evaluation set, the weight set is WB1 = (ωC1, . . . , ωC4), WB2 = (ωC5, . . . , ωC10), WB3 = (ωC11, . . . , ωC15). Then, the formula based on the indicator fuzzy evaluation set can be calculated: Bi = WBi × VBi, i = 1, 2, 3 (1) (1) A two-level fuzzy evaluation set for the factors aﬀecting the environmental risk characteristics of the project can be expressed as: A two-level fuzzy evaluation set for the factors aﬀecting the environmental risk characteristics of the project can be expressed as: B1 = WB1 × VB1 = (ωC1, . . . , ωC4)   vc11 · · · vc41 ... ... ... vc15 · · · vc45   = (vC1, vC2, . . . , vC4) (2) (2) The fuzzy assessment set of personal factors and social factors are evaluated in the same way with a fuzzy evaluation set of the ﬁrst-level indicators. The fuzzy assessment set of personal factors and social factors are evaluated in the same way with a fuzzy evaluation set of the ﬁrst-level indicators. (5) Risk perception value synthesis calculation. (5) Risk perception value synthesis calculation. (5) Risk perception value synthesis calculation. The calculation formula based on the risk perception value can be expressed as: (3) P(i) = P(a) × α (3) where P(i) is the risk perception value, P(a) is the risk perception base value of 7 × 10−5, α is the risk perception degree coeﬃcient, and the risk perception degree according to the fuzzy comprehensive evaluation method corresponds to the selected coeﬃcient (Table 1). As shown in Table 2, the risk perception value is small as the degree of risk perception is weak, which indicates that the group can accept more risks. If the degree of risk perception were strong, the group would have a risk-resisting mood. Table 1. Single factor membership and evaluation level of each factor. Factor Single Factor Membership Evaluation Level V1 V2 V3 V4 V5 Risk factor membership assessment Project environmental risk characteristics B1 Health risk C1 0.212 0.420 0.231 0.086 0.051 Accident risk C2 0.223 0.416 0.342 0.018 0.001 Pollution risk C3 0.076 0.052 0.345 0.327 0.200 Security risk C4 0.041 0.368 0.227 0.234 0.134 Personal inﬂuence factor B2 Career C5 0.030 0.024 0.621 0.113 0.212 Age C6 0.010 0.002 0.101 0.527 0.360 Education C7 0.050 0.057 0.574 0.202 0.117 Accept the risk willingness C8 0.001 0.227 0.442 0.320 0.010 Risk experience C9 0.010 0.531 0.337 0.112 0.010 Risk Education C10 0.045 0.112 0.531 0.212 0.100 Social inﬂuence factor B3 Government supervision level C11 0.030 0.621 0.212 0.117 0.020 Government assistance support capacity C12 0.015 0.312 0.527 0.116 0.030 Chemical Park Information Disclosure C13 0.010 0.114 0.312 0.447 0.117 Chemical Park Accident Emergency Rescue Capability C14 0.212 0.628 0.117 0.023 0.020 Media credibility C15 0.050 0.062 0.515 0.213 0.160 Table 2. Risk perception degree coeﬃcient and perceived value. Risk Perception Stronger Strong Normal Weak Weaker Risk perception coeﬃcient 0.01 0.1 1 10 100 Risk perception value 7 × 10−7 7 × 10−6 7 × 10−5 7 × 10−4 7 × 10−3 Table 1. Single factor membership and evaluation level of each factor. Table 1. Single factor membership and evaluation level of each factor. Table 2. Risk perception degree coeﬃcient and perceived value. Sustainability 2020, 12, 2723 9 of 18 3.2.4. Social Total Risk Perception Calculation 3.2.4. Social Total Risk Perception Calculation According to the fuzzy comprehensive evaluation method and the principle of maximum membership degree, the risk perception results of each group were analyzed. The risk perception value P(i), i = 1, 2, 3, 4 of each group was obtained by the risk perception calculation formula. The total risk perception value P of society was then calculated using the following equation: (4) P = P(i) × W = P(1) × ω1 + P(2) × ω2 + P(3) × ω3 + P(4) × ω4 (4) where W = (ω1, ω2, ω2, ω4) is the weight value of each group. 3.3. Social Risk Perception Assessment 3.3. Social Risk Perception Assessment 3.3.1. Survey and Data Sources Document research was conducted on four groups, taking residents as an example. Data collection was conducted by issuing a public participation questionnaire. The participants of this survey were residents living within 10 km of the boundary of the petrochemical base. We carried out the survey in seven regions in Xuwei, and on average, 76 samples were conducted in each. The residents living in these regions included farmers, industry workers, and people who attained both fundamental and elite education. The questionnaire was distributed by means of direct investigation and entrusting units. A total of 532 questionnaires were distributed and 516 questionnaires were collected, of which 500 were valid questionnaires. The eﬀective recovery rate was 94.1%. The content of the questionnaire design consisted of the risk assessment factors that were selected at diﬀerent evaluation levels of residents’ risk perception assessments. 3.3.2. Social Risk Perception Assessment of Resident Groups The data collected by the questionnaire were analyzed to obtain each factor’s membership degree as well as the evaluation grade result for the individual factors of risk perception, which in turn inﬂuence the factors of the resident group. According to the fuzzy comprehensive evaluation method and the principle of maximum membership degree, the residents’ judgment on the risk perception factors was further analyzed. Among the project’s characteristic risk factors, residents’ risks perceptions of health risks, accident risks, and safety risks were relatively strong (0.420, 0.416, and 0.368, respectively). This highlighted that the health risks in the park are important factors aﬀecting the health of residents. The outbreak of accidents is a risk that has the potential to directly lead to residents’ panic and aﬀect social stability. Similarly, security risks can trigger the outbreak of panic in residents’ groups. This indicates that residents’ risk perception of these three factors is strong. The risk of pollution may involve more ecological and environmental impacts. It takes a long time for pollution to accumulate to a level at which it has a signiﬁcant impact on the environment. Accordingly, residents’ perception of pollution risks is at a normal level (0.345). Among the personal inﬂuence factors, the risk experience is relatively strong for residents’ risk perception (0.531), because the residents are more sensitive to the risk and the resistance to the equipment will be stronger after the risk accident. According to the calculation formula of the fuzzy evaluation set of the second-level index, and according to the fuzzy comprehensive evaluation of the maximum membership degree, the maximum membership degree of B1 is 0.355, and the evaluation grade is V2, indicating that the residents’ perceptions of the project’s environmental risk characteristics is strong. The degree of B2 is at a normal level. The results of this indicator demonstrate that the impact of personal characteristics on residents’ risk perception is at a normal level. The fuzzy matrix based on the ﬁrst-level indexes obtained above constitutes the evaluation matrix VB of the target layer. The fuzzy matrix of the target layer is obtained by calculation, A = [0.170 0.342 0.317 0.149 0.071]. According to the maximum criterion of fuzzy comprehensive rating membership, 10 of 18 Sustainability 2020, 12, 2723 the residents’ groups have a strong level of environmental risk perception of the petrochemical project. 4.1. Social Risk Control Model Construction Risk control research along with the largest risk-aware group is divided into three parts, namely: the conﬁrmation of risk control research subjects; the selection of risk control factors and the degree of inﬂuence; and the design of risk control policies. 4.2. Survey Object and Data Source The survey scope of the social risk control investigation was streets, villages, enterprises, and institutions within the scope of evaluation of petrochemical bases. The design of the questionnaire was divided into three aspects: (1) basic personal characteristics, including gender, age, education level, occupation, etc.; (2) information on cognitive factors, including environmental quality satisfaction, petrochemical project understanding, and information disclosure trust; and (3) project inﬂuencing factors, including project planning rationality, quality of life improvement, economic development satisfaction, etc. You can see the results in Table 3 and see more details in Section 3.3.1. 3.3.2. Social Risk Perception Assessment of Resident Groups According to the calculation formula for residents’ risk perception value, the perceived value of the resident group is 7 × 10−6, which indicates that the resident group has a strong sense of risk of the petrochemical project, and that residents have a resistance to risk. In the same way, the perceived value of the expert group is 7 × 10−5, which demonstrates that the risk perception level of the expert group for the petrochemical project is normal. This indicates that experts rely more than residents on professional risk analysis for petrochemical projects via knowledge and professional ability to assess the risks of the project itself and the combined impact on society. The risk perception value of the government department is 7 × 10−6, indicating that the government’s risk perception level for the petrochemical project is at a relatively high level. The risk perception value of the enterprise groups is 7 × 10−4, indicating that the enterprise group’s risk perception of the Xuwei Petrochemical Project is weak, and the enterprise can accept more risks. It shows that enterprises are willing to accept greater risks and have a lesser degree of risk perception when they grasp the maximization of their own information and corporate interests. 3.3.3. Total Social Risk Perception Based on the analysis of the expert questionnaire, the multi-level structure weights of the residents, expert groups, government departments, and enterprise groups were determined (Equation (4)). The total social risk perception value is about 7.252 × 10−4, which is the result of the standard value of acceptable social risk perception. After the group risk perception and social risk perception are obtained, the results can be used as a reference for the planning of the park project. The risk of the petrochemical project should not exceed the standard value acceptable for a certain group and society. 4.3. Variable Setting The residents’ attitudes were set as the dependent variable Y, which was measured by the support rate of the measurement model. In the selection of the independent variable X, the key independent variables of this paper were set as information cognitive factors and project inﬂuencing factors according to our previous survey results of risk attitude evaluation of waste incineration plant with the model hypothesis [50]. Among them, information cognitive factors include environmental quality satisfaction, petrochemical project understanding, and information disclosure trust. Project inﬂuencing factors include project planning rationality, quality of life improvement, and economic development satisfaction. Sustainability 2020, 12, 2723 11 of 18 4.4. Probit Model Construction The constructed Probit regression model was used to measure the probability of residents supporting the construction of petrochemical projects, and then to assess the main inﬂuencing factors of residents’ attitudes. The Probit regression model is expressed as: Y∗ i = α + βXi + µ (5) (5) where Xi is the independent variable vector, which refers to the basic personal eigenvector (X1), information cognitive factor vector (X2), and project inﬂuence factor vector (X3). Yi is the explanatory variable, indicating whether the residents support the petrochemical project construction, µ is the random interference term and it obeys the standard positive distribution. When Yi = 1, the probability that the residents support the project construction can be expressed as: P(Yi = 1\|Xi = x) = P Y∗ i > 0 x = P[µ > −(α + βx) x] = 1 −P[µ ≤−(α + βx) x] = 1 −Φ[−(α + βx)] = Φ(α + βx) The analyzed variables are then incorporated into the above equation to obtain the following formula: P(Yi = 1\|Xi) = Φ(α0 + β1X1 + β2X2 + β3X3 + µ) = Φ(α0 + β11x11 + β12x12 + β13x13 + β14x14 + β21x21 +β22x22 + β23x23 + β31x31 + β32x32 + β33x33 + µ) According to the inﬂuencing factors of residents’ attitudes summarized above, x11 represents the ﬁrst independent variable under the basic personal eigenvector, namely the resident’s gender, x12 is the age, x13 is the education level, and x14 is the occupation; x21 is the ﬁrst independent variable under the information cognition factor of the environmental quality satisfaction, x22 is the degree of petrochemical project understanding, and x23 is the degree of trust in information publicity; x31 is the ﬁrst independent variable under the project inﬂuencing factors—the project planning rationality, x32 is the degree of quality of life improvement, and x33 is the degree of economic development satisfaction. 4.5. Model Analysis Results The probit regression model was used to analyze the inﬂuencing factors aﬀecting residents’ attitudes. The results are shown in Table 4. In the regression results of the model, the factors aﬀecting the basic personal characteristics are not signiﬁcantly related, except for the signiﬁcant degree of cultural levels and residents’ attitudes. Among the information cognitive factors, petrochemical project understanding, trust in information publicity, and residents’ attitudes are signiﬁcant. Further relevant factors are project planning rationality, quality of life improvement, and economic development satisfaction, which are signiﬁcant factors aﬀecting residents’ attitudes. In the average marginal eﬀect of the probit model (Table 5), in terms of information cognitive factors, the more comprehensive the interviewee’s information cognition, the more support they had for the petrochemical project construction. This is embodied in the following two aspects: the resident’s understanding of the petrochemical project can increase the project support rate by 5% for each level of increase; the resident’s conﬁdence in the project’s information publicity rises by one level, which indicates that the probability of residents supporting the project is 18% higher than the probability of residents opposing the projects. This shows that the higher the trust in information disclosure and the higher the understanding of petrochemical projects, the more inclined residents are to support the construction of the project. This means that the government can understand and support the project construction when the residents fully understand and trust the petrochemical project. 12 of 18 Sustainability 2020, 12, 2723 Table 3. Descriptive statistics of questionnaire survey results. Variable Number of People Proportion (%) Information cognition factor Environmental quality satisfaction Very satisﬁed 141 28.2 Satisﬁed 312 62.4 Not satisﬁed 47 9.4 Petrochemical project understanding understanding 242 48.4 Know a little 172 34.4 Do not understand 86 17.2 Information disclosure trust Trust 298 59.6 Does not matter 156 31.2 Distrust 46 9.2 Project impact factors Project planning rationality More reasonable 168 33.6 Reasonable 314 62.8 Unreasonable 18 3.6 Quality of life improvement Increase 344 68.8 No eﬀect 122 24.4 Reduce 34 6.8 Economic development satisfaction Advantageous 441 88.2 No eﬀect 52 10.4 Unfavorable 7 1.4 Attitude towards the petrochemical project Support 442 88.4 Oppose 58 11.6 Table 3. Descriptive statistics of questionnaire survey results. Table 4. Model generalized linear regression results. 4.5. Model Analysis Results Variables Probit Model Logistic Model Basic personal characteristics Gender −0.0643 −0.0900 [0.147] [0.255] Age −0.0488 −0.259 [0.104] [0.212] Educational level 0.546 * 0.992 * [0.083] [0.159] Career 0.0488 0.109 [0.048] [0.090] Information cognition factor Environmental quality satisfaction 0.151 0.350 [0.129] [0.241] Petrochemical project understanding 0.223 * 0.557 * [0.186] [0.382] Information disclosure trust 0.813 * 1.591 * [0.181] [0.380] Project inﬂuencing factors Project planning rationality 0.444 0.810 [0.193] [0.365] Quality of life improvement 0.414 0.885 [0.209] [0.415] Economic development satisfaction 0.668 * 1.541 * [0.214] [0.485] Constant −0.757 * −1.442 * [0.226] [0.400] Observations 500 500 * p < 0.01, p < 0.05, * p < 0.1. Table 4. Model generalized linear regression results. Table 5. Average marginal eﬀect of the probit model. Table 5. Average marginal eﬀect of the probit model. Mean dy/dx Std.Err. z P > \|z\| 95% Conf. Interval Gender 0.514 −0.0143 0.0328 −0.4400 0.6620 −0.0786 0.0499 Age 1.248 −0.0109 0.0226 −0.4800 0.6310 −0.0552 0.0335 Educational level 1.982 0.1215 0.0182 6.6700 0.0000 0.0858 0.1573 Career 2.304 0.0109 0.0106 1.0200 0.3060 −0.0100 0.0317 Environmental quality satisfaction 1.070 0.0336 0.0283 1.1900 0.2350 −0.0218 0.0890 Petrochemical project understanding 1.128 0.0496 0.0406 1.2200 0.0220 −0.0300 0.1291 Information disclosure trust 1.044 0.1811 0.0367 4.9400 0.0000 0.1092 0.2531 Project planning rationality 1.052 0.0990 0.0426 2.3300 0.0200 0.1824 0.0155 Quality of life improvement 1.020 0.0923 0.0444 2.0800 0.0380 0.0053 0.1793 Economic development satisfaction 0.944 0.1488 0.0412 3.6100 0.0000 0.2297 0.0680 Number of Obs: 500. 13 of 18 Sustainability 2020, 12, 2723 For project impact factors, it is considered that the more reasonable the project planning, the greater the improvement in the quality of life, the higher the satisfaction with the economic development, and the more positive the attitudes of the supporters towards the project. Among them, given the other variables, project planning rationality, quality of life improvement, and economic development satisfaction increased by 10%, 9%, and 15% for each level of increase in residents’ project support rate, respectively. Given the other variables, among the personal factors, as the level of education increased, the support rate for the project increased by 12%. The results show that people with higher levels of education are more likely to support petrochemical projects, which may be related to the extent to which they receive information. 4.5. Model Analysis Results In order to improve the situation of information asymmetry, the higher the education level of residents, the more actively they can understand various information, and hence rational judgment is improved. The analysis shows that there was no signiﬁcant correlation between the personal factors of residents’ gender, occupation, and age and residents’ attitudes. Within the personal factors, the higher the level of education, the more support for the petrochemical project, which may be related to the extent to which residents receive information. The judgment on the risk of the facility increased with a higher the level of education. This is attributed to greater knowledge and the ability to understand information, which increase the individual’s rational assessment. Factors significantly related to residents’ attitudes are information cognitive factors and project influencing factors. Information cognitive factors include trust in information publicity and petrochemical project understanding, while environmental quality satisfaction has no significant correlation with residents’ attitudes. Moreover, the most influential factors affecting residents’ attitudes are trust in information publicity followed by the education level. The economic development satisfaction factor has the lowest effect on the attitude to the project. 5.1. Risk Perception Diﬀerences between Various Groups There are many factors that can impact on risk perception. In 2019, Ul-Abdin and Zainwritten investigated various attributes of users’ formation of risk perception [51]. The results suggested that risk formation among users evolves around tangible and non-tangible attributes. The spectrum of risk perception was developed, which visualizes risk evolution by considering various attributes. Diﬀerent groups have diﬀerent risk perceptions of the chemical Park. Residents’ groups are more aware of risks and have a resistance to risk. This is because residents have a general knowledge of the risks of chemical projects, and there are few intellectual and scientiﬁc considerations. The factors aﬀecting risk estimates and tolerance among persons were closely associated with judged beneﬁts of the hazard source, acceptance or denial of vulnerability, judgments of exposure voluntariness, and environmental attitudes [52]. If the health risks, accident risks, and safety risks are very serious, this leads to risk perception being stronger and to a reduction in risk acceptance. According to the analysis results, the residents’ groups have a resistance to the risk of petrochemical projects, which is the largest risk source group of all social groups. Residents are also the participants of group events. Therefore, in subsequent research on social risk control, the resident group will be taken as the research subject of risk control, and the risk control will be analyzed. The risk perception level of the expert group on the project is at a normal level. This is because the experts have comprehensive information on chemical projects and the risk assessment is more scientiﬁc and reasonable due to the accumulation of their own professional knowledge [53]. In terms of risk perception, it relies on more comprehensive risk project information, risk education, and personal experience [14]. The risk assessment will comprehensively consider the inﬂuencing factors of the accident risk characteristics and objectively evaluate the risks. When government departments conduct risk management, the information asymmetry relies more on the judgment of experts. The decision-making also considers more political achievements. However, 14 of 18 14 of 18 Sustainability 2020, 12, 2723 for accident risks, the government shows a tendency to avoid risks. For government departments, health risk accidents, chemical accidents, security risks, and media transmission will lead to a passive situation. At this time, there will be a strong risk perception. In most cases of risk management, the government’s own interests will become an important decision-making factor for risk perception. 5.1. Risk Perception Diﬀerences between Various Groups Enterprise groups are less aware of the risks of the project and can accept greater risks. Under the comprehensive risk information, enterprises have a comprehensive understanding of the project’s environmental risks. Despite this, they have a risk-aware weak decision-making cognition that is willing to accept more risks as a result of economic interests. Among the risk-aware factors, the frequent appearance of project accident risks and the occurrence of major accidents will result in resistance to risks [54]. 5.2. The Risk Control of the Residents’ Attitudes The evaluation of the risk control was taken to represent the residents’ attitudes to the petrochemical project (support/objection), which is considered as the criterion for judging whether or not to participate in the group event. In 2017, Huan investigated the status of knowledge, attitudes, and behaviors with regard to schistosomiasis control among rural residents in the Wanjiang River region after a ﬂood, so as to provide a reference for targeted health education [55]. The conclusion was that targeted health education should be strengthened to decrease the risk of schistosomiasis transmission. Hong developed an extended technology acceptance model (TAM) to explain residents’ intention to adopt green labeled residential buildings (GLRBs), and examined it in a survey conducted in Tianjin City, China [56]. The results showed that subjective knowledge about GLRBs, social trust in organizations responsible for GLRBs, perceived usefulness of GLRBs, attitude towards GLRBs, and general environmental attitude measured by the new ecological paradigm (NEP) scale are the signiﬁcant psychological determinants of the intention to adopt GLRBs. According to the analysis results, educational level, information cognitive factors, and project inﬂuencing factors are signiﬁcantly related to the residents’ attitudes. In the present study, the information cognitive factors included the residents’ trust in information publicity and their petrochemical project understanding. The project inﬂuencing factors included project planning rationality and quality of life improvement, as well as economic development satisfaction. The degree of information publicity had the highest degree of inﬂuence on residents’ attitudes, followed by the level of education, and the satisfaction with economic development had the lowest degree of inﬂuence. Improving the level of trust in residents through information disclosure is the core of the government’s risk control policy. In the design and formulation of risk control policies, it is recommended that the government protect the nature of the information that residents receive through the information disclosure of enterprises and governments, as well as the guidance of media and experts. This is proposed to improve the information trust in residents, and to improve the support and understanding for chemical projects. 6. Conclusions This paper outlines a systematic study on the social risks of potential high environmental risk chemical park projects. Social risk assessment shows that diﬀerent groups have both diﬀerent perceptions of risk and diﬀerent factors aﬀecting risk perception. Taking the resident group as an example, the resident group has a general knowledge of the risks of chemical projects, and there are few intellectual and scientiﬁc considerations. According to the residents’ social risk perception value (7 × 10−6), it can be seen that the residents’ groups have a strong sense of risk of petrochemical projects and a strong resistance to risk. The risk perception level of the expert group for the petrochemical project is normal. The government’s risk perception value (7 × 10−6) shows that the government’s risk perception of the project is at a relatively high level, and that there is resistance to the project risk. Furthermore, according to the social risk perception value of the project (7 × 10−5), it is indicated that the enterprise group is weakly aware of the risk of the project and can accept more risks. Finally, the total social risk perception value of the petrochemical project is about 7.252 × 10−4, which is the result of the standard value of acceptable social risk perception. Therefore, in order to avoid the social risk of the park project, the risk of a petrochemical project should not exceed a standard value acceptable to a group and society’s total risk perception. The social risk control research was based on the risk perception judgment conclusion. The risk participation group was further analyzed by the established social risk control research model. The results of the analyses indicated that the individual factors of gender, occupation, and age of residents are not signiﬁcantly related to residents’ attitudes. Factors that are signiﬁcantly related to residents’ attitudes are information cognitive factors and project inﬂuencing factors. Information cognitive factors include trust in information publicity and petrochemical project understanding. Project impact factors include project planning rationality, quality of life improvement, and economic development satisfaction. The degree of trust in information disclosure has the highest contribution of inﬂuence, followed by the level of education; the satisfaction with economic development has lowest degree of inﬂuence. Therefore, improving residents’ trust in information disclosure is the key point with regard to government risk control policies. The government’s risk control policy is formulated according to the results of risk control research. 5.3. Risk Control Policy In order to eﬀectively prevent social risks and maintain social stability, the government should assume a key role in risk control. Li et al. studied soil pollution, summarizing the existing law, action plan, regulations, and risk control rules regarding soil pollution prevention in China [57]. In 2015, Zhao established ultra high voltage (UHV) power transmission construction projects, which seek to improve the risk control level and the sustainable development of UHV power transmission construction projects [58]. Ioannou investigated uncertainties present during the operation of oﬀshore wind (OW) energy assets with a view of informing risk control policies for hedging of the incurring losses [59]. As a risk regulator, the government needs to supervise and guide the risks for participants, namely residents, media, experts, and enterprises. As the main source of risk concerns, residents are the main subject of government risk control. The government needs to protect residents’ safety risks and health beneﬁts from the perspective of the residents. Through the analyses above, the core of risk control policy formulation is to ensure information transparency and information disclosure to improve residents’ information trust, by requiring that enterprises disclose information, using the information 15 of 18 15 of 18 Sustainability 2020, 12, 2723 transmission capabilities of expert media, diversifying and expanding residents’ participation, and so on. Therefore, more information needs to be taken into account and information guidance is required in the government’s risk control policy. The media and experts are connected to enterprises and residents as the mediums for risk information, hence the government needs to guide them to maintain social stability [60]. The government should pay attention to the role of the media and experts in the transmission of risk information, use the media and experts to disseminate correct risk knowledge, report government and enterprise risk information in a timely manner, and prevent the unwarranted ampliﬁcation of risks caused by the distortion of risk information [28]. Moreover, social software, such as network channels, is a tool for residents to communicate information. The government needs to promptly intervene to use the oﬃcial information publicity platform to enable the public to obtain correct risk perception. In addition, the petrochemical enterprise, as the main body responsible for risks, is an important part of the government’s risk control system and the object of supervision [5]. 5.3. Risk Control Policy The government’s supervision of enterprises not only requires the publicity and transparency of enterprise protection information, but also needs to comprehensively formulate risk control policies from both internal and external levels. Internal risk control includes the application of advanced facilities, implementation of cleaner production, improvement of emergency response capabilities, emphasis on diversiﬁcation of information disclosure, and awareness of corporate social responsibility; external risk control includes policies and regulations, reward and punishment mechanisms, and regulatory measures. In short, in the formulation of the government’s risk control policy, it is necessary to coordinate the relationship between various risk factors in order to truly control the social risks. 6. Conclusions As a risk regulator, the government needs to supervise 16 of 18 16 of 18 Sustainability 2020, 12, 2723 and guide the risks to participants, namely residents, media, experts, and enterprises. Residents, as the main source of risk concerns, are the main subject of government risk control. The government needs to maintain residents’ safety risks and health beneﬁts from the perspective of residents. To improve the residents’ perceptions of the trustworthiness of information disclosure, it is recommended that the government use experts to disclose information. The media has the ability to transmit information, diversify and expand residents’ participation, and improve residents’ knowledge of information to increase residents’ trust in government information disclosure, thereby enhancing residents’ support and understanding of chemical projects. Author Contributions: Conceptualization, Y.N.; Methodology, J.Z. (Jinbu Zhao); Stata and Investigation, J.Z. (Jinbu Zhao); Writing—Original Draft Preparation, Y.N.; Writing—Review and Editing, Y.Z.; Supervision, J.Z. (Jizhi Zhou); All authors have read and agreed to the published version of the manuscript. Funding: This work is supported by Shanghai Philosophy and Social Science Planning Project (2016BJB0 ding: This work is supported by Shanghai Philosophy and Social Science Planning Project (2016BJB008). Acknowledgments: We appreciate Yanjing Wu in the School of Economics and Zeyuan Liu in the School of Environmental and Chemical Engineering for their data collection. Acknowledgments: We appreciate Yanjing Wu in the School of Economics and Zeyuan Liu in the Schoo Environmental and Chemical Engineering for their data collection. Acknowledgments: We appreciate Yanjing Wu in the School of Economics and Zeyuan Liu in the School of Environmental and Chemical Engineering for their data collection. Conﬂicts of Interest: The authors declare no conﬂict of interest. Conﬂicts of Interest: The authors declare no conﬂict of interest. Conﬂicts of Interest: The authors declare no conﬂict of interest. References 1. Ike, G.N.; Usman, O.; Sarkodie, S.A. Testing the role of oil production in the environmental Kuznets curve of oil producing countries: New insights from Method of Moments Quantile Regression. Sci. Total Environ. 2020, 711, 135208. [CrossRef] [PubMed] 1. Ike, G.N.; Usman, O.; Sarkodie, S.A. Testing the role of oil production in the environmental Kuznets curve of oil producing countries: New insights from Method of Moments Quantile Regression. Sci. Total Environ. 2020, 711, 135208. [CrossRef] [PubMed] 2. Chen, T.-L.; Pei, S.-L.; Pan, S.-Y.; Yu, C.-Y.; Chang, C.-L.; Chiang, P.-C. An engineering-environmental-economic-energy assessment for integrated air pollutants reduction, CO2 capture and utilization exemplified by the high-gravity process. J. Environ. Manag. 2020, 255, 109870–109878. [CrossRef] [PubMed] 2. Chen, T.-L.; Pei, S.-L.; Pan, S.-Y.; Yu, C.-Y.; Chang, C.-L.; Chiang, P.-C. An engineering-environmental-economic-energy assessment for integrated air pollutants reduction, CO2 capture and utilization exemplified by the high-gravity process. J. Environ. Manag. 2020, 255, 109870–109878. [CrossRef] [PubMed] 3. Aghadadashi, V.; Molaei, S.; Mehdinia, A.; Mohammadi, J.; Moeinaddini, M.; Riyahi Bakhtiari, A. Using GIS, geostatistics and Fuzzy logic to study spatial structure of sedimentary total PAHs and potential eco-risks; An Eastern Persian Gulf case study. Mar. Pollut. Bull. 2019, 149, 110489–110501. [CrossRef] [PubMed] 3. Aghadadashi, V.; Molaei, S.; Mehdinia, A.; Mohammadi, J.; Moeinaddini, M.; Riyahi Bakhtiari, A. Using GIS, geostatistics and Fuzzy logic to study spatial structure of sedimentary total PAHs and potential eco-risks; An Eastern Persian Gulf case study. Mar. Pollut. Bull. 2019, 149, 110489–110501. [CrossRef] [PubMed] 4. Shaygan, M.; Yazdanpanah, M. Prevalence and Predicting Factors of Chronic Pain among Workers of Petrochemical and Petroleum Reﬁnery Plants. Int. J. Occup. Environ. Med. 2020, 11, 3–14. [CrossRef] 4. Shaygan, M.; Yazdanpanah, M. Prevalence and Predicting Factors of Chronic Pain among Workers of Petrochemical and Petroleum Reﬁnery Plants. Int. J. Occup. Environ. Med. 2020, 11, 3–14. [CrossRef] y p 5. Signorino, G. Proximity and risk perception. Comparing risk perception ‘proﬁles’ in two petrochemical areas of Sicily (Augusta and Milazzo). J. Risk Res. 2012, 15, 1223–1243. [CrossRef] 5. Signorino, G. Proximity and risk perception. Comparing risk perception ‘proﬁles’ in two petrochemical areas of Sicily (Augusta and Milazzo). J. Risk Res. 2012, 15, 1223–1243. [CrossRef] 6. Tortosa-Edo, V.; Lopez-Navarro, M.A.; Llorens-Monzonis, J.; Rodriguez-Artola, R.M. The antecedent role of personal environmental values in the relationships among trust in companies, information processing and risk perception. J. Risk Res. 2014, 17, 1019–1035. [CrossRef] 6. Tortosa-Edo, V.; Lopez-Navarro, M.A.; Llorens-Monzonis, J.; Rodriguez-Artola, R.M. References The antecedent role of personal environmental values in the relationships among trust in companies, information processing and risk perception. J. Risk Res. 2014, 17, 1019–1035. [CrossRef] 7. Geng, Z.; Zeng, R.; Han, Y.; Zhong, Y.; Fu, H. Energy eﬃciency evaluation and energy saving based on DEA integrated aﬃnity propagation clustering: Case study of complex petrochemical industries. Energy 2019, 179, 863–875. [CrossRef] 8. Nicolletti, M.; Lutti, N.; Souza, R.; Pagotto, L. Social and organizational learning in the adaptation to the process of climate change: The case of a Brazilian thermoplastic resins and petrochemical company. J. Clean. Prod. 2019, 226, 748–758. [CrossRef] 9. Choi, Y.; Lee, H.S.; Mastur, A. Are Sustainable Development Policies Really Feasible? Focused on the Petrochemical Industry in Korea. Sustainability 2019, 11, 3980. [CrossRef] 10. Kolla, G.; Strike, C.; Watson, T.M.; Jairam, J.; Fischer, B.; Bayoumi, A.M. Risk creating and risk reducing: Community perceptions of supervised consumption facilities for illicit drug use. Health Risk Soc. 2017, 19, 91–111. [CrossRef] 11. Pol, E.; Di Masso, A.; Castrechini, A.; Bonet, M.R.; Vidal, T. Psychological parameters to understand and manage the NIMBY eﬀect. Eur. Rev. Appl. Psychol. Rev. Eur. Psychol. Appl. 2006, 56, 43–51. [CrossRef] 12. Musmeci, L.; Falleni, F.; Cicero, M.; Carere, M. Environmental Pollution in Augusta-Priolo and Gela. In WHO Book: “Human Health in Areas with Industrial Contamination”; WHO Regional Oﬃce for Europe: København, Denmark, 2014; pp. 89–98. Sustainability 2020, 12, 2723 17 of 18 17 of 18 13. Zhang, L.; Wang, S.; Chen, C.; Yang, M.; She, X. Modeling lane-change risk in urban expressway oﬀ-ramp area based on naturalistic driving data. J. Test. Eval. 2020, 48. [CrossRef] 14. Yu, C.-H.; Huang, S.-K.; Qin, P.; Chen, X. Local residents’ risk perceptions in response to shale gas exploitation: Evidence from China. Energy Policy 2018, 113, 123–134. [CrossRef] 15. Leﬂey, F. What is Our Perception of Project Risk, and do the Current Theories Truly Reﬂect Our Pragmatic Interpretation of This Perception? IEEE Eng. Manag. Rev. 2018, 46, 65–73. [CrossRef] 16. Chiang, Y.-C. Exploring community risk perceptions of climate change—A case study of a ﬂood-prone urban area of Taiwan. Cities 2018, 74, 42–51. [CrossRef] 17. Zhu, W.; Wei, J.; Zhao, D. Anti-nuclear behavioral intentions: The role of perceived knowledge, information processing, and risk perception. Energy Policy 2016, 88, 168–177. [CrossRef] 18. Wang, H.Z.; Khan, F.; Ahmed, S.; Imtiaz, S. Dynamic quantitative operational risk assessment of chemical processes. Chem. Eng. Sci. 2016, 142, 62–78. References [CrossRef] 19. Gholipour, S.; Nikaeen, M.; Farhadkhani, M.; Nikmanesh, B. Survey of Listeria monocytogenes contamination of various environmental samples and associated health risks. Food Control 2020, 108, 6. [CrossRef] 0. Alileche, N.; Cozzani, V.; Reniers, G.; Estel, L. Thresholds for domino eﬀects and safety distances in process industry: A review of approaches and regulations. Reliab. Eng. Syst. Saf. 2015, 143, 74–84. [Cross 20. Alileche, N.; Cozzani, V.; Reniers, G.; Estel, L. Thresholds for domino eﬀects and safety distances in the process industry: A review of approaches and regulations. Reliab. Eng. Syst. Saf. 2015, 143, 74–84. [CrossRef] 21 Cozzani V; Gubinelli G ; Salzano E Escalation thresholds in the assessment of domino accidental events 20. Alileche, N.; Cozzani, V.; Reniers, G.; Estel, L. Thresholds for domino eﬀects and safety distances in the process industry: A review of approaches and regulations. Reliab. Eng. Syst. Saf. 2015, 143, 74–84. [CrossRef] 21. Cozzani, V.; Gubinelli, G.; Salzano, E. Escalation thresholds in the assessment of domino accidental events. J. Hazard. Mater. 2006, 129, 1–21. [CrossRef] 21. Cozzani, V.; Gubinelli, G.; Salzano, E. Escalation thresholds in the assessment of domino accidenta J. Hazard. Mater. 2006, 129, 1–21. [CrossRef] 22. Markowski, A.S.; Kotynia, A. “Bow-tie” model in layer of protection analysis. Process Saf. Environ. Protect. 2011, 89, 205–213. [CrossRef] 23. Fabbrocino, G.; Iervolino, I.; Orlando, F.; Salzano, E. Quantitative risk analysis of oil storage facilities in seismic areas. J. Hazard. Mater. 2005, 123, 61–69. [CrossRef] [PubMed] 24. Valencia-Barragan, L.; Martinez-Gomez, J.; Maria Ponce-Ortega, J. A quantitative risk analysis for the vegetable oil industry in Mexico. Clean Technol. Environ. Policy 2016, 18, 245–256. [CrossRef] 25. Zhang, G.; Fan, Y.; Jiang, X.; Fan, W.; Meng, T.; Xu, M. Assessing the impacts of signal coordination on the crash risks of various driving cohorts. J. Saf. Res. 2019, 70, 79–87. [CrossRef] [PubMed] 26. Fellenor, J.; Barnett, J.; Potter, C.; Urquhart, J.; Mumford, J.D.; Quine, C.P. The social ampliﬁcation of risk on Twitter: The case of ash dieback disease in the United Kingdom. J. Risk Res. 2018, 21, 1163–1183. [CrossRef] 27. Jagiello, R.D.; Hills, T.T. Bad News Has Wings: Dread Risk Mediates Social Ampliﬁcation in Risk Communication. Risk Anal. 2018, 38, 2193–2207. [CrossRef] 28. Wirz, C.D.; Xenos, M.A.; Brossard, D.; Scheufele, D.; Chung, J.H.; Massarani, L. Rethinking Social Ampliﬁcation of Risk: Social Media and Zika in Three Languages. Risk Anal. 2018, 38, 2599–2624. [CrossRef] 29. Frewer, L.J.; Miles, S.; Marsh, R. References Ma, J.; Ye, X.; Shi, C. Development of Multivariate Ordered Probit Model to Understand Household Vehicle Ownership Behavior in Xiaoshan District of Hangzhou, China. Sustainability 2018, 10, 3660. [CrossRef] 40. Ma, J.; Ye, X.; Shi, C. Development of Multivariate Ordered Probit Model to Understand Household Vehicle Ownership Behavior in Xiaoshan District of Hangzhou, China. Sustainability 2018, 10, 3660. [CrossRef] 41. James, M. Simpliﬁed Methods of Using Probit Analysis in Consequence Analysis. Process Saf. Prog. 2015, 34, 58–63. [CrossRef] 41. James, M. Simpliﬁed Methods of Using Probit Analysis in Consequence Analysis. Process Saf. Prog. 2015, 34, 58–63. [CrossRef] 42. Ignatowski, A.J.; Rosenthal, I. The chemical accident risk assessment thesaurus: A tool for analyzing and comparing diverse risk assessment processes and deﬁnitions. Risk Anal. 2001, 21, 513–532. [CrossRef] 43. Albaiges, J. Ecological risk assessment, 2nd edition. Int. J. Environ. Anal. Chem. 2008, 88, 44. Si, H.; Ji, H.; Zeng, X.H. Quantitative risk assessment model of hazardous chemicals leakage and application. Saf. Sci. 2012, 50, 1452–1461. [CrossRef] 45. Heo, S.; Kim, M.; Yu, H.; Lee, W.-K.; Sohn, J.R.; Jung, S.-Y.; Moon, K.W.; Byeon, S.H. Chemical accident hazard assessment by spatial analysis of chemical factories and accident records in South Korea. Int. J. Disaster Risk Reduct. 2018, 27, 37–47. [CrossRef] 46. Liu, Y.H.; Fang, P.P.; Bian, D.D.; Zhang, H.W.; Wang, S.X. Fuzzy comprehensive evaluation for the motion performance of autonomous underwater vehicles. Ocean Eng. 2014, 88, 568–577. [CrossRef] 47. Chu, W.W.; Li, Y.G.; Liu, C.Q.; Mou, W.P.; Tang, L.M. A manufacturing resource allocation method with knowledge-based fuzzy comprehensive evaluation for aircraft structural parts. Int. J. Prod. Res. 2014, 52, 3239–3258. [CrossRef] 48. Loh, H.S.; Zhou, Q.J.; Thai, V.V.; Wong, Y.D.; Yuen, K.F. Fuzzy comprehensive evaluation of port-centric supply chain disruption threats. Ocean Coast. Manag. 2017, 148, 53–62. [CrossRef] 49. Nie, Y.; Wu, Y.; Zhao, J.; Zhou, J.; Chen, X.J.; Maraseni, T.; Qian, G. Is the ﬁner the better for municipal solid waste (MSW) classiﬁcation in view of recyclable constituents? A comprehensive social, economic and environmental analysis. Waste Manag. 2018, 79, 472–480. [CrossRef] 50. Nie, Y.; Wu, Y.; Zhao, J.; Zhou, J.; Zhang, Y.; Zhao, J.; Maraseni, T.; Qian, G. Resident risk attitude analysis in the decision-making management of waste incineration construction. J. Environ. Manag. 2020, 258, 109946–109962. [CrossRef] 51. Ul-Abdin, Z.; De Winne, P.; De Backer, H. Risk-Perception Formation Considering Tangible and Non-Tangible Aspects of Cycling: A Flemish Case Study. Sustainability 2019, 11, 6474. References The media and genetically modiﬁed foods: Evidence in support of social ampliﬁcation of risk. Risk Anal. 2002, 22, 701–711. [CrossRef] 30. Moussaid, M.; Brighton, H.; Gaissmaier, W. The ampliﬁcation of risk in experimental diﬀusion chains. Proc. Natl. Acad. Sci. USA 2015, 112, 5631–5636. [CrossRef] 31. Wang, Y.N. Risk Control Cyber Physical System for Active Distributed Network. In Proceedings of the 2nd IEEE Conference on Energy Internet and Energy System Integration (EI2), Beijing, China, 20–22 October 2018; pp. 1–6. 2. Liu, X.; Li, J.; Li, X. Study of dynamic risk management system for ﬂammable and explosive danger chemicals storage area. J. Loss Prev. Process Ind. 2017, 49, 983–988. [CrossRef] 33. Feng, S.; Xu, L.D. Decision support for fuzzy comprehensive evaluation of urban development. Fuzzy Sets Syst. 1999, 105, 1–12. [CrossRef] 34. Chen, J.F.; Hsieh, H.N.; Do, Q.H. Evaluating teaching performance based on fuzzy AHP and comprehensive evaluation approach. Appl. Soft. Comput. 2015, 28, 100–108. [CrossRef] 35. Shi, S.; Cao, J.; Feng, L.; Liang, W.; Zhang, L. Construction of a technique plan repository and evaluation system based on AHP group decision-making for emergency treatment and disposal in chemical pollution accidents. J. Hazard. Mater. 2014, 276, 200–206. [CrossRef] [PubMed] 36. Zeng, D.; He, Q.; Yu, Z.; Jia, W.; Zhang, S.; Liu, Q. Risk assessment of sustained casing pressure in gas wells based on the fuzzy comprehensive evaluation method. J. Nat. Gas Sci. Eng. 2017, 46, 756–763. [CrossRef] 36. Zeng, D.; He, Q.; Yu, Z.; Jia, W.; Zhang, S.; Liu, Q. Risk assessment of sustained casing pressure in gas wells based on the fuzzy comprehensive evaluation method. J. Nat. Gas Sci. Eng. 2017, 46, 756–763. [CrossRef] 37. Xu, X.; Huang, D.; Guo, F. Addressing spatial heterogeneity of injury severity using Bayesian multilevel 37. Xu, X.; Huang, D.; Guo, F. Addressing spatial heterogeneity of injury severity using Bayesian multilevel ordered probit model. Res. Transp. Econ. 2019, 100748. [CrossRef] 18 of 18 Sustainability 2020, 12, 2723 18 of 18 38. Antunes, A.; Bonﬁm, D.; Monteiro, N.; Rodrigues, P.M.M. Forecasting banking crises with dynamic panel probit models. Int. J. Forecast. 2018, 34, 249–275. [CrossRef] 39. Gan, L.; Zhang, H.; Wen, Y.; Zou, Z. Risk Degree Analysis of Bridge Damage Caused by Collision of Disabled Ships Based on Ordered Probit Model. In Proceedings of the 21st 2011 International Oﬀshore and Polar Engineering Conference (ISOPE), Maui, HI, USA, 19–24 June 2011; pp. 871–875. 40. References [CrossRef] 52. Baird, B.N. Tolerance for environmental health risks: The inﬂuence of knowledge, beneﬁts, voluntariness, and environmental attitudes. Risk Anal. 1986, 6, 425–435. [CrossRef] 53. Sjoberg, L. The allegedly simple structure of experts’ risk perception: An urban legend in risk research. Sci. Technol. Hum. Values 2002, 27, 443–459. [CrossRef] 54. Karpuntsov, M.V. Enterprise risk-resistance. Actual Probl. Econ. 2008, 81, 71–76. 55. Huan, L.; Ai-Xia, W.; Yuan-Zhen, L.; Ming-Ming, Z. Knowledge, attitude and practice related to schistosomiasis control among rural residents in Wanjiang River region after a ﬂood. Chin. J. Schistosomiasis Cont. 2017, 29, 219–221. 56. Liu, Y.; Hong, Z.; Zhu, J.; Yan, J.; Qi, J.; Liu, P. Promoting green residential buildings: Residents’ environ attitude, subjective knowledge, and social trust matter. Energy Policy 2018, 112, 152–161. [CrossRef] 7. Li, T.; Liu, Y.; Lin, S.; Liu, Y.; Xie, Y. Soil Pollution Management in China: A Brief Introduction. Sustainab 2019, 11, 556. [CrossRef] 58. Zhao, H.; Li, N. Risk Evaluation of a UHV Power Transmission Construction Project Based on a Cloud Model and FCE Method for Sustainability. Sustainability 2015, 7, 2885–2914. [CrossRef] 59. Ioannou, A.; Angus, A.; Brennan, F. Informing parametric risk control policies for operational uncertainties of oﬀshore wind energy assets. Ocean Eng. 2019, 177, 1–11. [CrossRef] Á Á 60. Rutsaert, P.; Pieniak, Z.; Regan, Á.; McConnon, Á.; Kuttschreuter, M.; Lores, M.; Lozano, N.; Guzzon, A.; Santare, D.; Verbeke, W. Social media as a useful tool in food risk and beneﬁt communication? A strategic orientation approach. Food Policy 2014, 46, 84–93. [CrossRef] © 2020 by the authors. 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https://openalex.org/W2095227295	https://www2.ifrn.edu.br/ojs/index.php/HOLOS/article/download/249/253	Portuguese	null	Estudo de Caso: Análise de um Relatório de Controle Ambiental (RCA) Referente à Atividade de Levantamento Sísmico	Holos	2,010	cc-by	7,537	NASCIMENTO & REIS (2009) NASCIMENTO & REIS (2009) RESUMO Este trabalho aborda a análise de um Relatório de Controle Ambiental (RCA), da atividade de sísmica, submetido à obtenção de licenciamento ambiental junto ao Instituto de Defesa do Meio Ambiente – IDEMA. Realiza uma comparação dos itens exigidos no Termo de Referência (TR) do IDEMA com o Estudo ambiental citado, a fim de identificar se todos os itens exigidos legalmente foram contemplados no RCA e se os mesmos estão em conformidade com o Termo. Utiliza a técnica de estudo de impacto ambiental baseado em um contexto legal que estabelece requisitos a serem observados e procedimentos a serem cumpridos, tal técnica é a análise formal da qualidade de um estudo ambiental. Verifica, também, a repetição ou não das principais deficiências apontadas no elemento Mitigação e compensação de impactos com base no estudo do Ministério Público Federal - MPF, a partir da técnica de análise de conteúdo de estudo ambiental. PALAVRAS-CHAVE: Relatório de Controle Ambiental. Estudo Ambiental. Licenciamento Ambiental. PALAVRAS-CHAVE: Relatório de Controle Ambiental. Estudo Ambiental. Licenciamento Ambiental. IGOR LEONARDO DE MORAIS NASCIMENTO moraisnascimento@yahoo.com.br Pós-graduando em Licenciamento ambiental on shore - IFRN LECI MARTINS MENEZES REIS Profa. do IFRN leci@cefetrn.br KEY-WORDS: Report of Environmental Control. Environmental Study. Environmental License. INTRODUÇÃO É assegurado pela Constituição Federal de 1988 em seu art. 225 o direito a todos ao meio ambiente ecologicamente equilibrado, que seja bem comum do povo e essencial a qualidade de vida. No mesmo artigo, exige-se do poder público e da coletividade o dever de defender e preservar o meio ambiente para as presentes e futuras gerações. Ainda em seu parágrafo primeiro e em conjunto com o inciso IV do mesmo artigo, foi incumbido ao poder público para assegurar a efetividade desse direito a exigência, na forma da lei, para instalação de obra ou atividade potencialmente causadora de significativa degradação do meio ambiente, estudo prévio de impacto ambiental, o qual se deve dá publicidade. Com o exposto acima, podemos verificar que os estudos ambientais assumem uma grande importância no que tange a preservação ambiental. No entanto, apenas o estudo em si não garante o nosso direito a um meio ambiente ecologicamente equilibrado, é necessário que tenhamos estudos ambientais de qualidade, dotados de informações que subsidiem o processo de licenciamento ambiental dos órgãos ambientais. Dessa forma a qualidade do estudo ambiental torna-se um fator determinante para que tenhamos empreendimentos ecologicamente adequados. Mais do que isso, é necessário que os estudos sejam analisados e criticados por todas as partes envolvidas nesse processo (SÁNCHEZ, 2006), tais como: Empresas que contratam estudos de impacto ambiental podem analisá-los antes de submetê-los à aprovação dos órgãos governamentais ou de agentes financeiros; Associações que representam o público como organizações não governamentais e associações de moradores, podem analisar os estudos para buscar um melhor entendimento do projeto e de suas conseqüências; no caso de posturas contrárias ao empreendimento, a análise pode apontar falhas e lacunas que podem ser apresentadas como argumentos no debate; ela pode também indicar deficiências do projeto ou apontar para alternativas não estudadas, ou ainda sugerir novas medidas mitigadoras ou compensatórias, não consideradas no estudo; Membros do Ministério Público, assistentes técnicos e peritos judiciais, no caso de disputas judiciais envolvendo atividades sujeitas ao processo de avaliação de impacto ambiental; Agências setoriais reguladoras e outros órgãos governamentais interessados no empreendimento apresentado; Agentes financiadores públicos ou privados, cuja política inclua a discussão da viabilidade ambiental dos empreendimentos que lhes são submetidos; Órgãos governamentais com atribuições específicas, que devem ser ouvidos no licenciamento de uma atividade. ABSTRACT This paper aims to analysis of a report of environmental control (REC), of the sismic activity, submited to obtain the environmental license from Instituto de Defesa do Meio Ambiente – IDEMA. It compares the required items in the Reference Term (RT) of IDEMA with cited environmental study. In order to identify if all legal required items were achieved in the REC and if they are the way the Term states. It uses the study technique of environmental impact based on a legal context that estabilishes requirements to be observed and procedures to be done, such technique is the formal analysis of an environmental study’s quality. It also verifies whether there’s repetition of the main deficiencies pointed in the element Mitigation and compensation of impacts based on the study made by Ministério Público Federal - MPF, from the technique of analysis of the environmental study’s content. KEY-WORDS: Report of Environmental Control. Environmental Study. Environmental License. License. Holos, Ano 25, Vol. 3 16 NASCIMENTO & REIS (2009) INTRODUÇÃO A relevância do tema proposto neste estudo de caso consiste no fato de expor que a realidade dos estudos ambientais postos aos órgãos ambientais para o licenciamento ambiental não condiz com os conceitos teóricos estudados no curso de licenciamento ambiental on shore. A escassez de produção cientifica que aborde tal assunto, também deve ser utilizada como justificativa para esse trabalho, pretendendo-se que o produto final resultante do mesmo ultrapasse a esfera acadêmica e contribua na reavaliação das atuais práticas de licenciamento ambiental on shore. Sendo esse último, o principal motivo do estudo a seguir. g 2 REFERÊNCIAL TEÓRICO 17 Holos, Ano 25, Vol. 3 NASCIMENTO & REIS (2009) Geralmente, os estudos de impactos ambientais são realizados dentro de um contexto legal, no qual é feita uma comparação entre os pontos abordados, no estudo em questão, e os requisitos determinados pelo Termo de Referência (TR). Contudo, esse não é o único método. Tal estudo pode ser feito baseado numa prática recomendada, internacionalmente reconhecida, ou ainda seguindo recomendações de organismos internacionais ou órgão federais competentes. Independentemente do método utilizado, tal estudo deve garantir ao analista o atendimento aos requisitos mínimos estabelecidos pela regulamentação aplicável, assim como, apresentar qualidade técnica suficiente para subsidiar a tomada de decisões sobre o empreendimento. Em outras palavras, busca-se determinar se o estudo de impacto tem forma e conteúdo satisfatórios e adequados para a aprovação da concessão da licença ambiental. Se o estudo ambiental atende aos requisitos preestabelecidos, afirmamos que o mesmo apresenta a qualidade “formal”. Atendendo a critérios técnicos relevantes, afirmamos que este apresenta a qualidade “de conteúdo”. Normalmente este último está baseado nas melhores práticas adotadas internacionalmente (best practice). Vale salientar que a qualidade formal e a de conteúdo não são excludentes e tanto podem, como devem, ser complementares. De acordo com Lee (2000, p. 138), um bom estudo ambiental: "é aquele que apresenta, de uma forma apropriada para os usuários, constatações e conclusões que cubram todas as tarefas da avaliação, empregando métodos apropriados de coleta de informação, análise e comunicação". No Brasil, o conteúdo mínimo exigido em um estudo de impacto ambiental é determinado pela Resolução de no1 de 1986 do Conselho Nacional do Meio Ambiente – CONAMA, porém os órgãos licenciadores estaduais podem ter seus próprios critérios, desde que não sejam menos restritivos que aqueles estabelecidos pela norma federal e não a contrarie. INTRODUÇÃO Portanto, um estudo que não apresente o conteúdo mínimo exigido não deve ser aceito pelo órgão ambiental. Contudo, é desejável que o estudo apresente o atendimento aos requisitos legais e as práticas mundialmente reconhecidas. Com base nesse entendimento, o estudo de caso a seguir, não procura se limitar à verificação do atendimento aos requisitos mínimos estabelecidos pela lei, apesar das restrições de tempo e da complexidade e riqueza da abordagem da técnica de avaliação da qualidade de conteúdo, a mesma também será trabalhada. O trabalho a seguir tem como referência, o Termo de Referência do Instituto de Defesa do Meio Ambiente - IDEMA e um estudo feito em 2004 por uma equipe de analistas do Ministério Público Federal – MPF, em uma população de oitenta EIA’s Brasileiros de projetos postos ao licenciamento ambiental federal ou que implicaram, por diversas razões, no envolvimento do Ministério Público Federal. Como resultado do trabalho do MPF, tem- se o Quadro 1 que identifica todos os elementos do EIA, assim como as suas principais deficiências. Dessa forma, o estudo de caso proposto visa verificar a conformidade com o TR e a ocorrência ou não das principais deficiências apontadas pelo Quadro 1, no que tange ao elemento de Mitigação e Compensação de impactos, já que o mesmo pode ser considerado uma boa prática recomendada. Holos, Ano 25, Vol. 3 18 NASCIMENTO & REIS (2009) Holos, Ano 25, Vol. INTRODUÇÃO 3 19 Quadro 1 - Deficiências em estudos de impacto ambiental no Brasil Elemento do EIA Principais Deficiências Estudo de alternativas Ausência de proposição de alternativas; Apresentação de alternativas reconhecidamente inferiores à selecionada no EIA; Prevalência dos aspectos econômicos sobre os ambientais na escolha de Alternativas; Comparação de alternativas a partir de base de conhecimento diferenciada; Delimitação das áreas de influência Desconsideração da bacia hidrográfica; Delimitação das áreas de influência sem alicerce nas características e vulnerabilidades dos ambientes naturais e nas realidades sociais regionais; Diagnóstico ambiental Prazos insuficientes para a realização de pesquisas de campo; Caracterização da área baseada, predominantemente, em dados secundários; Ausência ou insuficiência de informações sobre a metodologia utilizada; Proposição de execução de atividades de diagnóstico em etapas do licenciamento posteriores à Licença Prévia; Falta de integração dos dados de estudos específicos; Diagnóstico ambiental - meios físico e biótico Ausência de mapas temáticos; Utilização de mapas em escala inadequada, desatualizados e/ou com ausência de informações; Ausência de dados que abarquem um ano hidrológico, no mínimo; Apresentação de informações inexatas, imprecisas ou contraditórias; Deficiências na amostragem para o diagnóstico; Caracterização incompleta de águas, sedimentos, solos, resíduos, ar, etc; Desconsideração da interdependência entre precipitação e escoamento superficial e subterrâneo; Superficialidade ou ausência de análise de eventos singulares em projetos envolvendo recursos hídricos; Ausência ou insuficiência de dados quantitativos sobre a vegetação; Ausência de dados sobre organismos de determinados grupos ou categorias; Ausência de diagnóstico de sítios de reprodução (criadouros) e alimentação de animais; Diagnóstico ambiental - meio antrópico Pesquisas insuficientes e metodologicamente ineficazes; Conhecimento insatisfatório dos modos de vida de coletividades socioculturais singulares e suas redes intercomunitárias; Ausência de estudos orientados pela ampla acepção do conceito de patrimônio cultural; Não-adoção de uma abordagem urbanística integrada em diagnósticos de áreas e populações urbanas afetadas; Caracterizações socioeconômicas regionais genéricas, não articuladas Holos, Ano 25, Vol. INTRODUÇÃO 3 19 NASCIMENTO & REIS (2009) às pesquisas diretas locais; Identificação, caracterização e análise dos impactos Não-identificação de determinados impactos (omissões em termos de impactos passíveis de previsão, impactos negativos indiretos sequer mecionados); Identificação parcial de impactos; Identificação de impactos genéricos (por vezes são tantos os impactos agrupados sob um único titulo que sua importância e significado não podem ser estabelecidos satisfatoriamente; Identificação de impactos mutuamente excludentes; Subutilização ou desconsideração de dados dos diagnósticos; Omissão de dados e/ou de justificativas quanto à metodologia utilizada para atribuir pesos aos atributos dos impactos; Cumulatividade e sinergia de impactos Aspectos desconsiderados; Mitigação e compensação de impactos Proposição de medidas que não são a solução para a mitigação do impacto; Indicação de medidas mitigadoras pouco detalhadas; Indicação de obrigações ou impedimentos, técnicos e legais, como propostas de medidas mitigadoras; Ausência de avaliação da eficiência das medidas mitigadoras propostas; Deslocamento compulsório de populações: propostas iniciais de compensações de perdas baseadas em diagnósticos inadequados; Não-incorporação de propostas dos grupos sociais afetados, na fase de formulação do EIA; Proposição de Unidade de Conservação da categoria de uso sustentável para a aplicação dos recursos, em casos não previstos pela legislação; Programa de monitoramento e acompanhamento ambiental Erros conceituais na indicação de monitoramento; Ausência de proposição de programa de monitoramento de impactos específicos; Rima O Rima é um documento incompleto; Emprego de linguagem inadequada à compreensão do público. Fonte: SÁNCHEZ (2006). Fonte: SÁNCHEZ (2006). Sendo assim, foi tomado como referência os ditames teóricos e legais que fundamentam o estudo aqui referido, apresentados nos itens a seguir. 2.1.1 Licença ambiental A Licença ambiental, segundo a Resolução CONAMA 237/97, é um ato administrativo pelo qual o órgão ambiental competente, estabelece as condições, restrições e medidas de controle ambiental que deverão ser obedecidas pelo empreendedor, pessoa física ou jurídica, para localizar, instalar, ampliar e operar empreendimentos ou atividades utilizadoras dos recursos ambientais consideradas efetiva ou potencialmente poluidoras ou aquelas que, sob qualquer forma, possam causar degradação ambiental (CONAMA, 1997). O licenciamento ambiental, em linhas gerais, compreende 3 tipos de licença: a Licença Prévia - LP, a Licença de Instalação - LI e a Licença de Operação – LO. 2.1 O LICENCIAMENTO AMBIENTAL O licenciamento ambiental, de acordo com Lima (2006), se constitui num procedimento complexo que envolve análises técnicas e discussão pública das informações produzidas, que tem como objetivo guiar o órgão competente pelo licenciamento na tomada de decisão quanto à implementação de atividades ou obras efetiva ou potencialmente causadora de impactos ambientais. Holos, Ano 25, Vol. 3 20 NASCIMENTO & REIS (2009) Outros atores como Fink et al. (2004), acrescentam ainda que o licenciamento não se limita a um simples ato, mas, sim, a uma série de passos encadeados com vistas à verificação de que certa atividade está dentro dos padrões ambientais permitidos. Segundo a Resolução Conama 237/97 o licenciamento ambiental é um procedimento administrativo pelo qual o órgão ambiental competente licencia a localização, instalação, ampliação e a operação de empreendimentos e atividades utilizadoras de recursos ambientais, consideradas efetiva ou potencialmente poluidoras ou daquelas que, sob qualquer forma, possam causar degradação ambiental, considerando as disposições legais e regulamentares e as normas técnicas aplicáveis ao caso. 2.1.2 O Licenciamento ambiental on shore O licenciamento de atividades relacionadas à exploração e lavra de jazidas de combustíveis líquidos e gás natural, são estabelecidos de acordo com a Resolução CONAMA 23/94. 2.1.3 Estudo Ambiental Um estudo técnico que possui como intuito fornecer as informações e análises técnicas de uma atividade causadora de impactos ambientais, desde a localização, implantação, operacionalização e ampliação, para subsidiar o processo de licenciamento é considerado um estudo ambiental (SÁNCHEZ, 2006). Já para a Resolução CONAMA 237/97, os estudos ambientais são todos e quaisquer estudos relativos aos aspectos ambientais relacionados à localização, instalação, operação e ampliação de uma atividade ou empreendimento, apresentado como subsídio para a análise da licença requerida, tais como: relatório ambiental, plano e projeto de controle ambiental, relatório ambiental preliminar, diagnóstico ambiental, plano de manejo, plano de recuperação de área degradada e análise preliminar de risco (CONAMA, 1997). O licenciamento de atividades relacionadas à exploração e lavra de jazidas de combustíveis líquidos e gás natural, são estabelecidos de acordo com a Resolução CONAMA 23/94. O licenciamento ambiental das atividades petrolíferas no mar (off shore), com base na localização do empreendimento e no alcance de seu impacto ambiental, cumpre ao IBAMA a competência de seu licenciamento de acordo com o art. 4º da Resolução CONAMA 237/97. Por outro lado, o licenciamento das atividades de exploração e produção de petróleo em terra (on shore) compete aos órgãos estaduais de meio ambiente. No Rio Grande do Norte, o licenciamento ambiental é baseado na Política Estadual do Meio Ambiente (PEMA) estabelecida na Lei Complementar Estadual nº 272 de 03 de março de 2004 e pela Lei Complementar Estadual nº 336 de 12 de dezembro de 2006. Para a atividade petrolífera on shore, exigem-se as seguintes licenças: Licença Prévia para Perfuração (LPper), Licença Prévia de Produção para Pesquisa (LPpro), Licença de Instalação (LI) e Licença de Operação (LO), de acordo com a Lei Complementar nº 272/2004 (ver Figura 1). Holos, Ano 25, Vol. 3 21 NASCIMENTO & REIS (2009) Figura 1 – Licenças da atividade on shore. Fonte: SILVA et al. (2008), adaptado da Lei complementar 272/04 - RN. Figura 1 – Licenças da atividade on shore. Figura 1 – Licenças da atividade on shore. Fonte: SILVA et al. (2008), adaptado da Lei complementar 272/04 - RN. Figura 1 – Licenças da atividade on shore. Fonte: SILVA et al. (2008), adaptado da Lei complementar 272/04 - RN. No processo de licenciamento ambiental on shore, o órgão ambiental responsável, que no caso do nosso Estado é o IDEMA, utiliza-se de alguns estudos ambientais, como: Estudo de Impacto Ambiental (EIA) com o seu respectivo Relatório de Impacto Ambiental (RIMA), Relatório de Controle Ambiental (RCA), Estudo de Viabilidade Ambiental (EVA), Relatório de Avaliação Ambiental (RAA) e Projeto de Controle Ambiental (PCA). 2.2 IMPACTO AMBIENTAL O impacto ambiental pode ser definido, segundo Sánchez (2006, p. 462), como “a alteração da qualidade ambiental que resulta da modificação de processos naturais ou sociais provocada por ação humana”. Considera-se impacto ambiental, segundo a Resolução CONAMA 1/86, qualquer alteração das propriedades físicas, químicas e biológicas do meio ambiente, causada por qualquer forma de matéria ou energia resultante das atividades humanas que, direta ou indiretamente, afetam: Holos, Ano 25, Vol. 3 22 NASCIMENTO & REIS (2009) I. a saúde, a segurança e o bem-estar da população; I. a saúde, a segurança e o bem-estar da população; II. as atividades sociais e econômicas; III. a biota; IV. as condições estéticas e sanitárias do meio ambiente; V. a qualidade dos recursos ambientais. V. a qualidade dos recursos ambientais. Entende-se por impacto ambiental, de acordo com a NBR ISO 14001:2004, qualquer conseqüência, negativa ou positiva, que resulte ou que possa resultar da interação dos aspectos ambientais ou elementos de processo, operações, serviços e produtos de uma organização com o meio ambiente durante as atividades do empreendimento. 3 METODOLOGIA A estratégia para alcançar as metas relacionadas com este estudo passa pela pesquisa bibliográfica, com leitura e discussão de textos centrais à questão e legislações pertinentes, e a revisão crítica de documentos e publicações da área, a fim de fundamentar o tom do debate e as questões levantadas. Foram feitas leituras de estudos que analisaram a qualidade formal e de conteúdo de estudos ambientais, assim como do TR do IDEMA para a atividade de sísmica. Como a análise técnica de um estudo de impacto ambiental não é de interesse exclusivo do agente decisório, e sim de todos, este trabalho se propõe a analisar um Relatório de Controle Ambiental (RCA) com o intuito de verificar se a prática existente retrata os conceitos teóricos. Portanto, o trabalho fará uma comparação dos itens exigidos no Termo de Referência do IDEMA com os que foram trabalhados no RCA, assim como foi feita uma pesquisa para a identificação da existência de boas práticas. No que tange as boas práticas, destaca-se o estudo realizado pelo MPF que foi tomado como a principal referencia nesse aspecto. O mesmo retrata de maneira muito rica a temática a ser desenvolvida nesse trabalho. A metodologia utilizada foi um estudo de caso de um RCA utilizado para a atividade de sísmica. Dessa forma, foi feita uma comparação dos itens exigidos no Termo de Referência do IDEMA com um Relatório de Controle Ambiental usado num processo de licenciamento ambiental pelo mesmo órgão, ou seja, foi visto se todos os itens exigidos legalmente foram contemplados no RCA e se os mesmo estão em conformidade. Esta técnica de estudo de impacto ambiental baseado dentro de um contexto legal que estabelece requisitos a serem observados e procedimentos a serem cumpridos, é conhecida como análise formal da qualidade de um estudo ambiental. Foi verificada, também, a repetição ou não das principais deficiências apontadas no elemento Mitigação e compensação de impactos, sendo trabalhado o conceito de qualidade de conteúdo, haja vista que o trabalho do MPF deve ser considerado uma referência. 4 ESTUDO DE CASO O estudo foi iniciado fazendo-se a comparação dos pontos abordados no RCA de sísmica com os exigidos pelo Termo de Referência do IDEMA para a mesma atividade, em seguida, o Quadro 2 adiante, composto pelo elemento Mitigação e Compensação de Impactos e suas principais deficiências, foi utilizado como referência para análise do Holos, Ano 25, Vol. 3 23 NASCIMENTO & REIS (2009) mesmo RCA. Observa-se que nesse estudo, os impactos previstos foram considerados a cada atividade do levantamento sísmico (Mobilização dos Recursos Materiais e Humanos, Abertura e Marcação de Picadas, Implantação de Pontos de Tiro, Detonação e Recuperação dos Pontos de Tiro) sendo cada atividade trabalhada nos meios físicos, bióticos e antrópicos, separadamente. Além disso, foi utilizada ferramentas de análise de impacto ambiental, sendo identificado impacto ambiental positivo. No entanto, quando se estuda a proposição de medidas mitigadoras, identificam-se pontos a serem melhorados no estudo. Com relação ao Termo de Referência, constata-se que na matriz de Leopold1, disposta no capítulo cinco, os impacto ambientais são identificados e analisados segundo a componente do sistema ambiental e a fase de ocorrência, porém alguns merecem uma consideração no que diz respeito às conformidades exigidas no TR fornecido pelo IDEMA para a elaboração de um RCA referente à atividade de levantamento sísmico. O estudo investigado não apresenta o detalhamento exigido no TR, na elaboração das medidas de mitigação. Exige-se o detalhamento dos processos, métodos, tecnologias e ações que permitam a prevenção, redução e/ou eliminação dos danos ambientais causados pelo empreendimento, ou ainda, a proposição de compensações ambientais que neutralizem os efeitos causados. Nem na matriz do capítulo cinco, nem as medidas propostas no capítulo seis apresentam de forma aprofundada os detalhes exigidos para a concessão da licença. O estudo considerado não cita a natureza das medidas, se elas são preventivas ou corretivas, nem tampouco a eficiência dos equipamentos de controle de poluição, quanto aos padrões de emissão de efluentes líquidos, emissões atmosféricas e resíduos sólidos. Outra conformidade legal exigidas é a classificação em relação à responsabilidade pela implementação, se seria o empreendedor, o poder público ou outrem e em relação ao custo total. Porém, no estudo nenhum desses aspectos são considerados, ficando a dúvida de quem deve implementar a medida e a viabilidade econômica da mesma. 1 A matriz de Leopold tem sido uma das mais utilizadas nos EIA/RIMA realizados no Brasil, sendo freqüentemente tomada como o método padrão para a elaboração desses estudos (IBAMA, 1985). Holos, Ano 25, Vol. 3 24 1 A matriz de Leopold tem sido uma das mais utilizadas nos EIA/RIMA realizados no Brasil, sendo freqüentemente tomada como o método padrão para a elaboração desses estudos (IBAMA, 1985). Holos, Ano 25, Vol. 3 4 ESTUDO DE CASO A partir dessa etapa será elencado todas as medidas mitigadoras propostas, considerando cada meio individualmente, e será feita uma análise critica de uma por uma com o intuito de observarmos ou não as principais deficiências apontadas pelo estudo do MPF. Como veremos em nenhuma medida mitigadora proposta, foi feita a avaliação de sua eficiência, ou seja, o ponto ausência de avaliação da eficiência das medidas mitigadoras propostas não foi trabalhado. 24 Holos, Ano 25, Vol. 3 NASCIMENTO & REIS (2009) Quadro 2 – Principais deficiências do elemento Mitigação e compensação de impactos. Mitigação e compensação de impactos Proposição de medidas que não são a solução para a mitigação do impacto; Indicação de medidas mitigadoras pouco detalhadas; Indicação de obrigações ou impedimentos, técnicos e legais, como propostas de medidas mitigadoras; Ausência de avaliação da eficiência das medidas mitigadoras propostas; Deslocamento compulsório de populações: propostas iniciais de compensações de perdas baseadas em diagnósticos inadequados; Não-incorporação de propostas dos grupos sociais afetados, na fase de formulação do EIA; Proposição de Unidade de Conservação da categoria de uso sustentável para a aplicação dos recursos, em casos não previstos pela legislação. Fonte: SÁNCHEZ (2006). Fonte: SÁNCHEZ (2006). 4.1 MEDIDAS MITIGADORAS PARA O MEIO FÍSICO Como medidas mitigadoras preventivas (ver gráfico 1) quanto aos impactos negativos nesse meio, propõe-se: 1. Realizar a eventual umidificação das vias não asfaltadas utilizadas para acesso à área de modo a evitar a dispersão de poeira; Não se observa nenhuma deficiência. 2. Realizar a regulagem periódica dos motores dos equipamentos (veículos, perfuratrizes, etc.) envolvidos no levantamento sísmico; Não se observa nenhuma deficiência. 2. Realizar a regulagem periódica dos motores dos equipamentos (veículos, perfuratrizes, etc.) envolvidos no levantamento sísmico; Não se observa nenhuma deficiência. Não se observa nenhuma deficiência. 3. Apenas utilizar explosivos sismográficos licenciados pelo Exército Brasileiro; Proposição de medidas que não são a solução para a mitigação do impacto, Indicação de obrigações ou impedimentos, técnicos e legais como propostas de medidas mitigadoras. 4. Armazenar os explosivos e detonantes com as devidas precauções de segurança, para evitar acidentes ou roubos; Proposição de medidas que não são a solução para a mitigação do impacto, Indicação de obrigações ou impedimentos, técnicos e legais como propostas de medidas mitigadoras. 5. Restringir a manipulação dos explosivos estritamente ao pessoal capacitado; Proposição de medidas que não são a solução para a mitigação do impacto, Indicação de obrigações ou impedimentos, técnicos e legais como propostas de medidas mitigadoras. 6. Manter um inventário diário detalhado dos explosivos utilizados, para evitar que sejam deixados no campo; P i ã d did ã ã l ã iti ã d i t I di ã d deixados no campo; Proposição de medidas que não são a solução para a mitigação do impacto, Indicação de obrigações ou impedimentos, técnicos e legais como propostas de medidas mitigadoras. roposição de medidas que não são a solução para a mitigação do impacto, Indicação d brigações ou impedimentos, técnicos e legais como propostas de medidas mitigadoras. 7. Este item não foi contemplado no estudo analisado; 7. Este item não foi contemplado no estudo analisado; 8. Manter uma distância de 100 metros do curso d’água sem promover corte na mata. Nesses locais, devem ser abertas picadas manualmente, sem corte de árvores, preservando a mata ciliar como barreira de proteção contra erosões e assoreamentos dos cursos d’água; Não se observa nenhuma deficiência. 8. Manter uma distância de 100 metros do curso d’água sem promover corte na mata. 4.1 MEDIDAS MITIGADORAS PARA O MEIO FÍSICO Nesses locais, devem ser abertas picadas manualmente, sem corte de árvores, preservando a mata ciliar como barreira de proteção contra erosões e assoreamentos dos cursos d’água; Não se observa nenhuma deficiência. 25 Holos, Ano 25, Vol. 3 NASCIMENTO & REIS (2009) 9. Não realizar picadas ao longo de margens de drenagens muito encaixadas com barrancos (caso existam), evitando represamentos em épocas chuvosas e mesmo erosão às circunvizinhanças; Não se observa nenhuma deficiência. 10. Abrir apenas áreas estritamente necessárias ao trabalho, tais como, áreas para manobras e estacionamentos; Não se observa nenhuma deficiência. 11. Não realizar picadas em encostas íngremes (caso existam). Proceder nesses locais a abertura manual, sem corte de árvores, evitando processos erosivos; Não se observa nenhuma deficiência. 12. A abertura das linhas sísmicas deve ser dimensionada com a largura mínima necessária ao trabalho, sendo no máximo de 4 metros; Indicação de obrigações ou impedimentos, técnicos e legais, como propostas de medidas mitigadoras. ao trabalho, sendo no máximo de 4 metros; Indicação de obrigações ou impedimentos, técnicos e legais, como propostas de medidas mitigadoras. 13. Não furar, carregar e detonar nas margens de recursos hídricos superficiais (rios, lagos, represas), mantendo-se uma distância de segurança de no mínimo 30 metros, de acordo com cada caso; Não se observa nenhuma deficiência. 13. Não furar, carregar e detonar nas margens de recursos hídricos superficiais (rios, lagos, represas), mantendo-se uma distância de segurança de no mínimo 30 metros, de acordo com cada caso; Não se observa nenhuma deficiência. 14. Evitar furar, carregar e detonar em áreas alagadas; 14. Evitar furar, carregar e detonar em áreas alagadas; Não se observa nenhuma deficiência. Não se observa nenhuma deficiência. 15. Promover o imediato tamponamento dos buracos eventualmente gerados pelas detonações dos explosivos; Não se observa nenhuma deficiência. 15. Promover o imediato tamponamento dos buracos eventualmente gerados pelas detonações dos explosivos; Não se observa nenhuma deficiência. 16. Os resíduos oriundos do acampamento-base (caso seja construído), e os gerados em campo, devem ser acondicionados em cestos específicos para cada tipo de material: 1. Lixo orgânico, 2. Plásticos, Papéis, Metais e Vidros e; 3. Óleos e graxas. Os resíduos orgânicos devem ser encaminhados ao vazadouro municipal, os inorgânicos à usina de reciclagem mais próxima e óleos e graxas para empresas licenciadas e capacitadas para dar destinação final a esses resíduos; Não se observa nenhuma deficiência. 17. 4.1 MEDIDAS MITIGADORAS PARA O MEIO FÍSICO Em caso de vazamento acidental dispor de recipiente capaz de acumular com folga todo o óleo/combustível armazenado nas máquinas utilizadas evitando a contaminação do solo; ; Indicação de medidas mitigadoras pouco detalhadas. ; Indicação de medidas mitigadoras pouco detalhadas. Holos, Ano 25, Vol. 3 26 NASCIMENTO & REIS (2009) Resumo das medidas mitigadoras do Meio Físico 16 4 1 1 10 0 2 4 6 8 10 12 14 16 18 1 Quantidade total de Medidas Mitigadoras Proposição de medidas que não são a solução para a mitigação do impacto; Indicação de medidas mitigadoras pouco detalhadas; Indicação de obrigações ou impedimentos, técnicos e legais, como propostas de medidas mitigadoras; Não se observa nenhuma deficiência. Gráfico 1 - Resumo das medidas mitigadoras do meio físico. Fonte: De acordo com a análise das medidas mitigadoras. 1 Gráfico 1 - Resumo das medidas mitigadoras do meio físico. Fonte: De acordo com a análise das medidas mitigadoras. 4.2 MEDIDAS MITIGADORAS PARA O MEIO BIÓTICO Os procedimentos para minimizar os impactos ambientais para o meio biótico (ver gráfico 2) são: 1. Diminuição da Faixa de Remoção da Vegetação. Essa é limitada ao máximo de 4,0 metros para o caso de abertura manual das picadas, a largura será inferior a 1,5m; Não se observa nenhuma deficiência. 1. Diminuição da Faixa de Remoção da Vegetação. Essa é limitada ao máximo de 4,0 metros para o caso de abertura manual das picadas, a largura será inferior a 1,5m; Não se observa nenhuma deficiência. 2. Não remover árvores consideradas nobres e/ou frutíferas, tais como: angico, umburana, juazeiro, oiticica, imburana, quixabeira, carnaubeira, cajueiro e outras; Não se observa nenhuma deficiência. 3. Não cortar árvores de grande porte, ou seja, tronco de diâmetro, à altura do peito, igual ou maior a 15 centímetros; Não se observa nenhuma deficiência. 4. Recolhimento dos materiais utilizados. Todo o material é recolhido após as operações, preservando as condições encontradas antes dos trabalhos; Não se observa nenhuma deficiência. 5. Não realizar aberturas em encostas íngremes. Nesses locais, recomendasse abrir picadas manualmente, sem corte de árvores, evitando processos erosivos; Não se observa nenhuma deficiência. 6. Não abrir em margens de drenagens muito encaixadas com barrancos, evitando represamentos em épocas chuvosas e mesmo erosão nas áreas circunvizinhas; Não se observa nenhuma deficiência. 27 Holos, Ano 25, Vol. 3 27 NASCIMENTO & REIS (2009) 7. Não abrir áreas, além das estritamente necessárias ao trabalho, tais como, áreas para manobras e estacionamentos; 7. Não abrir áreas, além das estritamente necessárias ao trabalho, tais como, áreas para manobras e estacionamentos; Repetição da recomendação do item 10 do meio físico. Proposição de medidas que não são é a solução para a mitigação do impacto. Não abrir áreas, além das estritamente necessárias ao trabalho, tais como, áreas par anobras e estacionamentos; epetição da recomendação do item 10 do meio físico. Proposição de medidas que não sã a solução para a mitigação do impacto. 8. Evitar raspar o solo orgânico e formar amontoados de terras ao longo das linhas sísmicas. Na medida do possível utilizar o trator de esteira com a lâmina afastada do solo apenas para tombar a vegetação sem a retirada da camada orgânica do solo. Isto facilitará a recomposição vegetal e microbiana mais rapidamente, evitando deixar o solo exposto aos processos erosivos por longo tempo; Não se observa nenhuma deficiência. 9. 4.2 MEDIDAS MITIGADORAS PARA O MEIO BIÓTICO Evitar o corte de vegetações que constituam abrigos de animais silvestres (ninhos, cortiços, tocas, etc.); 9. Evitar o corte de vegetações que constituam abrigos de animais silvestres (ninhos, cortiços, tocas, etc.); Não se observa nenhuma deficiência. Não se observa nenhuma deficiência. 10. Nas margens de estradas, fechadas por vegetação, por onde cruzar uma linha sísmica deverá ser construída uma cerca (falsa cerca), ao término dos trabalhos de Levantamento Sísmico, com o objetivo de evitar a utilização da linha sísmica como via de acesso para caçadores, carros, carroças, etc.; Não se observa nenhuma deficiência. ão se observa nenhuma deficiência. 11. Os amontoados de vegetação que, porventura forem deixados pela abertura das linhas com trator de esteira, deverão ser espalhados ao longo da linha sísmica; Não se observa nenhuma deficiência. 11. Os amontoados de vegetação que, porventura forem deixados pela abertura das linhas com trator de esteira, deverão ser espalhados ao longo da linha sísmica; Não se observa nenhuma deficiência. 11. Os amontoados de vegetação que, porventura forem deixados pela abertura das linhas com trator de esteira, deverão ser espalhados ao longo da linha sísmica; Nã b h d fi iê i 12. Limitar a limpeza do terreno e as obras somente a área física do empreendimento a ser implantado como forma de redução de locais geradores de ruídos; quanto mais próximos à atividade geradora destes ambientes, maiores são os impactos; Deveria está no meio físico, fator ruído, Indicação de medidas mitigadoras pouco detalhadas. 13. Tanto a abertura das linhas sísmicas quanto as detonações devem ser atividades de curta duração como forma de evitar prolongar as fontes de ruídos (máquinas, equipamentos e explosivos), restabelecendo o mais rápido o ambiente natural; Não se observa nenhuma deficiência. 14. Evitar o corte de vegetações que constituam abrigos de animais silvestres (ninhos, cortiços, tocas, etc.); Não se observa nenhuma deficiência Não se observa nenhuma deficiência. 15. Resgate e transferência da fauna criticamente exposta às atividades para áreas isentas das operações; N b h d fi iê i ão se observa nenhuma deficiência. Não se observa nenhuma deficiência. 16. Sinalização de trânsito e orientação dos operários nas estradas que dão acesso às obras como forma de prudência e redução de velocidade evitando acidentes com animais que estejam transitando pela área; Holos, Ano 25, Vol. 4.2 MEDIDAS MITIGADORAS PARA O MEIO BIÓTICO 3 28 NASCIMENTO & REIS (2009) Não trabalha o meio biótico, Proposição de medidas que não são a solução para a mitigação do impacto e Indicação de medidas mitigadoras pouco detalhadas. 17. Desenvolver ações de educação ambiental e divulgação de métodos de identificação de animais peçonhentos e de prevenção de acidentes com ofídios; Proposição de medidas que não são a solução para a mitigação do impacto e Indicação de medidas mitigadoras pouco detalhadas. Proposição de medidas que não são a solução para a mitigação do impacto e Indicação de medidas mitigadoras pouco detalhadas. 16. Não será permitida a caça, comercialização, aprisionamento, destruição de ninhos, coleta de ovos e maus tratos a animais silvestres, pelos membros da equipe sísmica. Cabe a empresa exercer forte fiscalização sobre seu Quadro funcional2; 16. Não será permitida a caça, comercialização, aprisionamento, destruição de ninhos, coleta de ovos e maus tratos a animais silvestres, pelos membros da equipe sísmica. Cabe a empresa exercer forte fiscalização sobre seu Quadro funcional2; Indicação de obrigações ou impedimentos, técnicos e legais, como propostas de medidas mitigadoras. Indicação de obrigações ou impedimentos, técnicos e legais, como propostas de medidas mitigadoras. 18. Quando oportuno, aplicar um programa de educação ambiental, incentivando a população circunvizinha a preservação da fauna; I di ã d did iti d d t lh d 18. Quando oportuno, aplicar um programa de educação ambiental, incentivando a população circunvizinha a preservação da fauna; Indicação de medidas mitigadoras pouco detalhadas. Indicação de medidas mitigadoras pouco detalhadas. ação de medidas mitigadoras pouco detalhadas. 19. Mediante constatação de prática ilegal, comunicar ao IDEMA a fim de que sejam tomadas as devidas providências; Indicação de medidas mitigadoras pouco detalhadas e Indicação de obrigações ou impedimentos, técnicos e legais, como propostas de medidas mitigadoras. 20. Evitar o corte de vegetação que constituem abrigos de animais silvestres (ninhos, tocas, etc); Repetição da medida 14. 19. Mediante constatação de prática ilegal, comunicar ao IDEMA a fim de que sejam tomadas as devidas providências; Indicação de medidas mitigadoras pouco detalhadas e Indicação de obrigações ou impedimentos, técnicos e legais, como propostas de medidas mitigadoras. 20. Evitar o corte de vegetação que constituem abrigos de animais silvestres (ninhos, tocas, etc); 19. 4.2 MEDIDAS MITIGADORAS PARA O MEIO BIÓTICO Mediante constatação de prática ilegal, comunicar ao IDEMA a fim de que sejam tomadas as devidas providências; tomadas as devidas providências; Indicação de medidas mitigadoras pouco detalhadas e Indicação de obrigações ou impedimentos, técnicos e legais, como propostas de medidas mitigadoras. 20. Evitar o corte de vegetação que constituem abrigos de animais silvestres (ninhos, tocas, etc); Repetição da medida 14. Indicação de medidas mitigadoras pouco detalhadas e Indicação de obrigações ou impedimentos, técnicos e legais, como propostas de medidas mitigadoras. 21. Diminuição da Faixa de Remoção da Vegetação. Essa é limitada ao máximo de 4,0 metros; Não se observa nenhuma deficiência. 22. Limitar a limpeza do terreno e as obras somente a área física do empreendimento a ser implantado como forma de redução de locais geradores de ruídos; quanto mais próximos à atividade geradora destes ambientes, maiores são os impactos; Repetição da medida 12. 23. Tanto a abertura das linhas sísmicas quanto as detonações devem ser atividades de curta duração como forma de evitar prolongar as fontes de ruídos (máquinas, equipamentos e explosivos), restabelecendo o mais rápido o ambiente natural; Repetição da medida 13. 24. Realizar campanhas de conscientização sobre o assunto com trabalhadores das obras e moradores da região; Proposição de medidas que não é a solução para a mitigação do impacto e Indicação de 24. Realizar campanhas de conscientização sobre o assunto com trabalhadores das obras e moradores da região; moradores da região; Proposição de medidas que não é a solução para a mitigação do impacto e Indicação de medidas mitigadoras pouco detalhadas. 5. Verificar os estoques de soro antiofídico nos postos de saúde e hospitais da região; 25. Verificar os estoques de soro antiofídico nos postos de saúde e hospitais da região; Proposição de medidas que não é a solução para a mitigação do impacto e Indicação de medidas mitigadoras pouco detalhadas. Proposição de medidas que não é a solução para a mitigação do impacto e Indicação de medidas mitigadoras pouco detalhadas. 29 Holos, Ano 25, Vol. 3 NASCIMENTO & REIS (2009) Resumo das medidas mitigadoras do Meio Biótico 26 4 6 2 14 3 0 5 10 15 20 25 30 1 Quantidade total de Medidas Mitigadoras Proposição de medidas que não são a solução para a mitigação do impacto; Indicação de medidas mitigadoras pouco detalhadas; Indicação de obrigações ou impedimentos, técnicos e legais, como propostas de medidas mitigadoras; Não se observa nenhuma deficiência. 4.2 MEDIDAS MITIGADORAS PARA O MEIO BIÓTICO Repetição de medidas Gráfico 2 - Resumo das medidas mitigadoras do meio biótico. Fonte: De acordo com a análise das medidas mitigadoras. Gráfico 2 - Resumo das medidas mitigadoras do meio biótico. Fonte: De acordo com a análise das medidas mitigadoras. 4.3 MEDIDAS MITIGADORAS PARA O MEIO ANTRÓPICO São propostas as seguintes medidas mitigadoras para meio antrópico (ver gráfico 3) relativas à implantação do Empreendimento: 1. Isolamento das linhas sísmicas quando estas cruzarem margem de estradas, para evitar que as mesmas se tornem acesso para os moradores do entorno do projeto; Não se observa nenhuma deficiência. 2.Utilizar o mínimo de carga de explosivos necessários ao levantamento sísmico, bem como reuniões com pessoal residente próximo à área para esclarecimento sobre o projeto; Não se observa nenhuma deficiência. 3.Realizar reuniões para esclarecimento às pessoas residentes próximas ao Empreendimento, quanto ao acesso à área do mesmo; Semelhante a medida 2. 4. Destinação adequada para acondicionamento do material retirado; Não relacionada ao meio antrópico. Proposição de medidas que não é a solução para a mitigação do impacto. Indicação de medidas mitigadoras pouco detalhadas. 5. Desenvolver plano de ação para evitar possíveis alterações ambientais dessa natureza; Não relacionada ao meio antrópico. Indicação de obrigações ou impedimentos, técnicos e legais como propostas de medidas mitigadoras. 5. Desenvolver plano de ação para evitar possíveis alterações ambientais dessa natureza; Não relacionada ao meio antrópico. Indicação de obrigações ou impedimentos, técnicos e legais como propostas de medidas mitigadoras. Holos, Ano 25, Vol. 3 30 NASCIMENTO & REIS (2009) 6. Destinação adequada para os resíduos gerados durante o processo de Levantamento Sísmico; Indicação de medidas mitigadoras pouco detalhadas. dicação de medidas mitigadoras pouco detalhadas. 7. Utilização de equipamentos de proteção individual (EPI’s); Indicação de obrigações ou impedimentos, técnicos e legais como propostas de medidas mitigadoras. 7. Utilização de equipamentos de proteção individual (EPI’s); Indicação de obrigações ou impedimentos, técnicos e legais como propostas de medidas mitigadoras. 7. Utilização de equipamentos de proteção individual (EPI’s); Indicação de obrigações ou impedimentos, técnicos e legais como propostas de medidas mitigadoras. 8. Indenização da safra agrícola; Não se observa nenhuma deficiência. Resumo das medidas mitigadoras do meio antrópico 8 1 1 2 3 1 0 1 2 3 4 5 6 7 8 9 1 Quantidade total de Medidas Mitigadoras Proposição de medidas que não são a solução para a mitigação do impacto; Indicação de medidas mitigadoras pouco detalhadas; Indicação de obrigações ou impedimentos, técnicos e legais, como propostas de medidas mitigadoras; Não se observa nenhuma deficiência. Repetição de medidas Gráfico 3 - Resumo das medidas mitigadoras do meio antrópico. Fonte: De acordo com a análise das medidas mitigadoras. Gráfico 3 - Resumo das medidas mitigadoras do meio antrópico. 4.3 MEDIDAS MITIGADORAS PARA O MEIO ANTRÓPICO Fonte: De acordo com a análise das medidas mitigadoras. 5 CONSIDERAÇÕES FINAIS O RCA apresenta como pontos positivos o detalhamento das medidas mitigadoras a cada fase, sendo cada uma trabalhada nos diversos meios (físico, biótico e antrópico), separadamente, assim como foi utilizada ferramenta de analise de impacto ambiental indicada pelo IBAMA, tal como a matriz de Leopold. Conforme se observa no estudo de caso, o RCA utilizado para a obtenção da concessão de licença ambiental da atividade de sísmica, não contemplou todos os itens legais exigidos no Termo de Referência do IDEMA, não apresentando o detalhamento exigido no TR, na elaboração das medidas de mitigação, não cita a natureza das medidas, se elas são preventivas ou corretivas, nem tampouco a eficiência dos equipamentos de controle de poluição, quanto aos padrões de emissão de efluentes líquidos, emissões atmosféricas e resíduos sólidos, além de não definir a responsabilidade de quem deve implementar a medida e a viabilidade econômica da mesma. Holos, Ano 25, Vol. 3 31 NASCIMENTO & REIS (2009) Nessa situação, é oportuno o detalhamento das medidas de mitigação, caracterizando a sua natureza em preventiva ou corretiva, descrever a eficiência dos equipamentos de controle de poluição, definindo as responsabilidades e a relação custo/beneficio. Acrescentando-se ao que foi mencionado, o RCA em estudo, apresentou as deficiências apontadas pelo MPF, ou seja, foram indicadas medidas que não mitiga o impacto, as medidas trabalhadas foram pouco detalhadas, foram utilizadas obrigações legais como medida mitigadora além erros na enumeração das medidas. Nesse caso, é cabível a utilização de medidas que de fato mitiguem o impacto, as medidas devem ser detalhadas e não se devem utilizar obrigações legais como medida mitigadora, fato que ocorreu na indicação de utilização de EPI, por exemplo. Desse modo, o RCA não garante ao analista, o atendimento aos requisitos mínimos estabelecidos pela regulamentação aplicável, assim como, não apresenta qualidade técnica suficiente para subsidiar a tomada de decisão sobre o empreendimento sendo necessárias as correções para que o mesmo possa ser melhor apreciado pelo órgão ambiental competente. Assim, acreditamos que este trabalho desperta a consciência daqueles que se interessam pelas questões ligadas ao processo de obtenção de licença ambiental e proporciona uma leitura interessante aos profissionais que lidam com esse tema. REFERÊNCIAS 1. ASSOCIAÇÃO BRASILEIRA DE NORMAS TÉCNICAS. NBR 13030: Elaboração e apresentação de projeto de reabilitação de áreas degradadas pela mineração. Rio de Janeiro, 1999. 2. ______. NBR ISO 14001:2004. Sistemas de gestão ambiental: requisitos com orientações para uso. Rio de Janeiro, 2004. 3. BARBIERI, José Carlos. Gestão ambiental empresarial: conceitos, modelos e instrumentos – 2. ed. atual e ampliada – São Paulo: Saraiva, 2007. 4. BRANCO, Samuel Murgel. O meio ambiente em debate – São Paulo: Moderna, 1988 – (Coleção polemica) 4. BRANCO, Samuel Murgel. O meio ambiente em debate – São Paulo: Moderna, 1988 – (Coleção polemica) 5. BRASIL. Constituição (1988). Constituição da República Federativa do Brasil. Brasília, DF: Senado, 1988. 5. BRASIL. Constituição (1988). Constituição da República Federativa do Brasil. Brasília, DF: Senado, 1988. 6. BRASIL. Congresso Nacional. Lei No 6938, de 31 de Agosto de 1981.Dispõe sobre a Política Nacional do Meio Ambiente, seus fins e mecanismos de formulação e aplicação, e dá outras providências. Diário Oficial da União, Poder Executivo, Brasília, DF, 02 de Setembro de 1981. P. 16509 7. CONSELHO NACIONAL DO MEIO AMBIENTE. Resolução 237. Brasil: CONAMA, 1997. Holos, Ano 25, Vol. 3 32 NASCIMENTO & REIS (2009) 8. FINK, Roberto Daniel; ALONSO, Hamilton Jr.; DAWALIBI, Marcelo. Aspectos jurídicos do licenciamento ambiental. Rio de Janeiro: Forense Universitária, 2004. 9. LIMA, Maíra Luísa Milani de. Licenciamento ambiental e Gestão de Riscos: o caso da usina hidrelétrica de barra grande (RS). 2006. Dissertação (Mestrado em Direito) – Centro de Pós-Graduação em Direito, Universidade Federal de Santa Catarina, Florianópolis, 2006. 10. PHILIPPI Jr. A, Pelicioni MCF, editores. Educação ambiental e sustentabilidade – Barueri, São Paulo: Manole, 2005 – (Coleção Ambiental; 3). 11. POLÍTICA ESTADUAL DE MEIO AMBIENTE DO ESTADO DO RIO GRANDE DO NORTE (PEMA). Lei complementar nº 272, de 03 de março de 2004. Disponível em: http://www.rn.gov.br/secretarias/idema/legislacao.asp >. Acesso em 03 de jun. de 2009. 12. SÁNCHEZ, Luiz Henrique. Avaliação de impacto ambiental: conceitos e métodos – São Paulo: Oficina de Textos, 2006. 12. SÁNCHEZ, Luiz Henrique. Avaliação de impacto ambiental: conceitos e métodos – São Paulo: Oficina de Textos, 2006. 13. SILVA, Robson Garcia da; PEGADO, Erika Araújo da Cunha; SILVA, Valdenildo Pedro da. A exploração e produção on shore e o licenciamento ambiental no Rio Grande do Norte. In: V CONGRESSO DE INICIAÇÃO CIENTÍFICA, 2008, Natal. Anais... Natal: CEFET-RN, 2008. Disponível em: <http://www.ifrn.edu.br/secoes/pesquisa/arquivos/anais-congic- 2008.pdf/view?searchterm=congic> Acesso em: 03 de jun. 2009. Holos, Ano 25, Vol. 3 33
https://openalex.org/W2569184182	https://bib-pubdb1.desy.de/record/321069/files/4.pdf	English	null	Search for massive resonances decaying into WW, WZ or ZZ bosons in proton-proton collisions at s = 13 $$ \sqrt{s}=13 $$ TeV	The Journal of high energy physics/The journal of high energy physics	2,017	cc-by	31,227	Search for massive resonances decaying into WW, WZ or ZZ bosons in proton-proton collisions at √s = 13 TeV JHEP03(2017)162 The CMS collaboration Open Access, Copyright CERN, Published for SISSA by Springer Published for SISSA by Springer Received: December 29, 2016 Revised: March 5, 2017 Accepted: March 13, 2017 Published: March 30, 2017 Received: December 29, 2016 Revised: March 5, 2017 Accepted: March 13, 2017 Published: March 30, 2017 Received: December 29, 2016 Revised: March 5, 2017 Accepted: March 13, 2017 Published: March 30, 2017 1 Introduction Several theories beyond the standard model (SM) predict the existence of heavy particles that preferentially decay to pairs of vector bosons V, where V represents a W or Z. These models usually aim to clarify open questions in the SM such as the apparently large dif- ference between the electroweak and the gravitational scales. Notable examples of such models include the bulk scenario [1–3] of the Randall-Sundrum warped extra-dimensions (RS1) [4, 5] and a heavy vector-triplet (HVT) model [6]. The bulk graviton model is de- scribed by two free parameters: the mass of the ﬁrst Kaluza-Klein (KK) excitation of a spin-2 boson (the KK bulk graviton Gbulk) and the ratio ˜k ≡k/MPl, where k is the un- known curvature scale of the extra dimension and MPl ≡MPl/ √ 8π is the reduced Planck mass. The HVT generalises a large number of models that predict spin-1 charged (W′) and neutral (Z′) resonances. Such models can be described in terms of just a few parameters: two coeﬃcients cF and cH, scaling the couplings to fermions, and to the Higgs and longi- tudinally polarized SM vector bosons respectively, and the strength gV of the new vector boson interaction. Two benchmark models are considered in the HVT scenario. In the ﬁrst one, referred to as HVT model A, with gV = 1, weakly coupled vector resonances arise from extensions of the SM gauge group, such as the sequential standard model (SSM) [7], that have comparable branching fractions to fermions and gauge bosons. In HVT Model B with gV = 3, the new resonances have large branching fractions to pairs of bosons, while their fermionic couplings are suppressed. This scenario is most representative of composite models of Higgs bosons. JHEP03(2017)162 Searches for diboson resonances have been previously performed in many diﬀerent ﬁnal states, placing lower limits on the masses of these resonances above the TeV scale [8–19]. Searches performed with proton-proton collisions at √s = 8 TeV indicated deviations from background expectations at resonance masses of about 2 TeV. The largest excesses of events were observed in the searches in the dijet WW, WZ or ZZ [12, 16] channels, as well as in the semi-leptonic WH →ℓνbb ﬁnal state [13], and have local signiﬁcances of 3.4σ and 2.2σ, respectively. The CMS collaboration E-mail: cms-publication-committee-chair@cern.ch E-mail: cms-publication-committee-chair@cern.ch E-mail: cms-publication-committee-chair@cern.ch Abstract: A search is presented for new massive resonances decaying to WW, WZ or ZZ bosons in ℓνq¯q and q¯qq¯q ﬁnal states. Results are based on data corresponding to an integrated luminosity of 2.3–2.7 fb−1 recorded in proton-proton collisions at √s = 13 TeV with the CMS detector at the LHC. Decays of spin-1 and spin-2 resonances into two vector bosons are sought in the mass range 0.6–4.0 TeV. No signiﬁcant excess over the standard model background is observed. Combining the results of the ℓνq¯q and q¯qq¯q ﬁnal states, cross section and mass exclusion limits are set for models that predict heavy spin-1 and spin-2 resonances. This is the ﬁrst search for a narrow-width spin-2 resonance at √s = 13 TeV. Keywords: Beyond Standard Model, Hadron-Hadron scattering (experiments), Particle and resonance production, proton-proton scattering Keywords: Beyond Standard Model, Hadron-Hadron scattering (experiments), Particle and resonance production, proton-proton scattering ArXiv ePrint: 1612.09159 ArXiv ePrint: 1612.09159 ArXiv ePrint: 1612.09159 doi:10.1007/JHEP03(2017)162 Contents 1 Introduction 2 2 The CMS detector 3 3 Simulated samples 4 4 Reconstruction and selection of events 4 4.1 Trigger and preliminary oﬄine selection 4 4.2 Jet reconstruction 5 4.3 Final reconstruction and selection of leptons and missing transverse momentum 6 4.4 The identiﬁcation of W/Z →qq using jet substructure 6 4.5 The reconstruction and identiﬁcation of W →ℓν 9 4.6 Final event selection and categorization 9 5 Modeling of background and signal 11 5.1 Multijet background 11 5.2 Top quark production 12 5.3 The W+jets background 12 5.4 Signal modelling 16 6 Systematic uncertainties 17 6.1 Systematic uncertainties in the background estimation 17 6.2 Systematic uncertainties in the signal prediction 18 7 Statistical interpretation 19 7.1 Limits on narrow-width resonance models 20 7.2 Model-independent limits 23 8 Summary 26 A Instructions and additional material for generic interpretation of the re- sults 27 The CMS collaboration 37 JHEP03(2017)162 5.1 Multijet background A Instructions and additional material for generic interpretation of the re- sults 2 – 1 – 1 Introduction The most stringent lower mass limit for a W′ (Z′) is set at 2.3 (2.0) TeV by a combination of searches in semi-leptonic and all-hadronic ﬁnal states performed with proton-proton collisions at √s = 13 TeV [9]. The same searches provide the current lower mass limit of 2.6 TeV for a HVT. This paper presents a search for resonances with masses above 0.6 TeV decaying into a pair of vector bosons. The analysis is based on data collected in proton-proton collisions at √s = 13 TeV with the CMS experiment at the CERN LHC during 2015, corresponding to an integrated luminosity of 2.3 and 2.7 fb−1 for the ℓνqq, where ℓ= µ or e, and qqqq ﬁnal state, respectively. The ℓν+jet search also includes the W →τν contribution from the decay τ →ℓνν. The gain in sensitivity from τ leptons is limited by the small branching fractions involved. The key challenge of the analyses is the reconstruction of the highly energetic decay products. Since the resonances under study have masses of order 1 TeV, their decay prod- ucts, i.e. the bosons, have on average transverse momenta (pT) of several hundred GeV or more. As a consequence, the particles emerging from the boson decays are very collimated. – 2 – In particular, the jet-decay products of the bosons cannot be resolved using the standard algorithms, but are instead reconstructed as a single jet object. Dedicated techniques, called jet “V tagging” techniques, are applied to exploit the substructure of such objects, to help resolve jet decays of massive bosons [20, 21]. The V tagging also helps suppress SM backgrounds, which mainly originate from the production of multijet, W+jets, and nonresonant VV events. In particular, the jet-decay products of the bosons cannot be resolved using the standard algorithms, but are instead reconstructed as a single jet object. Dedicated techniques, called jet “V tagging” techniques, are applied to exploit the substructure of such objects, to help resolve jet decays of massive bosons [20, 21]. The V tagging also helps suppress SM backgrounds, which mainly originate from the production of multijet, W+jets, and nonresonant VV events. The ﬁnal states considered are either ℓνqq or qqqq, where the hadronic decay products of the V decay are reconstructed in a single jet. They result in events with either a charged lepton, a neutrino, and a single reconstructed jet (ℓν+jet channel), or two reconstructed jets (dijet channel). 1 Introduction As in the analyses of previous data [11, 12], the aim is to reconstruct all decay products of the new resonance to be able to search for a localized enhancement in the diboson invariant mass spectrum. While the analyses in general aim at large resonance masses, we conduct two exclusive searches in the ℓν+jet ﬁnal state, separately optimized for the mass ranges 0.6–1.0 TeV (“low-mass”) and > 1 TeV (“high-mass”). JHEP03(2017)162 This paper is organized as follows. Section 2 brieﬂy describes the CMS detector. Section 3 gives an overview of the simulations used in this analysis. Section 4 provides a detailed description of the reconstruction and event selection. Section 5 describes the background estimation and the signal modelling procedures. Systematic uncertainties are discussed in section 6. The results of the search for a spin-2 bulk graviton and for spin-1 resonances as predicted by HVT models are presented in section 7, and section 8 provides a brief summary. 3 Simulated samples The bulk graviton model and HVT models are used as benchmark signal processes. In these models, the vector gauge bosons are produced with a longitudinal polarization in more than 99% of the cases. For each resonance hypothesis, we consider masses in the range 0.6 to 4.0 TeV. Simulated signal events are generated at leading order (LO) accuracy with MadGraph5 amc@nlo v2.2.2 [25] with a width of 0.1% of the resonance mass. JHEP03(2017)162 The Monte Carlo (MC) generated samples of SM backgrounds are used to optimize the analyses. The W+jets SM process is simulated with MadGraph5 amc@nlo, while tt and single top quark events are generated with both powheg v2 [26–31] and Mad- Graph5 amc@nlo. Diboson (WW, WZ, and ZZ) processes are generated with pythia v8.205 [32, 33]. Parton showering and hadronization are implemented through pythia using the CUETP8M1 tune [34, 35]. The NNPDF 3.0 [36] parton distribution functions (PDFs) are used for all simulated samples, except for diboson ones (WW, WZ and ZZ) for which NNPDF 2.3LO is used. All events are processed through a Geant4-based [37] simulation of the CMS detector. The simulated background is normalized using inclusive cross sections calculated at next-to-leading order (NLO), or next-to-NLO order in quantum chromodynamics (QCD) where available, using mcfm v6.6 [38–41] and fewz v3.1 [42]. Additional simulated minimum-bias interactions are added to the generated events to match the additional particle production observed in the large number of overlapping proton-proton interactions within the same or nearby bunch crossings (pileup). The sim- ulated events are corrected for diﬀerences between data and simulation in the eﬃciencies of the lepton trigger [43], lepton identiﬁcation and isolation [43], and selection of jets orig- inating from hadronization of b quarks (b jets) [44]. 2 The CMS detector The central feature of the CMS apparatus is a superconducting solenoid of 6 m internal diameter, providing a magnetic ﬁeld of 3.8 T. Contained within the solenoid volume are a silicon pixel and strip tracker, a lead tungstate crystal electromagnetic calorimeter (ECAL), and a brass and scintillator hadron calorimeter (HCAL), each composed of a barrel and two endcap sections. Extensive forward calorimetry complements the coverage provided by the barrel and endcap detectors. The forward hadron (HF) calorimeter uses steel as an absorber and quartz ﬁbers as the sensitive material. The two halves of the HF are located 11.2 m from the interaction region, one on each end, and together they provide coverage in the pseudorapidity range 3.0 < \|η\| < 5.2. Muons are measured in gas-ionization detectors embedded in the steel ﬂux-return yoke outside the solenoid. A particle-ﬂow (PF) event algorithm [22, 23] reconstructs and identiﬁes each individ- ual particle with an optimized combination of information from the various elements of the CMS detector. The energy of photons is obtained from the ECAL measurement, corrected for suppression of small readout signals. The energy of electrons is determined from a combination of the electron momentum at the primary interaction vertex as determined by the tracker, the energy of the corresponding ECAL cluster, and the energy sum of all bremsstrahlung photons spatially compatible with originating from the electron track. The momentum of muons is obtained from the curvature of the corresponding track. The en- ergy of charged hadrons is determined from a combination of their momentum measured in the tracker and the matching of energies deposited in ECAL and HCAL, also corrected for – 3 – suppression of small signals and for the response function of the calorimeters to hadronic showers. Finally, the energy of neutral hadrons is obtained from the corresponding cor- rected ECAL and HCAL energy. A more detailed description of the CMS detector, together with a deﬁnition of the coordinate system and the kinematic variables, can be found in ref. [24]. 4.1 Trigger and preliminary oﬄine selection In the ℓν+jet channel, events are collected with a trigger requiring either one muon or one electron. For the low-mass ℓν+jet analysis, both triggers have a pT requirement of 27 GeV. The muons and electrons selected online also satisfy both isolation requirements and identiﬁcation criteria. The selection eﬃciency of these triggers for leptons satisfying the oﬄine requirements described in section 4.3, varies in the range 90–95% for the single-muon trigger, depending on the η of the muon, and it is >94% for the single-electron trigger. In the high-mass ℓν+jet analysis, muons selected online must have pT > 45 GeV and \|η\| < 2.1, while the minimum pT threshold for electrons is 105 GeV. There are no requirements on the isolation and loose identiﬁcation criteria are used, since these introduce ineﬃciencies at high resonance masses. The selection eﬃciencies with respect to the oﬄine requirements – 4 – of the single-muon trigger vary between 90% and 95%. The eﬃciency is above 98% for the single-electron trigger. In the dijet channel, events are selected online using a variety of diﬀerent hadronic triggers based on the scalar pT sum of all jets in the event (HT) or the presence of at least one jet with loose substructure requirements; the details of jet substructure are described in section 4.4. Events must satisfy at least one of the following four requirements. The ﬁrst requirement is simply HT > 800 GeV. The second requirement is HT > 650 GeV and a diﬀerence in η between the two leading jets in the event satisfying the condition ∆η < 1.5. The accepted jets are further required to have a dijet invariant mass > 900 GeV. The third criterion is that at least one jet with pT > 360 GeV and a trimmed mass (as deﬁned in section 4.4) mjet > 30 GeV is present in the event. Fourthly, events with HT > 700 GeV and at least one jet with mjet > 50 GeV are also selected for analysis. JHEP03(2017)162 j The pp data collected by CMS with the detector in its fully operational state corre- spond to 2.3 fb−1 of integrated luminosity [45]. 4.1 Trigger and preliminary oﬄine selection Additional data equivalent of 0.37 fb−1 of integrated luminosity were collected with the HF running in suboptimal conditions; those data are used only for the dijet channel, since jets reconstructed online and used for the cal- culation of HT are in the range of \|η\| < 3.0. The trigger eﬃciency is found to be unaﬀected by the condition of the HF. Oﬄine, all events are required to have at least one primary interaction vertex recon- structed within a 24 cm window along the beam axis, with a transverse distance from the mean pp interaction point of less than 2 cm [46]. In the presence of more than one vertex passing these requirements, the primary interaction vertex is chosen to be the one with the highest total p2 T, summed over all the associated tracks. 4.3 Final reconstruction and selection of leptons and missing transverse mo- mentum 4.3 Final reconstruction and selection of leptons and missing transverse mo- mentum 4.3 Final reconstruction and selection of leptons and missing transverse mo- mentum Muons are reconstructed through a ﬁt to hits in both the inner tracking system and the muon spectrometer [51]. Muons must satisfy identiﬁcation requirements on the impact parameters of the track, the number of hits reconstructed in both the silicon tracker and the muon detectors, and the uncertainty in the pT. These quality criteria ensure a precise measurement of the four-momentum and rejection of misreconstructed muons. An isolation requirement is applied to suppress background from multijet events where jet constituents are identiﬁed as muons. A cone of radius ∆R = 0.3 is constructed around the muon direction, and the isolation parameter is deﬁned as the scalar sum of the pT of all the additional reconstructed tracks within the cone, divided by the muon pT. The eﬃciency of this muon selection has been measured through a “tag-and-probe” method using Z bosons [43], and it has a negligible dependence on the pileup. In the high-mass ℓν+jet analysis, events must have exactly one isolated muon with pT > 53 GeV and \|η\| < 2.1. A looser pT requirement of 40 GeV is used for the low resonance mass range. JHEP03(2017)162 Electron candidates are required to have a match between energy deposited in the ECAL and momentum determined from the reconstructed track [52]. To suppress multi- jet background, electron candidates must pass stringent identiﬁcation and isolation crite- ria [53]. Those criteria include requirements on the geometrical matching between ECAL depositions and position of reconstructed tracks, the ratio of the energies deposited in the HCAL and ECAL, the distribution of the ECAL depositions, the impact parameters of the track, and the number of reconstructed hits in the silicon tracker. In the high-mass ℓν+jet analysis, we require exactly one electron with pT > 120 GeV and \|η\| < 2.5. A looser pT requirement of 45 GeV is used for the low resonance mass range. Reconstructed electrons must also be located outside of the transition region between the ECAL barrel and endcaps (1.44 < \|η\| < 1.57), because the reconstruction of an electron object in this region is not optimal. The missing transverse momentum pmiss T is deﬁned as the magnitude of the vector sum of the transverse momenta of the reconstructed PF objects. 4.2 Jet reconstruction Jets are clustered from the four-momenta of the particles reconstructed using the CMS PF algorithm, from the FastJet software package [47]. In the jet clustering procedure charged PF candidates not associated with the primary interaction vertex are excluded. Jets used for identifying the W and Z boson decays to qq are clustered using the anti-kT algorithm [48] with distance parameter R = 0.8 (“AK8 jets”). To identify b jets, the anti- kT jet clustering algorithm is used with R = 0.4 (“AK4 jets”), along with the inclusive combined secondary vertex b tagging algorithm [44, 49]. The chosen algorithm working point provides a misidentiﬁcation rate of ≈1% and eﬃciency of ≈70%. A correction based on the area of the jet projected on the front face of the calorimeter is used to take into account the extra energy clustered in jets due to neutral particles coming from pileup. Jet energy corrections are obtained from simulation and from dijet and photon+jet events in data, as discussed in ref. [50]. Additional quality criteria are applied to the jets to remove spurious jet-like features originating from isolated noise patterns in the calorimeters or the tracker. The eﬃciency of these requirements for signal events is above 99%. In the ℓν+jet channel, the AK8 and AK4 jets are required to be separated from any well-identiﬁed muon or electron by ∆R = √ (∆φ)2 + (∆η)2 > 0.8 and >0.3, respectively. All AK4 and AK8 jets must have pT > 30 GeV and >200 GeV, respectively, and \|η\| < 2.4 to be considered in the subsequent steps of the analysis. – 5 – 4.3 Final reconstruction and selection of leptons and missing transverse mo- mentum The value of pmiss T is modiﬁed to account for corrections to the energy scale of all the reconstructed AK4 jets in the event. More details on the pmiss T performance in CMS can be found in refs. [54, 55]. Requirements of pmiss T > 40 and > 80 GeV are applied, respectively, in the muon and electron channels in the ℓν+jet analysis. The threshold is higher in the electron channel to further suppress the larger background from multijet processes. Since the pmiss T calculation requires the detector to provide complete geometric coverage, events in data without fully operational HF calorimeter are not considered for the ℓν+jet channel. 4.4 The identiﬁcation of W/Z →qq using jet substructure The AK8 jets are used to reconstruct the W jet and Z jet candidates from their decays to highly boosted quark jets. To discriminate against multijet backgrounds, we exploit both the reconstructed jet mass, which is required to be close to the W or Z boson mass, and the two-prong jet substructure produced by the particle cascades of two high-pT quarks – 6 – that merge into one jet [21]. Jets that are identiﬁed as arising from the merged decay products of a V boson are hereafter referred to as “V jets”. As a ﬁrst step in exploring potential substructure, the jet constituents are subjected to a jet grooming algorithm that improves the resolution in the jet mass and reduces the eﬀect of pileup [56]. The goal of the algorithm is to recluster the jet constituents, while applying additional requirements that eliminate soft, large-angle QCD radiation that increases the jet mass relative to the initial V boson mass. Diﬀerent jet grooming algorithms have been explored at CMS, and their performance on jets in multijet processes has been studied in detail [56]. In this analysis, we use the jet pruning [57, 58] algorithm for the main analysis and the jet trimming algorithm [59] at the trigger level as well as for cross checks. Jet pruning reclusters each AK8 jet starting from all its original constituents, through the implementation of the Cambridge-Aachen (CA) algorithm [60, 61] to discard “soft” recombinations in each step of the iterative CA procedure. The pruned jet mass, mjet, is computed from the sum of the four-momenta of the constituents that are not removed by the pruning; it is then scaled by the same factor as that used to correct the original jet pT. The jet is considered as a V jet candidate if mjet falls in the range 65 < mjet < 105 GeV, which we deﬁne as the signal jet mass window. In the low-mass analysis, only W jet candidates are considered, thus the mass window applied is 65 < mjet < 95 GeV. JHEP03(2017)162 Additional discrimination against jets from gluon and single-quark hadronization is obtained from the quantity called N-subjettiness [62]. The constituents of the jet be- fore the pruning procedure are reclustered using the kT algorithm [60, 63], until N joint objects (subjets) remain in the iterative combination procedure of the algorithm. 4.4 The identiﬁcation of W/Z →qq using jet substructure Data-to-simulation scale factors for the eﬃciency of the τ21 selection used in the analyses, as extracted from top quark enriched data and from simulation. τ21 < 0.45 ⟨mjet⟩(GeV) σ (GeV) Data 84.6 ± 0.7 8.2 ± 0.7 Simulation 85.1 ± 0.2 7.8 ± 0.3 Table 2. The W jet mass peak position and resolution, as extracted from top quark enriched data and from simulation. These results are used to apply corrections in the V tagging procedure. τ21 < 0.45 ⟨mjet⟩(GeV) σ (GeV) Data 84.6 ± 0.7 8.2 ± 0.7 Simulation 85.1 ± 0.2 7.8 ± 0.3 Table 2. The W jet mass peak position and resolution, as extracted from top quark enriched data and from simulation. These results are used to apply corrections in the V tagging procedure. JHEP03(2017)162 Table 2. The W jet mass peak position and resolution, as extracted from top quark enriched da and from simulation. These results are used to apply corrections in the V tagging procedure. Table 2. The W jet mass peak position and resolution, as extracted from top quark enriched data and from simulation. These results are used to apply corrections in the V tagging procedure. method, the pruned jet mass distributions of events that pass and fail the τ21 selection are ﬁtted simultaneously to separate the W boson signal from the combinatorial components in the top quark enriched sample in both data and simulation. The scale factors are listed in table 1 and are used to correct the total signal eﬃciency and the VV background nor- malization predicted by the simulation. The uncertainties in the scale factors quoted for the τ21 selection include two systematic uncertainties. One comes from the modelling of the nearby jets and pT spectrum in tt MC events, obtained by comparing LO and NLO tt simulation. The other is due to the choice of the models used to ﬁt signal and back- ground. The quadratic sum of these systematic uncertainties is found to be smaller than half of the statistical uncertainty in the scale factor. An additional uncertainty is calcu- lated to account for the extrapolation of the scale factor from tt events with an average jet pT ≈200 GeV to higher momenta. This is estimated from the diﬀerence between pythia and HERWIG++ [64] showering models with resulting factors of 4.5% ln(pT/200 GeV) and 5.9% ln(pT/200 GeV) for τ21 < 0.6 and τ21 < 0.45, respectively. 4.4 The identiﬁcation of W/Z →qq using jet substructure The N-subjettiness, τN, is then deﬁned as τN = 1 d0 X k pT,k min(∆R1,k, ∆R2,k, . . . , ∆RN,k), (4.1) (4.1) where the index k runs over the PF constituents of the jet, and the distances ∆Rn,k are calculated relative to the axis of the n-th subjet. The normalization factor d0 is calculated as d0 = P k pT,kR0, setting R0 to the distance parameter used in the clustering of the original jet. The variable τN quantiﬁes the compatibility of the jet clustering with the hypothesis that exactly N subjets are present, with small values of τN indicating greater compatibility. The ratio between 2-subjettiness and 1-subjettiness, τ21 = τ2/τ1, is found to be a powerful discriminant between jets originating from hadronic V decays and from gluon and single-quark hadronization. Jets from W or Z decays in signal events are characterized by lower values of τ21 relative to SM backgrounds. We reject V jet candidates with τ21 > 0.75. The remaining events are further categorized according to their value of τ21 to enhance the sensitivity of the analysis, as summarized in table 1. Since data/simulation discrepancies in the jet substructure variables mjet and τ21 can bias the signal eﬃciency estimated from simulated samples, the modelling of signal ef- ﬁciency is cross-checked in a signal-free sample with jets having characteristics that are similar to those expected for a genuine signal. A sample of high-pT W bosons that decay to quarks, and are reconstructed as single AK8 jets, is studied in tt and single top quark events. Scale factors for the τ21 selection eﬃciency are extracted following ref. [21]. In this – 7 – τ21 selection Eﬃciency scale factor τ21 < 0.45 0.95 ± 0.06 0.45 < τ21 < 0.75 1.25 ± 0.32 τ21 < 0.6 1.01 ± 0.03 Table 1. Data-to-simulation scale factors for the eﬃciency of the τ21 selection used in the analyses, as extracted from top quark enriched data and from simulation. τ21 selection Eﬃciency scale factor τ21 < 0.45 0.95 ± 0.06 0.45 < τ21 < 0.75 1.25 ± 0.32 τ21 < 0.6 1.01 ± 0.03 Table 1. Data-to-simulation scale factors for the eﬃciency of the τ21 selection used in the analyses, as extracted from top quark enriched data and from simulation. Table 1. 4.6 Final event selection and categorization JHEP03(2017)162 After reconstructing the two vector bosons, we apply the ﬁnal criteria in the search. For all channels, any V boson candidate is required to have pT > 200 GeV. In addition, there are speciﬁc selection criteria chosen for the ℓν+jet and dijet analyses. For the ℓν+jet channel, we reject events with more than one well-identiﬁed muon or electron. We also require that the two V bosons from the decay of a massive resonance are approximately back-to-back: the ∆R between the lepton and the V jet is greater than 1.6; the ∆φ between the vector ⃗p miss T and the W jet, as well as between the W →ℓν and V jet candidates, are both greater than 2 radians. To further reduce the level of the tt background in the ℓν+jet channel, events are rejected if they contain one or more b-tagged AK4 jets. This veto preserves about 90% of the signal events. For the dijet analysis, we require the two AK8 jets to have \|∆ηjj\| < 1.3, while the dijet system invariant mass mjj must be above 1 TeV. To enhance the analysis sensitivity, events are categorized according to the character- istics of the V jet. For the dijet and high-mass ℓν+jet channels, the V jet is deemed a W or Z boson candidate if its pruned mass falls in the range 65–85 or 85–105 GeV. This leads to three categories for the dijet channel (WW, WZ, and ZZ), and two categories for the ℓν+jet channel (WW and WZ). For the low-mass ℓν+jet channel, only W jets are considered in the signal region 65 < mjet < 95 GeV. In addition, in the low- and high-mass ℓν+jet channels, V jets are selected to have τ21 ≤0.45 and ≤0.6, respectively. A tighter selection is required for the low-mass analysis as more background is expected in that mass range. In the dijet channel, we select “high-purity” (HP) and “low-purity” (LP) V jets by requiring τ21 ≤0.45 and 0.45 < τ21 < 0.75, respectively. Events are always required to have one HP V jet, and are divided into HP or LP events, depending on whether the other V jet is of high or low purity. Although the HP category dominates the total sensitivity of the analysis, the LP category is retained since for heavy resonances it can improve the signal eﬃciency with only moderate background contamination. 4.5 The reconstruction and identiﬁcation of W →ℓν In the ℓν+jet channel, identiﬁed muons and electrons are associated with W →ℓν can- didates. The pT of the undetected neutrino is assumed to be equal to the pmiss T . The longitudinal momentum of the neutrino (pz) is obtained by solving a quadratic equation that sets the ℓν invariant mass to the known W boson mass [65]. In the case of two real so- lutions, we choose the one with smaller pz; in the case of two complex solutions, we use their real part. The four-momentum of the neutrino is used to reconstruct the four-momentum of the W →ℓν candidate. 4.4 The identiﬁcation of W/Z →qq using jet substructure For the 0.45 < τ21 < 0.75 selection, this uncertainty is increased by the ratio of the uncertainties in the scale fac- tors shown in table 1 (0.32/0.06), and treated as anti-correlated with the uncertainty for τ21 < 0.45. The mean ⟨mjet⟩and resolution σ value of the Gaussian component of the ﬁtted W jet mass are also extracted to obtain corrections that are applied to the simulated pruned jet mass. The values are listed in table 2, where the quoted uncertainties are sta- tistical. The mass peak position is slightly shifted relative to the W boson mass because of the extra energy deposited in the jet cone from pileup, underlying event, and initial-state radiation not completely removed in the jet pruning procedure. For events with top quarks, additional energy contributions arise also from the possible presence of a b jet close to the W jet candidate. Because the kinematic properties of W jets and Z jets are very similar, the same corrections are also used when the V jet is assumed to arise from a Z boson. – 8 – 4.6 Final event selection and categorization The ﬁnal categorization is therefore based on two and four classes of events for the low- and high-mass ℓν+jet channels, respectively, depending on their lepton ﬂavor (muon or electron), and V jet mass category (W or Z). For the dijet analysis, categorization in V jet purity and mass category (WW, WZ, and ZZ) yields a total of 6 orthogonal classes of events. The two boson candidates are combined into a diboson candidate, with presence of signal then inferred from the observation of localized excesses in the mVV distribution. – 9 – Selection Value Lepton selections Electron pT > 120 (45) GeV \|η\| < 2.5 (except 1.44 < \|η\| < 1.57) Muon pT > 53 (40) GeV \|η\| < 2.1 Number of electrons exactly 1 Number of muons exactly 1 AK4 jet selections Jet pT pT > 30 GeV Jet η \|η\| < 2.4 Number of b-tagged AK4 jets 0 pmiss T selections pmiss T (electron channel) pmiss T > 80 GeV pmiss T (muon channel) pmiss T > 40 GeV Boson selections W →ℓν pT > 200 GeV V →qq (AK8 jet) pT > 200 GeV \|η\| < 2.4 Back-to-back topology ∆R(ℓ, Vqq) > 1.6 ∆φ(Vqq, pmiss T ) > 2 ∆φ(Vqq, Wℓν) > 2 Pruned jet mass 65 < mjet < 105 (95) GeV 2- to 1-subjettiness ratio τ21 < 0.60 (0.45) mjet categories (only for high-mass analysis) WW 65 < mjet < 85 GeV WZ 85 < mjet < 105 GeV able 3. Summary of the ﬁnal selections and categories for the ℓν+jet channel. The value ndicated in parentheses correspond to the low-mass analysis. JHEP03(2017)162 Boson selections Table 3. Summary of the ﬁnal selections and categories for the ℓν+jet channel. The valu indicated in parentheses correspond to the low-mass analysis. When several diboson resonance candidates are present in the same event, only the one with the highest pT V jet (ℓν+jet analyses) or the two highest mass V jets (dijet analysis) are retained. A summary of the ﬁnal event selections and categories is presented in table 3 for the ℓν+jet analyses and in table 4 for the dijet analysis. 4.6 Final event selection and categorization – 10 – Selection Value Boson selections V →qq (2 AK8 jets) pT > 200 GeV \|η\| < 2.4 Pruned jet mass 65 < mjet1, mjet2 < 105 GeV Topology \|∆ηjj\| < 1.3 Dijet invariant mass mjj > 1 TeV 2- to 1-subjettiness ratio τ21 < 0.75 mjet categories WW 65 < mjet1 < 85 GeV, 65 < mjet2 < 85 GeV WZ 65 < mjet1 < 85 GeV, 85 < mjet2 < 105 GeV ZZ 85 < mjet1 < 105 GeV, 85 < mjet2 < 105 GeV τ21 categories High-purity τ21, jet1 < 0.45, τ21, jet2 < 0.45 Low-purity τ21, jet1 < 0.45, 0.45 < τ21, jet2 < 0.75 Table 4. Summary of the ﬁnal selections and categories for the dijet analyses. JHEP03(2017)162 Table 4. Summary of the ﬁnal selections and categories for the dijet analyses. Table 4. Summary of the ﬁnal selections and categories for the dijet analyses. 5 Modeling of background and signal The mVV distribution observed in data is dominated by SM background processes where single quark or gluon jets are falsely identiﬁed as V jets. Depending on the ﬁnal state, the dominant processes are multijets (dijet channel) and inclusive W boson production (ℓν+jet channel). Subdominant backgrounds include tt, single top quark, and nonresonant diboson SM production. 5.1 Multijet background In the ℓν+jet channel, the multijet background is predicted to be negligible from MC simu- lation, whereas it represents the major contribution in the dijet analysis. For the latter, we assume that the SM background can be described by a smooth, parametrizable, monotoni- cally decreasing distribution. The search is performed by separately ﬁtting the background function to each search region and simultaneously adding resonant Breit-Wigner (BW) forms across all search regions to represent the signal. The background probability func- tion is deﬁned by empirical functional forms of 3 and 2 parameters, respectively: dN dmjj = P0(1 −mjj/√s)P1 (mjj/√s)P2 and dN dmjj = P0 (mjj/√s)P2 , (5.1) (5.1) where mjj is the dijet invariant mass (equivalent to the diboson candidate mass mVV for this channel), √s is the pp collision energy in the centre of mass, P0 is a normalization parameter, and P1 and P2 parametrize the shape of the mVV distribution. Starting from – 11 – τ21 selection Muon channel Electron channel τ21 < 0.6 (high-mass) 0.87 ± 0.04 0.83 ± 0.07 τ21 < 0.45 (low-mass) 0.85 ± 0.05 0.86 ± 0.08 Table 5. Data-to-simulation scale factors for tt and single top quark background processes, ex- tracted from the comparison between data and simulation in the top quark enriched control sample. τ21 selection Muon channel Electron channel τ21 < 0.6 (high-mass) 0.87 ± 0.04 0.83 ± 0.07 τ21 < 0.45 (low-mass) 0.85 ± 0.05 0.86 ± 0.08 Table 5. Data-to-simulation scale factors for tt and single top quark background processes, ex- tracted from the comparison between data and simulation in the top quark enriched control sample. Table 5. Data-to-simulation scale factors for tt and single top quark background processes, ex- tracted from the comparison between data and simulation in the top quark enriched control sample. Table 5. Data-to-simulation scale factors for tt and single top quark background processes, ex- tracted from the comparison between data and simulation in the top quark enriched control sample. Table 5. Data-to-simulation scale factors for tt and single top quark background processes, ex- tracted from the comparison between data and simulation in the top quark enriched control sample. the two-parameter functional form, a Fisher F-test is used to check at 10% conﬁdence level (CL) if additional parameters are needed to model the background distribution. 5.2 Top quark production The backgrounds from tt and single top quark production in the ℓν+jet channel are es- timated from data-based correction factors in the normalization of the simulation. A top quark enriched control sample is selected by applying all the analysis requirements in ℓν+jet events except that the b jet veto is inverted by requiring, instead, at least one b-tagged AK4 jet in the event. From the comparison between data and simulation, normalization correction factors for tt and single top quark background processes are evaluated in the pruned jet mass regions 65 < mjet < 105 GeV and 65 < mjet < 95 GeV, for the electron and muon channels, and for the low- and high-mass selections, separately. The scale factors, summarized in table 5, include both the W boson signal and the combinatorial components mainly due to events where the extra b jet from the top quark decay is in the proximity of the W, and are used to correct the normalization of the tt and single top quark simu- lated background predictions in the signal regions. The mjet distribution in the top quark enriched sample is shown in the right plot of ﬁgure 2, while the left plot shows the τ21 distribution. The mjet distribution shows a clear peak for events with a W boson decaying to hadrons, including the combinatorial background. 5.1 Multijet background For the WW categories and the WZ HP category, the two-parameter form is found to describe the data spectrum suﬃciently well, while for all other channels the three-parameter functional form is preferable. Alternative parametrizations and functions with up to ﬁve parameters are also studied as a cross-check. JHEP03(2017)162 The binning chosen for the ﬁt reﬂects the detector resolution. The ﬁt range is chosen to start where the trigger eﬃciency reaches its plateau, as this minimizes bias from trigger ineﬃciency, and to extend to the bin after the highest mjj mass point. The results are shown in ﬁgure 1. The solid curve represents the maximum likelihood ﬁt to the data, ﬁxing the number of expected signal events to zero, while the bottom panels show the corresponding pull distributions, quantifying the agreement between the background-only ﬁt and the data. The expected contributions from bulk graviton and W′ resonances with a mass of 2 TeV, scaled to their corresponding cross sections, are given by the dashed curves. 5.3 The W+jets background The W+jets background in the ℓν+jet channel is estimated through the α ratio method. 5.3 The W+jets background This method assumes that the correlation between mjet and mVV for the dominant W+jets – 12 – Events / ( 0.1 TeV ) 1 10 2 10 3 10 4 10 data 2 parameter fit =0.5 k~ WW, → G(2 TeV) WW, high-purity > 200 GeV T \| < 2.4, p η\| \| < 1.3 jj η ∆ > 1 TeV, \| jj m (13 TeV) -1 2.7 fb CMS Dijet invariant mass (TeV) 1 1.5 2 2.5 3 3.5 data σ Data-Fit -2 0 2 Events / ( 0.1 TeV ) 1 10 2 10 3 10 4 10 data 2 parameter fit =0.5 k~ WW, → G(2 TeV) WW, low-purity > 200 GeV T \| < 2.4, p η\| \| < 1.3 jj η ∆ > 1 TeV, \| jj m (13 TeV) -1 2.7 fb CMS Dijet invariant mass (TeV) 1 1.5 2 2.5 3 3.5 data σ Data-Fit -2 0 2 Events / ( 0.1 TeV ) 1 10 2 10 3 10 4 10 data 2 parameter fit =3) V (g B WZ, HVT → W'(2 TeV) WZ, high-purity > 200 GeV T \| < 2.4, p η\| \| < 1.3 jj η ∆ > 1 TeV, \| jj m (13 TeV) -1 2.7 fb CMS Dijet invariant mass (TeV) 1 1.5 2 2.5 3 3.5 data σ Data-Fit -2 0 2 Events / ( 0.1 TeV ) 1 10 2 10 3 10 4 10 data 3 parameter fit =3) V (g B WZ, HVT → W'(2 TeV) WZ, low-purity > 200 GeV T \| < 2.4, p η\| \| < 1.3 jj η ∆ > 1 TeV, \| jj m (13 TeV) -1 2.7 fb CMS Dijet invariant mass (TeV) 1 1.5 2 2.5 3 3.5 data σ Data-Fit -2 0 2 Events / ( 0.1 TeV ) 1 10 2 10 3 10 4 10 data 3 parameter fit (default) 2 parameter fit (alt.) =0.5 k~ ZZ, → G(2 TeV) ZZ, high-purity > 200 GeV T \| < 2.4, p η\| \| < 1.3 jj η ∆ > 1 TeV, \| jj m (13 TeV) -1 2.7 fb CMS Dijet invariant mass (TeV) 1 1.5 2 2.5 3 3.5 data σ Data-Fit -2 0 2 Events / ( 0.1 TeV ) 1 10 2 10 3 10 4 10 data 3 parameter fit =0.5 k~ ZZ, → G(2 TeV) ZZ, low-purity > 200 GeV T \| < 2.4, p η\| \| < 1.3 jj η ∆ > 1 TeV, \| jj m (13 TeV) -1 2.7 fb CMS Dijet invariant mass (TeV) 1 1.5 2 2.5 3 3.5 data σ Data-Fit -2 0 2 Figure 1. 5.3 The W+jets background Events / ( 0.1 TeV ) 1 10 2 10 3 10 4 10 data 2 parameter fit =0.5 k~ WW, → G(2 TeV) WW, low-purity > 200 GeV T \| < 2.4, p η\| \| < 1.3 jj η ∆ > 1 TeV, \| jj m (13 TeV) -1 2.7 fb CMS Dijet invariant mass (TeV) 1 1.5 2 2.5 3 3.5 data σ Data-Fit -2 0 2 Events / ( 0.1 TeV ) 1 10 2 10 3 10 4 10 data 2 parameter fit =0.5 k~ WW, → G(2 TeV) WW, high-purity > 200 GeV T \| < 2.4, p η\| \| < 1.3 jj η ∆ > 1 TeV, \| jj m (13 TeV) -1 2.7 fb CMS Dijet invariant mass (TeV) 1 1.5 2 2.5 3 3.5 data σ Data-Fit -2 0 2 JHEP03(2017)162 j ( ) Events / ( 0.1 TeV ) 1 10 2 10 3 10 4 10 data 3 parameter fit =3) V (g B WZ, HVT → W'(2 TeV) WZ, low-purity > 200 GeV T \| < 2.4, p η\| \| < 1.3 jj η ∆ > 1 TeV, \| jj m (13 TeV) -1 2.7 fb CMS Dijet invariant mass (TeV) 1 1.5 2 2.5 3 3.5 data σ Data-Fit -2 0 2 j ( ) Events / ( 0.1 TeV ) 1 10 2 10 3 10 4 10 data 2 parameter fit =3) V (g B WZ, HVT → W'(2 TeV) WZ, high-purity > 200 GeV T \| < 2.4, p η\| \| < 1.3 jj η ∆ > 1 TeV, \| jj m (13 TeV) -1 2.7 fb CMS Dijet invariant mass (TeV) 1 1.5 2 2.5 3 3.5 data σ Data-Fit -2 0 2 j ( ) Events / ( 0.1 TeV ) 1 10 2 10 3 10 4 10 data 3 parameter fit =0.5 k~ ZZ, → G(2 TeV) ZZ, low-purity > 200 GeV T \| < 2.4, p η\| \| < 1.3 jj η ∆ > 1 TeV, \| jj m (13 TeV) -1 2.7 fb CMS Dijet invariant mass (TeV) 1 1.5 2 2.5 3 3.5 data σ Data-Fit -2 0 2 j ( ) Events / ( 0.1 TeV ) 1 10 2 10 3 10 4 10 data 3 parameter fit (default) 2 parameter fit (alt.) =0.5 k~ ZZ, → G(2 TeV) ZZ, high-purity > 200 GeV T \| < 2.4, p η\| \| < 1.3 jj η ∆ > 1 TeV, \| jj m (13 TeV) -1 2.7 fb CMS Dijet invariant mass (TeV) 1 1.5 2 2.5 3 3.5 data σ Data-Fit -2 0 2 Figure 1. 5.3 The W+jets background Final mjj distributions for the dijet analysis in six signal regions. The high-purity (on the left) and the low-purity (on the right) categories are shown for the WW (top row), WZ (central row), and ZZ (bottom row) mjet regions. The solid curve represents a background-only ﬁt to the data distribution, where the ﬁlled red area corresponds to the ±1 standard deviation statistical un- certainties of the ﬁt. The data are represented by the black points. For the ZZ high-purity category (bottom left), we also show the background-only ﬁt using the two-parameter functional form (blue solid line), for comparison. Signal benchmarks for a mass of 2 TeV are also shown with black dashed lines. In the lower panel of each plot, the bin-by-bin ﬁt residuals, (Ndata −Nﬁt)/σdata, are shown. 5.3 The W+jets background Final mjj distributions for the dijet analysis in six signal regions. The high-purity (on the left) and the low-purity (on the right) categories are shown for the WW (top row), WZ (central row), and ZZ (bottom row) mjet regions. The solid curve represents a background-only ﬁt to the data distribution, where the ﬁlled red area corresponds to the ±1 standard deviation statistical un- certainties of the ﬁt. The data are represented by the black points. For the ZZ high-purity category (bottom left), we also show the background-only ﬁt using the two-parameter functional form (blue solid line), for comparison. Signal benchmarks for a mass of 2 TeV are also shown with black dashed lines. In the lower panel of each plot, the bin-by-bin ﬁt residuals, (Ndata −Nﬁt)/σdata, are shown. – 13 – Events / ( 0.04 ) 50 100 150 200 250 300 350 ν µ data tt Single t VV W+jets MC stat. (13 TeV) -1 2.3 fb CMS 21 τ N-subjettiness ratio 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 MC Data 0.5 1 1.5 Events / ( 5 GeV ) 50 100 150 200 250 300 350 ν µ data tt Single t VV W+jets MC stat. (13 TeV) -1 2.3 fb CMS Pruned jet mass (GeV) 40 60 80 100 120 140 MC Data 0.5 1 1.5 Figure 2. Distributions in N-subjettiness ratio τ21 (left) and pruned mjet (right) from the top quark enriched control sample in the muon channel. The tt background is rescaled such that the total number of background events matches the number of events in data. In the lower panel of each plot, the ratio between data and simulation is shown together with the statistical uncertainty in the simulation normalized by its central value. Events / ( 0.04 ) 50 100 150 200 250 300 350 ν µ data tt Single t VV W+jets MC stat. (13 TeV) -1 2.3 fb CMS 21 τ N-subjettiness ratio 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 MC Data 0.5 1 1.5 Events / ( 5 GeV ) 50 100 150 200 250 300 350 ν µ data tt Single t VV W+jets MC stat. (13 TeV) -1 2.3 fb CMS Pruned jet mass (GeV) 40 60 80 100 120 140 MC Data 0.5 1 1.5 JHEP03(2017)162 21 τ N-subjettiness ratio Figure 2. 5.3 The W+jets background Distributions in N-subjettiness ratio τ21 (left) and pruned mjet (right) from the top quark enriched control sample in the muon channel. The tt background is rescaled such that the total number of background events matches the number of events in data. In the lower panel of each plot, the ratio between data and simulation is shown together with the statistical uncertainty in the simulation normalized by its central value. background can be adequately modelled by simulation. A signal-depleted control region (sideband) is deﬁned by requiring the mass of the V jet to lie below or above the nominal selection; the mVV distribution observed in this region is then extrapolated to the nominal region through a transfer function estimated from simulation. Other minor sources of background, such as tt, single top quark, and SM diboson production, are estimated from simulation after applying correction factors based on control regions in data, as described in sections 4.4 and 5.2. The sideband region is deﬁned around the jet mass window described in section 4. The lower and upper sidebands correspond to the mjet ranges 40–65 and 135– 150 GeV, respectively. The Higgs boson mass region, deﬁned by the range 105–135 GeV, corresponds to the signal region of searches for diboson in ﬁnal states with highly Lorentz- boosted Higgs bosons [66], and is therefore not used to estimate the background. The overall normalization of the W+jets background in the signal region is determined from a ﬁt to the mjet distribution in the lower and upper sidebands of the data. The analyt- ical form of the ﬁtting function is chosen from simulation studies, as are the contributions from minor backgrounds. Figure 3 shows the result of this ﬁt for the low- and high-mass ℓν+jet channels. 5.3 The W+jets background The form of the mVV distribution for the W+jets background in the signal region (SR) is determined from the lower mjet sideband (SB), through the transfer function αMC(mVV) obtained from the W+jets simulation, and deﬁned as: αMC(mVV) = F W+jets MC,SR (mVV) F W+jets MC,SB (mVV) , (5.2) (5.2) – 14 – Pruned jet mass (GeV) 40 60 80 100 120 140 data σ Data-Fit -2 0 2 Events / ( 5 GeV ) 50 100 150 200 250 300 350 signal region → ← → Higgs ← ν µ data W+jets WW/WZ tt Single t Uncertainty +jet ν l Low-mass (13 TeV) -1 2.3 fb CMS Pruned jet mass (GeV) 40 60 80 100 120 140 data σ Data-Fit -2 0 2 Events / ( 5 GeV ) 50 100 150 200 250 300 350 → signal region ← → Higgs ← WW enriched WZ enriched ν µ data W+jets WW/WZ tt Single t Uncertainty +jet ν l High-mass (13 TeV) -1 2.3 fb CMS Pruned jet mass (GeV) 40 60 80 100 120 140 data σ Data-Fit -2 0 2 Events / ( 5 GeV ) 50 100 150 200 250 300 350 signal region → ← → Higgs ← ν µ data W+jets WW/WZ tt Single t Uncertainty +jet ν l Low-mass (13 TeV) -1 2.3 fb CMS Figure 3. Distributions of the pruned jet mass mjet in the ℓν+jet high-mass (left) and low-mass (right) analyses in the muon channel. All selections are applied except the requirement on mjet signal window. Data are shown as black points. The signal regions and mjet categories of the analyses are indicated by the vertical dotted lines. The shaded mjet region 105–135 GeV is not used in these analyses. In the lower panel of each plot, the bin-by-bin ﬁt residuals, (Ndata −Nﬁt)/σdata, are shown together with the uncertainty band of the ﬁt normalized by the statistical uncertainty of data points, σdata. Pruned jet mass (GeV) 40 60 80 100 120 140 data σ Data-Fit -2 0 2 Events / ( 5 GeV ) 50 100 150 200 250 300 350 → signal region ← → Higgs ← WW enriched WZ enriched ν µ data W+jets WW/WZ tt Single t Uncertainty +jet ν l High-mass (13 TeV) -1 2.3 fb CMS JHEP03(2017)162 Figure 3. 5.3 The W+jets background Distributions of the pruned jet mass mjet in the ℓν+jet high-mass (left) and low-mass (right) analyses in the muon channel. All selections are applied except the requirement on mjet signal window. Data are shown as black points. The signal regions and mjet categories of the analyses are indicated by the vertical dotted lines. The shaded mjet region 105–135 GeV is not used in these analyses. In the lower panel of each plot, the bin-by-bin ﬁt residuals, (Ndata −Nﬁt)/σdata, are shown together with the uncertainty band of the ﬁt normalized by the statistical uncertainty of data points, σdata. where F(mVV) is the probability density function used to describe the mVV spectrum in diﬀerent regions. The upper mjet sideband is not considered in this ﬁt since the expected mVV distribution is diﬀerent here, displaying a threshold eﬀect not present in the lower sideband and signal regions. The adopted parameterization for the mVV spectrum in both SR and SB regions is of the form f(x) ∝ec0x+c1/x, which is found to adequately describe the simulation. Tests are performed with alternative functional forms, and the prediction for the backgrounds is found to agree with the one of the default function within the uncertainties. The mVV distribution observed in the lower sideband region is corrected for the pres- ence of minor backgrounds to have an estimate of the W+jets contribution in the control region of the data, F W+jets DATA,SB(mVV). The W+jets background distribution in the signal re- gion is then obtained by rescaling F W+jets DATA,SB(mVV) by αMC(mVV). The minor backgrounds are then added to the W+jets background to obtain the total SM prediction in the sig- nal region. Figure 4 shows the ﬁnal spectrum in mVV for events in all categories for the low- and high-mass analyses. The observed data and the predicted background agree. The highest mass events in the ℓν+jet channel are at mVV = 2.95 and 3.15 TeV for the muon and electron categories, respectively. 5.3 The W+jets background Signal benchmarks for a mass of 2 TeV (0.75 TeV) are also shown with black dashed lines for the upper (lower) plots. In the lower panel of each plot are the bin-by-bin ﬁt residuals, (Ndata −Nﬁt)/σdata, shown together with the uncertainty band of the ﬁt normalized by the statistical uncertainty of data, σdata. Figure 4. (Upper plots) Final mVV distributions for data and expected backgrounds in the high- mass analysis obtained from the combined muon and electron channels in the WW-enriched (left) and WZ-enriched (right) signal regions. (Lower plot) Final mVV distributions for data and expected backgrounds in the signal region of the low-mass analysis obtained from the combined muon and electron channels. In each plot the solid curve represents the background estimation provided by the α ratio method. The hatched band includes both statistical and systematic uncertainties. The data are shown as black points. Signal benchmarks for a mass of 2 TeV (0.75 TeV) are also shown with black dashed lines for the upper (lower) plots. In the lower panel of each plot are the bin-by-bin ﬁt residuals, (Ndata −Nﬁt)/σdata, shown together with the uncertainty band of the ﬁt normalized by the statistical uncertainty of data, σdata. 5.3 The W+jets background – 15 – JHEP03(2017)162 (TeV) VV m 1 1.5 2 2.5 3 3.5 4 data σ Data-Fit -2 0 2 Events / ( 0.1 TeV ) -2 10 -1 10 1 10 2 10 3 10 4 10 5 10 ν data l W+jets WW/WZ tt Single t Uncertainty 100) × =0.5 ( k~ G(2 TeV), WW enriched (13 TeV) -1 2.3 fb CMS (TeV) VV m 1 1.5 2 2.5 3 3.5 4 data σ Data-Fit -2 0 2 Events / ( 0.1 TeV ) -2 10 -1 10 1 10 2 10 3 10 4 10 5 10 ν data l W+jets WW/WZ tt Single t Uncertainty =3) V (g B W'(2 TeV), HVT WZ enriched (13 TeV) -1 2.3 fb CMS (TeV) VV m 0.6 0.7 0.8 0.9 1 1.1 1.2 1.3 1.4 1.5 data σ Data-Fit -2 0 2 Events / ( 0.05 TeV ) 100 200 300 400 500 600 700 ν data l W+jets WW/WZ tt Single t Uncertainty 20) × =0.5 ( k~ G(0.75 TeV), (13 TeV) -1 2.3 fb CMS Figure 4. (Upper plots) Final mVV distributions for data and expected backgrounds in the high- mass analysis obtained from the combined muon and electron channels in the WW-enriched (left) and WZ-enriched (right) signal regions. (Lower plot) Final mVV distributions for data and expected backgrounds in the signal region of the low-mass analysis obtained from the combined muon and electron channels. In each plot the solid curve represents the background estimation provided by the α ratio method. The hatched band includes both statistical and systematic uncertainties. The data are shown as black points. Signal benchmarks for a mass of 2 TeV (0.75 TeV) are also shown with black dashed lines for the upper (lower) plots. In the lower panel of each plot are the bin-by-bin ﬁt residuals, (Ndata −Nﬁt)/σdata, shown together with the uncertainty band of the ﬁt normalized by the statistical uncertainty of data, σdata. 5.3 The W+jets background (TeV) VV m 1 1.5 2 2.5 3 3.5 4 data σ Data-Fit -2 0 2 Events / ( 0.1 TeV ) -2 10 -1 10 1 10 2 10 3 10 4 10 5 10 ν data l W+jets WW/WZ tt Single t Uncertainty 100) × =0.5 ( k~ G(2 TeV), WW enriched (13 TeV) -1 2.3 fb CMS (TeV) VV m 1 1.5 2 2.5 3 3.5 4 data σ Data-Fit -2 0 2 Events / ( 0.1 TeV ) -2 10 -1 10 1 10 2 10 3 10 4 10 5 10 ν data l W+jets WW/WZ tt Single t Uncertainty =3) V (g B W'(2 TeV), HVT WZ enriched (13 TeV) -1 2.3 fb CMS (TeV) VV m 1 1.5 2 2.5 3 3.5 4 data σ Data-Fit -2 0 2 Events / ( 0.1 TeV ) -2 10 -1 10 1 10 2 10 3 10 4 10 5 10 ν data l W+jets WW/WZ tt Single t Uncertainty =3) V (g B W'(2 TeV), HVT WZ enriched (13 TeV) -1 2.3 fb CMS (TeV) VV m 1 1.5 2 2.5 3 3.5 4 data σ Data-Fit -2 0 2 Events / ( 0.1 TeV ) -2 10 -1 10 1 10 2 10 3 10 4 10 5 10 ν data l W+jets WW/WZ tt Single t Uncertainty 100) × =0.5 ( k~ G(2 TeV), WW enriched (13 TeV) -1 2.3 fb CMS JHEP03(2017)162 (TeV) VV m 0.6 0.7 0.8 0.9 1 1.1 1.2 1.3 1.4 1.5 data σ Data-Fit -2 0 2 Events / ( 0.05 TeV ) 100 200 300 400 500 600 700 ν data l W+jets WW/WZ tt Single t Uncertainty 20) × =0.5 ( k~ G(0.75 TeV), (13 TeV) -1 2.3 fb CMS Figure 4. (Upper plots) Final mVV distributions for data and expected backgrounds in the high- mass analysis obtained from the combined muon and electron channels in the WW-enriched (left) and WZ-enriched (right) signal regions. (Lower plot) Final mVV distributions for data and expected backgrounds in the signal region of the low-mass analysis obtained from the combined muon and electron channels. In each plot the solid curve represents the background estimation provided by the α ratio method. The hatched band includes both statistical and systematic uncertainties. The data are shown as black points. 5.4 Signal modelling Figure 5 shows the simulated mjj and mℓν+jet distributions for diﬀerent resonance masses from 0.8 to 4.0 TeV. The experimental resolution for the dijet channel is around 4%, while it ranges from 6% at 1 TeV to 4% at 4 TeV in the ℓν+jet channel. We adopt an analytical – 16 – Dijet invariant mass (TeV) 1 1.5 2 2.5 3 3.5 4 4.5 5 Arbitrary scale 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 WZ (MADGRAPH) → W' WW (MADGRAPH) → Bulk G ZZ (MADGRAPH) → Bulk G 13 TeV CMS Simulation (TeV) +jet νl m 1 1.5 2 2.5 3 3.5 4 4.5 Arbitrary scale 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 WZ (MADGRAPH) → W' WW (MADGRAPH) → bulk G WW (MADGRAPH) → Z' 13 TeV CMS Simulation Figure 5. Dijet invariant mass (left) and mℓν+jet (right) distributions expected for diﬀerent signal mass hypotheses. (TeV) +jet νl m 1 1.5 2 2.5 3 3.5 4 4.5 Arbitrary scale 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 WZ (MADGRAPH) → W' WW (MADGRAPH) → bulk G WW (MADGRAPH) → Z' 13 TeV CMS Simulation Dijet invariant mass (TeV) 1 1.5 2 2.5 3 3.5 4 4.5 5 Arbitrary scale 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 WZ (MADGRAPH) → W' WW (MADGRAPH) → Bulk G ZZ (MADGRAPH) → Bulk G 13 TeV CMS Simulation JHEP03(2017)162 Figure 5. Dijet invariant mass (left) and mℓν+jet (right) distributions expected for diﬀerent signal mass hypotheses. description of the signal, choosing a double-sided Crystal Ball (CB) function [67] (i.e. a Gaussian core with power law tails on both sides) to describe the simulated resonance distributions. A linear interpolation between a set of reference distributions (corresponding to masses of 0.6, 0.7, 0.8, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0, 2.5, 3.0, 3.5, and 4.0 TeV) is used to estimate the expected distributions for intermediate values of resonance mass. Table 6 summarizes the overall event-selection eﬃciency for our chosen analysis channels and signal models. All channels are used in the statistical analysis of each signal. 6.1 Systematic uncertainties in the background estimation For the dijet analysis, the background estimation is obtained from a ﬁt to the data. As such, the only relevant uncertainty is the statistical one as represented by the covariance matrix of the ﬁt to the dijet function. Diﬀerent parameterizations of the ﬁtting function have been studied, and the diﬀerences observed are well within the bounds of the aforementioned uncertainty and are assumed to pose no additional contribution. For the ℓν+jet analyses, uncertainties in both the distribution and normalization of the background prediction can be important. The uncertainty in the distribution is dominated by the statistical uncertainties in the simultaneous ﬁts to the data of the sideband region, and the simulation in signal and sideband regions. An eﬀect of almost equal magnitude is due to the uncertainties in the modelling of the transfer function α(mVV) between the sideband and the signal region. 6.1 Systematic uncertainties in the background estimation The uncertainty in the normalization of the background has three sources: the W+jets component, dominated by the statistical uncertainty of the events in the pruned jet mass sideband, varying from 5 to 9%; the tt/single top quark component, dominated by the scale factor obtained from the top quark enriched control – 17 – Eﬃciency (%) Dijet channel ℓν+jet channel Signal Mass (TeV) WW WZ ZZ WW WZ HP LP HP LP HP LP e µ e µ Gbulk →WW 0.75 — — — — — — 4.4 5.3 — — Gbulk →WW 1.2 4.9 5.6 3.6 3.9 0.6 0.6 5.7 7.4 1.7 2.1 Gbulk →WW 2.0 6.5 9.1 2.1 2.9 0.2 0.3 7.3 8.0 1.4 1.5 Gbulk →WW 3.0 4.9 7.8 2.3 3.3 0.3 0.3 7.0 7.5 1.5 1.7 Gbulk →WW 4.0 4.2 8.0 2.8 3.9 0.3 0.6 7.0 7.0 2.0 1.9 Gbulk →ZZ 1.2 1.1 1.2 5.3 5.1 6.1 4.6 — — — — Gbulk →ZZ 2.0 1.3 2.3 5.0 6.7 4.7 4.5 — — — — Gbulk →ZZ 3.0 1.1 2.5 4.3 7.2 3.8 4.5 — — — — Gbulk →ZZ 4.0 0.9 2.7 3.7 7.2 3.7 4.3 — — — — HVT W ′ →WZ 0.75 — — — — — — 1.3 1.6 — — HVT W ′ →WZ 1.2 2.7 3.0 7.2 6.8 1.5 1.4 1.2 1.6 2.8 3.4 HVT W ′ →WZ 2.0 3.0 4.7 6.0 6.7 0.8 0.8 1.8 2.0 3.0 3.3 HVT W ′ →WZ 3.0 2.3 4.5 5.0 6.8 1.0 0.8 1.9 2.0 3.1 3.2 HVT W ′ →WZ 4.0 1.9 4.2 4.2 6.4 1.0 1.2 1.9 2.0 3.1 3.0 HVT Z′ →WW 0.75 — — — — — — 4.1 5.1 — — HVT Z′ →WW 1.2 7.2 7.6 3.3 3.6 0.4 0.4 6.0 7.7 1.6 2.0 HVT Z′ →WW 2.0 6.1 8.1 2.0 2.3 0.1 0.2 7.9 8.8 1.3 1.5 HVT Z′ →WW 3.0 4.7 8.0 2.1 2.8 0.3 0.2 7.5 8.1 1.6 1.5 HVT Z′ →WW 4.0 3.8 6.7 2.1 3.0 0.3 0.3 7.4 7.6 1.9 1.9 Table 6. Summary of signal eﬃciencies for all analysis channels and all signal models. The quoted eﬃciencies are in percent, and include the branching fractions of the two vector bosons to the ﬁnal state of the analysis channel, eﬀects from detector acceptance, as well as reconstruction and selection eﬃciencies. 6.1 Systematic uncertainties in the background estimation Values are not indicated for categories and masses where the analysis channel has no sensitivity. JHEP03(2017)162 Table 6. Summary of signal eﬃciencies for all analysis channels and all signal models. The quoted eﬃciencies are in percent, and include the branching fractions of the two vector bosons to the ﬁnal state of the analysis channel, eﬀects from detector acceptance, as well as reconstruction and selection eﬃciencies. Values are not indicated for categories and masses where the analysis channel has no sensitivity. region, amounting to about 5–7% and 8% in the muon and electron channels, respectively; and the diboson component, dominated by the V tagging uncertainty, which varies in the range of 3–25%. region, amounting to about 5–7% and 8% in the muon and electron channels, respectively; and the diboson component, dominated by the V tagging uncertainty, which varies in the range of 3–25%. 6.2 Systematic uncertainties in the signal prediction The dominant uncertainty in the signal selection eﬃciency arises from uncertainties in data- to-simulation scale factors for the V tagging eﬃciency derived from a top quark enriched control sample, as described in section 4.4. The normalization uncertainties are summarized in tables 7 and 8 for the dijet and ℓν+jet channels, respectively. Uncertainties in the reconstruction of jets aﬀect both the signal eﬃciency and the distribution in the reconstructed resonance mass. The four-momenta of the reconstructed jets are rescaled or smeared according to the uncertainties in the respective jet energy scale or resolution. The selection eﬃciencies are recalculated on these modiﬁed events, – 18 – with the resulting changes taken as systematic uncertainties that depend on the resonance mass. The induced changes on the reconstructed resonances are propagated as uncertainties in the peak position and width of the Gaussian core. In addition, the induced relative migration among V jet mass categories is evaluated, and found not to aﬀect the overall signal eﬃciency. The correlations in these uncertainties between the diﬀerent categories are taken into account. The uncertainty in the lepton energy scale is correlated with the obtained signal eﬃ- ciency. Changes in lepton energy are propagated to the reconstructed pmiss T , and through the entire analysis. The relative change in the number of selected signal events is taken as a systematic uncertainty in the signal normalization. For both lepton ﬂavors, the uncertain- ties are smaller than 1%, and are uncorrelated for diﬀerent lepton ﬂavors, but correlated for diﬀerent pruned jet mass and τ21 categories. In addition, the induced change in peak position and its width are added as systematic uncertainties in the distribution of the signal. Again, for both lepton ﬂavors, the uncertainties are below 1%. JHEP03(2017)162 The systematic uncertainties in the lepton trigger, identiﬁcation, and isolation eﬃcien- cies are obtained using a tag-and-probe method in Z →ℓℓevents [43], and are used only for the ℓν+jet channel. An uncertainty of 1–3% is assigned to the trigger eﬃciency for both lepton ﬂavors, depending on the lepton pT and η. For lepton identiﬁcation and isolation eﬃciency, the systematic uncertainty is estimated to be 1–2% for the muon and 3% for electron ﬂavors. The 2.7% uncertainty in the integrated luminosity [45] applies to the normalization of signal events. 6.2 Systematic uncertainties in the signal prediction Uncertainties in the signal yield due to the choice of PDFs and the values chosen for the factorization (µf) and renormalization (µr) scales are also taken into account. The PDF uncertainties are evaluated using the NNPDF 3.0 [36] PDFs. The uncertainty related to the choice of µf and µr scales is evaluated following the proposal in refs. [68, 69] by varying the default choice of scales in the following 6 combinations of factors: (µf, µr) × (1/2, 1/2), (1/2, 1), (1, 1/2), (2, 2), (2, 1), and (1, 2). The uncertainty in the signal cross section from the choice of PDFs and of factorization and renormalization scales ranges from 4 to 77%, and from 1 to 22%, respectively, depending on the resonance mass, particle type and its production mechanism. These uncertainties are fully correlated among the ℓν+jet and dijet channels. Tables 7 and 8 summarize the systematic uncertainties in the dijet and ℓν+jet channels, respectively. 7 Statistical interpretation The mVV distribution observed in data and the SM background prediction are compared to check for the presence of a new resonance decaying to vector bosons. No bins with an excess with signiﬁcance larger than three standard deviations are observed. We set upper limits on the production cross section of such resonances by combining the event categories of the dijet and ℓν+jet analyses. We follow the asymptotic approximation [70] of the CLS criterion described in refs. [71, 72]. The limits computed following this approach are found to agree with the results obtained using the modiﬁed frequentist prescription [71, 72]. For each – 19 – Source Relevant quantity HP uncertainty (%) LP uncertainty (%) Jet energy scale Resonance shape 2 2 Jet energy resolution Resonance shape 10 10 Jet energy and mjet scale Signal yield 0.1–4 Jet energy and mjet resolution Signal yield 0.1–1.4 Pileup Signal yield 2 Integrated luminosity Signal yield 2 PDFs (W ′) Signal yield 4–19 PDFs (Z′) Signal yield 4–13 PDFs (Gbulk) Signal yield 9–77 Scales (W ′) Signal yield 1–14 Scales (Z′) Signal yield 1–13 Scales (Gbulk) Signal yield 8–22 Jet energy and mjet scale Migration 1–50 V tagging τ21 Migration 14 21 V tagging pT-dependence Migration 7–14 5–11 Table 7. Summary of the systematic uncertainties in the contribution from signal in the dijet anal- ysis and their impact on the event yield in the signal region and on the reconstructed distribution in mVV (mean and width). The last three uncertainties result in migrations between event categories, but do not aﬀect the overall signal eﬃciency. JHEP03(2017)162 Table 7. Summary of the systematic uncertainties in the contribution from signal in the dijet anal- ysis and their impact on the event yield in the signal region and on the reconstructed distribution in mVV (mean and width). The last three uncertainties result in migrations between event categories, but do not aﬀect the overall signal eﬃciency. Table 7. Summary of the systematic uncertainties in the contribution from signal in the dijet anal- ysis and their impact on the event yield in the signal region and on the reconstructed distribution in mVV (mean and width). The last three uncertainties result in migrations between event categories, but do not aﬀect the overall signal eﬃciency. channel and each signal hypothesis a likelihood function is built from the reconstructed mVV mass distribution observed in data, the background prediction, and the signal resonance shape. 7 Statistical interpretation A maximum-likelihood ﬁt to the data is then performed to obtain the best estimate of the signal cross section. Systematic uncertainties are proﬁled [73] as log-normal nuisance parameters in the statistical interpretation, and for each possible value of the ﬁtted signal cross section they are all reﬁtted to maximize the likelihood. 7.1 Limits on narrow-width resonance models Exclusion limits are set in the context of the bulk graviton model and of the HVT Models A and B, under the assumption of a natural width negligible compared to the experimen- tal resolution (narrow-width approximation). To maximize the sensitivity of the search we combine the results from all the analysis channels in each of the considered signal hy- potheses. In the combination, the systematic uncertainties in jet momentum scale and resolution, V tagging eﬃciency scale factors, and integrated luminosity are assumed to be 100% correlated. Figure 6 shows the resulting expected and observed exclusion limits at 95% CL on the signal cross section as a function of the resonance mass for all signal hypotheses. The limits are compared with the product of cross section and branching fraction (σB) to WW and ZZ for a bulk graviton with k/MPl = 0.5, and with σB for WZ and WW for spin-1 particles predicted by the HVT Models A and B. In this context, we consider a scenario where we expect the W ′and Z′ bosons to be degenerate in mass (triplet hypothesis). In addition, we consider also a scenario where only a charged (W ′) or a neutral (Z′) resonance is expected at a given mass (singlet hypothesis). Combining the analyses leads to about 10–30% – 20 – Source Relevant quantity Uncertainty (%) Lepton trigger (µ/e) Signal yield 1–3 / 1–3 Lepton identiﬁcation (µ/e) Signal yield 1–2 / 3 Jet energy and mjet scale Signal yield 0.2–4 Jet energy and mjet resolution Signal yield 0.1–2 Integrated luminosity Signal yield 2.7 PDFs (W ′) Signal yield 4–19 PDFs (Z′) Signal yield 4–13 PDFs (Gbulk) Signal yield 9–77 Scales (W ′) Signal yield 1–14 Scales (Z′) Signal yield 1–13 Scales (Gbulk) Signal yield 8–22 Jet energy scale Resonance shape (mean) 1.3 Jet energy scale Resonance shape (width) 2–3 Jet energy resolution Resonance shape (mean) 0.1 Jet energy resolution Resonance shape (width) 4 Jet energy and mjet scale Migration 2–24 V tagging τ21 (0.45/0.6) Migration 7 / 3 V tagging pT-dependence (0.45/0.6) Migration 3–6 / 6–10 Table 8. Summary of the signal systematic uncertainties for the ℓν+jet analyses and their impact on the event yield in the signal region and on the reconstructed mVV shape (mean and width) for both muon and electron channels. The last three uncertainties result in migrations between event categories, but do not aﬀect the overall signal eﬃciency. 7.1 Limits on narrow-width resonance models The correlations among diﬀerent categories are taken into account. JHEP03(2017)162 Table 8. Summary of the signal systematic uncertainties for the ℓν+jet analyses and their impact on the event yield in the signal region and on the reconstructed mVV shape (mean and width) for both muon and electron channels. The last three uncertainties result in migrations between event categories, but do not aﬀect the overall signal eﬃciency. The correlations among diﬀerent categories are taken into account. Table 8. Summary of the signal systematic uncertainties for the ℓν+jet analyses and their impact on the event yield in the signal region and on the reconstructed mVV shape (mean and width) for both muon and electron channels. The last three uncertainties result in migrations between event categories, but do not aﬀect the overall signal eﬃciency. The correlations among diﬀerent categories are taken into account. Table 8. Summary of the signal systematic uncertainties for the ℓν+jet analyses and their impact on the event yield in the signal region and on the reconstructed mVV shape (mean and width) for both muon and electron channels. The last three uncertainties result in migrations between event categories, but do not aﬀect the overall signal eﬃciency. The correlations among diﬀerent categories are taken into account. more stringent expected upper limits on the cross section compared to the most sensitive individual channel, depending on the resonance mass and the signal hypothesis. For Gbulk, Z′ and triplet signals (W’ signal) with masses <0.8 TeV (<0.75 TeV), the limits are obtained from the low-mass ℓν+jet channel, while for the higher masses they are obtained from the high-mass ℓν+jet and dijet channels. The dijet analysis provides more stringent expected upper limits on the cross sections than the ℓν+jet analysis for resonance masses above 1.7 TeV for Z′ and >1.3 TeV for W ′, because of the larger branching fractions: B(WW → qqqq) = 44%, B(WW →ℓνqq) = 31%, B(WZ →qqqq) = 46%, and B(WZ →ℓνqq) = 16%. In fact, the combination of high- and low-purity categories, together with the weak dependence of tagging eﬃciency on pT [20] improves the sensitivity for most potential signal models. In the narrow-width bulk graviton model, the combined sensitivity of the searches is not large enough to set mass limits, however, cross sections are excluded in the range 3–1200 fb. 7.1 Limits on narrow-width resonance models For HVT Model A (B), the combined data exclude singlet W ′resonances with masses <2.0 (2.2) TeV and Z′ resonances with masses below <1.6 (1.7) TeV. Under the triplet hypothesis, spin-1 resonances with masses <2.3 and <2.4 TeV are excluded for HVT Models A and B, respectively. – 21 – JHEP03(2017)162 (TeV) Z' M 1 1.5 2 2.5 3 3.5 4 (pb) WW → Z' Β × σ -3 10 -2 10 -1 10 1 10 (13 TeV) -1 2.3-2.7 fb CMS observed S Asympt. CL 1 s.d. ± expected S Asympt. CL 2 s.d. ± expected S Asympt. CL =3) V (g B HVT =1) V (g A HVT lvqq + qqqq channels (TeV) W' M 1 1.5 2 2.5 3 3.5 4 (pb) WZ → W' Β × σ -3 10 -2 10 -1 10 1 10 (13 TeV) -1 2.3-2.7 fb CMS observed S Asympt. CL 1 s.d ± expected S Asympt. CL 2 s.d ± expected S Asympt. CL =3) V (g B HVT =1) V (g A HVT lvqq + qqqq channels (TeV) V' M 1 1.5 2 2.5 3 3.5 4 (pb) WV → V' Β × σ -3 10 -2 10 -1 10 1 10 (13 TeV) -1 2.3-2.7 fb CMS observed S Asympt. CL 1 s.d. ± expected S Asympt. CL 2 s.d. ± expected S Asympt. CL =3) V (g B HVT =1) V (g A HVT lvqq + qqqq channels (TeV) bulk G M 1 1.5 2 2.5 3 3.5 4 (pb) VV → bulk G Β × σ -3 10 -2 10 -1 10 1 10 Observed S Asympt. CL 1 s.d. ± Expected S Asympt. CL 2 s.d. ± Expected S Asympt. CL =0.5 k~ , VV → bulk G BR × TH σ (13 TeV) -1 2.3-2.7 fb CMS observed S Asympt. CL 1 s.d. ± expected S Asympt. CL 2 s.d. ± expected S Asympt. CL =0.5 k~ , bulk G lvqq + qqqq channels Figure 6. Observed (black solid) and expected (black dashed) 95% CL upper limits on the produc- tion of a narrow-width resonance decaying to a pair of vector bosons for diﬀerent signal hypotheses. In the upper plots, limits are set in the context of a spin-1 neutral Z′ (left) and charged W ′(right) resonances, and compared with the prediction of the HVT Models A and B. 7.1 Limits on narrow-width resonance models In the lower left plot, limits are set in the same model under the triplet hypothesis (W ′and Z′). In the lower right plot, limits are set in the context of a bulk graviton with k/M Pl = 0.5 and compared with the predic- tion. For Gbulk, Z′ and triplet signals (W’ signal) with masses <0.8 TeV (<0.75 TeV), the limits are obtained from the low-mass ℓν+jet channel, while for the higher masses they are obtained from the (TeV) W' M 1 1.5 2 2.5 3 3.5 4 (pb) WZ → W' Β × σ -3 10 -2 10 -1 10 1 10 (13 TeV) -1 2.3-2.7 fb CMS observed S Asympt. CL 1 s.d ± expected S Asympt. CL 2 s.d ± expected S Asympt. CL =3) V (g B HVT =1) V (g A HVT lvqq + qqqq channels (TeV) Z' M 1 1.5 2 2.5 3 3.5 4 (pb) WW → Z' Β × σ -3 10 -2 10 -1 10 1 10 (13 TeV) -1 2.3-2.7 fb CMS observed S Asympt. CL 1 s.d. ± expected S Asympt. CL 2 s.d. ± expected S Asympt. CL =3) V (g B HVT =1) V (g A HVT lvqq + qqqq channels JHEP03(2017)162 (TeV) W' M (TeV) bulk G M 1 1.5 2 2.5 3 3.5 4 (pb) VV → bulk G Β × σ -3 10 -2 10 -1 10 1 10 Observed S Asympt. CL 1 s.d. ± Expected S Asympt. CL 2 s.d. ± Expected S Asympt. CL =0.5 k~ , VV → bulk G BR × TH σ (13 TeV) -1 2.3-2.7 fb CMS observed S Asympt. CL 1 s.d. ± expected S Asympt. CL 2 s.d. ± expected S Asympt. CL =0.5 k~ , bulk G lvqq + qqqq channels (TeV) V' M 1 1.5 2 2.5 3 3.5 4 (pb) WV → V' Β × σ -3 10 -2 10 -1 10 1 10 (13 TeV) -1 2.3-2.7 fb CMS observed S Asympt. CL 1 s.d. ± expected S Asympt. CL 2 s.d. ± expected S Asympt. CL =3) V (g B HVT =1) V (g A HVT lvqq + qqqq channels Figure 6. Observed (black solid) and expected (black dashed) 95% CL upper limits on the produc- tion of a narrow-width resonance decaying to a pair of vector bosons for diﬀerent signal hypotheses. 7.1 Limits on narrow-width resonance models In the upper plots, limits are set in the context of a spin-1 neutral Z′ (left) and charged W ′(right) resonances, and compared with the prediction of the HVT Models A and B. In the lower left plot, limits are set in the same model under the triplet hypothesis (W ′and Z′). In the lower right plot, limits are set in the context of a bulk graviton with k/M Pl = 0.5 and compared with the predic- tion. For Gbulk, Z′ and triplet signals (W’ signal) with masses <0.8 TeV (<0.75 TeV), the limits are obtained from the low-mass ℓν+jet channel, while for the higher masses they are obtained from the high-mass ℓν+jet and dijet channels. Figure 7 shows a scan of the coupling parameters and the corresponding observed 95% CL exclusion contours in the HVT model for the combined analyses. The parameters are deﬁned as gVcH and g2cF/gV, related to the coupling strengths of the new resonance to the Higgs boson and to fermions. The range of the scan is limited by the assumption – 22 – H c V g -3 -2 -1 0 1 2 3 V /g F c 2 g -1 -0.5 0 0.5 1 3.5 TeV 1.5 TeV 2 TeV A B > 5% M th Γ (13 TeV) -1 2.3-2.7 fb CMS Figure 7. Exclusion regions in the plane of the HVT couplings (g2cF/gV, gVcH) for three resonance masses, 1.5, 2.0, and 3.5 TeV. Model points A and B of the benchmarks used in the analysis are also shown. The solid, dashed, and dashed-dotted lines represent the boundaries of the regions excluded by this search for diﬀerent resonance masses (the region outside these lines is excluded). The areas indicated by the solid shading correspond to regions where the resonance width is predicted to be more than 5% of the resonance mass and the narrow-resonance assumption is not satisﬁed. H c V g -3 -2 -1 0 1 2 3 V /g F c 2 g -1 -0.5 0 0.5 1 3.5 TeV 1.5 TeV 2 TeV A B > 5% M th Γ (13 TeV) -1 2.3-2.7 fb CMS JHEP03(2017)162 Figure 7. Exclusion regions in the plane of the HVT couplings (g2cF/gV, gVcH) for three resonance masses, 1.5, 2.0, and 3.5 TeV. Model points A and B of the benchmarks used in the analysis are also shown. 7.1 Limits on narrow-width resonance models The solid, dashed, and dashed-dotted lines represent the boundaries of the regions excluded by this search for diﬀerent resonance masses (the region outside these lines is excluded). The areas indicated by the solid shading correspond to regions where the resonance width is predicted to be more than 5% of the resonance mass and the narrow-resonance assumption is not satisﬁed. that the new resonance is narrow. A contour is overlaid, representing the region where the theoretical width is larger than the experimental resolution of the searches, and hence where the narrow-resonance assumption is not satisﬁed. This contour is deﬁned by a predicted resonance width of 5%, corresponding to the narrowest resonance mass resolution of the searches. 7.2 Model-independent limits The above analysis is speciﬁc to a narrow bulk graviton and HVT models, but these are not the only extension of the SM that predicts resonances decaying to vector bosons. It is therefore useful to reinterpret these results through a more generic model. In this section, we present the exclusion limits on the number of events that remain after modifying the analysis and greatly simplifying its structure, at a moderate cost in performance. Together with the upper limits on the number of signal events, we provide tables on reconstruction and identiﬁcation eﬃciencies for vector bosons emitted in the kinematic acceptance of the analysis. Following the instructions detailed in appendix A, it is possible to estimate the number of events expected in a generic signal that would be detected in CMS with the present data set, and to compare it with the upper limit on the number of signal events. To avoid the dependence on assumptions in the construction of the separate categories, we perform a simpliﬁed analysis, reducing the event classiﬁcation to two (ℓν+jet) and one (dijet) categories, respectively. This is done by eliminating the low-purity categories and combining the jet mass categories in the analyses. The loss in performance is very small – 23 – for a large range of masses. The eﬀect of dropping the LP category is observed only at very high masses, where the upper limit on the cross section becomes less stringent. A generic model cannot be restricted to narrow signals, and we therefore provide limits as a function of both mass (MX) and natural width (ΓX) of the new resonance. The generated line shape is parametrized with a BW function and its full width at half maximum is deﬁned as the Γ parameter of the BW function. The BW line shape is convolved with a double sided CB function describing the detector resolution in the ℓν+jet analysis, and with a sum of a Gaussian and CB functions for the dijet analysis. As ΓX is varied, the parameters of the double-CB function are kept ﬁxed to the values determined under the narrow-width approximation. It has been checked that the parametrization of detector eﬀects factorizes from the natural width of the resonance and is stable as ΓX increases. 7.2 Model-independent limits The width is scanned at regular steps of the relative width, ΓX/MX, which spans from the zero-width approximation (as in the nominal analysis), up to ΓX/MX = 0.30, in steps of 0.05. For high masses, the resonance shape is distorted from the BW shape owing to PDF eﬀects creating a tail towards low masses. The line shape is corrected for this by a linear function that works well for quark induced processes. However, the shape description using this approach is unsatisfactory for gluon induced processes at very high masses and widths. JHEP03(2017)162 We provide the eﬃciency as a function of the kinematic variables of the vector boson, as the eﬃciency can depend signiﬁcantly on the production and decay kinematic quantities of the new resonance. The eﬃciencies are extracted from the bulk graviton samples generated for the baseline analysis. The eﬃciencies are calculated by ﬁrst preselecting simulated signal events according to the acceptance requirements of the analysis. The tables are therefore valid only within this kinematic region, as summarized in tables 9 and 10 of appendix A for the ℓν+jet and dijet analyses, respectively. For preselected events, the reconstructed V candidates are then required to pass all the analysis selections. The eﬃciencies are presented as a function of the pT and η of the V boson prior to any simulation of detector eﬀects. All the reweighting and rescaling eﬀects (including lepton identiﬁcation and trigger eﬃciencies, and V tagging scale factors) are included in the eﬃciencies. The eﬃciencies of requiring no additional well-identiﬁed leptons and b-tagged jets in the ℓν+jet analysis are found to be independent of the diboson event kinematics. We use a constant eﬃciency of 95% for the combined vetoes. Similarly, the ∆η requirement in the dijet analysis is taken into account as a global eﬃciency factor of 98%. It has been checked that the dependence of the total signal eﬃciency and acceptance on the width of the generated sample is very weak. We include this eﬀect in the systematic uncertainties of the procedure, as discussed below. Special consideration is given to cases where the boson is transversely polarized, be- cause the calculated eﬃciencies are based on longitudinally polarized bosons, as in the case of the reference bulk graviton model. The eﬃciency of the V tagging selections depend sig- niﬁcantly on the degree of polarization of the vector boson [21]. 7.2 Model-independent limits This eﬀect is investigated using RS1 gravitons produced with the MadGraph generator. The V bosons originating from the decays of RS1 gravitons are transversely polarized in about 90% of the cases. For bosons decaying leptonically, the tables are still valid because of the generator-level selec- tion on individual leptons, which guarantees that polarization eﬀects for the leptonic boson – 24 – (TeV) X M 1 2 3 4 X /M X Γ 0.00 0.05 0.10 0.15 0.20 0.25 0.30 +jet) ν (l events N 4 5 10 20 30 40 2 10 2 10 × 2 (13 TeV) -1 2.3 fb CMS (TeV) X M 1.5 2.0 2.5 3.0 3.5 4.0 X /M X Γ 0.00 0.05 0.10 0.15 0.20 0.25 0.30 (dijet) events N 3 4 5 10 20 30 40 2 10 (13 TeV) -1 2.7 fb CMS Figure 8. Observed exclusion limits at 95% CL on the number of events for a WV →ℓν +jet (left) and a VV →dijet (right) resonance, as a function of its mass and normalized width. The dark shaded area denotes the kinematic regime where the limit is valid only for the quark-antiquark annihilation processes. (TeV) X M 1.5 2.0 2.5 3.0 3.5 4.0 X /M X Γ 0.00 0.05 0.10 0.15 0.20 0.25 0.30 (dijet) events N 3 4 5 10 20 30 40 2 10 (13 TeV) -1 2.7 fb CMS (TeV) X M 1 2 3 4 X /M X Γ 0.00 0.05 0.10 0.15 0.20 0.25 0.30 +jet) ν (l events N 4 5 10 20 30 40 2 10 2 10 × 2 (13 TeV) -1 2.3 fb CMS JHEP03(2017)162 Figure 8. Observed exclusion limits at 95% CL on the number of events for a WV →ℓν +jet (left) and a VV →dijet (right) resonance, as a function of its mass and normalized width. The dark shaded area denotes the kinematic regime where the limit is valid only for the quark-antiquark annihilation processes. are included in the acceptance. As shown in ref. [21], the eﬃciency of the jet substructure selection is found to be smaller for transversely polarized V bosons that tend to have more asymmetric subjet pT, resulting in a higher probability for the subjet with lower pT to be rejected by the pruning algorithm. 8 Summary A search has been presented for new resonances decaying to WW, ZZ, or WZ boson pairs in which at least one of the bosons decays into quarks. The ﬁnal states involve dijet and ℓν+jet events with ℓ= µ or e. The results include the W →τν contribution with subsequent decay τ →ℓνν. The W and Z bosons that decay to quarks are selected by requiring a jet with mass compatible with the W or Z boson mass, respectively. Additional information from jet substructure is used to suppress background from W+jets and multijet processes. No evidence for a signal is found. In particular, the excesses at a resonance mass of 2 TeV observed in previous searches [12, 16] are not conﬁrmed. The result is interpreted as an upper limit on the production cross section of a narrow-width resonance as a function its mass, in the context of the bulk graviton model (with decays to WW or ZZ), heavy vector- triplet Models A and B, and W ′ and Z′ singlet models. The upper limits are based on the statistical combination of the two channels. For the heavy vector-triplet, we exclude W ′and Z′ resonances with respective masses <2.0 and <1.6 TeV for Model A, <2.2 and <1.7 TeV for Model B. Under the triplet hypothesis, spin-1 resonances with masses below 2.3 and 2.4 TeV are excluded for heavy vector-triplet Model A and B, respectively. In the narrow- width bulk graviton model, cross sections are excluded in the range of 3–1200 fb. This is the ﬁrst search for a narrow-width bulk graviton with ˜k = 0.5 at √s = 13 TeV. Tabulated eﬃciencies for the reconstruction of the vector bosons within the kinematic acceptance of the analysis are also provided, allowing for a reintepretation of the exclusion limits in a generic phenomenological model. JHEP03(2017)162 7.2 Model-independent limits Studies of simulated RS1 graviton samples show that the loss in eﬃciency is largely independent of the V kinematic variables, so that the eﬀect of the transverse polarization can be adequately modelled by a constant scale factor of 0.76, independent of the pT and η of the V →qq decays. To validate the above procedure, the resulting parametrized eﬃciencies (including the event veto eﬃciencies) are used to predict the total eﬃciency for reconstructing resonances of diﬀerent spin and width. The estimation is compared to the exact number obtained from performing the baseline analysis directly on the simulated events. In all cases, the agreement between the nominal and parametrized eﬃciencies are of the order of 10–20% for the majority of the parameter space, but grow up to 40% for very low resonance masses, were migration eﬀects over selection boundaries cannot be treated in our parametrization approach. Various approximations and uncertainties contribute to the ﬁnal additional sys- tematic uncertainty in the eﬃciency; the main ones are unaccounted correlations between the physical objects, statistical uncertainties due to limited numbers of simulated events, and residual dependence on natural width. We assign an additional systematic uncertainty which ranges from 20% at high masses to 40% at low masses in the total signal eﬃciency for calculating the model-independent limits. This additional systematic uncertainty addresses the remaining imperfections in the parametrization of eﬃciencies. Figure 8 shows the observed limits on the number of events extracted from the sim- pliﬁed analysis, independently for the ℓν+jet and dijet analyses, which are not combined in order to avoid assumptions on branching fractions of a resonance decaying to both WW and ZZ channels. The limits are calculated using an asymptotic approximation of the CLS method. All systematic uncertainties considered in the baseline analysis are included in the calculation of these limits, together with the additional uncertainty related to the – 25 – approximations for parametrizing eﬃciencies. The main features of the observed limits presented above are still visible. With increasing width, the overall sensitivity degrades. The shaded area denotes where the limit is valid only for quark-antiquark annihilation pro- cesses, because in this region the mass distribution resulting from gluon-fusion processes can no longer be approximated by a peaking resonance. Acknowledgments We congratulate our colleagues in the CERN accelerator departments for the excellent performance of the LHC and thank the technical and administrative staﬀs at CERN and at other CMS institutes for their contributions to the success of the CMS eﬀort. In ad- dition, we gratefully acknowledge the computing centres and personnel of the Worldwide LHC Computing Grid for delivering so eﬀectively the computing infrastructure essential to our analyses. Finally, we acknowledge the enduring support for the construction and operation of the LHC and the CMS detector provided by the following funding agencies: BMWFW and FWF (Austria); FNRS and FWO (Belgium); CNPq, CAPES, FAPERJ, and FAPESP (Brazil); MES (Bulgaria); CERN; CAS, MoST, and NSFC (China); COL- CIENCIAS (Colombia); MSES and CSF (Croatia); RPF (Cyprus); SENESCYT (Ecuador); MoER, ERC IUT, and ERDF (Estonia); Academy of Finland, MEC, and HIP (Finland); CEA and CNRS/IN2P3 (France); BMBF, DFG, and HGF (Germany); GSRT (Greece); – 26 – OTKA and NIH (Hungary); DAE and DST (India); IPM (Iran); SFI (Ireland); INFN (Italy); MSIP and NRF (Republic of Korea); LAS (Lithuania); MOE and UM (Malaysia); BUAP, CINVESTAV, CONACYT, LNS, SEP, and UASLP-FAI (Mexico); MBIE (New Zealand); PAEC (Pakistan); MSHE and NSC (Poland); FCT (Portugal); JINR (Dubna); MON, RosAtom, RAS, RFBR and RAEP (Russia); MESTD (Serbia); SEIDI and CPAN (Spain); Swiss Funding Agencies (Switzerland); MST (Taipei); ThEPCenter, IPST, STAR, and NSTDA (Thailand); TUBITAK and TAEK (Turkey); NASU and SFFR (Ukraine); STFC (United Kingdom); DOE and NSF (USA). Individuals have received support from the Marie-Curie programme and the Euro- pean Research Council and EPLANET (European Union); the Leventis Foundation; the A. P. Acknowledgments Sloan Foundation; the Alexander von Humboldt Foundation; the Belgian Federal Science Policy Oﬃce; the Fonds pour la Formation `a la Recherche dans l’Industrie et dans l’Agriculture (FRIA-Belgium); the Agentschap voor Innovatie door Wetenschap en Technologie (IWT-Belgium); the Ministry of Education, Youth and Sports (MEYS) of the Czech Republic; the Council of Science and Industrial Research, India; the HOM- ING PLUS programme of the Foundation for Polish Science, coﬁnanced from European Union, Regional Development Fund, the Mobility Plus programme of the Ministry of Science and Higher Education, the National Science Center (Poland), contracts Har- monia 2014/14/M/ST2/00428, Opus 2014/13/B/ST2/02543, 2014/15/B/ST2/03998, and 2015/19/B/ST2/02861, Sonata-bis 2012/07/E/ST2/01406; the Thalis and Aristeia pro- grammes coﬁnanced by EU-ESF and the Greek NSRF; the National Priorities Research Program by Qatar National Research Fund; the Programa Clar´ın-COFUND del Principado de Asturias; the Rachadapisek Sompot Fund for Postdoctoral Fellowship, Chulalongkorn University and the Chulalongkorn Academic into Its 2nd Century Project Advancement Project (Thailand); and the Welch Foundation, contract C-1845. JHEP03(2017)162 A Instructions and additional material for generic interpretation of the results This appendix presents a technical description of the procedure for calculating the signal yield expected to be observed in the CMS detector in a scenario with a new resonance, X, decaying to two vector bosons in the ℓν+jet ﬁnal state (WW, WZ), as well as the dijet ﬁnal state (WW, WZ, and ZZ). The eﬃciencies are calculated using the reference bulk graviton samples described in section 3 and listed in tables 11–13. These eﬃciencies can be applied to a generic model with the following procedure: 1. Generate a sample of events for a given mass and width of the X resonance; the simulated process must include the decay of the X resonance to leptons and quarks (including W→τν →ℓννν decays). 1. Generate a sample of events for a given mass and width of the X resonance; the simulated process must include the decay of the X resonance to leptons and quarks (including W→τν →ℓννν decays). 2. Split the sample into ℓν+jet and dijet decays. 3. Filter the events according to the criteria listed in table 9 (for ℓν+jet WW decays) and table 10 (for dijet WW decays). If the resonance decays to WZ →ℓνqq, the criteria for a hadronically decaying W boson in table 9 should be applied to the generated – 27 – Objects Requirements Muons pT > 53 GeV \|η\| < 2.1 Electrons pT > 120 GeV \|η\| < 2.5 P ⃗pT,ν pT > 40 GeV (muon channel) pT > 80 GeV (electron channel) W →ℓν or W →τν →ℓννν pT > 200 GeV V →qq pT > 200 GeV \|η\| < 2.4 WV system 0.7 < mWV < 5.0 TeV ∆φ(Vqq, Wlν) > 2 ∆φ(Vqq, P ⃗pT,ν) > 2 ∆R(Vqq, ℓ) > π/2 JHEP03(2017)162 Table 9. Generator-level requirements for the ℓν+jet analysis, to be used for the computation of the eﬃciency parametrization. The vector sum of the transverse neutrino momenta P ⃗pT,ν is taken over all the neutrinos in the ﬁnal state, coming either from W →ℓν or W →τν →ℓννν decays with ℓ= µ or e. Objects Requirements V →qq pT > 200 GeV \|η\| < 2.4 VV system mVV > 1 TeV \|ηV1 −ηV2\| < 1.3 Table 10. Generator-level requirements for the dijet analysis, to be used for the computation of the eﬃciency parametrization. Table 10. Generator-level requirements for the dijet analysis, to be used for the computation the eﬃciency parametrization. hadronically decaying Z boson. A Instructions and additional material for generic interpretation of the results If the resonance decays to ZW or ZZ in the dijet channel, the criteria in table 10 should be applied to the generated hadronically decaying Z bosons as well. hadronically decaying Z boson. If the resonance decays to ZW or ZZ in the dijet channel, the criteria in table 10 should be applied to the generated hadronically decaying Z bosons as well. 4. For each of the remaining events, calculate the eﬃciency for reconstructing the chan- nels W →µν and W →τν →µννν, and W →eν and W →τν →eννν, using table 11. The table provides the eﬃciency parametrized as a function of pT and η of the W. 5. In a similar way, in the ℓν+jet channel calculate the eﬃciency of the hadronically decaying W or Z bosons using the values in table 12. For the dijet decays, compute the eﬃciency for each boson from the values in table 13. 6. Weight each accepted event with the product of the two eﬃciencies found at steps 3 and 4. In the case of a X resonance decaying to WV (ℓν+jet channel), also multiply by the combined eﬃciency of the second-lepton and b jet vetoes, equal to 95%. A 6. Weight each accepted event with the product of the two eﬃciencies found at steps 3 and 4. In the case of a X resonance decaying to WV (ℓν+jet channel), also multiply by the combined eﬃciency of the second-lepton and b jet vetoes, equal to 95%. A Instructions and additional material for generic interpretation of the results A – 28 – W →µν and W →τν →µννν W →µν and W →τν →µννν pW T range (GeV) \|ηW\| range 0–0.2 0.2–0.4 0.4–0.6 0.6–0.8 0.8–1.0 1–1.25 1.25–1.5 1.5–2.0 2–2.5 200–250 0.82 0.79 0.79 0.80 0.85 0.82 0.81 0.82 0.78 250–300 0.89 0.90 0.88 0.86 0.90 0.86 0.91 0.86 0.91 300–400 0.90 0.89 0.90 0.90 0.89 0.90 0.89 0.90 0.87 400–500 0.88 0.89 0.91 0.90 0.88 0.89 0.90 0.89 0.89 500–600 0.90 0.90 0.92 0.90 0.88 0.89 0.91 0.87 0.88 600–700 0.91 0.90 0.92 0.91 0.88 0.90 0.92 0.88 0.87 700–800 0.91 0.89 0.92 0.91 0.89 0.89 0.91 0.90 0.82 800–1000 0.92 0.89 0.92 0.91 0.88 0.88 0.90 0.88 0.94 1000–1200 0.91 0.89 0.92 0.91 0.89 0.88 0.89 0.85 0.75 1200–1500 0.91 0.88 0.92 0.91 0.87 0.87 0.89 0.87 — 1500–2000 0.90 0.87 0.92 0.91 0.86 0.88 0.87 — — 2000–2500 0.91 0.86 0.91 0.90 0.83 0.82 — — — 2500–3000 0.88 0.79 0.90 0.82 — — — — — 3000–4000 0.78 0.88 0.80 1.00 — — — — — W →eν and W →τν →eννν 200–250 0.78 0.75 0.82 0.81 0.79 0.80 0.71 0.79 0.68 250–300 0.79 0.79 0.77 0.80 0.78 0.82 0.79 0.73 0.78 300–400 0.82 0.82 0.82 0.83 0.82 0.82 0.80 0.81 0.80 400–500 0.82 0.82 0.81 0.84 0.81 0.81 0.82 0.82 0.80 500–600 0.83 0.83 0.84 0.84 0.83 0.81 0.82 0.84 0.85 600–700 0.83 0.84 0.84 0.83 0.85 0.84 0.82 0.84 0.88 700–800 0.84 0.83 0.84 0.85 0.84 0.84 0.82 0.82 0.94 800–1000 0.83 0.84 0.84 0.84 0.85 0.86 0.82 0.85 0.78 1000–1200 0.83 0.84 0.84 0.83 0.84 0.85 0.84 0.86 0.33 1200–1500 0.84 0.84 0.84 0.84 0.85 0.84 0.85 0.81 — 1500–2000 0.83 0.85 0.84 0.84 0.86 0.84 0.86 0.95 — 2000–2500 0.83 0.85 0.84 0.85 0.84 0.79 — — — 2500–3000 0.78 0.82 0.78 0.69 — — — — — 3000–4000 0.80 0.81 0.67 1.00 — — — — — Table 11. Reconstruction and identiﬁcation eﬃciency for the (upper table) W →µν and W → τν →µννν, and (lower table) W →eν and W →τν →eννν decays as function of generated pW T and \|ηW\|. Uncertainties in the eﬃciencies are included in the generic limit calculation as discussed in the text. JHEP03(2017)162 Table 11. A Instructions and additional material for generic interpretation of the results Reconstruction and identiﬁcation eﬃciency for the (upper table) W →µν and W → τν →µννν, and (lower table) W →eν and W →τν →eννν decays as function of generated pW T and \|ηW\|. Uncertainties in the eﬃciencies are included in the generic limit calculation as discussed in the text. Table 11. Reconstruction and identiﬁcation eﬃciency for the (upper table) W →µν and W → τν →µννν, and (lower table) W →eν and W →τν →eννν decays as function of generated pW T and \|ηW\|. Uncertainties in the eﬃciencies are included in the generic limit calculation as discussed in the text. correction factor amounting to 98% should be applied to events in the dijet category to take into account the eﬃciency of the ∆η requirement. 7. The resulting sum of weighted events for the ℓν+jet and dijet subsamples, divided by the total number of events, provides an approximation to the total eﬃciency for the given model in each of the two channels. The ﬁnal numbers of events can be directly compared to the observed limits in ﬁgure 8 and table 14, in order to assess the exclusion power of the present data with respect to the model considered. A Instructions and additional material for generic interpretation of the results – 29 – WL →qq pW T range (GeV) \|ηW\| range 0–0.2 0.2–0.4 0.4–0.6 0.6–0.8 0.8–1.0 1.0–1.25 1.25–1.5 1.5–2.0 2.0–2.5 200–250 0.31 0.36 0.33 0.28 0.37 0.38 0.30 0.25 0.26 250–300 0.54 0.48 0.57 0.46 0.50 0.54 0.47 0.48 0.56 300–400 0.71 0.70 0.72 0.70 0.70 0.65 0.66 0.63 0.59 400–500 0.65 0.65 0.66 0.64 0.64 0.67 0.62 0.63 0.70 500–600 0.72 0.71 0.73 0.72 0.73 0.70 0.66 0.69 0.72 600–700 0.74 0.75 0.74 0.73 0.72 0.71 0.71 0.72 0.78 700–800 0.73 0.74 0.73 0.75 0.72 0.71 0.67 0.68 0.65 800–1000 0.73 0.74 0.74 0.74 0.73 0.71 0.65 0.66 0.62 1000–1200 0.69 0.71 0.71 0.71 0.69 0.66 0.57 0.65 0.67 1200–1500 0.68 0.69 0.69 0.70 0.68 0.67 0.54 0.63 — 1500–2000 0.69 0.69 0.69 0.68 0.67 0.65 0.47 0.11 — 2000–2500 0.68 0.68 0.69 0.69 0.67 0.66 0.50 — — 2500–3000 0.76 0.63 0.77 0.53 0.67 — — — — 3000–4000 0.77 0.43 1.00 1.00 — — — — — ZL →qq 200–250 0.26 0.48 0.27 0.37 0.33 0.41 0.37 0.36 0.28 250–300 0.64 0.56 0.62 0.58 0.60 0.57 0.56 0.61 0.53 300–400 0.76 0.75 0.77 0.75 0.74 0.72 0.71 0.73 0.67 400–500 0.75 0.75 0.76 0.74 0.76 0.77 0.73 0.74 0.71 500–600 0.80 0.81 0.82 0.80 0.78 0.79 0.76 0.78 0.73 600–700 0.81 0.83 0.80 0.81 0.82 0.80 0.76 0.77 0.72 700–800 0.81 0.80 0.79 0.79 0.80 0.78 0.74 0.75 0.77 800–1000 0.81 0.81 0.81 0.81 0.79 0.77 0.72 0.74 0.72 1000–1200 0.78 0.78 0.79 0.78 0.77 0.75 0.66 0.71 0.77 1200–1500 0.77 0.77 0.76 0.77 0.75 0.73 0.60 0.65 0.66 1500–2000 0.74 0.74 0.73 0.74 0.72 0.67 0.52 0.57 — 2000–2500 0.72 0.73 0.73 0.73 0.69 0.66 0.53 — — 2500–3000 0.80 0.85 0.69 0.76 — — — — — 3000–4000 1.0 0.50 1.0 — — — — — — Table 12. Reconstruction and identiﬁcation eﬃciency for the (upper table) WL →qq and (lower table) ZL →qq decay as a function of generated pV T and \|ηV\| applying the V tagging requirements used in the ℓν+jet analysis (τ21 < 0.6). Uncertainties in the eﬃciencies are included in the generic limit calculation as discussed in the text. JHEP03(2017)162 Table 12. Reconstruction and identiﬁcation eﬃciency for the (upper table) WL →qq and (lower table) ZL →qq decay as a function of generated pV T and \|ηV\| applying the V tagging requirements used in the ℓν+jet analysis (τ21 < 0.6). A Instructions and additional material for generic interpretation of the results Uncertainties in the eﬃciencies are included in the generic limit calculation as discussed in the text. The numbers provided refer to longitudinally polarized bosons. For transversely po- larized bosons that decay leptonically, the same numbers are valid, as long as they are applied after the kinematic acceptance requirements. If the boson decays to quarks and has a transverse polarization, the eﬃciency must be scaled down by a factor of 0.76 for each hadronically decaying boson in the event. A Instructions and additional material for generic interpretation of the results – 30 – WL →qq pW T range (GeV) \|ηW\| range 0.0–0.2 0.2–0.3 0.3–0.4 0.4–0.6 0.6–0.8 0.8–1.0 1.00–1.25 1.2–1.5 1.5–2.0 2.0–2.4 200–250 0.27 0.34 0.23 0.25 0.35 0.32 0.31 0.30 0.32 250–300 0.55 0.50 0.55 0.51 0.54 0.58 0.52 0.56 0.54 300–400 0.74 0.74 0.75 0.73 0.73 0.69 0.69 0.67 0.63 400–500 0.69 0.68 0.70 0.69 0.68 0.69 0.65 0.65 0.71 500–600 0.72 0.72 0.74 0.73 0.74 0.70 0.66 0.70 0.75 600–700 0.74 0.75 0.75 0.74 0.73 0.72 0.71 0.73 0.78 700–800 0.74 0.75 0.74 0.75 0.73 0.72 0.68 0.69 0.66 800–1000 0.74 0.75 0.75 0.75 0.73 0.71 0.66 0.66 0.58 1000–1200 0.70 0.71 0.72 0.72 0.69 0.67 0.59 0.63 0.40 1200–1500 0.69 0.70 0.70 0.70 0.68 0.65 0.54 0.59 — 1500–2000 0.68 0.69 0.68 0.68 0.67 0.65 0.47 — — 2000–2500 0.69 0.69 0.69 0.69 0.67 0.69 — — — 2500–3000 0.74 0.66 0.73 0.60 — — — — — 3000–4000 0.74 0.74 1.00 1.00 — — — — — ZL →qq 200–250 — — 0.25 — — 0.50 — 0.50 — — 250–300 0.30 — 0.33 0.25 0.18 — 0.33 0.67 — — 300–350 0.46 0.15 0.33 0.44 0.45 0.21 0.41 0.12 0.44 — 350–400 0.38 0.40 0.47 0.47 0.43 0.41 0.26 0.35 0.36 0.50 400–500 0.50 0.46 0.51 0.47 0.45 0.51 0.41 0.38 0.45 0.59 500–600 0.59 0.60 0.61 0.60 0.58 0.55 0.52 0.44 0.51 0.67 600–700 0.63 0.61 0.62 0.59 0.59 0.56 0.50 0.45 0.48 0.53 700–800 0.60 0.62 0.61 0.60 0.60 0.58 0.50 0.40 0.41 0.75 800–1000 0.60 0.60 0.59 0.60 0.56 0.52 0.48 0.38 0.46 1.00 1000–1200 0.55 0.52 0.57 0.52 0.53 0.48 0.43 0.25 0.40 1.00 1200–1500 0.53 0.52 0.53 0.52 0.50 0.44 0.39 0.25 0.16 — 1500–2000 0.49 0.49 0.48 0.47 0.46 0.42 0.34 0.24 — — 2000–2500 0.47 0.49 0.48 0.44 0.43 0.42 0.41 0.33 — — 2500–3000 0.43 0.36 0.47 0.47 0.38 0.17 — — — — 3000–4000 0.44 0.50 — — — — — — — — Table 13. Reconstruction and identiﬁcation eﬃciency for the (upper table) WL →qq and (lower table) ZL →qq decays as a function of generated pV T and \|ηV\| applying the V tagging requirements used in the dijet analysis (τ21 < 0.45). Uncertainties in the eﬃciencies are included in the generic limit calculation as discussed in the text. JHEP03(2017)162 Table 13. A Instructions and additional material for generic interpretation of the results Reconstruction and identiﬁcation eﬃciency for the (upper table) WL →qq and (lower table) ZL →qq decays as a function of generated pV T and \|ηV\| applying the V tagging requirements used in the dijet analysis (τ21 < 0.45). Uncertainties in the eﬃciencies are included in the generic limit calculation as discussed in the text. A Instructions and additional material for generic interpretation of the results – 31 – MX (TeV) ℓν+jet channel dijet channel ΓX/MX ΓX/MX 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.8 139.9 173.5 189.2 192.7 185.7 173.1 157.8 — — — — — — — 0.9 66.9 87.8 104.4 115.5 120.4 120.6 117.1 — — — — — — — 1.0 46.9 61.4 72.4 81.6 87.9 91.0 91.4 — — — — — — — 1.1 35.2 47.1 58.2 66.7 72.2 75.3 76.3 — — — — — — — 1.2 50.7 56.5 59.7 62.0 63.8 65.1 65.9 38.3 61.7 88.6 84.8 84.3 82.8 78.1 1.3 22.7 29.4 34.9 40.4 45.5 49.9 53.0 39.6 54.9 68.9 77.8 82.2 83.0 79.0 1.4 15.1 20.5 26.3 32.1 37.9 43.0 46.9 29.8 41.9 57.3 66.7 82.7 86.3 85.7 1.5 18.2 22.4 27.1 32.1 37.2 41.7 45.1 19.7 31.0 45.6 89.1 127.4 116.0 93.4 1.6 20.1 24.1 28.4 33.4 38.3 42.3 44.9 22.4 34.0 65.7 114.8 100.5 90.1 77.3 1.7 14.2 19.0 24.4 30.6 36.7 41.3 44.0 22.1 29.1 57.6 70.9 70.9 64.6 57.2 1.8 11.8 17.7 24.5 31.6 37.0 40.0 40.6 13.0 15.4 24.2 34.6 40.4 41.1 39.7 1.9 11.6 16.6 23.1 29.8 35.1 38.4 39.7 7.7 11.8 17.2 23.7 27.8 29.3 29.5 2.0 14.7 20.4 26.7 32.0 35.2 36.8 37.2 7.7 10.6 14.5 18.7 21.5 23.1 23.9 2.1 15.4 20.8 26.4 30.6 32.7 33.6 33.9 6.2 9.0 12.5 15.6 17.5 18.8 19.5 2.2 13.2 18.5 23.9 27.5 29.4 30.2 30.6 5.1 7.8 10.9 13.4 15.1 15.9 16.5 2.3 9.8 15.4 20.7 24.2 26.1 27.1 27.3 4.6 7.8 10.5 12.2 13.2 13.8 14.3 2.4 7.9 13.3 18.4 21.4 23.0 24.2 25.1 5.9 8.4 10.2 11.1 11.8 12.2 12.6 2.5 8.5 13.7 17.4 19.5 20.6 21.6 22.6 6.4 8.4 9.5 10.1 10.6 11.0 11.3 2.6 11.0 14.6 16.7 18.0 18.8 19.5 20.3 5.5 7.8 8.7 9.2 9.6 9.9 10.2 2.7 11.9 14.6 16.0 16.8 17.3 17.7 18.4 4.8 7.0 7.8 8.3 8.6 8.9 9.2 2.8 12.3 14.1 15.0 15.5 16.0 16.2 16.7 4.8 6.2 6.8 7.3 7.7 8.0 8.3 2.9 11.9 13.1 13.8 14.3 14.6 14.9 15.2 4.6 5.5 6.1 6.5 6.9 7.2 7.4 3.0 9.5 11.0 11.7 12.0 12.4 12.5 12.6 4.5 5.1 5.6 5.9 6.3 6.6 6.9 3.1 7.5 9.2 10.1 10.7 11.2 11.6 11.9 4.1 4.5 4.9 5.3 5.6 5.9 6.2 3.2 5.6 7.1 8.0 8.8 9.4 9.9 10.3 2.9 3.7 4.2 4.6 5.0 5.3 5.6 3.3 4.0 5.3 6.2 7.0 7.7 8.3 8.8 2.4 3.1 3.6 4.0 4.4 4.8 5.1 3.4 3.4 4.3 5.1 5.8 6.5 7.1 7.6 2.3 2.7 3.1 3.5 4.0 4.3 4.6 3.5 3.2 3.9 4.5 5.1 5.6 6.2 6.8 2.2 2.5 2.8 3.2 3.6 3.9 4.2 3.6 3.0 3.6 4.1 4.6 5.1 5.6 6.1 2.2 2.5 2.7 3.0 3.5 3.7 4.0 3.7 3.0 3.5 3.9 4.3 4.7 5.2 5.7 2.2 2.4 2.6 2.9 3.3 3.5 3.8 4.0 3.1 3.4 3.7 4.0 4.4 4.8 5.3 2.1 2.2 2.3 2.6 3.0 3.2 3.4 4.1 3.3 3.6 3.9 4.2 4.6 5.0 5.5 — — — — — — — 4.5 3.4 3.7 4.0 4.3 4.8 5.3 6.1 — — — — — — — Table 14. A Instructions and additional material for generic interpretation of the results Simpliﬁed limits on the number of visible events from generic resonances decaying to pairs of V bosons in the ℓν+jet (left) and dijet (right) channels as a function of resonance mass, MX, and normalized width, ΓX/MX. Shown are limits on the visible number of events at 95% CL using the asymptotic CLS approach. Results with ΓX/MX = 0 are obtained using the resolution function only. JHEP03(2017)162 Table 14. Simpliﬁed limits on the number of visible events from generic resonances decaying to pairs of V bosons in the ℓν+jet (left) and dijet (right) channels as a function of resonance mass, MX, and normalized width, ΓX/MX. Shown are limits on the visible number of events at 95% CL using the asymptotic CLS approach. Results with ΓX/MX = 0 are obtained using the resolution function only. – 32 – Open Access. This article is distributed under the terms of the Creative Commons Attribution License (CC-BY 4.0), which permits any use, distribution and reproduction in any medium, provided the original author(s) and source are credited. References [1] K. Agashe, H. Davoudiasl, G. Perez and A. Soni, Warped gravitons at the LHC and beyond, Phys. Rev. D 76 (2007) 036006 [hep-ph/0701186] [INSPIRE]. [1] K. Agashe, H. Davoudiasl, G. Perez and A. Soni, Warped gravitons at the LHC and beyond, Phys. Rev. D 76 (2007) 036006 [hep-ph/0701186] [INSPIRE]. [2] A.L. Fitzpatrick, J. Kaplan, L. Randall and L.-T. Wang, Searching for the Kaluza-Klein graviton in bulk RS models, JHEP 09 (2007) 013 [hep-ph/0701150] [INSPIRE]. [2] A.L. Fitzpatrick, J. Kaplan, L. Randall and L.-T. Wang, Searching for the Kaluza-Klein graviton in bulk RS models, JHEP 09 (2007) 013 [hep-ph/0701150] [INSPIRE]. JHEP03(2017)162 [3] O. Antipin, D. Atwood and A. Soni, Search for RS gravitons via WLWL decays, Phys. Lett. B 666 (2008) 155 [arXiv:0711.3175] [INSPIRE]. [3] O. Antipin, D. Atwood and A. Soni, Search for RS gravitons via WLWL decays, Phys. Lett. B 666 (2008) 155 [arXiv:0711.3175] [INSPIRE]. [4] L. Randall and R. Sundrum, A Large mass hierarchy from a small extra dimension, Phys. Rev. Lett. 83 (1999) 3370 [hep-ph/9905221] [INSPIRE]. [4] L. Randall and R. Sundrum, A Large mass hierarchy from a small extra dimension, Phys. Rev. Lett. 83 (1999) 3370 [hep-ph/9905221] [INSPIRE]. [5] L. Randall and R. Sundrum, An Alternative to compactiﬁcation, Phys. Rev. Lett. 83 (1999) 4690 [hep-th/9906064] [INSPIRE]. [5] L. Randall and R. Sundrum, An Alternative to compactiﬁcation, Phys. Rev. Lett. 83 (1999) 4690 [hep-th/9906064] [INSPIRE]. [6] D. Pappadopulo, A. Thamm, R. Torre and A. Wulzer, Heavy vector triplets: bridging theory and data, JHEP 09 (2014) 060 [arXiv:1402.4431] [INSPIRE]. [6] D. Pappadopulo, A. Thamm, R. Torre and A. Wulzer, Heavy vector triplets: bridging theory and data, JHEP 09 (2014) 060 [arXiv:1402.4431] [INSPIRE]. [7] G. Altarelli, B. Mele and M. Ruiz-Altaba, Searching for new heavy vector bosons in p¯p colliders, Z. Phys. C 45 (1989) 109 [Erratum ibid. C 47 (1990) 676] [INSPIRE]. [7] G. Altarelli, B. Mele and M. Ruiz-Altaba, Searching for new heavy vector bosons in p¯p colliders, Z. Phys. C 45 (1989) 109 [Erratum ibid. C 47 (1990) 676] [INSPIRE]. [8] ATLAS collaboration, Search for new resonances decaying to a W or Z boson and a Higgs boson in the ℓ+ℓ−b¯b, ℓνb¯b and ν¯νb¯b channels with pp collisions at √s = 13 TeV with the ATLAS detector, Phys. Lett. B 765 (2017) 32 [arXiv:1607.05621] [INSPIRE]. References [9] ATLAS collaboration, Searches for heavy diboson resonances in pp collisions at √s = 13 TeV with the ATLAS detector, JHEP 09 (2016) 173 [arXiv:1606.04833] [INSPIRE [10] CMS collaboration, Search for new resonances decaying via WZ to leptons in proton-proton collisions at √s = 8 TeV, Phys. Lett. B 740 (2015) 83 [arXiv:1407.3476] [INSPIRE]. [11] CMS collaboration, Search for massive resonances decaying into pairs of boosted bosons in semi-leptonic ﬁnal states at √s = 8 TeV, JHEP 08 (2014) 174 [arXiv:1405.3447] [INSPIRE]. [12] CMS collaboration, Search for massive resonances in dijet systems containing jets tagged as W or Z boson decays in pp collisions at √s = 8 TeV, JHEP 08 (2014) 173 [arXiv:1405.1994] [INSPIRE]. [13] CMS collaboration, Search for massive WH resonances decaying into the ℓνbb ﬁnal state at √s = 8 TeV, Eur. Phys. J. C 76 (2016) 237 [arXiv:1601.06431] [INSPIRE]. [14] CMS collaboration, Search for a massive resonance decaying into a Higgs boson and a W or Z boson in hadronic ﬁnal states in proton-proton collisions at √s = 8 TeV, JHEP 02 (2016) 145 [arXiv:1506.01443] [INSPIRE]. [15] CMS collaboration, Search for Narrow High-Mass Resonances in Proton-Proton Collisions at √s = 8 TeV Decaying to a Z and a Higgs Boson, Phys. Lett. B 748 (2015) 255 [arXiv:1502.04994] [INSPIRE]. [16] ATLAS collaboration, Search for high-mass diboson resonances with boson-tagged jets in proton-proton collisions at √s = 8 TeV with the ATLAS detector, JHEP 12 (2015) 055 [arXiv:1506.00962] [INSPIRE]. – 33 – [17] ATLAS collaboration, Search for production of WW/WZ resonances decaying to a lepton, neutrino and jets in pp collisions at √s = 8 TeV with the ATLAS detector, Eur. Phys. J. C 75 (2015) 209 [Erratum ibid. C 75 (2015) 370] [arXiv:1503.04677] [INSPIRE]. [18] ATLAS collaboration, Search for WZ resonances in the fully leptonic channel using pp collisions at √s = 8 TeV with the ATLAS detector, Phys. Lett. B 737 (2014) 223 [arXiv:1406.4456] [INSPIRE]. [19] ATLAS collaboration, Search for a new resonance decaying to a W or Z boson and a Higgs boson in the ℓℓ/ℓν/νν + b¯b ﬁnal states with the ATLAS detector, Eur. Phys. J. C 75 (2015) 263 [arXiv:1503.08089] [INSPIRE]. [20] CMS collaboration, V Tagging Observables and Correlations, CMS-PAS-JME-14-002 (2014 JHEP03(2017)162 [21] CMS collaboration, Identiﬁcation techniques for highly boosted W bosons that decay into hadrons, JHEP 12 (2014) 017 [arXiv:1410.4227] [INSPIRE]. References [22] CMS collaboration, Particle-Flow Event Reconstruction in CMS and Performance for Jets, Taus and MET, CMS-PAS-PFT-09-001 (2009). [23] CMS collaboration, Commissioning of the Particle-ﬂow Event Reconstruction with the ﬁrst LHC collisions recorded in the CMS detector, CMS-PAS-PFT-10-001 (2010). [24] CMS collaboration, The CMS Experiment at the CERN LHC, 2008 JINST 3 S08004 [INSPIRE]. [25] J. Alwall et al., The automated computation of tree-level and next-to-leading order diﬀerential cross sections and their matching to parton shower simulations, JHEP 07 (2014) 079 [arXiv:1405.0301] [INSPIRE]. [26] P. Nason, A New method for combining NLO QCD with shower Monte Carlo algorithms, JHEP 11 (2004) 040 [hep-ph/0409146] [INSPIRE]. [27] S. Frixione, P. Nason and C. Oleari, Matching NLO QCD computations with Parton Shower simulations: the POWHEG method, JHEP 11 (2007) 070 [arXiv:0709.2092] [INSPIRE]. [28] S. Alioli, P. Nason, C. Oleari and E. Re, A general framework for implementing NLO calculations in shower Monte Carlo programs: the POWHEG BOX, JHEP 06 (2010) 043 [arXiv:1002.2581] [INSPIRE]. [29] S. Alioli, P. Nason, C. Oleari and E. Re, NLO single-top production matched with shower in POWHEG: s- and t-channel contributions, JHEP 09 (2009) 111 [Erratum ibid. 1002 (2010) 011] [arXiv:0907.4076] [INSPIRE]. [30] E. Re, Single-top Wt-channel production matched with parton showers using the POWHEG method, Eur. Phys. J. C 71 (2011) 1547 [arXiv:1009.2450] [INSPIRE]. [30] E. Re, Single-top Wt-channel production matched with parton showers using the POWHEG method, Eur. Phys. J. C 71 (2011) 1547 [arXiv:1009.2450] [INSPIRE]. [31] S. Alioli, S.-O. Moch and P. Uwer, Hadronic top-quark pair-production with one jet and parton showering, JHEP 01 (2012) 137 [arXiv:1110.5251] [INSPIRE]. [31] S. Alioli, S.-O. Moch and P. Uwer, Hadronic top-quark pair-production with one jet and parton showering, JHEP 01 (2012) 137 [arXiv:1110.5251] [INSPIRE]. [32] T. Sj¨ostrand, S. Mrenna and P.Z. Skands, PYTHIA 6.4 Physics and Manual, JHEP 05 (2006) 026 [hep-ph/0603175] [INSPIRE]. [32] T. Sj¨ostrand, S. Mrenna and P.Z. Skands, PYTHIA 6.4 Physics and Manual, JHEP 05 (2006) 026 [hep-ph/0603175] [INSPIRE]. [33] T. Sj¨ostrand, S. Mrenna and P.Z. Skands, A brief introduction to PYTHIA 8.1, Comput. Phys. Commun. 178 (2008) 852 [arXiv:0710.3820] [INSPIRE]. [33] T. Sj¨ostrand, S. Mrenna and P.Z. Skands, A brief introduction to PYTHIA 8.1, Comput. Phys. Commun. 178 (2008) 852 [arXiv:0710.3820] [INSPIRE]. [34] P. Skands, S. Carrazza and J. Rojo, Tuning PYTHIA 8.1: the Monash 2013 Tune, Eur. Phys. J. C 74 (2014) 3024 [arXiv:1404.5630] [INSPIRE]. References [35] CMS collaboration, Event generator tunes obtained from underlying event and multiparton scattering measurements, Eur. Phys. J. C 76 (2016) 155 [arXiv:1512.00815] [INSPIRE]. – 34 – [36] R.D. Ball et al., Impact of Heavy Quark Masses on Parton Distributions and LHC Phenomenology, Nucl. Phys. B 849 (2011) 296 [arXiv:1101.1300] [INSPIRE]. [37] GEANT4 collaboration, S. Agostinelli et al., GEANT4: A Simulation toolkit, Nucl. Instrum. Meth. A 506 (2003) 250 [INSPIRE]. [38] J.M. Campbell, R.K. Ellis and D.L. Rainwater, Next-to-leading order QCD predictions for W + 2 jet and Z + 2 jet production at the CERN LHC, Phys. Rev. D 68 (2003) 094021 [hep-ph/0308195] [INSPIRE]. [39] J.M. Campbell, R.K. Ellis and C. Williams, Vector boson pair production at the LHC, JHEP 07 (2011) 018 [arXiv:1105.0020] [INSPIRE]. [40] J.M. Campbell and R.K. Ellis, Top-quark processes at NLO in production and decay, J. Phys. G 42 (2015) 015005 [arXiv:1204.1513] [INSPIRE]. JHEP03(2017)162 JHEP03(2017)162 [41] J.M. Campbell, R.K. Ellis and F. Tramontano, Single top production and decay at next-to-leading order, Phys. Rev. D 70 (2004) 094012 [hep-ph/0408158] [INSPIRE]. [42] Y. Li and F. Petriello, Combining QCD and electroweak corrections to dilepton production in FEWZ, Phys. Rev. D 86 (2012) 094034 [arXiv:1208.5967] [INSPIRE]. [43] CMS collaboration, Measurements of Inclusive W and Z Cross Sections in pp Collisions at √s = 7 TeV, JHEP 01 (2011) 080 [arXiv:1012.2466] [INSPIRE]. [44] CMS collaboration, Identiﬁcation of b quark jets at the CMS Experiment in the LHC Run 2, CMS-PAS-BTV-15-001 (2016). [45] CMS collaboration, CMS luminosity measurement for the 2015 data-taking period, CMS-PAS-LUM-15-001 (2015). [46] CMS collaboration, Description and performance of track and primary-vertex reconstruction with the CMS tracker, 2014 JINST 9 P10009 [arXiv:1405.6569] [INSPIRE]. [47] M. Cacciari, G.P. Salam and G. Soyez, FastJet User Manual, Eur. Phys. J. C 72 (2012) 1896 [arXiv:1111.6097] [INSPIRE]. [48] M. Cacciari, G.P. Salam and G. Soyez, The Anti-kt jet clustering algorithm, JHEP 04 (2008) 063 [arXiv:0802.1189] [INSPIRE]. [49] CMS collaboration, Identiﬁcation of b-quark jets with the CMS experiment, 2013 JINST 8 P04013 [arXiv:1211.4462] [INSPIRE]. [50] CMS collaboration, Determination of Jet Energy Calibration and Transverse Momentum Resolution in CMS, 2011 JINST 6 P11002 [arXiv:1107.4277] [INSPIRE]. [51] CMS collaboration, Performance of CMS muon reconstruction in pp collision events at √s = 7 TeV, 2012 JINST 7 P10002 [arXiv:1206.4071] [INSPIRE]. References [52] CMS collaboration, Performance of Electron Reconstruction and Selection with the CMS Detector in Proton-Proton Collisions at √s = 8 TeV, 2015 JINST 10 P06005 [arXiv:1502.02701] [INSPIRE]. [53] CMS collaboration, Search for leptonic decays of W ′ bosons in pp collisions at √s = 7 TeV, JHEP 08 (2012) 023 [arXiv:1204.4764] [INSPIRE]. [54] CMS collaboration, Missing transverse energy performance of the CMS detector, 2011 JINST 6 P09001 [arXiv:1106.5048] [INSPIRE]. [55] CMS collaboration, Performance of the CMS missing transverse momentum reconstruction in pp data at √s = 8 TeV, 2015 JINST 10 P02006 [arXiv:1411.0511] [INSPIRE]. [56] CMS collaboration, Studies of jet mass in dijet and W/Z + jet events, JHEP 05 (2013) 090 [arXiv:1303.4811] [INSPIRE]. – 35 – [57] S.D. Ellis, C.K. Vermilion and J.R. Walsh, Techniques for improved heavy particle searches with jet substructure, Phys. Rev. D 80 (2009) 051501 [arXiv:0903.5081] [INSPIRE]. [58] S.D. Ellis, C.K. Vermilion and J.R. Walsh, Recombination Algorithms and Jet Substructure: Pruning as a Tool for Heavy Particle Searches, Phys. Rev. D 81 (2010) 094023 [arXiv:0912.0033] [INSPIRE]. [59] D. Krohn, J. Thaler and L.-T. Wang, Jet Trimming, JHEP 02 (2010) 084 [arXiv:0912.1342] [INSPIRE]. [60] S. Catani, Y.L. Dokshitzer, M.H. Seymour and B.R. Webber, Longitudinally invariant Kt clustering algorithms for hadron hadron collisions, Nucl. Phys. B 406 (1993) 187 [INSPIRE]. [61] Y.L. Dokshitzer, G.D. Leder, S. Moretti and B.R. Webber, Better jet clustering algorithms, JHEP 08 (1997) 001 [hep-ph/9707323] [INSPIRE]. JHEP03(2017)162 JHEP03(2017)162 [62] J. Thaler and K. Van Tilburg, Identifying Boosted Objects with N-subjettiness, JHEP 03 (2011) 015 [arXiv:1011.2268] [INSPIRE]. [63] S.D. Ellis and D.E. Soper, Successive combination jet algorithm for hadron collisions, Phys. Rev. D 48 (1993) 3160 [hep-ph/9305266] [INSPIRE]. [64] M. Bahr et al., HERWIG++ physics and manual, Eur. Phys. J. C 58 (2008) 639 [arXiv:0803.0883] [INSPIRE]. [65] Particle Data Group collaboration, K.A. Olive et al., Review of Particle Physics, Chin. Phys. C 38 (2014) 090001 [INSPIRE]. [66] CMS collaboration, Search for heavy resonances decaying into a vector boson and a Higgs boson in ﬁnal states with charged leptons, neutrinos and b quarks, Phys. Lett. 768 B (2017) 137 [arXiv:1610.08066] [INSPIRE]. [67] M.J. Oreglia, A study of the reactions ψ′ →γγψ, Ph.D. Thesis, Stanford University, Stanford U.S.A. (1980), SLAC Report SLAC-R-236. [68] M. Cacciari, S. Frixione, M.L. Mangano, P. Nason and G. The CMS collaboration The CMS collaboration The CMS collaboration The CMS collaboration Yerevan Physics Institute, Yerevan, Armenia A.M. Sirunyan, A. Tumasyan Institut f¨ur Hochenergiephysik, Wien, Austria W. Adam, E. Asilar, T. Bergauer, J. Brandstetter, E. Brondolin, M. Dragicevic, J. Er¨o, M. Flechl, M. Friedl, R. Fr¨uhwirth1, V.M. Ghete, C. Hartl, N. H¨ormann, J. Hrubec, M. Jeitler1, A. K¨onig, I. Kr¨atschmer, D. Liko, T. Matsushita, I. Mikulec, D. Rabady, N. Rad, B. Rahbaran, H. Rohringer, J. Schieck1, J. Strauss, W. Waltenberger, C.-E. Wulz1 Institute for Nuclear Problems, Minsk, Belarus V. Chekhovsky, O. Dvornikov, Y. Dydyshka, I. Emeliantchik, A. Litomin, V. Makarenko, V. Mossolov, R. Stefanovitch, J. Suarez Gonzalez, V. Zykunov National Centre for Particle and High Energy Physics, Minsk, Belarus N. Shumeiko Universiteit Antwerpen, Antwerpen, Belgium Yerevan Physics Institute, Yerevan, Armenia A.M. Sirunyan, A. Tumasyan Institut f¨ur Hochenergiephysik, Wien, Austria W. Adam, E. Asilar, T. Bergauer, J. Brandstetter, E. Brondolin, M. Dragicevic, J. Er¨o, M. Flechl, M. Friedl, R. Fr¨uhwirth1, V.M. Ghete, C. Hartl, N. H¨ormann, J. Hrubec, M. Jeitler1, A. K¨onig, I. Kr¨atschmer, D. Liko, T. Matsushita, I. Mikulec, D. Rabady, N. Rad, B. Rahbaran, H. Rohringer, J. Schieck1, J. Strauss, W. Waltenberger, C.-E. Wulz1 Institute for Nuclear Problems, Minsk, Belarus V. Chekhovsky, O. Dvornikov, Y. Dydyshka, I. Emeliantchik, A. Litomin, V. Makarenko, V M l R St f it h J S G l V Z k Yerevan Physics Institute, Yerevan, Armenia A.M. Sirunyan, A. Tumasyan Institut f¨ur Hochenergiephysik, Wien, Austria W. Adam, E. Asilar, T. Bergauer, J. Brandstetter, E. Brondolin, M. Dragicevic, J. Er¨o, M. Flechl, M. Friedl, R. Fr¨uhwirth1, V.M. Ghete, C. Hartl, N. H¨ormann, J. Hrubec, M. Jeitler1, A. K¨onig, I. Kr¨atschmer, D. Liko, T. Matsushita, I. Mikulec, D. Rabady, N. Rad, B. Rahbaran, H. Rohringer, J. Schieck1, J. Strauss, W. Waltenberger, C.-E. Wulz1 Institute for Nuclear Problems Minsk Belarus Institut f¨ur Hochenergiephysik, Wien, Austria W. Adam, E. Asilar, T. Bergauer, J. Brandstetter, E. Brondolin, M. Dragicevic, J. Er¨o, M. Flechl, M. Friedl, R. Fr¨uhwirth1, V.M. Ghete, C. Hartl, N. H¨ormann, J. Hrubec, M. Jeitler1, A. K¨onig, I. Kr¨atschmer, D. Liko, T. Matsushita, I. Mikulec, D. Rabady, N. Rad, B. Rahbaran, H. Rohringer, J. Schieck1, J. Strauss, W. Waltenberger, C.-E. Wulz1 JHEP03(2017)162 V. Chekhovsky, O. Dvornikov, Y. Dydyshka, I. Emeliantchik, A. Litomin, V. Makarenko, V. Mossolov, R. Stefanovitch, J. Suarez Gonzalez, V. Zykunov National Centre for Particle and High Energy Physics, Minsk, Belarus N. References Ridolﬁ, The t¯t cross-section at 1.8-TeV and 1.96-TeV: A Study of the systematics due to parton densities and scale dependence, JHEP 04 (2004) 068 [hep-ph/0303085] [INSPIRE]. [69] S. Catani, D. de Florian, M. Grazzini and P. Nason, Soft gluon resummation for Higgs boson production at hadron colliders, JHEP 07 (2003) 028 [hep-ph/0306211] [INSPIRE]. [70] G. Cowan, K. Cranmer, E. Gross and O. Vitells, Asymptotic formulae for likelihood-based tests of new physics, Eur. Phys. J. C 71 (2011) 1554 [Erratum ibid. C 73 (2013) 2501] [arXiv:1007.1727] [INSPIRE]. [71] A.L. Read, Presentation of search results: The CLS technique, J. Phys. G 28 (2002) 2693 [INSPIRE]. [72] T. Junk, Conﬁdence level computation for combining searches with small statistics, Nucl. Instrum. Meth. A 434 (1999) 435 [hep-ex/9902006] [INSPIRE]. [73] ATLAS and CMS collaboration, Combined Measurement of the Higgs Boson Mass in pp Collisions at √s = 7 and 8 TeV with the ATLAS and CMS Experiments, Phys. Rev. Lett. 114 (2015) 191803 [arXiv:1503.07589] [INSPIRE]. – 36 – The CMS collaboration Shumeiko Universiteit Antwerpen, Antwerpen, Belgium S. Alderweireldt, E.A. De Wolf, X. Janssen, J. Lauwers, M. Van De Klundert, H. Van Haevermaet, P. Van Mechelen, N. Van Remortel, A. Van Spilbeeck Vrije Universiteit Brussel, Brussel, Belgium S. Abu Zeid, F. Blekman, J. D’Hondt, N. Daci, I. De Bruyn, K. Deroover, S. Lowette, S. Moortgat, L. Moreels, A. Olbrechts, Q. Python, K. Skovpen, S. Tavernier, W. Van Doninck, P. Van Mulders, I. Van Parijs Universit´e Libre de Bruxelles, Bruxelles, Belgium H. Brun, B. Clerbaux, G. De Lentdecker, H. Delannoy, G. Fasanella, L. Favart, R. Goldouzian, A. Grebenyuk, G. Karapostoli, T. Lenzi, A. L´eonard, J. Luetic, T. Maer- schalk, A. Marinov, A. Randle-conde, T. Seva, C. Vander Velde, P. Vanlaer, D. Vannerom, Q. Wang, R. Yonamine, F. Zenoni, F. Zhang2 Ghent University, Ghent, Belgium A. Cimmino, T. Cornelis, D. Dobur, A. Fagot, M. Gul, I. Khvastunov, D. Poyraz, S. Salva, R. Sch¨ofbeck, M. Tytgat, W. Van Driessche, E. Yazgan, N. Zaganidis Universit´e Catholique de Louvain, Louvain-la-Neuve, Belgium H. Bakhshiansohi, C. Beluﬃ3, O. Bondu, S. Brochet, G. Bruno, A. Caudron, S. De Visscher, C. Delaere, M. Delcourt, B. Francois, A. Giammanco, A. Jafari, M. Komm, G. Krin- tiras, V. Lemaitre, A. Magitteri, A. Mertens, M. Musich, C. Nuttens, K. Piotrzkowski, L. Quertenmont, M. Selvaggi, M. Vidal Marono, S. Wertz Universit´e de Mons, Mons, Belgium N. Beliy Brazil S. Ahujaa, C.A. Bernardesa, S. Dograa, T.R. Fernandez Perez Tomeia, E.M. Gregores S. Ahujaa, C.A. Bernardesa, S. Dograa, T.R. Fernandez Perez Tomeia, E.M. Gregoresb, P.G. Mercadanteb, C.S. Moona, S.F. Novaesa, Sandra S. Padulaa, D. Romero Abadb, J.C. Ruiz Vargasa JHEP03(2017)162 J.C. Ruiz Vargasa Institute for Nuclear Research and Nuclear Energy, Soﬁa, Bulgaria A. Aleksandrov, R. Hadjiiska, P. Iaydjiev, M. Rodozov, S. Stoykova, G. Sultanov, M. Vu- tova Universit´e de Mons, Mons, Belgium N. Beliy Centro Brasileiro de Pesquisas Fisicas, Rio de Janeiro, Brazil W.L. Ald´a J´unior, F.L. Alves, G.A. Alves, L. Brito, C. Hensel, A. Moraes, M.E. Pol, P. Rebello Teles – 37 – Universidade do Estado do Rio de Janeiro, Rio de Janeiro, Brazil E. Belchior Batista Das Chagas, W. Carvalho, J. Chinellato4, A. Cust´odio, E.M. Da Costa, G.G. Da Silveira5, D. De Jesus Damiao, C. De Oliveira Martins, S. Fonseca De Souza, L.M. Huertas Guativa, H. Malbouisson, D. Matos Figueiredo, C. Mora Herrera, L. Mundim, H. Nogima, W.L. Prado Da Silva, A. Santoro, A. Sznajder, E.J. Tonelli Manganote4, A. Vilela Pereira Universidade do Estado do Rio de Janeiro, Rio de Janeiro, Brazil H. Nogima, W.L. Prado Da Silva, A. Santoro, A. Sznajder, E.J. Tonelli Manganote4, A. Vilela Pereira Universidade Estadual Paulista a, Universidade Federal do ABC b, S˜ao Paulo, Brazil University of Soﬁa, Soﬁa, Bulgaria University of Soﬁa, Soﬁa, Bulgaria A. Dimitrov, I. Glushkov, L. Litov, B. Pavlov, P. Petkov Beihang University, Beijing, China W. Fang6 Beihang University, Beijing, China W. Fang6 Institute of High Energy Physics, Beijing, China Institute of High Energy Physics, Beijing, China M. Ahmad, J.G. Bian, G.M. Chen, H.S. Chen, M. Chen, Y. Chen7, T. Cheng, C.H. Jiang, M. Ahmad, J.G. Bian, G.M. Chen, H.S. Chen, M. Chen, Y. Chen7, T. Cheng, C.H. Jiang, D. Leggat, Z. Liu, F. Romeo, M. Ruan, S.M. Shaheen, A. Spiezia, J. Tao, C. Wang, Z. Wang, H. Zhang, J. Zhao State Key Laboratory of Nuclear Physics and Technology, Peking University, Beijing, China Y. Ban, G. Chen, H. Huang, Q. Li, S. Liu, Y. Mao, S.J. Qian, D. Wang, Z. Xu Universidad de Los Andes, Bogota, Colombia C. Avila, A. Cabrera, L.F. Chaparro Sierra, C. Florez, J.P. Gomez, C.F. Gonz´alez Hern´andez, J.D. Ruiz Alvarez, J.C. Sanabria University of Split, Faculty of Electrical Engineering, Mechanical Engineering and Naval Architecture, Split, Croatia N. Godinovic, D. Lelas, I. Puljak, P.M. Ribeiro Cipriano, T. Sculac N. Godinovic, D. Lelas, I. Puljak, P.M. Ribeiro Cipriano, T. Sculac University of Split, Faculty of Science, Split, Croatia Z. Antunovic, M. Kovac University of Split, Faculty of Science, Split, Croatia Z. Antunovic, M. Kovac Institute Rudjer Boskovic, Zagreb, Croatia V. Brigljevic, D. Ferencek, K. Kadija, B. Mesic, T. Susa Institute Rudjer Boskovic, Zagreb, Croatia Universidad San Francisco de Quito, Quito, Ecuador E. Carrera Jarrin Academy of Scientiﬁc Research and Technology of the Arab Republic of Egypt, Egyptian Network of High Energy Physics, Cairo, Egypt A. Ellithi Kamel9, M.A. Mahmoud10,11, A. Radi11,12 National Institute of Chemical Physics and Biophysics, Tallinn, Estonia M. Kadastik, L. Perrini, M. Raidal, A. Tiko, C. Veelken JHEP03(2017)162 Department of Physics, University of Helsinki, Helsinki, Fin P. Eerola, J. Pekkanen, M. Voutilainen Helsinki Institute of Physics, Helsinki, Finland J. H¨ark¨onen, T. J¨arvinen, V. Karim¨aki, R. Kinnunen, T. Lamp´en, K. Lassila-Perini, S. Lehti, T. Lind´en, P. Luukka, J. Tuominiemi, E. Tuovinen, L. Wendland Helsinki Institute of Physics, Helsinki, Finland Lappeenranta University of Technology, Lappeenranta, Finland J. Talvitie, T. Tuuva IRFU, CEA, Universit´e Paris-Saclay, Gif-sur-Yvette, France M. Besancon, F. Couderc, M. Dejardin, D. Denegri, B. Fabbro, J.L. Faure, C. Favaro, F. Ferri, S. Ganjour, S. Ghosh, A. Givernaud, P. Gras, G. Hamel de Monchenault, P. Jarry, I. Kucher, E. Locci, M. Machet, J. Malcles, J. Rander, A. Rosowsky, M. Titov, A. Zghiche Laboratoire Leprince-Ringuet, Ecole Polytechnique, IN2P3-CNRS, Palaiseau, France A. Abdulsalam, I. Antropov, S. Baﬃoni, F. Beaudette, P. Busson, L. Cadamuro, E. Chapon, C. Charlot, O. Davignon, R. Granier de Cassagnac, M. Jo, S. Lisniak, P. Min´e, M N C O h d G O t P P i i P Pi d S R d R S l A. Abdulsalam, I. Antropov, S. Baﬃoni, F. Beaudette, P. Busson, L. Cadamuro, E. Chapon, C. Charlot, O. Davignon, R. Granier de Cassagnac, M. Jo, S. Lisniak, P. Min´e, M. Nguyen, C. Ochando, G. Ortona, P. Paganini, P. Pigard, S. Regnard, R. Salerno, Y. Sirois, T. Strebler, Y. Yilmaz, A. Zabi E. Chapon, C. Charlot, O. Davignon, R. Granier de Cassagnac, M. Jo, S. Lisniak, P. Mine, M. Nguyen, C. Ochando, G. Ortona, P. Paganini, P. Pigard, S. Regnard, R. Salerno, Y. Sirois, T. Strebler, Y. Yilmaz, A. Zabi Institut Pluridisciplinaire Hubert Curien (IPHC), Universit´e de Strasbourg, CNRS-IN2P3 J.-L. Agram13, J. Andrea, A. Aubin, D. Bloch, J.-M. Brom, M. Buttignol, E.C. Chabert, N. Chanon, C. Collard, E. Conte13, X. Coubez, J.-C. Fontaine13, D. Gel´e, U. Goerlach, A.-C. Le Bihan, P. Van Hove Centre de Calcul de l’Institut National de Physique Nucleaire et de Physique des Particules, CNRS/IN2P3, Villeurbanne, France S. Gadrat Universit´e de Lyon, Universit´e Claude Bernard Lyon 1, CNRS-IN2P3, Institut de Physique Nucl´eaire de Lyon, Villeurbanne, France S. Beauceron, C. Bernet, G. Boudoul, C.A. Carrillo Montoya, R. Chierici, D. Contard S. Beauceron, C. Institute Rudjer Boskovic, Zagreb, Croatia V. Brigljevic, D. Ferencek, K. Kadija, B. Mesic, T. Susa V. Brigljevic, D. Ferencek, K. Kadija, B. Mesic, T. Susa University of Cyprus, Nicosia, Cyprus A. Attikis, G. Mavromanolakis, J. Mousa, C. Nicolaou, F. Ptochos, P.A. Razis, H. Rykaczewski, D. Tsiakkouri – 38 – Charles University, Prague, Czech Republic M. Finger8, M. Finger Jr.8 M. Finger8, M. Finger Jr.8 Universidad San Francisco de Quito, Quito, Ecuador E. Carrera Jarrin Universidad San Francisco de Quito, Quito, Ecuador E. Carrera Jarrin Bernet, G. Boudoul, C.A. Carrillo Montoya, R. Chierici, D. Contardo, B. Courbon, P. Depasse, H. El Mamouni, J. Fan, J. Fay, S. Gascon, M. Gouzevitch, B. Courbon, P. Depasse, H. El Mamouni, J. Fan, J. Fay, S. Gascon, M. Gouzevitch, – 39 – G. Grenier, B. Ille, F. Lagarde, I.B. Laktineh, M. Lethuillier, L. Mirabito, A.L. Pequegnot, S P A P 14 D S b V S d M V d D k P V d S V Grenier, B. Ille, F. Lagarde, I.B. Laktineh, M. Lethuillier, L. Mirabito, A.L. Pequegnot, renier, B. Ille, F. Lagarde, I.B. Laktineh, M. Lethuillier, L. Mirabito, A.L. Pequegnot, rries, A. Popov14, D. Sabes, V. Sordini, M. Vander Donckt, P. Verdier, S. Viret Perries, A. Popov14, D. Sabes, V. Sordini, M. Vander Donckt, P. Verdier, S. Viret Georgian Technical University, Tbilisi, Georgia T. Toriashvili15 Tbilisi State University, Tbilisi, Georgia D. Lomidze RWTH Aachen University, I. Physikalisches Institut, Aachen, Germany C. Autermann, S. Beranek, L. Feld, M.K. Kiesel, K. Klein, M. Lipinski, M. Preuten, C. Schomakers, J. Schulz, T. Verlage JHEP03(2017)162 RWTH Aachen University, III. Physikalisches Institut A, Aachen, Germany A. Albert, M. Brodski, E. Dietz-Laursonn, D. Duchardt, M. Endres, M. Erdmann, S. Erd- weg, T. Esch, R. Fischer, A. G¨uth, M. Hamer, T. Hebbeker, C. Heidemann, K. Hoepfner, S. Knutzen, M. Merschmeyer, A. Meyer, P. Millet, S. Mukherjee, M. Olschewski, K. Padeken, T. Pook, M. Radziej, H. Reithler, M. Rieger, F. Scheuch, L. Sonnenschein, D. Teyssier, S. Th¨uer RWTH Aachen University, III. Physikalisches Institut B, Aachen, Germany V. Cherepanov, G. Fl¨ugge, B. Kargoll, T. Kress, A. K¨unsken, J. Lingemann, T. M¨uller, A. Nehrkorn, A. Nowack, C. Pistone, O. Pooth, A. Stahl16 Deutsches Elektronen-Synchrotron, Hamburg, Germany Deutsches Elektronen-Synchrotron, Hamburg, Germany M. Aldaya Martin, T. Arndt, C. Asawatangtrakuldee, K. Beernaert, O. Behnke, U. Behrens, A.A. Bin Anuar, K. Borras17, A. Campbell, P. Connor, C. Contreras- Campana, F. Costanza, C. Diez Pardos, G. Dolinska, G. Eckerlin, D. Eckstein, T. Eichhorn, E. Eren, E. Gallo18, J. Garay Garcia, A. Geiser, A. Gizhko, J.M. Grados Luyando, A. Grohsjean, P. Gunnellini, A. Harb, J. Hauk, M. Hempel19, H. Jung, A. Kalogeropoulos, O. Karacheban19, M. Kasemann, J. Keaveney, C. Kleinwort, I. Korol, D. Kr¨ucker, W. Lange, A. Lelek, J. Leonard, K. Lipka, A. Lobanov, W. Lohmann19, R. Mankel, I.- A. Melzer-Pellmann, A.B. Meyer, G. Mittag, J. Mnich, A. Mussgiller, E. Ntomari, D. Pitzl, R. Placakyte, A. Raspereza, B. Roland, M. ¨O. Sahin, P. Saxena, T. Schoerner-Sadenius, C. Seitz, S. Spannagel, N. Stefaniuk, G.P. Van Onsem, R. Walsh, C. Wissing y g, g, y V. Blobel, M. Centis Vignali, A.R. Draeger, T. Dreyer, E. Garutti, D. Gonzalez, J. Haller, M. Hoﬀmann, A. Junkes, R. Klanner, R. Kogler, N. Kovalchuk, T. Lapsien, T. Lenz, I. Marchesini, D. Marconi, M. Meyer, M. Niedziela, D. Nowatschin, F. Pantaleo16, T. Peiﬀer, A. Perieanu, J. Poehlsen, C. Sander, C. Scharf, P. Schleper, A. Schmidt, S. Schumann, J. Schwandt, H. Stadie, G. Steinbr¨uck, F.M. Stober, M. St¨over, H. Tholen, D. Troendle, E. Usai, L. Vanelderen, A. Vanhoefer, B. Vormwald V. Blobel, M. Centis Vignali, A.R. Draeger, T. Dreyer, E. Garutti, D. Gonzalez, J. Haller, M. Hoﬀmann, A. Junkes, R. Klanner, R. Kogler, N. Kovalchuk, T. Lapsien, T. Lenz, I. Marchesini, D. Marconi, M. Meyer, M. Niedziela, D. Nowatschin, F. Pantaleo16, T. Peiﬀer, A. Perieanu, J. Poehlsen, C. Sander, C. Scharf, P. Schleper, A. Schmidt, S. Schumann, J. Schwandt, H. Stadie, G. Steinbr¨uck, F.M. Stober, M. St¨over, H. Tholen, D. Troendle, E. Usai, L. Vanelderen, A. Vanhoefer, B. Vormwald Institut f¨ur Experimentelle Kernphysik, Karlsruhe, Germany M. Akbiyik, C. Barth, S. Baur, C. Baus, J. Berger, E. Butz, R. Caspart, T. Chwalek, F. Colombo, W. De Boer, A. Dierlamm, S. Fink, B. Freund, R. Friese, M. Giﬀels, A. Gilbert, M. Akbiyik, C. Barth, S. Baur, C. Baus, J. Berger, E. Butz, R. Caspart, T. Chwale F C l b W D B A Di l S Fi k B F d R F i M Giﬀl A Gilb M. Akbiyik, C. Barth, S. Baur, C. Baus, J. Berger, E. Butz, R. Caspart, T. Chwalek, F. Colombo, W. De Boer, A. Deutsches Elektronen-Synchrotron, Hamburg, Germany Dierlamm, S. Fink, B. Freund, R. Friese, M. Giﬀels, A. Gilbert, y , , , , g , , p , , F. Colombo, W. De Boer, A. Dierlamm, S. Fink, B. Freund, R. Friese, M. Giﬀels, A. Gilbert, – 40 – P. Goldenzweig, D. Haitz, F. Hartmann16, S.M. Heindl, U. Husemann, I. Katkov14, P. Goldenzweig, D. Haitz, F. Hartmann16, S.M. Heindl, U. Husemann, I. Katkov14, S. Kudella, H. Mildner, M.U. Mozer, Th. M¨uller, M. Plagge, G. Quast, K. Rabbertz, S. R¨ocker, F. Roscher, D. Sch¨afer, M. Schr¨oder, I. Shvetsov, G. Sieber, H.J. Simonis, S. Kudella, H. Mildner, M.U. Mozer, Th. M¨uller, M. Plagge, G. Quast, K. Rabbert S. Kudella, H. Mildner, M.U. Mozer, Th. M¨uller, M. Plagge, G. Quast, K. Rabbertz, S. R¨ocker, F. Roscher, D. Sch¨afer, M. Schr¨oder, I. Shvetsov, G. Sieber, H.J. Simonis, R. Ulrich, S. Wayand, M. Weber, T. Weiler, S. Williamson, C. W¨ohrmann, R. Wolf S. R¨ocker, F. Roscher, D. Sch¨afer, M. Schr¨oder, I. Shvetsov, G. Sieber, H.J. Simonis, S. R¨ocker, F. Roscher, D. Sch¨afer, M. Schr¨oder, I. Shvetsov, G. Sieber, H.J. Simonis, R Ulrich S Wayand M Weber T Weiler S Williamson C W¨ohrmann R Wolf R. Ulrich, S. Wayand, M. Weber, T. Weiler, S. Williamson, C. W¨ohrmann, R. Wolf Institute of Nuclear and Particle Physics (INPP), NCSR Demokritos, Aghia Paraskevi, Greece G. Anagnostou, G. Daskalakis, T. Geralis, V.A. Giakoumopoulou, A. Kyriakis, D. Loukas, I. Topsis-Giotis National and Kapodistrian University of Athens, Athens, Greece National and Kapodistrian University of Athens, Athens, Greece S. Kesisoglou, A. Panagiotou, N. Saoulidou, E. Tziaferi JHEP03(2017)162 JHEP03(2017)162 National and Kapodistrian University of Athens, Athens, Greece S. Kesisoglou, A. Panagiotou, N. Saoulidou, E. Tziaferi S. Kesisoglou, A. Panagiotou, N. Saoulidou, E. Tziaferi University of Io´annina, Io´annina, Greece University of Io´annina, Io´annina, Greece I. Evangelou, G. Flouris, C. Foudas, P. Kokkas, N. Loukas, N. Manthos, I. Papadopoulos, E. Paradas MTA-ELTE Lend¨ulet CMS Particle and Nuclear Physics Group, E¨otv¨os Lor´and University, Budapest, Hungary N. Filipovic Wigner Research Centre for Physics, Budapest, Hungary G. Bencze, C. Hajdu, D. Horvath20, F. Sikler, V. Veszpremi, G. Vesztergombi21, A.J. Zsig- mond Institute of Nuclear Research ATOMKI, Debrecen, Hungary N. Beni, S. Czellar, J. Karancsi22, A. Makovec, J. Molnar, Z. Szillasi Institute of Physics, University of Debrecen M. Bart´ok21, P. Raics, Z.L. Trocsanyi, B. Ujvari National Institute of Science Education and Research, Bhubaneswar, India S. Bahinipati, S. Choudhury23, P. Mal, K. Mandal, A. Nayak24, D.K. Sahoo, N. Deutsches Elektronen-Synchrotron, Hamburg, Germany Sahoo, S.K. Swain Panjab University, Chandigarh, India Panjab University, Chandigarh, India S. Bansal, S.B. Beri, V. Bhatnagar, R. Chawla, U.Bhawandeep, A.K. Kalsi, A. Kaur, M. Kaur, R. Kumar, P. Kumari, A. Mehta, M. Mittal, J.B. Singh, G. Walia Panjab University, Chandigarh, India S. Bansal, S.B. Beri, V. Bhatnagar, R. Chawla, U.Bhawandeep, A.K. Kalsi, A. Kaur, M. Kaur, R. Kumar, P. Kumari, A. Mehta, M. Mittal, J.B. Singh, G. Walia University of Delhi, Delhi, India M. Kaur, R. Kumar, P. Kumari, A. Mehta, M. Mittal, J.B. Singh, G. Walia University of Delhi, Delhi, India Ashok Kumar, A. Bhardwaj, B.C. Choudhary, R.B. Garg, S. Keshri, S. Malhotra, M. Naimuddin, N. Nishu, K. Ranjan, R. Sharma, V. Sharma Saha Institute of Nuclear Physics, Kolkata, India Bhabha Atomic Research Centre, Mumbai, India R. Chudasama, D. Dutta, V. Jha, V. Kumar, A.K. Mohanty16, P.K. Netrakanti, L.M. Pant, P. Shukla, A. Topkar Tata Institute of Fundamental Research-A, Mumbai, India Tata Institute of Fundamental Research-A, Mumbai, India T. Aziz, S. Dugad, G. Kole, B. Mahakud, S. Mitra, G.B. Mohanty, B. Parida, N. Sur, B. Sutar T. Aziz, S. Dugad, G. Kole, B. Mahakud, S. Mitra, G.B. Mohanty, B. Parida, N. Sur, B. Sutar Saha Institute of Nuclear Physics, Kolkata, India Saha Institute of Nuclear Physics, Kolkata, India R. Bhattacharya, S. Bhattacharya, K. Chatterjee, S. Dey, S. Dutt, S. Dutta, S. Ghosh, N. Majumdar, A. Modak, K. Mondal, S. Mukhopadhyay, S. Nandan, A. Purohit, A. Roy, D. Roy, S. Roy Chowdhury, S. Sarkar, M. Sharan, S. Thakur y , , Bhattacharya, S. Bhattacharya, K. Chatterjee, S. Dey, S. Dutt, S. Dutta, S. Ghosh, Bhattacharya, S. Bhattacharya, K. Chatterjee, S. Dey, S. Dutt, S. Dutta, S. Ghosh, Majumdar, A. Modak, K. Mondal, S. Mukhopadhyay, S. Nandan, A. Purohit, A. Roy, Roy, S. Roy Chowdhury, S. Sarkar, M. Sharan, S. Thakur Indian Institute of Technology Madras, Madras, India P.K. Behera P.K. Behera – 41 – Bhabha Atomic Research Centre, Mumbai, India Tata Institute of Fundamental Research-B, Mumbai, India S. Banerjee, S. Bhowmik25, R.K. Dewanjee, S. Ganguly, M. Guchait, Sa. Jain, S. Kumar, M. Maity25, G. Majumder, K. Mazumdar, T. Sarkar25, N. Wickramage26 JHEP03(2017)162 Indian Institute of Science Education and Research (IISER), Pune, India S. Chauhan, S. Dube, V. Hegde, A. Kapoor, K. Kothekar, S. Pandey, A. Rane, S. Sharma Institute for Research in Fundamental Sciences (IPM), Tehran, Iran Institute for Research in Fundamental Sciences (IPM), Tehran, Iran S. Chenarani27, E. Eskandari Tadavani, S.M. Etesami27, M. Khakzad, M. Mohammadi Najafabadi, M. Naseri, S. Paktinat Mehdiabadi28, F. Rezaei Hosseinabadi, B. Safarzadeh29, M. Zeinali S. Chenarani27, E. Eskandari Tadavani, S.M. Etesami27, M. Khakzad, M. Mohamm Najafabadi, M. Naseri, S. Paktinat Mehdiabadi28, F. Rezaei Hosseinabadi, B. Safarzadeh2 M. Zeinali University College Dublin, Dublin, Ireland M. Felcini, M. Grunewald Italy JHEP03(2017)162 S. Buontempoa, N. Cavalloa,c, G. De Nardo, S. Di Guidaa,d,16, M. Espositoa,b, F. Fabozzia,c, F. Fiengaa,b, A.O.M. Iorioa,b, G. Lanzaa, L. Listaa, S. Meolaa,d,16, P. Paoluccia,16, C. Sciaccaa,b, F. Thyssena INFN Sezione di Padova a, Universit`a di Padova b, Padova, Italy, Universit`a di Trento c, Trento, Italy University College Dublin, Dublin, Ireland M. Felcini, M. Grunewald INFN Sezione di Bari a, Universit`a di Bari b, Politecnico di Bari c, Bari, Italy M. Abbresciaa,b, C. Calabriaa,b, C. Caputoa,b, A. Colaleoa, D. Creanzaa,c, L. Cristellaa,b, N. De Filippisa,c, M. De Palmaa,b, L. Fiorea, G. Iasellia,c, G. Maggia,c, M. Maggia, G. Minielloa,b, S. Mya,b, S. Nuzzoa,b, A. Pompilia,b, G. Pugliesea,c, R. Radognaa,b, A. Ranieria, G. Selvaggia,b, A. Sharmaa, L. Silvestrisa,16, R. Vendittia,b, P. Verwilligena INFN Sezione di Bologna a, Universit`a di Bologna b, Bologna, Italy G. Abbiendia, C. Battilana, D. Bonacorsia,b, S. Braibant-Giacomellia,b, L. Brigliadoria,b, R. Campaninia,b, P. Capiluppia,b, A. Castroa,b, F.R. Cavalloa, S.S. Chhibraa,b, G. Codispotia,b, M. Cuﬃania,b, G.M. Dallavallea, F. Fabbria, A. Fanfania,b, D. Fasanellaa,b, P. Giacomellia, C. Grandia, L. Guiduccia,b, S. Marcellinia, G. Masettia, A. Montanaria, F.L. Navarriaa,b, A. Perrottaa, A.M. Rossia,b, T. Rovellia,b, G.P. Sirolia,b, N. Tosia,b,16 P. Giacomellia, C. Grandia, L. Guiduccia,b, S. Marcellinia, G. Masettia, A. Montanar F.L. Navarriaa,b, A. Perrottaa, A.M. Rossia,b, T. Rovellia,b, G.P. Sirolia,b, N. Tosia,b,16 INFN Sezione di Catania a, Universit`a di Catania b, Catania, Italy S. Albergoa,b, S. Costaa,b, A. Di Mattiaa, F. Giordanoa,b, R. Potenzaa,b, A. Tricomia,b, C. Tuvea,b INFN Sezione di Firenze a, Universit`a di Firenze b, Firenze, Italy G. Barbaglia, V. Ciullia,b, C. Civininia, R. D’Alessandroa,b, E. Focardia,b, P. Lenzia,b, M. Meschinia, S. Paolettia, L. Russoa,30, G. Sguazzonia, D. Stroma, L. Viliania,b,16 INFN Laboratori Nazionali di Frascati, Frascati, Italy L. Benussi, S. Bianco, F. Fabbri, D. Piccolo, F. Primavera16 INFN Sezione di Genova a, Universit`a di Genova b, Genova, Italy V. Calvellia,b, F. Ferroa, M.R. Mongea,b, E. Robuttia, S. Tosia,b – 42 – INFN Sezione di Milano-Bicocca a, Universit`a di Milano-Bicocca b, Milano, Italy L. Brianzaa,b,16, F. Brivioa,b, V. Cirioloa,b, M.E. Dinardoa,b, S. Fiorendia,b,16, S. Gennai A. Ghezzia,b, P. Govonia,b, M. Malbertia,b, S. Malvezzia, R.A. Manzonia,b, D. Menasce L. Moronia, M. Paganonia,b, D. Pedrinia, S. Pigazzinia,b, S. Ragazzia,b, T. Tabarelli de Fatisa,b INFN Sezione di Napoli a, Universit`a di Napoli ’Federico II’ b, Napoli, Italy, Universit`a della Basilicata c, Potenza, Italy, Universit`a G. Marconi d, Roma, Italy Trento c, Trento, Italy P. Azzia,16, N. Bacchettaa, L. Benatoa,b, D. Biselloa,b, A. Bolettia,b, R. Carlina P. Azzia,16, N. Bacchettaa, L. Benatoa,b, D. Biselloa,b, A. Bolettia,b, R. Carlina,b, P. Checchiaa, M. Dall’Ossoa,b, P. De Castro Manzanoa, T. Dorigoa, U. Dossellia, F. Gasparinia,b, U. Gasparinia,b, A. Gozzelinoa, M. Margonia,b, A.T. Meneguzzoa,b, M. Michelottoa, J. Pazzinia,b, M. Pegoraroa, N. Pozzobona,b, P. Ronchesea,b, E. Torassaa, M. Zanettia,b, P. Zottoa,b, G. Zumerlea,b INFN Sezione di Pavia a, Universit`a di Pavia b, Pavia, Italy A. Braghieria, F. Fallavollitaa,b, A. Magnania,b, P. Montagnaa,b, S.P. Rattia,b, V. Rea, C. Riccardia,b, P. Salvinia, I. Vaia,b, P. Vituloa,b INFN Sezione di Perugia a, Universit`a di Perugia b, Perugia, Italy L. Alunni Solestizia,b, G.M. Bileia, D. Ciangottinia,b, L. Fan`oa,b, P. Laricciaa,b, R. Leonardia,b, G. Mantovania,b, M. Menichellia, A. Sahaa, A. Santocchiaa,b INFN Sezione di Pisa a, Universit`a di Pisa b, Scuola Normale Superiore di Pisa c, Pisa, Italy INFN Sezione di Pisa a, Universit`a di Pisa b, Scuola Normale Superiore di Pisa c, Pisa, Italy K. Androsova,30, P. Azzurria,16, G. Bagliesia, J. Bernardinia, T. Boccalia, R. Castaldia, M.A. Cioccia,30, R. Dell’Orsoa, S. Donatoa,c, G. Fedi, A. Giassia, M.T. Grippoa,30, F. Ligabuea,c, T. Lomtadzea, L. Martinia,b, A. Messineoa,b, F. Pallaa, A. Rizzia,b, A. Savoy- Navarroa,31, P. Spagnoloa, R. Tenchinia, G. Tonellia,b, A. Venturia, P.G. Verdinia K. Androsova,30, P. Azzurria,16, G. Bagliesia, J. Bernardinia, T. Boccalia, R. Castaldia, M.A. Cioccia,30, R. Dell’Orsoa, S. Donatoa,c, G. Fedi, A. Giassia, M.T. Grippoa,30, F. Ligabuea,c, T. Lomtadzea, L. Martinia,b, A. Messineoa,b, F. Pallaa, A. Rizzia,b, A. Savoy- Navarroa,31, P. Spagnoloa, R. Tenchinia, G. Tonellia,b, A. Venturia, P.G. Verdinia INFN Sezione di Roma a, Universit`a di Roma b, Roma, Italy L. Baronea,b, F. Cavallaria, M. Cipriania,b, D. Del Rea,b,16, M. Diemoza, S. Gellia,b, E. Longoa,b, F. Margarolia,b, B. Marzocchia,b, P. Meridiania, G. Organtinia,b, R. Paramattia, F. Preiatoa,b, S. Rahatloua,b, C. Rovellia, F. Santanastasioa,b INFN Sezione di Torino a, Universit`a di Torino b, Torino, Italy, Universit`a del Piemonte Orientale c, Novara, Italy N. Amapanea,b, R. Arcidiaconoa,c,16, S. Argiroa,b, M. Arneodoa,c, N. Bartosika, Amapanea,b, R. Arcidiaconoa,c,16, S. Argiroa,b, M. Arneodoa,c, N. Bartosika, ellana,b, C. Biinoa, N. Cartigliaa, F. Cennaa,b, M. Costaa,b, R. Covarellia,b, Deganoa,b, N. Demariaa, L. Fincoa,b, B. Kiania,b, C. Mariottia, S. Masellia, Amapanea,b, R. Arcidiaconoa,c,16, S. Argiroa,b, M. Arneodoa,c, N. Bartosika, Bellana,b, C. Biinoa, N. Cartigliaa, F. Cennaa,b, M. Costaa,b, R. Covarellia,b, b b b N. Amapane , , R. Arcidiacono , , , S. Argiro , , M. Hanyang University, Seoul, Korea Hanyang University, Seoul, Korea J.A. Brochero Cifuentes, T.J. Kim Korea University, Seoul, Korea S. Cho, S. Choi, Y. Go, D. Gyun, S. Ha, B. Hong, Y. Jo, Y. Kim, K. Lee, K.S. Lee, S. Lee, J. Lim, S.K. Park, Y. Roh J. Almond, J. Kim, H. Lee, S.B. Oh, B.C. Radburn-Smith, S.h. Seo, U.K. Yang, H.D. Yoo, G.B. Yu Trento c, Trento, Italy Arneodo , , N. Bartosik R. Bellana,b, C. Biinoa, N. Cartigliaa, F. Cennaa,b, M. Costaa,b, R. Covarellia R. Bellana,b, C. Biinoa, N. Cartigliaa, F. Cennaa,b, M. Costaa,b, R. Covarellia,b, A. Deganoa,b, N. Demariaa, L. Fincoa,b, B. Kiania,b, C. Mariottia, S. Masellia, A. Deganoa,b, N. Demariaa, L. Fincoa,b, B. Kiania,b, C. Mariottia, S. Masellia, – 43 – E. Migliorea,b, V. Monacoa,b, E. Monteila,b, M. Montenoa, M.M. Obertinoa,b, L. Pachera,b, N P t a M P lli i ia G L Pi A i ia b F R a b A R a b E. Migliorea,b, V. Monacoa,b, E. Monteila,b, M. Montenoa, M.M. Obertinoa,b, L. Pachera N. Pastronea, M. Pelliccionia, G.L. Pinna Angionia,b, F. Raveraa,b, A. Romeroa M. Ruspaa,c, R. Sacchia,b, K. Shchelinaa,b, V. Solaa, A. Solanoa,b, A. Staianoa, P. Traczyka,b P. Traczyka,b INFN Sezione di Trieste a, Universit`a di Trieste b, Trieste, Italy S. Belfortea, M. Casarsaa, F. Cossuttia, G. Della Riccaa,b, A. Zanettia Kyungpook National University, Daegu, Korea y gp y, g , D.H. Kim, G.N. Kim, M.S. Kim, S. Lee, S.W. Lee, Y.D. Oh, S. Sekmen, D.C. Son, Y.C. Yang JHEP03(2017)162 Chonbuk National University, Jeonju, Korea A. Lee Chonbuk National University, Jeonju, Korea A. Lee Chonnam National University, Institute for Universe and Elementary Particles, Kwangju, Korea H Ki Laborat´orio de Instrumenta¸c˜ao e F´ısica Experimental de Part´ıculas, Lisboa, Portugal Laborat´orio de Instrumenta¸c˜ao e F´ısica Experimental de Part´ıculas, Lisboa, Portugal University of Seoul, Seoul, Korea M. Choi, H. Kim, J.H. Kim, J.S.H. Lee, I.C. Park, G. Ryu, M.S. Ryu Sungkyunkwan University, Suwon, Korea Y. Choi, J. Goh, C. Hwang, J. Lee, I. Yu Sungkyunkwan University, Suwon, Korea Y. Choi, J. Goh, C. Hwang, J. Lee, I. Yu Sungkyunkwan University, Suwon, Korea Vilnius University, Vilnius, Lithuania V. Dudenas, A. Juodagalvis, J. Vaitkus Vilnius University, Vilnius, Lithuania V. Dudenas, A. Juodagalvis, J. Vaitkus National Centre for Particle Physics, Universiti Malaya, Kuala Lumpur, Malaysia National Centre for Particle Physics, Universiti Malaya, Kuala Lumpur, Malaysia I. Ahmed, Z.A. Ibrahim, J.R. Komaragiri, M.A.B. Md Ali32, F. Mohamad Idris33, W.A.T. Wan Abdullah, M.N. Yusli, Z. Zolkapli I. Ahmed, Z.A. Ibrahim, J.R. Komaragiri, M.A.B. Md Ali32, F. Mohamad Idris33, W.A.T. Wan Abdullah, M.N. Yusli, Z. Zolkapli Centro de Investigacion y de Estudios Avanzados del IPN, Mexico City, Mexico H. Castilla-Valdez, E. De La Cruz-Burelo, I. Heredia-De La Cruz34, A. Hernandez-Almada, R. Lopez-Fernandez, R. Maga˜na Villalba, J. Mejia Guisao, A. Sanchez-Hernandez Centro de Investigacion y de Estudios Avanzados del IPN, Mexico City, Mexico H. Castilla-Valdez, E. De La Cruz-Burelo, I. Heredia-De La Cruz34, A. Hernandez-Almada, R. Lopez-Fernandez, R. Maga˜na Villalba, J. Mejia Guisao, A. Sanchez-Hernandez g y , y, H. Castilla-Valdez, E. De La Cruz-Burelo, I. Heredia-De La Cruz34, A. Hernandez-Almada, R. Lopez-Fernandez, R. Maga˜na Villalba, J. Mejia Guisao, A. Sanchez-Hernandez Universidad Iberoamericana, Mexico City, Mexico S. Carrillo Moreno, C. Oropeza Barrera, F. Vazquez Valencia – 44 – Benemerita Universidad Autonoma de Puebla, Puebla, Mexico S. Carpinteyro, I. Pedraza, H.A. Salazar Ibarguen, C. Uribe Estrada Universidad Aut´onoma de San Luis Potos´ı, San Luis Potos´ı, Mexico A. Morelos Pineda University of Auckland, Auckland, New Zealand D. Krofcheck University of Canterbury, Christchurch, New Zealand P.H. Butler JHEP03(2017)162 JHEP03(2017)162 National Centre for Physics, Quaid-I-Azam University, Islamabad, Pakistan A. Ahmad, M. Ahmad, Q. Hassan, H.R. Hoorani, W.A. Khan, A. Saddique, M.A. Shah, M. Shoaib, M. Waqas National Centre for Nuclear Research, Swierk, Poland H. Bialkowska, M. Bluj, B. Boimska, T. Frueboes, M. G´orski, M. Kazana, K. Nawrocki, K. Romanowska-Rybinska, M. Szleper, P. Zalewski Institute of Experimental Physics, Faculty of Physics, University of Warsaw, Warsaw, Poland K. Bunkowski, A. Byszuk35, K. Doroba, A. Kalinowski, M. Konecki, J. Krolikowski, M. Misiura, M. Olszewski, M. Walczak Portugal P. Bargassa, C. Beir˜ao Da Cruz E Silva, B. Calpas, A. Di Francesco, P. Faccioli, P.G. Fer- reira Parracho, M. Gallinaro, J. Hollar, N. Leonardo, L. Lloret Iglesias, M.V. Nemallapudi, J. Rodrigues Antunes, J. Seixas, O. Toldaiev, D. Vadruccio, J. Varela, P. Vischia P.N. Lebedev Physical Institute, Moscow, Russia P.N. Lebedev Physical Institute, Moscow, Russia V. Andreev, M. Azarkin37, I. Dremin37, M. Kirakosyan, A. Leonidov37, A. Terkulov Skobeltsyn Institute of Nuclear Physics, Lomonosov Moscow State University, Moscow, Russia Moscow, Russia A. Baskakov, A. Belyaev, E. Boos, M. Dubinin41, L. Dudko, A. Ershov, A. Gribushin, A. Baskakov, A. Belyaev, E. Boos, M. Dubinin41, L. Dudko, A. Ershov, A. Gribushin, V. Klyukhin, O. Kodolova, I. Lokhtin, I. Miagkov, S. Obraztsov, S. Petrushanko, V. Savrin, A. Snigirev JHEP03(2017)162 V. Klyukhin, O. Kodolova, I. Lokhtin, I. Miagkov, S. Obraztsov, S. Petrushanko, V. Savrin, A. Snigirev Novosibirsk State University (NSU), Novosibirsk, Russia V. Blinov42, Y.Skovpen42, D. Shtol42 State Research Center of Russian Federation, Institute for High Energy Physics, Protvino, Russia State Research Center of Russian Federation, Institute for High Energ Physics, Protvino, Russia I. Azhgirey, I. Bayshev, S. Bitioukov, D. Elumakhov, V. Kachanov, A. Kalinin, D. Kon- stantinov, V. Krychkine, V. Petrov, R. Ryutin, A. Sobol, S. Troshin, N. Tyurin, A. Uzunian, A. Volkov University of Belgrade, Faculty of Physics and Vinca Institute of Nuclear Sciences, Belgrade, Serbia P. Adzic43, P. Cirkovic, D. Devetak, M. Dordevic, J. Milosevic, V. Rekovic P. Adzic43, P. Cirkovic, D. Devetak, M. Dordevic, J. Milosevic, V. Rekovic Centro de Investigaciones Energ´eticas Medioambientales y Tec- nol´ogicas (CIEMAT), Madrid, Spain Joint Institute for Nuclear Research, Dubna, Russia Joint Institute for Nuclear Research, Dubna, Russia Joint Institute for Nuclear Research, Dubna, Russia P. Bunin, M. Gavrilenko, I. Golutvin, I. Gorbunov, A. Kamenev, V. Karjavin, A. Lanev, A. Malakhov, V. Matveev36,37, V. Palichik, V. Perelygin, M. Savina, S. Shmatov, S. Shulha, N. Skatchkov, V. Smirnov, N. Voytishin, A. Zarubin Petersburg Nuclear Physics Institute, Gatchina (St. Petersburg), Russia L. Chtchipounov, V. Golovtsov, Y. Ivanov, V. Kim38, E. Kuznetsova39, V. Murzin, V. Oreshkin, V. Sulimov, A. Vorobyev Institute for Nuclear Research, Moscow, Russia Yu. Andreev, A. Dermenev, S. Gninenko, N. Golubev, A. Karneyeu, M. Kirsanov, N. Krasnikov, A. Pashenkov, D. Tlisov, A. Toropin Institute for Theoretical and Experimental Physics, Moscow, Russia V. Epshteyn, V. Gavrilov, N. Lychkovskaya, V. Popov, I. Pozdnyakov, G. Safronov, A. Spiridonov, M. Toms, E. Vlasov, A. Zhokin Moscow Institute of Physics and Technology, Moscow, Russia A. Bylinkin37 – 45 – National Research Nuclear University ’Moscow Engineering Physics Insti- tute’ (MEPhI), Moscow, Russia R. Chistov40, S. Polikarpov, E. Tarkovskii P.N. Lebedev Physical Institute, Moscow, Russia V. Andreev, M. Azarkin37, I. Dremin37, M. Kirakosyan, A. Leonidov37, A. Terkulov Centro de Investigaciones Energ´eticas Medioambientales y Te nol´ogicas (CIEMAT), Madrid, Spain J. Alcaraz Maestre, M. Barrio Luna, E. Calvo, M. Cerrada, M. Chamizo Llatas, N. Col- ino, B. De La Cruz, A. Delgado Peris, A. Escalante Del Valle, C. Fernandez Bedoya, J.P. Fern´andez Ramos, J. Flix, M.C. Fouz, P. Garcia-Abia, O. Gonzalez Lopez, S. Goy Lopez, J.M. Hernandez, M.I. Josa, E. Navarro De Martino, A. P´erez-Calero Yzquierdo, J. Puerta Pelayo, A. Quintario Olmeda, I. Redondo, L. Romero, M.S. Soares Universidad Aut´onoma de Madrid, Madrid, Spain J.F. de Troc´oniz, M. Missiroli, D. Moran Universidad Aut´onoma de Madrid, Madrid, Spain J.F. de Troc´oniz, M. Missiroli, D. Moran Universidad de Oviedo, Oviedo, Spain J. Cuevas, J. Fernandez Menendez, I. Gonzalez Caballero, J.R. Gonz´alez Fern´andez, E. Palencia Cortezon, S. Sanchez Cruz, I. Su´arez Andr´es, J.M. Vizan Garcia Universidad de Oviedo, Oviedo, Spain J. Cuevas, J. Fernandez Menendez, I. Gonzalez Caballero, J.R. Gonz´alez Fern´andez, E. Palencia Cortezon, S. Sanchez Cruz, I. Su´arez Andr´es, J.M. Vizan Garcia Cuevas, J. Fernandez Menendez, I. Gonzalez Caballero, J.R. Gonz´alez Fern´andez, Palencia Cortezon, S. Sanchez Cruz, I. Su´arez Andr´es, J.M. Vizan Garcia Instituto de F´ısica de Cantabria (IFCA), CSIC-Universidad de Cantabria, Santander, Spain I.J. Cabrillo, A. Calderon, E. Curras, M. Fernandez, J. Garcia-Ferrero, G. Gomez, A. Lopez Virto, J. Marco, C. Martinez Rivero, F. Matorras, J. Piedra Gomez, T. Rodrigo, A. Ruiz- Jimeno, L. Scodellaro, N. Trevisani, I. Vila, R. Vilar Cortabitarte I.J. Cabrillo, A. Calderon, E. Curras, M. Fernandez, J. Garcia-Ferrero, G. Gomez, A. Lopez Virto, J. Marco, C. Martinez Rivero, F. Matorras, J. Piedra Gomez, T. Rodrigo, A. Ruiz- Jimeno, L. Scodellaro, N. Trevisani, I. Vila, R. Vilar Cortabitarte – 46 – CERN, European Organization for Nuclear Research, Geneva, Switzerland D. Abbaneo, E. Auﬀray, G. Auzinger, M. Bachtis, P. Baillon, A.H. Ball, D. Barney, P. Bloch, A. Bocci, C. Botta, T. Camporesi, R. Castello, M. Cepeda, G. Cerminara, Y. Chen, D. d’Enterria, A. Dabrowski, V. Daponte, A. David, M. De Gruttola, A. De Roeck, E. Di Marco44, M. Dobson, B. Dorney, T. du Pree, D. Duggan, M. D¨unser, N. Dupont, A. Elliott-Peisert, P. Everaerts, S. Fartoukh, G. Franzoni, J. Fulcher, W. Funk, D. Gigi, K. Gill, M. Girone, F. Glege, D. Gulhan, S. Gundacker, M. Guthoﬀ, P. Har- ris, J. Hegeman, V. Innocente, P. Janot, J. Kieseler, H. Kirschenmann, V. Kn¨unz, A. Kornmayer16, M.J. Kortelainen, K. Kousouris, M. Krammer1, C. Lange, P. Lecoq, C. Louren¸co, M.T. Lucchini, L. Malgeri, M. Mannelli, A. Martelli, F. Meijers, J.A. Merlin, S. Centro de Investigaciones Energ´eticas Medioambientales y Te nol´ogicas (CIEMAT), Madrid, Spain Mersi, E. Meschi, P. Milenovic45, F. Moortgat, S. Morovic, M. Mulders, H. Neugebauer, S. Orfanelli, L. Orsini, L. Pape, E. Perez, M. Peruzzi, A. Petrilli, G. Petrucciani, A. Pfeiﬀer, M. Pierini, A. Racz, T. Reis, G. Rolandi46, M. Rovere, H. Sakulin, J.B. Sauvan, C. Sch¨afer, C. Schwick, M. Seidel, A. Sharma, P. Silva, P. Sphicas47, J. Steggemann, M. Stoye, Y. Takahashi, M. Tosi, D. Treille, A. Triossi, A. Tsirou, V. Veckalns48, G.I. Veres21, M. Verweij, N. Wardle, H.K. W¨ohri, A. Zagozdzinska35, W.D. Zeuner CERN, European Organization for Nuclear Research, Geneva, Switzerland D. Abbaneo, E. Auﬀray, G. Auzinger, M. Bachtis, P. Baillon, A.H. Ball, D. Barney, P. Bloch, A. Bocci, C. Botta, T. Camporesi, R. Castello, M. Cepeda, G. Cerminara, Y. Chen, D. d’Enterria, A. Dabrowski, V. Daponte, A. David, M. De Gruttola, A. De Roeck, E. Di Marco44, M. Dobson, B. Dorney, T. du Pree, D. Duggan, M. D¨unser, N. Dupont, A. Elliott-Peisert, P. Everaerts, S. Fartoukh, G. Franzoni, J. Fulcher, W. Funk, D. Gigi, K. Gill, M. Girone, F. Glege, D. Gulhan, S. Gundacker, M. Guthoﬀ, P. Har- ris, J. Hegeman, V. Innocente, P. Janot, J. Kieseler, H. Kirschenmann, V. Kn¨unz, A. Kornmayer16, M.J. Kortelainen, K. Kousouris, M. Krammer1, C. Lange, P. Lecoq, C. Louren¸co, M.T. Lucchini, L. Malgeri, M. Mannelli, A. Martelli, F. Meijers, J.A. Merlin, S. Mersi, E. Meschi, P. Milenovic45, F. Moortgat, S. Morovic, M. Mulders, H. Neugebauer, S. Orfanelli, L. Orsini, L. Pape, E. Perez, M. Peruzzi, A. Petrilli, G. Petrucciani, A. Pfeiﬀer, M. Pierini, A. Racz, T. Reis, G. Rolandi46, M. Rovere, H. Sakulin, J.B. Sauvan, C. Sch¨afer, C. Schwick, M. Seidel, A. Sharma, P. Silva, P. Sphicas47, J. Steggemann, M. Stoye, Y. Takahashi, M. Tosi, D. Treille, A. Triossi, A. Tsirou, V. Veckalns48, G.I. Veres21, M. Verweij, N. Wardle, H.K. W¨ohri, A. Zagozdzinska35, W.D. Zeuner JHEP03(2017)162 Paul Scherrer Institut, Villigen, Switzerland Institute for Particle Physics, ETH Zurich, Zurich, Switzerland F. Bachmair, L. B¨ani, L. Bianchini, B. Casal, G. Dissertori, M. Dittmar, M. Doneg`a, C. Grab, C. Heidegger, D. Hits, J. Hoss, G. Kasieczka, W. Lustermann, B. Mangano, M. Marionneau, P. Martinez Ruiz del Arbol, M. Masciovecchio, M.T. Meinhard, D. Meister, F. Micheli, P. Musella, F. Nessi-Tedaldi, F. Pandolﬁ, J. Pata, F. Pauss, G. Perrin, L. Perrozzi, M. Quittnat, M. Rossini, M. Sch¨onenberger, A. Starodumov49, V.R. Tavolaro, K. Theoﬁlatos, R. Wallny F. Bachmair, L. B¨ani, L. Bianchini, B. Casal, G. Dissertori, M. Dittmar, M. Doneg C. Grab, C. Heidegger, D. Hits, J. Hoss, G. Kasieczka, W. Lustermann, B. Mangan M. Marionneau, P. Martinez Ruiz del Arbol, M. Masciovecchio, M.T. Meinhard, D. Meister, F. Micheli, P. Musella, F. Nessi-Tedaldi, F. Pandolﬁ, J. Pata, F. Pauss, G. Perrin, M. Marionneau, P. Martinez Ruiz del Arbol, M. Masciovecchio, M.T. Meinhard, D. Meiste F. Micheli, P. Musella, F. Nessi-Tedaldi, F. Pandolﬁ, J. Pata, F. Pauss, G. Perri L. Perrozzi, M. Quittnat, M. Rossini, M. Sch¨onenberger, A. Starodumov49, V.R. Tavolaro, K. Theoﬁlatos, R. Wallny Universit¨at Z¨urich, Zurich, Switzerland Paul Scherrer Institut, Villigen, Switzerland W. Bertl, K. Deiters, W. Erdmann, R. Horisberger, Q. Ingram, H.C. Kaestli, D. Kotlinski, U. Langenegger, T. Rohe Institute for Particle Physics, ETH Zurich, Zurich, Switzerland Universit¨at Z¨urich, Zurich, Switzerland T.K. Aarrestad, C. Amsler50, L. Caminada, M.F. Canelli, A. De Cosa, C. Galloni, A. Hinzmann, T. Hreus, B. Kilminster, J. Ngadiuba, D. Pinna, G. Rauco, P. Robmann, D. Salerno, Y. Yang, A. Zucchetta National Central University, Chung-Li, Taiwan National Central University, Chung-Li, Taiwan V. Candelise, T.H. Doan, Sh. Jain, R. Khurana, M. Konyushikhin, C.M. Kuo, W. Lin, Y.J. Lu, A. Pozdnyakov, S.S. Yu National Taiwan University (NTU), Taipei, Taiwan Arun Kumar, P. Chang, Y.H. Chang, Y. Chao, K.F. Chen, P.H. Chen, F. Fiori, W.-S. Hou, Y. Hsiung, Y.F. Liu, R.-S. Lu, M. Mi˜nano Moya, E. Paganis, A. Psallidas, J.f. Tsai National Taiwan University (NTU), Taipei, Taiwan run Kumar, P. Chang, Y.H. Chang, Y. Chao, K.F. Chen, P.H. Chen, F. Fiori, W.-S. Hou, H i Y F Li R S L M Mi˜ M E P i A P llid J f T i Y. Hsiung, Y.F. Liu, R.-S. Lu, M. Mi˜nano Moya, E. Paganis, A. Psallidas, J.f. Tsai Chulalongkorn University, Faculty of Science, Department of Physics, Bangkok, Thailand B. Asavapibhop, G. Singh, N. Srimanobhas, N. Suwonjandee – 47 – Cukurova University - Physics Department, Science and Art Faculty A. Adiguzel, S. Damarseckin, Z.S. Demiroglu, C. Dozen, E. Eskut, S. Girgis, G. Gokbulut, Y. Guler, I. Hos51, E.E. Kangal52, O. Kara, A. Kayis Topaksu, U. Kiminsu, M. Oglakci, G. Onengut53, K. Ozdemir54, S. Ozturk55, A. Polatoz, B. Tali56, S. Turkcapar, I.S. Zor- bakir, C. Zorbilmez Middle East Technical University, Physics Department, Ankara, Turkey B. Bilin, S. Bilmis, B. Isildak57, G. Karapinar58, M. Yalvac, M. Zeyrek Bogazici University, Istanbul, Turkey E. G¨ulmez, M. Kaya59, O. Kaya60, E.A. Yetkin61, T. Yetkin62 JHEP03(2017)162 Istanbul Technical University, Istanbul, Turkey A. Cakir, K. Cankocak, S. Sen63 Institute for Scintillation Materials of National Academy of Science of Ukraine, Kharkov, Ukraine B. Grynyov National Scientiﬁc Center, Kharkov Institute of Physics and Technology, Kharkov, Ukraine L. Levchuk, P. Sorokin L. Levchuk, P. Sorokin University of Bristol, Bristol, United Kingdom R. Aggleton, F. Ball, L. Beck, J.J. Brooke, D. Burns, E. Clement, D. Cussans, H. Flacher, J. Goldstein, M. Grimes, G.P. Heath, H.F. Heath, J. Jacob, L. Kreczko, C. Lucas, D.M. Newbold64, S. Paramesvaran, A. Poll, T. Sakuma, S. Seif El Nasr-storey, D. Smith, V.J. Smith R. Aggleton, F. Ball, L. Beck, J.J. Brooke, D. Burns, E. Clement, D. Cussans, H. Flache Rutherford Appleton Laboratory, Didcot, United Kingdom Rutherford Appleton Laboratory, Didcot, United Kingdom K.W. Bell, A. Belyaev65, C. Brew, R.M. Brown, L. Calligaris, D. Cieri, D.J.A. Cockerill, J.A. Coughlan, K. Harder, S. Harper, E. Olaiya, D. Petyt, C.H. Shepherd-Themistocleous, A. Thea, I.R. Tomalin, T. Williams Imperial College, London, United Kingdom M. Baber, R. Bainbridge, O. Buchmuller, A. Bundock, D. Burton, S. Casasso, M. Citron, D. Colling, L. Corpe, P. Dauncey, G. Davies, A. De Wit, M. Della Negra, R. Di Maria, P. Dunne, A. Elwood, D. Futyan, Y. Haddad, G. Hall, G. Iles, T. James, R. Lane, C. Laner, R. Lucas64, L. Lyons, A.-M. Magnan, S. Malik, L. Mastrolorenzo, J. Nash, A. Nikitenko49, J. Pela, B. Penning, M. Pesaresi, D.M. Raymond, A. Richards, A. Rose, C. Seez, S. Summers, A. Tapper, K. Uchida, M. Vazquez Acosta66, T. Virdee16, J. Wright, S.C. Zenz C. Seez, S. Summers, A. Tapper, K. Uchida, M. Vazquez Acosta66, T. Virdee16, J. Wright, S.C. Zenz Brunel University, Uxbridge, United Kingdom Brunel University, Uxbridge, United Kingdom J.E. Cole, P.R. Hobson, A. Khan, P. Kyberd, I.D. Reid, P. Symonds, L. Teodorescu, M. Turner Baylor University, Waco, U.S.A. A. Borzou, K. Call, J. Dittmann, K. Hatakeyama, H. Liu, N. Pastika – 48 – The University of Alabama, Tuscaloosa, U.S.A. S.I. Cooper, C. Henderson, P. Rumerio, C. West The University of Alabama, Tuscaloosa, U.S.A. S.I. Cooper, C. Henderson, P. Rumerio, C. West The University of Alabama, Tuscaloosa, U.S.A. S.I. Cooper, C. Henderson, P. Rumerio, C. West Boston University, Boston, U.S.A. D. Arcaro, A. Avetisyan, T. Bose, D. Gastler, D. Rankin, C. Richardson, J. Rohlf, L. Sulak, D. Zou Brown University, Providence, U.S.A. G. Benelli, D. Cutts, A. Garabedian, J. Hakala, U. Heintz, J.M. Hogan, O. Jesus, K.H.M. Kwok, E. Laird, G. Landsberg, Z. Mao, M. Narain, S. Piperov, S. Sagir, E. Spencer, R. Syarif JHEP03(2017)162 University of California, Davis, Davis, U.S.A. R. Breedon, D. Burns, M. Calderon De La Barca Sanchez, S. Chauhan, M. Chertok, J. Conway, R. Conway, P.T. Cox, R. Erbacher, C. Flores, G. Funk, M. Gardner, W. Ko, R. Lander, C. Mclean, M. Mulhearn, D. Pellett, J. Pilot, S. Shalhout, J. Smith, M. Squires, D. Stolp, M. Tripathi University of California, Los Angeles, U.S.A. University of California, Los Angeles, U.S.A. C. Bravo, R. Cousins, A. Dasgupta, A. Florent, J. Hauser, M. Ignatenko, N. Mccoll, D. Saltzberg, C. Schnaible, V. Valuev, M. Weber University of California, Riverside, Riverside, U.S.A. University of California, Riverside, Riverside, U.S.A. E. Bouvier, K. Burt, R. Clare, J. Ellison, J.W. Gary, S.M.A. Ghiasi Shirazi, G. Han- son, J. Heilman, P. Jandir, E. Kennedy, F. Lacroix, O.R. Long, M. Olmedo Negrete, M.I. Paneva, A. Shrinivas, W. Si, H. Wei, S. Wimpenny, B. R. Yates University of California, San Diego, La Jolla, U.S.A. J.G. Branson, G.B. Cerati, S. Cittolin, M. Derdzinski, R. Gerosa, A. Holzner, D. Klein, V. Krutelyov, J. Letts, I. Macneill, D. Olivito, S. Padhi, M. Pieri, M. Sani, V. Sharma, S. Simon, M. Tadel, A. Vartak, S. Wasserbaech67, C. Welke, J. Wood, F. W¨urthwein, A. Yagil, G. Zevi Della Porta University of California, Santa Barbara - Department of Physics, Santa Bar- bara, U.S.A. N. Amin, R. Bhandari, J. Bradmiller-Feld, C. Campagnari, A. Dishaw, V. Dutta, M. Franco Sevilla, C. George, F. Golf, L. Gouskos, J. Gran, R. Heller, J. Incandela, S.D. Mullin, A. Ovcharova, H. Qu, J. Richman, D. Stuart, I. Suarez, J. University of Colorado Boulder, Boulder, U.S.A. University of Colorado Boulder, Boulder, U.S.A. J.P. Cumalat, W.T. Ford, F. Jensen, A. Johnson, M. Krohn, T. Mulholland, K. Stenson, S.R. Wagner Cornell University, Ithaca, U.S.A. J. Alexander, J. Chaves, J. Chu, S. Dittmer, K. Mcdermott, N. Mirman, G. Nicolas Kaufman, J.R. Patterson, A. Rinkevicius, A. Ryd, L. Skinnari, L. Soﬃ, S.M. Tan, Z. Tao, J. Thom, J. Tucker, P. Wittich, M. Zientek Fairﬁeld University, Fairﬁeld, U.S.A. Fairﬁeld University, Fairﬁeld, U.S.A. D. Winn Fairﬁeld University, Fairﬁeld, U.S.A. D. Winn JHEP03(2017)162 D. Winn Fermi National Accelerator Laboratory, Batavia, U.S.A. S. Abdullin, M. Albrow, G. Apollinari, A. Apresyan, S. Banerjee, L.A.T. Bauerdick, A. Beretvas, J. Berryhill, P.C. Bhat, G. Bolla, K. Burkett, J.N. Butler, H.W.K. Cheung, F. Chlebana, S. Cihangir†, M. Cremonesi, V.D. Elvira, I. Fisk, J. Freeman, E. Gottschalk, L. Gray, D. Green, S. Gr¨unendahl, O. Gutsche, D. Hare, R.M. Harris, S. Hasegawa, J. Hirschauer, Z. Hu, B. Jayatilaka, S. Jindariani, M. Johnson, U. Joshi, B. Klima, B. Kreis, S. Lammel, J. Linacre, D. Lincoln, R. Lipton, T. Liu, R. Lopes De S´a, J. Lykken, K. Maeshima, N. Magini, J.M. Marraﬃno, S. Maruyama, D. Mason, P. McBride, P. Merkel, S. Mrenna, S. Nahn, V. O’Dell, K. Pedro, O. Prokofyev, G. Rakness, L. Ristori, E. Sexton- Kennedy, A. Soha, W.J. Spalding, L. Spiegel, S. Stoynev, N. Strobbe, L. Taylor, S. Tkaczyk, N.V. Tran, L. Uplegger, E.W. Vaandering, C. Vernieri, M. Verzocchi, R. Vidal, M. Wang, H.A. Weber, A. Whitbeck, Y. Wu N.V. Tran, L. Uplegger, E.W. Vaandering, C. Vernieri, M. Verzocchi, R. Vidal, M. Wang, H.A. Weber, A. Whitbeck, Y. Wu Brunel University, Uxbridge, United Kingdom Yoo California Institute of Technology, Pasadena, U.S.A. D. Anderson, J. Bendavid, A. Bornheim, J. Bunn, J. Duarte, J.M. Lawhorn, A. Mott, H.B. Newman, C. Pena, M. Spiropulu, J.R. Vlimant, S. Xie, R.Y. Zhu Carnegie Mellon University, Pittsburgh, U.S.A. g y, g , M.B. Andrews, T. Ferguson, M. Paulini, J. Russ, M. Sun, H. Vogel, I. Vorobiev, M. Wein- berg – 49 – The University of Kansas, Lawrence, U.S.A. The University of Kansas, Lawrence, U.S.A. JHEP03(2017)162 A. Al-bataineh, P. Baringer, A. Bean, S. Boren, J. Bowen, J. Castle, L. Forthomm A. Al-bataineh, P. Baringer, A. Bean, S. Boren, J. Bowen, J. Castle, L. Forthomme, R.P. Kenny III, S. Khalil, A. Kropivnitskaya, D. Majumder, W. Mcbrayer, M. Murray, S. Sanders, R. Stringer, J.D. Tapia Takaki, Q. Wang Kansas State University, Manhattan, U.S.A. Kansas State University, Manhattan, U.S.A. A. Ivanov, K. Kaadze, Y. Maravin, A. Mohammadi, L.K. Saini, N. Skhirtladze, S. Toda Lawrence Livermore National Laboratory, Livermore, U.S.A. F. Rebassoo, D. Wright University of Maryland, College Park, U.S.A. University of Maryland, College Park, U.S.A. C. Anelli, A. Baden, O. Baron, A. Belloni, B. Calvert, S.C. Eno, C. Ferraioli, J.A. Gomez, N.J. Hadley, S. Jabeen, R.G. Kellogg, T. Kolberg, J. Kunkle, Y. Lu, A.C. Mignerey, F. Ricci-Tam, Y.H. Shin, A. Skuja, M.B. Tonjes, S.C. Tonwar University of Florida, Gainesville, U.S.A. University of Florida, Gainesville, U.S.A. Acosta, P. Avery, P. Bortignon, D. Bourilkov, A. Brinkerhoﬀ, A. Carnes, M. Carver, Curry S Das R D Field I K Furic J Konigsberg A Korytov J F Low P Ma D. Acosta, P. Avery, P. Bortignon, D. Bourilkov, A. Brinkerhoﬀ, A. Carnes, M. Carver, D. Curry, S. Das, R.D. Field, I.K. Furic, J. Konigsberg, A. Korytov, J.F. Low, P. Ma, D. Acosta, P. Avery, P. Bortignon, D. Bourilkov, A. Brinkerhoﬀ, A. Carnes, M. Carve D. Curry, S. Das, R.D. Field, I.K. Furic, J. Konigsberg, A. Korytov, J.F. Low, P. M K. Matchev, H. Mei, G. Mitselmakher, D. Rank, L. Shchutska, D. Sperka, L. Thomas, J. Wang, S. Wang, J. Yelton Florida International University, Miami, U.S.A. S. Linn, P. Markowitz, G. Martinez, J.L. Rodriguez Florida International University, Miami, U.S.A. S. Linn, P. Markowitz, G. Martinez, J.L. Rodriguez Florida State University, Tallahassee, U.S.A. A. Ackert, T. Adams, A. Askew, S. Bein, S. Hagopian, V. Hagopian, K.F. Johnson, H. Prosper, A. Santra, R. Yohay Florida Institute of Technology, Melbourne, U.S.A. Florida Institute of Technology, Melbourne, U.S.A. M.M. Baarmand, V. Bhopatkar, S. Colafranceschi, M. Hohlmann, D. Noonan, T. Roy, F. Yumiceva M.M. Baarmand, V. Bhopatkar, S. Colafranceschi, M. Hohlmann, D. Noonan, T. Roy, F. Yumiceva University of Illinois at Chicago (UIC), Chicago, U.S.A. M.R. Adams, L. Apanasevich, D. Berry, R.R. Betts, I. Bucinskaite, R. Cavanaugh, O. Evdokimov, L. Gauthier, C.E. Gerber, D.J. Hofman, K. Jung, I.D. Sandoval Gonzalez, N. Varelas, H. Wang, Z. Wu, M. Zakaria, J. Zhang – 50 – The University of Iowa, Iowa City, U.S.A. B. Bilki68, W. Clarida, K. Dilsiz, S. Durgut, R.P. Gandrajula, M. Haytmyradov, V. Khris- tenko, J.-P. Merlo, H. Mermerkaya69, A. Mestvirishvili, A. Moeller, J. Nachtman, H. Ogul, Y. Onel, F. Ozok70, A. Penzo, C. Snyder, E. Tiras, J. Wetzel, K. Yi Johns Hopkins University, Baltimore, U.S.A. Johns Hopkins University, Baltimore, U.S.A. I. Anderson, B. Blumenfeld, A. Cocoros, N. Eminizer, D. Fehling, L. Feng, A.V. Gritsan, P. Maksimovic, C. Martin, M. Osherson, J. Roskes, U. Sarica, M. Swartz, M. Xiao, Y. Xin, I. Anderson, B. Blumenfeld, A. Cocoros, N. Eminizer, D. Fehling, L. Feng, A.V. Gritsa I. Anderson, B. Blumenfeld, A. Cocoros, N. Eminizer, D. Fehling, L. Feng, A.V. Gritsan, P. Maksimovic, C. Martin, M. Osherson, J. Roskes, U. Sarica, M. Swartz, M. Xiao, Y. Xin, C. You P. Maksimovic, C. Martin, M. Osherson, J. Roskes, U. Sarica, M. Swartz, M. Xiao, Y. Xin, C. You Massachusetts Institute of Technology, Cambridge, U.S.A. D. Abercrombie, B. Allen, A. Apyan, V. Azzolini, R. Barbieri, A. Baty, R. Bi, K. Bierwagen, S. Brandt, W. Busza, I.A. Cali, M. D’Alfonso, Z. Demiragli, L. Di Matteo, G. Gomez Ceballos, M. Goncharov, D. Hsu, Y. Iiyama, G.M. Innocenti, M. Klute, D. Kovalskyi, K. Krajczar, Y.S. Lai, Y.-J. Lee, A. Levin, P.D. Luckey, B. Maier, A.C. Marini, C. Mcginn, C. Mironov, S. Narayanan, X. Niu, C. Paus, C. Roland, G. Roland, J. Salfeld-Nebgen, G.S.F. Stephans, K. Tatar, M. Varma, D. Velicanu, J. Veverka, J. Wang, T.W. Wang, B. Wyslouch, M. Yang B. Wyslouch, M. Yang University of Minnesota, Minneapolis, U.S.A. A.C. Benvenuti, R.M. Chatterjee, A. Evans, P. Hansen, S. Kalafut, S.C. Kao, Y. Kubota, Z. Lesko, J. Mans, S. Nourbakhsh, N. Ruckstuhl, R. Rusack, N. Tambe, J. Turkewitz University of Minnesota, Minneapolis, U.S.A. y , p , A.C. Benvenuti, R.M. Chatterjee, A. Evans, P. Hansen, S. Kalafut, S.C. Kao, Y. Kubota, Z. Lesko, J. Mans, S. Nourbakhsh, N. Ruckstuhl, R. Rusack, N. Tambe, J. Turkewitz University of Mississippi, Oxford, U.S.A. J.G. Acosta, S. Oliveros University of Mississippi, Oxford, U.S.A. J.G. Acosta, S. Oliveros University of Nebraska-Lincoln, Lincoln, U.S.A. University of Nebraska-Lincoln, Lincoln, U.S.A. E. Avdeeva, R. Bartek71, K. Bloom, D.R. Claes, A. Dominguez71, C. Fangmeier, R. Gon- zalez Suarez, R. Kamalieddin, I. Kravchenko, A. Malta Rodrigues, F. Meier, J. Monroy, J.E. Siado, G.R. Snow, B. Stieger – 51 – State University of New York at Buﬀalo, Buﬀalo, U.S.A. M. Alyari, J. Dolen, A. Godshalk, C. Harrington, I. Iashvili, J. Kaisen, A. Kharchilava, A. Parker, S. Rappoccio, B. Roozbahani Northeastern University, Boston, U.S.A. G. Alverson, E. Barberis, A. Hortiangtham, A. Massironi, D.M. Morse, D. Nash, T. Ori- moto, R. Teixeira De Lima, D. Trocino, R.-J. Wang, D. Wood Northwestern University, Evanston, U.S.A. Northwestern University, Evanston, U.S.A. y, , S. Bhattacharya, O. Charaf, K.A. Hahn, A. Kumar, N. Mucia, N. Odell, B. Pollack, M.H. Schmitt, K. Sung, M. Trovato, M. Velasco JHEP03(2017)162 University of Notre Dame, Notre Dame, U.S.A. University of Notre Dame, Notre Dame, U.S.A. N. Dev, M. Hildreth, K. Hurtado Anampa, C. Jessop, D.J. Karmgard, N. Kellams, K. Lannon, N. Marinelli, F. Meng, C. Mueller, Y. Musienko36, M. Planer, A. Reinsvold, R. Ruchti, G. Smith, S. Taroni, M. Wayne, M. Wolf, A. Woodard The Ohio State University, Columbus, U.S.A. J. Alimena, L. Antonelli, B. Bylsma, L.S. Durkin, S. Flowers, B. Francis, A. Hart, C. Hill, R. Hughes, W. Ji, B. Liu, W. Luo, D. Puigh, B.L. Winer, H.W. Wulsin The Ohio State University, Columbus, U.S.A. y, , J. Alimena, L. Antonelli, B. Bylsma, L.S. Durkin, S. Flowers, B. Francis, A. Hart, C. Hill, R. Hughes, W. Ji, B. Liu, W. Luo, D. Puigh, B.L. Winer, H.W. Wulsin Princeton University, Princeton, U.S.A. S. Cooperstein, O. Driga, P. Elmer, J. Hardenbrook, P. Hebda, D. Lange, J. Luo, D. Mar- low, T. Medvedeva, K. Mei, J. Olsen, C. Palmer, P. Pirou´e, D. Stickland, A. Svyatkovskiy, C. Tully University of Puerto Rico, Mayaguez, U.S.A. S. Malik Purdue University, West Lafayette, U.S.A. A. Barker, V.E. Barnes, S. Folgueras, L. Gutay, M.K. Jha, M. Jones, A.W. Jung, A. Khatiwada, D.H. Miller, N. Neumeister, J.F. Schulte, X. Shi, J. Sun, F. Wang, W. Xie Purdue University Calumet, Hammond, U.S.A. N. Parashar, J. Stupak Rice University, Houston, U.S.A. Rice University, Houston, U.S.A. A. Adair, B. Akgun, Z. Chen, K.M. Ecklund, F.J.M. Geurts, M. Guilbaud, W. Li, B. Michlin, M. Northup, B.P. Padley, J. Roberts, J. Rorie, Z. Tu, J. Zabel University of Rochester, Rochester, U.S.A. B. Betchart, A. Bodek, P. de Barbaro, R. Demina, Y.t. University of Nebraska-Lincoln, Lincoln, U.S.A. Duh, T. Ferbel, M. Galanti, A. Garcia-Bellido, J. Han, O. Hindrichs, A. Khukhunaishvili, K.H. Lo, P. Tan, M. Verzetti Rutgers, The State University of New Jersey, Piscataway, U.S.A. A. Agapitos, J.P. Chou, Y. Gershtein, T.A. G´omez Espinosa, E. Halkiadakis, M. Heindl, E. Hughes, S. Kaplan, R. Kunnawalkam Elayavalli, S. Kyriacou, A. Lath, K. Nash, H. Saka, S. Salur, S. Schnetzer, D. Sheﬃeld, S. Somalwar, R. Stone, S. Thomas, P. Thomassen, M. Walker A. Agapitos, J.P. Chou, Y. Gershtein, T.A. G´omez Espinosa, E. Halkiadakis, M. Heindl, E. Hughes, S. Kaplan, R. Kunnawalkam Elayavalli, S. Kyriacou, A. Lath, K. Nash, H. Saka, S S l S S h t D Sh ﬃld S S l R St S Th P Th S. Salur, S. Schnetzer, D. Sheﬃeld, S. Somalwar, R. Stone, S. Thomas, P. Thomassen, M. Walker – 52 – University of Tennessee, Knoxville, U.S.A. A.G. Delannoy, M. Foerster, J. Heideman, G. Riley, K. Rose, S. Spanier, K. Thapa A.G. Delannoy, M. Foerster, J. Heideman, G. Riley, K. Ro Texas A&M University, College Station, U.S.A. O. Bouhali72, A. Celik, M. Dalchenko, M. De Mattia, A. Delgado, S. Dildick, R. Eusebi, J. Gilmore, T. Huang, E. Juska, T. Kamon73, R. Mueller, Y. Pakhotin, R. Patel, A. Perloﬀ, L. Perni`e, D. Rathjens, A. Safonov, A. Tatarinov, K.A. Ulmer J. Gilmore, T. Huang, E. Juska, T. Kamon73, R. Mueller, Y. Pakho L. Perni`e, D. Rathjens, A. Safonov, A. Tatarinov, K.A. Ulmer Texas Tech University, Lubbock, U.S.A. Texas Tech University, Lubbock, U.S.A. N. Akchurin, C. Cowden, J. Damgov, F. De Guio, C. Dragoiu, P.R. Dudero, J. Faulkner, N. Akchurin, C. Cowden, J. Damgov, F. De Guio, C. Dragoiu, P.R. Dudero, J. Faulkner, E. Gurpinar, S. Kunori, K. Lamichhane, S.W. Lee, T. Libeiro, T. Peltola, S. Undleeb, I. Volobouev, Z. Wang JHEP03(2017)162 I. Volobouev, Z. Wang Vanderbilt University, Nashville, U.S.A. S. Greene, A. Gurrola, R. Janjam, W. Johns, C. Maguire, A. Melo, H. Ni, P. Sheldon, S. Tuo, J. Velkovska, Q. Xu University of Virginia, Charlottesville, U.S.A. M.W. Arenton, P. Barria, B. Cox, J. Goodell, R. Hirosky, A. Ledovskoy, H. Li, C. Neu, T. Sinthuprasith, X. Sun, Y. Wang, E. Wolfe, F. Xia Wayne State University, Detroit, U.S.A. C. Clarke, R. Harr, P.E. Karchin, J. Sturdy University of Wisconsin - Madison, Madison, WI, U.S.A. D.A. Belknap, J. Buchanan, C. Caillol, S. Dasu, L. Dodd, S. Duric, B. Gomber, M. Groth M. Herndon, A. Herv´e, P. Klabbers, A. University, Tehran, Iran 30: Also at Universit`a degli Studi di Siena, Siena, Italy 31: Also at Purdue University, West Lafayette, U.S.A. 31: Also at Purdue University, West Lafayette, U.S.A. 32: Also at International Islamic University of Malaysia, Kuala Lumpur, Malaysia 32: Also at International Islamic University of Malaysia, Kuala Lumpur, Malaysia 33: Also at Malaysian Nuclear Agency, MOSTI, Kajang, Malaysia 33: Also at Malaysian Nuclear Agency, MOSTI, Kajang, Malaysia 34: Also at Consejo Nacional de Ciencia y Tecnolog´ıa, Mexico city, Mexico 34: Also at Consejo Nacional de Ciencia y Tecnolog´ıa, Mexico city, Mexico 35: Also at Warsaw University of Technology, Institute of Electronic Systems, Warsaw, Poland 35: Also at Warsaw University of Technology, Institute of Electronic Systems, Warsaw, Poland 36: Also at Institute for Nuclear Research, Moscow, Russia 37: Now at National Research Nuclear University ’Moscow Engineering Physics Insti- tute’ (MEPhI), Moscow, Russia 37: Now at National Research Nuclear University ’Moscow Engineering Physics Insti- tute’ (MEPhI), Moscow, Russia 38: Also at St. Petersburg State Polytechnical University, St. Petersburg, Russia 38: Also at St. Petersburg State Polytechnical University, St. Petersburg, Russia 39: Also at University of Florida, Gainesville, U.S.A. 40: Also at P.N. Lebedev Physical Institute, Moscow, Russia 41: Also at California Institute of Technology, Pasadena, U.S.A. University of Nebraska-Lincoln, Lincoln, U.S.A. Physikalisches Institut A, Aachen, Germany 18: Also at University of Hamburg, Hamburg, Germany 19: Also at Brandenburg University of Technology, Cottbus, Germany 20: Also at Institute of Nuclear Research ATOMKI, Debrecen, Hungary 21: Also at MTA-ELTE Lend¨ulet CMS Particle and Nuclear Physics Group, E¨otv¨os Lor´and University, Budapest, Hungary 22: Also at Institute of Physics, University of Debrecen, Debrecen, Hungary 23: Also at Indian Institute of Science Education and Research, Bhopal, India Also at Institute of Physics, Bhubaneswar, India JHEP03(2017)162 JHEP03(2017)162 25: Also at University of Visva-Bharati, Santiniketan, India 26: Also at University of Ruhuna, Matara, Sri Lanka 27: Also at Isfahan University of Technology, Isfahan, Iran 27: Also at Isfahan University of Technology, Isfahan, Iran 28: Also at Yazd University, Yazd, Iran 29: Also at Plasma Physics Research Center, Science and Research Branch, Islamic Azad University, Tehran, Iran University of Nebraska-Lincoln, Lincoln, U.S.A. Lanaro, A. Levine, K. Long, R. Loveless, I. Ojalvo, T. Perry, G.A. Pierro, G. Polese, T. Ruggles, A. Savin, N. Smith, W.H. Smith, D. Taylor, N. Woods †: Deceased 1: Also at Vienna University of Technology, Vienna, Austria 2: Also at State Key Laboratory of Nuclear Physics and Technology, Peking University, Beijing, China 3: Also at Institut Pluridisciplinaire Hubert Curien (IPHC), Universit´e de Strasbourg, CNRS/IN2P3, Strasbourg, France / 4: Also at Universidade Estadual de Campinas, Campinas, Brazil 4: Also at Universidade Estadual de Campinas, Campinas, Brazil 5: Also at Universidade Federal de Pelotas, Pelotas, Brazil 5: Also at Universidade Federal de Pelotas, Pelotas, Brazil 6: Also at Universit´e Libre de Bruxelles, Bruxelles, Belgium 6: Also at Universit´e Libre de Bruxelles, Bruxelles, Belgium 7: Also at Deutsches Elektronen-Synchrotron, Hamburg, Germany 7: Also at Deutsches Elektronen-Synchrotron, Hamburg, Germany 8: Also at Joint Institute for Nuclear Research, Dubna, Russia 8: Also at Joint Institute for Nuclear Research, Dubna, Russia 8: Also at Joint Institute for Nuclear Resea 9: Now at Cairo University, Cairo, Egypt 10: Also at Fayoum University, El-Fayoum, Egypt 10: Also at Fayoum University, El-Fayoum, Egypt 11: Now at British University in Egypt, Cairo, Egypt 11: Now at British University in Egypt, Cairo, Egypt 12: Now at Ain Shams University, Cairo, Egypt 12: Now at Ain Shams University, Cairo, Egypt 13: Also at Universit´e de Haute Alsace, Mulhouse, France 13: Also at Universit´e de Haute Alsace, Mulhouse, France 14: Also at Skobeltsyn Institute of Nuclear Physics, Lomonosov Moscow State University, Moscow, Russia Moscow, Russia – 53 – 15: Also at Tbilisi State University, Tbilisi, Georgia 16: Also at CERN, European Organization for Nuclear Research, Geneva, Switzerland Also at CERN, European Organization for Nuclear Research, Geneva, Switzerland 17: Also at RWTH Aachen University, III. University, Tehran, Iran 42: Also at Budker Institute of Nuclear Physics, Novosibirsk, Russia 42: Also at Budker Institute of Nuclear Physics, Novosibirsk, Russia 43: Also at Faculty of Physics, University of Belgrade, Belgrade, Serbia 43: Also at Faculty of Physics, University of Belgrade, Belgrade, Serbia 44: Also at INFN Sezione di Roma; Universit`a di Roma, Roma, Italy 44: Also at INFN Sezione di Roma; Universit`a di Roma, Roma, Italy 45: Also at University of Belgrade, Faculty of Physics and Vinca Institute of Nuclear Sciences, Belgrade, Serbia 45: Also at University of Belgrade, Faculty of Physics and Vinca Institute of Nuclear Science Belgrade, Serbia 46: Also at Scuola Normale e Sezione dell’INFN, Pisa, Italy 46: Also at Scuola Normale e Sezione dell’INFN, Pisa, Italy 47: Also at National and Kapodistrian University of Athens, Athens, Greece 47: Also at National and Kapodistrian University of Athens, Athens, Greece 48: Also at Riga Technical University, Riga, Latvia 49: Also at Institute for Theoretical and Experimental Physics, Moscow, Russia 49: Also at Institute for Theoretical and Experimental Physics, Moscow, Russia 50: Also at Albert Einstein Center for Fundamental Physics, Bern, Switzerland 51: Also at Istanbul Aydin University, Istanbul, Turkey 52: Also at Mersin University, Mersin, Turkey 53: Also at Cag University, Mersin, Turkey 54: Also at Piri Reis University, Istanbul, Turkey 55: Also at Gaziosmanpasa University, Tokat, Turkey 55: Also at Gaziosmanpasa University, Tokat, Turkey 56: Also at Adiyaman University, Adiyaman, Turkey 57: Also at Ozyegin University, Istanbul, Turkey 58: Also at Izmir Institute of Technology, Izmir, Turkey 58: Also at Izmir Institute of Technology, Izmir, Turkey – 54 – 59: Also at Marmara University, Istanbul, Turkey 59: Also at Marmara University, Istanbul, Turkey 60: Also at Kafkas University, Kars, Turkey 61: Also at Istanbul Bilgi University, Istanbul, Turkey 61: Also at Istanbul Bilgi University, Istanbul, Turkey 62: Also at Yildiz Technical University, Istanbul, Turkey 62: Also at Yildiz Technical University, Istanbul, Turkey 63: Also at Hacettepe University, Ankara, Turkey 63: Also at Hacettepe University, Ankara, Turkey 64: Also at Rutherford Appleton Laboratory, Didcot, U.K. 64: Also at Rutherford Appleton Laboratory, Didcot, U.K. 65: Also at School of Physics and Astronomy, University of Southampton, Southampton, U.K 65: Also at School of Physics and Astronomy, University of Southampto 66: Also at Instituto de Astrof´ısica de Canarias, La Laguna, Spain 66: Also at Instituto de Astrof´ısica de Canarias, La Laguna, Spain 7: Also at Utah Valley University, Orem, U.S.A. University, Tehran, Iran 68: Also at Argonne National Laboratory, Argonne, U. 68: Also at Argonne National Laboratory, Argonne, U.S.A. 69: Also at Erzincan University, Erzincan, Turkey JHEP03(2017)162 70: Also at Mimar Sinan University, Istanbul, Istanbul, Turkey 70: Also at Mimar Sinan University, Istanbul, Istanbul, Turkey 71: Now at The Catholic University of America, Washington, U.S.A. 71: Now at The Catholic University of America, Washington, U.S.A. 72: Also at Texas A&M University at Qatar, Doha, Qatar 72: Also at Texas A&M University at Qatar, Doha, Qatar 73: Also at Kyungpook National University, Daegu, Korea 73: Also at Kyungpook National University, Daegu, Korea – 55 –
https://openalex.org/W2106739087	https://seer.sis.puc-campinas.edu.br/seer/index.php/reflexao/article/download/3231/2142	Portuguese	null	O messianismo político da teologia latino-americana da libertação	Reflexão	2,015	cc-by	9,708	The political messianism of Latin American liberation theology The political messianism of Latin American liberation theology Claudio de Oliveira RIBEIRO1 Claudio de Oliveira RIBEIRO1 Resumo As reflexões apresentam os resultados de pesquisa sobre o desenvolvimento histórico da Teologia Latino-Americana da Libertação. O objetivo foi elaborar uma crítica à essa destacada corrente do pensamento a partir da identificação de aspectos de messianismo político nela presentes. Metodologicamente, a tarefa foi realizada a partir de uma aproximação da Teologia da Libertação com outras duas expressões teológicas. A primeira, ad intra, com autores que criticaram certas visões de messianismo político nela presentes e priorizaram a relação com a economia para oferecer tal crítica, especialmente as contribuições de Hugo Assmann, Julio de Santa Ana e Jung Mo Sung. A segunda, ad extra, com o pensamento teológico de Paul Tillich, que também valorizou o debate com a economia e indicou aspectos críticos a formas de messianismos. Entre os resultados da pesquisa, destacamos a necessidade de identificação constante do fato maior que marca a conjuntura socioeconômica e política, para, a partir dele, se refletir teologicamente. A realidade precisa ser compreendida com profundidade, especialmente considerando a complexidade social. A visão bipolar dominados x dominantes, por exemplo, além de favorecer messianismos políticos e simplificações, é insuficiente para se compreender as questões relativas ao contexto social. As reflexões apresentadas requerem uma elucidação, a mais transparente possível, da categoria teológica do Reino de Deus como a referência utópico-escatológica para todos os projetos. Palavras-chave: Messianismo. Reino de Deus. Teologia da libertação. alavras-chave: Messianismo. Reino de Deus. Teologia da libertação. 1 Universidade Metodista de São Paulo, Faculdade de Teologia, Curso de Teologia. R. do Sacramento, 230, Rudge Ramos, 09640-000, São Bernardo do Campo, SP, Brasil. E-mail: <claudio.ribeiro@metodista.br>. O messianismo político da teologia latino-americana da libertação O messianismo político da teologia latino-americana da libertação Abstract The reflections introduce the findings of the research into the historical development of Latin American liberation theology. The goal is to develop a critique of this important current of thought from the identification of aspects of political messianism present in it. Methodologically, the task was carried out 1 Universidade Metodista de São Paulo, Faculdade de Teologia, Curso de Teologia. R. do Sacramento, 230, Rudge Ramos, 09640-000, São Bernardo do Campo, SP, Brasil. E-mail: <claudio.ribeiro@metodista.br>. Recebido em 31/3/2015 e aprovado para publicação em 11/6/2015. Recebido em 31/3/2015 e aprovado para publicação em 11/6/2015. Reflexão, Campinas, 40(1):41-57, jan./jun., 2015 Reflexão, Campinas, 40(1):41-57, jan./jun., 2015 Reflexão, Campinas, 40(1):41-57, jan./jun., 2015 42 C.O. RIBEIRO from an approximation of Liberation Theology with two other theological expressions. The first, ad intra, was based on authors who have criticized certain views of political messianism in this theology, and who have prioritized the relationship with economy to offer such criticism, especially the contributions of Hugo Assmann, Julio Santa Ana and Jung Mo Sung. The second, ad extra, with the theological thought of Paul Tillich, who had also appreciated the debate with economy and indicated critical aspects of messianic forms. Among the results of our research, we highlight the need for the constant identification of the important fact that marks the socioeconomic and political situation to reflect theologically. Reality must be understood in depth, especially considering the social complexity. The bipolar perspective dominated x dominants, for example, in addition to promoting political messianism and simplifications, is insufficient to understand the issues related to the social context. Our reflections require an elucidation, as transparent as possible, of the theological category of the Kingdom of God as the utopian-eschatological vision for all projects. Keywords: Messianism. Kingdon of God. Liberation thelogy. Introdução Desde o início dos anos de 1990, tenho procurado fazer ad intra uma avaliação crítica da Teologia Latino-Americana da Libertação, referência teológica de sublime importância para mim, pela qual procuro seguir, mesmo com limitações, para pautar minha vida pessoal, minha inserção pastoral e meu trabalho acadêmico. Trata-se de avaliações efetuadas “de dentro” e em compromisso com os princípios práticos e teóricos fundamentais desta visão teológica, sobretudo a preferência que o Evangelho nos exige que se dê às pessoas pobres. Não obstante a isso, o objetivo de nossa pesquisa, nesse momento, é fazer uma crítica ao destacar aspectos de messianismo político presentes nessa corrente de pensamento. Desejamos sinalizar, com elementos críticos, possibilidades de renovação teórica e metodológica da Teologia da Libertação, dentro do quadro de desafios que a reflexão latino-americana sobre o campo religioso enfrenta. Realizaremos tal tarefa a partir de uma aproximação da Teologia da Libertação com outras duas expressões teológicas. A primeira, interna, com autores que criticaram certas visões de messianismo político nela presentes e priorizaram a relação com a economia para oferecer tal crítica. A segunda, ad extra, com o pensamento teológico do renomado teólogo protestante Paul Tillich, que também valorizou o debate com a economia e indicou aspectos de crítica a formas de messianismos e de idolatria. Perspectivas autocríticas O forte acento sociológico que a Teologia da Libertação assumiu, sobretudo nos anos de 1980, redundou em certos reducionismos políticos que acabaram oferecendo a ela um perfil messiânico. Alguns autores propuseram a valorização da dimensão econômica para que tal perfil pudesse ser revisto. Assim se deram as críticas que foram formuladas no contexto da Escola do Departamento Ecumênico de Investigação (DEI), de Costa Rica, e grupos afins. Nelas se destacam, especialmente, os trabalhos de Hinkelammert, Assmann, Santa Ana e Sung. Reflexão, Campinas, 40(1):41-57, jan./jun., 2015 eflexão, Campinas, 40(1):41-57, jan./jun., 2015 43 TEOLOGIA LATINO-AMERICANA DA LIBERTAÇÃO Em conexão com o contexto e o pensamento latino-americanos de uma ‘teologia da vida’, Hinkelammert (1983) já nos indicava, como o título de seu livro sugere: “As Armas Ideológicas da Morte”. Com ele, o teólogo brasileiro Hugo Assmann (1989) denunciava “A Idolatria do Mercado”. Da mesma forma, o uruguaio Julio de Santa Ana também fazia a “Crítica Teológica à Economia Política”, subtítulo de seu livro “O Amor e as Paixões” (1989). Tal perspectiva oferecia ao pensamento teológico a possibilidade de ir além dos fatos conjunturais da política e da dimensão eclesiástica. Tratava-se de uma contraposição aos imediatismos políticos e aos pragmatismos pastorais, em geral com acentos messiânicos. É fato que são muitas as limitações em nosso contexto latino-americano para uma compreensão mais apurada da complexidade que a economia traz para a vida e para o campo teológico. Embora seja também verdade que tal limite começou a ser superado quando Jung Mo Sung (1994) identificou, com maior precisão, certa “anomalia no paradigma da Teologia da Libertação”, uma vez que essa destacava as dominações sociais que geravam pobreza, mas não compreendia muito bem o fascínio que as formas de consumo exercem sobre as pessoas pobres. As transformações econômicas ocorridas na sociedade, tanto em âmbito mundial como continental, desafiam, portanto, fortemente as avaliações científicas e teológicas em especial em relação às formulações teóricas e as práticas pastorais inovadoras que se destacaram nas últimas décadas do século XX e que hoje parecem não serem mais os fatores que caracterizam a vivência religiosa em nossas terras. Como repetidas vezes tenho afirmado, a teologia e a pastoral latino-americanas não ficaram isentas dos impactos proporcionados pelas mudanças socioeconômicas e políticas no final do século passado simbolizadas pela queda do “muro de Berlim”. Retomando aspectos históricos e olhando o quadro presente O surgimento da Teologia da Libertação nas décadas de 1960 e 1970 pode ser sintetizado em pelo menos cinco pontos característicos. Em todos eles a relação entre teologia e economia se destaca. O primeiro deles é a práxis de libertação dos pobres e o compromisso evangélico de outros setores sociais com eles. A consciência dessa realidade gerou uma nova linguagem religiosa e teológica, fruto da relação dialética entre práxis e teoria presente na metodologia desse novo pensar teológico. Um segundo aspecto foi a necessidade de análise científica da realidade social com o recurso da teoria da dependência e, posteriormente, com o que se denominou mediações socioanalíticas. Um terceiro é a consciência do condicionamento socioeconômico da teologia e da igreja e a crítica de ambos a partir da ótica da libertação histórica dos pobres. Um quarto é a perspectiva de a reflexão teológica estar a serviço da transformação da sociedade, com indicações práticas e concretas de caminhos históricos de libertação sociopolítica. Nesse sentido, a Teologia da Libertação não se esgota no âmbito acadêmico. E, por fim, o lugar central da economia na reflexão teológica para, entre outros aspectos, estabelecer uma crítica ao messianismo tecnologista, às relações entre capital e trabalho, e vislumbrar alternativas de cunho socialista (SUNG, 1994). Em contraposição à visão desenvolvimentista, surgiram diferentes interpretações da realidade social, em especial a produção teórica de Celso Furtado, Theotonio dos Santos, Fernando Henrique Cardoso e Enzo Faletto. A teoria da dependência, como esses autores consagraram, compreendia a realidade de uma forma peculiar. Nesse contexto, a Teologia da Libertação, como elaboração teórica, procura(va) compreender a realidade por meio de mediações científicas, julgá-la mediante a tradição bíblica, com destaque para o aspecto profético, e indicar uma nova inserção dos cristãos. Hinkelammert (1996a), em uma análise que fez da Teologia da Libertação destacou a experiência do movimento “Cristãos para o Socialismo”, especialmente a do Chile dos anos de 1960 e início de 1970, e analisou também como o “império” produziu, nos anos de 1980, uma “teologia” que cooptou expressões (embora distorcidas e ideologizadas) da Teologia da Libertação, como a questão dos pobres. Para isso, ele analisou, por exemplo, discursos do secretário-geral do Fundo Monetário Internacional na época sobre o Reino de Deus. Perspectivas autocríticas Em função disso, novos referenciais precisaram ser descobertos para que a produção teológica pudesse ser aprofundada e que novos estágios cada vez mais relevantes no contexto do século XXI fossem adquiridos. A pressuposição com qual trabalhamos é que a tensão entre compreender e transformar o mundo não ficou isenta de simplificações para todos aqueles que temos trabalhado com a herança do marxismo ou formas similares de racionalismo político e social. O entusiasmo messiânico pelos esforços de transformação social impediu fortemente uma percepção mais definida de que o mundo mudou. Para refletir sobre os atuais desafios que se apresentam à teologia e à pastoral no contexto latino-americano é necessário pressupor, ao menos, quatro aspectos. O primeiro deles trata-se das já referidas transformações nos campos político, social, econômico e cultural ocorridas na virada para os anos de 1990 e que até hoje exigem melhor compreensão. Tais mudanças fortaleceram o neoliberalismo econômico e desordenaram significativamente os processos de produção de conhecimento. Ao mesmo tempo vivemos o crescimento e o fortalecimento institucional de novos movimentos religiosos, em especial do pentecostalismo no campo cristão e das experiências de avivamento religioso em diferentes religiões. Este pressuposto evidenciou um segundo aspecto, já igualmente engendrado desde os anos de 1980, relacionado com certa crise teórica nos setores teológicos. Ela precisa ser refletida em função das conexões necessárias que advogamos entre teoria (teológica) e prática (pastoral). Um terceiro pressuposto reside no fato de que as práticas pastorais sobrevivem, indubitavelmente, sob impasses de diferentes naturezas e carecem de novos referenciais para um processo de renovação. Um último aspecto são os desafios e possibilidades de refazimento de utopias. Trata da crise em seu aspecto dialético, ou seja, portadora de novas realidades e de novos caminhos de aprofundamento. Reflexão, Campinas, 40(1):41-57, jan./jun., 2015 44 C.O. RIBEIRO Retomando aspectos históricos e olhando o quadro presente Nessa ocasião, os teólogos da libertação e grupos de cristãos identificados com os mesmos postulados, ao afirmarem suas opções pastorais e políticas, se viram em confronto com os setores eclesiásticos – que estavam norteados por uma teologia “dogmática” e “conservadora” –, e com os setores militares de segurança nacional que assumiram ditatorialmente o governo chileno em 1973. Hinkelammert segue em sua análise apresentando, além do impasse latente entre a Teologia da Libertação e a teologia oficial das igrejas, o conflito com o “império”. Esse confronto se tornou mais evidente a partir das preocupações presentes no “Documento de Santa Fé” (1980), que formulou a plataforma política do presidente norte-americano, Ronald Reagan, na qual a Teologia da Libertação e as práticas pastorais dela decorrentes eram consideradas como questões de segurança nacional dos Estados Unidos da América (HINKELAMMERT, 1996b). Para os grupos referenciados pela Teologia da Libertação, os tempos que se seguiram dos anos de 1970 ao início dos 1980 foram marcados por confrontos e por fortes expectativas de mudanças econômicas. Assmann (1994), um dos teólogos que produziu os primeiros e mais destacados escritos da Teologia da Libertação, indica o otimismo e as condições que caracterizam a passagem dos anos de 1970 para os de 1980: Reflexão, Campinas, 40(1):41-57, jan./jun., 2015 45 TEOLOGIA LATINO-AMERICANA DA LIBERTAÇÃO Os que viveram esse período no Brasil, ainda sob o regime militar, mas com o lento alvorecer na ‘transição para a democracia’, certamente não podem riscar da memória as projeções utópicas lançadas sobre o futuro do país (e de outros países). Creio, aliás, que no Brasil se deram circunstâncias peculiares para que se fosse particularmente intenso o sonho de uma conversão da Igreja aos pobres. As características, ademais bastante singulares, assumidas pela Teologia da Libertação no contexto brasileiro, não se explicam sem o clima de projeções otimistas sobre a Conferência Nacional dos Bispos do Brasil (CNBB), sobre as (magníficas) comunidades eclesiais de base e, no plano político, sobre o Partido dos Trabalhadores (PT). (ASSMANN, 1994, p.16). No entanto, os anos de 1990 não confirmaram tais expectativas. Eles foram marcados por sinais mais efetivos da globalização econômica e pela exclusão social. Não é possível, pelos limites desse trabalho, uma abordagem detalhada do quadro econômico. Todavia, é importante, destacar ao menos alguns consensos de diferentes e recorrentes análises do campo social dessa época. Retomando aspectos históricos e olhando o quadro presente As práticas políticas e econômicas vistas no Brasil e na América Latina são coerentes com as políticas neoliberais estabelecidas em todo o mundo. A própria expressão “Terceiro Mundo” não se constituiu mais como forma adequada para descrever o mundo pobre, em função do fato de a internacionalização do mercado ter desenhado um mapa inteiramente novo. Na atualidade, novas fronteiras de uma ordem econômica estão sendo estabelecidas e essas fronteiras reforçam a exclusão social. A força dominante no mundo é o mercado. Os países que são capazes para participar no mundo do mercado são aqueles aptos a produzir e consumir. Caso contrário, eles estão fora da dinâmica econômica. Os Estados têm sido incapazes de mudar as leis de mercado ou influenciar o sistema global. A ideologia neoliberal, disseminada por intermédio da globalização da informação, faz com que os povos acreditem que o mercado ou o consumo é a solução da humanidade. Isso leva as pessoas a não priorizarem os laços de solidariedade, tornando-as mais individualistas e fortalecendo, assim, preconceitos contra os pobres. A globalização econômica, por ser baseada em monopólios sustentados por grupos (e nações) dominantes, é, portanto, uma forma de um sistema assimétrico. No Brasil, a mesma lógica prevalece: as pessoas que são capazes de produzir e consumir estão dentro da lógica do mercado; aquelas tidas por incapazes se tornam obstáculos ao “sucesso” do sistema. Elas não são “necessárias” e, dessa forma, são simplesmente excluídas. A tendência na sociedade é não se prover recursos financeiros nem mesmo tempo social para se dedicar à reflexão e ação sobre a situação na qual a massa crescente de pessoas pobres vive. Canclini (1996, p.18), na conhecida obra Consumidores e Cidadãos: conflitos multiculturais da globalização, mostra que: A maneira neoliberal de fazer globalização consiste em reduzir empregos para reduzir custos, competindo entre empresas transnacionais, cuja direção se faz desde um ponto desconhecido, de modo que os interesses sindicais e nacionais quase não podem ser exercidos. A conseqüência de tudo isto é que mais de 40% da população latino-americana se encontra privada de trabalho estável e de condições mínimas de segurança, que sobreviva nas aventuras também globalizadas do comércio informal, da eletrônica japonesa vendida junto a roupas do sudoeste asiático, junto a ervas esotéricas e artesanato local. Retomando aspectos históricos e olhando o quadro presente Desde a derrocada do sistema socialista soviético, o neoliberalismo, o novo estágio que o capitalismo experimentou no final do século XX, tem sido apresentado como o único caminho para se organizar a sociedade. As conhecidas e controvertidas teses de Fukuyama (1992) afirmam que o triunfo do capitalismo como um sistema político e econômico significou que o mundo teria alcançado o “fim da história”. Reflexão, Campinas, 40(1):41-57, jan./jun., 2015 46 C.O. RIBEIRO Esse estágio do sistema capitalista acentua a desvalorização da força de trabalho em função da automação e da especialização técnica e em detrimento das políticas sociais públicas. Forma-se, portanto, um enorme contingente de massas humanas excluído do sistema econômico e destinado a situações desumanas de sobrevivência ou passível de ser eliminado pela morte. Os ajustes sociais e econômicos implementados pelas políticas neoliberais geram degradação humana, perda do sentido de dignidade e consequentes problemas sociais das mais variadas naturezas. Contraditoriamente, em meio ao processo de globalização da economia e da informação, emergem, com maior intensidade, os conflitos étnicos, raciais e regionais no mundo inteiro. No campo social, as sociedades latino-americanas vivem processos que, embora variados, possuem em comum uma série de obstáculos para o exercício da cidadania e para a sustentabilidade da vida. Além da realidade política e econômica, está o desenvolvimento de uma cultura da violência que, além da dimensão social, envolve os aspectos ecológicos, étnicos, raciais e de gênero. O Brasil e os demais países da América Latina vivem tal realidade intensamente. Soma-se a isto a violência a partir das ações do crime organizado, de justiceiros e de grupos de extermínio, e a degradação da vida humana com tráfico de crianças, comércio de órgãos humanos, prostituição e reforço de formas de violência simbólica contra grupos subalternos e estigmatizados na sociedade. É fato que, em termos políticos, há sinais que contradizem tal tendência. Mesmo que cada grupo ou opção política tenha diferentes avaliações em relação às suas atuações, é consenso afirmar que, nos últimos anos, diversos governos na América Latina assumiram e têm desenvolvido políticas cujo perfil se enquadra em um espectro mais ‘à esquerda’ do que seus antecessores. É o caso do Brasil, da Venezuela, do Chile, da Argentina, da Bolívia e do Equador. As repercussões de tais políticas requerem uma análise à parte, mas elas têm gerado expectativas de mudança social. Reflexão, Campinas, 40(1):41-57, jan./jun., 2015 Retomando aspectos históricos e olhando o quadro presente Esta, como se sabe, é marcada por elementos mágicos e místicos, fruto de uma simbiose das religiões indígenas, africanas e do catolicismo ibérico. Em primeiro lugar, é necessário destacar que o processo de secularização vivido em meio à modernidade não produziu, como se esperava, o desaparecimento ou a atenuação das experiências religiosas. Ao contrário, no campo cristão, por exemplo, as formas pentecostais e carismáticas ganharam apego popular, espaço social e base institucional, tanto no mundo evangélico como no católico. Outras religiões também vivenciam, no Brasil e no mundo, momentos de reflorescimento. Sobre a “explosão religiosa” atual há outro aspecto relevante. Trata-se da influência na vivência religiosa de aspectos, não explicitamente religiosos, que formam a mentalidade da sociedade moderna no final do século XX, como é o caso das ênfases no consumo, na vida privada, na ascensão social e aspectos similares. Talvez isto explique, pelo menos em parte, o sucesso dos livros e ideias de autores bastante difundidos como Paulo Coelho e Lair Ribeiro, entre outros. São muitos os detalhes dessa perspectiva e diversas são as práticas à ela relacionada, o que dificulta as sínteses. Sob o nome de Teologia da Prosperidade – correndo o risco de simplificações –, podemos agrupar visões religiosas como a “Confissão Positiva” (não- aceitação da fragilidade humana), o “Rhema” (poder direto de Deus concedido pessoalmente aos crentes), a “Batalha Espiritual” (deslocamento religioso para explicações dos projetos históricos) e a “Vida na Bênção” ou “na Graça” (transferência da escatologia para a vida terrena). Neste sentido, destacam-se as “religiões de mercado” bastante evidenciadas em propostas no campo pentecostal, tanto nas vertentes evangélicas como católica. No entanto, não é somente no campo cristão que esse fenômeno se manifesta. Diferentes religiões, incluindo as de natureza afro-brasileira, possuem vertentes que advogam formas de uma “espiritualidade de consumo”, cujo caráter intimista, individualista e marcado pela busca de respostas imediatas para problemas pessoais ou familiares concretos revela-se na troca de esforços humanos (ofertas materiais e financeiras, atos religiosos como orações, bênção de objetos materiais e outros) por um retorno favorável aos desejos e necessidades humanas por parte do divino. Uma simples observação dos meios de comunicação social possibilita constatar o aumento do número de programas que utilizam os sistemas “0800” e “0900” para fins religiosos. Todo esse quadro está em sintonia com as transformações sociopolíticas, econômicas e culturais em todo o mundo. Retomando aspectos históricos e olhando o quadro presente O mesmo se dá com alguns movimentos sociais, como, por exemplo, o Movimento de Trabalhadores Rurais Sem Terra (MST) no Brasil, articulações de povos indígenas na Bolívia e mobilizações populares diversas, em especial as que integram o Fórum Social Mundial em suas diferentes versões no Brasil e em outros países, cuja referência básica é que “um outro mundo é possível”. Mesmo assim, diante desse quadro, todos os agrupamentos que tinham direta ou indiretamente como referência as experiências e as utopias socialistas chegaram a, pelo menos, duas constatações: a primeira trata da ausência de um projeto global alternativo ao neoliberalismo; e a segunda refere-se ao conjunto de perplexidades em diferentes campos do conhecimento que, usualmente, passou a ser denominado “crise dos paradigmas”. No campo religioso também encontramos intensas transformações. As últimas décadas do século XX e a primeira do corrente desafiaram os cientistas da religião e teólogos, em especial pelas mudanças socioeconômicas e as implicações delas na esfera religiosa. O leque de influências filosóficas e teológicas é tamanho que se torna árdua tarefa até mesmo descrever o cotidiano doutrinário, teológico e prático de uma comunidade religiosa. O fato é que a vivência religiosa no Brasil sofreu, nas últimas décadas, fortes mudanças. Alguns aspectos do novo perfil devem-se à multiplicação dos grupos orientais; à afirmação religiosa afro-brasileira; ao fortalecimento institucional dos movimentos católicos de renovação carismática; às expressões espiritualistas e mágicas que se configuram em torno da chamada Nova Era; e ao crescimento evangélico, em especial, o das igrejas e movimentos pentecostais. Todas estas expressões, além de outras, formam um quadro complexo e de matizes as mais diferenciadas. Os limites deste trabalho, obviamente, permitem apenas uma visão panorâmica da situação religiosa no Brasil. Teólogos e cientistas da religião, ao analisarem especificamente Reflexão, Campinas, 40(1):41-57, jan./jun., 2015 Reflexão, Campinas, 40(1):41-57, jan./jun., 2015 47 TEOLOGIA LATINO-AMERICANA DA LIBERTAÇÃO o campo das igrejas e dos movimentos cristãos, indicam que há no crescimento numérico dos grupos uma incidência intensa e direta de vários elementos provenientes da matriz religiosa e cultural brasileira. Esta, como se sabe, é marcada por elementos mágicos e místicos, fruto de uma simbiose das religiões indígenas, africanas e do catolicismo ibérico. o campo das igrejas e dos movimentos cristãos, indicam que há no crescimento numérico dos grupos uma incidência intensa e direta de vários elementos provenientes da matriz religiosa e cultural brasileira. Retomando aspectos históricos e olhando o quadro presente A multiplicação de grupos e expressões religiosas não-cristãs e o crescimento vertiginoso do pentecostalismo têm motivado pesquisas e mobilizado a opinião pública, uma vez que incidem diretamente no comportamento econômico, social e cultural do povo brasileiro. Para o tempo presente, maiores esforços de compreensão e análise precisam ser realizados. A Teologia da Libertação diante da conjuntura econômica e da estrutura social Assmann (1994), com o propósito de aprofundar questões em torno dos fundamentos da teologia latino-americana, já indicava, entre outros aspectos, a dificuldade dos setores mais Reflexão, Campinas, 40(1):41-57, jan./jun., 2015 Reflexão, Campinas, 40(1):41-57, jan./jun., 2015 48 C.O. RIBEIRO hegemônicos dessa corrente teológica em compreender que o “fato maior” que originou e motivou as primeiras reflexões teológicas alterou-se significativamente a partir do final dos anos de 1980. hegemônicos dessa corrente teológica em compreender que o “fato maior” que originou e motivou as primeiras reflexões teológicas alterou-se significativamente a partir do final dos anos de 1980. O autor, não obstante reconhecer certas simplificações dicotômicas, recorda que a ênfase para se compreender a realidade era o esquema opressão-libertação, com um abismo cada vez maior ente ricos e pobres e entre países ricos e pobres. Esse esquema era identificado pelas análises ancoradas nas causas estruturais e contraposto pelos movimentos populares, a partir da concepção messiânica de que os pobres eram o novo sujeito histórico. Esse “fato maior” possuía uma versão eclesiológica evidenciada na irrupção da “Igreja dos Pobres”. Assmann indica que boa parte dos teólogos da libertação não soube considerar devidamente o “fato maior” da realidade que se seguiu, em especial na apreciação das estratégias de confrontação, teórica e prática, com ele. Para o teólogo, “em síntese, o fato maior no mundo atual, e mais acentuadamente em nosso país, é a adoção consentida e celebrada como ‘modernização’, de uma férrea lógica da exclusão, que produz e perpetua uma assustadora ‘massa sobrante’ de seres humanos, tidos como economicamente inaproveitáveis e, portanto, objetivamente descartáveis” (ASSMANN, 1994, p.20). Assmann (1994) afirma estar fazendo uma leitura “não polêmica” da Teologia da Libertação, em continuidade e aprofundamento de seus postulados básicos. No entanto, as reflexões que o autor faz requerem uma ruptura com as formas majoritárias de elaboração dessa teologia. Retomando aspectos históricos e olhando o quadro presente Dessa forma, perdeu-se a percepção da possibilidade de um desenvolvimento autônomo, não excludente. Ao lado disso, o autor apresenta as limitações dos teólogos da libertação, ao não discernirem devidamente as transformações da realidade socioeconômica, em especial a substituição do desenvolvimentismo pelo neoliberalismo e o declínio da teoria da dependência. Entre as consequências disso está a compreensão equivocada, já presente na visão burguesa, assim como na socialista-marxista, de que a modernidade se caracteriza pela secularização e não – como o autor defende –, pela idolatria. Sung (1994, p.269) conclui que: Só uma teologia que introduz explicitamente a cláusula escatológica contra a ilusão transcendente da modernidade, abrindo espaço para uma transcendência teológica fundada na fé e na ressurreição de Jesus, e assume como sua tarefa fundamental a crítica anti-idolátrica a todas as instituições sacralizadas – sejam capitalistas ou socialistas – que exigem sacrifícios de vidas humanas pode reivindicar um lugar relevante no mundo moderno e servir eficazmente às lutas de libertação dos oprimidos sem perder a identidade de discurso teológico. Para o autor, as correntes mais destacadas da Teologia da Libertação não cumpriram adequadamente essa tarefa. A crítica à ilusão transcendente, fundamental para os que lutam contra o capitalismo, não foi efetuada, em especial pelo fato de o marxismo, na sua visão hegemônica, também compartilhar dessa ilusão de que é possível construir o Reino (da liberdade) em plenitude no interior da história. Setores importantes da teologia latino-americana, em especial os grupos que se dedicam à reflexão sobre as relações entre teologia e economia, têm empreendido esforços para melhor elucidação da temática da idolatria. Sung (1992, p.124), por exemplo, no início dos anos de 1990, já indicara que: A sacralização ou a absolutização de um sistema, seja capitalista ou socialista, significa a gestação de um totalitarismo. A distinção entre o projeto histórico e a utopia transcendental (ou, na linguagem de Dussel, a utopia histórica e a utopia escatológica) é fundamental na luta por uma sociedade mais humana. A utopia transcendental, não factível historicamente, deve acompanhar o projeto histórico, sendo uma fonte de inspiração e o fim a ser aproximado, mas não atingido; e, ao mesmo tempo, fonte de crítica ao projeto e às estratégias históricos. O que está em debate é o caráter de uma escatologia acentuadamente intra-histórica. Retomando aspectos históricos e olhando o quadro presente Nesse sentido, ele afirma que se essa reflexão “inovadora” for compreendida como mera “continuidade linear”, Corre-se o perigo de não analisar, com a devida atenção, as razões que levaram a Teologia da Libertação a incorrer em determinadas ingenuidades (por exemplo, a idealização dos oprimidos como o ‘novo sujeito histórico emergente’, assim como a exagerada aposta no surgimento de uma “Igreja dos pobres” etc.), e os motivos por que a teologia da Libertação apresenta certas lacunas (como a ausência de uma conjugação entre necessidade e desejos humanos, a escassa análise crítica do capitalismo enquanto sedução e simulação do prazer e da felicidade, enfim, toda a cadeia de vazios relacionados com uma confrontação, crítica mas também positiva, com a vigência de uma economia-com-mercado) (ASSMANN, 1994, p.14). Para analisar as diferentes lacunas, Assmann apresenta pressupostos antropológicos, políticos, econômicos, teológicos e eclesiológico-pastorais. Nesse conjunto de questões, o autor identifica o reducionismo antropológico que superestima a satisfação das necessidades elementares em detrimento da dinâmica dos desejos humanos, a visão política que mitifica a “força histórica dos pobres” e a “Igreja dos Pobres”, o descuido da temática “economia e teologia” e as generalidades em torno da concepção de “Deus dos pobres” que permitiam, entre outros aspectos, a manutenção de ideologias machistas e patriarcais, e de messianismos. Outra análise surgiu nos estudos de Sung (1994). Ele identificou na corrente interna mais divulgada social e eclesialmente da Teologia da Libertação uma “anomalia” que, no desenvolvimento dessa proposta teológica, causou um “esvaziamento”, em especial no seu conceito de libertação. Isso ficou mais evidenciado para o autor na “ausência de temas importantes e vitais para as lutas populares – tais como a questão do capitalismo- socialismo, a substituição do desenvolvimentismo pelo neoliberalismo como ideologia hegemônica na América Latina e o problema da dívida externa” (SUNG, 1994, p.265). Ou seja, a Teologia da Libertação, que pressupõe a análise da realidade como passo metodológico fundamental, relegou a uma “quase ausência” – pelo menos nos setores hegemônicos dessa teologia –, a economia como tema central de análise. Reflexão, Campinas, 40(1):41-57, jan./jun., 2015 Reflexão, Campinas, 40(1):41-57, jan./jun., 2015 49 TEOLOGIA LATINO-AMERICANA DA LIBERTAÇÃO O autor critica a falta de um aprofundamento por parte dos teólogos da libertação no tocante à teoria da dependência, especialmente ao adotarem uma visão bipolar, marcada pela simplificação “dominados x dominantes”, e por uma mera rejeição do crescimento econômico como se fosse sinônimo de desenvolvimento. Retomando aspectos históricos e olhando o quadro presente Para o contexto latino-americano, tanto para as versões teológicas relacionadas com a Teologia da Libertação como para as propostas religiosas vinculadas à Teologia da Prosperidade, com fortes similaridades com as formas de consumo capitalista, ressalta-se a demanda de melhor interpretação teológica da história, a fim de evitar messianismos, distorções ou reducionismos na compreensão do Reino. Outro aspecto de nossa análise é a necessidade de revisão teológica a partir do “círculo hermenêutico”. Santa Ana faz parte também do grupo de teólogos que, no interior da Teologia da Libertação, reflete sobre as questões entre teologia e economia e que também faz críticas às formas messiânicas presentes nessa corrente teológica. Nesse sentido, as avaliações feitas por Assmann e Sung – e acrescente-se também Hinkelammert –, são total ou parcialmente partilhadas por Santa Ana. Além desses aspectos, Santa Ana (1991) analisa também certo enrijecimento metodológico em setores da Teologia da Libertação, especialmente por não implementarem devidamente o “círculo hermenêutico” proposto por Gustavo Gutierrez em seus primórdios. A Teologia da eflexão, Campinas, 40(1):41-57, jan./jun., 2015 Reflexão, Campinas, 40(1):41-57, jan./jun., 2015 50 C.O. RIBEIRO Libertação, em sua proposta metodológica, considerou um círculo hermenêutico a partir das perguntas oriundas da experiência prática dos cristãos. Estas deveriam passar pelo crivo da crítica, para identificar sua validade e ajudar a enquadrá-las na realidade sociopolítica – que necessitava ser compreendida. Esse primeiro momento foi denominado como o das mediações socioanalíticas e foi privilegiado o marxismo como instrumental científico para as análises. Seguiam-se os momentos hermenêutico, prático/pastoral e de verificação na própria vida da comunidade dos pobres (a práxis). Percorrer esse círculo hermenêutico só faz sentido a partir de uma postura de suspeita de que as respostas dadas num momento anterior não sejam necessariamente válidas no seguinte. Cabe perguntar, portanto, se o conjunto de questões e de respostas com o qual a Teologia da Libertação trabalhava nas décadas de 1970 e início de 1980 é compatível com as necessidades da produção teológica nos anos que se seguiram. As motivações utópicas inerentes à Teologia da Libertação, por exemplo – referenciadas indiretamente às experiências do socialismo –, indicavam uma articulação da esfera pastoral com a esfera política. Como decorrência, era formulada uma sequência de perguntas no campo das relações entre fé e política. Estarão tais perguntas, ainda hoje, em sintonia com a experiência dos cristãos pobres, ou são necessárias novas sínteses? Retomando aspectos históricos e olhando o quadro presente Como já referido, a Teologia da Libertação surge como reflexão das práticas de libertação. Estas foram vividas na década de 1960 até a primeira metade da década de 1980. O que se seguiu foram “práticas de reajuste”, nas quais o povo pobre, em especial por motivos de sobrevivência, aceita, na maioria das vezes, resignadamente, as políticas econômicas e sociais nos diferentes países latino-americanos. As perguntas, portanto, não passaram a ser feitas em um contexto de libertação e, sim, de reajuste, o que altera substancialmente a forma de orientar as práticas pastorais. Sobre a compreensão da realidade, Santa Ana (1991) reafirma que as análises de corte teórico marxista demonstraram não ser suficientes para as mediações socioanalíticas da produção teológica. Tais análises, ao partir de contradições que se dão em plano socioeconômico, encontram dificuldades em desvelar outros aspectos da realidade, em especial os marcados pela dinâmica cultural. Soma-se a isso na América Latina o crescimento de importância dos conflitos sociais que não são de classes, como os étnicos, os raciais e os de gênero. Isso parece indicar a necessidade de se complementarem as análises marxistas com elementos da teoria sistêmica, das ciências antropológicas e da psicologia social. Sobre a questão hermenêutica, Santa Ana (1991) destaca duas necessidades, entre outras. Um aprofundamento bíblico do tema da idolatria e uma produção simbólica que coopere com a relativização dos “sagrados sociológicos” – onde a elementos meramente humanos é atribuída artificialmente uma dimensão sagrada –, a partir de uma penetração, nos limites do que seja possível, no “sagrado religioso”, que é mistério de Deus, interpelador e impulsionador do ser humano. Nesse sentido, o autor, ao retomar as intuições e perguntas centrais do teólogo Richard Shaull, precursor da Teologia da Libertação, como “o que Deus está fazendo no mundo, hoje?”, afirma que Grande parte da reflexão teológica ou chamada teológica, dos últimos tempos, não é uma pergunta sobre Deus, mas sobre outras coisas, sobre a igreja, sobre as formas da igreja, sobre a legitimidade da igreja. Não é uma pergunta sobre Deus, é uma pergunta sobre nós. Isto não é teologia. O fato de que haja um capítulo da teologia que se chama eclesiologia não significa que aí estejamos falando sobre Deus. Existe uma luta pela igreja tão forte neste momento, que muitas vezes nos leva a esquecer de Deus (SANTA ANA, 1991, p.35). A categoria teológica do Reino de Deus e a relativização de projetos políticos Após esse o diálogo com setores internos da Teologia da Libertação, que fizeram a crítica ad intra pelo viés da relação com a economia, vamos estender a reflexão, agora, a partir do diálogo com uma vertente do Norte: a teologia política de Tillich (1977). O ponto específico a ser refletido refere-se à prática política como referência fundamental dos cristãos e ao mesmo tempo o combate à todas as formas messiânicas e de idolatria. Daí a importância de tal aproximação para as críticas ao messianismo político presente da Teologia da Libertação. A noção de combate à idolatria, forte na perspectiva de Tillich e na teologia latino-americana, não pode se constituir em inércia, imobilismo ou formas de isenção e absenteísmo político. As teologias de Tillich e da Libertação fundamentam-se na proposição de envolvimentos políticos concretos. Para o exercício de combate a posturas idolátricas ou aos riscos de elas surgirem ou se fortalecerem serão apresentados na sequência três polos de críticas: aos idealismos e às práticas impositivas deles decorrentes, às formas político-pastorais que não articulam as dimensões de concretude e transcendência, e à exclusão social. A reflexão teológica que tende a relativizar os projetos e iniciativas históricas não pode tornar-se uma teoria que iniba ou não proponha ações políticas concretas. Ao contrário, Tillich teve a sua produção filosófica e teológica marcada pelos envolvimentos políticos que marcaram a trajetória dele. A renovação teológica latino-americana, por sua vez, caracterizou-se fortemente por possuir uma mediação prática com ênfase no aspecto da inserção política dos cristãos. O movimento pastoral orientado pela Teologia da Libertação formulou uma crítica ad extra ao questionar o funcionamento e a estruturação da sociedade. Essa perspectiva, tanto em termos prático-pastorais como em termos teológicos, trouxe o tema da Libertação para dentro do conjunto da sociedade. O movimento eclesial e teológico latino-americano formulou também uma crítica ad intra, ao estabelecer políticas pastorais distintas em relação aos modelos eclesiásticos oficiais. Trata-se de uma “nova forma de ser igreja” que, ao longo das últimas três décadas do século XX como até os dias de hoje, oportuniza experiências comunitárias inéditas de fé, com desdobramentos pastorais, por vezes intensamente conflitivos. Os conflitos pastorais decorrentes da prática política proposta pela teologia latino- americana baseiam-se, entre outros aspectos, na polaridade ideológica presente na sociedade entre capitalismo e socialismo. Retomando aspectos históricos e olhando o quadro presente Do ponto de vista prático-pastoral, ainda sobre a produção simbólica, surgem diferentes implicações, todas firmadas na busca de canais férteis de proclamação da mensagem Reflexão, Campinas, 40(1):41-57, jan./jun., 2015 51 TEOLOGIA LATINO-AMERICANA DA LIBERTAÇÃO evangélica. Entre os limites está o fato de a racionalidade da pastoral popular dificultar que a tarefa de Deus seja refeita: “ouvir o sofrimento do povo”, consolá-lo, seduzi-lo. Como escutar o povo se ele não fala? Os pobres, para manifestarem sua resistência, o fazem a partir de uma produção simbólica. Essa é, portanto, a linguagem dos oprimidos. Os projetos de conscientização – e aí se inclui a Teologia da Libertação –, estarão destinados ao insucesso se não mergulharem na tensão com a produção simbólica no âmbito popular. evangélica. Entre os limites está o fato de a racionalidade da pastoral popular dificultar que a tarefa de Deus seja refeita: “ouvir o sofrimento do povo”, consolá-lo, seduzi-lo. Como escutar o povo se ele não fala? Os pobres, para manifestarem sua resistência, o fazem a partir de uma produção simbólica. Essa é, portanto, a linguagem dos oprimidos. Os projetos de conscientização – e aí se inclui a Teologia da Libertação –, estarão destinados ao insucesso se não mergulharem na tensão com a produção simbólica no âmbito popular. A categoria teológica do Reino de Deus e a relativização de projetos políticos Nesse sentido, as aproximações entre a teologia latino-americana e a de Tillich são intensas e pertinentes, pois ambas as produções colocam em questão o capitalismo e estão relacionadas direta ou indiretamente ao socialismo (RICHARD, 1994). Reflexão, Campinas, 40(1):41-57, jan./jun., 2015 52 C.O. RIBEIRO Quanto à relação entre fé cristã e as práticas políticas concretas relacionadas ao socialismo, como é o caso histórico da Teologia da Libertação, da mesma forma, há da parte de Tillich uma substancial contribuição. Isso se dá pela crítica do teólogo às formas dogmáticas e messiânicas de socialismo, formulada simultaneamente aos compromissos políticos concretos firmados pelo autor por uma sociedade justa e igualitária com inspiração socialista. Nesse sentido, para se pensar possíveis indicações prático-pastorais, especialmente no campo político, serão priorizados nesse momento os aspectos relacionados ao socialismo. Trata-se das necessidades – que são pressupostas na reflexão teológica latino-americana –, de crítica ao sistema capitalista cuja versão atual se expressa no neoliberalismo econômico, em função de seu caráter excludente, e ao mesmo tempo, de busca efetiva de uma sociedade igualitária, participativa e firmada nos princípios da justiça social. Crítica aos idealismos e às práticas impositivas deles decorrentes Reflexão, Campinas, 40(1):41-57, jan./jun., 2015 Crítica aos idealismos e às práticas impositivas deles decorrentes O princípio socialista gera uma expectativa em relação à realização Reflexão, Campinas, 40(1):41-57, jan./jun., 2015 Reflexão, Campinas, 40(1):41-57, jan./jun., 2015 53 TEOLOGIA LATINO-AMERICANA DA LIBERTAÇÃO de todo e qualquer empreendimento sociopolítico. Quais seriam as implicações práticas dessa perspectiva? de todo e qualquer empreendimento sociopolítico. Quais seriam as implicações práticas dessa perspectiva? As análises sobre a realidade teológico-pastoral latino-americana, especialmente os aspectos principais de certos messianismos identificados em setores da Teologia da Libertação nos anos de 1980, revelaram a necessidade de uma reflexão mais aprofundada sobre a tensão entre realização e expectativa. No tocante à prática política, o conceito de expectativa formulado por Tillich representa singular contribuição para o contexto latino-americano. A partir dele é possível intuir diferentes atitudes concretas que possam refutar formas de idealismos (os pobres como sujeitos de uma iminente transformação da sociedade nos moldes teoricamente idealizados, por exemplo), de imobilismos (atitudes que não consideram as possibilidades históricas ou o surgimento de novos sujeitos sociais e alternativas políticas), de utopismos (como, por exemplo, a desconsideração dos processos históricos e do desenvolvimento paulatino de mudanças sociais) e de dogmatismos (a não-aceitação de formas revisionistas e críticas das propostas políticas que visem manter ou implementar o socialismo, ou mesmo a não-consideração do fato de que os pobres reproduzem também aspectos da ideologia dominante). Os quatro pontos seguintes, contidos no conceito de expectativa que Tillich (1997, p.97) formulou em sua filosofia social, iluminam essas indicações: (i) O socialismo posiciona-se decisivamente por uma atitude de expectativa. Ele conhece as frustrações da história e não espera que a existência humana e a realidade histórica sejam transformadas miraculosamente. (ii) Expectativa é a tensão com a meta que está adiante, é algo que se dirige ao novo, ao inesperado. Não se trata de atitude subjetivista, mas de algo firmado no movimento dos próprios eventos históricos. A expectativa inclui ação, pois algo de incondicional está sendo demandado. (iii) O que é esperado não está em contradição absoluta com a realidade presente, mas é o significado pleno de sua origem que há de ser cumprida no futuro. O que é demandado não são normas abstratas de justiça sem relação com a origem, mas o cumprimento da própria origem. Assim, passado (origem), presente (realidade) e futuro (expectativa) mantêm-se intimamente correlacionados. Crítica aos idealismos e às práticas impositivas deles decorrentes A expectativa, portanto, está no presente, mas com as tarefas de unir passado e futuro, de olhar para dentro do próprio socialismo e de sua realização a partir de uma nova ordem social e compreender, dessa forma, que o socialismo não é o fim da luta socialista. (iv) Esta seria a contribuição do que Tillich chamou de “socialismo religioso”, em especial pelo conceito de Kairos, que procura explicitar os limites assim como a validade e o significado da expectativa concreta. O socialismo requer uma atitude a mais realista possível, mas totalmente envolvida em uma expectativa (“realismo crente”) (believing realism). A expectativa é sempre relacionada ao concreto, mas ao mesmo tempo transcende cada instância do concreto. Crítica aos idealismos e às práticas impositivas deles decorrentes Crítica aos idealismos e às práticas impositivas deles decorrentes Considerando o contexto latino-americano, uma das abordagens a ser vista trata da concepção de ser humano e as implicações que a perspectiva socialista requer. Para Tillich, as propostas prático-pastorais, para serem relevantes bíblica e teologicamente, necessitam romper com a visão burguesa do ser humano. A manutenção dessa visão burguesa pode fazer com que o socialismo viva, pelo menos, quatro conflitos internos: (a) a supremacia da transformação social ante a pessoal; (b) a desvalorização dos carismas pessoais; (c) uma ausência de símbolos; (d) refutação de valores e sentimentos humanos como nostalgia, segurança pessoal, familiar e comunitária. Aqui e na sequência estamos tendo como base a obra The Socialist Decision, publicada em 1933 e que gerou conflitos com o governo nazista alemão (TILLICH, 1977). Portanto, o aprofundamento da reflexão antropológica nas atividades teológicas e pastorais é tarefa necessária e de fundamental importância. Sem ela, corre-se o risco da presença de perspectivas reducionistas, racionalistas e, nesse sentido, desconectadas da globalidade que a experiência humana representa. As propostas orientadas por essa visão, geralmente, tornam-se discursos e iniciativas políticas e pastorais “para o povo” e não “do povo”, ainda que marcadas por expressões ou ideias vinculadas às necessidades populares. Ao lado desse aspecto está uma série de outras práticas político-pastorais que, em função de equívocos na compreensão das dimensões pedagógicas, comunicacionais e organizacionais, tornam-se marcadas por simplificações e cristalizações absolutistas, portanto, idolátricas. Visando uma prática política que fuja dessas características, Tillich (1977) oferece como referência teórica para a prática política o conceito por ele elaborado do princípio socialista. O princípio socialista é um conceito dinâmico, de acordo com o próprio caráter da história, na medida em que ele contém as possibilidades de tornar compreensíveis novas e inesperadas realizações de determinadas origens históricas. O princípio socialista contém a possibilidade, a dinâmica e o poder do socialismo como realidade histórica. Tal princípio sempre se situa em uma perspectiva de crítica e de julgamento a essas realizações. Ele é, nas palavras do autor, “a situação proletária interpretada pela sua própria dinâmica, e por isso é obtido somente por uma decisão socialista e torna-se o ponto de vista por excelência para a interpretação e julgamento da realidade socialista” (TILLICH, 1977, p.47). Crítica às formas que não articulam concretude e transcendência A visão de Tillich (1977) pressupõe uma cristologia que articule as dimensões de concretude e de transcendência. A base cristológica da prática política como referência fundamental dos cristãos encontra-se em três aspectos da prática de Jesus: ser processual (histórica e desenvolvida a partir de ações e de reações concretas), situada (encarnada na realidade Reflexão, Campinas, 40(1):41-57, jan./jun., 2015 Reflexão, Campinas, 40(1):41-57, jan./jun., 2015 54 C.O. RIBEIRO econômica, política e religiosa) e conflitiva (não desejada, mas inevitável, em função da contradição entre o Reino de Deus e a realidade social da época) (BRAVO GALLARDO, 1986). econômica, política e religiosa) e conflitiva (não desejada, mas inevitável, em função da contradição entre o Reino de Deus e a realidade social da época) (BRAVO GALLARDO, 1986). Como no campo da teologia cristã, há, como se sabe, uma constante tensão no Evangelho entre os códigos da Aliança e da Pureza. O primeiro retoma o Êxodo, a experiência do deserto e a corrente profética, e o segundo refere-se ao Templo, à perspectiva do sacerdócio real e à oposição à reforma deuteronômica. A prática jesuânica é a personificação do código da Aliança. O conhecimento e a sabedoria de Jesus vêm do deserto e não da sinagoga. As interpretações teológicas mais substanciais revelam que Jesus confrontou as autoridades religiosas pela centralização do poder, pela cristalização das doutrinas, pela dogmatização e absolutização das ideias teológicas (a Lei) e pela supremacia da dimensão institucional em detrimento da vida humana. Tanto a teologia de Tillich quanto a da Libertação realçam o aspecto profético presente nessa perspectiva cristológica. A dimensão profética reafirma o caráter concreto da fé cristã e dos respectivos compromissos com a história e com a vida humana. A transcendência se dá a partir dessa perspectiva e contribui para redimensioná-la permanentemente, para evitar assim simplificações, messianismos e reducionismos da fé. Há, portanto, a necessidade de articulação entre concretude e transcendência. Essa relação traduz, em termos teológicos, para o contexto teológico latino-americano, a relação também necessária e urgente entre libertação e liberdade. A Teologia da Libertação necessita aprofundar a relação entre as temáticas da libertação e da liberdade, para não se tornar refém dos reducionismos e da efemeridade dos fenômenos sociais. A reflexão sobre a liberdade pressupõe as bases bíblicas e as experiências na história da Igreja, em especial a Reforma, e o diálogo com a modernidade, com destaque para a ideia de diversidade (COMBLIN, 1996). Crítica às formas que não articulam concretude e transcendência A efetivação desses aspectos se dá de forma coletiva, como a tradição teológico-pastoral latino-americana tem consagrado, ou seja, vida comunitária e prática política são dimensões necessariamente associadas. Tillich, desde os seus escritos sobre filosofia social e política que deram base para as noções do socialismo religioso, afirma, de forma semelhante, o valor da comunidade nas ações políticas. Ao analisar as aproximações entre a visão política de Tillich e a Teologia da Libertação, Richard (1994, p.154) apresenta a concepção do teólogo, ao indicar que “a dimensão religiosa do marxismo – expressa em uma linguagem não-religiosa –, consiste precisamente de dois elementos de qualquer pensamento religioso: a responsabilidade de ação (ou praxis) e a fé e a crença de que pelo que se está lutando necessariamente virá” e conclui que “a compreensão religiosa do socialismo gera finalmente uma comunidade espiritual que se assemelha ao movimento de comunidades de base” (p.155). Nesse sentido, há uma avaliação positiva da articulação entre concretude e transcendência no contexto teológico latino-americano, não obstante a necessidade de atenção constante a esses aspectos. Crítica à exclusão social Crítica à exclusão social Os conceitos formulados por Tillich contribuem, sobretudo, para a “crítica teológica da economia política”, também elaborada no contexto latino-americano, como visto. Aqui retomamos as reflexões entre teologia e economia que se desenvolveram, com ênfase, em especial no final dos anos de 1980. Elas, como já indicado, procuraram responder Reflexão, Campinas, 40(1):41-57, jan./jun., 2015 Reflexão, Campinas, 40(1):41-57, jan./jun., 2015 55 TEOLOGIA LATINO-AMERICANA DA LIBERTAÇÃO a determinada “anomalia” na Teologia da Libertação que, em seu desenvolvimento, não aprofundou a própria previsão da centralidade da questão econômica nas reflexões teológicas. Por outro lado, tais reflexões contribuem substancialmente para o debate de questões suscitadas pelas críticas às propostas religiosas identificadas com os tipos de teologias de prosperidade já mencionados, em função das associações quase que diretas entre aspectos econômicos da vida pessoal ou familiar com a bênção de Deus. A realidade de exclusão social requer uma crítica teológica consistente. As profundas mudanças no sistema capitalista estabeleceram, como se sabe, a mais recente e sofisticada etapa do sistema econômico: o neoliberalismo. Seguindo a própria lógica do sistema, foram geradas, nas últimas décadas, massas consideráveis da população excluídas do mercado de trabalho e das possibilidades de aquisição de bens materiais, até mesmo os de natureza básica para a sobrevivência. Em função desse novo aspecto da realidade, os círculos teológicos e pastorais tiveram que se debruçar sobre a temática dos “excluídos do sistema”. Com isso, novos desafios de compreensão da realidade surgiram, uma vez que os referidos círculos, no contexto latino-americano, em regra geral, utilizavam como referência o binômio dominador/dominados para compreender a realidade, o que, não dá conta da complexidade dos processos atuais de exclusão social. A crítica teológica da economia política representa, portanto, um salto de qualidade nas reformulações necessárias da Teologia da Libertação, especialmente em função das transformações sociopolíticas e econômicas, como a revolução tecnológica, e a nova ordem econômica mundial, além da crise de paradigmas da modernidade. “Salto de qualidade” e “limitação teórica” por vezes convivem. Isso porque, como visto, nem todos os setores da Teologia da Libertação percebem os aspectos que apontam para uma alteração do processo de produção teológica e de novas orientações pastorais. Em primeiro lugar, as práticas dos pobres (cristãos ou não-cristãos) não têm-se constituído em um processo efetivo de libertação. Na atualidade, na América Latina existem manifestações de práticas libertadoras, todavia sem representar um projeto articulado. Reflexão, Campinas, 40(1):41-57, jan./jun., 2015 Considerações Finais O objetivo com as reflexões que apresentamos foi elaborar uma crítica à Teologia Latino-Americana da Libertação a partir da identificação de aspectos de messianismo político presentes nessa corrente de pensamento. Nosso esforço foi realizar tal tarefa a partir de uma aproximação da Teologia da Libertação com outras duas visões teológicas. A primeira, ad intra, com autores que criticaram certas visões de messianismo político nela presentes e priorizaram a relação com a economia para oferecer tal crítica, especialmente Hugo Assmann, Julio de Santa Ana e Jung Mo Sung. A segunda, ad extra, com o pensamento teológico de Paul Tillich, que também valorizou o debate com a economia e indicou aspectos críticos a formas de messianismos. Entre diversos desafios apresentados, destacamos a necessidade de identificação constante do “fato maior” que marca a conjuntura socioeconômica e política, para, a partir dele, se refletir teologicamente. Com isso, nas reflexões críticas e nas análises científicas da realidade é de vital importância o destaque para a economia e os efeitos dela nos processos sociais, culturais e políticos. A realidade precisa ser compreendida com profundidade, especialmente considerando a complexidade social. A visão bipolar dominados x dominantes, por exemplo, além de favorecer messianismos políticos e simplificações, é insuficiente para se compreender as questões relativas ao contexto social. No campo religioso, igualmente, a discussão entre religião e neoliberalismo econômico apresenta diferentes questões. Destacamos as transformações no campo religioso que enfatizam a “religiosidade de consumo”, a visão social e política consumista, individualista e excludente reforçada pelo sistema econômico neoliberal, e o reflexo desse caráter no plano das práticas e das ideias religiosas, cristãs e não cristãs. As reflexões apresentadas requerem uma elucidação, a mais transparente possível, da categoria teológica do Reino de Deus como a referência utópico-escatológica para todos os projetos. Isso ganha destaque, especialmente em função de confusões e de simplificações no contexto prático entre Reino de Deus e projetos históricos, tanto na pastoral popular baseada em algumas formulações da Teologia da Libertação, como em certos ambientes marcados pelas teologias de prosperidade, que direta ou indiretamente associam a bênção de Deus com os valores e práticas do neoliberalismo econômico. Referimo-nos, com essa reflexão, à prática política como referência fundamental dos cristãos, em especial as propostas que visem à justiça social, a igualdade, a liberdade e a solidariedade humana. Crítica à exclusão social Embora haja práticas de resistência – inegáveis –, o que prevalece são as de reajuste, e nestas se encontra a maioria do povo pobre. Portanto, as perguntas feitas, como passo metodológico inicial, não são orientadas ou formuladas num contexto de práticas libertadoras, mas de reajuste, de sobrevivência. A nova expressão “excluídos”, comum nos ambientes pastorais nas últimas décadas, não pode simplesmente ser mera substituição de “oprimidos” e tornar-se um novo jargão de militantes cristãos. Há que se aprofundar os processos de análise da realidade e superar, dessa forma, as referidas anomalias que marcaram o desenvolvimento da Teologia da Libertação. Para isso, sobretudo com a inspiração dos pressupostos teológicos de Tillich já vistos, impõe-se uma reflexão teológica sobre a economia. Esta se dá num contexto de confronto entre dimensões que reivindicam sacralidade. O discurso e a prática do neoliberalismo remontam a exigências de sacrifícios humanos, a perspectivas absolutistas e globalizantes e a promessas de retribuição dos investimentos e de prosperidade, o que possibilitou aos estudiosos a criação da expressão deus-mercado. Não há necessidade de legitimação religiosa para esse sistema, pois ele, em si, é religioso. Do ponto de vista político-pastoral, urge, portanto, reforçar as ações de solidariedade e de afirmação da dignidade humana, uma vez que a lógica sistêmica considera tais ações como empecilho ao funcionamento autorregulador do mercado. Ou seja, as ações humanas de solidariedade e de luta pela dignidade dificultariam a “mão invisível” do sistema. Reflexão, Campinas, 40(1):41-57, jan./jun., 2015 56 C.O. RIBEIRO Considerações Finais A partir dos pressupostos da produção teológica de Tillich e da teologia latino-americana, foi ressaltado que o combate à idolatria e aos messianismos políticos não pode se constituir em inércia prático-pastoral, imobilismo das igrejas ou de grupos dentro delas, ou atitudes de isenção e de absenteísmo político. O Reino de Deus, em sua intra-historicidade, requer envolvimentos políticos concretos. No entanto, há de se retomar tal categoria, especialmente como destacou Tillich, no sentido de ser o grande e único horizonte para as ações políticas e pastorais. Nesse sentido, a partir de conceitos de Tillich como o princípio socialista e o de expectativa, ao lado da vocação ao agir própria da Teologia Latino-americana da Libertação, o combate a posturas messiânicas ou aos riscos delas surgirem ou se fortalecerem necessita de, pelo menos, três posturas críticas: aos idealismos e às práticas impositivas, às formas político-pastorais que não articulam as dimensões de concretude e transcendência, e à exclusão social. Para pensar a teologia e a pastoral no contexto latino-americano, portanto, é necessário admitir e compreender, o mais profundamente possível, a força, as influências diretas Reflexão, Campinas, 40(1):41-57, jan./jun., 2015 57 TEOLOGIA LATINO-AMERICANA DA LIBERTAÇÃO e indiretas e a capacidade de mobilização e de sedução do neoliberalismo. Não serão concepções messiânicas que cooperarão para a superação da força destrutiva sistêmica. Todavia, a esperança está no bojo da vocação teológica e não deve ser descartada mesmo que a realidade seja sombria. Não será esquecida a força histórica dos pobres, como indicou Gustavo Gutiérrez, ainda que seja necessário, como por sua vez afirmou Juan Luis Segundo, tratar da fraqueza histórica dos pobres. e indiretas e a capacidade de mobilização e de sedução do neoliberalismo. Não serão concepções messiânicas que cooperarão para a superação da força destrutiva sistêmica. Todavia, a esperança está no bojo da vocação teológica e não deve ser descartada mesmo que a realidade seja sombria. Não será esquecida a força histórica dos pobres, como indicou Gustavo Gutiérrez, ainda que seja necessário, como por sua vez afirmou Juan Luis Segundo, tratar da fraqueza histórica dos pobres. Reflexão, Campinas, 40(1):41-57, jan./jun., 2015 Referências ASSMANN, H. Crítica à lógica da exclusão: ensaios sobre economia e teologia. São Paulo: Paulus, 1994. ASSMANN, H.; HINKELAMMERT, F. A idolatria do mercado: ensaio sobre economia e teologia. Petrópolis: Vozes, 1989. RAVO GALLARDO, C. Jesús hombre en conflicto. Ciudad de México: Centro de Reflexão Teológ BRAVO GALLARDO, C. Jesús hombre en conflicto. Ciudad de México: Centro de Reflexão Teológica, 1986. CANCLINI, N. Consumidores e cidadãos. Rio de Janeiro: UFRJ, 1996. BRAVO GALLARDO, C. Jesús hombre en conflicto. Ciudad de México: Centro de Reflexão Teológica, 1986. ANCLINI, N. Consumidores e cidadãos. Rio de Janeiro: UFRJ, 1996. COMBLIN, J. Cristãos rumo ao Século XXI: nova caminhada de libertação. São Paulo: Paulus, 1996. FUKUYAMA, F. O fim da história e o último homem. Rio de Janeiro: Rocco, 1992. HINKELAMMERT, F. As armas ideológicas da morte. São Paulo: Paulinas, 1983. HINKELAMMERT, F. A teologia da libertação no contexto econômico-social da América Latina: economia e teologia ou a irracionalidade do racionalizado (I). Revista Eclesiástica Brasileira, v.56, n.221, p.45-61, 1996a. HINKELAMMERT, F. A teologia da libertação no contexto cconômico-social da América Latina: economia e teologia ou a irracionalidade do racionalizado (II). Revista Eclesiástica Brasileira, v.56, n.222, p.331-347, 1996b. RICHARD, J. The socialist Tillich and liberation theology. In: Bulman, R.F.; Parrela, F.J. (Ed.). Paul Tillich: A new catholic assessment. Collegeville: The Liturgical Press, 1994. p.148-173. SANTA ANA, J. Questões atuais da reflexão pastoral e teológica da libertação. Papos, v.3, n.5, p.20- 40, 1991. SUNG, J.M. Deus numa economia sem coração. São Paulo: Paulinas, 1992. SUNG, J.M. Teologia & economia: repensando a teologia da libertação e utopias. Petrópolis: Vozes, 1994. TILLICH, P. The socialist decision. New York: Harper & How Publishers, 1977. Reflexão, Campinas, 40(1):41-57, jan./jun., 2015
https://openalex.org/W4288060279	https://figshare.com/articles/thesis/Sovereign_Sense/20388042/1/files/36446013.pdf	English	null	Sovereign Sense	null	2,022	cc-by	26,173	III. III. ABSTRACT chitectural advantages of atoll environments. How the preservation of social, cultural identity and order can be maintained through a contemporary evolutionary process. Throughout this changing context it is imperative to maintain a sense of hu- man scale within this small populace of Tuvaluan’s. The South Pacific Island of Funafuti, Tuvalu is at threat of becoming one of the first countries glob- ally to be Inundated due to rising sea levels. The likely result is that the people of this country will lose a sense of place and culture and be unable to sustain their National Sovereignty in the face of impending climate change and refugee status. Willi Telavi Tuvalu’s Prime Minister states ‘reloca- tion is not seen as an option but as a last resort, rights to land and culture are held with the utmost importance’ (McNamara and Gibson, 2009). Relo- cation will result in a loss of sovereignty however architectural intervention can insure that a sense of sovereignty is maintained on the islands during the drastic climate change transformations that they face. The intention of this Architectural The- sis is to design a solution that actively engages with sea level rise so that Tuvalu and other low-lying atoll nations can maintain a minimum of subsis- tence dwelling, economy and sovereignty. The process begins with analysing a series of archi- tectural design experiments. They are design led research experiments with themes of impending reality. They are similar to Tuvalu’s vernacular and built environment by their inherent characteristics and layout design Sourcing concrete current ideas and findings on Tuvalu itself are scarce as to the nature of fluxes of the global climate change predictions. Therefore research will be provided on the current environ- mental conditions of the island and the current problems the Tuvaluan’s face, The predictions for sea level rise will be compared on a Funafuti cross section. This will show impact on the islands in- formal dwellings over time periods and how im- provements can be made to mitigate the exacerbat- ed conditions of climate change and the potential future problems that Funafuti Island will face. Dylan Majurey Dylan Majurey A 120-point thesis submitted to the Victoria University of Wellington in partial fulfill- ment for the requirements of the degree of Master of Architecture (Professional) IV. ABSTRACT ‘The spirit of invention that makes survival possible under the extreme conditions cre- ated by destruction makes possible the new ways of living in a city that will, in a sense, always remain in a paradoxical state of destruction and construction.’ The current problems the Capital Island of Funa- futi face are crippling with the loss of coastal areas and increased tidal flooding. This results with not only a loss in land area but also permanent salini- sation in areas traditionally used for crop harvest- ing. This salinisation will only increase in severity with the projected future sea level rise. It will force the population of Funafuti to become climate ref- ugees before it is fully submerged (IPCC, 2013). - Lebbeus Woods (Radical Reconstruction, 1997) The main question this thesis aims to address is; How can architecture maintain a sense of sover- eignty within a disappearing context. And what are the implications of habitation, culture and con- tested territories for the Tuvaluan’s? This critical reflection aims to investigate the ar- VI. DISCOURSE ACKNOWLEDGMENTS To my supervisors Maibritt Pedersen Zari and Carles Martinez Almoyna Gual. Without your commitments as supervision I wouldn’t have got nearly this far. Thank you to my family and friends for all the sup- port and wishes along the way. To my mother thank you for the infinite encour- agement to push me across the line. And to my step dad Steve, thank you for knowing not to ask how everything was going along the way. To my dad thank you for always being there and knowing when to take a step back or when to secretly help me out. To my partner Michelle, sorry for being such a nuisance to deal with throughout this process. But I wouldn’t have made it to the otherside without you, picking me up along the way. Figure 01: VIII. CHAPTER ONE: THESIS INTRODUCTION 12 Research Discourse Reflection 26 Defining Site Atoll Environment Climatic Conditions Cultural Conditions Reflection CHAPTER TWO: THEORETICAL FRAMEWORK 075 Literature review Introduction Reconstruction Active participants Spatial Activation Narrative environment Incrementalism & the informal Reflection 92 Precedent Review Introduction Quinta Monroy Housing Makoko floating school Boat house Wetland park Reflection CHAPTER THREE: RESEARCH THROUGH DESIGN 118 Design process 121 Site Analysis Vernacular Dwellings Preliminary Design Concept Design CONTENTS CHAPTER ONE: THESIS INTRODUCTION CHAPTER FOUR: CLOSING DISCOURSE 190 Discussion CHAPTER FIVE: THESIS REFERENCES 225 Figure References 267 Bibliography 270 Appendix XI. X. Research Question, Aims and Objectives: How can architecture maintain a sense of sover- eignty within a disappearing context and what are the implications of habitation, culture and deterri- torialisation? be given to subsistence living with the use of local resources and the connection to the Tuvaluan’s en- vironment. Tzonis suggests that the human in its effort to adapt throughout millennia uses objects as extensions of the human body. These tools are created as a direct response of the human physiol- ogy and the natural environment. ( Tzonis, pg.7). However within the Tuvaluan evolution there are divides between past, post-colonial and present typologies within the landscape. The architectur- al fabric is lacking in a successful evolution from the vernacular dwelling. Initial architectural de- sign approaches will be implemented by acknowl- edging the successful architectural characteristics and regenerate design opportunities that main- tains territorial and evolutional integrity. I suggest this will also maintain a sense of sovereignty for the disappearing landmass, which can provide for the remediation of slums and impoverished areas thereby harnessing temporal growth and survival against inundation. This thesis proposes that a sense of Sovereignty and cultural values can be entrenched in the de- velopment of architecture that is specific to the material culture, community and environment in which it is established. The aims outlined be- low allow for a continued occupation of Funafuti atoll whilst accounting for future habitation, crop production and subsistent community roles in its ever-changing landscape. xiv. 373 - POPULATION DENSITY 1763 URBAN 90 RURAL 11, 636 - POPULATION (July 2005 est) 275 Km2 - LAGOON AREA 2.4 Km2 - LAND AREA 5 PEOPLE - AVERAGE HOUSEHOLD SIZE LAND AREA - 2.4 KM2 100m 100m 100m 100m Figure 02: Tuvalu housing statistics presenting the architectural problem Tuvalu’s relatively small size and oceanic loca- tion create a situation whereby it is isolated from proximal assistance. It has a harsh environment that produces significant constraints to the coun- tries development. Funafuti atoll is the capital and home to 6,200 inhabitants. This accounts for 57.2 percent of the nation’s population. It is Tuvalu’s most populated atoll being a narrow sliver of land circling a large lagoon that is 18 km long and 14 km wide (surface area 275 km2). It is encompassed by 33 islet aggregates with a land area of 2.4 km2, which is under one percent of the total Atolls area (see fig. 5). With an average height of just under 2 meters it is extremely vulnerable to sea level rise. a populations potential and the support of local stewardship encourages resilience and adaptation for the population (Thaman, 2014). Therefore ad- dressing these current social, and economic prob- lems through architecture is not only crucial to the mental and social well being of Tuvaluan peo- ple. But along with other factors this will mitigate some of the effects of perceived hardship in squat- ter settlements such as the burrow pits of Funa- futi. The use of design principles that supplement low-income atoll settlements and pave the way for architectural change that is needed to combat the issues that a rising sea level brings. In 2007 Tuvalu developed and released a Nation- al Adaptation Programme of Action Plan. The plan outlines the impact of droughts and deluge in relation to the amount of salt inundation al- ready found in coral Atolls. These Atolls, which were once used for growing crops, are now unable to harness the available plant nutrients previous- ly found in swamp areas. Beachhead erosion in conjunction with the use of sand as a building re- source means that viable land is being depleted at a heightened rate. The current overpopulation and an already scarred landscape place large amounts of pressure on the remaining land area. The lack of arable land is continually challenged due to bur- row pit inundation with sea water as a result of predicted climate change. The access to sanitation, solid waste removal and land management, which due to poor provision and lack of resources are also problems that impact Funafuti’s increasingly fragile environment. The principle aims of this design led thesis are: 1. To discover the Architects role in low-lying atoll environments and reconfiguring the typical hier- archical approach to design. 2. To sustain a sense of community and belonging, whilst addressing sea level rise and its challenges. 3.To Improve the standard of living by restoring the relationship between contemporary urban settlement and the social, cultural and ecological conditions of site. 4. To critically engage in the preservation of the cultural and social identity of a low-income strata and to provide a ‘sense of place’ 4. To critically engage in the preservation of the cultural and social identity of a low-income strata and to provide a ‘sense of place’ 5. To adapt typologies for predicted sea level rise. 6. To provide a sense of human-scale, order, evolu- tion and sovereignty within this changing context. 6. To provide a sense of human-scale, order, evolu- tion and sovereignty within this changing context. This investigation engages a position that observes past and present structures of an Island commu- nity that are reflective of the vernacular and its architectural adaption. Historical testament must xiii. % OF PERSONS ENGAUGED IN SUBSISTENT ACTIVITIES ETHNIC MAKE-UP 88% 12% 93.6% 4.6% 0.6% 0.3% 0.9% 7% 36% 57% 9% 10% 81% ACTIVITIES OTHERS OUTER ISLANDS FUNAFUTI OTHER PACIFIC I-KIRIBATI PART TUVALUAN TUVALUAN - FORMALLY EMPLOYED - FORMALLY EMPLOYED - UN-EMPLOYED - UN-EMPLOYED - FISHING,AGRICULTURE, HANDICRAFTS - FISHING,AGRICULTURE, HANDICRAFTS TUVALU ACTIVITY STATUS FUNAFUTI ACTIVITY STATUS Figure 07: Pie charts by Author displaying the local make up and activity status to understand community roles 93.6% 4.6% 0.6% 0.3% 0.9% OTHERS OTHER PACIFIC I-KIRIBATI PART TUVALUAN TUVALUAN 88% 12% OUTER ISLANDS FUNAFUTI xiv. 373 - POPULATION DENSITY 1763 URBAN 90 RURAL 11, 636 - POPULATION (July 2005 est) 275 Km2 - LAGOON AREA 2.4 Km2 - LAND AREA 5 PEOPLE - AVERAGE HOUSEHOLD SIZE LAND AREA - 2.4 KM2 100m 100m 100m 100m Figure 02: Tuvalu housing statistics presenting the architectural problem The current and future impacts on Funafuti in- clude the loss of coastal margins with frequent tidal flooding and soil salinisation damaging crops and polluting fresh water sources. A higher sea level has already begun to threaten the coun- tries porous underground water table. This has substantially impacted the environment and the population of Funafuti. The current development challenges facing the population of Tuvalu are the lack of adequate infrastructure for housing, and the instability within the political system and civil service. Amongst the general populace there is a perception that the current hardship Tuvalu faces is caused by Limited access to quality basic services. Limited access to quality basic services. Limited opportunities to earn money, particularly for women and youth. Overcrowding and overpopulation of households and communities(see fig. 2). Deteriorating social support systems Idleness and a dependency attitude. Limited opportunities to earn money, particularly for women and youth. y Overcrowding and overpopulation of households y Overcrowding and overpopulation of households and communities(see fig. 2). y Overcrowding and overpopulation of households and communities(see fig. 2). Thaman proposes that in adaptive management and in the development of local security, one must provide a basis for a response to develop- ing threats. He suggests that the understanding of Figure 02: DEFINING SITE xv. III. 0 2KM 4KM HARBOUR ACCESS ROUTE FUNAFUTI CONSERVATION AREA Figure 05: Author map showing the capital Funafuti highlighted with lagoon depths, coral substrata and Funafuti con- servation area. NEW ZEALAND NIUE TONGA W. SAMOA A. SAMOA KIRIBATI 8.5000° S, 179.1833° E FIJI VANUATU NEW CALEDONIA SOLOMON ISLANDS PAPUA NEW GUINEA FEDERATED STATES OF MICRONESIA MARSHALL ISLANDS NAURU TOKELAU AUSTRALIA EQUATOR AUSTRALIA FUNAFUTI ATOLL TUVALU & FUTUNA WALLACE Figure 04: Map showing Tuvalu’s isolated location and bearing of Funafuti island relative to New Zealand. Flights t T l f S Fiji FEDERATED STATES OF MICRONESIA Figure 04: Map showing Tuvalu’s isolated location and bearing of Funafuti island relative to New Zealand. Flights Figure 05: Author map showing the capital Funafuti highlighted with lagoon depths, coral substrata and Funafuti con- servation area. to Tuvalu run from Suva, Fiji. III. II. % OF PERSONS ENGAUGED IN SUBSISTENT ACTIVITIES Figure 08: Some of Tuvalu’s locals are overcome by the cocerns of climate change as the landscape deteriates around them. If they leave the island, cultural and social identity hangs in the balance. OPPORTUNITY FOR DESIGN RESEARCH The design research endeavours to achieve a speculative solution that contradicts the social, cultural and architectural norms currently at- tributed to climate responsive design. Migration is seen as the saviour for countries facing climate change. Due to the financial feasibility to migrate compared to adaption this neglects the cultural implication of sovereignty. Therefore I intend to investigate imaginative and efficient architectural solutions for the maintenance of territory and na- tionhood as a possible solution for the low-income settlements of Funafuti that inhabit a continuously changing context. Unveiling the current and po- tential impacts for the local inhabitants that will effect their communal life. The research focuses on the current neglect of cul- tural, social and political values and how they can be adapted and implemented in the developmen- tal process. The scope has varied limitations due to the sites isolation and lack of available information on the current built environment including topo- graphic information and states of building degra- dation. Design experiments are based on the in- formation available and requires the application of speculation to apply an environmental framework that accounts for sovereignty and a small populous to safe guard it. Figure 07: Pie charts by Author displaying the local make up and activity status to understand community roles Figure 07: IV. V. Summary The Tuvaluan culture has existed on these islands for over 2000 years and is at threat due to rising sea levels. Many of the islanders do not want to leave as their nationhood and sovereignty would be challenged. ‘Tuvaluan’s become climate change refugees when the land of Tuvalu becomes unin- habitable. With this last resort adaptation to cli- mate change we Tuvaluan’s lose our sovereignty, our traditional customs. I think you all know how important these are to us as native landholders’ (Siuila Toloa) board member of Island Care, a Tu- valu environmental group). This thesis aims to investigate the architectural ad- vantages of atoll environments and how the preser- vation of social, cultural identity and order can be maintained through a contemporary evolutionary process. The intention of this architectural thesis is to design a solution that actively engages with sea level rise so that Tuvalu and other low-lying atoll nations can maintain a sense of sovereignty with- in a disappearing context (see fig. 8). Throughout this changing context it is imperative to maintain a sense of human scale within this small populace of Tuvaluan’s and what the implications of habi- tation, culture and contested territories are for the Tuvaluan’s in order to maintain a minimum of subsistence dwelling, economy and sovereign- ty. The focus will be on the ‘dwelling’, developing architectural characteristics to inform the design within the prior outlined aims and objectives. VI. VII. The thin slither of land that is Funafuti Island, Tuvalu’s capital. As displayed the vulnerability of land is evi- dent with no more than several hundred meters separating the internal lagoon from a vast ocean. Figure 11: FUNAFUTI ATOLL Tuvalu’s capital Funafuti Atoll, consists of 33 islets that encircle a lagoon. The largest is Fongfale islet which has been selected for investigation. 0m 5000m 10000m S N E W WS 150 120 60 30 330 300 240 210 30º 20º 10º 40º 50º 60º 70º 80º SS EQUINOX -1000m -1000m -1000m -1000m -500m -100m -500m -100m -100m -100m -500m -500m Figure 13: Author map displaying the topography and landmass of the site selected for research IX. Figure 10: Statistics Tuvalu, displaying the architectural distribution and dominance in private dwelling and urban living. Figure 10: Statistics Tuvalu, displaying the architectural distribution and dominance in private dwelling and urban living. IX. “This case suggests that accelerated and heightened human insecurity, generated from biophysical climate changes, could have significant and lasting consequences for the global community. For example, given the number of communities that are extremely vulnerable to shifting environmen- tal conditions, growing local human insecurity from increasing vulnerability and declining adaptive capacity due to climate change may represent a significant challenge to all levels of governance and must become a prominent consideration for future global climate policy” ( Fisher, pg.295). ( Fisher, pg.295). The thin slither of land that is Funafuti Island, Tuvalu’s capital. As displayed the vulnerability of land is evi- dent with no more than several hundred meters separating the internal lagoon from a vast ocean. 10. 11. INTRODUCTION Tuvalu is a western pacific nation, formally known as the Ellice Islands. It is Located north of Fiji ap- proximately halfway between Australia and Ha- waii. At a bearing of 8.5000° S and 179.1833° E. Funafuti one of the six atoll islands, out of the nine located in the region of Tuvalu has been selected for research. Funafuti with only 2.4 square kilome- ters of land accounts for less than one percent of the total atoll area (Fig. 13). It is extremely vulner- able to tidal surge with an average elevation just under 2-3 meters and is common to poor soil con- ditions. It is of concern within the International Panel on Climate Change (IPCC). The sovereignty of the island is challenged through a global battle with sea level rise that has scarred the landscape. 10000m Figure 13: Author map displaying the topography and landmass of the site selected for research Figure 13: 12. 13. DEFINING SITE ATOLL PROCESS 15 FRINGING BARRIER REEF ATOLL VERTICAL GROWTH OF CORAL VERTICAL GROWTH OF CORAL SUBSIDENCE SUBSIDENCE SUBSIDENCE LAGOON SUBSIDENCE ATOLL FORMATION Figure 14: Author diagram displaying subsidence process, formation of a coral Atoll. SUBSIDENCE ATOLL FORMATION FRINGING BARRIER REEF ATOLL VERTICAL GROWTH OF CORAL VERTICAL GROWTH OF CORAL SUBSIDENCE SUBSIDENCE SUBSIDENCE LAGOON SUBSIDENCE ATOLL FORMATION Figure 14: Author diagram displaying subsidence process, formation of a coral Atoll. SETTLEMENT TIME LINE 1568 1781 15th of Januray 1568, Alvaro de Mendana first white visitor to the waters, hovering just of an atoll to which he produced the name ‘Isla de Jesus: it’s position donated to that of Nukufetau. (Pg. 12, Koch). The area was again visited by european explorers in 1781 Fran- cisco Maurelle 1819 Arent Schuyler de Peyster’ captin of the brittish brigantine ‘Rebecca’ arrived in 1819. It was Pesyster who gave the archipel- ego from which he called funafuti atoll “ellice group” after patron Edward Ellice ( Pg. 12, Koch). 1850 Blackbirders appeeared between 1850 - 1870, raiding the accessi- ble atoll islands oif Funaftti and Nukufetau. 1865 Samoan missionaries began a systematic Christiani9sation of the islands’. Pg. 12, Koch. This led to the introduction of a puritanical way of life and which mixed values from Old time reglion and orders in society familiar to that of the tuvaluan people. This broguht cultural changes in p[-articular regarding to clothing and ornaments. 1915 Tuvalu became a crown collony. 1896 In 1896 the archipelego became a brittish protectorate . ME LINE 15th of Januray 1568, Alvaro de Mendana first white visitor to the waters, hovering just of an atoll to which he produced the name ‘Isla de Jesus: it’s position donated to that of Nukufetau. (Pg. 12, Koch). Figure 17: Pre-contact woman on Funafuti island Tuva- lu 1781 Figure 16: Missionary settlement on Funafuti tucked in a clearing The area was again visited by european explorers in 1781 Fran- cisco Maurelle Arent Schuyler de Peyster’ captin of the brittish brigantine ‘Rebecca’ arrived in 1819. It was Pesyster who gave the archipel- ego from which he called funafuti atoll “ellice group” after patron Edward Ellice ( Pg. 12, Koch). Figure 16: Missionary settlement on Funafuti tucked in a clearing Figure 17: Pre-contact woman on Funafuti island Tuva- lu lu 1865 Samoan missionaries began a systematic Christiani9sation of the islands’. Pg. 12, Koch. This led to the introduction of a puritanical way of life and which mixed values from Old time reglion and orders in society familiar to that of the tuvaluan people. This broguht cultural changes in p[-articular regarding to clothing and ornaments. Samoan missionaries began a systematic Christiani9sation of the islands’. Pg. 12, Koch. Understanding the atoll environment FRINGING BARRIER REEF ATOLL VERTICAL GROWTH OF CORAL VERTICAL GROWTH OF CORAL SUBSIDENCE SUBSIDENCE SUBSIDENCE LAGOON A coral atoll is a ring shaped reef that has a coral rim that fragilely encircles a lagoon in part or completely. Many atolls have coral islets or cays, which are also found at the rim edges. The coral substrata sit a top the rim of an extinct volcano that has subsided and eroded over time to partially submerge beneath the water (see fig. 15). A lagoon generally forms over a volcanic crater, whilst other parts of the rim are raised above water or at shallow depths forming reefs and permitting coral to develop and grow. For an atoll to remain above sea level, subsidence or erosion must be occurring at a rate slower than the growth of the coral. A strata projects upwards and outwards in order to replace the lost height due to sub- sidence (Atoll Formation). This delicate ecological process is now exacerbated due to rapidly changing global climatic condi- tions. Note: ‘Many communities and regions that are vulnerable to climate change are also under pressure from forces such as popula- tion growth, resource depletion, and pover- ty. (McCarthy, pg.8) ATOLL Figure 14: Author diagram displaying subsidence process, formation of a coral Atoll. Figure 14: 14. 15. DEFINING SITE SETTLEMENT TIME LINE SETTLEMENT TIME LINE SETTLEMENT TIME LINE This led to the introduction of a puritanical way of life and which mixed values from Old time reglion and orders in society familiar to that of the tuvaluan people. This broguht cultural changes in p[-articular regarding to clothing and ornaments. 16. 17. 1942 1967 United States landed on Funafuti on the 2nd of October 1942. Naval Construction Battalion (Seabees) built Funafuti Airfield. A Constitution was introduced, which created a House of Repre- sentatives for the Gilbert and Ellice Islands Colony that comprised 7 appointed officials and 23 members elected by the islanders. 1972 21 & 22 October 1972 Funafuti was severely damaged by Cyclone Bebe. 1976 1 January 1976 The Gilbert and Ellice Islands Colony ceased to exist and the separate British colonies of Kiribati and Tuvalu came into exist- ence. 1978 1 October 1978, Tuvalu became fully independent within the Commonwealth. 2000 17 September 2000 Tuvalu became the 189th member of the United Nations. 1999 The Funafuti Conservation Area was created for the conservation of the marine and land based biodiversity (plants, animals and ecosystems) within the protected area. 2013 5 September 2013, Tuvalu signed the Majuro Declaration which is intended to spark a “new wave of climate leadership” and high- light the impact of climate change in the Pacific Ocean. Pre- contact washing hole Funafuti., Locals bathing (Andrew Thomas 1886). 1942 United States landed on Funafuti on the 2nd of October 1942. Naval Construction Battalion (Seabees) built Funafuti Airfield. A Constitution was introduced, which created a House of Repre- sentatives for the Gilbert and Ellice Islands Colony that comprised 7 appointed officials and 23 members elected by the islanders. 21 & 22 October 1972 Funafuti was severely damaged by Cyclone Bebe. Figure 18: Aerial view of Fongafale Islet, showing the seaplane ramp and gun bunkers built by the Americans. Figure 19: Aerial view of Funafuti’s Fongafale Islet, with the newly constructed airstrip clearly visible. 1976 1 January 1976 The Gilbert and Ellice Islands Colony ceased to exist and the separate British colonies of Kiribati and Tuvalu came into exist- ence. Figure 19: Aerial view of Funafuti’s Fongafale Islet, with the newly constructed airstrip clearly visible. Figure 18: Aerial view of Fongafale Islet, showing the seaplane ramp and gun bunkers built by the Americans. Figure 18: Figure 18: seaplan The Funafuti Conservation Area was created for the conservation of the marine and land based biodiversity (plants, animals and ecosystems) within the protected area. The Funafuti Conservation Area was created for the conservation of the marine and land based biodiversity (plants, animals and ecosystems) within the protected area. 17 September 2000 5 September 2013, Tuvalu signed the Majuro Declaration which is intended to spark a “new wave of climate leadership” and high- light the impact of climate change in the Pacific Ocean. 19. 18. DEFINING SITE Figure 20: Burrow pits Due to Tuvalu’s geographical location and Spartan like existence ‘colonial rule’ touched Tuvalu, light- ly in comparison with many other countries, it did, nevertheless have a significant impact’ (Koch, pg.134). Colonial rule was a mixture of negligence and petty acts of interference. Past human activities on Tuvalu’s landscape have also had adverse impacts. The construction of Fu- nafuti airstrip in WWII, reduced fertile crops and produced burrow pits that are now toxic. A large proportion of the land formerly used for growing Pulaka and Taro was in filled by the American construction battalion the ‘Seabees’. The airfield was finished before the end of 1942 for missions against the Japanese. ‘From Kiribati the Japanese intended moving south to Tuvalu but their losses at the battle of midway delayed them. This enabled the Americans to get to Tuvalu first on 2 October 1942’(Koch, pg.140). Tuvalu’s separation from Kiribati in 1976 focused on higher education. ‘A year after separation, the new government, having taken full responsibili- ty for the training of its own people, sent the first group of prospective teachers and nurses to study in Fiji’ (Faaniu pg.150). As with education, health was another major focus of the government. Since the war modern medicine and equipment have been supplied to the outer islands along with trained staff. In 1978 New Zealand aid helped to bring up to date hospital facilities to the islanders. Princess Margaret hospital was completed but was not the only notable development. ‘On Funafuti nearly every household has come to possess a mo- torcycle, a bicycle and an outboard motor’(Faaniu, pg.152). ‘The airfield took up one-sixth of the land area, to make it the Americans destroyed nearly half the coconut trees, 22,000 out of 54,000. Moreover ef- forts to replant that land have not been very suc- cessful. The coral is packed too hard for the trees to grow properly’(Koch, pg.143). In these dugouts poverty finds a home. Although not admitted by locals, current living conditions are below acceptable world standards. A priority will be to rehouse the inhabitants most at risk of poverty (see fig. 22). Prior to the war inhabitants used to walk varied distances carrying loads of coconuts or firewood. A regular air service now arrives from Suva for both passengers and freight, while an overseas ship calls at the capital at least every three months to deliver cargo and collect Copra. Isolation Due to Tuvalu’s geographical location it can be a strategic advantage or disadvantage to the occu- pants of the island. Their sea of islands render the Tuvaluan people with a unique lifestyle, culture, language and sense of identity. In early settlement communication between islands was restricted to access via vessel. The traditions are predominantly culturally derivative of Samoa however it is known that inhabitants did arrive on voyages from the Tokelau Islands and Central Polynesia, possibly unintentionally driven by the easterly winds and the westerly ocean current (Koch, pg.11). The Tuvaluan’s landed over two thousand years ago, and in small groups. Each clan lived by it- self on its own land with one or more communal dwelling houses, a kitchen hut, a storehouse, and canoe house ( Koch, pg.141). Settlements of ver- nacular were to be found clustered along coastal shoreline and sustained by the fertile ocean. This connected individual settlements to a central spine and between each other by informal path- ways. Houses were also found on islets of Nu- kufetau atoll and in the interior of Naitao, Nanu- maga and on Fale. During pre European times there was frequent canoe voyaging between the nearer islands. The Tuvaluan islanders are very much at one with the sea and obtain a major part of their sub- sistence from it, canoe building is particularly important for their everyday existence (Koch, pg.143). Their communal life is simple and less formal than other island cultures . They settled and worked in family groups under the leader- ship of the oldest people in the clan, the Chiefs (Aliki). The Chiefs ruled the islands, with the as- sistance of the Council of Elders. Their culture is very simple and in an environment where there is no solid rock they live a rather Spartan existence. They produce fewer articles then they might, to make their life more comfortable (Koch, pg.11). Figure 20: 20. 21. e 2 DEFINING SITE 0m -20m > WATER BODIES & BURROW PIT LOCATIONS (SCARRED LANDSCAPE) >LAKE (TARASAL) >ROAD NETWORK >BUILDING FOOTPRINTS -30m -10m S N E W WS 150 120 60 30 330 300 240 210 30º 20º 10º 40º 50º 60º 70º 80º SS EQUINOX Figure 22: Author map showing location island infrastructure, burrow pits and water body locations. Burrow pits A deep-water wharf that was constructed in 1981 enables ship- ping links with the greater Pacific. The develop- ment of shipping between the islands has become more frequent and of great importance to the local economy. Figure 22: Author map showing location island infrastructure, burrow pits and water body locations. Author map showing location island infrastructure, burrow pits and water body locations. Figure 22: 22. 23. 23. DEFINING SITE S N E W WINTER SOULTICE EQUINOX SUMMER SOULTICE 150 120 40 30 330 300 240 210 30º 20º 10º 40º 50º 60º 70º 80º N AVERAGE WIND SPEED 13km/h AVERAGE SUN HOURES DOMINANT WIND DIRECTION (YEAR%) SUN PATH DIAGRAM 20 15 10 5 E W S SSE SE ESE ENE NE NNE NNW NW WNW WSW SW SSW 23% Author diagram displaying the local conditions, sun path, angle and Average wind speed a S N E W WINTER SOULTICE EQUINOX SUMMER SOULTICE 150 120 40 30 330 300 240 210 30º 20º 10º 40º 50º 60º 70º 80º AVERAGE SUN HOURES SUN PATH DIAGRAM CLIMATIC CONSIDERATIONS SUN PATH DIAGRAM SUN PATH DIAGRAM N This section assesses the climatic conditions pres- ent across Funafuti Island, the site for selected re- search. This will allow informed design decisions that promote local technology and design respons- es to climate Tuvalu’s Climate On Funafuti, the capital of Tuvalu there is little variation in temperature throughout the year. The maximum temperature is between 31–32°C and the minimum temperature between 25–26°C all year round. Air temperatures are responsive to the ocean temperatures surrounding the islands and atolls of the country. 210 150 AVERAGE SUN HOURES The country has two significant seasons, A wet season from November to April A dry season from May to October 200 mm of Rainfall averages each month of the year in Funafuti alone (Figure 24). This is due to the location of Tuvalu Islands near the West Pa- cific Warm Pool. The wet season is affected by the movement and strength of the South Pacific Con- vergence Zone. DOMINANT WIND DIRECTION (YEAR%) NNW NNE This band of heavy rainfall is caused by air rising over warm water where winds converge, result- ing in thunderstorm activity. It extends across the South Pacific Ocean and is most intense during Tuvalu’s wet season. The West Pacific Monsoon can also bring heavy rainfall to Tuvalu during the wet season. The Monsoon is driven by large dif- ferences in temperature between the land and the ocean, and its arrival usually brings a switch from very dry to very wet conditions (Ralston, pg.3). WNW AVERAGE WIND SPEED 13km/h Author diagram displaying the local conditions, sun path, angle and Average wind speed and direction Figure 23: Author diagram displaying the local conditions, sun path, angle and Average wind speed and direction 24. 25. DEFINING SITE Figure 25: Low-lying stretches of atoll islands such as Funafuti, Tuvalu’s most populated island, are over washed by king tides the highest of spring tides, which occur a few times a year. AV MAX MIN 31-32º C 200MM AV 25-26º C AV 26º C - Monthly average Funafuti - Per year (Average sea level rise) - Average temperature range - Sea level rise (High emissions senario IPCC) LEGEND: 2100 0.19-0.58M 5MM 5MM Figure 24: Author Infograhpics displaying climate conditions AV MAX M 31-32º C AV 25-26º C Figure 25: Figure 25: Figure 25: 26. 27. 0m 5000m 10000m S N E W WS 150 120 60 30 330 300 240 210 30º 20º 10º 40º 50º 60º 70º 80º SS EQUINOX Figure 27: Author diagram displaying dominant winter wind direction and percent 0m 5000m 10000m S N E W WS 150 120 60 30 330 300 240 210 30º 20º 10º 40º 50º 60º 70º 80º SS EQUINOX Figure 28: Author diagram displaying summer dominant wind direction and percent Figure 27: Author diagram displaying dominant winter wind direction and percent Author diagram displaying summer dominant wind direction and percent Figure 28: 28. 29. (IPCC, pg. 847 and 855, UNFCCC, pg. 21) (IPCC, pg. 847 and 855, UNFCCC, pg. 21) Figure 33: Author diagram displaying emission scenarios for future sea level rise predictions. For the Purpose of this investigation the ‘High emissions scenario’ will be used. AV MAX MIN 31-32º C 200MM AV 25-26º C AV 26º C - Monthly average Funafuti - Per year (Average sea level rise) - Average temperature range - Sea level rise (High emissions senario IPCC) LEGEND: 2100 0.19-0.58M 5MM 5MM Figure 24: Author Infograhpics displaying climate conditions 0m 5000m 10000m S N E W WS 150 120 60 30 330 300 240 210 30º 20º 10º 40º 50º 60º 70º 80º SS EQUINOX 60º NOTE: In summer the dominant wave condition (occuring often) is slight, the waves are almost never calm and almost never rough and the principal wave direction is from the Northeast ( 60º) (SPC, pg.5) Figure 29: Author diagram displaying dominant Swell direction summer 0m 5000m 10000m S N E W WS 150 120 60 30 330 300 240 210 30º 20º 10º 40º 50º 60º 70º 80º SS EQUINOX 120º NOTE: In winter the dominant wave condition (occuring often) is moderate, the waves are almost never calm and almost never rough and the principal wave direction is from the South- east (120º) (SPC, pg.5) Figure 30: Author diagram displaying dominant Swell direction winter NOTE: In summer the dominant wave condition (occuring often) is slight, the waves are almost never calm and almost never rough and the principal wave direction is from the Northeast ( 60º) (SPC, pg.5) NOTE: In summer the dominant wave condition (occuring often) is slight, the waves are almost never calm and almost never rough and the principal wave direction is from the Northeast ( 60º) (SPC, pg.5) NOTE: In winter the dominant wave condition (occuring often) is moderate, the waves are almost never calm and almost never rough and the principal wave direction is from the South- east (120º) (SPC, pg.5) NOTE: In winter the dominant wave condition (occuring often) is moderate, the waves are almost never calm and almost never rough and the principal wave direction is from the South- east (120º) (SPC, pg.5) Author diagram displaying dominant Swell direction winter Author diagram displaying dominant Swell direction summer Figure 29: Author diagram displaying dominant Swell direction summer Figure 30: Author diagram displaying dominant Swell direction winter 31. 30. CLIMATE CHANGE: RISING TIDES SEA-LEVEL PREDICTIONS Figure 33: Author diagram displaying emission scenarios for future sea level rise predictions. For the Purpose of this investigation the ‘High emissions scenario’ will be used. 2030 4-14cm 9-28cm 19-58cm 5-14cm 10-29cm 19-56cm 2055 2090 2030 2055 HIGH EMISSIONS SCENARIO MEDIUM EMISSIONS SCENARIO 2090 4-14cm 9-25cm 16-45cm 2030 2055 LOW EMISSIONS SCENARIO 2090 Sea-level rise process 2030 4-14cm 9-28cm 19-58cm 2055 2090 HIGH EMISSIONS SCENARIO than that of the past 100 years (IPCC, pg. 847). than that of the past 100 years (IPCC, pg. 847). than that of the past 100 years (IPCC, pg. 847). Sea level rise is caused by thermal expansion of the ocean and degradation of the polar ice sheets due to global warming. Small low-lying atoll na- tions such as Tuvalu, Kiribati and the Maldives are just a few at risk of their populace becoming ‘cli- mate refugees’, forced to migrate within the next 50 - 100 years. The inundation experienced and predicted due to rising sea levels will cripple the local subsistent economy and force a population of 6,000 alone on the capital to become ‘climate refugees’. The sea is inextricably linked to Tuvalu’s environ- ment, culture and social system sustaining life on the islands. A change in sea level is therefore not an abstract risk but a challenge to the every day life of Tuvaluan’s. ‘As most island homes, infrastructure, and commercial activities are along the coasts...sea level rise is a high risk to the life and health of the inhabitants ‘(IPCC, pg. 847). 5-14cm 10-29cm 19-56cm 2030 2055 MEDIUM EMISSIONS SCENARIO 2090 4-14cm 9-25cm 16-45cm 2030 2055 LOW EMISSIONS SCENARIO 2090 The principal impacts anticipated fall into three main categories: Global warming is no longer a myth for small atoll nations and a common reality has struck. Our planet is getting hotter with the help of the earths natural periodic cycle and human driv- en emissions & wastes. The International Panel on Climate Change (IPCC) states that out of all concerns challenging island nations from the discourse of climate change, Sea level rises is ‘by far the greatest,’ both economically and socially (IPCC pg.855). a) Loss of coastal lands b) Flooding and soil salinisation in addition to harm to crops c) Ground water sources, land and marine biodi- versity c) Ground water sources, land and marine biodi- versity Figure 31: IPCC graph displaying observed and projected relative sea level rise near Tuvalu. Shaded in green are the projected ranges for a medium emission scenario High tide and King tide flooding in the borrow pits of Funafuti. A runway was constructed on the main islet during WWII borrowing coral and sand from the unpopulated areas of the island. Figure 36: Flooding during king tides is a problem and not uncommon with extreme weather events only predicted to Figure 37: Erosion eats away at the periphery of the coral atolls. Old tin drums are filled with concrete, which is set and backfilled with compressed rubbish on the inside of the seawall. increase. Water percolates up through the coral matrix and laps eat the door of the meteorological office of Tuvalu. Figure 35: Figure 36: What the future holds The island environments are considered to have a Spartan like existence at present but also it is likely to become more inhospitable overtime. If sea lev- els continue to rise without remediation or rede- velopment of the Architectural condition at hand Tuvalu could be lost. It is possible to design for the worst case scenario of high global emissions with a large range of projected rise, which is appropriate for the design discussion within this thesis. The future of Tuvalu will be determined by the ca- pacity of local inhabitants to react to natural and human factors. Consideration into a range of pos- sible future conditions with outcomes are predict- ed by IPCC climate models. They produce a se- ries of scenarios based on a set of assumptions as listed. 1) Future population change. 2) Economic development and technological advances. The climate projections for Tuvalu are based on three IPCC emissions scenarios: low, medium and high, for time periods around 2030, 2055 and 2090 (see Fig. 33). Individual models give different re- sults; the projections are presented as a range of values. The IPCC states that sea level rise by the end of this century will be at the rate of 5 mm an average per year, which is two to four times higher Figure 33: Author diagram displaying emission scenarios for future sea level rise predictions. For the Purpose of this investigation the ‘High emissions scenario’ will be used. 32. 33. 33. DEFINING SITE Figure 36: Flooding during king tides is a problem and not uncommon with extreme weather events only predicted to increase. Water percolates up through the coral matrix and laps eat the door of the meteorological office of Tuvalu. Figure 37: Erosion eats away at the periphery of the coral atolls. Old tin drums are filled with concrete, which is set and backfilled with compressed rubbish on the inside of the seawall. Figure 35: 34. 35. Figure 39: Water percolates up through the coral matrix. Niu Loane lost his main food source and income when a king tide destroyed his Pulaka plantation in Funafuti, Tuvalu. What the future holds 0m -20m -30m -10m S N E W WS 150 120 60 30 330 300 240 210 30º 20º 10º 40º 50º 60º 70º 80º SS EQUINOX A B Figure 42: Author map showing location of sections and urban layout of the island S N WS 150 120 60 30 330 300 240 210 30º 20º 10º 40º 50º 60º 70º 80º SS EQUINOX Figure 41: Burrow pit on Funafuti islet showing informal dwellings and proximity to flood waters Figure 41: Figure 41: Burrow pit on Funafuti islet showing informal dwellings and proximity to flood waters A Figure 39: Water percolates up through the coral matrix. Niu Loane lost his main food source and income when a king tide destroyed his Pulaka plantation in Funafuti, Tuvalu. Figure 42: Author map showing location of sections and urban layout of the island 36. 37. What the future holds LEGEND VL – VEGETATION LINE BF – BEACH FACE SD – SAND CR – CORAL RUBBLE BR – BEACH ROCK CP – CORAL PATCHES RF – REEF FLAT BRB – BEACH ROCK BOULDERS BRP – BEACH ROCK PEBBLES BRG – BEACH ROCK GRAVEL RB – RUBBLES L – LAND SP – SAND PIT BP – BORROW PIT SD RB L TARO PATCH L RUNWAY L MANGROVES SWAMP L RB BR RF SECTION F Average King Tide + Sea Level Rise (2090, 3.76m) Average King Tide before sea level rise 3.2m Average Spring Tide 2.7m Average Sea Level 2.0m Average Sea Level 2.0m Average Spring Tide 2.7m Average Low Water Level 1.37m 1 2 3 4 5 6 1 2 3 4 5 6 SEA LEVEL RISE SECTION SHOWING VARRYING LEVELS OF HIGH WATER LEGEND VL – VEGETATION LINE BF – BEACH FACE SD – SAND CR – CORAL RUBBLE BR – BEACH ROCK CP – CORAL PATCHES RF – REEF FLAT BRB – BEACH ROCK BOULDERS BRP – BEACH ROCK PEBBLES BRG – BEACH ROCK GRAVEL RB – RUBBLES L – LAND SP – SAND PIT BP – BORROW PIT L TARO PATCH L RUNWAY L MANGROVES SWAMP L RB BR RF SECTION F Sea Level Rise (2090, 3.76m) ore sea level rise 3.2m .7m 0m el 2.0m Tide 2.7m evel 1.37m 1 2 3 4 5 6 TION SHOWING F HIGH WATER Average King Tide + Sea Level Rise (2090, 3.76m) Average King Tide before sea level rise 3.2m Average Spring Tide 2.7m Average Sea Level 2.0m Average Low Water Level 1.37m 6 SEA LEVEL RISE SECTION SHOWING VARRYING LEVELS OF HIGH WATER SECTION F SECTION F Author sections of Funafuti modeled from SOPAC site survey report showing projected sea level rise in relation to the Atoll cross section Figure 45: 39. 39. 38. CONTESTED TERRITORY Figure 47: Many in this strongly Christian country hold to the scriptural promise of God to Noah, that floods would never again destroy the Earth. Summary By effectively noting the climatic and Atoll condi- tions it is easy to see how applicable, maintenance of a Tuvaluan way of life is paramount. If architec- tural design experimentation can provide personal ownership and subsistence, then this will improve the informal dwellings of Funafuti to serve as sov- ereign possessions reflective of the occupant. The principal impacts anticipated fall into three main categories: a) Loss of coastal lands b) Flooding and soil salinisation c) Harm to ground water sources, land and marine biodiversity. These impacts along with the thesis aims will allow a reflective report to the indigenous landscape 2030 - 4 - 14 cm max projected rise 2055 - 10-29 cm max projected rise 2090 - 19-58 cm max projected rise To account for climate change impacts; 1. Sea Level rise rate by IPCC - Proposed a High emission scenario, which has been selected for the purpose of investigation. 2030 - 4 - 14 cm max projected rise 2055 - 10-29 cm max projected rise 2090 - 19-58 cm max projected rise 40. 41. CONTESTED TERRITORY INTRODUCTION This chapter explores the applications of contem- porary theorists that provide influence in address- ing the Architectural fabric of Funafuti Island. Suggesting how to materialise cultural and social values of the Tuvaluan people. THEORETICAL FOUNDATION 2.0 This will allow the agency of design to develop a theoretical basis teasing successful techniques from each exemplar to inform a new understand- ing that can be reflected through design progres- sions later in the document. The aim is to obtain new thought in design led methods and principles to sculpt objectives in response to the thesis ques- tion. Theories reviewed have been discussed to supple- ment one another to create a body of research that will provide extended knowledge in further itera- tions and propositions. 43. FOUNDATION RADICAL RECONSTRUCTION LEBBEUS WOODS cipatory design beyond bounds of the catastrophe for growth and healing. Lebbeus Woods was one of the most prominent and critiqued architects of this century, using ar- chitecture to explore considerations into social and political implications in the face of adversity. His work entailed how architecture could react to the individual and collective whilst reflecting so- cial and ideological conditions. The modes of ar- chitectural experimentation displayed by Woods explore architectonic process. This thesis therefore seeks to pull individual tectonic responses from the context of the site detailed by cultural, social and political ties of Tuvaluan life. In Tuvalu’s case Woods approach serves to suggest ways in which the complexity of sovereignty can be constructed with shifts from nostalgic emotions toward new prototypes of renewal and dwelling. Imposed conditions can form positive reflec- tions, which commonly may be embedded with emotion. A switch in approaches due to necessity changes the manner by which they make advanc- es in knowledge and survival. The intention is to memorialise the ‘event’ through the integration of form and structure within the degraded condition (see fig. 53 & 54). Woods thought of architecture as the ability to en- gage and learn from catastrophe ‘By confronting the extreme conditions brought about by willful destruction, particularly as it affects urban life and its structures, architects will learn much about the practice of architecture within stable conditions, which they will never learn by unquestionably ac- cepting the often illusionary appearances and as- sumptions of stability. ‘ (Woods, pg.22) By doing so architecture can become more flexible and respon- sive for the inhabitants. Not only to generate new conditions but to sustain spiritual and ruminative connections to moments of memorialisation from hardship. ‘Architecture has always been concerned with imbuing the ordinary with something of the extraordinary. It is concerned, as a practice, with enhancing the normative, with giving routine be- havior and cultural conventions aesthetic qualities that elevate them beyond the power of their own monotony’ (Woods, pg.23) A Exhibition for Lebbues woods san Francisco bay project Woods sketch concepts for the planning of drawings shown above and below Figure 51: San Francisco bay project lebbeus woods, Two towers Figure 52: Lebbeus wood sketch concepts for the Francisco bay project after earthquake damage Summary Woods explains his ideologies through metaphors as vehicles of design, which are applicable to the preservation and healing of cultural and social identity that this thesis investigates. His uncon- ventional tectonic theory presents the possible, implications of evolving architecture to memori- alise and integrate into a degraded condition or system. Tuvalu’s remediation to sea level rise im- pacts begins with the role of the architect to see past the degraded condition, improve and evolve the architectural state presented. Suggesting that Architecture offers possibility of reintegrating functions to a system that has lost equilibrium, but only by analysing the complexi- ties of that system are we able to achieve the edifice. His work doesn’t provoke to erase the catastrophe in which they intervene, but to suggest an eman- 44. 45. FOUNDATION DESIGN CATASTROPHE INFORMS Design Principle extracted Figure 53: Simpson-Lee house House showing pitched high ceiling allowing environmental drivers to add large amount of natural light filtering into the interior. The building also has a tranquil walkway to enter next to a body off water, radiant cooling for the end of the building. Figure 54: Marika-Alderton House ventilation openings can be seen along the side of the building, raised off the ground to reduce moister and increase cooling efficiency. 1. Radical reconstruction > Radical reconstruction of the normative, gives routine behavior and cultural conventions aesthet- ic qualities that can elevate them beyond the power of their own monotony. Tuvalu’s burrow pits are a system that has lost equilibrium due to inundated conditions. Only by analysing the complexities of that system is it possible to reconstruct new condi- tions to sustain spiritual or ruminative connection to moments of hardship (slums), sovereignty and site. Figure 51: Figure 52: 46. FOUNDATION 47. ACTIVE ENVIRONMENT GLEN MURCUTT ENVIRONMENT INFORMS INFORMS CULTURE principles of integration within the landscape. To respond to Tuvalu’s warmer climate, engaging the user with simple adjustments of airflow and shad- ing screens to control internal comfort (see Fig. 84). Moreover to discover a design that enhances sus- tainability, way of life, serenity and mitigates mon- umentality improving an occupants sense of place and well being. I suggest that this will produce a connection to the island of Funafuti through cli- matic consideration of the indigenous vernacular techniques. Enabling the discovery of design char- acteristics that produce a sense of calm, so inhab- itants feel a certain level of comfort, participation and human scale within the intervention. Glenn Murcutt’s principles of integration with- in the landscape respond to contexts, which are in need of a place to call ‘home’ less iconic, and more a place of function, belonging and well be- ing. Suggesting that cultures brought together may respond to a new place as ‘home’ if the architecture is considerate of its surroundings, integrated with landscape and not just of iconic status. Discussion will revolve around the work of Glenn Murcutt and the comprehensive level at which his archi- tecture adapts systems of nature to merge with the landscape and respond readily to climatic condi- tions (see fig. 55 & 56). ENVIRONMENT INFORMS INFORMS CULTURE ENVIRONMENT INFORMS INFORMS CULTURE INFORMS What sets Murcutt apart from his international peers is an architectural intelligence that accepts both autonomy of a building and the autonomy of the place where it sits. Paradoxically, he makes human habitats appear as part of their settings but distinct a quality of poise that is at once grounded in and elevated from the natural world’ (Pallas- ma). It is somewhat rusticated and sophisticated adaptions of local conditions are almost primal in sense. By achieving this connection to place and so forth his building becomes a part of the land, re- sponsive to the climate, prevailing winds, sunlight angles for winter and summer, views with con- nection to native plants and natural ventilation. The house for painter Marika Alderton, where the hinged facade of sliding panels function both as ‘equipment’ and ‘background’ (Lynch, pg.2). Design research experiments throughout this thesis will look to engage and replicate Murcutt’s 48. FOUNDATION 49. 2. Active participant >Allowing the building to perform under tropical conditions by involving natural and low mainte- nance use of climatic design principles and en- gagement of the occupant. 51. 50. 51. SPATIAL ACTIVATION KARIN JASCHKE Karin Jaschke is a distinguished lecturer at the University of Brighton, who explores relationships between the architect and user. Jaschke propos- es that spatial configuration constitutes a social and embodied activity that does not take place in space but in itself defines it (Jaschke, pg. 135). In the case of Tuvalu daily rituals and tasks constitute the space in which these activities such as fishing, weaving and water collection are performed. SPACE SOCIAL ACTIVITY INFORMS Indigenous cultural parameters can be integrat- ed into the narrative form and be reflective of the anthropology to organise subsequent spatial con- figuration. Jaschke explores that to produce spa- tial activation a configurative approach is required ‘Configurative design proposes that the user take possession of the built structure. It thus intro- duced a sort of alternative concept of ownership, a physiological ownership, which resonated with anthropological notions of property and phenom- enological ideas on inhabitation’ (Jaschke, pg.137). This approach encourages the user to explore a configurative setting, discover and redefining the space as their own. This offers a sense of personal identity by presenting architecture that is devoid of an atypical hierarchical architecture. Design Principles extracted 4. Spatial activation > Social activities preformed by various cultures can have an effect on the activation of internal space. Through cognitive design it is possible to involve social participants. These notions are used to supplement prior the- oretical implications brought to this discussion through Ganoe position who suggests that design through narrative can influence the inhabitants re- sponse to space as well. Woods sees architecture as an act of healing within contexts of catastrophe and Murcutt practices autonomy not only of a building but the autonomy of the place. 53. 52. 53. FOUNDATION NARRATIVE ENVIRONMENT CATHY J.GANOE based on the consciousness of the majority, rath- er than a series of personal mini narratives, cre- ate meaning through expression (Ganoe pg.10) Applying these narratives and formal gestures to interconnect inhabitants brings into question whether architecture can simultaneously reflect social and cultural identity through multiple nar- ratives within a formal concept. Cathy Ganoe Michigan University professor pres- ents key design principles that allow narrative space to concern itself with inhabitants, using space as a construct for the perception of the nar- rative environment. Although an interior focus on space is discussed a coherent analysis and ratio- nale allow interconnections between humans and space. Appropriate for design consideration when subjective narratives of the occupants are applied in architectural intervention. DESIGN NARRATIVE INFORMS INFORMS Design Principle extracted 4. Narrative environment > Connection to cultural dialogue through the narrative presence to analyse and connect with complex phenomenological conditions of social and cultural identity. Therefore connecting and creating a respectful dialogue between the human inhabitants and architecture. DESIGN NARRATIVE INFORMS INFORMS This thesis provokes an underlying narrative to express values attached to site, local community and national history of Tuvalu. Ganoe argues that design through narrative can influence the inhab- itant’s response to space. ‘Each criterion of design narrative theory defines an important aspect of design meaning and purpose, addresses silent de- sign issues and has various possible applications’ (Ganoe, p.g13). Design Principle extracted Design Principle extracted The criterion mechanisms are as follows: f The criterion mechanisms are as follows: a) Order b) Inclusion of differing psychological states of mind for occupants c) Symbolic meaning based on individual history d) Transformation of space to serve cultural and personal goals whilst accounting for subjectivity of experience. This will yield a cultural narrative that is responsive to the local inhabitants on site thus producing metaphors that enrich architecture by inviting exploration of narrative experiments in the proposed solutions. > Connection to cultural dialogue through the narrative presence to analyse and connect with complex phenomenological conditions of social and cultural identity. Therefore connecting and creating a respectful dialogue between the human inhabitants and architecture. The framework suggests that a design can go be- yond a single grand narrative, seeking to increase the degree to which human imagination is chal- lenged, resulting in more depth of contemplation and interpretation by the participant. A design 55. 54. 55. FOUNDATION Figure 56: Internal view of formalised housing project in the Santa Marta Fevelas Figure 55: Santa Marta Favela, Rio de Janeiro, Brazil. The informal urban vernacular is supplemented here with the recent addition of a funicular and formalised housing to the right; note the gentrification spreading upwards and banners showing resistance to eviction at the top. INCREMENTALISM & THE INFORMAL KIM DOVEY junction with dwelling typologies to form interre- lationships between inhabitants and elements of the landscape, sustaining connection and durabil- ity overtime. junction with dwelling typologies to form interre- lationships between inhabitants and elements of the landscape, sustaining connection and durabil- ity overtime. Architecture today often is a product of the ‘Mon- ey shot’ where the principal aim is to produce the smilingly realistic photorealism. Often neglect oc- curs when concerned with the elements of time. The once perfect envelopes are now degraded and in a declining state. 57 Figure 57: Author diagram displaying the new temporal phasing process to ignite incrementil- ism and the informal GENERAL APPROACH POTENTIAL INCREMENTALISATION Construction Phase 1 Construction Construction Phase 2 Design Design Built Design Phase 1 Final Built Design Built Design Phase 2 Built Design Phase 3 Construction Phase 3 TEMPORAL GROWTH: PROCESS REPEATS TIME POTENTIAL INCREMENTALISATION GENERAL APPROACH This failure to understand incremental change and existing morphologies has left communities detached from social, public and natural ecol- ogies. Kim Dovey discusses his thoughts on in- crementalism concerning ‘Informal settlements that embody informal practices of sociality and economic production that are not easily retained in a transformation to formal housing’. Switching the process from a final product to an agency for incremental development overtime or in the case of this thesis to maintain settlement and improve hardships faced on low lying atolls. Dovey also suggests ‘There is a need to invent new construction types that incorporate recycled materials, incremental process, adaptability and multi-functionality with greater efficiency, safe- ty and built density’ (Dovey, pg. 88). This allows the landscape and dwellings to become flexible for unforeseeable futures and can be built with a low economic footprint (see fig. 57 & 58). Therefore this approach embraces a level of understanding that is sensitive to informal settlements and eco- logical conditions that over time to produce often unknown complexities and regenerative formal- isation. Ultimately coinciding with the view of landscape as a living ecology to be used in con- TEMPORAL GROWTH: PROCESS REPEATS Figure 57: Author diagram displaying the new temporal phasing process to ignite incrementil- ism and the informal 57. FOUNDATION 56. Design Principles extracted Design Principles extracted 2. Active participant REFLECTION Literature is a tool to unlock previous and future design queries offering solutions that provoke insight into how one might resolve the research question. By understanding the narrative and an- thropological environment we can define a sense of place thereby beginning to develop a sense of sovereignty within a disappearing context. 5. Incrementalism (Time & process)i 5. Incrementalism (Time & process)i > Allowing the building to perform under tropical conditions by involving natural and low mainte- nance use of climatic design principles and the en- gagement of the user. ( p ) > Looking past the final product to generate a framework for phasing regenerative growth of the local landscape and architecture to flourish over- time as opposed to degradation. Also acknowledg- ing the position of the informal and how it can be developed over time. ( p ) > Looking past the final product to generate a framework for phasing regenerative growth of the local landscape and architecture to flourish over- time as opposed to degradation. Also acknowledg- ing the position of the informal and how it can be developed over time. 3. Spatial activation p >Social activities preformed by various cultures can have an effect on the activation of internal space. Through cognitive design it is possible to involve social participants . 4. Narrative environment 1. Radical reconstruction > Reintegrating functions of a system that have lost equilibrium. > Reintegrating functions of a system that have lost equilibrium. Design Principles extracted; Design Principles extracted; 1. Radical reconstruction 5. Incrementalism (Time & process) > Looking past the final product to generate a framework for phasing regenerative growth of the local landscape and architecture to flourish over- time as opposed to degradation. Also acknowledg- ing the position of the informal and how it can be developed over time. 4. Narrative environment > Connection to cultural dialogue through the narrative presence. By using narrative to analyse and connect with complex phenomenological conditions of social and cultural identity. 5. Incrementalism (Time & process)i 5. Incrementalism (Time & process)i > Looking past the final product to generate a framework for phasing regenerative growth of the > Looking past the final product to generate a framework for phasing regenerative growth of the 58. 59. INTRODUCTION The following precedents I suggest utilise the pre- vious design principles extracted from the litera- ture reviews. The architectural solution I endeav- our to propose looks beyond replicating previous models. But instead applies a multidisciplinary approach to processes that informs design by dis- covering appropriate responses within the thesis framework. I propose that a pragmatic and exploratory re- sponse to ‘climate’ and anthropology of the site al- lows a theoretical basis to invigorate a new agency in design which addresses the sense of sovereignty within a disappearing context and the implications of habitation, culture and contested territories for the Tuvaluan’s? 61. Figure 58: Top Left: Interior view of finished housing project, wood and concrete interior. Figure 59: Top Right: Exterior view of finished homes showing potential. Figure 60: Bottom Left: External view of the occupants additions to proposed framework Figure 61: Bottom Right: Interior view of lounge/dinning area showing the before and after Quinta Monroy Housing FRAMEWORK DESIGN (small scale) This project was selected because it changed the way in which architects approached social hous- ing. The transformation from social housing into middle class dwellings by way of the occupant. For this to take place people are removed from slums and placed into facilitated living conditions which allow personal temporal construction and addi- tional construction value. The project is a unique simple concrete housing framework for low-income communities. A core house is setup and left for the occupants to estab- lish and construct key areas themselves . It is pro- grammed as a mixed use housing option, as seen in figures 62 & 63. The house provide personal pos- session of the building shell and extended devel- opment adding additional rooms and value to the project. The temporal condition is held in mind when considering, promoting expansion, custom- isation and investment. Thus allowing freedom for inhabitants to attach a sense of custodianship and ownership to the dwelling. This solution for low-income housing is innovative in terms of ownership that is placed with the occu- pant to react. It also addresses a key design direc- tive of this thesis, that of providing a human scale; order and evolution imposed which improves the standard of living. This sense of human scale can be extrapolated into territories of low-lying atoll architecture, appropriate for the low-income com- munity of Tuvalu (‘burrow pits’). Its success is in the sense of community and belonging that add a personal conversation with the development and construction of social housing (see fig. 64). This is applicable to the community of Tuvalu with sup- plementation of local technologies. 62. 63. CASE STUDY > Mixed use space multi level > Framework allows a range of additions to the ‘core’ of house > Can be multiplied to form neighbouring units forming a community > Simple prefabricated concrete framework > Can be multiplied to form neighbouring units forming a community > Can be multiplied to form neighbouring units forming a community > Simple prefabricated concrete framework > Simple prefabricated concrete framework > Simple prefabricated concrete framework > Simple prefabricated concrete framework > Can be multiplied to form neighbouring units forming a community > Framework allows a range of additions to the ‘core’ of house Extrapolated Design Principle 1. Provide the framework for temporal growth, al- lowing the community to take control of the built form, creating a sense of involvement and person- al connection to the architecture itself to maintain sanitary conditions. > Mixed use space multi level > Framework allows a range of additions to the ‘core’ of house > Vertical living is adopted rather than sprawl > Small scale dwellings 10M 6M 10M 6M > Vertical living is adopted rather than sprawl > Vertical living is adopted rather than sprawl > Small scale dwellings Figure 62: Author Architectural characteristics Author Architectural characteristics Figure 62: 65. 65. 64. Figure 63: Makoko Floating school a community project Figure 64: School in context with surrounding slum area of Makoko, Nigeria. Figure 65: Top level interior space showing the view out across makoko. Figure 66: Kids play in the internal class room, material finish from locally sourced products MAKOKO FLOATING SCHOOL KUNLE ADEYEMI The Makoko floating school was designed to cel- ebrate the informal settlement of Makoko, coined as rebel architecture by Kunle Adeyemi. It places an architectonic layering of local resources and in- genuity to the traditional typology of the original settlement, a simplistic revelry with provocative acquisitions and framework for development. Kunle’s design replicates the influence of informal environments and local resources for inhabitants to integrate into adapted technologies from local materials. It is a remedial structure for the current lack of arable land in densified urban areas of Ni- geria (see fig. 66). It was commissioned by the gov- ernment of Nigeria in conjunction with the com- munity to provide school facilities within the slum area of Makoko and within informal coastal settle- ments that are threatened due to rising sea levels. The design presents successful architectural char- acteristics (see fig. 70) that are applicable to my thesis research. Such as similar typologies, local resourcing, adapted local construction character- istics, low cost and maintenance and a back to ba- sics approach to formal design. (Rise, pg.3) 68. CASE STUDY 69. > Can be duplicated as seperate mixed use programs to provide more formal housing settlement on water > Simple materials, easily replacible and flexable interior space to allow multiple solutions > Technology developed from local waste material to provide a floating platform > Simple local frame construction > Technology developed from local waste material to provide a floating platform > Simple local frame construction > Simple local frame construction > Simple local frame construction > Simple materials, easily replacible and flexable interior space to allow multiple solutions duplicated as seperate mixed use programs to ore formal housing settlement on water > Simple materials, easily replacible and flexable interior space to allow multiple solutions gy developed from local waste material to oating platform > Technology developed from local waste material to provide a floating platform > Can be duplicated as seperate mixed use programs to provide more formal housing settlement on water Figure 67: Author diagram showing arhcitectural charateristics Extrapolated Design Principle 3. Provide the framework for a respectful dialogue between the indigenous cultural environments, fa- cilitating future development through local sourc- ing, environmental sensitivity and innovation (see fig. 69). > Can be duplicated as seperate mixed use programs to provide more formal housing settlement on water > Simple materials, easily replacible and flexable interior space to allow multiple solutions $ > Small scale and low construction cost > Ability to mitigate against sea level rise via floatation to adjust to varrying tidal levels. > Small scale and low construction cost > Ability to mitigate against sea level rise via floatation to adjust to varrying tidal levels. Figure 67: Author diagram showing arhcitectural charateristics Figure 67: 70. 71. Figure 68: Boathouse main level floor plan Figure 69: Boathouse front view of dwelling raised above water Figure 70: Walkway swing bridge which leads occupants into the site Figure 71: Line of sight as you approach the retreat Figure 68: Figure 72: Author diagram showing extracted architectural charateristics BOAT HOUSE ANDERSON WISE ARCHITECTS This structure is located on the steeply sloped bank of Lake Austin; it was designed as a retreat from the nearby hustle and bustle. A path leads the oc- cupant to the retreat that extends a kilometre long, over a suspension bridge spanning a ravine, slop- ing down to the boathouse. The simple and elegant design rises above the plane of water off-the-grid domicile, exerting a minimal impact on surround- ing conditions, a mixed-use dwelling that serves multiple functions (see fig. 70 & 74) . Wind and water are combined to provide nat- ural cooling for the entire envelope. Full height openings can be activated by users to swing out on the North and Eastern side that also serve as impromptu diving platforms. Revisiting this con- cept not only provides precedent for the low-in- come strata with viable small-scale flexibility but provides a housing solution that provides potential mitigation to tidal level increases with minimal ex- cavation and is of low cost construction. ‘Its structure, fabricated into a single framework of steel and barged to the site, is anchored into rock beneath the water. A floating carpenter shop was used to complete the construction from the water side’(Wise, pg.30) The boathouse provides a respite from the exhaus- tions of heat and the exertion of swimming, but it constantly reminds occupants of their proximi- ty to the waters edge and the cross programmatic space provided. Flexible in almost every way the design presents an additional view on contested territory architecture. 74. 75. > Small footprint, programatic reponses stacked verticaly > Simple fixed foundations underwaters surface > Small footprint, programatic reponses stacked verticaly > > Simple fixed foundations underwaters surface Derived Design Principle Mixed typologies 4. Relationships between different ecological conditions and social typologies may form connections between people and place. Figure 73: Sectional elevation and site plan Figure 74: Boathouse dock level floor plan Figure 74: Boathouse dock level floor plan Figure 73: Sectional elevation and site plan > Small footprint, programatic reponses stacked verticaly > Simple fixed foundations underwaters surface > User controlled climatic devices, ventilation and light access etc > Single floor dwelling, open plan and mixed use space > Single floor dwelling, open plan and mixed use space > User controlled climatic devices, ventilation and light access etc > Components are easily fixed to steel shell > Simple steel framed construction, light weight and highly durable. BOAT HOUSE ANDERSON WISE ARCHITECTS Figure 74: Boathouse dock level floor plan Figure 73: Sectional elevation and site plan > Simple steel framed construction, light weight and highly durable. Derived Design Principle Derived Design Principle 4. Relationships between different ecological conditions and social typologies may form connections between people and place. > Components are easily fixed to steel shell > Components are easily fixed to steel shell > Simple steel framed construction, light weight and highly durable. 77. 76. Figure 76: Left top: Beginning construction of wetland succession and topography Figure 77: Left middle: Close up of demolition debris left around the original site, which once was a shooting range Figure 78: Left Bottom: Existing topography and site conditions before temporal process was undertaken Figure 79: Right top: Wetlands have been succulently planted within the topography to provide regenerative land- scapes Figure 80: Right Middle: Series of park seating integrated within the landscape to provide moments of rest Figure 81: Right Bottom: Boardwalk protrude intro grass wetlands allowing the immersion of visitors Figure 75: Deep Water pond in winter months Tianjin Qiaoyuan Wetland Park CHINA - TIANJIN CITY BY TURENSCAPE for Adaptation Palettes, which were designed to let the nature do the work of rejuvenation (see fig 80,81 and 82). Tianjin Oiaoyuan Wetland Park is a network of pathways, pools and naturescapes successfully displaying the application of regenerative design. A wetland with changing landforms is revitalised from a neglected site, transgressing into a plant adaptive landscape in a community evolved pro- cess. BEFORE AFTER AFTER Untidy forms, unplanned biodiversity and nature’s ‘messiness’ keep ongoing, letting plants live and expose their genuine beauty to enrich the land- scape. The ecology-driven Adaptation Palettes have become a valued and unmistakable site of the community of Tianjin. This project helps to define the new aesthetics of landscape, defined by a con- tinuous evolving process. Temporal growth and phasing a design can build a relationship between community and design, and subsequently an affili- ation between people and place( see fig. 83). It is located in 22 ha in the coastal city of Tianjin, China. Urbanisation in Tianjin over the century has been tireless with re-purposing from one type of land use to another such as a former shooting range becoming a garbage dump and drainage sink for storm water. Now a contaminated site with little life left but a deserted and polluted landscape. It’s surroundings are dotted by squatters and tempo- rary structures. ‘Qiaoyuan Wetland Park implicitly asserts that the beauty of wild grass landscapes has been undervalued, especially in terms of the sus- tainable services they provide’. (Saunders, pg.116) A low-maintenance urban park with adaptive plant palettes, altering and phasing the landforms has been developed. This allows the natural processes of plant community adaptation and evolution to take place. Diversification flourishes and natural resources for the city are produced, including re- taining and purifying storm water, improvements in soil quality,and offering opportunities for envi- ronmental education by creating refreshing expe- riences. Its is derived from the adaptive vegetation communities that dot the landscape in this region of China. The solution for this park was developed Figure 75: 80. CASE STUDY 81. HABITAT EARTH CREATE CLEAN EARTH CLEAN HABITAT EARTH CREATE CLEAN VEGETATE INCREASE PRESERVE TEMPORAL PROCESS MANAGE WATER CLEAN PHASE 1: Cconstruction, topograhy and pond development. Figure 83: Author diagram showing combined regenrative design layers and process Tianjin Qiaoyuan Wetland Park CHINA - TIANJIN CITY BY TURENSCAPE Depression are constructed at varying depths which can both collect varing amounts of stormwater and fill with groundwater depending on elevation PHASE 3: Architectural elements are built.Water both infiltraits via the depressions and creates hospitable soil for vegetation growth. PHASE4: Vegetive planting and wetland succession. Planting can occur via seed dispersal, responding to the ecological conditions of the surrounding area, or be planted with specific species, creating habitat as required(Adaption palette) PHASE 2: construction of pathways and wetland links. Figure 82: Author diagram showing regenrative design process PHASE 2: construction of pathways and wetland links. PHASE 1: Cconstruction, topograhy and pond development. Depression are constructed at varying depths which can both collect varing amounts of stormwater and fill with groundwater depending on elevation PHASE 1: Cconstruction, topograhy and pond development. Depression are constructed at varying depths which can both collect varing amounts of stormwater and fill with groundwater depending on elevation PHASE 2: construction of pathways and wetland links. PHASE 2: construction of pathways and wetland links. HABITAT EARTH CREATE CLEAN EARTH CLEAN HABITAT EARTH CREATE CLEAN VEGETATE INCREASE PRESERVE TEMPORAL PROCESS MANAGE WATER CLEAN PHASE 3: Architectural elements are built.Water both infiltraits via the depressions and creates hospitable soil for vegetation growth. PHASE4: Vegetive planting and wetland succession. Planting can occur via seed dispersal, responding to the ecological conditions of the surrounding area, or be planted with specific species, creating habitat as required(Adaption palette) Figure 82: Author diagram showing regenrative design process EARTH CLEAN MANAGE WATER CLEAN MANAGE WATER EARTH WATER CLEAN CREATE CLEAN CLEAN MANAGE CLEAN HABITAT EARTH CREATE CLEAN VEGETATE HABITAT EARTH CREATE CLEAN PHASE 3: Architectural elements are built.Water both infiltraits via the depressions and creates hospitable soil for vegetation growth. PHASE 3: Architectural elements are built.Water both infiltraits via the depressions and creates hospitable soil for vegetation growth. PHASE4: Vegetive planting and wetland succession. Planting can occur via seed dispersal, responding to the ecological conditions of the surrounding area, or be planted with specific species, creating habitat as required(Adaption palette) PHASE4: Vegetive planting and wetland succession. Planting can occur via seed dispersal, responding to the ecological conditions of the surrounding area, or be planted with specific species, creating habitat as required(Adaption palette) Figure 83: Figure 82: Figure 83: Author diagram showing combined regenrative design layers and process 83. 82. 83. CASE STUDY function. Through this design led research it is possible to unlock the understanding of the role of the archi- tect. The relevance of permanence and the ability to provide remediation and curate change through expression of the local community knowledge process. When combined with the potential im- pacts for low lying atoll communities it enables the involvement on a human level attaching not just physical properties of building mass but the iden- tity of the architecture proposed by an outsider to be influenced by the vernacular setting. This gen- erates a connection to sovereignty and ownership as propositioned in the thesis question. Derived Design Principle The way to improve the situation of the squatter within the design must also take into consideration the people, not just a sense of community, but also an improvement in social standing and desire for self-sufficiency. The ability to directly influence the future of inhabitants positively through the built environment. Note: ‘Without the positive experience of nature, people will not be inclined to commit the necessary energy, emotions, and resources to sustaining buildings and constructed land- scapes over time’ (Kellert, pg.93) a. Temporal growth and regenerative design can build a relationship between community and project, which then forms auxiliary agency between people and place. The housing situation in Funafuti leaves large areas for improvement to adopt the positive attributes of the previously discussed theoretical & practical participatory designs (Figure 86). Security of land tenure through shared environment and critical understanding of the inhabitants historical, finan- cial and physical needs. Ecological functions to inform design’; The selected case studies have presented relevant themes for the architectural conditions, address- ing the dwelling requirements of the low-income strata on an architectural level, as opposed to fo- cusing on the social. HABITAT EARTH CREATE CLEAN MANAGE WATER CLEAN ECOLOGICAL FUNCTIONS VEGETATE INCREASE PRESERVE EARTH CLEAN VEGETATE INCREASE HABITAT CREATE HABITAT CREATE PRESERVE VEGETATE INCREASE MANAGE ATER CLEAN PRESERVE The five practical applications identified in the precedent studies will form the architectural basis for the proposed housing solution, framework for agencies that seeks to reinvent living conditions, accounting for the worst case scenario of future sea inundation and future growth. Key themes are as follows: 1. Simple construction 2. Ability to expand 3. Vernacular, cheap, locally sourced materials 4. Traditional themes in form and function 5. Collective/community construction It is these themes that will form the basis for devel- opment when considering architectural form and 1. Simple construction 2. Ability to expand 3. Vernacular, cheap, locally sourced materials 4. Traditional themes in form and function 5. Collective/community construction CREATE CLEAN MANAGE INCREASE CLEAN PRESERVE Key design principles extracted address the infor- mal settlement criteria 4. Traditional themes in form and function 5. Collective/community construction 1. Insecure residential status 2. Poor structural quality of housing 3. Inadequate access to safe water 4. Sanitation and other infrastructure 5. Overcrowding It is these themes that will form the basis for devel- opment when considering architectural form and 1. Insecure residential status 2. Poor structural quality of housing 3. Derived Design Principle Inadequate access to safe water 4. Sanitation and other infrastructure 5. Overcrowding 84. 85. CASE STUDY DESIGN PROCESS This chapter documents the iterative process, which was undertaken through this design led thesis. This is displayed through three preliminary design experiments in order to show the progres- sion of a ‘place for the forgotten’. These experiments touch on climate imposed is- sues and possible architectural solutions to main- tain subsistent dwelling, therefore increasing the period of habitation on the island. The design be- gins with the analysis of site the conditions to as- certain which areas of the island need immediate attention. 3.0 Experiment one and two look to progress in a se- quential order by identifying points of adjustment. The first two proposals are large program based entities for future mass inundation which link to the thesis question by suggesting a floatable settle- ment. Design experiments look to bridge the gap between adaption and action using an intimate scale, such as the dwelling, to allow cultural her- itage, sovereignty and vernacular techniques that can be extracted through architectural form. The final design objectives and formal outcomes, integrate with the remediation of land loss by ac- tively removing habitat from unstable conditions engaging the natural process of biological conser- vation. Exploring how the edifice is key to express- ing a traditional dialogue to climatic design and reaction to new environmental conditions whilst protecting the sovereignty of Tuvalu. 87. Figure 85: Funafuti island has many degraded shelters and pig pens near areas of low water. DESIGN AGENCIES 100m 100m 100m 100m SITE SPECIFICALLY TEMPORAL FRAMEWORK ENGAUGING LANDSCAPE HYBRID TYPOLOGIES LOCAL TECHNOLOGY & SOURCING Figure 84: Informatics associated with the theoretical and case study principles in the document LOCAL TECHNOLOGY & SOURCING LOCAL TECHNOLOGY & SOURCING ENGAUGING LANDSCAPE HYBRID TYPOLOGIES Figure 84: Informatics associated with the theoretical and case study principles in the document Figure 85: Funafuti island has many degraded shelters and pig pens near areas of low water. 88. 89. PRELIMINARY FIGURE GROUND 0m -20m > WATER BODIES & BURROW PIT LOCATIONS (SCARRED LANDSCAPE) >LAKE (TARASAL) >ROAD NETWORK >BUILDING FOOTPRINTS -30m -10m S N E W WS 150 120 60 30 330 300 240 210 30º 20º 10º 40º 50º 60º 70º 80º SS EQUINOX Figure 87: Author diagram looking at the water-body locations, road network and important buildings of Funafuti Figure 86: Author diagram looking at the categorisation of the figure ground into sizes to analyse the most common size grain to establish how the architecture should respond. Not to scale. S Figure 86: Author diagram looking at the categorisation of the figure ground into sizes to analyse the most common size grain to establish how the architecture should respond. Not to scale. Figure 87: Author diagram looking at the water-body locations, road network and important buildings of Funafuti 90. 91. ON SITE RISING TIDES: BURROW PIT SECTION Figure 89: Author diagram displaying the current cross section of the most at risk area in Funafuti and the typical sectional elevation of dwelling in these locations Figure 88: Author diagram displaying the current ‘burrow pits’(water bodies) of Funafuti, inundated areas were inhabitants live in slum like con- ditions. Figure 91: The most applicable typologies were selected to progress design thought as a means to engauge with se alevel rise BURROW PITS TUVALU 92. A. B. C. D. N N N N Figure 88: Author diagram displaying the current ‘burrow pits’(water bodies) of Funafuti, inundated areas were inhabitants live in slum like con- ditions. A. N B. N L BP L RB BR BR RF RB AVERAGE SPRING TIDE 2.7m AVERAGE KING TIDE 3.2m KING TIDE + SEA LEVEL RISE (2090, 3.76m) MEAN SEA LEVEL HEIGHT 2m > Typical section of burrow pits housing elevated 1 metre above ground level 4.00m 3.50m 3.64m 3.80m 3.60m L BP L RB BR BR RF RB AVERAGE SPRING TIDE 2.7m AVERAGE KING TIDE 3.2m KING TIDE + SEA LEVEL RISE (2090, 3.76m) MEAN SEA LEVEL HEIGHT 2m B. A. B. N N C. N D. N > Typical section of burrow pits housing elevated 1 metre above ground level 4.00m 3.50m 3.64m 3.80m 3.60m 4.00m N N N C. D. > Typical section of burrow pits housing elevated 1 metre above ground level Figure 88: Author diagram displaying the current ‘burrow pits’(water bodies) of Funafuti, inundated areas were inhabitants live in slum like con- ditions. 92. 93. WATER-ADAPTIVE BUILDING MATRIX Wet proof Residence Peak storage Seasonal storage Disater storage High water Groundwa- ter flooding Dry proof Residence Neutral Not Recommended Highly Recommended Highly Recommended Most Applicable to context Most Aplicable Shoreline Residence Column Residence Floating Residence Amphibious Residence Low drainage Residence Figure 90: Author diagram displaying extracted analysis of Dutch water building techniques to establish an effective solution for flood mitigation Wet proof Residence Peak storage Seasonal storage Disater storage Dry proof Residence Shoreline Residence Column Residence Floating Residence Amphibious Residence Low drainage Residence High water Groundwa- ter flooding Most Aplicable Disater storage High water Peak storage Groundwa- ter flooding Most Applicable to context Adaption for Resilience Most Applicable to context Adaption for Resilience Most Applicable to context Adaption for Resilience Adaption for Resilience Low drainage Residence Most Aplicable Most Aplicable Not Recommended Not Recommended Highly Recommended Neutral Neutral Most Applicable to context Highly Recommended Figure 90: Author diagram displaying extracted analysis of Dutch water building techniques to establish an effective solution for flood mitigation 95. 94. 96. BURROW PITS TUVALU 0.0- 1.0m 1.1- 2.0m 2.1- 3.0m 3.1- 4.0m 0 100 200 300 400 500 S N E W WS 150 120 60 30 330 300 240 210 30º 20º 10º 40º 50º 60º 70º 80º SS EQUINOX A A B B C C D D E E F F G G H H I I J Figure 92: Author diagram displaying cross section locations and urban layout A 97. L BP L RB BR BR RF RB SECTION A LEGEND VL – VEGETATION LINE BF – BEACH FACE SD – SAND CR – CORAL RUBBLE BR – BEACH ROCK CP – CORAL PATCHES RF – REEF FLAT BRB – BEACH ROCK BOULDERS BRP – BEACH ROCK PEBBLES BRG – BEACH ROCK GRAVEL RB – RUBBLES L – LAND SP – SAND PIT BP – BORROW PIT 2090 Sea Level 2050 Sea Level 2030 Sea Level Current Sea Level L SP RB BR RF RB BR RF SECTION B L BP L RB BR RF RB SD RF SECTION C RB BRB BP L RB BR L SECTION D Figure 93: Cross section of the main islet of Funafuti displaying the effects of sea level rise in high emissions and potential king tide heights that threaten to wash Tuvaluans away L BP L RB BR BR RF RB SECTION A LEGEND VL – VEGETATION LINE BF – BEACH FACE SD – SAND CR – CORAL RUBBLE BR – BEACH ROCK CP – CORAL PATCHES RF – REEF FLAT BRB – BEACH ROCK BOULDERS BRP – BEACH ROCK PEBBLES BRG – BEACH ROCK GRAVEL RB – RUBBLES L – LAND SP – SAND PIT BP – BORROW PIT 2090 Sea Level 2050 Sea Level 2030 Sea Level Current Sea Level L SP RB BR RF RB BR RF SECTION B B B C D D E SECTION A F E SECTION B SECTION B L BP L RB BR RF RB SD RF SECTION C RB BRB BP L RB BR L SECTION Df SECTION B L BP L RB BR RF RB SD RF SECTION C G F SECTION C G Figure 93: Cross section of the main islet of Funafuti displaying the effects of sea level rise in high emissions and potential king tide heights that threaten to wash Tuvaluans away 96. Figure 95: Author impression of porjected sea level rise 2090 inundating the land Figure 94: Burrow pits Funafuti BURROW PITS TUVALU Figure 92: Author diagram displaying cross section locations and urban layout Figure 92: Author diagram displaying cross section locations and urban layout 97. 96. Figure 94: Burrow pits Funafuti Figure 94: Burrow pits Funafuti Figure 95: Author impression of porjected sea level rise 2090 inundating the land 98. 99. 100 0.0- 1.0m 1.1- 2.0m 2.1- 3.0m AVERAGE KING TIDE FLOODING 3.2m (1.2m increase) 3.1- 4.0m 0 100 200 300 400 500 S N E W WS 150 120 60 30 330 300 240 210 30º 20º 10º 40º 50º 60º 70º 80º SS EQUINOX FUN149 FUN148 FUN147 FUN146 FUN FUN153 FUN157 FUN158 FUN156 FUN155 A A B B C C D D E E F F G G H H I I J Figure 96: Author diagram showing cross section locations and average estimated king tide inundation 1.2m increase in sea level. BURROW PITS TUVALU Displaying the vulnerability of Funafuti A FUN FUN1 B SD RB L TARO PATCH L RUNWAY L MANGROVES SWAMP L RB BR RF SECTION F SD RIP RAP L RUNWAY L LAKE BRB RB BR SECTION E LEGEND VL – VEGETATION LINE BF – BEACH FACE SD – SAND CR – CORAL RUBBLE BR – BEACH ROCK CP – CORAL PATCHES RF – REEF FLAT BRB – BEACH ROCK BOULDERS BRP – BEACH ROCK PEBBLES BRG – BEACH ROCK GRAVEL RB – RUBBLES L – LAND SP – SAND PIT BP – BORROW PIT 2090 Sea Level 2050 Sea Level 2030 Sea Level Current Sea Level RF RIP RAP L RB BR RF SECTION G L RB SD RB BR RF SECTION I Figure 97: Island cross sections displaying project sea level rise increments and expected king tide level N WS 150 120 60 30 330 300 240 210 30º 20º 10º 40º 50º 60º 70º 80º SS EQUINOX LEGEND VL – VEGETATION LINE BF – BEACH FACE SD – SAND CR – CORAL RUBBLE BR – BEACH ROCK CP – CORAL PATCHES RF – REEF FLAT BRB – BEACH ROCK BOULDERS BRP – BEACH ROCK PEBBLES BRG – BEACH ROCK GRAVEL RB – RUBBLES L – LAND SP – SAND PIT BP – BORROW PIT B 60º 70º 80º E C FUN C D FUN D SECTION E F SECTION F FU E G FUN F SECTION G L RB SD RB BR RF SECTION I FUN G Figure 97: Island cross sections displaying project sea level rise increments and expected king tide level Figure 96: Author diagram showing cross section locations and average estimated king tide inundation 1.2m increase in sea level. Displaying the vulnerability of Funafuti 101. 100. Figure 101: Basic amenities needed per person BURROW PITS TUVALU 0m -20m > WATER BODIES & BURROW PIT LOCATIONS (SCARRED LANDSCAPE) >LAKE (TARASAL) >ROAD NETWORK >BUILDING FOOTPRINTS >BEACH RIDGE LAST LAND TO GO -30m A B C D ZONE 2 SETTLEMENT ZONE -10m S N E W WS 150 120 60 30 330 300 240 210 30º 20º 10º 40º 50º 60º 70º 80º SS EQUINOX 0m 5000m 10000m S N E W WS 150 120 60 30 330 300 240 210 30º 20º 10º 40º 50º 60º 70º 80º SS EQUINOX NOTE: Habitable zone for lagoon settlement in black Figure 98: Author diagram showing showing site locations at an auxilary scale S N WS 150 120 60 30 330 300 240 210 30º 20º 10º 40º 50º 60º 70º 80º SS EQUINOX N S NOTE: Habitable zone for lagoon settlement in black NOTE: Habitable zone for lagoon settlement in black Figure 98: Author diagram showing showing site locations at an auxilary scale Figure 99: Author diagram dislaying otential site zones for habitation and location of most at risk settlements Figure 99: Author diagram dislaying otential site zones for habitation and location of most at risk settlements 102. 103. Figure 102: Tuvaluan vernacular shoreline dwellings Introduction KASO (Fala pandanus) TILOFA (Fala pandanus) KAUKAUUI (Fala pandanus) POU (Fala pandanus) IWI (Fala pandanus) UTUPOTO (Fala pandanus) SASAGA (Fala pandanus) KAUTU (Fala pandanus) TAOMAGA (Fala pandanus) TELETELEKIMONA (Fala pandanus) This section discusses the organisational make up of the Tuvaluan vernacular which is reflective of the environment in which they are prescribed. These are systems of construction, engagement in local hierarchies and the relationship to social val- ues, status and municipal materials. Sovereignty is an aim that denotes the social elements of appli- cable programs and can be symbolically tested to manipulate an intervention reflective of cultural and social values of the inhabitants. This section discusses the organisational make up of the Tuvaluan vernacular which is reflective of the environment in which they are prescribed. These are systems of construction, engagement in “Taumatafenua” type of sleeping house (Common) (Koch, pg.115). Figure 104: Traditional construction typologies: >Sleeping houses (see fig. 105 & 107) >Sleeping houses (see fig. 105 & 107) >Oven huts >Storehouses >Chief houses >Meeting houses and houses for the gods. >Oven huts >Oven huts Figure 105: “Fale Poutasi” type of sleeping house (Koch,pg.118). >Storehouses >Chief houses >Chief houses >Meeting houses and houses for the gods. >Meeting houses and houses for the gods. Following organisational patterns that arrange public and private space within a hierarchy for gathering and communal configurations. The traditional typology for most houses has a rectangular ground plan and gable roof with a straight ridge consisting of a fala pandanus post and beam structure. As the ‘Taumatafenua’ type (Observe Islands), when translated means ‘to look at distant lands’ Its construction was robust but flexible, a typical structure would span seven meters wide and ten meters long, which could be lifted from its posts and repositioned as necessary. Thatch ribs came down to a meter off the ground, with ends pointed like spears for protection against intruders. The roofs would consist of Pandanus leaves joined in groups of 15-20 to make a piece of thatch (lau). Approximately 1400 lau would be re- quired for a house of similar dimensions (Knoch). A large storage area, which doubles as a sleeping platform is built into the roof structure at a specif- ic height, it is said that while sleeping, a smoky fire could be lit and protect against mosquitoes. Figure 105: “Fale Poutasi” type of sleeping house (Koch,pg.118). Figure 106: a. Fakasala Lua type screens(Pola). b. 105. WATER CONSUMPTION = 2-4 LITRES(PER PERSON A DAY) LOCATION, SIZE, AND DENSITY THE BASICS; MEAT CONSUMPTION = 38.8KG(PER PERSON A YEAR) FRUIT & VEGTABLE CONSUMPTION = 384GRAMS(PER PERSON A DAY) 5-6 People per household Approximate WATER FOOD SHELTER Key themes are as follows: 1. Simple construction 2. Ability to expand 3. Vernacular, cheap, locally-sourced materials 4. Traditional themes in form and function 5. Collective/community construction 6. Small scale Figure 101: Basic amenities needed per person MOST AT RISK THE BASICS; C. D. N N A. B. N N Figure 100: Author diagram presenting the most at risk settlements ‘Burrow pits’ B. N A. N N B. C. N D. N N D. Figure 100: Author diagram presenting the most at risk settlements ‘Burrow pits’ Figure 101: Basic amenities needed per person 104. 105. Figure 102: Tuvaluan vernacular shoreline dwellings 107. VERNACULAR DWELLINGS Figure 104: “Taumatafenua” type of sleeping house (Common) (Koch, pg.115). Figure 107: Model made by Author displaying traditional fale construction(Taumatafenua, type of sleeping Figure 107: Model made by Author displaying traditional fale construction(Taumatafenua, type of sleeping house) and given names for structural members 109. Figure 107: Model made by Author displaying traditional fale construction(Taumatafenua, type of sleeping house) and given names for structural members Introduction Laga Fakalaulau type wall screens(Pola). c. Faksala tasi type wall screens(Pola)(Koch,pg.118). Figure 106: a. Fakasala Lua type screens(Pola). b. Laga Fakalaulau type wall screens(Pola). c. Faksala tasi type wall screens(Pola)(Koch,pg.118). 108. 109. VERNACULAR DISCOURSE Vernacular and Climatic sensitivity: The structure places high importance regarding airflow by the use of permeable materials; and large openings. Pola (Woven Screens) form the shell of the envelope, resulting in the large steep thatched roof becoming the dominant feature, with wide overhangs mitigating solar radiation (see fig. 108). In addition, the (relatively) thin vertical supports allow the air to freely flow, further mediating the climate (while nowadays, adding a memorable correlation with the forest of vertical pinnacles). These features will be incorporated into the design research experiments as tested means of respond- ing to the site conditions. Figure 110: Pre-contact village scene on Tuvaluan island, Niutao Figure 111: Post-contact village scene on Tuvalu, displaying a hybridisation of the vernacular propping the traditional thatched roof on masonry walls adopting a European Figure 113: Tuvaluan Vernacular Characteristics Current situation: Prior to the arrival of missionaries that brought Christianity to Tuvalu, homes were of vernacular construction (see fig. 110 & 111), with rectangular open structures, pitched thatch roofs, posts, and roll-up mats around the perimeter with little to no walls. Today constructions are typically raised on posts one meter above ground for protection from flooding, corrugated metal sheet roofing, 2 x 4 pine framing, and cement board cladding are all utilised. Each home has distinctive spaces of varying enclosure and exposure, some are paint- ed bright colours, mostly greens and blues, a few salmon, yellow and red. The living, bedrooms and kitchen are the most en- closed and secondary to that is an outdoor aper- ture with a simple raised floor and roof with little walls and low railings. This semi-enclosed space near the main house or attached to it. This space is frequently used during the day, whether it be as a gathering or working space for the family. Struc- tures are laid out in close proximity to one another, Figure 108: More exposed vernacular houses use coconut fronds as wind breaks Figure 109: Historic photo showing native islanders dancing in front of traditional dwellings on Funafuti, Tuvalu dwellings on Funafuti, Tuvalu Figure 110: Pre-contact village scene on Tuvaluan island, Niutao Figure 111: Post-contact village scene on Tuvalu, displaying a hybridisation of the vernacular propping the traditional thatched roof on masonry walls adopting a European 111. 110. VERNACULAR DISCOURSE > Sloped walls/facade > Principle Airflow > Seperate kitchen hutt generally detatched in traditional times > Small scale and mixed use space > Sloped walls/facade > Principle Airflow > Principle Airflow > Floor Seperation for storage and sleeping > Permeable facade > Simple post and beam construction form local wood > Transportable, light weight materials > Sloped walls/facade > Floor Seperation for storage and sleeping > Sloped walls/facade > Permeable facade > Principle Airflow > Floor Seperation for storage and sleeping > Floor Seperation for storage and sleeping > Seperate kitchen hutt generally detatched in traditional times > Small scale and mixed use space > Small scale and mixed use space > Seperate kitchen hutt generally detatched in traditional times > Simple post and beam construction form local wood > Transportable, light weight materials 113. 112. Figure 115: Inland dwelling typology on Funafuti, Tuvalu Figure 114: Traditional thatched roof Fale con- struction process. Figure 118: Typical plan layouts and average Nº of people (Not to scale) Current situation: in an apparently random fashion, with settlements shaped into a spinal setting by the landscape allow- ing transitional movement from the ocean to the street front. The lines of property become blurred as people often cross into each others yards for ca- sual visits. A two story home often has most of the first floor unenclosed, shaded by the upper floor of the house, it is open to cool breezes. If flooding oc- curs possessions are popped up onto the table and chairs to remain above water level. On a typical day ‘People can be seen weaving, sewing, sleeping, playing cards, cooking, and children are playing in this versatile, open plan space. The rafters of the building are used as a laundry line. In some places fabric, vinyl or tarps are used as curtains for pro- tection and privacy screens surrounding the space and can either be pulled to the side or rolled up(- see fig.118 & 119). Figure 115: Inland dwelling typology on Funafuti, Tuvalu Figure 116: Burrow pit dwelling typologies raised generally on poorly constructed post foundations, fibre cement board walls lining the exterior. Some homes have a third outdoor space which is simply a raised platform used for drying meat or as a work space or to recline and gaze at the night- time sky. Privacy does not seem to be an issue with doors and windows left open and curtains pulled back. Spaces are illuminated in the evening and one can view domestic life from the street. Win- dows are mostly 12 cm slats with no screens, but some have awning-style boards propped up, which are lowered to seal the inhabitants in during storms (Tribal link, pg.7). Figure 114: Traditional thatched roof Fale con- struction process. Figure 116: Burrow pit dwelling typologies raised generally on poorly constructed post foundations, fibre cement board walls lining the exterior. 114. 115. TYPICAL DWELLING LAYOUTS WALL DIVIDE SINGLE ROOM OPEN PLAN Figure 118: Typical plan layouts and average Nº of people (Not to scale) Figure 119: Vernacular housing on Vaitupu island, 24 foot long by 16 feet wide, high open sides, thatched roof of pandanus. Figure 120: This shows an adopted more modern vernacular, with coral limestone foundations Figure 121: Stilted vernacular house built over lagoon Figure 122: Author sketch displaying pre contact village layout with clearing in middle for con- gressions Figure 122: 116 = 7m 9m 10m 12m PRE- COLONIAL POST-URBANISATION Figure 117: Opportunity for design; large difference between vernacular typology and current construction TYPICAL DWELLING LAYOUTS WALL DIVIDE SINGLE ROOM OPEN PLAN 18: Typical plan layouts and average Nº of people (Not to scale) PRE- COLONIAL TYPICAL DWELLING LAYOUTS WALL DIVIDE SINGLE ROOM OPEN PLAN TYPICAL DWELLING LAYOUTS 10m OPEN PLAN POST-URBANISATION OPEN PLAN 116. 117. Figure 122: Figure 122: Author sketch displaying pre contact village layout with clearing in middle for con- gressions 119. 119. 118. 118. PRELIMINARY DESIGN 123. EXPERIMENT ONE The initial preliminary design experiments dis- played explore how a potentially new settlement pattern, structure and habitable surface can be de- veloped in order to extend the period of habita- tion on Funafuti island. These sketch experiments and models look to respond to the prior contextual analysis of Funafuti island, developing a tectonic connection to the vernacular architecture. Al- lowing a continued sovereignty imposed through dwelling in the face of land loss. Therefore con- ditions of resistance begin to develop responding how the people can reclaim the land and protect- ing a traditional dialogue between sovereignty and dwelling. LEGEND VL – VEGETATION LINE BF – BEACH FACE SD – SAND CR – CORAL RUBBLE BR – BEACH ROCK CP – CORAL PATCHES RF – REEF FLAT BRB – BEACH ROCK BOULDERS BRP – BEACH ROCK PEBBLES BRG – BEACH ROCK GRAVEL RB – RUBBLES L – LAND SP – SAND PIT BP – BORROW PIT RUNWAY L MANGROVES SWAMP L RB BR RF 1 2 3 4 5 6 Land Above Water PRELIMINARY DESIGN Figure 123: Aurhor cross section showing land left over and levels of projected flooding RISING SEAS SD RB L TARO P Average King Tide + Sea Level Rise (2090, 3.76m) Average King Tide before sea level rise 3.2m Average Spring Tide 2.7m Average Sea Level 2.0m Average Sea Level 2.0m Average Spring Tide 2.7m Average Low Water Level 1.37m 1 2 3 4 5 6 Land Above Water Design Setting: >Its the year 2090 Sea level has risen to 2.56m, ex- pected average king tide is now estimated at 3.76m. Displayed on the cross-section below are the levels and leftover land. Due to Tuvalu’s limestone base this land is expected to be severly inundated (see fig. 126). Research Question: How can architecture maintain a sense of sover- eignty within a disappearing context and the im- plications of habitation, culture and deterritorial- isation? Summary The vernacular presence is one that I suggest should be acknowledged in the development of ar- chitecture, as it is ‘architecture without architects’. It’s respective of the local climatic conditions, the availability of locally sourced materials, of low cost and with high construction efficiency with mini- mal material waste and timely. Through design led research the key vernacular characteristics have been extracted and are out- lined below 1. Large roof 2. Air flow 3. Open plan 4.Built from local resources (See fig. 115) 1. Large roof 2. Air flow 3. Open plan 4.Built from local resources (See fig. 115) 1. Large roofl 2. Air flow l 3. Open plan p p 4.Built from local resources i (See fig. 115) 120. Figure 124: Author diagram showing the proximities of programmes and island make up Figure 125: Author initial sketch showing the tectonic nature of proposed designs mimicking successful vernacular typologies Figure 125: 126. Figure 125: Author initial sketch showing the tectonic nature of proposed designs mimicking successful vernacular typologies EXPERIMENT ONE The initial preliminary design experiments dis- played explore how a potentially new settlement pattern, structure and habitable surface can be de- veloped in order to extend the period of habita- tion on Funafuti island. These sketch experiments and models look to respond to the prior contextual analysis of Funafuti island, developing a tectonic connection to the vernacular architecture. Al- lowing a continued sovereignty imposed through dwelling in the face of land loss. Therefore con- ditions of resistance begin to develop responding how the people can reclaim the land and protect- ing a traditional dialogue between sovereignty and dwelling. 122. 123. BF – BEACH FACE SD – SAND CR – CORAL RUBBLE BR – BEACH ROCK CP – CORAL PATCHES RF – REEF FLAT BRP – BEACH ROCK PEBBLES BRG – BEACH ROCK GRAVEL RB – RUBBLES L – LAND SP – SAND PIT BP – BORROW PIT SD RB L TARO PATCH L RUNWAY L MANGROVES SWAMP L RB BR RF Average King Tide before sea level rise 3.2m Average Spring Tide 2.7m Average Sea Level 2.0m Average Sea Level 2.0m Average Spring Tide 2.7m Average Low Water Level 1.37m 1 2 3 4 5 6 1 2 3 4 5 6 Land Above Water Land Above Water Figure 123: Aurhor cross section showing land left over and levels of projected flooding 6 Land Above Wate Land Above Water 123. 1KM PROXIMITY INTERSECTIONS 2KM RUBBISH DUMP HIGH SCHOOL FUNAFUTI PORT MAINAGA FOU CHURCH PRIMARY SCHOOL GOVERNMENT DESALINATION PLANT 2KM 1KM PROXIMITY INTERSECTIONS COMBINED: INSTERSECTIONS AND PROXIMITY ZONES 2KM RUBBISH DUMP HIGH SCHOOL FUNAFUTI PORT MAINAGA FOU CHURCH PRIMARY SCHOOL GOVERNMENT DESALINATION PLANT 2KM 2KM COMBINED: INSTERSECTIONS AND PROXIMITY ZONES 2KM INTERSECTIONS FROM ONE FOCAL LOCATION TO REMANDER + 2KM ZONES 2KM COMBINED: INSTERSECTIONS AND PROXIMITY ZONES 2KM INTERSECTIONS 1KM PROXIMITY INTERSECTIONS FROM ONE FOCAL LOCATION TO REMANDER + 2KM ZONES COMBINED: INSTERSECTIONS AND PROXIMITY ZONES Figure 124: 125. 124. 127. 126. EXPERIMENT ONE DOCKING AREA DOCKING AREA CIRCULATION S DESALINISATION PLANT SEED VAULT COMMUNAL EVENTS CENTRE HYDROPONIC FARM GOVERNMENT FACILITIES RESIDENTIAL ZONE SEWAGE FACILITY Figure 127: Author initial sketch showing a secondary possible layout of a floatable rig SK2 DOCKING AREA CIRCULATION SPINES SEWAGE FACILITY RAIN WATER CATCHMENT FACILITY RESIDENTIAL ZONE HYDROPONIC FARM CHURCH/COMMUNITY CENTRE Figure 126: Author initial sketch showing the possible layout of a floatable rig SK1 DOCKING AREA CIRCULATION SPINES SEWAGE FACILITY HYDROPONIC FARM DESALINISATION PLANT RAIN WATER CATCHMENT FACILITY RAIN WATER CATCHMENT FACILITY RESIDENTIAL ZONE HYDROPONIC FARM GOVERNMENT FACILITIES RESIDENTIAL ZONE COMMUNAL EVENTS CENTRE DOCKING AREA Figure 127: Author initial sketch showing a secondary possible layout of a floatable rig SK2 Author initial sketch showing a secondary possible layout of a floatable rig Figure 126: Author initial sketch showing the possible layout Author initial sketch showing the possible layout of a floatable rig S Figure 126: Author initial sketch showing the possible layout of a floatable rig SK1 129. 128. 131. Figure 129: Author preliminary design showing the ability to grow much like a cell multiplying to account for populous SK4 Figure 128: Author preliminary speculative concept SK3 Figure 128: Author preliminary speculative concept SK3 Author preliminary speculative concept SK3 Figure 129: Author preliminary design showing the ability to grow much like a cell multiplying to account for populous SK4 130. 131. 132 Figure 130: Author preliminary sketch displaying the development of potential community hub in spinal alignment arranged to mimic the islands proximities between communities 133. Figure 131: Author sketch planning of a potential fish farming rig for sustainable food manage- ment Figure 132: Author sketch perspective view of potential walkways and typologies raised above swell level in tension but able to flex . Figure 131: Author sketch planning of a potential fish farming rig for sustainable food manage- ment Figure 132: Author sketch perspective view of potential walkways and typologies raised above swell level in tension but able to flex . Figure 130: Author preliminary sketch displaying the development of potential community hub in spinal alignment arranged to mimic the islands proximities between communities 133. 132. Reflection: This preliminary design experiment is a specula- tive design suggesting a growing urban fabric that extends into the lagoon area. This takes advantage of the shelter from Funafuti’s main islet. The con- cept serves as means to address the objective of incrementalism and formalism within a changing context. Remaining mindful of the changing fac- tors of overcrowding, subsistence and ruminative design. Figure 134: Author diagrams extracting the urban fabric looking at the hospital layout, func- tions (Top) and also the primary school located on the island(Bottom). Figure 133: Author diagrams extracting the urban fabric if necessity requires,looking at how current urban fabric can be tested to inform formal gestures of community layout. Top is the govern- ment of Tuvalu and below Funafuti port. Experiment two: ‘Fluxes’ Experiment two: ‘Fluxes’ The site for these concepts is at a depth range be- tween 8 - 12 metres deep to reduce the period of oscillation between incoming swells. These first two sketch experiments are abstract perceptions of the retention of tuvalu’s sovereignty might look like in the uncertain future. It Gives opportunities to explore how the functions of a system that has lost equilibrium might be reintegrated by looking at prior social activities to suggest new ways of re- structuring the loss. The site for these concepts is at a depth range be- tween 8 - 12 metres deep to reduce the period of oscillation between incoming swells. These first two sketch experiments are abstract perceptions of the retention of tuvalu’s sovereignty might look like in the uncertain future. It Gives opportunities to explore how the functions of a system that has lost equilibrium might be reintegrated by looking at prior social activities to suggest new ways of re- structuring the loss. This experiment draws directly from Funafuti’s urban fabric, extracting key programs to integrate with low income housing to suggest a habitable concept. These iterations propose how one might safe guard the urban fabric, social and cultural ex- istence of Funafuti. The programmes were selected and arranged into a spinal settlement, replicating Funafuti’s vernacular characteristics and lagoon living. The floating rigs were arranged to propose flexibility and adaptability within the waters con- text. The success is in the approach to reflect pri- or architectural language of the island for future planned communities or metabolisms. These ex- periments use the current formal fabric control spatial activation, allowing reconstruction to oc- cur on a larger scale, providing a sense of order and evolution to maintain sovereignty. While this design suggests how settlement could inhabit the lagoon it doesn’t take note of the cul- tural practices and use of speculative architecture to add additional meaning of nationhood and sov- ereignty, which this experiment lacks. 135. 134. 1. 25M 0 1. 25M 0 re 134: Author diagrams extracting the urban fabric looking at the hospital layout, fu tions (Top) and also the primary school located on the island(Bottom). 2. 1. 50M 0 1. 2. 50M 0 1. 2. 50M 0 1. 25M 0 Figure 133: Author diagrams extracting the urban fabric if necessity requires,looking at how current urban fabric can be tested to inform formal gestures of community layout. Experiment two: ‘Fluxes’ Top is the govern- ment of Tuvalu and below Funafuti port. Figure 134: 136. 137. SETTLEMENT PATTERNS INFORMAL PATTERNS Figure 135: Author diagrams of formal settlement patterns of the Figure 136: Author diagrams extracting the urban fabric looking at the hospital layout and functions and also the primary school located on the island. INFORMAL PATTERNS Figure 135: Author diagrams of formal settlement patterns of the A. B. C. D. N N N N 139. Figure 136: Author diagrams extracting the urban fabric looking at the hospital layout and functions and also the primary school located on the island. C. D. N N A. B. N N A. N B. N B. N A. N A. B. A. N N N A. B. B. D. N D. N C. N C. N N N N D. D. C. Figure 135: Author diagrams of formal settlement patterns of the Figure 136: Author diagrams extracting the urban fabric looking at the hospital layout and functions and also the primary school located on the island. 138. 139. 140. Figure 137: Author concepts displaying how the urban grain of funafuti’s may inhabit the la- goon, the first is a concept that produces a formal gestures the provides a curved spine settlement that links into the coral substrata. Below the concept slips through between coral patch reefs providing extra swell shelter. 141. Figure 138: Author concepts, direct replication of the exiting important building urban fabric, aligned and anchored from the shore of funafuti. This final concept test how the rig can be situated between coral substrata to also help facilitate growth by using local to trasplant coral substract to form growing patch reeefs 140. Figure 137: Author concepts displaying how the urban grain of funafuti’s may inhabit the la- goon, the first is a concept that produces a formal gestures the provides a curved spine settlement that links into the coral substrata. Below the concept slips through between coral patch reefs providing extra swell shelter. 141. Figure 138: Author concepts, direct replication of the exiting important building urban fabric, aligned and anchored from the shore of funafuti. This final concept test how the rig can be situated between coral substrata to also help facilitate growth by using local to trasplant coral substract to form growing patch reeefs Figure 138: Reflection: By selecting important buildings and settlement patterns from the site, it maintains familiarity to the urban setting and we can begin to understand how they inhabit the space. The architect can re- spond and mimic familiar urban surroundings and social conditions. However the response lacks a connection to the environment and the influ- ences of indigenous culture with little narrative or connection to sovereignty. 142. 143. Figure 139: Author model , perspective showing the walkway floatation of the design Figure 139: CONCEPT DESIGN This experiment responds from prior implications from previous preliminary experiments and is de- veloped to suggest a structure that can flex and elevate the user well above the water plain. The circular hub serves as the connection to the land anchoring it’s self in place. The idea is constructed for mixed use, a food production hub that could potentially be used for reliable crop harvesting using simplified hydroponics to provide food all year round. The walkway modeled was designed in mind to provide shelter from the wave conditions below, yet suggest another surface of habitation. ‘The stability of Tuvaluan society and the happiness and welfare of the people of Tuvalu, Both present and future, depend very largely on the maintenance of Tuvaluan values, culture and Tradition, including the vitality and the sense of identity of island communities and attitudes of cooper- ation, self-help and unity within and amongst those communities’, (Papoutsaki, pg.262) This design phase allowed a more in depth solu- tion that is the ‘Hub for Habitation‘ , placed within the zone of habitation outlined by prior design re- search. The research objectives were met through the creation of a floatable settlement framework which allows continued logic taking a more pri- mordial practical step back, responding to local technologies. The structures have been modeled to represent future typologies that may be required to mitigate sea level rise. Whilst trying to improve a sense of community, belonging and reduce uncer- tainty to maintain sovereignty. 145. Figure 140: Author model, perspective looking down the line of sea level below the contructed hub Figure 140: Author model, perspective looking down the line of sea level below the contructed hub Figure 140: Author model, perspective looking down the line of sea level below the contructed hub uthor model, perspective looking down the line of sea level below the contru 146. 147. 148. CONCEPT DESIGN Figure 141: Author model , perspective showing the walkway bridge and suggestive tension sup- ports Figure 142: Perspective showing the hubs potential structural floatation and design Figure 143: Author model, perspective looking down the walkways facing the proposed commu- nity hub, a plane above the rest 148 Figure 141: Author model , perspective showing the walkway bridge and suggestive tension sup- ports Figure 142: Perspective showing the hubs potential structural floatation and design 141: Author model , perspective showing the walkway bridge and suggestive tension sup- ports Figure 142: Perspective showing the hubs potential structural floatation and design Figure 141: Author model, perspective looking down the walkways facing the proposed commu- nity hub, a plane above the rest Figure 142: 148. 149. 150 151 Figure 144: Author model, perspective showing the length of the construction and replication and redevelopment of vernacular tectonic Figure 144: Figure 144: Author model, perspective showing the length of the construction and replication and redevelopment of vernacular tectonic Author model, perspective showing the length of the construction and replication and redevelopment of vernacular tectonic 150. 151. Figure 147: Author diagram showing an orthograhic projection of the construction coponents of the floating habitat, displaying the complexity of the model Figure 148: Author Level one plan of the proposed hub concept Figure 148: DEVELOPING TYPOLOGIES 152. 153. 154. 155. Figure 147: Author diagram showing an orthograhic projection of the construction coponents of the floating habitat, displaying the complexity of the model Figure 147: Author diagram showing an orthograhic projec the floating habitat displaying the complexi 154. 155. 157. 156. Figure 149: Auther, Level one plan of designed dwelling for habitation, respojnsive of vernacular angency Figure 152: Author perspective of the walkway and dwelling units at the end of the circulation spine, anchored to the ground, allowing flexibility in the horizontal direction and acoount for large variations in sea levels overtime Figure 156: Tuvaluan fisherman, fish depletion is a consenting issue for Tuvaluans in recent times there has been a decline in numbers due to climate change and human depletion of resources. 159. DYLAN MAJUREY Figure 150: Author diagram showing the integration of sustainable resourcese for future surviv- ial 159. DYLAN MAJUREY Figure 150: Author diagram showing the integration of sustainable resourcese for future surviv- ial 158. AA AA BB BB Figure 149: Auther, Level one plan of designed dwelling for habitation, respojnsive of vernacular angency BB Figure 149: Auther, Level one plan of designed dwelling for habitation, respojnsive of vernacular angency hor diagram showing the integration of sustainable resourcese for future survi 158. 160. 161. Figure 151: Author, Elevations of the hub, top is the west and below is the eastern elevation 160. 161. Figure 151: Author, Elevations of the hub, top is the west and below is the eastern elevation 160. Figure 151: Author, Elevations of the hub, top is the west and below is the eastern elevation Figure 151: Author, Elevations of the hub, top is the west and below is the eastern elevation 161. 161. 160. 163. DYLAN MAJUREY Figure 153: Author perspective showing the canoe/boat platform and the back of the dwelling Figure 153: 162. 164. 164. 165. 165. Figure 154: Author perspective of the overall rig on a calm day displaying the possiblity of recy- clying building material for structure most at risk before tidal encrochment. Figure 155: Author perspective showing the rig in rough weather cpnditions, each dwelling was design to have a wash level which allows water to flow over the pontoon without pooling or sinking the dwelling. Figure 155: Author perspective showing the rig in rough weather cpnditions, each dwelling was design to have a wash level which allows water to flow over the pontoon without pooling or sinking the dwelling. Author perspective of the overall rig on a calm day displaying the possiblity of recy- clying building material for structure most at risk before tidal encrochment. 167. 166. STRENGTHS & WEAKNESSES Although this model conveys a formal gesture of space to suggest how the Tuvaluan’s could inhab- it structures above the water plane. It is requires further development to include a more sovereign connection to the active environment and vernac- ular. Considering the social implications of Look- ing into the development of recycled materials for construction and subsistent programmes within a floatable context, the hub includes a collapsible walkway that allows minimal damage in storms a technique that will be continued, dwellings are de- rivative of vernacular typologies but do not display order and evolution from the vernacular process. A community hub has been produced for garden- ing and social activities. Overall a stiff approach to the material object but high function to mitigate sea level rise. 168. 169. Adaption for Resilience Figure 160: Burrow pit pollution of Funafuti, Tuvalu Figure 161: Perspective view showing how the removal of human habitation could allow re- generative natural landscapes Figure 160: A PLACE FOR THE FORGOTTEN is positioned in the locale of the last remaining piece of land that is habitable and allows a frame- work for future development. The process of min- eral deposition otherwise known as ‘bio-rock’ may be used to convert the steel structures holding wa- ter tanks into coral branches facilitating marine life environment under the rig. The bio-rock may also be used in conjunction with the foundation to provide coral cement for holding the piles in place rather concrete. A place for the forgotten is sited within the lagoon area of Funafuti island as a response to, and exam- ination of the research aims and objectives of the thesis ‘How can architecture maintain a sense of sovereignty within a disappearing context and the implications of habitation, culture and contested territories.’ By activating the concepts identified, of reconstruction, active environment, spatial activa- tion, narrative , incrementalism and formalism, in the development of the architectural solution. The materiality of the project aims to draw from tradi- tional post and beam construction using a variety of split ‘Pandanus’ logs to form the framework for architectural implementation. The structure of the design for flotation is to be constructed out of wa- ter tanks recycled from the islands at risk homes, to be stacked and surrounded by a wire frame. Then filled with fresh water to provide ballast for the pontoons and water storage, at cool tempera- tures. The design is anchored into the atoll base to provide durability for weather events. The shelter This responding to the problem of a contest- ed context, of salt inundated land and threats of overcrowding. As habitants from outer islands are forced to migrate to the capital, Funafuti due to the decline of resources. The extracted speculative design solution provides an approach to the impli- cations of maintaining a sense of sovereignty with- in architecture through the integration of a object and utilising the discussed theoretical principles. Figure 158: Tuvaluan tribesmen outside vernacular dwellings Figure 158: Tuvaluan tribesmen outside vernacular dwellings 171. Figure 160: Figure 161: Perspective view showing how the removal of human habitation could allow re- generative natural landscapes 173. 172. Figure 162: Author model showing a more abstract dwelling concept that deals with architec- tural characteristics to mitigate conditions on site. Form is influenced to increase density of dwelling and provide higher wind resistance A PLACE FOR THE FORGOTTEN Figure 163: Perspective view showing multiple levels and the verticality of space suggested Figure 164: Perspective view showing possible sturctural components of the dwelling post and beam Figure 164: Perspective view showing possible sturctural components of the dwelling post and beam Figure 164: Perspective view showing possible sturctural components of the dwelling post and beam : Perspective view showing possible sturctural components of the dwelling post and beam Figure 164: Figure 163: Perspective view showing multiple levels and the verticality of space suggested Figure 163: Perspective view showing multiple levels and the verticality of space suggested Perspective view showing multiple levels and the verticality o Figure 163: 174. 175. Figure 166: Perspective view showing a high density typology for overcrowded conditions and protection Figure 165: Author model, aerial view of the proposed typology, displaying the roof as a domi- nant architectural feature much like vernacular examples Figure 165: Author model, aerial view of the proposed typology, displaying the roof as a domi- nant architectural feature much like vernacular examples Figure 166: Perspective view showing a high density typology for overcrowded conditions and protection 177. 176. Figure 167: Historic photo of Traditional dwellings Funafuti islet Figure 167: Historic photo of Traditional dwellings Funafuti islet Figure 169: Dwelling development appling a framework for growth, order and evolution Figure 171: Dwelling development appling a framework for flotation water storage and privacy to neigbouring units Figure 170: Dwelling perferated screen are developed much like traditional facades allowing constant but subtle airflow. Awnings are able to be pulled up to provide shade and more vigous airflow A place for the forgotten; a narrative extract set in 2100 from my design journal. by abundant ‘Pulaka’ leaves and various vegetables at each level. People could be seen almost at the top collecting food from various beds. The boys heard a voice, ‘You boys after some fish for your mother? Uncle Pau said. ‘Uncle! Where did you come from!, I just paddled back from fishing on the distant side of the lagoon, I’ll make you a deal you boys get me a couple of roots from the top and ill consider trading for two fish?. Tuame shouted to Motomoto ‘race you to the top, and the boys took off to complete th trade. ‘As the breeze blew a salty spray across the face of Tuame, who looked back towards the once abun- dant landmass of Funafuti islet. He shouted to Motomoto who had just jumped off the roof of the house ‘Do you think we will ever return to the is- land? Motomoto, I don’t know Tuame, but we must try to safe guard the sovereignty of our culture and land. This rig that we now call home allows the po- tential for our island to regenerate… Uncle Pau laughed, enjoying watching the boys race. Some of the elders were rotating vegetable trays to the top of the rig for maturing as the boys raced past almost knocking them over. Tuame was the first there ripping a bunch out of the soil bed trailing dirt behind. Motomoto wasn’t far behind but still not enough to win. Clutching the plants above his head, he cannon balled into the water next to his uncle. His head popped out of the wa- ter and the submerged taro. Motomoto said ‘I won Uncle! Taume reached the pontoon shortly after puffing from the remaining run. Well done boys here are your fish, tell your mother I’ll be over at the community hub if she needs anything else be- fore tea (Dylan Majurey 2015) All right, come on, lets go get some Pulaka to have with dinner, Motomoto pulled himself up out of the water and onto the pontoon and shook out his hair at Tuame, Tuame, eh! stop that, the boys gig- gled. Tuame began to walk along the suspended walkway leading the boys towards the communal garden with coconut trees planted down the cen- tre with soil obtained from the island. A planter bed with just enough from for a singular tree. DWELLING This section responds to prior experiments that addressed the urban fabric, zone of habitation and the active environment,. It examines how architec- ture can be culturally rich and that development accounts for a lengthened period to continue in- habiting sovereign land.The Ability to expand and develop organically to cope for the most at risk in- habitants and future overcrowding. A speculative approach to a new dwelling frame- work that provokes interpretation for traditional dwelling characteristics that have been developed over the iterative process. Displayed is the re- sponse to this process, a vertically compact typol- ogy that is based on the vernacular characteristic with the traditional imposing roof structure as the largest architectural feature (see fig. 165 & 166). Maintaining a connection to the derived design principles and the vernacular themes allows archi- tecture to evoke sovereign qualities of the culture represented in incremental development. Figure 167: Historic photo of Traditional dwellings Funafuti islet 7: Historic photo of Traditional dwellings Funafuti islet 178. 179. 179. Figure 168: Dwelling typology development showing the various levels and verticality of space distribution Figure 168: 180. 181. 182. Figure 170: Dwelling perferated screen are developed much like traditional facades allowing constant but subtle airflow. Awnings are able to be pulled up to provide shade and more vigous airflow Figure 171: Dwelling development appling a framework for flotation water storage and privacy to neigbouring units Figure 171: Figure 170: Dwelling perferated screen are developed much like traditional facades allowing constant but subtle airflow. Awnings are able to be pulled up to provide shade and more vigous airflow 183. 182. Figure 172: Dwelling development appling a framework for flotation experiments 185. Figure 173: Design development looking at how the dwelling can be placed onto 22,000L water tanks with the selected sectional development and structure required Figure 172: Dwelling development appling a framework for flotation experiments Dwelling development appling a framework for flotation experime Design development looking at how the dwelling can be placed onto 22,000L water tanks with the selected sectional development and structure required Figure 173: 184. 185. NARRATIVE EXTRACT A place for the forgotten; a narrative extract set in 2100 from my design journal. Figure 174: Author Diagram displaying the site context for settlement development A place for the forgotten; a narrative extract set in 2100 from my design journal. As they reached the end of the walkway the two boys jumped down to the floating pontoons that sur- round the communal gardens ( marine access), splashing water up onto the platform as the boys landed together. Looking up the sun was blocked 186. 187. 188. WATER PLANE SAND AND CORAL SUBSTRATA BASE ZONE FOR HABITATION Figure 174: Author Diagram displaying the site context for settlement development Figure 175: Author Diagram displaying the site context for settlement development Figure 177: Author Plan level three of rig Figure 178: Author Site plan showing complete rig Figure 178: Figure 179: Author Main level plan of community centre and emergency housing Figure 180: Author section BB showing the internal make up of the community Hub Figure 183: Author Plan Level one, dwelling unit Figure 183: Author Plan L Author Plan Level one, dwelling unit Figure 183: 188. WATER PLANE SAND AND CORAL SUBSTRATA BASE ZONE FOR HABITATION Figure 174: Author Diagram displaying the site context for settlement development WATER PLANE SAND AND CORAL SUBSTRATA BA ZONE FOR HABITATION SAND AND CORAL SUBSTRATA BASE ZONE FOR HABITATION 189. 188. 191. 190. BB - 50M 0 HOUSING COMMUNITY GARDEN COMMUNITY GARDEN MMUNITY HUB/ EMER- NCY FACILITIES EMERGENCY HOUSING & COMMUNITY SPACE Figure 176: Author Plan level Two of rig 0 L2 PLAN COMMUNITY GARDEN COMMUNITY HUB/ EMER- GENCY FACILITIES EMERGENCY HOUSING & COMMUNITY SPACE Author Plan level Two of rig 193. 192. L3 PLAN HOUSING COMMUNITY GARDEN COMMUNITY HUB/ EMER- GENCY FACILITIES 50M 0 0 50M 50M L3 PLAN COMMUNITY HUB/ EMER- GENCY FACILITIES COMMUNITY HUB/ EMER- GENCY FACILITIES Figure 177: Author Plan level three of rig 195. 194. Figure 178: 197. 196. ACTIVITY SPACE STAIR UNITS WALKWAY/HALL EMERGANCY DWELLING UNITS AND SOTRAGE WC CHURCH CONGRESSIONAL SEATING 5M 10M 0 Figure 179: Author Main level plan of community centre and emergency housing CHURCH CONGRESSIONAL SEATING EMERGANCY DWELLING UNITS AND SOTRAGE WALKWAY/HALL Figure 179: Author Main level plan of community centre and emergency housing 199. 198. 5M 10M 0 Figure 180: Author section BB showing the internal make up of the community Hub 201. 200. 5M 10M 0 Figure 182: Author Transverse section of the proposed dwellings SECTION AA 5M 10M 0 Figure 181: Author section AA showing the internal make up in the transverse direction, circu- lation and air flow SECTION AA 10M Figure 182: Author Transverse section of the proposed dwellings Figure 181: Author section AA showing the internal make up in the transverse direction, circu- lation and air flow 202. 203. Floating deck Stair 3 Stair 1 Storage Area Stair 2 Light Well Wash area W/C AA AA BB BB BB Stair 3 Storage Area Floating deck Light Well Wash area Stair 1 W/C BB 205. 204. Floating deck Stair Stair Kitchen Area Stair Light Well B1 B2 AA AA BB BB Figure 184: Author Plan Level Two of the proposed dwelling unit BB Floating deck BB BB BB Figure 184: Author Plan Level Two of the proposed dwelling unit Figure 184: Author Plan Level Two of the proposed dwelling unit 207. 206. Figure 185: Author section of multiple dwelling unit suggesting a framework for evolution and habitation Figure 191: Author Internal perspective, looking down through the centre of the community hub, church congressional seating on left Figure 190: Author exterior perspective, looking at the vegtable hub and marine connection Figure 192: Author exterior perspective, looking down the proposed walkways of the design, planter beds for coconut trees line the centre of the spine Figure 193: Author External perspective, looking out towards the lagoon and dwelling unit pro- tuding into view 217. Figure 193: Author External perspective, looking out towards the lagoon and dwelling unit pro- tuding into view 188. WATER PLANE SAND AND CORAL SUBSTRATA BASE ZONE FOR HABITATION Figure 174: Author Diagram displaying the site context for settlement development 5M 10M 0 < LEVEL TWO (SLEEPING & COOKING) < LEVEL THREE (SLEEPING) < LEVEL ONE (MIXED USE SPACE & WC 5M 10M 0 < LEVEL TWO (SLEEPING & COOKING) < LEVEL THREE (SLEEPING) < LEVEL ONE (MIXED USE SPACE & WC Figure 185: Author section of multiple dwelling unit suggesting a framework for evolution and habitation LEVEL THREE (SLEEPING) WO (SLEEPING & COOKING) LEVEL TWO (SLEEPING & COOKING) < LEVEL ONE Figure 185: Author section of multiple dwelling unit suggesting a framework for evolution and habitation 209. 208. Figure 186: Author section, circulation, temporal dwelling units and the integration of coconut plantation, structure and architecture 211. + ACCRETION ON STEEL AFTER FIRST NIGHT OF BIOROCK + ACCRETION ON STEEL AFTER 21 DAYS OF BIOROCK + PHOTO: R.INGEBO + PHOTO: TPS + ACCRETION ON STEEL AFTER FIRST NIGHT OF BIOROCK + ACCRETION ON STEEL AFTER 21 DAYS OF BIOROCK + PHOTO: R.INGEBO GROWTH SELF-REPAIR + - + - + - GROWTH RATE = 5CM PER YEAR 12 X 12M GRID = 3WATTS PER SQUARE METRE -ENERGY SOURCE SOLAR PANELS BIOROCK Figure 187: Author Transverse section of the community centre displaying circulation in red and floatation on watertank constructed platforms Figure 186: Author section, circulation, temporal dwelling units and the integration of coconut plantation, structure and architecture Figure 187: Author Transverse section of the community centre displaying circulation in red and floatation on watertank constructed platforms 210. 211. 212. Figure 188: Author section displaying circulation through the community hub and also the church terraced seating within the centre of the complex, and outdoor mixed use space 213. Figure 189: Author orthograhpic section diagraming the vegtable hub design, circulation and garden bed construction Figure 189: Author orthograhpic section diagraming the vegtable hub design, circulation and garden bed construction Figure 189: Author orthograhpic section diagraming the vegtable hub design, circulation and garden bed construction re 188: Author section displaying circulation through the community hub and also the church terraced seating within the centre of the complex, and outdoor mixed use space 212. 213. 214. 215. 217. 216. Figure 194: Author exterior perspective looking at the entire rig from flooded shoreline supple- menting the land lossed to provide a sense of soveraignty with a changing context by incorporating the ‘material object’ (Vernacular) 219. 218. FINAL PRODUCT This thesis produces links to the vernacular tec- tonic producing speculative outcomes that directs attention to the loss of sovereignty and landmass that low lying atolls face, due to a rising sea level and climatic change impacts. futi atoll. The dilemma of Tuvalu’s situation is that migra- tion as seen as the only form of option for many of these low lying atoll communities. No effort has been placed in the development of habitation for future generations because without it there is no defined territory therefore no sovereignty or rights to culture and land. In Tuvalu’s case a loss of the Tuvaluan way of life will occur if they are forced to leave, with nothing except the memories of a distant homeland. Tuvalu is an ever-changing complex ecosystem which struggles to maintain sovereignty against global sea level rise. At the outset a multi disciplinary approach was analysed for successful architectural characteris- tics and theoretical principles that would combat the issue of sovereignty. These principles of recon- struction, active environment, spatial activation, narrative , incrementalism and formalism have been applied to an iterative process which are a direct response to the aims and objectives of this thesis. Through a series of preliminary design ex- periments that iterate conceptual outcomes of how habitation can be developed to relieve pressure on the atoll environment. Whilst the uncertain phys- ical boundaries require the adaptation of percep- tual parameters to be reflective of the inhabitants. A transformation can occur in architecture from a ‘Culture-less’, edifice into one that is ‘Cultural- ly rich’. As such it has the ability to maintain cus- todianship over a scarred landscape allowing the user to occupy, re-connect and repair with their environment to escape the effects of sea level rise. Therefore the architectural solution must provide an extension to the indigenous culture by facili- tating engagement with this natural environment, predicted sea level rise and provide low-income dwellings for the ‘most at risk’ occupants of Funa- The dilemma of Tuvalu’s situation is that migra- tion as seen as the only form of option for many of these low lying atoll communities. No effort is placed in the development of habitation for future generations because without it there is no defined territory therefore no sovereignty or rights to cul- ture and land. In Tuvalu’s case a loss of the Tuvalu- an way of life will occur if they are forced to leave, with nothing except the memories of a distant homeland. Tuvalu is an ever-changing complex ecosystem in which struggles to maintain sover- eignty against global sea level rise. 221. REFLECTION 223 5M 10M 0 < 2055 - 2056 FLOAT CORAL STRUCTURE USING 10,000 WATER TANKS & PREPARE PIPING FOR FRESH WATER STORAGE < 2051 - 2055 WELD STEEL STRUCTURAL UNITS TO BE SUBMERGED & START MINERAL ACCRETION (2-3 years). < 2050 START LAYING FOUNDATION PILES ( 1 year) < 2056 - 2063 ABOVE SEA LEVEL CONSTRUCTION, ERECT WALKWAYS, FRAMING & ROOFS FOR EACH PROGRAM < 2063 - 2066 INTERNAL WORKS & FINISHES PHASE 1 > PHASE 2 > PHASE 3 > PHASE 4 > PHASE 5 > BUILDING WORK: Figure 195: Author Diagram showing possible temporal framework for construction phasing of dwellings 5M 10M 0 < 2055 - 2056 FLOAT CORAL STRUCTURE USING 10,000 WATER TANKS & PREPARE PIPING FOR FRESH WATER STORAGE < 2056 - 2063 ABOVE SEA LEVEL CONSTRUCTION, ERECT WALKWAYS, FRAMING & ROOFS FOR EACH PROGRAM < 2063 - 2066 INTERNAL WORKS & FINISHES PHASE 3 > PHASE 4 > PHASE 5 > BUILDING WORK: BUILDING WORK: drawings and their links to the theoretical research at the time that was less provocative. The following design methods are significant as they advanced the knowledge and reflection re- quired in addressing the concerns of this design project. It seeks to challenge and develop a con- temporary settlement framework that maintains sovereignty of Tuvalu through habitation and architectural characteristics. The proposal is to lengthen the period of habitation by reviving im- poverished areas that people can inhabit, develop lagoon based habitable typologies to mitigate sea level rise. The methods were selected to recognise and reinforce the cultural relationships of the Tu- valuan’s and their architecture. The concept design phase allowed a detailed solu- tion that is the ‘Hub for Habitation’,. This is placed within the established zone of habitation outlined by prior design research. The research objectives were met through the creation of a floatable set- tlement framework which allows continued habi- tation. The logic behind the design was developed from the preliminary stages taking a primordial step back by firstly responding to local conditions. The initial stage of design began by acknowledg- ing the issue of overcrowding and the degrading conditions of a contested site. This has produced a housing concept that is complex but it requires further refinement and testing to ensure it contin- ues to meet the thesis aims of maintaining the con- nection to sovereignty. Design Principles extracted 1. Radical reconstruction. 2. Active participant 3. REFLECTION Spatial activation 4. Narrative environment 5. Incrementalism (Time & process) 4. Narrative environment 5. Incrementalism (Time & process The concurrent design is a speculative formal re- sponse to the current impacts of sea level rise and the sovereign implications involved. By allowing the architecture to maintain a sense of nationhood within an inundated context through the use of design led research tools extracted throughout the process of this thesis. Due to the nature of its speculation its shortcomings are in regards to fea- sibility. Key architectural characteristics are as follows: Key architectural characteristics are as follows: Key architectural characteristics are as follows: 1. Simple construction 2. Ability to expand 3. Vernacular, locally-sourced materials 4. Traditional themes in form and function 5. Collective/community construction Key architectural characteristics are as follow 1. Simple construction 2. Ability to expand 3. Vernacular, locally-sourced materials 4. Traditional themes in form and function 5. Collective/community construction 1. Simple construction y 4. Traditional themes in form and function 5. Collective/community construction The intention is to maintain territory through speculative design, formal settlement and im- proved sanitary living conditions on the low-lying atoll. A theoretical basis looks to establish progres- sive tools in which designs could be tested and cri- tiqued to their success or demise. q Preliminary designs involved the mapping and drawing of distilled ideas relating to speculative outcomes of the vernacular tectonic and future habitation. The speculative concept that followed the vernacular analysis resulted in a less robust concept. This was due to the intuitive nature of the Figure 195: Author Diagram showing possible temporal framework for construction phasing of dwellings 222. 223. valuan’s’ challenges the permanence of culture that is attached to architecture and its ability to con- struct sovereign narratives for the informal settle- ments of Funafuti, Tuvalu. ‘a place for the forgotten’ provides an Architectural solution that transitions from slum conditions to constructed dwellings maintaining the sovereignty of their nation. There is potential for further development and an ex- panded scope within exploration into the architec- tural sovereignty through relocation or migration looking towards ruminative connections to their homeland. The formation of sovereignty and po- tential sovereign regeneration in distant lands may be a future challenge for the Tuvaluan’s themselves. Over a wider time frame and further engagement with onsite reflection I would look to develop a deeper connection to the narrative of the site by becoming an active participant and residing on the site. I would experience the climatic variations, lo- cal routines and the island lifestyle that is absent from the abstract viewpoint of literature. Therefore the development of a larger ‘opportunity for de- sign research’ would expand the speculative pro- cess undertaken within this thesis. To explore the temporal and modular requirements of such an isolated environment by working with the locals to test the feasibility of an on site housing project. However if the process becomes restrained by a ‘cost effective’ bias the ideas embodied have poten- tial to be overlooked. Which will result in a design process and outcome that is not reflective of the narrative vernacular tectonic or the inhabitants themselves. Habitation would enable the architect to extract elements to engage with the landscape, community and dwelling. A temporal framework could then be tested much more rigorously by us- ing this multidisciplinary approach and consulta- tion to remediate atoll growth. The thesis question of ‘How can architecture main- tain a sense of sovereignty within a disappearing context. And what are the implications of habita- tion, culture and contested territories for the Tu- 225. 224. Carlo Scarpa. “Can Architecture Be Poetry.” from Peter Nover, Ed. The Other City Carlo Scarpa: The Architect’s Working Method as Shown by the Brion Cemetery in San Vito D’Avitole. p17-18. Dovey, Kim, Mosley, Jonathan, and Sara, Rachel. “Informalising Architecture: The Challenge of Informal Settlements.” Architectural Design 83.6 (2013): 82-89. Web. 12 Feb. 2016 Faaniu, Simati, and Hugh Laracy. Tuvalu, a History. Suva, Fiji: Institute of Pacific Stud- ies and Extension Services, U of the South Pacific and the Ministry of Social Services, Government of Tuvalu, 1983. Print Fisher, Aled Dilwyn. “Climate Change and Indigenous Peoples: The Search for Legal Remedies.” Nordic Journal of Human Rights32.4 (2014): 403-05. Web. 16 Feb. 2016. Ganoe, Cathy J. “Design Narrative: A Theory of Inhabiting Interior Space.” Journal Of Interior Design 25.2 (1999): 1. Hartoonian, Gevork. “Kenneth Frampton, David Malouf and Juhani Pallasma, Glenn Murcutt, Architect.” Architectural Theory Review12.2 (2007): 212-16. Web. 12 August 2015. (IPCC, 2013) Church, J.A., P.U. Clark, A. Cazenave, J.M. Gregory, S. Jevrejeva, A. Lev- ermann, M.A. Merrifield, G.A. Milne, R.S. Nerem, P.D. Nunn, A.J. Payne, W.T. Pfeffer, D. Stammer and A.S. Unnikrishnan, 2013: Sea Level Change. In: Climate Change 2013: The Physical Science Basis. Contribution of Working Group I to the Fifth Assessment Report of the Intergovernmental Panel on Climate Change [Stocker, T.F., D. Qin, G.-K. Plattner, M. Tignor, S.K. Allen, J. Boschung, A. Nauels, Y. Xia, V. Bex and P.M. Midgley (eds.)]. Cambridge University Press, Cambridge, United Kingdom and New York, NY, USA. Jaschke, Karin. “Architecture as Artifice.” The Journal of Architecture 6.2 (2001): 135- 44. Web. 12 August 2015. Koch, Gerd., and University of the South Pacific. Institute of Pacific Studies. The Ma- terial Culture of Tuvalu / by Gerd Koch. Suva, Fiji: Institute of Pacific Studies, U of the South Pacific, 1984. Print. “Let Nature Do Her Work: The Adaptation Palettes of Qiaoyuan Wetland Park Tianjin, China, 2008.” Designed Ecologies The Landscape Architecture of Kongjian Yu (n.d.): n. pag. Web. 12 Feb. 2016. <http://landscapeperformance.org/sites/default/files/Qiaoyu- an%20Park%20Methodology.pdf>. 227. Lynch, P. (2008). Glenn murcutt. The Architects’ Journal, 227(2), 46-47. Web. 12 Feb 2016. 12 Feb. 2016. “Vienna Declaration and Programme of Action.” Verfassung Und Recht in Übersee / Law and Politics in Africa Asia and Latin America.Vol. 27, No. 1 (1994): 129-54. Web. 13 Jan. 2016. McCarthy, James J. Climate Change 2001: Impacts, Adaptation, and Vulnerability: Con- tribution of Working Group II to the Third Assessment Report of the Intergovernmen- tal Panel on Climate Change. Cambridge: Cambridge UP, 2001. Print. Woods, Lebbeus, Clare Jacobson, and Aleksandra Wagner. Radical Reconstruction. New York, NY: Princeton Architectural, 1997. Print. Mcnamara, and Gibson. “‘We Do Not Want to Leave Our Land’: Pacific Ambassadors at the United Nations Resist the Category of ‘climate Refugees’.” Geoforum 40.3 (2009): 475-83. Web. Murcutt, Glenn. Glenn Murcutt, Architect. Collector’s ed. Woods, Lebbeus, Clare Jacobson, and Aleksandra Wagner. Radical Reconstruction. New York, NY: Princeton Architectural, 1997. Print. SOURCES OF FIGURES 6.0 Fig. 20: Andrew, Thomas. Web. 12 Feb. 2016 <http://collections.tepapa.govt.nz/search?searchTerm=tuvalu&scope=all&state=(op:(e nd:204,start:193))> Fig. 25: Carlin, Jocelyn. Web. 12 Feb. 2016 <http://pacificvoyagers.org/will-pacific-island-nations-disappear-as-seas-rise-maybe- not/> Fig. 35. Carlin, Jocelyn. Web. 12 Feb. 2016 <http://www.carlin.co.nz/gallery.php?gid=24&ds=1> Fig. 35. Carlin, Jocelyn. Web. 12 Feb. 2016 <http://www.carlin.co.nz/gallery.php?gid=24&ds=1> Fig. 36. Carlin, Jocelyn. Web. 12 Feb. 2016 <http://pacificvoyagers.org/will-pacific-island-nations-disappear-as-seas-rise-maybe- not/> Fig. 37. Carlin, Jocelyn. Web. 12 Feb. 2016 <http://www.carlin.co.nz/gallery.php?gid=24&ds=1> Fig. 37. Carlin, Jocelyn. Web. 12 Feb. 2016 <http://www.carlin.co.nz/gallery.php?gid=24&ds=1> Fig. 39. Hobgood, Nick.Web. 12 Feb 2016 <https://www.flickr.com/photos/globalvoy- ager/10896961643/in/photolist> Fig. 41.Dekker, Rodney. Web 12 Feb 2016. Fig. 41.Dekker, Rodney. Web 12 Feb 2016. <www.rodneydekker.com> Rozelle, NSW: 01 Editions Pty Ltd, 2006. Print. Vol. 8 of 8 (Arthur and Yvonne Boyd Education Centre, Riversdale, 1999). Ralston, Holley, Britta Horstmann, and Carina Holl. “Climate Change.”Challenges Tu- valu (2004): 1-19. Web. 12 Nov. 2015. Riise, and Adeyemi. “Case Study: Makoko Floating School.”Current Opinion in Envi- ronmental Sustainability 13 (2015): 58-60. Web. y ( ) Thaman, John Campbell, Joeli Veitayaki, Thomas Giambelluca, Salesa Nihmei, Etika Rupeni, Lucille Apis-Overhoff, William Aalbersberg, and Dan F. Orcherton. “Small Is- lands, Valuable Insights: Systems of Customary Resource Use and Resilience to Climate Change in the Pacific.” Ecology and Society 19.4 (2014): 44. Web. ROBERTS, R. G.. “TE ATU TUVALU: A Short History of the Ellice Islands”. The Jour- nal of the Polynesian Society 67.4 (1958): 394–423. Print. Saunders, William S., ed. Birkhäuser Generalstandingorder : Designed Ecologies : The Landscape Architecture of Kongjian Yu. Basel/Berlin/Boston, DE: Birkhäuser, 2013. ProQuest ebrary. Web. 14 June 2016. Schalk, Meike. “The Architecture Of Metabolism. Inventing A Culture Of Resilience”. Arts 3.2 Schalk, Meike. “The Architecture Of Metabolism. Inventing A Culture Of Resilience”. Arts 3.2 (2014): 279-297. Web. 18 Oct. 2015. Secretariat of the Pacific Community. 2005. Tuvalu 2002: Population and Housing Cen- sus. Volume 1. Analytical report. Noumea, New Caledonia: Secretariat of the Pacific Community. Web. 6 JAN. 2016. Secretariat of the Pacific Community. 2005. Tuvalu 2002: Population and Housing Cen- sus. Volume 1. Analytical report. Noumea, New Caledonia: Secretariat of the Pacific Community. Web. 6 JAN. 2016. The United Nation’s Convention on the Law of the Sea (Treaty Doc. 103-39): Hearings before the Committee on Foreign Relations, United States Senate, One Hundred Tenth Congress, First Session, September 27 and October 4, 2007. Washington: U.S. G.P.O., 2008. Print. The United Nation’s Convention on the Law of the Sea (Treaty Doc. 103-39): Hearings before the Committee on Foreign Relations, United States Senate, One Hundred Tenth Congress, First Session, September 27 and October 4, 2007. Washington: U.S. G.P.O., 2008. Print. Tzonis, and White. “Introduction.” Automation in Construction 2.1 (1993): Ix-Xii. Web. Tzonis, and White. “Introduction.” Automation in Construction 2.1 (1993): Ix-Xii. Web. 229. 228. g , y <https://www.flickr.com/photos/unitednationsdevelopmentprogramme/17019489907/ in/photolist-rVXkpa> Fig.8: Soeparyono, Astri. Web. 12 Feb 2016. <http://kawankumagz.com/Feature/Playground/Pulau-Pulau-Yang-Terancam-Teng- gelam> g p y <http://kawankumagz.com/Feature/Playground/Pulau-Pulau-Yang-Terancam-Teng- gelam> Fig.11 Hobgood, Nick.Web. 12 Feb 2016 <https://www.flickr.com/photos/globalvoyag- er/10896961643/in/photolist> er/10896961643/in/photolist> Fig. 16: Andrew, Thomas. Web. 12 Feb. 2016 <http://collections.tepapa.govt.nz/search?searchTerm=tuvalu&scope=all&state=(op:(e nd:204,start:193))> Fig. 17: Fasset, Clifford Harry. Web. 12 Feb 2016 <https://en.wikipedia.org/wiki/Women_in_Tuvalu> Fig. 18: Barret, Bill. Web 12 Feb 2016. <http://www.tuvaluislands.com/ww2/ww2-frag-ss.htm> Fig. 19: Barret, Bill. Web 12 Feb 2016. <http://www.tuvaluislands.com/ww2/ww2-frag-ss.htm> Fig. 20: Andrew, Thomas. Web. 12 Feb. 2016 <http://collections.tepapa.govt.nz/search?searchTerm=tuvalu&scope=all&state=(op:(e nd:204,start:193))> Fig. 25: Carlin, Jocelyn. Web. 12 Feb. 2016 <http://pacificvoyagers.org/will-pacific-island-nations-disappear-as-seas-rise-maybe- not/> Fig. 35. Carlin, Jocelyn. Web. 12 Feb. 2016 <http://www.carlin.co.nz/gallery.php?gid=24&ds=1> Fig. 36. Carlin, Jocelyn. Web. 12 Feb. 2016 <http://pacificvoyagers.org/will-pacific-island-nations-disappear-as-seas-rise-maybe- not/> Fig. 37. Carlin, Jocelyn. Web. 12 Feb. 2016 <http://www.carlin.co.nz/gallery.php?gid=24&ds=1> Fig. 39. Hobgood, Nick.Web. 12 Feb 2016 <https://www.flickr.com/photos/globalvoy- ager/10896961643/in/photolist> Fig. 41.Dekker, Rodney. Web 12 Feb 2016. <www.rodneydekker.com> g ,h <http://collections.tepapa.govt.nz/search?searchTerm=tuvalu&scope=all&state=(op:(e nd:204,start:193))>f gf y <https://en.wikipedia.org/wiki/Women_in_Tuvalu> 231. >http://www.archdaily.com/10775/quinta-monroy-elemental/50102e- 3228ba0d4222001003-quinta-monroy-elemental-image> Fig. 47. Carlin, Jocelyn. Web. 12 Feb. 2016 <http://www.carlin.co.nz/gallery.php?gid=24&ds=1> Fig. 49. Hill, John. Web 12 Feb. 2016 <https://www.flickr.com/photos/archidose/13876945485> Fig. 50. Hill, John. Web 12 Feb. 2016 <https://www.flickr.com/photos/archidose/13876945484> Fig. 51. Hill, John. Web 12 Feb. 2016 <https://www.flickr.com/photos/archidose/13876945483> Fig. 52. Hill, John. Web 12 Feb. 2016 <https://www.flickr.com/photos/archidose/13876945485> Fig. 53. Browell, Anthonyii Fig. 47. Carlin, Jocelyn. Web. 12 Feb. 2016 Fig. 47. Carlin, Jocelyn. Web. 12 Feb. 2016 <http://www.carlin.co.nz/gallery.php?gid=24&ds=1> g y <http://www.carlin.co.nz/gallery.php?gid=24&ds=1> Fig.61. Jalocha , Tadeuz Fig.61. Jalocha , Tadeuz Fig. 49. Hill, John. Web 12 Feb. 2016 <https://www.flickr.com/photos/archidose/13876945485> g J Web. 12 Feb 2016. g Web. 12 Feb 2016. <http://www.archdaily.com/10775/quinta-monroy-elemental> <http://www.archdaily.com/10775/quinta-monroy-elemental> <http://www.archdaily.com/10775/quinta-monroy-elemental> Fig. 63. NLE Architects. Web. 12 Feb 2016. <http://www.archdaily.com/344047/makoko-floating-school-nle-architects> Fig. 63. NLE Architects. Web. 12 Feb 2016. Fig. 50. Hill, John. Web 12 Feb. 2016 <https://www.flickr.com/photos/archidose/13876945484> <https://www.flickr.com/photos/archidose/13876945484> <http://www.archdaily.com/344047/makoko-floating-school-nle-architects> Fig. 51. Hill, John. Web 12 Feb. 2016 <https://www.flickr.com/photos/archidose/13876945483> Fig. 64. NLE Architects. Web. 12 Feb 2016. <http://www.archdaily.com/344047/makoko-floating-school-nle-architects> Fig. 65. NLE Architects. Web. 12 Feb 2016. <http://www.archdaily.com/344047/makoko-floating-school-nle-architects> Fig. 52. Hill, John. Web 12 Feb. 2016 <https://www.flickr.com/photos/archidose/13876945485> Fig. 52. Hill, John. Web 12 Feb. 2016 <https://www.flickr.com/photos/archidose/13876945485> Fig. 66. NLE Architects. Web. 12 Feb 2016. <http://www.archdaily.com/344047/makoko-floating-school-nle-architects> g y <http://www.residentialarchitect.com/practice/firm-profiles/ra50-glenn-murcutt_o> <http://www.archdaily.com/344047/makoko-floating-school-nle-architects> Fig. 68. Anderson wise Architect . Web 12 Feb. 2016 <http://www.archdaily.com/479419/lake-house-andersson-wise-architects/5306911e- c07a80c45f00008d-lake-house-andersson-wise-architects-upper-floor-plan> Fig. 69. Bardagjy, Paul. Web 12 Feb 2016 http://www.archdaily.com/479419/lake-house-andersson-wise-architects Fig. 70. Bardagjy, Paul. Web 12 Feb 2016 <http://www.archdaily.com/479419/lake-house-andersson-wise-architects> Fig. 71. Bardagjy, Paul. Web 12 Feb 2016 -<http://www.archdaily.com/479419/lake-house-andersson-wise-architects/5306906f- c07a80c45f00008a-lake-house-anderss> - Fig. 74. Anderson wise Architects. Web 12 Feb. 2016 <http://www.archdaily.com/479419/lake-house-andersson-wise-architects/ 53069109c07a806b06000086-lake-house-andersson-wise-architects-section> Fig. 73. Anderson wise Architects. Web 12 Feb. 2016 <http://www.archdaily.com/479419/lake-house-andersson-wise-architects/ 53069106c07a80c45f00008c-lake-house-andersson-wise-architects-lower-floor-plan> Fig. 75. Turenscape. 11 Feb. 2016 <http://www.turenscape.com/english/projects/project.php?id=339> Fig. 76. Turenscape. 11 Feb. 2016 <http://www.turenscape.com/english/projects/project.php?id=339> Fig. 54. Lassen, Seyama. Web 12 Oct. 2015 Fig. 68. Anderson wise Architect . Web 12 Feb. 2016 <http://www.archdaily.com/479419/lake-house-andersson-wise-architects/5306911e- c07a80c45f00008d-lake-house-andersson-wise-architects-upper-floor-plan> g y <http://www.ozetecture.org/2012/marika-alderton-house/> g y <http://www.ozetecture.org/2012/marika-alderton-house/> Fig. 69. Bardagjy, Paul. Web 12 Feb 2016 http://www.archdaily.com/479419/lake-house-andersson-wise-architects g <https://www.google.co.nz/url?sa=i&rct=j&q=&esrc=s&source=im- a g e s & c d = & v e d = 0 a h U K E w i e 4 P O S y u L NA h V F p Z Q K H e g k D L A Q - j x w IAw & u r l = ht t p % 3 A % 2 F % 2 F w w w. e b c . c om . br % 2 Fn ot i c i a s % 2 F- brasil%2F2013%2F12%2Fbolsa-familia-quer-beneficiar-600-mil-brasile- iros-nao-atendidos-por&psig=AFQjCNGPzsAit0qkDHJ3XEqX9LL9at_ Dmg&ust=1468023044332956> Fig. 70. Bardagjy, Paul. 231. Web 12 Feb 2016 <http://www.archdaily.com/479419/lake-house-andersson-wise-architects> Fig. 71. Bardagjy, Paul. Web 12 Feb 2016 -<http://www.archdaily.com/479419/lake-house-andersson-wise-architects/5306906f- c07a80c45f00008a-lake-house-anderss> Fig. 71. Bardagjy, Paul. Web 12 Feb 2016 -<http://www.archdaily.com/479419/lake-house-andersson-wise-architects/5306906f- c07a80c45f00008a-lake-house-anderss> Fig. 56. Schwarz, Stefanie. Web 12 Jan. 2016 <https://www.google.co.nz/url?sa=i&rct=j&q=&esrc=s&source=im- a g e s & c d = & v e d = 0 a h U K E w i e 4 P O S y u L NA h V F p Z Q K H e g k D L A Q - j x w IAw & u r l = ht t p % 3 A % 2 F % 2 F w w w. e b c . c om . br % 2 Fn ot i c i a s % 2 F- brasil%2F2013%2F12%2Fbolsa-familia-quer-beneficiar-600-mil-brasile- iros-nao-atendidos-por&psig=AFQjCNGPzsAit0qkDHJ3XEqX9LL9at_ Dmg&ust=1468023044332956> Fig. 74. Anderson wise Architects. Web 12 Feb. 2016 <http://www.archdaily.com/479419/lake-house-andersson-wise-architects/ 53069109c07a806b06000086-lake-house-andersson-wise-architects-section> Fig. 58. Palma, Cristobal Web. 12 Feb 2016. Fig. 58. Palma, Cristobal Web. 12 Feb 2016. Fig. 58. Palma, Cristobal Web. 12 Feb 2016. g. 58. a a, C stoba Web. eb 0 6. <http://www.archdaily.com/10775/quinta-monroy-elemental> Fig. 59. Palma, Cristobal Web. 12 Feb 2016. Fig. 59. Palma, Cristobal Web. 12 Feb 2016. <http://www.archdaily.com/10775/quinta-monroy-elemental/50102dd- 828ba0d4222000ff3 quinta monroy elemental image> Fig. 60. Palma, Cristobal Web. 12 Feb 2016. Fig. 60. Palma, Cristobal Web. 12 Feb 2016. 232. 233. Fig. 77. Turenscape. 11 Feb. 2016 <http://www.turenscape.com/english/projects/project.php?id=339> Fig. 110. Andrew, Thomas. Web. 15 Jan 2016. gh <http://collections.tepapa.govt.nz/search?searchTerm=tuvalu&scope=all&state=(op:(e nd:204,start:193))> h <http://collections.tepapa.govt.nz/search?searchTerm=tuvalu&scope=all&state=(op:(e nd:204,start:193))> Fig. 78. Turenscape. 12 Feb. 2016 <http://www.turenscape.com/english/projects/project.php?id=339> Fig. 114. Koch, Gerd., and University of the South Pacific. Institute of Pacific Studies. The Material Culture of Tuvalu / by Gerd Koch. Suva, Fiji: Institute of Pacific Studies, U of the South Pacific, 1984. Print. Fig. 79. Turenscape. 12 Feb. 2016 <http://www.turenscape.com/english/projects/project.php?id=339> Fig. 115. United Nations Development. Web. 12 November 2015. <https://www.flickr.com/photos/unitednationsdevelopmentprogramme/17040728649/ in/photolist> Fig. 115. United Nations Development. Web. 12 November 2015. <https://www.flickr.com/photos/unitednationsdevelopmentprogramme/17040728649/ in/photolist> Fig. 80. Turenscape. 12 Feb. 2016 <http://www.turenscape.com/english/projects/project.php?id=339> <https://www.flickr.com/photos/unitednationsdevelopmentprogramme/17040728649/ in/photolist> Fig. 81.Turenscape. 12 Feb. 2016 <http://www.turenscape.com/english/projects/project.php?id=339> Fig. 116. United Nations Development. Web.. 12 November 2015. <https://www.flickr.com/photos/unitednationsdevelopmentprogramme/17040728649/ in/photolist> Fig. 85. New Zealand Ministry of Foreign Affairs and Trade. 15 Jan. 2016 Fig. 85. New Zealand Ministry of Foreign Affairs and Trade. 15 Jan. 2016 <-https://mfatimages.smugmug.com/CompleteLibrary/Approved-Images/i-bmhCp- cP> Fig. 167. Andrew, Thomas. Web. 15 Jan 2016. <http://collections.tepapa.govt.nz/search?searchTerm=tuvalu&scope=all&state=(op:(e nd:204,start:193))> Fig. 167. Andrew, Thomas. Web. 15 Jan 2016. f <-https://mfatimages.smugmug.com/CompleteLibrary/Approved-Images/i-bmhCp- cP> gh J <http://collections.tepapa.govt.nz/search?searchTerm=tuvalu&scope=all&state=(op:(e nd:204,start:193))> Fig. 94. Lebedev, Artemy. 15 Jan. 2016 <http://www.tema.ru/travel/tuvalu/> Fig. 102. Royal Society Coral Reef committee. 231. 15 Jan. 2016 <https://www.flickr.com/photos/internetarchivebookimages/14765813295/in/pho- tolist> g y y J <https://www.flickr.com/photos/internetarchivebookimages/14765813295/in/pho- tolist> Fig. 104. Koch, Gerd., and University of the South Pacific. Institute of Pacific Studies. The Material Culture of Tuvalu / by Gerd Koch. Suva, Fiji: Institute of Pacific Studies, U of the South Pacific, 1984. Print. Fig. 105. Koch, Gerd., and University of the South Pacific. Institute of Pacific Studies. The Material Culture of Tuvalu / by Gerd Koch. Suva, Fiji: Institute of Pacific Studies, U of the South Pacific, 1984. Print. Fig. 106. Koch, Gerd., and University of the South Pacific. Institute of Pacific Studies. The Material Culture of Tuvalu / by Gerd Koch. Suva, Fiji: Institute of Pacific Studies, U of the South Pacific, 1984. Print. Fig. 108. Andrew, Thomas. Web. 15 Jan 2016. <http://collections.tepapa.govt.nz/search?searchTerm=tuvalu&scope=all&state=(op:(e nd:204,start:193))> Fig. 109. Koch, Gerd., and University of the South Pacific. Institute of Pacific Studies. The Material Culture of Tuvalu / by Gerd Koch. Suva, Fiji: Institute of Pacific Studies, U of the South Pacific, 1984. Print. Fig. 109. Koch, Gerd., and University of the South Pacific. Institute of Pacific Studies. The Material Culture of Tuvalu / by Gerd Koch. Suva, Fiji: Institute of Pacific Studies, U of the South Pacific, 1984. Print. 235. 234.
https://openalex.org/W1496807543	https://europepmc.org/articles/pmc4419676?pdf=render	English	null	NAC-MYB-based transcriptional regulation of secondary cell wall biosynthesis in land plants	Frontiers in plant science	2,015	cc-by	17,587	NAC-MYB-based transcriptional regulation of secondary cell wall biosynthesis in land plants Yoshimi Nakano 1, Masatoshi Yamaguchi 2, 3, Hitoshi Endo 1, Nur Ardiyana Rejab 1, 4 and Misato Ohtani 1, 5* 1 Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, Japan, 2 Division of Strategic Research and Development, Graduate School of Science and Engineering, Saitama University, Saitama, Japan, 3 PRESTO (Precursory Research for Embryonic Science and Technology), Japan Science and Technology Agency, Kawaguchi, Japan, 4 Faculty of Science, Institute of Biological Sciences, University of Malaya, Kuala Lumpur, Malaysia, 5 Biomass Engineering Program Cooperation Division, RIKEN Center for Sustainable Resource Science, Yokohama, Japan Plant cells biosynthesize primary cell walls (PCW) in all cells and produce secondary cell walls (SCWs) in speciﬁc cell types that conduct water and/or provide mechanical support, such as xylem vessels and ﬁbers. The characteristic mechanical stiffness, chemical recalcitrance, and hydrophobic nature of SCWs result from the organization of SCW-speciﬁc biopolymers, i.e., highly ordered cellulose, hemicellulose, and lignin. Synthesis of these SCW-speciﬁc biopolymers requires SCW-speciﬁc enzymes that are regulated by SCW-speciﬁc transcription factors. In this review, we summarize our current knowledge of the transcriptional regulation of SCW formation in plant cells. Advances in research on SCW biosynthesis during the past decade have expanded our understanding of the transcriptional regulation of SCW formation, particularly the functions of the NAC and MYB transcription factors. Focusing on the NAC-MYB-based transcriptional network, we discuss the regulatory systems that evolved in land plants to modify the cell wall to serve as a key component of structures that conduct water and provide mechanical support. Edited by: Masaru Fujimoto, The University of Tokyo, Japan e U e s ty o o yo, Japa Reviewed by: Nobutaka Mitsuda, National Institute of Advanced Industrial Science and Technology, Japan Jacqueline Grima-Pettenati, Centre National Recherche Scientiﬁque, France Reviewed by: Nobutaka Mitsuda, National Institute of Advanced Industrial Science and Technology, Japan Jacqueline Grima-Pettenati, Centre National Recherche Scientiﬁque, France *Correspondence: Misato Ohtani, Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama-cho, Ikoma 630-0192, Japan misato@bs.naist.jp Keywords: land plant evolution, MYB transcription factor, NAC transcription factor, secondary cell wall, netwo misato@bs.naist.jp Introduction Specialty section: This article was submitted to Plant Physiology, a section of the journal Frontiers in Plant Science Received: 14 February 2015 Accepted: 09 April 2015 Published: 05 May 2015 Specialty section: This article was submitted to Plant Physiology, a section of the journal Frontiers in Plant Science The cell wall, a characteristic feature of plant cells, consists of biopolymers, such as polysaccha- rides, phenolic compounds, and various proteins, which impart mechanical strength and rigidity. The structure of the cell wall determines the characteristics of plant cells, thus directly aﬀecting organ development and responses to environmental stimuli (Hamant and Traas, 2010; Wolf et al., 2012). Received: 14 February 2015 Accepted: 09 April 2015 Published: 05 May 2015 Plant cells have two types of cell wall, primary cell wall (PCW) and secondary cell wall (SCW), based on their biosynthetic composition and cellular location (Figure 1). Every plant cell has a PCW, a relatively thin and extensible wall that the cell synthesizes during cell division. The force generated by the PCW functions as a critical regulator of cell elongation and expansion; thus, PCW biosynthesis fundamentally conditions the shape and size of cells (Geitmann, 2010; Hamant and Traas, 2010). By contrast, the relatively thick and rigid SCW forms in speciﬁc types of cells, such as xylem cells, and cells of valve margin and anther endothecium. The cell produces the SCW between the PCW and the plasma membrane during cell diﬀerentiation, and the SCW imparts additional REVIEW published: 05 May 2015 doi: 10.3389/fpls.2015.00288 Edited by: Edited by: Masaru Fujimoto, The University of Tokyo, Japan Citation: (B) Model of the secondary cell wall, which is deposited between the primary cell wall and the plasma membrane. The secondary cell wall mainly contains relatively long and thick cellulose microﬁbrils, hemicellulosic xylan, and lignin. (C) Cross section of an Arabidopsis inﬂorescence stem stained with Safranin, which stains lignin red, and Astra blue. co, cortex; ep, epidermis; if, interfascicular ﬁber; xv, xylem vessel. Bar = 50 µm. mechanical stiﬀness and/or hydrophobicity to the cell (Cosgrove and Jarvis, 2012). Cellulose, also called (1,4)-β-D-glucan, contains >500 β-D-glucose residues polymerized with glycosidic bonds into a chain; cellulose microﬁbrils contain ∼40 cellulose chains formed into bundles. Cellulose constitutes the main component of the PCW and SCW, but the cellulose of the PCW and SCW shows key structural diﬀerences. In the PCW, cellulose has a rela- tively low degree of polymerization (e.g., 2000–6000 β-D-glucose residues in cotton, Gossypium hirsutum) and microﬁbril widths of 2–2.5 nm. By contrast, in the SCW, cellulose has a high degree of polymerization (e.g., 13,000 β-D-glucose residues in cotton and approximately 8000 in wood; Marx-Figini, 1969) and microﬁbril widths of 5–10 nm (Heyn, 1955, 1965, 1966). Other components of the cell wall also diﬀer between the PCW and SCW. For exam- ple, the PCW typically contains xyloglucan as the major hemi- cellulose (i.e., the polysaccharide component that is soluble in alkali), but the SCW contains xylan. In addition, the PCW is rich in gel-like pectin, whereas lignin and speciﬁc phenolic polymers are more abundant in the SCW (Figure 1). These diﬀerences in cell wall composition impart diﬀerent physical properties to the cell wall, rendering the PCW ﬂexible and the SCW mechanically and biologically robust. mechanical stiﬀness and/or hydrophobicity to the cell (Cosgrove and Jarvis, 2012). Cellulose, also called (1,4)-β-D-glucan, contains >500 β-D-glucose residues polymerized with glycosidic bonds into a chain; cellulose microﬁbrils contain ∼40 cellulose chains formed into bundles. Cellulose constitutes the main component of the PCW and SCW, but the cellulose of the PCW and SCW shows key structural diﬀerences. In the PCW, cellulose has a rela- tively low degree of polymerization (e.g., 2000–6000 β-D-glucose residues in cotton, Gossypium hirsutum) and microﬁbril widths of 2–2.5 nm. By contrast, in the SCW, cellulose has a high degree of polymerization (e.g., 13,000 β-D-glucose residues in cotton and approximately 8000 in wood; Marx-Figini, 1969) and microﬁbril widths of 5–10 nm (Heyn, 1955, 1965, 1966). Citation: Cellulose microﬁbrils in the primary cell wall are relatively short and thin, compared with those in the secondary cell wall, and hemicellulose in the primary cell wall is composed of xyloglucan. The primary cell wall is rich in pectin. (B) Model of the secondary cell wall, which is deposited between the primary cell wall and the plasma membrane. The secondary cell wall mainly contains relatively long and thick cellulose microﬁbrils, hemicellulosic xylan, and lignin. (C) Cross section of an Arabidopsis inﬂorescence stem stained with Safranin, which stains lignin red, and Astra blue. co, cortex; ep, epidermis; if, interfascicular ﬁber; xv, xylem vessel. Bar = 50 µm. In 2005, a milestone year for SCW biosynthesis research, multiple studies identiﬁed transcriptional regulators of SCW biosynthesis. For example, Kubo et al. used an in vitro cell culture system to identify the plant-speciﬁc NAM, ATAF1,2, and CUC2 (NAC) transcription factors VASCULAR-RELATED NAC-DOMAIN1-7 (VND1-7) as master regulators of xylem ves- sel cell diﬀerentiation (Kubo et al., 2005). Also, Mitsuda et al. reported that NAC SECONDARY WALL THICKENING PRO- MOTING FACTOR1 (NST1) and NST2, members of a sister group to the VNDs, regulate SCW formation in anther cells (Mitsuda et al., 2005). Subsequent work showed that NST1 and NST3 (also called SECONDARY WALL-ASSOCIATED NAC DOMAIN PROTEIN1 [SND1]) function as master switches of ﬁber cell diﬀerentiation in Arabidopsis (Zhong et al., 2006; Mit- suda et al., 2007). These ﬁndings revealed the regulation of SCW biosynthesis at the molecular level, showing that plants have spe- ciﬁc transcriptional switches that regulate SCW biosynthesis, and these factors belong to the NAC family, including VND and NST transcription factors (Yamaguchi and Demura, 2010; Zhong et al., 2010a; Wang and Dixon, 2011; Hussey et al., 2013). After these reports on NAC proteins, additional reports implicated sev- eral MYB-type transcription factors as secondary master regula- tors of SCW formation (McCarthy et al., 2009; Ko et al., 2012, 2014; Zhong and Ye, 2012; Hussey et al., 2013), and proposed an intricate network of transcription factors that regulate SCW formation in Arabidopsis (Figure 2). FIGURE 1 \| Plant cell walls.(A) Model of the primary cell wall. Cellulose microﬁbrils in the primary cell wall are relatively short and thin, compared with those in the secondary cell wall, and hemicellulose in the primary cell wall is composed of xyloglucan. The primary cell wall is rich in pectin. Citation: Nakano Y, Yamaguchi M, Endo H, Rejab NA and Ohtani M (2015) NAC-MYB-based transcriptional regulation of secondary cell wall biosynthesis in land plants. Front. Plant Sci. 6:288. doi: 10.3389/fpls.2015.00288 May 2015 \| Volume 6 \| Article 288 Frontiers in Plant Science \| www.frontiersin.org 1 Regulation of secondary wall biosynthesis Nakano et al. The diﬀerences in cell wall components between the SCW and PCW suggest that plants have a set of SCW-speciﬁc biosynthetic genes. Indeed, transcriptome analysis of xylem tissues in tree species identiﬁed many genes thought to be involved in the biosynthesis of SCW-speciﬁc polymers dur- ing xylem development in loblolly pine (Pinus taeda, Allona et al., 1998; Lorenz and Dean, 2002), poplar (Populus, spp., Sterky et al., 2004), white spruce (Picea glauca, Pavy et al., 2005), and eucalyptus (Eucalyptus gunnii, Rengel et al., 2009). A series of molecular genetic studies on Arabidopsis thaliana irregular xylem (irx) mutants, in which xylem cells are dis- rupted due to stunted SCW formation (Turner and Somerville, 1997), expanded our knowledge of SCW-speciﬁc enzymes. For example, functional molecular and co-expression analysis of IRX genes identiﬁed SCW-speciﬁc cellulose synthase subunit A (CesA) genes (IRX1/CesA8, IRX3/CesA7, and IRX5/CesA4), SCW-speciﬁc hemicellulose biosynthetic genes (IRX7, IRX8, IRX9, IRX10, IRX14, and IRX15), and lignin biosynthetic genes (IRX4 and IRX12) (Turner and Somerville, 1997; Jones et al., 2001; Brown et al., 2005, 2009, 2011; Lee et al., 2007; Peña et al., 2007; Wu et al., 2009; Jensen et al., 2011). These ﬁndings also suggested that the upregulation of SCW-speciﬁc enzyme genes promotes SCW formation, and that SCW formation requires this SCW-speciﬁc transcriptional regulatory system. FIGURE 1 \| Plant cell walls.(A) Model of the primary cell wall. Cellulose microﬁbrils in the primary cell wall are relatively short and thin, compared with those in the secondary cell wall, and hemicellulose in the primary cell wall is composed of xyloglucan. The primary cell wall is rich in pectin. (B) Model of the secondary cell wall, which is deposited between the primary cell wall and the plasma membrane. The secondary cell wall mainly contains relatively long and thick cellulose microﬁbrils, hemicellulosic xylan, and lignin. (C) Cross section of an Arabidopsis inﬂorescence stem stained with Safranin, which stains lignin red, and Astra blue. co, cortex; ep, epidermis; if, interfascicular ﬁber; xv, xylem vessel. Bar = 50 µm. FIGURE 1 \| Plant cell walls.(A) Model of the primary cell wall. Citation: Other components of the cell wall also diﬀer between the PCW and SCW. For exam- ple, the PCW typically contains xyloglucan as the major hemi- cellulose (i.e., the polysaccharide component that is soluble in alkali), but the SCW contains xylan. In addition, the PCW is rich in gel-like pectin, whereas lignin and speciﬁc phenolic polymers are more abundant in the SCW (Figure 1). These diﬀerences in cell wall composition impart diﬀerent physical properties to the cell wall, rendering the PCW ﬂexible and the SCW mechanically and biologically robust. In this review, we describe the transcriptional regulation of SCW formation based on information accumulated in the decade since 2005, focusing on the well-studied NAC and MYB tran- scription factors. An analysis of the NAC-MYB-based transcrip- tional regulatory system of the SCW reveals clues to how plant cells modify cell wall biosynthesis to conduct water (xylem ves- sels) and/or provide support (ﬁbers). May 2015 \| Volume 6 \| Article 288 Frontiers in Plant Science \| www.frontiersin.org 2 Regulation of secondary wall biosynthesis Nakano et al. gene family with more than 100 members in A. thaliana (Ooka et al., 2003), Oryza sativa (Ooka et al., 2003; Nuruzzaman et al., 2010), Glycine max (Le et al., 2011), Populus trichocarpa (Hu et al., 2010), and Eucalyptus grandis (Hussey et al., in press). The NAC proteins have been reported to participate in many devel- opmental processes (Krizek and Fletcher, 2005; Olsen et al., 2005; Petricka et al., 2012), including SCW formation (Yamaguchi and Demura, 2010) and biotic and abiotic stress responses (Fang et al., 2008; Nakashima et al., 2012; Puranik et al., 2012). FIGURE 2 \| Transcriptional network regulating secondary cell wall formation. (A) NAC-MYB-based transcriptional regulation of secondary cell wall biosynthesis. Some metabolic genes for secondary cell wall biosynthesis are targeted by both NACs and MYBs, producing “feed-forward” regulation. (B) Transcriptional regulatory network around VNS proteins, ﬁrst-layer master switches for secondary cell wall formation, based on work in Arabidopsis. (C) Transcriptional regulatory network around MYB proteins, second-layer master switches for secondary cell wall formation, based on work in Arabidopsis. The ﬁrst clear indication of NAC protein function in SCW for- mation came from studies of in vitro transdiﬀerentiation of trac- heary elements using Zinnia elegans mesophyll cells. Demura and co-workers found that expression of the NAC domain transcrip- tion factor Z567 increased during transdiﬀerentiation (Demura et al., 2002). Citation: (A) NAC-MYB-based transcriptional regulation of secondary cell wall biosynthesis. Some metabolic genes for secondary cell wall biosynthesis are targeted by both NACs and MYBs, producing “feed-forward” regulation. (B) Transcriptional regulatory network around VNS proteins, ﬁrst-layer master switches for secondary cell wall formation, based on work in Arabidopsis. (C) Transcriptional regulatory network around MYB proteins, second-layer master switches for secondary cell wall formation, based on work in Arabidopsis. formation. (A) NAC-MYB-based transcriptional regulation of secondary cell wall biosynthesis. Some metabolic genes for secondary cell wall biosynthesis are targeted by both NACs and MYBs, producing “feed-forward” regulation. (B) Transcriptional regulatory network around VNS proteins, ﬁrst-layer master switches for secondary cell wall formation, based on work in Arabidopsis. (C) Transcriptional regulatory network around MYB proteins, second-layer master switches for secondary cell wall formation, based on work in Arabidopsis. The Function of NAC (NAM, ATAF1,2 and CUC2) Proteins in SCW Formation VNS (VND, NST/SND, and SMB Related) Proteins Function as Master Regulators of SCW Formation Citation: They further established an in vitro system for xylem vessel cell diﬀerentiation with Arabidopsis suspension cul- ture cells, and showed that expression of seven NAC transcrip- tion factors with high sequence similarity to Z567 also increased, beginning in the early stages of cell diﬀerentiation (Kubo et al., 2005). They named these proteins VASCULAR-RELATED NAC- DOMAIN1 (VND1) to VND7 (Figure 3A, Table 1). All the VND genes are preferentially expressed in developing vascular tissues, although their expression patterns diﬀer; promoter analysis sug- gested that VND7 regulates all types of xylem vessels in roots and shoots, whereas the other VND proteins might diﬀerentially reg- ulate vessel formation (Kubo et al., 2005; Yamaguchi et al., 2008). Overexpression of VND genes induces ectopic deposition of pat- terned SCW, which is characteristic of xylem vessel cells (Kubo et al., 2005; Zhou et al., 2014; Endo et al., 2015; Figures 3B–G). Conversely, overexpression of a dominant chimeric repressor constructed by fusing VND6 or VND7 to the SRDX transcrip- tional repression domain, severely inhibited xylem vessel cell dif- ferentiation (Kubo et al., 2005; Reusche et al., 2012). Together, these ﬁndings indicate that the VND proteins act as master regulators of xylem vessel cell diﬀerentiation. g y In A. thaliana, the VND-related proteins NAC SECONDARY WALL THICKENING PROMOTING FACTOR1 (NST1), NST2, and NST3/SECONDARY WALL-ASSOCIATED NAC DOMAIN PROTEIN 1 (SND1)/ARABIDOPSIS NAC DOMAIN CON- TAINING PROTEIN012 (ANAC012) (Figure 3A, Table 1) regu- late the diﬀerentiation of SCW-containing cells other than xylem vessel cells, such as anther endothecium (NST1 and NST2; Mit- suda et al., 2005), ﬁber cells (NST1 and NST3; Zhong et al., 2006, 2007b; Mitsuda et al., 2007; Figures 3H,I, Table 1), and silique cells (NST1 and NST3; Mitsuda and Ohme-Takagi, 2008). More- over, other VND-related Arabidopsis proteins, namely SOM- BRERO (SMB), BEARSKIN1 (BRN1), and BRN2, induce ectopic SCW deposition when overexpressed, although in wild type cells, they are expressed in root cap regions where SCW is not deposited (Willemsen et al., 2008; Bennett et al., 2010; Figure 3A, Table 1). These results indicate that the capacity to induce SCW biosynthesis is conserved among the VND, NST, SMB, and BRN proteins, and that these genes likely evolved from a common ancestral gene, acquiring the capacity to regulate wall modiﬁca- tion during the diﬀerentiation of speciﬁc cell types. FIGURE 2 \| Transcriptional network regulating secondary cell wall FIGURE 2 \| Transcriptional network regulating secondary cell wall formation. VNS (VND, NST/SND, and SMB Related) Proteins Function as Master Regulators of SCW Formation Members of the NAC domain transcription factor family have a highly conserved N-terminal NAC domain, which has been implicated in nuclear localization, DNA binding, and homo- and/or heterodimer formation with other NAC domain proteins (Olsen et al., 2005). NAC transcription factors consist of a large The NAC protein subfamily, including VND, NST, SMB and BRN of Arabidopsis has been termed the VNS (VND, NST/SND, May 2015 \| Volume 6 \| Article 288 Frontiers in Plant Science \| www.frontiersin.org 3 Regulation of secondary wall biosynthesis Nakano et al. RE 3 \| The VNS genes function as ﬁrst-layer master switches for ndary cell wall formation. (A) Phylogenetic tree of VNS proteins. The oted phylogenetic tree was constructed with amino acid sequences of AC domain (sequences provided in Table S1) by the mum-likelihood method. Numbers indicate bootstrap values for the s that received support values of over 70% (1000 resamplings). Scale represents a 10% change in sequences. Based on the tree, the VNS ins are classiﬁed into four groups, VND, NST/SND, SMB, and Ancestral ps. (B–G) Seven-day-old Arabidopsis roots of wild type (wt, B) and genic plants, in which AtVND1 (C), AtVND2 (D), AtVND3 (E), AtVND6 (F), and AtVND7 (G) were overexpressed by an inducible system. Ectopic xylem elements formed in the transgenic roots (white arrowheads). Data were adapted from Endo et al. (2015). (H,I) Cross sections of Arabidopsis inﬂorescence stems stained with Safranin, which stains lignin red, and Astra blue. In the wild type (wt), both xylem vessel cells and interfascicular ﬁber cells have lignin-containing secondary cell wall, thus they stain red (H). By contrast, the mutant nst1 snd1/nst3 lacks secondary cell wall in interfascicular ﬁber cells, thus only xylem vessel cells were stained by Safranin (I), as described in Mitsuda et al. (2007). co, cortex; ep, epidermis; if, interfascicular ﬁber; xv, xylem vessel. Bars = 100 µm (B–I). (F), and AtVND7 (G) were overexpressed by an inducible system. Ectopic xylem elements formed in the transgenic roots (white arrowheads). Data were adapted from Endo et al. (2015). (H,I) Cross sections of Arabidopsis inﬂorescence stems stained with Safranin, which stains lignin red, and Astra blue. In the wild type (wt), both xylem vessel cells and interfascicular ﬁber cells have lignin-containing secondary cell wall, thus they stain red (H). VNS (VND, NST/SND, and SMB Related) Proteins Function as Master Regulators of SCW Formation By contrast, the mutant nst1 snd1/nst3 lacks secondary cell wall in interfascicular ﬁber cells, thus only xylem vessel cells were stained by Safranin (I), as described in Mitsuda et al. (2007). co, cortex; ep, epidermis; if, interfascicular ﬁber; xv, xylem vessel. Bars = 100 µm (B–I). (F), and AtVND7 (G) were overexpressed by an inducible system. Ectopic xylem elements formed in the transgenic roots (white arrowheads). Data were adapted from Endo et al. (2015). (H,I) Cross sections of Arabidopsis inﬂorescence stems stained with Safranin, which stains lignin red, and Astra blue. In the wild type (wt), both xylem vessel cells and interfascicular ﬁber cells have lignin-containing secondary cell wall, thus they stain red (H). By contrast, the mutant nst1 snd1/nst3 lacks secondary cell wall in interfascicular ﬁber cells, thus only xylem vessel cells were stained by Safranin (I), as described in Mitsuda et al. (2007). co, cortex; ep, epidermis; if, interfascicular ﬁber; xv, xylem vessel. Bars = 100 µm (B–I). (F), and AtVND7 (G) were overexpressed by an inducible system. Ectopic xylem elements formed in the transgenic roots (white arrowheads). Data were adapted from Endo et al. (2015). (H,I) Cross sections of Arabidopsis inﬂorescence stems stained with Safranin, which stains lignin red, and Astra blue. In the wild type (wt), both xylem vessel cells and interfascicular ﬁber cells have lignin-containing secondary cell wall, thus they stain red (H). By contrast, the mutant nst1 snd1/nst3 lacks secondary cell wall in interfascicular ﬁber cells, thus only xylem vessel cells were stained by Safranin (I), as described in Mitsuda et al. (2007). co, cortex; ep, epidermis; if, interfascicular ﬁber; xv, xylem vessel. Bars = 100 µm (B–I). FIGURE 3 \| The VNS genes function as ﬁrst-layer master switches for secondary cell wall formation. (A) Phylogenetic tree of VNS proteins. The unrooted phylogenetic tree was constructed with amino acid sequences of the NAC domain (sequences provided in Table S1) by the maximum-likelihood method. Numbers indicate bootstrap values for the clades that received support values of over 70% (1000 resamplings). Scale (0.1) represents a 10% change in sequences. Based on the tree, the VNS proteins are classiﬁed into four groups, VND, NST/SND, SMB, and Ancestral groups. VNS (VND, NST/SND, and SMB Related) Proteins Function as Master Regulators of SCW Formation (B–G) Seven-day-old Arabidopsis roots of wild type (wt, B) and transgenic plants, in which AtVND1 (C), AtVND2 (D), AtVND3 (E), AtVND6 May 2015 \| Volume 6 \| Article 288 Frontiers in Plant Science \| www.frontiersin.org 4 Regulation of secondary wall biosynthesis Nakano et al. TABLE 1 \| Known VNS genes. VNS GENES References VND NST SMB Ancestral Arabidopsis thaliana AtVND1 AtNST1 AtBRN1 – Kubo et al., 2005 AtVND2 AtNST2 AtBRN2 Mitsuda et al., 2005, 2007 AtVND3 AtNST3/AtSND1 AtSMB Zhong et al., 2006 AtVND4 Bennett et al., 2010 AtVND5 AtVND6 AtVND7 Carica papaya CpNAC028 CpNAC037 CpNAC011 – Zhu et al., 2012 CpNAC030 CpNAC048 Populus trichocarpa PtVNS01/PtrWND5A/PtrVND6-C1 PtVNS09/PtrWND2A/PtrSND1-B1 PtVNS13/PtrSND1-L-1 – Zhong et al., 2010b PtVNS02/PtrWND5B/PtrVND6-C2 PtVNS10/PtrWND2BPtrSND1-B2 PtVNS14/PtrSND1-L-2 Ohtani et al., 2011 PtVNS03/PtrWND4A/PtrVND6-B2 PtVNS11/PtrWND1B/PtrSND1-A2 PtVNS15 Li et al., 2012b PtVNS04/PtrWND4B/PtrVND6-B1 PtVNS12/PtrWND1A/PtrSND1-A1 PtVNS16 PtVNS05/PtrWND3A/PtrVND6-A1 PtVNS06/PtrWND3B/PtrVND6-A2 PtVNS07/PtrWND6A/PtrVND7-2 PtVNS08/PtrWND6B/PtrVND7-1 Medicago truncatula Medtr4g101680.1 MtNST1 Medtr2g062730.1 – Zhao et al., 2010 Medtr5g012080.1 Medtr4g035590.1 Phytozome v9.1 Medtr5g021710.1 (http://www.phytozome.net/) Medtr8g076110.1 Eucalyptus grandis EgrNAC26 EgrNAC49 EgrNAC81 - Hussey et al., in press EgrNAC50 EgrNAC61 EgrNAC75 EgrNAC146 Vitis vinifera VvNAC012 VvNAC002 VvNAC006 – Zhu et al., 2012 VvNAC057 VvNAC049 VvNAC061 VvNAC060 VvNAC067 Zea mays ZmSWN3 ZmSWN1 Zm2g041746 – Zhong et al., 2011 ZmSWN4 ZmSWN2 Zm2g099144 Zhu et al., 2012 ZmSWN5 Zm2g091490 Zm2g104074 ZmSWN6 ZmSWN7 Zm2g048826 Sorghum bicolor SbNAC002 SbNAC043 SbNAC020 – Zhu et al., 2012 SbNAC030 SbNAC046 SbNAC040 SbNAC065 SbNAC107 SbNAC069 TABLE 1 \| Known VNS genes. PtVNS01/PtrWND5A/PtrVND6-C1 PtVNS02/PtrWND5B/PtrVND6-C2 PtVNS03/PtrWND4A/PtrVND6-B2 PtVNS04/PtrWND4B/PtrVND6-B1 PtVNS05/PtrWND3A/PtrVND6-A1 PtVNS06/PtrWND3B/PtrVND6-A2 PtVNS07/PtrWND6A/PtrVND7-2 PtVNS08/PtrWND6B/PtrVND7-1 PtVNS06/PtrWND3B/PtrVND6-A2 PtVNS07/PtrWND6A/PtrVND7-2 PtVNS08/PtrWND6B/PtrVND7-1 Medicago truncatula Medtr4g101680.1 MtNST1 Medtr2g062730.1 – Zhao et al., 2010 Medtr5g012080.1 Medtr4g035590.1 Phytozome v9.1 Medtr5g021710.1 (http://www.phytozome.net/) Medtr8g076110.1 Eucalyptus grandis EgrNAC26 EgrNAC49 EgrNAC81 - Hussey et al., in press EgrNAC50 EgrNAC61 EgrNAC75 EgrNAC146 Vitis vinifera VvNAC012 VvNAC002 VvNAC006 – Zhu et al., 2012 VvNAC057 VvNAC049 VvNAC061 VvNAC060 VvNAC067 Zea mays ZmSWN3 ZmSWN1 Zm2g041746 – Zhong et al., 2011 ZmSWN4 ZmSWN2 Zm2g099144 Zhu et al., 2012 ZmSWN5 Zm2g091490 Zm2g104074 ZmSWN6 ZmSWN7 Zm2g048826 Sorghum bicolor SbNAC002 SbNAC043 SbNAC020 – Zhu et al., 2012 SbNAC030 SbNAC046 SbNAC040 SbNAC065 SbNAC107 SbNAC069 SbNAC089 (Continued) May 2015 \| Volume 6 \| Article 288 May 2015 \| Volume 6 \| Article 288 Frontiers in Plant Science \| www.frontiersin.org Nakano et al. VNS (VND, NST/SND, and SMB Related) Proteins Function as Master Regulators of SCW Formation trichocarpa; Pt or Ptr in diﬀerent studies), the ﬁrst tree species with a high-quality, annotated genomic sequence (Tuskan et al., 2006), contains 16 PtVNS genes, including eight genes of the VND group, four genes of the NST group, and four genes of the SMB group (Zhong et al., 2010b; Ohtani et al., 2011; Li et al., 2012b; Figure 3A, Table 1). The PtVNS genes include PtrWND, PtrVND6, PtrVND7, and SMB related protein) family (Ohtani et al., 2011; Xu et al., 2014). The same subfamily was called “subfamily Ic” by Zhu et al. (2012), and is conserved among wide range of plant species, including non-vascular land plant species such as Bryophytes (Zhu et al., 2012; Xu et al., 2014; Figure 3A, Table 1). classiﬁed into VND, NST, or SMB groups by phylogenetic analy- sis (Figure 3A), suggesting that the diversiﬁcation of VNS genes occurred within the vascular plant lineage. Black cottonwood (P. trichocarpa; Pt or Ptr in diﬀerent studies), the ﬁrst tree species with a high-quality, annotated genomic sequence (Tuskan et al., 2006), contains 16 PtVNS genes, including eight genes of the VND group, four genes of the NST group, and four genes of the SMB group (Zhong et al., 2010b; Ohtani et al., 2011; Li et al., 2012b; Figure 3A, Table 1). The PtVNS genes include PtrWND, PtrVND6, PtrVND7, and VNS (VND, NST/SND, and SMB Related) Proteins Function as Master Regulators of SCW Formation Regulation of secondary wall biosynthesis TABLE 1 \| Continued VNS GENES References VND NST SMB Ancestral SbNAC101 SbNAC102 Oryza sativa OsSWN3 OsSWN1 LOC_Os02g15340.1 – Zhong et al., 2011 OsSWN4 OsSWN2 LOC_Os06g33940.1 Zhu et al., 2012 OsSWN5 OsSWN6 OsSWN7 LOC_Os02g42970.1 LOC_Os03g03540.1 LOC_Os04g59470.1 Brachypodium distachyon BdSWN1 BdSWN7 Bradi1g38730.1 – Valdivia et al., 2013 BdSWN2 BdSWN8 Bradi3g09520.1 Phytozome v9.1 BdSWN3 Bradi5g11247.1 (http://www.phytozome.net/) BdSWN4 BdSWN5 BdSWN6 Picea abies Pa_comp83767_c0_seq8 – Pa138461p0010 – Nystedt et al., 2013 Pa6777p0010 Pa18939p0010 Picea glauca PgNAC7 – PgNAC4 – Duval et al., 2014 Selaginella moellendorfﬁi – – – Sm36139 Xu et al., 2014 Sm71404 Sm74950 Sm89986 Physcomitrella patens – – – PpVNS1 Xu et al., 2014 PpVNS2 PpVNS3 PpVNS4 PpVNS5 PpVNS6 PpVNS7 PpVNS8 Marchantia polymorpha – – – MpoJPYU-9533 Xu et al., 2014 SMB related protein) family (Ohtani et al., 2011; Xu et al., 2014). The same subfamily was called “subfamily Ic” by Zhu et al. (2012), and is conserved among wide range of plant species, including classiﬁed into VND, NST, or SMB groups by phylogenetic analy- sis (Figure 3A), suggesting that the diversiﬁcation of VNS genes occurred within the vascular plant lineage. Regulation of secondary wall biosynthesis Regulation of secondary wall biosynthesis Nakano et al. References Pa_comp83767_c0_seq8 – Pa138461p0010 – Nystedt et al., 2013 Pa6777p0010 Pa18939p0010 Picea glauca PgNAC7 – PgNAC4 – Duval et al., 2014 Selaginella moellendorfﬁi – – – Sm36139 Xu et al., 2014 Sm71404 Sm74950 Sm89986 Physcomitrella patens – – – PpVNS1 Xu et al., 2014 PpVNS2 PpVNS3 PpVNS4 PpVNS5 PpVNS6 PpVNS7 PpVNS8 Marchantia polymorpha – – – MpoJPYU-9533 Xu et al., 2014 Physcomitrella patens Marchantia polymorpha classiﬁed into VND, NST, or SMB groups by phylogenetic analy- sis (Figure 3A), suggesting that the diversiﬁcation of VNS genes occurred within the vascular plant lineage. Black cottonwood (P. trichocarpa; Pt or Ptr in diﬀerent studies), the ﬁrst tree species with a high-quality, annotated genomic sequence (Tuskan et al., 2006), contains 16 PtVNS genes, including eight genes of the VND group, four genes of the NST group, and four genes of the SMB group (Zhong et al., 2010b; Ohtani et al., 2011; Li et al., 2012b; Figure 3A, Table 1). The PtVNS genes include PtrWND, PtrVND6, PtrVND7, and classiﬁed into VND, NST, or SMB groups by phylogenetic analy- sis (Figure 3A), suggesting that the diversiﬁcation of VNS genes occurred within the vascular plant lineage. Black cottonwood (P. VNS Proteins Are Well-Conserved Among Vascular Plants Monocot VNS proteins have been also studied in terms of their expression patterns and molecular functions (Zhong et al., 2011; Valdivia et al., 2013; Yoshida et al., 2013). The mem- bers of the VND and NST groups in rice, maize (Zea mays), and Brachypodium distachyon are named SECONDARY WALL- ASSOCIATED NAC (SWN) proteins, and are expressed in SCW- forming cells such as xylem vessels, cortical ﬁbers, and bundle sheath ﬁbers (Zhong et al., 2011; Valdivia et al., 2013; Yoshida et al., 2013). Heterologous overexpression of the SWN genes can induce ectopic SCW deposition (Zhong et al., 2011; Valdivia et al., 2013), like the AtVNS and PtVNS genes; thus the SWN pro- teins are suﬃcient to promote the downstream events of SCW formation. Around 2010, several groups independently identiﬁed the direct target genes of AtVND6, AtVND7, and AtSND1/NST3 (Ohashi-Ito et al., 2010; Zhong et al., 2010c; Yamaguchi et al., 2011). These studies did not identify identical sets of genes, reﬂecting the diﬀerent genes and diﬀerent experimental strate- gies, but the sets showed some overlap. First, common targets of AtVND and AtSND1/NST3 include transcription factors such as MYB and ASYMMETRIC LEAVES2-LIKE/LATERAL ORGAN BOUNDARIES DOMAIN (ASL/LBD) (for details, please see sec- tions below; Figure 2), and the genes encoding enzymes involved in SCW formation, such as the IRX genes (Taylor et al., 2003). Second, the VND proteins preferentially target genes involved in programmed cell death (nucleases, proteases, and metacaspases), and signal transduction (receptor-like kinases). Additionally, even for common targets, the transcriptional activation activi- ties of AtVND and AtSND1/NST3 sometimes diﬀer (Ohashi-Ito et al., 2010; Zhong et al., 2010c; Yamaguchi et al., 2011). In sil- ico and in vitro analyses of cis-elements targeted by VNS revealed the 11-bp tracheary element-regulating cis-elements (TEREs; Pyo et al., 2007) and the 19-bp secondary wall NAC-binding elements (SNBEs; Zhong et al., 2010c), which partly overlap. Both cis- elements are enriched in promoter regions of the genes directly regulated by AtVND and AtSND1/NST3 (Pyo et al., 2007; Ohashi-Ito et al., 2010; Zhong et al., 2010c), and are recognized by both AtVND and AtSND1/NST3 in transient expression experi- ments (Zhong et al., 2010c), suggesting that the determination of target preference between the VND group and the NST group must be regulated by cis-elements other than TERE or SNBE. In addition to poplar and monocots, comparative genomics research has identiﬁed VNS genes in many plant species. VNS Proteins Are Well-Conserved Among Vascular Plants As shown in Table 1, many VNS proteins have been identiﬁed in land plant species. The VNS proteins in vascular plants can be May 2015 \| Volume 6 \| Article 288 Frontiers in Plant Science \| www.frontiersin.org 6 Regulation of secondary wall biosynthesis Nakano et al. Yamaguchi et al., 2010a; Ohtani et al., 2011; Valdivia et al., 2013; Endo et al., 2015; Xu et al., 2014; Figures 3B–G). This observa- tion demonstrates that VNS proteins likely share target genes to produce SCW. Indeed, the overexpression of AtVND and AtNST commonly upregulates the genes involved in the biosynthesis of SCW components, such as cellulose, hemicellulose and lignin (Kubo et al., 2005; Mitsuda et al., 2005, 2007; Zhong et al., 2006; Ko et al., 2007). However, xylem vessel cells and ﬁber cells diﬀer in speciﬁc cell wall characteristics, such as the syringyl/guaiacyl (S/G) ratio of lignin subunits (Saito et al., 2012). In addition, expression of AtVND7 under the control of the AtSND1/NST3 promoter in the nst1 snd1/nst3 double mutant rescues the lack of SCW in mutant ﬁber cells (Mitsuda et al., 2007; Zhong et al., 2007b; Yamaguchi et al., 2011; Figures 3H,I). However, the SCW formed in the ﬁbers diﬀers from that produced by expression AtNST3; expression of AtVND7 causes formation of the pat- terned SCW characteristic of xylem vessel cells, even in the ﬁber cells (Yamaguchi et al., 2011). Accordingly, parts of the down- stream SCW-related pathway appear to diﬀer between AtVND and AtNST. PtrSND1 and all PtVNS genes of the VND and NST groups are expressed in developing xylem and phloem ﬁber regions, although in primary vessels in the stem, only PtVNS genes of the VND group are expressed (Zhong et al., 2010b; Ohtani et al., 2011). The SMB-group PtVNS genes are not expressed in the xylem tissues, but rather are expressed in root tissues (Zhong et al., 2010b; Ohtani et al., 2011), indicating that the molecular functions of VNS genes in the SMB group in root tissue development might be conserved in Arabidopsis and poplar. Overexpression of PtVNS genes of the VND and NST groups in Arabidopsis and poplar caused ectopic deposition of SCW (Zhong et al., 2010b; Ohtani et al., 2011), and AtNST3 promoter-driven PtVNS genes can rescue ﬁber cell formation in nst1 snd1/nst3 double mutant stems (Zhong et al., 2010b; Figures 3H,I). Thus, the PtVNS proteins appear to possess the full potential to induce SCW biosynthesis. VNS Proteins Are Well-Conserved Among Vascular Plants By contrast, in poplar, rice, and maize, the VNS genes of both VND and NST groups are expressed in vessels and ﬁbers (Zhong et al., 2010b, 2011; Ohtani et al., 2011). Thus, SCW formation in xylem vessels and ﬁbers in those species must involve distinct layers of regulation for downstream events, in addition to transcriptional control. Work on the spatial organization of SCW in xylem vessel cells could inform our understanding of this. In the current model, the patterns of SCW deposition in xylem vessel cells can be reg- ulated by the balance of assembly and disassembly of the cor- tical microtubule array, determining the spatial orientation of the CesA complex (Oda and Fukuda, 2013b). The interactions of speciﬁc proteins with microtubules and/or ROP-GTPase activ- ities control this balance (Oda and Fukuda, 2012, 2013a). Certain SCW-related enzymes likely function in the apoplastic regions of plant cells (Schuetz et al., 2014), so we might have to think about the regulation of spatial activities of enzymes in each plant species. induction of VND activity through many types of transcription factors. Infection by the soil-born fungal pathogen Verticillium longisporum induces AtVND7 expression, resulting in the trans- diﬀerentiation of vascular bundle cells into tracheary elements (Reusche et al., 2012). Recent work also showed that AtVND6 and AtVND7 expression increased in response to high salin- ity and iron depletion (Taylor-Teeples et al., 2015). Such abiotic and/or biotic stress signals might be mediated by speciﬁc types of transcription factors functioning upstream of VNS genes. In addition to many positive regulators of VNS genes, as described above, other studies have identiﬁed negative regulators of VNS, including the WRKY-type transcription factor, WRKY12 (Wang et al., 2010). In the loss-of-function wrky12 mutant, ectopic SCW formation occurred in pith parenchyma cells of inﬂorescence stems, and the expression of NST2 increased. Recombinant WRKY12 protein can bind to the NST2 pro- moter sequence in vitro; thus, WRKY12 negatively regulates SCW formation by directly inhibiting NST2 expression in pith parenchyma cells (Wang et al., 2010). Additionally, protein-DNA interaction analysis in xylem-expressed transcription factors of Arabidopsis showed that E2Fc, a member of a transcription factor family conserved in eukaryotes and a negative regulator of endoreduplication in plants, may function as a key regula- tor of SCW formation (Taylor-Teeples et al., 2015). Notably, E2Fc seems to function as both an activator and a repressor of AtVND6 and AtVND7 expression, depending on the situa- tion. Transcriptional- and Post-Transcriptional Regulation of VNS Genes Following the upregulation of VNS genes, the cells begin to dif- ferentiate as SCW-forming cells, such as xylem vessel cells and ﬁbers. These cells are dead at maturity; therefore, VNS expres- sion and/or activity must be well regulated in accordance with developmental programs and/or environmental signals. p p g g During plant development, xylem vessel cells diﬀerentiate from vascular stem cells of the procambium and cambium (Fukuda, 1997; Demura and Fukuda, 2007). Phytohormones, especially auxin, provide one of ﬁrst cues for vascular stem cell initiation. Several transcription factors function in the regulation of initiation, maintenance, and diﬀerentiation of vascular stem cells downstream of auxin signals (De Rybel et al., 2013; Ohashi- Ito et al., 2013a,b). Screening for factors upstream of AtVND7 in a transient expression system recently identiﬁed one such tran- scription factor, REV, a member of the Class III HD-Zip proteins (Carlsbecker et al., 2010; Miyashima et al., 2011; Endo et al., 2015). In addition, LBD18/ASL20 and LBD30/ASL19, which are expressed in xylem vessels, can induce ectopic SCW deposition in various types of cells through the upregulation of AtVND6 and AtVND7 (Soyano et al., 2008). LBD18/ASL20 and LBD30/ASL19 are upregulated by auxin and by AtVND6 and AtVND7 (Soy- ano et al., 2008), and LBD15/ASL11 and LBD30/ASL19 are also direct targets of AtVND6 and/or AtVND7 (Ohashi-Ito et al., 2010; Zhong et al., 2010c; Yamaguchi et al., 2011). This indicates the existence of auxin-mediated feedback regulation between VND and LBD/ASL (Figure 2). Moreover, all AtVND genes can induce AtVND7 expression by direct interaction with the VND7 promoter region through regions containing the SMBE/TERE motif (Zhou et al., 2014; Endo et al., 2015). Thus, once the precursor cells initiate xylem vessel cell diﬀerentiation, the pos- itive feedback transcriptional regulation eﬃciently upregulates VND activity. Transient expression assays identiﬁed GATA fam- ily members (GATA5 and GATA12), and other NAC proteins (ANAC075 and SND2) as upstream factors of AtVND7 (Endo et al., 2015), suggesting that multiple signals contribute to the Post-transcriptional regulation also likely plays an important role in modulating VNS activity. A VNS gene in the poplar P. trichocarpa, PtrWND1B/PtVNS11/PtrSND1-A2, has alternative splicing variants that vary in abundance in diﬀerent tissues (Li et al., 2012b; Zhao et al., 2014). The predicted protein product of the short and minor variant lacks the C-terminal region, but can bind to full-length PtVNS proteins. VNS Proteins Are Well-Conserved Among Vascular Plants The numbers of VNS gene vary by plant species without apparent cor- relation to genome size or the presence of woody tissues (Zhu et al., 2012; Figure 3A, Table 1). Recently, genome sequencing has been completed for other tree species, such as Picea abies (Nystedt et al., 2013) and E. grandis (Myburg et al., 2014). These tree species have only a few VNS genes: four in P. abies (Nyst- edt et al., 2013), six in E. grandis (Hussey et al., in press; Myburg et al., 2014), and two in P. glauca (Duval et al., 2014). It is note- worthy that no VNS genes of the NST group have been identiﬁed in gymnosperms at present (Figure 3A); thus, the NST group might have evolved within the angiosperm lineage, or lost in the gymnosperm lineage. The wood of gymnosperms is composed of single cell tracheids that function in water conduction and provide mechanical strength to the axis, whereas the wood of angiosperms is composed of xylem vessels and ﬁbers, cells that are specialized for water conduction and providing mechanical strength, respectively (Pallady, 2008). Phylogenetic analysis sug- gests that gymnosperm tracheid cell diﬀerentiation could be reg- ulated by the VND-type VNS genes (Figure 3A). Further studies on gymnosperm VNS genes will give insights into how woody cells developed during land plant evolution. The overall characteristics of VNS targets identiﬁed in Ara- bidopsis, including the cis-elements TERE and SNBE, are basi- cally conserved in poplar, rice, maize, B. distachyon, and Med- icago truncatula (Zhao et al., 2010; Zhong et al., 2010b, 2011; Ohtani et al., 2011; Valdivia et al., 2013). However, notably, the expression speciﬁcity depending on gene group that occurs in Arabidopsis was not detected in the other plant species. For example, in Arabidopsis stems, AtVND genes are prefer- entially expressed in xylem vessel cells, while AtNST genes are Target Genes of VNS for SCW Formation As mentioned above, the overexpression of VNS genes induces ectopic SCW deposition (Kubo et al., 2005; Mitsuda et al., 2005, 2007; Zhong et al., 2006, 2010a,b, 2011; Bennett et al., 2010; May 2015 \| Volume 6 \| Article 288 Frontiers in Plant Science \| www.frontiersin.org 7 Regulation of secondary wall biosynthesis Nakano et al. expressed in interfascicular ﬁbers (Mitsuda et al., 2005, 2007; Zhong et al., 2006; Yamaguchi et al., 2008). VNS Proteins Are Well-Conserved Among Vascular Plants E2Fc also can bind to the promoters of many kinds of xylem-expressed transcription factors in addition to the AtVND promoters (Taylor-Teeples et al., 2015). These complex interac- tions among transcription factors suggest that vascular plants have developed a robust transcriptional regulation system to pro- mote xylem vessel cell diﬀerentiation, which is vital for land plants. AtMYB46 and AtMYB83: Second-Layer Master Switches of SCW Biosynthesis y After the emergence of the ﬁrst sets of evidence showing the involvement of pine and Eucalyptus MYBs in SCW formation in woody species (Patzlaﬀet al., 2003a,b; Goicoechea et al., 2005), their Arabidopsis closest functional orthologs MYB46 and MYB83 (AtMYB46 and AtMYB83) were reported to function as key regulators of SCW formation (Zhong et al., 2007a; Ko et al., 2009; McCarthy et al., 2009; Table 2). These genes are preferentially expressed in xylem tissues, and their overexpres- sion induced ectopic deposition of SCW. Conversely, expression of the chimeric repressors for AtMYB46 or AtMYB83 inhib- ited SCW deposition in xylem (Zhong et al., 2007a; McCarthy et al., 2009). Promoter activity of AtMYB46 was found in both protoxylem-type and metaxylem-type vessel cells, suggesting the involvement of these MYBs in xylem vessel cell formation (Nakano et al., 2010). Consistent with this observation, in the double mutant myb46 myb83, SCW deposition in vessel cells is severely aﬀected, leading to seedling growth arrest in the mutant (McCarthy et al., 2009; Figure 4). These ﬁndings indicate that AtMYB46 and AtMYB83 redundantly regulate SCW formation in Arabidopsis (McCarthy et al., 2009). Importantly, AtVND and/or AtNST/SND, the master regulators of the diﬀerentiation of SCW-containing cells, directly target these MYB genes (Zhong et al., 2007a, 2010c; McCarthy et al., 2009; Ohashi-Ito et al., 2010; Yamaguchi et al., 2011). Thus, AtMYB46 and AtMYB83 act as second layer-master switches of SCW biosynthesis (Figure 2). Transcriptional- and Post-Transcriptional Regulation of VNS Genes As a result, the truncated PtrWND1B/PtVNS11/PtrSND1-A2 inhibits transcriptional acti- vation by PtVNS proteins (Li et al., 2012b), and can suppress the SCW thickening of ﬁber cells in poplar (Zhao et al., 2014). This alternative splicing regulation is completely dependent on the intron sequence of PtrWND1B/PtVNS11/PtrSND1-A2, and such regulation could be speciﬁc to poplar. Understanding the contri- butions of regulation of splicing to the control of VNS activity will require further survey of VNS genes. NAC domain proteins form homo- and/or hetero-dimers (Olsen et al., 2005; Weiner et al., 2012). Indeed, yeast two- hybrid screens showed that AtVND and AtNST can bind each other to form hetero-dimers as well as forming homo-dimers (Yamaguchi et al., 2008; Li et al., 2012b). Transient expression assays on poplar VNS genes indicate that transactivation activ- ity varies by PtVNS, even between “twin” genes that possess more than 90% similarity in amino acid sequence (Ohtani et al., 2011). Thus, the VNS hetero-dimers and homo-dimers should May 2015 \| Volume 6 \| Article 288 Frontiers in Plant Science \| www.frontiersin.org 8 Regulation of secondary wall biosynthesis Nakano et al. have diﬀerent transactivation activities and we should consider the eﬀects of dimerization, particularly when multiple VNS are expressed. Yeast two-hybrid screening for proteins that inter- act with VND7 also identiﬁed the NAC domain protein VND- INTERACTING2 (VNI2) as a key regulator of VND7 activity (Yamaguchi et al., 2010b). VNI2 acts solely as a transcriptional repressor and inhibits transcriptional activation activities of AtVND7 and xylem vessel cell diﬀerentiation, probably through direct interaction via the NAC domain regions (Yamaguchi et al., 2010b). A Recent work also reported ANAC103 as a possible interactor with AtVND7 in vascular tissues (Yamaguchi et al., in press). Taken together with the fact that proteasome-mediated proteolysis actively regulates AtVND7 (Yamaguchi et al., 2008), these ﬁndings show that multiple layers of regulation also aﬀect AtVND7 at the protein level. biosynthesis genes through the AC elements. Moreover, func- tional analysis of MYB genes expressed in secondary xylem of P. taeda (PtMYB4) and E. gunnii (EgMYB2) demonstrated that they can enhance lignin biosynthesis and/or SCW thickening when overexpressed (Patzlaﬀet al., 2003a; Goicoechea et al., 2005; Table 2). MYB proteins have been reported to be involved in lignin biosynthesis in many plant species (Grima-Pettenati et al., 2012); in this review we limit our focus to Arabidopsis MYB genes. MYB Transcription Factors as Lignin Biosynthesis Regulators y g MYB transcription factors occur widely in eukaryotes and have characteristic, highly conserved DNA-binding domains, called the R1, R2, and R3 domains, at their N-termini. In plants, the majority of MYB proteins have only two domains and thus are called R2R3-MYB proteins; R2R3-MYB proteins are encoded by 126 genes in Arabidopsis (Stracke et al., 2001; Dubos et al., 2010), 109 in rice (Yanhui et al., 2006), 141 in eucalyptus (Soler et al., in press), and 192 in poplar (Wilkins et al., 2009). These R2R3-MYB family genes function in a wide range of developmental processes, stress responses, and metabolism (Jin and Martin, 1999; Stracke et al., 2001; Larkin et al., 2003; Grotewold, 2006; Lepiniec et al., 2006; Valliyodan and Nguyen, 2006; Bergmann and Sack, 2007; Chinnusamy et al., 2007; Ishida et al., 2008; Dubos et al., 2010; De Geyter et al., 2012; Grima-Pettenati et al., 2012; Muñoz-Nortes et al., 2014). Studies in the 1990s revealed the involvement of MYBs in biosynthesis of phenylpropanoids. Since then, promoter analysis of phenylpropanoid biosynthetic genes, including PAL (encoding phenylalanine ammonia-lyase) and 4CL (encoding 4-coumarate CoA ligase), revealed several important cis-elements in their pro- moters (Lois et al., 1989; Ohl et al., 1990; Becker-André et al., 1991; Leyva et al., 1992; Hauﬀe et al., 1993; Hatton et al., 1995; Wanner et al., 1995). One of them is the AC element, also known as the C1-motif, PAL-box, or H-box, which is rich in the sequence AC and is critical for xylem-speciﬁc expression of PAL and 4CL (Leyva et al., 1992; Hauﬀe et al., 1993; Bell-Lelong et al., 1997). Bioinformatic and biochemical analyses showed that the AC elements share sequence similarity to the motif rec- ognized by MYB transcription factors, and that, indeed, some MYBs bind to the AC elements to regulate gene expression (Romero et al., 1998). These ﬁndings led to genome-wide in sil- ico analysis of presumed lignin biosynthesis genes of Arabidop- sis, which found that almost all lignin biosynthesis genes have AC elements in their promoter regions (Weisshaar and Jenk- ins, 1998; Rogers and Campbell, 2004). Taken together, these ﬁndings suggest that MYB genes coordinately regulate lignin Downstream Genes of Second-Layer Master Switch MYBs Biosynthetic Genes of SCW Components The regulatory target genes of AtMYB46 include many down- stream factors involved in SCW formation (Zhong et al., 2007a; Zhong and Ye, 2012; Ko et al., 2009, 2012; Kim et al., 2012, 2013a,b, 2014a,b). Overexpression analysis of AtMYB46 and time-course transcriptome analysis using an AtMYB46- dependent SCW formation induction system revealed that AtMYB46 can upregulate a number of SCW-biosynthesis genes (Zhong et al., 2007a; Zhong and Ye, 2012; Kim et al., 2013a,b, 2014a,b). Recently, independent groups deﬁned the cis-element sequence recognized by MYB46 and/or MYB83 as the secondary wall MYB-responsive element [SMRE; ACC(A/T)A(A/C)(T/C), Zhong and Ye, 2012] or MYB46-responsive cis-regulatory ele- ment [M46RE; (T/C)ACC(A/T)A(A/C)(T/C), Kim et al., 2012], both of which contain the AC element sequence originally iden- tiﬁed in the PAL gene promoter (Lois et al., 1989; Ohl et al., 1990; Leyva et al., 1992; Hatton et al., 1995; Wanner et al., May 2015 \| Volume 6 \| Article 288 Frontiers in Plant Science \| www.frontiersin.org 9 Regulation of secondary wall biosynthesis Nakano et al. TABLE 2 \| MYB genes related to SCW formation. Gene Name Locus or Transcript ID Accession No. Transcriptional activity References Arabidopsis thaliana AtMYB4 At4g38620 Repressor Jin et al., 2000 Wang and Dixon, 2011 AtMYB6 At4g09460 Unknown Zhong and Ye, 2012 AtMYB7 At2g16720 Repressor Wang and Dixon, 2011 Zhong and Ye, 2012 AtMYB20 At1g66230 Unknown Zhong et al., 2008 Nakano et al., 2010 AtMYB32 At4g34990 Repressor Preston et al., 2004 Wang and Dixon, 2011 AtMYB42 At4g12350 Unknown Zhong et al., 2008 AtMYB43 At5g16600 Unknown Zhong et al., 2008 Nakano et al., 2010 AtMYB46 At5g12870 Activator Ko et al., 2009 Zhong et al., 2007a Nakano et al., 2010 AtMYB52 At1g17950 Activator/ Zhong et al., 2008 Repressor Nakano et al., 2010 Cassan-Wang et al., 2013 AtMYB54 At1g73410 Activator Zhong et al., 2008 AtMYB58 At1g16490 Activator Zhou et al., 2009 AtMYB63 At1g79180 Activator Zhou et al., 2009 Nakano et al., 2010 AtMYB69 At4g33450 Activator Zhong et al., 2008 AtMYB83 At3g08500 Activator McCarthy et al., 2009 AtMYB85 At4g22680 Activator Zhong et al., 2008 Nakano et al., 2010 AtMYB99 At5g62320 Unknown Nakano et al., 2010 AtMYB103 At1g63910 Activator Zhong et al., 2008 TABLE 2 \| MYB genes related to SCW formation. Downstream Genes of Second-Layer Master Switch MYBs Eucalyptus gunnii EgMYB2 AJ576023 Activator Goicoechea et al., 2005 Oryza sativa OsMYB46 Os12g0515300/ LOC_Os12g33070 JN634084 Activator Zhong et al., 2011 Pinus taeda PtMYB1 AY356372 Activator Bomal et al., 2008 Patzlaff et al., 2003b PtMYB4 AY356371 Activator Patzlaff et al., 2003a PtMYB8 DQ399057 Activator Bomal et al., 2008 Populus trichocarpa PtrMYB002 Potri.001G258700 KF148677 Activator McCarthy et al., 2010 PtrMYB003 Potri.001G267300 KF148675 Activator Wilkins et al., 2009 PtrMYB020 Potri.009G061500 KF148676 Activator Zhong et al., 2013 PtrMYB021 Potri.009G053900 KF148678 Activator Zea mays ZmMYB46 JN634085 Activator Zhong et al., 2011 Eucalyptus gunnii EgMYB2 AJ576023 Activator Goicoechea et al., 2005 Oryza sativa OsMYB46 Os12g0515300/ LOC_Os12g33070 JN634084 Activator Zhong et al., 2011 Pinus taeda PtMYB1 AY356372 Activator Bomal et al., 2008 Patzlaff et al., 2003b PtMYB4 AY356371 Activator Patzlaff et al., 2003a PtMYB8 DQ399057 Activator Bomal et al., 2008 Populus trichocarpa PtrMYB002 Potri.001G258700 KF148677 Activator McCarthy et al., 2010 PtrMYB003 Potri.001G267300 KF148675 Activator Wilkins et al., 2009 PtrMYB020 Potri.009G061500 KF148676 Activator Zhong et al., 2013 PtrMYB021 Potri.009G053900 KF148678 Activator Zea mays ZmMYB46 JN634085 Activator Zhong et al., 2011 1995). A genomic survey of these cis-elements and DNA-protein binding assays suggest that the direct targets of AtMYB46/83 include genes encoding transcription factors (KNAT7, MYBs, and AtC3H14), the suite of SCW biosynthetic genes, includ- ing SCW-speciﬁc cellulose synthase genes (CesA4, CesA7, and CesA8), xylan biosynthetic genes (IRX7/FRA8, IRX8, IRX9, and IRX14), a mannan synthesis gene (CSLA9), and lignin biosyn- thetic genes (PAL1, C4H, 4CL1, C3H1, HCT, CCoAOMT, CCR1, F5H1, CAD6, and laccases), and genes related to cytoskeleton reg- ulation and signal transduction (Zhong and Ye, 2012; Kim et al., 2013a,b, 2014a,b,c). Of note, some AtMYB46/83 direct targets overlap with the direct targets of VNS proteins, such as CesA4 1995). A genomic survey of these cis-elements and DNA-protein binding assays suggest that the direct targets of AtMYB46/83 include genes encoding transcription factors (KNAT7, MYBs, and AtC3H14), the suite of SCW biosynthetic genes, includ- ing SCW-speciﬁc cellulose synthase genes (CesA4, CesA7, and CesA8), xylan biosynthetic genes (IRX7/FRA8, IRX8, IRX9, and IRX14), a mannan synthesis gene (CSLA9), and lignin biosyn- thetic genes (PAL1, C4H, 4CL1, C3H1, HCT, CCoAOMT, CCR1, F5H1, CAD6, and laccases), and genes related to cytoskeleton reg- ulation and signal transduction (Zhong and Ye, 2012; Kim et al., 2013a,b, 2014a,b,c). Downstream Genes of Second-Layer Master Switch MYBs Of note, some AtMYB46/83 direct targets overlap with the direct targets of VNS proteins, such as CesA4 May 2015 \| Volume 6 \| Article 288 Frontiers in Plant Science \| www.frontiersin.org 10 Regulation of secondary wall biosynthesis Nakano et al. and AtMYB7, close homologs of AtMYB4, also negatively reg- ulate several lignin biosynthesis genes (Jin et al., 2000; Preston et al., 2004). These repressor MYBs downregulate the expression of AtNST3/SND1 in vitro, and AtNST3/SND1 directly regulates AtMYB32 (Wang and Dixon, 2011). Based on this observation, negative feedback regulation of the VNS-MYB network for ﬁne- tuning of SCW formation has been suggested (Wang and Dixon, 2011; Figure 2). Moreover, the lignin-speciﬁc MYBs, AtMYB58, AtMYB63, and AtMYB85, regulate lignin biosynthesis, but not cellulose and hemicellulose deposition, because their overexpres- sion induced ectopic deposition of only lignin (Zhong et al., 2008; Zhou et al., 2009). These genes are expressed in lignifying cells such as xylem vessels and ﬁbers, and are probably regulated by both AtVNS and AtMYB46/83 (Zhong et al., 2006, 2007a,b; Ko et al., 2009; Kim et al., 2012; Zhong and Ye, 2012). AtMYB58 and AtMYB63 can bind the AC element, and AtMYB85 can activate the promoter activity of 4CL; thus they can activate the monolig- nol biosynthesis pathway (Zhong et al., 2008; Zhou et al., 2009). Recent work on AtMYB103 revealed that the myb103 mutant shows a strong reduction of syringyl lignin, possibly due to a decrease in F5H expression (Öhman et al., 2013). These results suggest that the primary function of AtMYB103 is in regulation of lignin biosynthesis and that AtMYB103 functions as one of the lignin-speciﬁc MYBs, although in vitro transient expression assays showed the AtMYB103 can upregulate CesA8 promoter activity (Zhong et al., 2008). FIGURE 4 \| The MYB46/83 genes function as second-layer master switches for secondary cell wall formation. (A) Ten-day-old Arabidopsis seedlings of wild type (wt), myb46, myb83, and myb46 myb83 mutants. The myb46 myb83 mutant shows growth inhibition in aerial parts. (B,C) Xylem vessels in the roots of the wild type (wt, B) and myb46 myb83 mutants (C). In the wild type, thick secondary cell wall is deposited in protoxylem-type (px) and metaxylem-type (mx) vessel cells (inset in B). In the myb46 myb83 mutant, secondary cell wall deposition in xylem vessel cells is strongly inhibited, as described in McCarthy et al. (2009). Bars = 1 cm (A) and 25 µm (B,C). Downstream Genes of Second-Layer Master Switch MYBs FIGURE 4 \| The MYB46/83 genes function as second-layer master switches for secondary cell wall formation. (A) Ten-day-old Arabidopsis seedlings of wild type (wt), myb46, myb83, and myb46 myb83 mutants. The myb46 myb83 mutant shows growth inhibition in aerial parts. (B,C) Xylem vessels in the roots of the wild type (wt, B) and myb46 myb83 mutants (C). In the wild type, thick secondary cell wall is deposited in protoxylem-type (px) and metaxylem-type (mx) vessel cells (inset in B). In the myb46 myb83 mutant, secondary cell wall deposition in xylem vessel cells is strongly inhibited, as described in McCarthy et al. (2009). Bars = 1 cm (A) and 25 µm (B,C). and CesA8 (Ohashi-Ito et al., 2010; Zhong et al., 2010c; Yam- aguchi et al., 2011). In vitro DNA-protein binding assays revealed that such “feed-forward” regulation often occurs in the transcrip- tional regulatory network in xylem cells (Taylor-Teeples et al., 2015). However, in the case of Arabidopsis, AtMYB46/83 appears to have a greater contribution than VNS, at least for cellulose synthase gene activation, as demonstrated by the severe defects in SCW deposition in xylem vessels of the myb46 myb83 double mutant (McCarthy et al., 2009; Figure 4) and the failed com- plementation of the cesa mutant phenotype by M46RE-mutated promoter-driven CesA genes (Kim et al., 2013a). Further study on the mechanisms by which VNS and MYB46/83 generate SCW will ﬁll the gap between the in vitro DNA-protein interactions and in vivo mutant phenotypes. The last group of MYB46/83-downstream MYBs contains AtMYB42, AtMYB43, AtMYB52, and AtMYB54, which are pref- erentially expressed in xylem tissues (Zhong et al., 2008; Nakano et al., 2010). However, their functions in SCW formation remain controversial. Overexpression of dominant-repressor forms of MYB52 and MYB54 inhibit SCW deposition in interfascicular ﬁbers and vessels, but overexpression of MYB52 and MYB54 produced no signiﬁcant changes (Zhong et al., 2008). Recently, AtMYB52 was suggested to negatively regulate SCW formation, because the myb52 mutant showed ectopic lignin deposition and the expression of AtMYB52 and SCW-related genes showed a high degree of correlation (Cassan-Wang et al., 2013). AtMYB46 and AtMYB83 can upregulate AtMYB43 (Nakano et al., 2010), but a detailed functional analysis remains to be performed. Frontiers in Plant Science \| www.frontiersin.org Other MYBs That Function in SCW Formation In addition to the genes for biosynthesis of SCW components, transcriptome analysis identiﬁed more than 40 transcription fac- tors downstream of AtMYB46/83 (Ko et al., 2009; Kim et al., 2012; Zhong and Ye, 2012). This list includes KNAT7, NACs, MYBs, and AtC3H14, some of which function in SCW formation (Zhong et al., 2008; Ko et al., 2009; Zhou et al., 2009; Li et al., 2011, 2012a; Wang and Dixon, 2011; Kim et al., 2012; Zhong and Ye, 2012; Cassan-Wang et al., 2013; Öhman et al., 2013). Here we focus on AtMYB transcription factors downstream of AtMYB46/83 and describe the other factors in subsequent sections. Other MYBs That Function in SCW Formation Several additional MYB transcription factors also participate in the regulation of SCW biosynthesis, probably in an MYB46/83- independent manner. Zhong et al. demonstrated that AtMYB20 and AtMYB69 also function downstream of AtNST1 and/or AtNST3/SND1, and are preferentially expressed in xylem cells (Zhong et al., 2006, 2007a,b, 2008). Dominant-repression analysis showed AtMYB69 is involved in the regulation of SCW forma- tion (Zhong et al., 2008), and AtMYB69 is among the top 60 genes co-expressed with AtMYB52 (Cassan-Wang et al., 2013). In addition, AtMYB75 has been shown to be involved in SCW formation of xylem tissues (Bhargava et al., 2010). AtMYB75 is also called PRODUCTION OF ANTHOCYANIN PIGMENT1 (PAP1), because this gene was earlier identiﬁed as a positive reg- ulator of anthocyanin biosynthesis (Borevitz et al., 2000). Pheno- types of loss-of-function mutant and overexpressor of AtMYB75 Before their identiﬁcation as direct targets of AtMYB46/83, several AtMYBs had been reported to function in lignin biosyn- thesis. AtMYB4, an active repressor, regulates the expression of C4H, which encodes a cinnamic acid 4-hydroxylase required for biosynthesis of all types of lignin monomers, and AtMYB32 May 2015 \| Volume 6 \| Article 288 Frontiers in Plant Science \| www.frontiersin.org 11 Regulation of secondary wall biosynthesis Nakano et al. 2008; Zhong et al., 2011, 2013; Zhao and Bartley, 2014). Com- monly, these MYBs can bind to the AC-rich elements, and over- expression of these MYBs upregulates lignin deposition (Patzlaﬀ et al., 2003a,b; Goicoechea et al., 2005; Bomal et al., 2008; Zhong et al., 2011, 2013). These facts strongly suggest evolutionary con- servation of second-layer master switch MYBs for SCW forma- tion among vascular plant species. Other MYBs That Function in SCW Formation However, given the diﬀerent characteristics of the SCW in diﬀerent plant species and the fact that MYBs represent one of the most-expanded gene families in plants, the numbers and functions of intermediate MYBs down- stream of MYB46/83 may have diversiﬁed among vascular plants. Engineering of lignin modiﬁcation is an important target for industrial uses of plant materials (Pauly and Keegstra, 2010; Sim- mons et al., 2010; Zeng et al., 2014); thus we anticipate further studies of MYBs in crop species, particularly in woody species and biofuels feedstocks. suggested that AtMYB75 negatively regulates SCW biosynthesis, especially for the branch of phenylpropanoid pathway connected to lignin biosynthesis (Bhargava et al., 2010). Nakano et al. (2010) identiﬁed AtMYB99 in a survey of factors upregulated during in vitro diﬀerentiation of xylem vessel cells. AtMYB99 is expressed in xylem vessel cells from early stages, suggesting its involvement in SCW formation in vessels (Nakano et al., 2010). However, the contribution of these genes to SCW formation in xylem tis- sues remains unknown. Further experiments will give us clues to elucidate the function of these MYBs. In addition to the MYBs described above, AtMYB26 has been known to regulate SCW formation in anther endothecium (Wil- son et al., 2011). Arabidopsis myb26 mutant, also known as male sterile35 mutant, lacks SCW in anther endothecium, resulting in a failure of anther dehiscence and male sterility (Dawson et al., 1999; Steiner-Lange et al., 2003). Similar phenotypes are found in nst1 nst2 double mutant (Mitsuda et al., 2005), and overex- pression of AtMYB26 induced ectopic deposition of SCW (Yang et al., 2007) as the cases of AtNST1 and AtNST2 overexpres- sion (Mitsuda et al., 2005). Interestingly, AtNST1 overexpres- sion can induce AtMYB26 expression (Mitsuda et al., 2005), and adversely AtMYB26 overexpression can upregulate AtNST1 and AtNST2 expressions (Yang et al., 2007). AtMYB26 shares rela- tively high sequence homology with AtMYB46/83, second-layer master switches of SCW biosynthesis in xylem (Zhao and Bartley, 2014). Therefore, these ﬁndings seem to suggest that in the case of anther endothecium, the relationship between NAC and MYB had been changed to make a positive transcriptional feedback loop rather than the transcriptional regulation cascade, proba- bly to make it possible to complete SCW biosynthesis in a short time of anther stage 11, which can be estimated up to 48 h (Smyth et al., 1990; Sanders et al., 1999), during another development. Frontiers in Plant Science \| www.frontiersin.org Other Transcription Factors Involved in SCW Formation Finally, we would like to mention additional important tran- scription factors involved in SCW formation. Two NAC tran- scription factors, AtSND2 and AtSND3, which are expressed in SCW-associated tissues, function downstream of VNS proteins for SCW formation (Zhong et al., 2008). Hussey and his co- workers showed that AtSND2 can inﬂuence almost all the reg- ulatory programs involved in SCW formation, i.e., biosynthesis of cellulose and hemicellulose, lignin polymerization, and sig- nal transduction, in addition to the expression of AtNST3/SND1 (Hussey et al., 2011). Overexpression of AtSND2 in Eucalyptus increased the thickness of the SCW in ﬁber cells; thus the molec- ular function of SND2 is basically conserved between herbaceous and woody plants (Hussey et al., 2011). However, overexpres- sion phenotypes diﬀer in the woody species and transgenic lines; the eﬀects of AtSND2 overexpression in Arabidopsis diﬀered between Zhong et al. (2008) and Hussey et al. (2011), and the AtSND2 overexpression in Eucalyptus showed the increase in SCW thickness, while overexpression of PopNAC154, one of poplar genes homologous to AtSND2, did not change SCW thick- ness in xylem tissues of poplar (Grant et al., 2010; Hussey et al., 2011). These observations suggest that AtSND2 and its orthologs function as key modulators of SCW formation, and the eﬀects of overexpression may change depending on the situation. Evolutionary Conservation of MYB46/83 Function as Second-Layer Master Switches for SCW Formation are now at a turning point in research on the transcriptional regulation of SCW biosynthesis: in addition to continuing our eﬀorts to identify genes involved in SCW formation and reveal the interactions between them on a one-on-one basis, we must move to the next steps. Thus, future research must aim to reveal the dynamism of the network itself based on observations of what happens in vivo, because modeling based on in vitro data only tells us the many possibilities of the network. The tran- scriptional regulatory network of SCW formation could become a good model for such advanced analysis in plants. Moreover, other types of transcription factors, namely the homeodomain protein KNOTTED ARABIDOPSIS THALIANA7 (KNAT7) and OVATE FAMILY PROTEIN 4 (OFP4), have been described as negative regulators of SCW biosynthesis. Arabidopsis KNAT7 was ﬁrst identiﬁed by co- expression analysis with SCW-related enzyme genes (Brown et al., 2005; Ehlting et al., 2005; Persson et al., 2005). The knat7 mutant showed a xylem phenotype similar to irx mutants; thus KNAT7 was also named IRX11 (Brown et al., 2005). AtNST3/SND1 and AtMYB46 directly target KNAT7 (Zhong et al., 2008; Ko et al., 2009). Li et al. revealed that KNAT7 func- tions as a transcriptional repressor, and that OFP4 can enhance KNAT7 activity via physical interaction. Both KANT7 and OFP4 are expressed in SCW-forming xylem cells (Li et al., 2011, 2012a), but, interestingly, the eﬀects of knat7 loss-of-function mutation diﬀer in xylem vessels and ﬁber cells. In xylem cells the SCW thickness decreased, leading to the irx phenotype (Brown et al., 2005; Li et al., 2012a). By contrast, the SCW thickness increased in ﬁber cells (Li et al., 2012a). Based on the identi- ﬁcation of several interaction partners of KNAT7, including OFP4, MYB75/PAP1, and BELL1-LIKE HOMEODOMAIN6 proteins (Hackbusch et al., 2005; Bhargava et al., 2010; Li et al., 2011; Liu et al., 2014), KNAT7 may regulate speciﬁc aspects of SCW formation, depending on cell type, through interaction with diﬀerent partners (Li et al., 2012a; Liu et al., 2014). The molecular function of KNAT7 is conserved with its poplar ortholog (Li et al., 2012a), and KNAT7 appears to have developed as a negative regulator to ﬁne-tune SCW biosynthesis to ﬁt the situation. Evolutionary Conservation of MYB46/83 Function as Second-Layer Master Switches for SCW Formation Second-Layer Master Switches for SCW Formation The data described above indicate a complex network of MYB-mediated transcriptional regulation of SCW formation (Figure 2). The VNS proteins act as the primary master switches of woody cell diﬀerentiation, and the MYB46/83 proteins act as secondary master switches of SCW formation. Downstream of the MYB46/83, several groups of MYBs mediate the tran- scriptional signals that regulate SCW biosynthetic processes; some MYBs speciﬁcally control lignin biosynthesis, and some MYBs repress or enhance SCW biosynthesis, at least partially (Figure 2). Signals can pass to the master switches from these MYBs (Wang and Dixon, 2011; Figure 2). SCW features, such as composition of cellulose, hemicellulose, and lignin, and S/G ratio of lignin, vary with tissue type, plant age, plant species, and environmental stress (Campbell and Sederoﬀ, 1996; Knox, 2008; Vogel, 2008; Pauly and Keegstra, 2010). Complex networks of MYBs likely operate as a ﬁne-tuning system for the formation of SCW with the appropriate composition for the speciﬁc situation. Work to date has identiﬁed several orthologs and putative functional homologs of AtMYB46 from vascular plants, includ- ing poplar, pine, spruce, rice, maize, and switchgrass (Panicum virgatum), in addition to PtrMYB4 and EgMYB2 (Patzlaﬀet al., 2003a,b; Goicoechea et al., 2005; Bedon et al., 2007; Bomal et al., Transcriptome analysis of an AtMYB46-overexpressing line identiﬁed the plant-speciﬁc tandem CCCH zinc-ﬁnger gene AtC3H14 as a direct target of AtNST3/SND1 and AtMYB46 (Ko et al., 2009). AtC3H14 can activate transcription of the genes for cellulose, hemicellulose, and lignin biosynthesis (Ko et al., 2009; Kim et al., 2014b). Also, a recent study proposed an addi- tional role of AtC3H14 in post-transcriptional regulation (Kim et al., 2014b). AtC3H14 can directly bind to mRNA in a target sequence-speciﬁc manner, similar to animal tandem CCCH zinc- ﬁnger proteins (Blackshear, 2002), and some cell wall-related genes seem to be binding targets of AtC3H14. Thus, AtC3H14 might participate in post-transcriptional and transcriptional reg- ulation of cell wall biosynthetic genes (Kim et al., 2014b). Further May 2015 \| Volume 6 \| Article 288 Frontiers in Plant Science \| www.frontiersin.org 12 Regulation of secondary wall biosynthesis Nakano et al. functional analysis of AtC3H14 might reveal a new regulatory layer in the current model of SCW formation by transcription factors. Evolutionary Conservation of MYB46/83 Function as Second-Layer Master Switches for SCW Formation g y p Comparative transcriptomic work revealed that the xylem transcriptomes of vascular plants are more highly conserved than the overall transcriptomes (Li et al., 2010), indicating the ancient origin of the xylem transcriptome. In accordance with this idea, recent work showed that the scheme of VNS-based transcrip- tional regulation of cell diﬀerentiation for wall thickening is con- served in the moss Physcomitrella patens (Figure 5), although P. patens does not have vascular plant-type SCW (Xu et al., 2014). The P. patens genome has eight VNS loci (Figure 3A), and the triple mutant ppvns1 ppvns6 ppvns7 of P. patens showed reduced wall thickness in stereid cells, which serve as supporting cells in mosses (Figure 5). Transcriptome analysis of P. patens over- expressing PpVNS7 indicated that PpVNS regulates many puta- tive orthologs of the direct targets of AtVNS, including putative orthologs of AtMYB46/83/103 and AtMYB85 (Xu et al., 2014). These ﬁndings suggest that the genes downstream of VNS are evolutionarily conserved, and that the VNS-MYB-based tran- scriptional regulatory system of wall modiﬁcation has an ancient root, at least at the common ancestors of mosses and vascular plants. Land plants would have developed this core regulatory scheme of cell wall modiﬁcation during evolution to adapt to new environments. As reviewed here, we now have extensive knowl- edge on the factors governing SCW formation in a wide range of plant species, which probably includes some species-speciﬁc ele- ments. What is the particular regulatory scheme for each species? Which parts of the common regulatory module are conserved Frontiers in Plant Science \| www.frontiersin.org References C., Douglas, C. J., and Ellis, B. E. (2010). MYB75 functions in regulation of secondary cell wall formation in the Arabidopsis inﬂorescence stem. Plant Physiol. 154, 1428–1438. doi: 10.1104/pp.110.162735 Demura, T., Tashiro, G., Horiguchi, G., Kishimoto, N., Kubo, M., Mat- suoka, N., et al. (2002). Visualization by comprehensive microarray anal- ysis of gene expression programs during transdiﬀerentiation of mesophyll cells into xylem cells. Proc. Natl. Acad. Sci. U.S.A. 99, 15794–15799. doi: 10.1073/pnas.232590499 Blackshear, P. J. (2002). Tristetraprolin and other CCCH tandem zinc-ﬁnger pro- teins in the regulation of mRNA turnover. Biochem. Soc. Trans. 30, 945–952. doi: 10.1042/BST0300945 De Geyter, N., Gholami, A., Goormachtig, S., and Goossens, A. (2012). Transcrip- tional machineries in jasmonate-elicited plant secondary metabolism. Trends Plant Sci. 17, 349–359. doi: 10.1016/j.tplants.2012.03.001 Bomal, C., Bedon, F., Caron, S., Mansﬁeld, S. D., Levasseur, C., Cooke, J. E. K., et al. (2008). Involvement of Pinus taeda MYB1 and MYB8 in phenylpropanoid metabolism and secondary cell wallbiogenesis: a comparative in planta analysis. J. Exp. Bot. 59, 3925–3939. doi: 10.1093/jxb/ern234 De Rybel, B., Möller, B., Yoshida, S., Grabowicz, I., Barbier de Reuille, P., Boeren, S., et al. (2013). A bHLH complex controls embryonic vascular tissue estab- lishment and indeterminate growth in Arabidopsis. Dev. Cell 24, 426–437. doi: 10.1016/j.devcel.2012.12.013 Borevitz, J. O., Xia, Y., Blount, J., Dixon, R. A., and Lamb, C. (2000). Activation tagging identiﬁes a conserved MYB regulator of phenylpropanoid biosynthesis. Plant Cell 12, 2383–2394. doi: 10.1105/tpc.12.12.2383 Dubos, C., Stracke, R., Grotewold, E., Weisshaar, B., Martin, C., and Lepiniec, L. (2010). MYB transcription factors in Arabidopsis. Trends Plant Sci. 15, 573–581. doi: 10.1016/j.tplants.2010.06.005 Brown, D. M., Zeef, L. A. H., Ellis, J., Goodacre, R., and Turner, S. R. (2005). Identi- ﬁcation of novel genes in Arabidopsis involved in secondary cell wall formation using expression proﬁling and reverse genetics. Plant Cell 17, 2281–2295. doi: 10.1105/tpc.105.031542 Duval, I., Lachance, D., Giguère, I., Bomal, C., Morency, M. J., Pelletier, G., et al. (2014). Large-scale screening of transcription factor–promoter interactions in spruce reveals a transcriptional network involved in vascular development. J. Exp. Bot. 65, 2319–2333. doi: 10.1093/jxb/eru116 Brown, D. M., Zhang, Z., Stephens, E., Dupree, P., and Turner, S. R. (2009). Char- acterization of IRX10 and IRX10-like reveals an essential role in glucuronoxy- lan biosynthesis in Arabidopsis. Plant J. 57, 732–746. doi: 10.1111/j.1365- 313X.2008.03729.x Ehlting, J., Mattheus, N., Aeschliman, D. S., Li, E., Hamberger, B., Cullis, I. F., et al. (2005). References Carlsbecker, A., Lee, J. Y., Roberts, C. J., Dettmer, J., Lehesranta, S., Zhou, J., et al. (2010). Cell signalling by microRNA165/6 directs gene dose-dependent root cell fate. Nature 465, 316–321. doi: 10.1038/nature08977 Allona, I., Quinn, M., Shoop, E., Swope, K., St Cyr, S., Carlis, J., et al. (1998). Anal- ysis of xylem formation in pine by cDNA sequencing. Proc. Natl. Acad. Sci. U.S.A. 95, 9693–9698. doi: 10.1073/pnas.95.16.9693 Carpita, N. C. (2012). Progress in the biological synthesis of the plant cell wall: new ideas for improving biomass for bioenergy. Curr. Opin. Biotechnol. 23, 330–337. doi: 10.1016/j.copbio.2011.12.003 Becker-André, M., Schulze-Lefert, P., and Hahlbrock, K. (1991). Structural com- parison, modes of expression, and putative cis-acting elements of the two 4-coumarate: CoA ligase genes in potato. J. Biol. Chem. 266, 8551–8559. Cassan-Wang, H., Goué, N., Saidi, M. N., Legay, S., Sivadon, P., Goﬀner, D., et al. (2013). Identiﬁcation of novel transcription factors regulating sec- ondary cell wall formation in Arabidopsis. Front. Plant Sci. 4:189. doi: 10.3389/fpls.2013.00189 Bedon, F., Grima-Pettenati, J., and Mackay, J. (2007). Conifer R2R3-MYB transcri- otion factors: sequence analyses and gene expression in wood-forming tissues of white spruce (Picea glauca). BMC Plant Biol. 7:17. doi: 10.1186/1471-2229- 7-17 Chinnusamy, V., Zhu, J., and Zhu, J. K. (2007). Cold stress regula- tion of gene expression in plants. Trends Plant Sci. 12, 444–451. doi: 10.1016/j.tplants.2007.07.002 Bell-Lelong, D. A., Cusumano, J. C., Meyer, K., and Chapple, C. (1997). Cinnamate-4-hydroxylase expression in Arabidopsis. Regulation in response to development and the environment. Plant Physiol. 113, 729–738. doi: 10.1104/pp.113.3.729 Cosgrove, D. J., and Jarvis, M. C. (2012). Comparative structure and biome- chanics of primary and secondary cell walls. Front. Plant Sci. 3:204. doi: 10.3389/fpls.2012.00204 Dawson, J., Sözen, E., Vizir, I., van Waeyenberge, S., Wilson, Z. A., and Mulli- gan, B. J. (1999). Characterization and genetic mapping of a mutation (ms35) which prevents anther dehiscence in Arabidopsis thaliana by aﬀecting sec- ondary wall thickening in the endothecium. New Phytol. 144, 213–222. doi: 10.1046/j.1469-8137.1999.00507.x Bennett, T., van den Toorn, A., Sanchez-Perez, G. F., Campiho, A., Willem- sen, V., Snel, B., et al. (2010). SOMBRERO, BEARSKIN1, and BEARSKIN2 regulate root cap maturation in Arabidopsis. Plant Cell 22, 640–654. doi: 10.1105/tpc.109.072272 Bergmann, D., and Sack, F. D. (2007). Stomatal development. Annu. Rev. Plant Biol. 58, 163–181. doi: 10.1146/annurev.arplant.58.032806.104023 Demura, T., and Fukuda, H. (2007). Transcriptional regulation in wood formation. Trends Plant Sci. 12, 64–70. doi: 10.1016/j.tplants.2006.12.006 Bhargava, A., Mansﬁeld, S. D., Hall, H. Conclusion and Perspectives During the last decade, extensive research has produced remark- able progress in our understanding of transcriptional regulation of SCW formation. Although some key modulators may remain unknown, the current model (Figure 2) covers the essential play- ers of SCW formation regulation. SCW in wood tissues provides a major source of land biomass, and SCW formation thus is an important target for biomass engineering. Several trials using the transcription factors described in this review to target SCW properties have been already reported (Carpita, 2012; Yang et al., 2013; Sakamoto and Mitsuda, 2015). Such applied research will assume growing importance in studies of the regulation of SCW formation. FIGURE 5 \| The PpVNS genes function in cell wall thickening in the moss P. patens. Stereid cells in leaf vein of the wild type (A) and ppvns1 ppvns6 pvns7 triple mutant (B). In the triple mutants, the stereid cell walls were signiﬁcantly less thick, suggesting the importance of PpVNS proteins in cell wall thickening in the moss. Data were adapted from Xu et al. (2014). Bar = 5 µm. In 2015, Taylor-Teeples et al. reported a protein–DNA interaction-based network between transcription factors and SCW metabolic genes of Arabidopsis (Taylor-Teeples et al., 2015). This map showed complex interactions among transcrip- tion factors and SCW metabolic genes, with many instances of feed-forward regulation (Figure 2A). In the protein–DNA interaction network, many kinds of transcription factors other than those previously reported could recognize the promoter sequence of transcription factors and SCW metabolic genes (Taylor-Teeples et al., 2015). This observation implies that we FIGURE 5 \| The PpVNS genes function in cell wall thickening in the moss P. patens. Stereid cells in leaf vein of the wild type (A) and ppvns1 ppvns6 pvns7 triple mutant (B). In the triple mutants, the stereid cell walls were signiﬁcantly less thick, suggesting the importance of PpVNS proteins in cell wall thickening in the moss. Data were adapted from Xu et al. (2014). Bar = 5 µm. May 2015 \| Volume 6 \| Article 288 13 Regulation of secondary wall biosynthesis Nakano et al. among land plants? Future work will provide clues to answer these questions. Conclusion and Perspectives to M.O., The Naito Foundation Subsidy for Female Researchers after Maternity Leave, and The Sumitomo Foundation for Grant for Basic Science Research Projects to M.O., and by Grants- in-Aid from the Japan Society for the Promotion of Science (grant numbers 24770052 and 25114520 to M.O, and 25840098 to M.Y.). Supplementary Material The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fpls.2015. 00288/abstract Acknowledgments We thank Dr. Taku Demura (Nara Institute of Science and Tech- nology) for critical discussions. We also thank Dr. Bo Xu and Mr. Nobuhiro Akiyoshi (Nara Institute of Science and Technol- ogy) for kindly providing the data of plant sections. This work was supported in part by Grants-in-Aid from the MEXT NC- CARP project to Y.N., and by a Start-up Grant for Women Researchers from the Nara Institute of Science and Technology References Revealing the structural and functional diversity of plant cell walls. Curr. Opin. Plant Biol. 11, 308–313. doi: 10.1016/j.pbi.2008.03.001 Hauﬀe, K. D., Lee, S. P., Subramaniam, R., and Douglas, C. J. (1993). Combinatorial interactions between positive and negative cis-acting elements control spatial patterns of 4CL-1 expression in transgenic tobacco. Plant J. 4, 235–253. doi: 10.1046/j.1365-313X.1993.04020235.x Ko, J. H., Jeon, H. W., Kim, W. C., and Han, K. H. (2014). The MYB46/MYB83- mediated transcriptional regulatory programme is a gatekeeper of secondary wall biosynthesis. Ann. Bot. 114, 1099–1107. doi: 10.1093/aob/mcu126 Ko, J. H., Kim, W. C., and Han, K. H. (2009). Ectopic expression of MYB46 identi- ﬁes transcriptional regulatory genes involved in secondary wall biosynthesis in Arabidopsis. Plant J. 60, 649–665. doi: 10.1111/j.1365-313X.2009.03989.x Heyn, A. N. J. (1955). Small particle X-ray scattering by ﬁbers, size and shape of microcrystallites. J. Appl. Phys. 26, 519–526. doi: 10.1063/1.1722032 Ko, J. H., Kim, W. C., Kim, J. Y., Ahn, S. J., and Han, K. H. (2012). MYB46- mediated transcriptional regulation of secondary cell wall biosynthesis. Mol. Plant 5, 961–963. doi: 10.1093/mp/sss076 Heyn, A. N. J. (1965). Observations on the size and shape of the cellulose micro- crystallite in cotton ﬁber by electron staining. J. Appl. Phys. 36, 2088. doi: 10.1063/1.1714417 Heyn, A. N. J. (1966). The microcrystalline structure of cellulose in cell walls of cotton, ramie, and jute ﬁbers as revealed by negative staining of sections. J. Cell Biol. 29, 181–197. doi: 10.1083/jcb.29.2.181 Ko, J. H., Yang, S. H., Park, A. H., Lerouxel, O., and Han, K. H. (2007). ANAC012, a member of the plant-speciﬁc NAC transcription factor family, negatively reg- ulates xylary ﬁber development in Arabidopsis thaliana. Plant J. 50, 1035–1048. doi: 10.1111/j.1365-313X.2007.03109.x Hu, R., Qi, G., Kong, Y., Kong, D., Gao, Q., and Zhou, G. (2010). Compre- hensive analysis of NAC domain transcription factor gene family in Populus trichocarpa. BMC Plant Biol. 10:145. doi: 10.1186/1471-2229-10-145 Krizek, B. A., and Fletcher, J. C. (2005). Molecular mechanisms of ﬂower develop- ment: an armchair guide. Nat. Rev. Genet. 6, 688–698. doi: 10.1038/nrg1675 Hussey, S. G., Mizrachi, E., Creux, N. M., and Myburg, A. A. (2013). Navigating the transcriptional roadmap regulating plant secondary cell wall deposition. Front. Plant Sci. 4:325. doi: 10.3389/fpls.2013.00325 Kubo, M., Udagawa, M., Nishikubo, N., Horiguchi, G., Yamaguchi, M., Ito, J., et al. (2005). Transcription switches for protoxylem and metaxylem vessel formation. Genes Dev. 19, 1855–1860. doi: 10.1101/gad.1331305 Larkin, J. C., Brown, M. References Global transcript proﬁling of primary stems from Arabidopsis thaliana identiﬁes candidate genes for missing links in lignin biosynthesis and transcriptional regulators of ﬁber diﬀerentiation. Plant J. 42, 618–640. doi: 10.1111/j.1365-313X.2005.02403.x Brown, D. M., Wightman, R., Zhang, Z., Gomez, L. D., Atanassov, I., Bukowski, J.- P., et al. (2011). Arabidopsis genes IRREGULAR XYLEM (IRX15) and IRX15L encode DUF579-containing proteins that are essential for normal xylan depo- sition in the secondary cell wall. Plant J. 66, 401–413. doi: 10.1111/j.1365- 313X.2011.04501.x Endo, H., Yamaguchi, M., Tamura, T., Nakano, Y., Nishikubo, N., Yoneda, A., et al. (2015). Multiple classes of transcription factors regulate the expression of VASCULAR-RELATED NAC-DOMAIN7, a master switch of xylem vessel diﬀerentiation. Plant Cell Physiol. 56, 242–254. doi: 10.1093/pcp/pcu134 Campbell, M. M., and Sederoﬀ, R. R. (1996). Variation in lignin content and composition. Plant Physiol. 110, 3–13. May 2015 \| Volume 6 \| Article 288 Frontiers in Plant Science \| www.frontiersin.org 14 Regulation of secondary wall biosynthesis Nakano et al. sunscreens in Arabidopsis. EMBO J. 19, 6150–6161. doi: 10.1093/emboj/19. 22.6150 Fang, Y., You, J., Xie, K., Xie, W., and Xiong, L. (2008). Systematic sequence analysis and identiﬁcation of tissue-speciﬁc or stress-responsive genes of NAC transcription factor family in rice. Mol. Genet. Genomics 280, 547–563. doi: 10.1007/s00438-008-0386-6 sunscreens in Arabidopsis. EMBO J. 19, 6150–6161. doi: 10.1093/emboj/19. 22.6150 Jin, H., and Martin, C. (1999). Multifunctionality and diversity within the plant MYB-gene family. Plant Mol. Biol. 41, 577–585. doi: 10.1023/A:1006319732410 Fukuda, H. (1997). Tracheary element diﬀerentiation. Plant Cell 9, 1147–1156. doi: 10.1105/tpc.9.7.1147 Jones, L., Ennos, A. R., and Turner, S. R. (2001). Cloning and characterization of irregular xylem4 (irx4): a severely lignin-deﬁcient mutant of Arabidopsis. Plant J. 2, 205–216. doi: 10.1046/j.1365-313x.2001.01021.x Geitmann, A. (2010). Mechanical modeling and structural analysis of the primary plant cell wall. Curr. Opin. Plant Biol. 13, 693–699. doi: 10.1016/j.pbi.2010.09.017 Kim, W. C., Kim, J. Y., Ko, J. H., Kim, J., and Han, K. H. (2013a). Transcrip- tion factor MYB46 is an obligate component of the transcriptional regula- tory complex for functional expression of secondary wall-associated cellu- lose synthases in Arabidopsis thaliana. J. Plant Physiol. 170, 1374–1378. doi: 10.1016/j.jplph.2013.04.012 Goicoechea, M., Lacombe, E., Legay, S., Mihaljevic, S., Rech, P., Jauneau, A., et al. (2005). EgMYB2, a new transcriptional activator from Eucalyptus xylem, regu- lates secondary cell wall formation and lignin biosynthesis. Plant J. 43, 553–567. doi: 10.1111/j.1365-313X.2005.02480.x Kim, W. C., Ko, J. H., and Han, K. H. (2012). References Identiﬁcation of a cis-acting reg- ulatory motif recognized by MYB46, a master regulator of secondary wall biosynthesis. Plant Mol. Biol. 78, 489–501. doi: 10.1007/s11103-012-9880-7 Grant, E. H., Fujino, T., Beers, E. P., and Brunner, A. M. (2010). Characteri- zation of NAC domain transcription factors implicated in control of vascu- lar cell diﬀerentiation in Arabidopsis and Populus. Planta 232, 337–352. doi: 10.1007/s00425-010-1181-2 Kim, W. C., Ko, J. H., Kim, J. Y., Kim, J., Bae, H. J., and Han, K. H. (2013b). MYB46 directly regulated the gene expression of secondary wall-associated cellulose synthases in Arabidopsis. Plant J. 73, 26–36. doi: 10.1111/j.1365- 313x.2012.05124.x Grima-Pettenati, J., Soler, M., Camargom, E., and Wang, H. (2012). “Transcrip- tional regulation of the lignin biosynthetic pathway revisited: new players and insights,” in Advances in Botanical Research, Vol. 61, eds L. Jouanin and C. Lapierre (Burlington, VT: Academic Press), 173–218. Kim, W. C., Kim, J. Y., Ko, J. H., Kang, H., and Han, K. H. (2014a). Identiﬁca- tion of direct targets of transcription factor MYB46 provides insights into the transcriptional regulation of secondary wall biosynthesis. Plant Mol. Biol. 85, 589–599. doi: 10.1007/s11103-014-0205-x Grotewold, E. (2006). The genetics and biochemistry of ﬂoral pigments. Ann. Rev. Plant Biol. 57, 761–780. doi: 10.1146/annurev.arplant.57.032905.105248 Hackbusch, J., Richter, K., Muller, J., Salamini, F., and Uhrig, J. F. (2005). A central role of Arabidopsis thaliana ovate family proteins in networking and subcellu- lar localization of 3-aa loop extension homeodomain proteins. Proc. Natl. Acad. Sci. U.S.A. 102, 4908–4912. doi: 10.1073/pnas.0501181102 Kim, W. C., Kim, J. Y., Ko, J. H., Kang, H., Kim, J., and Han, K. H. (2014b). AtC3H14, a plant-speciﬁc tandem CCCH zinc-ﬁnger protein, binds to its target mRNAs in a sequence-speciﬁc manner and aﬀects cell elongation in Arabidopsis thaliana. Plant J. 80, 772–784. doi: 10.1111/tpj.12667 Hamant, O., and Traas, J. (2010). The mechanics behind plant development. New Phytol. 185, 369–385. doi: 10.1111/j.1469-8137.2009.03100.x Kim, W. C., Reca, I. B., Kim, Y. S., Prk, S., Thomashow, M. F., Keegstra, K., et al. (2014c). Transcription factors that directly regulate the expression of CSLA9 encoding mannan synthase in Arabidopsis thaliana. Plant Mol. Biol. 84, 577–587. doi: 10.1007/s11103-013-0154-9 Hatton, D., Sablowski, R., Yung, M. H., Smith, C., Schuch, W., and Bevan, M. (1995). Two classes of cis sequences contribute to tissue-speciﬁc expres- sion of a PAL2 promoter in transgenic tobacco. Plant J. 7, 859–876. doi: 10.1046/j.1365-313X.1995.07060859.x Knox, J. P. (2008). References 56, 768–778. doi: 10.1111/j.1365-313X.2008.03633.x Patzlaﬀ, A., McInnis, S., Courtenay, A., Surman, C., Newman, L. J., Smith, C., et al. (2003a). Characterization of a pine MYB that regulates ligniﬁcation. Plant J. 36, 743–754. doi: 10.1046/j.1365-313X.2003.01916.x Mitsuda, N., Seki, M., Shinozaki, K., and Ohme-Takagi, M. (2005). The NAC tran- scription factors NST1 and NST2 of Arabidopsis regulate secondary wall thick- enings and are required for anther dehiscence. Plant Cell 17, 2993–3006. doi: 10.1105/tpc.105.036004 Patzlaﬀ, A., Mewman, L. J., Dubos, C., Whetten, R. W., Smith, C., McInnis, S., et al. (2003b). Characterization of PtMYB1, an R2R3-MYB from pine xylem. Plant Mol. Biol. 53, 597–608. doi: 10.1023/B:PLAN.0000019066.07933.d6 Pauly, M., and Keegstra, K. (2010). Plant cell wall polymers as precursors for biofuels. Curr. Opin. Plant Biol. 13, 304–311. doi: 10.1016/j.pbi.2009. 12.009 Miyashima, S., Koi, S., Hashimoto, T., and Nakajima, K. (2011). Non-cell- aoutonomous microRNA165 acts in a dose-dependent manner to regulate multiple diﬀerentiation status in the Arabidopsis root. Development 138, 2303–2313. doi: 10.1242/dev.060491 Pavy, N., Paule, C., Parsons, L., Crow, J. A., Morencey, M. J., Cooke, J., et al. (2005). Generation, annotation, analysis and database integration of 16,500 white spruce EST clusters. BMC Genomics 6:144. doi: 10.1186/1471-2164-6-144 Muñoz-Nortes, T., Wilson-Sánchez, D., Candela, H., and Micol, L. (2014). Symme- try, asymmetry, and the cell cycle in plants: known knowns and some known unknowns. J. Exp. Bot. 65, 2645–2655. doi: 10.1093/jxb/ert476 Peña, M. J., Zhong, R., Zhou, G. K., Richardson, E. A., O’Neil, M. A., Darvill, A. G., et al. (2007). Arabidopsis irregular xylem8 and irregular xylem9: implications for the complexity of glucuronoxylan biosynthesis. Plant Cell 19, 549–563. doi: 10.1105/tpc.106.049320 Myburg, A. A., Grattapaglia, D., Tuskan, G. A., Hellsten, U., Hayes, R. D., Grim- wood, J., et al. (2014). The genome of Eucalyptus grandis. Nature 510, 356–362. doi: 10.1038/nature13308 Persson, S., Wei, H., Milne, J., Page, G. P., and Somerville, C. R. (2005). Iden- tiﬁcation of genes required for cellulose synthesis by regression analysis of public microarray data sets. Proc. Natl. Acad. Sci. U.S.A. 102, 8633–8638. doi: 10.1073/pnas.0503392102 Nakano, Y., Nishikubo, N., Goué, N., Ohtani, M., Yamaguchi, M., Katayama, Y., et al. (2010). MYB transcription factors orchestrating the developmental pro- gram of xylem vessels in Arabidopsis roots. Plant Biotechnol. 27, 267–272. doi: 10.5511/plantbiotechnology.27.267 Petricka, J. J., Winter, C. M., and Benfey, P. N. (2012). Control of Arabidopsis root development. Annu. Rev. Plant Biol. 63, 563–590. References L., and Schiefelbein, J. (2003). How do cells know what they want to be when they grow up? Lessons from epider- mal patterning in Arabidopsis. Annu. Rev. Plant Biol. 54, 403–430. doi: 10.1146/annurev.arplant.54.031902.134823 Hussey, S. G., Mizrachi, E., Spokevicius, A. V., Bossinger, G., Berger, D. K., and Myburg, A. A. (2011). SND2, a NAC transcription factor gene, regulates genes involved in secondary cell wall development in Arabidopsis ﬁbres and increases ﬁbre cell area in Eucalyptus. BMC Plant Biol. 11:173. doi: 10.1186/1471-2229- 11-173 Le, D. T., Nishiyama, R., Watanabe, Y., Mochida, K., Yamaguchi-Shinozaki, K., Shinozaki, K., et al. (2011). Genome-wide survey and expression analysis of the plant-speciﬁc NAC transcription factor family in soybean during development and dehydration stress. DNA Res. 18, 263–276. doi: 10.1093/dnares/dsr015 Hussey, S. G., Saïdi, M. N., Hefer, C. A., Myburg, A. A., and Grima-Pettenati, J. (in press). Structural, evolutionary and functional analysis of the NAC domain protein family in Eucalyptus. New Phytol. doi: 10.1111/nph.13139 Lee, C., O’Neill, M. A., Tsumuraya, Y., Darvill, A. G., and Ye, Z. H. (2007). The irregular xylem9 mutant is deﬁcient in xylan xylosyltransferase activity. Plant Cell Physiol. 48, 1624–1634. doi: 10.1093/pcp/pcm135 Ishida, T., Kurata, T., Okada, K., and Wada, T. (2008). A genetic regulatory net- work in the development of trichomes and root hairs. Annu. Rev. Plant Biol. 59, 365–386. doi: 10.1146/annurev.arplant.59.032607.092949 Jensen, J. K., Kim, H., Cocuron, J. C., Orler, R., Ralph, J., and Wilkerson, C. G. (2011). The DUF579 domain containing proteins IRX15 and IRX15-L aﬀect xylan synthesis in Arabidopsis. Plant J. 66, 387–400. doi: 10.1111/j.1365- 313X.2010.04475.x Lepiniec, L., Debeaujon, I., Routaboul, J.-M., Baudry, A., Pourcel, L., Nesi, N., et al. (2006). Genetics and biochemistry of seed ﬂavonoids. Ann. Rev. Plant Biol. 57, 405–430. doi: 10.1146/annurev.arplant.57.032905.105252 Leyva, A., Liang, X., Pintor-Toro, J. A., Dixon, R. A., and Lamb, C. J. (1992). cis- Element combinations determine phenylalanine ammonia-lyase gene tissue- speciﬁc expression patterns. Plant Cell 4, 263–271. doi: 10.1105/tpc.4.3.263 Jin, H., Cominelli, E., Bailey, P., Parr, A., Mehrtens, F., Jones, J., et al. (2000). Trancriptional repression by AtMYB4 controls production of UV-protecting May 2015 \| Volume 6 \| Article 288 Frontiers in Plant Science \| www.frontiersin.org 15 Regulation of secondary wall biosynthesis Nakano et al. Li, E., Bhargava, A., Qiang, W., Friedmann, M. C., Forneris, N., Savidge, R. A., et al. (2012a). The Class II KNOX gene KNAT7 negatively regulates secondary wall formation in Arabidopsis and is functionally conserved in Populus. References New Phytol. 194, 102–115. doi: 10.1111/j.1469-8137.2011.04016.x Nystedt, B., Street, N. R., Wetterbom, A., Zuccolo, A., Lin, Y. C., Scoﬁeld, D. G., et al. (2013). The Norway spruce genome sequence and conifer genome evolution. Nature 497, 579–584. doi: 10.1038/nature12211 Oda, Y., and Fukuda, H. (2012). Initiation of cell wall pattern by a rho- and microtubule-driven symmetry breaking. Science 337, 1333–1336. doi: 10.1126/science.1222597 Li, E., Wang, S., Liu, Y., Chen, J. G., and Douglas, C. J. (2011). OVATE FAM- ILY PROTEIN4 (OFP4) interaction with KNAT7 regulates secondary cell wall formation in Arabidopsis thaliana. Plant J. 67, 328–341. doi: 10.1111/j.1365- 313X.2011.04595.x Oda, Y., and Fukuda, H. (2013a). Rho of plant GTPase signaling regulates the behavior of Arabidopsis kinesin-13A to establish secondary cell call patterns. Plant Cell 25, 4439–4450. doi: 10.1105/tpc.113.117853 Plant Cell 25, 4439–4450. doi: 10.1105/tpc.113.117853 Li, Q., Lin, Y. C., Sun, Y. H., Song, J., Chen, H., Zhang, X. H., et al. (2012b). Splice variant of the SND1 transcription factor is a dominant negative of SND1 mem- bers and their regulation in Populus trichocarpa. Proc. Natl. Acad. Sci. U.S.A. 109, 14699–14704. doi: 10.1073/pnas.1212977109 Oda, Y., and Fukuda, H. (2013b). The dynamic interplay of plasma membrane domains and cortical microtubules in secondary cell wall patterning. Front. Plant Sci. 4:511. doi: 10.3389/fpls.2013.00511 Li, X., Wu, H. X., and Southerton, S. G. (2010). Comparative genomics reveals con- servative evolution of the xylem transcriptome in vascular plants. BMC Evol. Biol. 10:190. doi: 10.1186/1471-2148-10-190 Ohashi-Ito, K., Matsukawa, M., and Fukuda, H. (2013a). An atypical bHLH tran- scription factor regulates early xylem development downstream of auxin. Plant Cell Physiol. 54, 398–405. doi: 10.1093/pcp/pct013 Ohashi-Ito, K., Oda, Y., and Fukuda, H. (2010). Arabidopsis VASCULAR- RELATED NAC-DOMAIN6 directly regulates the genes that govern pro- grammed cell death and secondary wall formation during xylem diﬀerentiation. Plant Cell 22, 3461–3473. doi: 10.1105/tpc.110.075036 Liu, Y., You, S., Taylor-Teeples, M., Li, W. L., Schuetz, M., Brady, S. M., et al. (2014). BEL1-LIKE HOMEODOMAIN6 and KNOTTED ARABIDOPSIS THALIANA7 interact and regulate secondary cell wall formation via repression of REVOLUTA. Plant Cell 26, 4843–4861. doi: 10.1105/tpc.114.128322 Lois, R., Dietrich, A., Hahlbrock, K., and Schulz, W. (1989). A phenylalanine ammonia-lyase gene from parsley: structure, regulation and identiﬁcation of elicitor and light responsive cis-acting elements. EMBO J. 8, 1641–1648. Ohashi-Ito, K., Oguchi, M., Kojima, M., Sakakibara, H., and Fukuda, H. (2013b). Auxin-associated initiation of vascular cell diﬀerentiation by LONESOME HIGHWAY. Development 140, 765–769. References doi: 10.1242/dev.087924 Ohl, S., Hedrick, S. A., Chory, J., and Lamb, C. J. (1990). Functional properties of a phenylalanine ammonia-lyase promoter from Arabidopsis. Plant Cell 2, 837–848. doi: 10.1105/tpc.2.9.837 Lorenz, W. W., and Dean, J. F. D. (2002). SAGE Proﬁling and demonstration of diﬀerential gene expression along the axial developmental gradient of lig- nifying xylem in loblolly pine (Pinus taeda). Tree Physiol. 22, 301–310. doi: 10.1093/treephys/22.5.301 Öhman, D., Demedts, B., Kumar, M., Gerber, L., Gorzsás, A., Goeminne, G., et al. (2013). MYB103 is required for FERULATE-5-HYDROXYLASE expression and syringyl lignin biosynthesis in Arabidopsis stems. Plant J. 73, 63–76. doi: 10.1111/tpj.12018 Marx-Figini, M. (1969). On the biosynthesis of cellulose in higher and lower plants. J. Polym. Sci. Part C 28, 57–67. doi: 10.1002/polc.5070280108 McCarthy, R. L., Zhong, R., Fowler, S., Lyskowski, D., Piyasena, H., Carleton, K., et al. (2010). The poplar MYB transcription factors, PtrMYB3 and PtrMYB20, are involved in the regulation of secondary wall biosynthesis. Plant Cell Physiol. 51, 1084–1090. doi: 10.1093/pcp/pcq064 j Ohtani, M., Nishikubo, N., Xu, B., Yamaguchi, M., Mitsuda, N., Goue, N., et al. (2011). A NAC domain protein family contributing to the regula- tion of wood formation in poplar. Plant J. 67, 499–512. doi: 10.1111/j.1365- 313X.2011.04614.x McCarthy, R. L., Zhong, R., and Ye, Z. H. (2009). MYB83 is a direct target of SND1 and acts redundantly with MYB46 in the regulation of secondary cell wall biosynthesis in Arabidopsis. Plant Cell Physiol. 50, 1950–1964. doi: 10.1093/pcp/pcp139 Olsen, A. N., Ernst, H. A., Leggio, L. L., and Skriver, K. (2005). NAC transcription factors: structurally distinct, functionally diverse. Trends Plant Sci. 10, 79–87. doi: 10.1016/j.tplants.2004.12.010 Mitsuda, N., Iwase, A., Yamamoto, H., Yoshida, M., Seki, M., Shinozaki, K., et al. (2007). NAC transcription factors, NST1 and NST3, are key regulators of the formation of secondary walls in woody tissues of Arabidopsis. Plant Cell 19, 270–280. doi: 10.1105/tpc.106.047043 Ooka, H., Satho, K., Doi, K., Nagata, T., Otomo, Y., Murakami, K., et al. (2003). Comprehensive analyisis of NAC family genes in Oryza sativa and Arabidopsis thaliana. DNA Res. 10, 239–247. doi: 10.1093/dnares/10.6.239 Pallady, S. G. (2008). “Chapter 2 – The woody plant body,” in Physiology of woody plants, 3rd Edn (San Diego, CA: Academic Press), 9–38. doi: 10.1016/B978- 012088765-1.50003-8 Mitsuda, N., and Ohme-Takagi, M. (2008). NAC transcription factors NST1 and NST3 regulate pod shattering in a partially redundant manner by promoting secondary wall formation after the establishment of tissue identity. Plant J. References 5, 297–303. doi: 10.1093/mp/ssr098 Rogers, L. A., and Campbell, M. M. (2004). The genetic control of lignin depo- sition during plant growth and development. New Phytol. 1, 17–30. doi: 10.1111/j.1469-8137.2004.01143.x Wanner, L. A., Li, G., Ware, D., Somssich, I. E., and Davis, K. R. (1995). The pheny- lalanine ammonia-lyase gene family in Arabidopsis thaliana. Plant Mol. Biol. 27, 327–338. doi: 10.1007/BF00020187 Romero, I., Fuertes, A., Benito, M. J., Malpica, J. M., Leyva, A., and Paz-Ares, J. (1998). More than 80 R2R3-MYB regulatory genes in the genome of Arabidopsis thaliana. Plant J. 14, 273–284. doi: 10.1046/j.1365-313X.1998.00113.x Weiner, D. H., Lindemose, S., Grossmann, J. G., Møllegaard, N. E., Olsen, A. N., Helgstrand, C., et al. (2012). DNA binding by the plant-speciﬁc NAC tran- scription factors in crystal and solution: a ﬁrm link to WRKY and GCM transcription factors. Biochem. J. 444, 395–404. doi: 10.1042/BJ20111742 Sakamoto, S., and Mitsuda, N. (2015). Reconstitution of a secondary cell wall in a secondary cell wall-deﬁcient Arabidopsis mutant. Plant Cell Physiol. 56, 299–310. doi: 10.1093/pcp/pcu208 Weisshaar, B., and Jenkins, G. I. (1998). Phenylpropanoid biosynthesis and its regulation. Curr. Opin. Plant Biol. 1, 251–257. doi: 10.1016/S1369-5266(98) 80113-1 Saito, K., Watanabe, Y., Shirakawa, M., Matsushita, Y., Imai, T., Koike, T., et al. (2012). Direct mapping of morphological distribution of syringyl and guaiacyl lignin in the xylem of maple by time-of-ﬂight secondary ion mass spectrometry. Plant J. 69, 542–552. doi: 10.1111/j.1365-313X.2011.04811.x Wilkins, O., Nahal, H., Foong, J., Provart, N. J., and Campbell, M. M. (2009). Expansion and diversiﬁcation of the Populus R2R3-MYB family of transcrip- tion factors. Plant Physiol. 149, 981–993. doi: 10.1104/pp.108.132795 Sanders, P. M., Bui, A. Q., Weterings, K., McIntire, K. N., Hsu, Y.-C., Lee, P. Y., et al. (1999). Anther developmental defects in Arabidopsis thaliana male-sterile mutants. Sex. Plant Reprod. 11, 297–322. Willemsen, V., Bauch, M., Bennett, T., Campiho, A., Wolkenfelt, H., Xu, J., et al. (2008). The NAC domain transcription factors FEZ and SOMBRERO control the orientation of cell division plane in Arabidopsis root stem cells. Dev. Cell 15, 923–922. doi: 10.1016/j.devcel.2008.09.019 Schuetz, M., Benske, A., Smith, R., Watanabe, Y., Tobimatsu, Y., Ralph, J., et al. (2014). Laccases direct ligniﬁcation in the discrete secondary cell wall domains of protoxylem. Plant Physiol. 166, 798–807. doi: 10.1104/pp.114.245597 Wilson, Z. A., Song, J., Taylor, B., and Yang, C. (2011). The ﬁnal split: the regulation of anther dehiscence. J. Exp. Bot. 62, 1633–1649. doi: 10.1093/jxb/err014 Simmons, B. References A., Loqué, D., and Ralph, J. (2010). Advances in modifying lignin for enhanced biofuel production. Curr. Opin. Plant Biol. 13, 312–319. doi: 10.1016/j.pbi.2010.03.001 Wolf, S., Hématy, K., and Höfte, H. (2012). Growth control and cell wall signaling in plants. Annu. Rev. Plant Biol. 63, 381–407. doi: 10.1146/annurev-arplant- 042811-105449 Smyth, D. R., Bowman, J. L., and Meyerowitz, E. M. (1990). Early ﬂower develop- ment in Arabidopsis. Plant Cell 2, 755–767. doi: 10.1105/tpc.2.8.755 Wu, A. M., Rihouey, C., Seveno, M., Hörnblad, E., Singh, S. K., Matsunaga, T., et al. (2009). The Arabidopsis IRX10 and IRX10-LIKE glycosyltransferases are criti- cal for glucuronoxylan biosynthesis during secondary cell wall formation. Plant J. 57, 718–731. doi: 10.1111/j.1365-313X.2008.03724.x Soler, M., Camargo, E. L. O., Carocha, V., Cassan-Wang, H., Clemente, H. S., Savelli, B., et al. (in press). The Eucalyptus grandis R2R3-MYB transcription factor family: evidence for woody growth-related evolution and function. New Phytol. doi: 10.1111/nph.13039 Xu, B., Ohtani, M., Yamaguchi, M., Toyooka, K., Wakazaki, M., Sato, M., et al. (2014). Contribution of NAC transcription factors of plant adaptation to land. Science 343, 1505–1508. doi: 10.1126/science.1248417 Soyano, T., Thitamadee, S., Machida, Y., and Chua, N. H. (2008). ASYMMET- RIC LEAVES2-LIKE19/LATERAL ORGAN BOUNDARIES DOMAIN30 and ASL20/LBD18 regulate tracheary element diﬀerentiation in Arabidopsis. Plant Cell. 20, 3359–3373. doi: 10.1105/tpc.108.061796 Yamaguchi, M., and Demura, T. (2010). Transcriptional regulation of secondary wall formation controlled by NAC domain proteins. Plant Biotechnol. 27, 237–242. doi: 10.5511/plantbiotechnology.27.237 Steiner-Lange, S., Unte, U. S., Eckstein, L., Yang, C., Wilson, Z. A., Schmelzer, E., et al. (2003). Disruption of Arabidopsis thaliana MYB26 results in male sterility due to non-dehiscent anthers. Plant J. 34, 519–528. doi: 10.1046/j.1365- 313X.2003.01745.x Yamaguchi, M., Goué, N., Igarashi, H., Ohtani, M., Nakano, Y., Mortimer, J. C., et al. (2010a). VASCULAR-RELATED NAC-DOMAIN6 and VASCULAR- RELATED NAC-DOMAIN7 eﬀectively induce transdiﬀerentiation into xylem vessel elements under control of an induction system. Plant Physiol. 153, 906–914. doi: 10.1104/pp.110.154013 Sterky, F., Bhalerao, R. R., Unneberg, P., Segerman, B., Nilsson, P., Brunner, A. M., et al. (2004). A populus EST resources for plant functional genomics. Proc. Natl. Acad. Sci. U.S.A. 101, 13951–13956. doi: 10.1073/pnas.0401641101 Yamaguchi, M., Kubo, M., Fukuda, H., and Demura, T. (2008). VASCULAR- RELATED NAC-DOMAIN7 is involved in the diﬀerentiation of all types of xylem vessels in Arabidopsis roots and shoots. Plant J. 55, 652–664. doi: 10.1111/j.1365-313X.2008.03533.x Stracke, R., Werber, M., and Weisshaar, B. (2001). The R2R3-MYB gene family in Arabidopsis thaliana. Curr. Opin. Plant Biol. 4, 447–456. References doi: 10.1146/annurev- arplant-042811-105501 Nakashima, K., Takasaki, H., Mizoi, J., Shinozaki, K., and Yamaguchi-Shinozaki, K. (2012). NAC transcription factors in plant abiotic stress responses. Biochim. Biophys. Acta. 1819, 97–103. doi: 10.1016/j.bbagrm.2011.10.005 Preston, J., Wheeler, J., Heazlewood, J., Li, S. F., and Parish, R. W. (2004). AtMYB32 is required for normal pollen development in Arabidopsis thaliana. Plant J. 40, 979–995. doi: 10.1111/j.1365-313X.2004.02280.x Nuruzzaman, M., Manimekalai, R., Sharoni, A. M., Satoh, K., Kondoh, H., Ooka, H., et al. (2010). Genome-wide analysis of NAC transcription factor family in rice. Gene 465, 30–44. doi: 10.1016/j.gene.2010.06.008 May 2015 \| Volume 6 \| Article 288 Frontiers in Plant Science \| www.frontiersin.org 16 Regulation of secondary wall biosynthesis Nakano et al. Puranik, S., Sahu, P. P., Srivastava, P. S., and Prasad, M. (2012). NAC proteins: regulation and role in stress tolerance. Trends Plant Sci. 17, 369–381. doi: 10.1016/j.tplants.2012.02.004 Valdivia, E. R., Herrera, M. T., Gianzo, C., Fidalgo, J., Revilla, G., Zarra, I., et al. (2013). Regulation of secondary wall synthesis and cell death by NAC tran- scription factors in the monocot Brachypodium distachyon. J. Exp. Bot. 64, 1333–1343. doi: 10.1093/jxb/ers394 Pyo, H., Demura, T., and Fukuda, H. (2007). TERE; a novel cis-element responsi- ble for a coordinated expression of genes related to programmed cell death and secondary wall formation during diﬀerentiation of tracheary elements. Plant J. 51, 955–965. doi: 10.1111/j.1365-313X.2007.03180.x Valliyodan, B., and Nguyen, H. (2006). Understanding regulatory networks and engineering for enhanced drought tolerance in plants. Curr. Opin. Plant Biol. 9, 189–195. doi: 10.1016/j.pbi.2006.01.019 Rengel, D., Clemente, H. S., Servant, F., Ladouce, N., Paux, E., Wincker, P., et al. (2009). A new genomic resource dedicated to wood formation in Eucalyptus. BMC Plant Biol. 9:36. doi: 10.1186/1471-2229-9-36 Vogel, J. (2008). Unique aspects of the grass cell wall. Curr. Opin. Plant Biol. 11, 301–307. doi: 10.1016/j.pbi.2008.03.002 Wang, H., Avci, U., Nakashima, J., Hahn, M. G., Chen, F., and Dixon, R. A. (2010). Mutation of WRKY transcription factors initiates pith secondary wall forma- tion and increases stem biomass in dicotyledonous plants. Proc. Natl. Acad. Sci. U.S.A. 107, 22338–22343. doi: 10.1073/pnas.1016436107 Reusche, M., Thole, K., Janz, D., Truskina, J., Rindﬂeisch, S., Drubert, C., et al. (2012). Verticillium infection triggers VASCULAR-RELATED NAC DOMAIN7-dependent de novo xylem formation and enhances drought toler- ance in Arabidopsis. Plant Cell 24, 3823–3837. doi: 10.1105/tpc.112.103374 U.S.A. 107, 22338–22343. doi: 10.1073/pnas.1016436107 Wang, H. Z., and Dixon, R. A. (2011). On-oﬀswitches for secondary cell wall biosynthesis. Mol. Plant. References doi: 10.1016/S1369- 5266(00)00199-0 Taylor, N. G., Howells, R. M., Huttly, A. K., Vickers, K., and Turner, S. R. (2003). Interactions among three distinct CesA proteins essential for cellulose synthe- sis. Proc. Natl. Acad. Sci. U.S.A. 100, 1450–1455. doi: 10.1073/pnas.0337628100 Yamaguchi, M., Mitsuda, N., Ohtani, M., Ohme-Takagi, M., and Demura, T. (2011). VASCULAR-RELATED NAC-DOMAIN 7 directly regulates the expres- sion of broad range of genes for xylem vessel formation. Plant J. 66, 579–590. doi: 10.1111/j.1365-313X.2011.04514.x Taylor-Teeples, M., Lin, L., de Lucas, M., Turco, G., Toal, T. W., Gaudinier, A., et al. (2015). An Arabidopsis gene regulatory network for secondary cell wall synthesis. Nature 517, 571–575. doi: 10.1038/nature14099 Yamaguchi, M., Nagahage, I. S. P., Ohtani, M., Ishikawa, T., Uchimiya, H., Kawai-Yamada, M., et al. (in press). Arabidopsis NAC domain proteins VND- INTERACTING1 and ANAC103 interact with multiple NAC domain proteins. Plant Biotechnol. doi: 10.5511/plantbiotechnology.15.0208a Turner, S. R., and Somerville, C. R. (1997). Collapsed xylem phenotype of Ara- bidopsis identiﬁes mutants deﬁcient in cellulose deposition in the secondary cell wall. Plant Cell 9, 689–701. doi: 10.1105/tpc.9.5.689 Yamaguchi, M., Ohtani, M., Mitsuda, N., Kubo, M., Ohme-Takagi, M., Fukuda, H., et al. (2010b). VND-INTERACTING2, a NAC domain transcription fac- tor, negatively regulates xylem vessel formation in Arabidopsis. Plant Cell 22, 1249–1263. doi: 10.1105/tpc.108.064048 Tuskan, G. A., DiFazio, S., Jansson, S., Bohlmann, J., Grigoriev,. I., Hellsten, U., et al. (2006). The genome of black cottonwood, Populus trichocarpa (Torr. & Gray). Science 15, 1596–1604. doi: 10.1126/science.1128691 May 2015 \| Volume 6 \| Article 288 Frontiers in Plant Science \| www.frontiersin.org 17 Regulation of secondary wall biosynthesis Nakano et al. Yang, C., Xu, Z., Song, J., Conner, K., Vizcay Barrena, G., and Wilson, Z. A. (2007). Arabidopsis MYB26/MALE STERILE35 regulates secondary thickening in the endothecium and is essential for anther dehiscence. Plant Cell 19, 534–548. doi: 10.1105/tpc.106.046391 Zhong, R., Lee, C., and Ye, Z. H. (2010c). Global analysis of direct targets of sec- ondary wall NAC master switches in Arabidopsis. Mol. Plant. 3, 1087–1103. doi: 10.1093/mp/ssq062 p q Zhong, R., Lee, C., Zhou, J., McCarthy, R. L., and Ye, Z. H. (2008). A battery of tran- scription factors involved in the regulation of secondary cell wall biosynthesis Zhong, R., Lee, C., Zhou, J., McCarthy, R. L., and Ye, Z. H. (2008). A battery of tran- scription factors involved in the regulation of secondary cell wall biosynthesis in Arabidopsis. Plant Cell 20, 2763–2782. References doi: 10.1105/tpc.108.061325 Yang, F., Mitra, P., Zhang, L., Prak, L., Verhertbruggen, Y., Kim, J. S., et al. (2013). Engineering secondary cell wall deposition in plants. Plant Biotechnol. J. 11, 325–335. doi: 10.1111/pbi.12016 Zhong, R., McCarthy, R. L., Haghighat, M., and Ye, Z. H. (2013). The poplar MYB master switches bind to the SMRE site and activate the secondary wall biosynthetic program during wood formation. PLoS ONE 8:e69219 doi: 10.1371/journal.pone.0069219 Yanhui, C., Xiaoyuan, Y., Kun, H., Meihua, L., Jigang, L., Zhaofeng, G., et al. (2006). The MYB transcription factor superfamily of Arabidopsis: expression analysis and phylogenetic comparison with the rice MYB family. Plant Mol. Biol. 60, 107–124. doi: 10.1007/s11103-005-2910-y Zhong, R., Richardson, E. A., and Ye, Z. H. (2007a). The MYB46 transcription fac- tor is a direct target of SND1 and regulates secondary cell wall biosynthesis in Arabidopsis. Plant Cell 19, 2776–2791. doi: 10.1105/tpc.107.053678 Yoshida, K., Sakamoto, S., Kawai, T., Kobayashi, Y., Sato, K., Ichinose, Y., et al. (2013). Engineering the Oryza sativa cell wall with rice NAC transcrip- tion factors regulating secondary wall formation. Front. Plant Sci. 4:383. doi: 10.3389/fpls.2013.00383 rabidopsis. Plant Cell 19, 2776–2791. doi: 10.1105/tpc.107.053678 Zhong, R., Richardson, E. A., and Ye, Z. H. (2007b). Two NAC domain tran- scription factors, SND1 and NST1, function redundantly in regulation of sec- ondary wall synthesis in ﬁbers of Arabidopsis. Planta 225, 1603–1611. doi: 10.1007/s00425-007-0498-y Zeng, Y., Zhao, S., Yang, S., and Ding, S. Y. (2014). Lignin plays a negative role in the biochemical process for producing lignocellulosic biofuels. Curr. Opin. Biotechnol. 27, 38–45. doi: 10.1016/j.copbio.2013.09.008 Zhong, R., and Ye, Z. H. (2012). MYB46 and MYB83 bind to the SMRE sites and directly activate a suit of transcription factors and secondary wall biosynthetic genes. Plant Cell Physiol. 53, 368–380. doi: 10.1093/pcp/pcr185 Zhao, K., and Bartley, L. E. (2014). Comparative genomic analysis of the R2R3 MYB secondary cell wall regulators of Arabidopsis, poplar, rice, maize, and switchgrass. BMC Plant Biol. 14:135. doi: 10.1186/1471-2229-14-135 Zhou, J., Lee, C., Zhong, R., and Ye, Z. H. (2009). MYB58 and MYB63 are tran- scriptional activators of the lignin biosynthetic pathway during secondary cell wall formation in Arabidopsis. Plant Cell 21, 248–266. doi: 10.1105/tpc.108. 063321 Zhao, Q., Wang, H., Yin, Y., Xu, Y., Chen, F., and Dixon, R. A. (2010). Syringyl lignin biosynthesis is directly regulated by a secondary cell wall master switch. Proc. Natl. Acad. Sci. U.S.A. 107, 14496–14501. Frontiers in Plant Science \| www.frontiersin.org References doi: 10.1073/pnas.1009170107 Zhou, J., Zhong, R., and Ye, Z. H. (2014). Arabidopsis NAC domain proteins, VND1 to VND5, are transcriptional regulators of secondary wall biosynthesis in vessels. PLoS ONE 9:e105726. doi: 10.1371/journal.pone.0105726 Zhao, Y., Sun, J., Xu, P., Zhang, R., and Li, L. (2014). Intron-mediated alternative splicing of WOOD-ASSOCIATED NAC TRANSCRIPTION FACTOR1B reg- ulates cell wall thickening during ﬁber development in Populus species. Plant Physiol. 164, 765–776. doi: 10.1104/pp.113.231134 Zhu, T., Nevo, E., Sun, D., and Peng, J. (2012). Phylogenic analyses unravel the evolutionary of NAC proteins in plants. Evolution 66, 1833–1848. doi: 10.1111/j.1558-5646.2011.01553.x Zhong, R., Demura, T., and Ye, Z. H. (2006). SND1, a NAC domain transcription factor, is a key regulator of secondary wall synthesis in ﬁbers of Arabidopsis. Plant Cell 18, 3158–3170. doi: 10.1105/tpc.106.047399 Zhong, R., Lee, C., McCarthy, R. L., Reeves, C. K., Jones, E. G., and Ye, Z. H. (2011). Transcriptional activation of secondary wall biosynthesis by rice and maize NAC and MYB transcription factors. Plant Cell Physiol. 52, 1856–1871. doi: 10.1093/pcp/pcr123 Conﬂict of Interest Statement: The authors declare that the research was con- ducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. Conﬂict of Interest Statement: The authors declare that the research was con- ducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. Zhong, R., Lee, C., and Ye, Z. H. (2010a). Evolutionary conservation of the tran- scriptional network regulating secondary cell wall biosynthesis. Trends Plant Sci. 15, 625–632. doi: 10.1016/j.tplants.2010.08.007 Copyright © 2015 Nakano, Yamaguchi, Endo, Rejab and Ohtani. This is an open- access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publica- tion in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Zhong, R., Lee, C., and Ye, Z. H. (2010b). Functional characterization of poplar wood associated NAC domain transcription factors. Plant Physiol. 152, 1044–1055. doi: 10.1104/pp.109.148270 May 2015 \| Volume 6 \| Article 288 Frontiers in Plant Science \| www.frontiersin.org 18
https://openalex.org/W4362579178	https://link.springer.com/content/pdf/10.1007/s00170-023-11251-1.pdf	English	null	The effect of geometric design and materials on section properties of additively manufactured lattice elements	The international journal of advanced manufacturing technology/International journal, advanced manufacturing technology	2,023	cc-by	10,431	The effect of geometric design and materials on section properties of additively manufactured lattice elements Received: 19 August 2022 / Accepted: 9 March 2023 © The Author(s) 2023 / Published online: 3 April 2023 https://doi.org/10.1007/s00170-023-11251-1 The International Journal of Advanced Manufacturing Technology (2023) 126:3555–3577 https://doi.org/10.1007/s00170-023-11251-1 The International Journal of Advanced Manufacturing Technology (2023) 126:3555–3577 https://doi.org/10.1007/s00170-023-11251-1 The International Journal of Advanced Manufacturing Technology (2023) 126:3555–3577 ORIIGINAL ARTICLE Abstract Keywords Laser-based powder bed fusion · Additive manufacturing · Geometric analysis · Micro-computed tomography · Additive manufacturing defects · Design for additive manufacturing Nomenclature AM Additive manufacturing BJT Binder jetting technology CAD Computer-aided design CSP Cold spray DED Directed energy deposition DFAM Design for additive manufacturing DOE Design of experiments IQR Interquartile range LB-PBF Laser-based powder bed fusion MAM Metal additive manufacturing MEX Material extrusion MJF Multi jet fusion MJT Material jetting technology PBF Powder bed fusion PBS Powder bed system PFS Powder feed system SEM Scanning electron microscope SHL Sheet lamination WFS Wire feed system µCT Micro-computed tomography Notation Term Definition Unit ACT Cross-sectional area of the as-manufactured case mm2 Aideal Cross-sectional area of the idealised case mm2 Cp Specific heat J/(kg.K) Deff Effective diameter mm h Thermal diffusivity m2/s I Second moment of area mm4 ICT Second moment of area of the as-manufactured case mm4 ICT,max Maximum second moment of area of the as- manufactured case mm4 ICT,min Minimum second moment of area of the as- manufactured case mm4 * Abduladheem Almalki s3795686@student.rmit.edu.au Martin Leary martin.leary@rmit.edu.au 1 RMIT Centre for Additive Manufacture, RMIT University, Melbourne, Australia 2 ARC Training Centre in Additive Biomanufacturing, Kelvin Grove, Australia 3 MD Anderson Cancer Center, Houston, TX, United States 4 Umm Al Qura University, Makkah, Saudi Arabia (0121 3456789) 3 3556 The International Journal of Advanced Manufacturing Technology (2023) 126:3555–3577 Iideal Second moment of area of the idealised case mm4 k Thermal conductivity W/K h Elastic bending shape factor mm4/mm4 ∅e B Elastic bending shape factor of the as-manufac- tured case mm4/mm4 ∅e B,ideal Elastic bending shape factor of the idealised case mm4/mm4 ∅f B Failure bending shape factor mm3/mm3 ∅f B,CT Failure bending shape factor of the as-manufac- tured case mm3/mm3 ∅f B,ideal Failure bending shape factor of the idealised case mm3/mm3 p Polygon order integer Rg Radius of gyration mm Rg,CT Radius of gyration of the as-manufactured case mm Rg,ideal Radius of gyration of the idealised case mm s Polygon side length mm Z Section modulus of any polygon shape mm3 Zideal Section modulus of the idealised case mm3 Zcircle Section modulus of the circle of equal area mm3 αap Apparent angle degree αed Edge angle degree αin Inclination angle degree 휃rotation Rotation angle degree 휌 Density kg/m3 geometric properties rather than the fundamental section properties that determine structural performance. Abstract In response to this shortcoming, a methodology is proposed to algorithmically quantify the section properties, including the second moment of area and shape factor of as-manufactured strut elements. This methodology is demonstrated with a design of experiments (DOE) that considers a range of design-relevant control factors for strut elements fabricated by laser-based powder bed fusion (LB-PBF), including various cross-section shapes (triangular face up, triangular face down, square, octagonal, and circle), materials (AlSi10Mg and Ti6Al4V), and strut element inclination angles, 훼in (35°, 45°, 90°). To enable the algorithmic characterisation of the geometric attributes of these as-manufactured lattice strut elements, micro- computed tomography (μCT) imaging techniques are applied to characterise the three-dimensional geometry of manufactured specimens [5]. This outcome contributes to the fundamental understanding of analytical and as-manufactured quantification methods for AM strut elements, as well as providing a robust DFAM tool for lattice certification and structure optimisation. 1.1 Metal additive manufacturing As a sub-classification of AM, MAM processes sequentially fabricate three-dimensional metal components based on dig- ital CAD data [6, 7]. Fabrication using MAM is typically achieved by the iterative fusion, or adhesion, of metallic input material over a series of layers based on cross-sec- tions taken from digital design data [7]. Prominent exam- ples include sheet lamination (SHL), binder jet technology (BJT), material jetting technology (MJT), material extrusion (MEX), powder bed fusion (PBF), and directed energy depo- sition (DED) [8]. MAM provides an opportunity to fabricate novel, mass-optimised, or high-value components such as medical implants and lightweight aerospace components. In this research, LB-PBF is used to fabricate individual strut element specimens due to the compatibility of this method with the fabrication of high-resolution features at small scales with a high degree of dimensional control [9]. Abstract Additive manufacturing (AM) technologies such as laser-based powder bed fusion (LB-PBF) facilitate the fabrication of complex lattice structures. However, these structures consistently display dimensional variation between the idealised and as-manufactured specimens. This research proposes a method to characterise the impact of common LB-PBF powders (alu- minium and titanium alloys) and geometric design parameters (polygon order, effective diameter, and inclination angle) on section properties relevant to stiffness and strength of as-manufactured strut elements. Micro-computed tomography (µCT) has been applied to algorithmically characterise the as-manufactured variation and identify a scale threshold below which additional geometric resolution does not influence the section properties of as-manufactured parts. This methodology pro- vides a robust and algorithmic design for additive manufacturing (DFAM) tool to characterise the effects of manufacturing and design parameters on the functional response of AM strut elements, as is required for certification and optimisation. 1.2 Mechanical response of lattice strut elements Lattice structures are commonly employed in applications subject to compressive or absorption loading conditions. The mechanical response of individual strut elements depends on the associated unit cell topology and loading conditions. The strut element loading response can be categorised as either bending-dominated or stretch-dominated [21], resulting in strut elements subjected predominantly to bending moments or axial loads, as illustrated in Fig. 1a,b, respectively. The structural response of the lattice can be predicted by Max- well’s stability criterion which considers pin-jointed struc- tures and predicts the determinacy of the structure based on the number of struts and nodes [22, 23]. Strut element cross-section properties contribute substantially to structural response, especially for bending and buckling modes. This research provides insight into the structural efficiency of as-manufactured strut elements according to the associated material and geometric design parameters. The primary commercial opportunity for AM production is typically high-value applications; consequently, methods to predict and quantify the magnitude and influence of AM defects in as-manufactured components are of critical impor- tance [19, 20]. Echeta et al. [11] comprehensively reviewed the primary measurement methods available for quantify- ing MAM defects. These methods include μCT, scanning 1 Introduction Insufficient energy density or using inappropriate process parameters may cause balling effects on the surface of the fabricated object. The main driving mechanism for a balling defect in LB-PBF is Rayleigh instability and a lack of wetting which produces a segmented melt pool with the associated formation of ball shapes [12, 13]. Residual stresses are formed during the PBF processes due to rapid temperature cycling rates that can cause cracking in lattice strut elements [14, 15]. Further- more, shrinkage occurs during the phase transition from a liquid melt pool to a solid structure, potentially leading to surface cracking. Surface texture variation is observed on MAM lattice structures, which is caused by several phe- nomena. Layer-wise fabrication methods employed during MAM inherently lead to the stair-stepping phenomenon [11, 16]. This is most clearly observed on surfaces inclined to the build direction, with a distinct characteristic surface rough- ness on both sides of the inclined surface of a lattice struc- ture [17]. The magnitude of roughness on both the upward and downward faces of struts is a function of the inclination angle as well as the associated process parameters. This as- manufactured roughness may provide advantages in biomed- ical applications where it can be beneficial as a biomimetic surface encouraging cellular adhesion [18]. electron microscope (SEM), physical inspection, optical microscopy, and Archimedes’ method. Each of these meth- ods has a specific set of capabilities for quantifying cer- tain attributes of observed manufacturing defects. For this research, μCT provides a robust tool to quantify the geomet- ric defects introduced during MAM and to characterise the associated section properties. 1 Introduction Additive manufacturing (AM) enables the layer-by-layer fab- rication of three-dimensional geometry directly from com- puter-aided design (CAD) data, thereby providing poten- tial advantages over conventional manufacturing [1]. For example, AM can fabricate complex, topology-optimised geometry as a single structure, whereas conventional manu- facturing may be constrained by the need for tooling access [2]. Despite these advantages, AM processes are subject to a series of technical challenges including relatively low production rates, thermal stresses, potentially high material costs, and geometric uncertainties in the as-manufactured geometry [3]. Metal additive manufacturing (MAM) ena- bles fabrication in a range of fusible metal alloys including light alloys, superalloys, and tool steels. As such, MAM is well suited for the fabrication of high-value lattice applica- tions including medical implants, aerospace components, and custom tooling [4]. While understanding MAM defects within individual lattice strut elements is required to opti- mise mechanical performance, few fundamental studies have been executed, and the existing studies typically focus on As with any manufacturing process, defects occur dur- ing the MAM process due to complex physical phenomena and process parameters such as laser power, scan speed, hatch spacing, powder features, powder packing arrange- ments, density distribution, morphology, and thickness. This results in a high possibility of defect formation [10]. Echeta et al. [11] classified LB-PBF defects into three categories: porosity or incomplete fusion, residual stresses, and surface texture. Zhang et al. [10] defined this porosity as spheroidal voids within the fused powder, where the pore diameter is up to approximately 100 µm. This porosity is formed due to high cooling rates during solidification, leading to dissolved 1 3 3 The International Journal of Advanced Manufacturing Technology (2023) 126:3555–3577 3557 Fig. 1 Bending-dominated (a) and stretch-dominated (b) mechanical response of lattice strut elements in response to an applied external load F [24] Fig. 1 Bending-dominated (a) and stretch-dominated (b) mechanical response of lattice strut elements in response to an applied external load F [24] Fig. 1 Bending-dominated (a) and stretch-dominated (b) mechanical response of lattice strut elements in respons [24] Fig. 1 Bending-dominated (a) and stretch-dominated (b) mechanical response of lattice strut elements in response to an applied external load F [24] gas that cannot escape from the melt pool which results in voids once the melt pool has solidified. Additionally, insuf- ficient input energy may lead to incomplete fusion and can result in pores in the order of 500 µm. 1 The isoperimetric quotient of a closed contour is the ratio of the contour area to the area of a circle of equal perimeter to the closed curve. It is a measure of ‘circularity’, where a circle yields an isoperi- metric quotient of unity [25]. 25.Jywe, W.-Y., C.-H. Liu, and C.o.-K. Chen, The min–max problem for evaluating the form error of a circle. Measurement, 1999. 26(4): p. 273–282. 2.1 Second moment of area The second moment of area is a measure of the capacity of a column to resist buckling and a beam to resist bending [25]. The planar second moment of area for a cross-section is defined as the integral sum of the squared distance, y , of infinitesimal area, dA , from the neutral axis (Eq. 1) [26]. For regular polygonal shapes, such as a circle of arbitrary radius, r (Eq. 2), the second moment of area can be calculated ana- lytically. A general equation (Eq. 3) is introduced to obtain the second moment of area for any n-sided polygon2 [27], where xi and yi represent the Cartesian coordinates of the i -th polygon vertex. (1) I = ∫y2dA (2) I퐜퐢퐫퐜퐥퐞= 흅 4 r4 (3) I퐩퐨퐥퐲퐠퐨퐧= 1 12 n ∑ i=1 (xiyi+1 −xi+1yi )(x2 i + xixi+1 + x2 i+1 ) (1) I = ∫y2dA 2 The polygon points should be ordered in a counter-clockwise direc- tion; voids are included by clockwise ordering. 1.3 Strut cross‑section manufacturability Alghamdi et al. [5] investigated the effect of polygon order, p (triangular, square, octagonal, and circular), on the geom- etry of as-manufactured lattice strut elements fabricated with LB-PBF in aluminium alloy AlSi10Mg, and titanium 1 The International Journal of Advanced Manufacturing Technology (2023) 126:3555–3577 3558 Table 1 Effect of apparent angle (blue faces) and edge angle (orange edges) on the manufacturability of triangular sections with the vertex point- ing upward and downward Table 1 Effect of apparent angle (blue faces) and edge angle (orange edges) on the manufacturability of triangular sections with the vertex point- ing upward and downward Table 1 Effect of apparent angle (blue faces) and edge angle (orange edges) on the manufacturability of triangular sections with the vertex point- ing upward and downward Table 1 Effect of apparent angle (blue faces) and edge angle (orange edges) on the manufacturability of triangular sec ng upward and downward alloy Ti6Al4V. They observed the tendency for nominally triangular and square cross-sections to become circular upon fabrication, as quantified by the isoperimetric quotient.1 This observed effect was particularly strong in the alumin- ium strut elements and for relatively small cross-sectional areas. Furthermore, triangular cross-sections were observed to have greater manufacturability when the triangle is ori- ented with a vertex pointing down (towards the build platen), compared to a vertex pointing upward (away from the build platen). The enhanced manufacturability observed for vertex down triangular sections appears to be due to a combination of cross-sectional area and apparent inclination angle, where the vertex down triangles show an increase in the appar- ent inclination angle of the associated facets (Table 1). This reflects to the manufacturability of triangular strut elements vertex downward has fewer defects compared with vertex upward. factor, ∅e B ; and failure shape factor, ∅f B . These properties will be calculated to analytically compare the efficiency of the specimens assessed in this research and are briefly defined below. 2 Geometric properties of polygon’s cross‑section (1) (2) I퐜퐢퐫퐜퐥퐞= 흅 4 r4 Section properties are the quantities that can be derived from the distribution of area in the cross-section of a given col- umn or beam. They can be used to characterise structural design efficiency, including the second moment of area, I ; the radius of gyration, Rg ; section modulus, Z ; elastic shape (2) (3) I퐩퐨퐥퐲퐠퐨퐧= 1 12 n ∑ i=1 (xiyi+1 −xi+1yi )(x2 i + xixi+1 + x2 i+1 ) (3) 2 The polygon points should be ordered in a counter-clockwise direc- tion; voids are included by clockwise ordering. 1 3 The International Journal of Advanced Manufacturing Technology (2023) 126:3555–3577 3559 Fig. 2 Radius of gyration schematic comparing (a) distances, yi , from the x-axis for infinitesimal areas, ai , taken from the cross-section and (b) equivalent area in an infinitesimal strip offset by the radius of gyration from the x-axis [ dy →0] Fig. 2 Radius of gyration schematic comparing (a) distances, yi , from the x-axis for infinitesimal areas, ai , taken from the cross-section and (b) equivalent area in an infinitesimal strip offset by the radius of gyration from the x-axis [ dy →0] 2.2 Radius of gyration of a solid circular cross-section are unity and changes with cross-section shape. When the shape factor increases, the resistance to bending and buckling increases. In addition, the dimensionless nature of shape factors allows for the com- parison of shapes independent of scale. The influence of cross-section shape on stiffness can be calculated using the elastic bending shape factor, ∅e B (Eq. 5). As strength depends on local stress, the section modulus, Z , must be calculated (Eq. 6), where ymax is the outermost fibre from the neutral axis subject to compression or tension to quantify the bend- ing failure shape factor, ∅f B . This is the ratio of any given section modulus over that of a circle of equal area (Eq. 7), Zcircle. The radius of gyration defines the theoretical distance from the cross-section centroid at which the cross-sectional area can be considered to be concentrated to achieve an equal second moment of area as the actual cross-section distribu- tion (Fig. 2b) [26]. It is a measure of the resistance of the cross-section to elastic buckling or bending [28, 29] and can therefore be useful to compare the resistance to buckling or bending of various sections with an equal cross-sectional area (Eq. 4). (4) Rg = √ I A Rg = √ I A Zcircle. (4) (5) ∅e B = 4흅I A2 (6) Z = I y퐦퐚퐱 (7) ∅f B = 4 √ 흅Z A3∕2 = Z Z퐜퐢퐫퐜퐥퐞 (5) For example, comparing Rg for a triangular cross-section with that of a circular cross-section shows that triangular sections have larger Rg , indicating they are more efficient when resisting elastic buckling or bending than a circular section with equal area. (6) (7) ∅f B = 4 √ 흅Z A3∕2 = Z Z퐜퐢퐫퐜퐥퐞 (7) 2.3 Elastic and failure shape factors for bending Structural performance in bending can therefore be char- acterised by selecting an appropriate combination of mate- rial and shape for both elastic and failure scenarios [30]. In the “3.3” section, it is shown that the solid triangular cross- section under bending load is stiffer than an equivalent solid The shape factors compare the mechanical performance (such as stiffness and failure under buckling or bending) of a cross-section of interest against a reference circular cross-section of the equivalent area [30]. The shape factors 1 The International Journal of Advanced Manufacturing Technology (2023) 126:3555–3577 3560 Fig. 3 Circles of effective diameter, D퐞퐟퐟 , superimposed onto polygons of equivalent area Fig. 3 Circles of effective diameter, D퐞퐟퐟 , superimposed onto polygons of equivalent area circular cross-section by 21%, whereas it has 23% lower strength. proposed method can be classified into the following steps: CAD design, analytical quantification, PB-LBF fabrication, μCT imaging, as-manufactured quantification, and statisti- cal analysis. 3.1 Design of experiments Effective diameter, Deff , represents the diameter of a circle that has an equivalent area to that of the cross-section of interest. In this research, four effective diameters are imple- mented (3.0 mm, 2.0 mm, 1.0 mm, and 0.5 mm) for four different cross-sections (circular, octagonal, square, and tri- angular) with equivalent areas [5] (Fig. 3). The methodology was implemented on a set of strut geom- etries designed, fabricated, and scanned in previous work [5]. The design of experiments (DOE) contains four control factors including two material types implemented separately as powder feedstock for the LB-PBF process (aluminium alloy AlSi10Mg and titanium alloy Ti6Al4V), three incli- nation angles ( 훼in = 90°, 45°, 35°), four nominal diameters (D = 3.0, 2.0, 1.0, 0.5 mm), and five polygonal cross-sections (circle, octagon, square, triangle vertex up, triangle vertex down). These specimens were arranged in rows dependent upon the inclination angle on a plate feature 3-mm thick and extruded from the plate by a length of 15 mm (Fig. 5), resulting in a total of 120 strut elements. A = 휋 4 D2 eﬀA = (2 + 2 √ 2)s 2 A = s2 A = √ 3 4 s2 3 Method Recent literature reviews have highlighted a lack of DFAM tools that can quantify the structural integrity of as-man- ufactured AM strut elements. Cross-section design has a significant impact on strut performance; thus, the shape factor of as-manufactured strut elements should be char- acterised to assess the strength and stiffness of proposed designs. This research proposes a fundamental methodology for the characterisation of as-manufactured strut elements to quantify the associated geometric and functional prop- erties. This generalisable DFAM tool is implemented spe- cifically on strut element specimens fabricated by PB-LBF. Figure 4 illustrates the proposed methodology’s workflow, which can be applied as a guideline to any manufacturing process to ensure optimal production and certification. The 3.2 Laser‑based powder bed fusion An SLM Solutions 250HL machine was implemented to fabricate the Ti6Al4V struts, and an SLM Solutions 125HL machine manufactured the AlSi10Mg struts. The operational parameters for each machine are displayed in Table 2. Fol- lowing fabrication, the parts were cooled within the machine to room temperature, and then electrical discharge machin- ing (EDM) was utilised to wire cut the strut specimens from the build plate. 1 3 3 The International Journal of Advanced Manufacturing Technology (2023) 126:3555–3577 3561 Fig. 4 Flow chart for the proposed method of quantifying cross-section properties for idealised and as-manufactured strut elements [5] Fig. 4 Flow chart for the proposed method of quantifying cross-section properties for idealised and as-manufactured strut elements [5] Fig. 5 CAD (a) and as-manufactured parts (b) of strut elements [5] Fig. 5 CAD (a) and as-manufactured parts (b) of strut elements [5] Fig. 5 CAD (a) and as-manufactured parts (b) of strut elements [5] 3.3 Laser scan strategy implemented to scan the remainder of the strut’s cross-sec- tional area. This hatch pattern is inset by 90 µm (Ti6Al4V) and 150 µm (AlSi10Mg) from the offset scan. To ensure complete melting, the hatching is rotated by 66.9 ◦ at each consecutive scan layer. Finally, a single contour scan tracing the hatch pattern perimeter is implemented to smooth the non-uniform edges of the hatch pattern. A standard hatch infill scan strategy was implemented to fabricate these struts, as displayed in Fig. 6. Initially, a scan path is applied that follows the slice perimeter of the STL file (this scan is referred to as a border scan). To achieve a high geometrical accuracy to the idealised model, the bor- der scan is inset from the nominal slice contour by 60 µm (Ti6Al4V) and 120 µm (AlSi10Mg) for melt pool com- pensation. An offset scan is then implemented, inset from the initial border scan by a further 90 µm (Ti6Al4V) and 150 µm (AlSi10Mg). A hatch pattern inclined at 90 ◦ is then 3.5 Algorithmic implementation The cross-section geometric properties for both idealised and as-manufactured cases are calculated using a MATLAB (R2020b) script [31] that was updated and customised for quantifying the section properties. The code starts by obtaining the idealised properties based on the polygon order from the DOE. Then, µCT cross-section images for the as-manufactured case are imported and converted into binary images. From this data, the geometric properties are calculated as a function of the angular orientation of the cross-section about its centroidal axis for the second moment of area (Fig. 7). To provide sta- tistical distributions of cross-section response, this method is repeated sequentially on images within the image stack. 3.4 Micro‑computed tomography To quantify the strut geometries, a Bruker SKYSCAN X-ray micro µCT machine (Bruker Pty Ltd.) was utilised. 1 3 The International Journal of Advanced Manufacturing Technology (2023) 126:3555–3577 3562 Table 2 LB-PBF parameters and associated powder size profile for both AlSi10Mg and Ti6Al4V Fi 6 L t t f th tit i d l i i t t d d li th h t h tt I th i th Table 2 LB-PBF parameters and associated powder size profile for both AlSi10Mg and Ti6Al4V 0Mg and Ti6Al4V 1 3 Fig. 6 Laser scan strategy for the titanium and aluminium struts over a range of inclines (35°, 45°, 90°) for the effective diameters of 3 mm, 2 mm, 1 mm, and 0.5 mm (right to left), where the red lines are bor- der scans, and green lines are the hatch pattern. In these images, the contour scan is coincidental with the hatch pattern and is not visible due to the overlap Fig. 6 Laser scan strategy for the titanium and aluminium struts over a range of inclines (35°, 45°, 90°) for the effective diameters of 3 mm, 2 mm, 1 mm, and 0.5 mm (right to left), where the red lines are bor- der scans, and green lines are the hatch pattern. In these images, the contour scan is coincidental with the hatch pattern and is not visible due to the overlap Fig. 6 Laser scan strategy for the titanium and aluminium struts over a range of inclines (35°, 45°, 90°) for the effective diameters of 3 mm, 2 mm, 1 mm, and 0.5 mm (right to left), where the red lines are bor- der scans, and green lines are the hatch pattern. In these images, the contour scan is coincidental with the hatch pattern and is not visible due to the overlap 1 3 3 The International Journal of Advanced Manufacturing Technology (2023) 126:3555–3577 3563 The International Journal of Advanced Manufacturing Technology (2023) 126:3555–35 Fig. 7 Algorithmic process for quantifying geometric properties of both idealised and as-manufactured cases Fig. 7 Algorithmic process for quantifying geometric properties of both idealised and as-manufactured cases 4 Observation and results The technology used an X-ray tube current of 100 μA, an acceleration voltage of 100 kV, and a pixel size of 8 µm. The AlSi10Mg samples employed an Al 1-mm filter, while the Ti6Al4V samples used a Cu 1-mm filter. To reconstruct the cross-section slices acquired from the μCT, nRECON shadow image reconstruction software (Bruker Pty Ltd.) was used. Reconstructed grey scale images were then used to identify the strut element boundary and quantify the as- manufactured section properties. The technology used an X-ray tube current of 100 μA, an acceleration voltage of 100 kV, and a pixel size of 8 µm. The AlSi10Mg samples employed an Al 1-mm filter, while the Ti6Al4V samples used a Cu 1-mm filter. To reconstruct the cross-section slices acquired from the μCT, nRECON shadow image reconstruction software (Bruker Pty Ltd.) was used. Reconstructed grey scale images were then used to identify the strut element boundary and quantify the as- manufactured section properties. Section properties of the proposed DOE for both idealised and as-manufactured cases are presented in this section. It is divided into seven subsections: The idealised case, which demonstrates how polygon order affects the geometric prop- erties; the comparison between idealised polygon cross-sec- tions and as-manufactured strut element cross-sections for both materials; the second moment of area; the radius of gyration; the elastic shape factor, used to evaluate the stiff- ness of the as-manufactured geometry; the failure bending shape factor, used to evaluate the strength of the as-manufac- tured geometry; and main effect plots of section properties. 4.1 Geometric properties of idealised strut elements The stiffness and strength of strut element specimens associ- ated with the same cross-sectional area may be characterised by quantifying the idealised geometric properties including the second moment of area, Iideal , the radius of gyration, Rg,ideal , elastic bending shape factor, ∅e B,ideal , section modu- lus, Zideal , and bending failure shape factor, ∅f B,ideal. 1 3 The International Journal of Advanced Manufacturing Technology (2023) 126:3555–3577 The International Journal of Advanced Manufacturing Technology (2023) 126:3555–3577 3564 3 3 1 3 The International Journal of Advanced Manufacturing Technology (2023) 126:3555–3577 3565 rectangular box representing the interquartile range (IQR) (25–75% percentiles), with the whiskers extending up to 1.5 × IQR and the median as the horizontal line within the box. The plots are presented in a graphical array as defined by material (columns) and effective diameter (rows), while cross-section shape and build inclination angle form the hor- izontal axis labels and the as-manufactured cross-sectional area forms the vertical axis labels. The expected value based on the idealised shape is presented as horizontal lines span- ning the plots. For consistency, each of the sectional charac- teristics discussed in later subsections is presented using the same graphical array, with only the vertical axis changing to reflect the relevant value. Fig. 8 Idealised geometric properties for polygon orders rotated by 휽퐫퐨퐭퐚퐭퐢퐨퐧 , where (a) circle, octagon, square, and triangle, associated with the same cross-sectional area, (b) second moment of area per- centage improvement, over the circle, for each shape, (c) radius of gyration, (d) elastic shape factor, (e) percentage improvement of sec- tion modulus, over the circle, and (f) failure shape factor ◂ These geometric properties are quantified in Fig. 8 for various regular polygonal cross-sections associated with the equal cross-sectional area, for a range of orientations achieved by incremental rotations, 휃rotation , about the cen- troid. Figure 8b–d shows that the second moment of area, radius of gyration, and elastic shape factor all remain con- stant while rotating these idealised polygonal shapes. The elastic shape factor of the octagonal cross-section has a very slight increase of 0.2% over the circular section, whereas square and triangular shapes indicate 4.7% and 20.9% increases, respectively. The section modulus and failure shape factor show a dependency on the polygon’s orientation that increases with lower-order polygons which is associated with vertex orientation. For example, the failure shape factor of the idealised triangular cross-section ranged from − 23% lower (pointing up, 휃rotation = 0◦, 120◦, 240◦ ) to 55% higher (pointing down, 휃rotation = 60◦, 180◦, 300◦ ), when compared to that of the circular cross-section. These results evaluate the section modulus and shape factor based on the extreme fibres on one side of the neutral axis only. The International Journal of Advanced Manufacturing Technology (2023) 126:3555–3577 When the extreme fibres are considered from both sides simultaneously, the failure shape factor for the triangle, although orientation dependent, is always less than the circle, as indicated by the solid line in Fig. 8f. Although the triangular cross-section is 20.9% stiffer than the circle, it tends to fail in the weak- est direction at 23% lower load compared with the circular cross-section (Fig. 8f). The concept of shape factors is well known in civil engineering but has yet been applied in the design of lattice structure elements fabricated by AM. The following subsections investigate how the as-manufactured cross-section varies from the idealised results for these important section properties. When comparing the aluminium (left column of Fig. 9a–d) to the titanium (right column of Fig. 9e–h) speci- mens, generally, there is greater variation within the indi- vidual aluminium specimens than in the titanium specimens, as indicated by the relative size of the IQR, the exception being at Deff = 0.5mm . Furthermore, for the aluminium, there is a trend that area (median) and variation in the area (box plot size) increases with decreasing inclination angle. For the titanium (Fig. 9e–h), there is an upward trend in the position of the box plots within each graph, indicating that the cross-sectional area of the strut elements increases as the shape changes from circular to triangular, i.e., as the polygon order decreases, the area increases. This suggests that more material may be accumulating on the as-manu- factured triangular shape than on the circular shape, even though they are intended to be the same area as indicated by the spanning horizontal line. For the aluminium strut ele- ments (Fig. 9a–d), the same upward trend across the shapes is not visible; however, within each shape (clusters of three), there is both a downward trend in the cross-sectional area and in the variation of the cross-sectional area with increas- ing build inclination angle. These trends indicate that the cross-sectional area of aluminium strut elements is strongly affected by inclination, accumulating more area and greater variation in the area along a strut element as the build incli- nation angle is decreased from 90° to 35°. Meanwhile, the titanium strut elements show far less variation in the area. 4.3 Second moment of area (ideal versus CT) The as-manufactured polygon cross-sectional area, ACT , is affected by manufacturing processes, leading to variation between the idealised and as-manufactured strut elements. The effective diameter, Deff , inclination angle, 훼in , polygon order, p , and material choice all significantly affect LB-PBF manufacturability. The second moment of area for as-manufactured case, ICT , is calculated based on extracted data from µCT cross-section images. This extracted data provides the outer boundary, centroid, and as-manufactured area, ACT , for each image. Each fabricated strut element is imaged many times along its length. Therefore, to quantify ICT with image orientation, the extracted boundary is incrementally rotated by a small rotation angle, 휃rotation , (3.6°), as shown in Fig. 10a. At each rotational angle, the ICT is calculated, as shown in Fig. 10b. Figure 9 shows the variation in the cross-sectional area within each of the as-manufactured strut elements. The box plots provide a graphical statistical summary for each cross-section image for the given strut. These include the 1 3 The International Journal of Advanced Manufacturing Technology (2023) 126:3555–3577 The International Journal of Advanced Manufacturing Technology (2023) 126:3555–3577 3566 n e te at o a Jou a o d a ced a u actu g ec o ogy ( 0 3) 6:3555 35 Fig. 9 Box plots of A퐂퐓 com- pared with A퐢퐝퐞퐚퐥 (green hori- zontal lines), where (a) to (d) are aluminium strut elements, with an effective diameter of 0.5 mm, 1.0 mm, 2.0 mm, and 3.0 mm, and (e) to (h) are titanium strut elements, with an effective diameter of 0.5 mm, 1.0 mm, 2.0 mm, and 3.0 mm (outliers hidden) Fig. 9 Box plots of A퐂퐓 com- pared with A퐢퐝퐞퐚퐥 (green hori- zontal lines), where (a) to (d) are aluminium strut elements, with an effective diameter of 0.5 mm, 1.0 mm, 2.0 mm, and 3.0 mm, and (e) to (h) are titanium strut elements, with an effective diameter of 0.5 mm, 1.0 mm, 2.0 mm, and 3.0 mm (outliers hidden) 1 3 The International Journal of Advanced Manufacturing Technology (2023) 126:3555–3577 3567 Fig. 10 Second moment of area of as-manufactured strut element where maximum I퐂퐓,퐦퐚퐱 and minimum I퐂퐓,퐦퐢퐧 are denoted with red dash lines; idealised I퐢퐝퐞퐚퐥 represented with a solid blue line; the col- oured lines represent I for each image in the stack at rotation 휽퐫퐨퐭퐚퐭퐢퐨퐧 ; (a) cross-section image showing key rotations in the graph; (b) sec- ond moment of area plotted over all possible rotations for as-manu- factured and idealised cases Fig. 10 Second moment of area of as-manufactured strut element where maximum I퐂퐓,퐦퐚퐱 and minimum I퐂퐓,퐦퐢퐧 are denoted with red dash lines; idealised I퐢퐝퐞퐚퐥 represented with a solid blue line; the col- oured lines represent I for each image in the stack at rotation 휽퐫퐨퐭퐚퐭퐢퐨퐧 ; (a) cross-section image showing key rotations in the graph; (b) sec- ond moment of area plotted over all possible rotations for as-manu- factured and idealised cases Figure 12 shows box plots for the radius of gyration of the as-manufactured strut elements, Rg,CT , compared with the idealised case, Rg,ideal , represented as a green horizontal line segment for both aluminium and titanium. It is useful to evaluate the efficiency of the actual shape versus the ide- alised shape as it does not consider the material. Figure 12 also evaluates the quality of fabrication and the effective- ness of controlling factors such as 휶퐢퐧퐜 . Overall, it can be observed that the variation in Rg,CT is larger in the aluminium (Fig. 12a–d) than the titanium (Fig. The International Journal of Advanced Manufacturing Technology (2023) 126:3555–3577 12a–d) strut elements. The variation within individual aluminium strut elements is largest for the 35° and 45° build inclination angle, while the 90° cases are similar to the titanium. All values of ICT are compared with those of the idealised case of a circular cross-section, Iideal , for each strut, catego- rised by material (AlSi10Mg and Ti6Al4V), Deff , shape, 훼in , as shown in Fig. 11. The distribution of ICT is presented in the form of box plots, using the same graphical array described in the “Idealised versus as-manufactured cross- sectional area” section. Iideal is shown as horizontal line seg- ments that increase with decreasing polygon order. g p yg Comparing the aluminium and titanium struts at the same effective diameter, there is significantly more variation in ICT across the aluminium struts than across the titanium struts, suggesting that titanium provides a more consistent stiffness. The aluminium strut elements seen in Fig. 11a–d show both ICT and ACT experience a similar trend. The magnitude and variation of ICT within the as-manufactured strut elements show a decreasing trend with increasing build inclination. This is seen by longer IQR boxes for 35° strut elements com- pared to the 90° struts. Considering the titanium strut ele- ments in Fig. 11e–h, the increase in ICT across the shapes is greater than expected, in comparison to the idealised cross- section. This corresponds with the previous observation that the as-manufactured area, ACT , increased with decreasing polygon order at a given Deff. 4.5 Elastic shape factor (ideal versus CT) The elastic shape factor, ∅e B , provides a measure of the stiff- ness efficiency of the cross-section shape, as discussed pre- viously in the “2.1” section. The as-manufactured elastic shape factor, ∅e B,CT , is compared with the idealised case, ∅e B,ideal , in Fig. 13. With the idealised shape factor for the circular cross-section being 1.0, the octagonal, square, and triangular cross-sections are 1.002, 1.047, and 1.209, respectively. Comparing ∅e B,CT to ∅e B,ideal shows the effect of manufacturing defects and control factors. The orienta- tional dependence observed in I is again observed in ∅e B . The shape factor removes size dependence, so comparisons can be made purely on the achieved shape and not be confounded with whether more or less material is contributing to the change. Comparing ∅e B between aluminium (Fig. 13a–d) 4.4 Radius of gyration (ideal versus CT) The efficiency of a cross-section shape of interest for elas- tic stability under compression can be evaluated using the radius of gyration, Rg , associated with the cross-sectional area of interest, as discussed in the “Introduction” section. 1 3 The International Journal of Advanced Manufacturing Technology (2023) 126:3555–3577 The International Journal of Advanced Manufacturing Technology (2023) 126:3555–3577 Fig. 11 Box plots of I퐂퐓 com- pared with I퐢퐝퐞퐚퐥 (green horizon- tal lines), where (a) to (d) are aluminium strut elements, with an effective diameter of 0.5 mm, 1.0 mm, 2.0 mm, and 3.0 mm, and (e) to (h) are titanium strut elements, with an effective diameter of 0.5 mm, 1.0 mm, 2.0 mm, and 3.0 mm (outliers hidden) Fig. 12 Box plots of R퐂퐓 com- pared with R퐢퐝퐞퐚퐥 (green hori- zontal lines), where(a) to (d) are aluminium strut elements, with an effective diameter of 0.5 mm, 1.0 mm, 2.0 mm, and 3.0 mm, and (e) to (h) are titanium strut elements, with an effective diameter of 0.5 mm, 1.0 mm, 2.0 mm, and 3.0 mm (outliers hidden) The International Journal of Advanced Manufacturing Technology (2023) 126:3555–3577 3568 - m, g gy Fig. 11 Box plots of I퐂퐓 com- pared with I퐢퐝퐞퐚퐥 (green horizon- tal lines), where (a) to (d) are aluminium strut elements, with an effective diameter of 0.5 mm, 1.0 mm, 2.0 mm, and 3.0 mm, and (e) to (h) are titanium strut elements, with an effective diameter of 0.5 mm, 1.0 mm, 2.0 mm, and 3.0 mm (outliers hidden) 1 3 The International Journal of Advanced Manufacturing Technology (2023) 126:3555–3577 The International Journal of Advanced Manufacturing Technology (2023) 126:3555–3577 Another observa- tion is that the median ∅e B for the titanium strut elements lies at or below the ideal value; however, in the aluminium, particularly at an Deff of 1.0 mm and 2.0 mm, the median ∅e B for the inclined circular, octagonal and square cross-sections lie above their ideal values. This appears to be an indication of defects introduced during MAM processes, suggesting that as-manufactured defects could increase local stiffness and failure response if they align favourably with loading conditions as illustrated in Fig. 8f; i.e., unintended additional material is deposited in such a way as to increase ymax , align- ing with the optimal orientation. and titanium (Fig. 13e–h), generally, there is less variability within individual titanium strut elements than within indi- vidual aluminium strut elements, as can be seen by the size of the IQR for individual box plots. This is most notable in the 35° and 45° cases. The exception appears to be in strut elements with smaller Deff of 0.5 mm, where the alu- minium and titanium both show relatively large variation in ∅e B . Also, at lower effective diameters, the elastic shape factors of the as-manufactured triangular cross-sections are producing results more in line with a circular cross-section. 4.6 Failure shape factor (ideal versus CT) The failure shape factor, ∅f B , can be used to evaluate the manufacturability of a strut element cross-section. With the idealised failure shape factor, ∅f B,ideal , for the circular cross-section being 1.0, the octagonal, square, and triangu- lar cross-sections experience an orientation dependence and range from 0.95 to 1.029, 0.83 to 1.18, and 0.77 to 1.55, respectively. The failure shape factors for the as-manufac- tured strut elements, ∅f B,CT , are compared to the ideal ranges in Fig. 14. When comparing the aluminium (Fig. 14a–d) and titanium (Fig. 14e–h) cases, the titanium strut elements show greater consistency for a given shape across the three incli- nation angles, and the square and triangular shapes tend to remain bound by the ideal range, with the distributions better matching the ideal range with increasing effective diameter. This trend is not observed in the aluminium strut elements which show greater variation and is particularly apparent for circular and octagonal shapes. The large variation in the 4.7 Main effect plots of section properties Main effect plots illustrate the influence of independent vari- ables, or factors, on the dependent variables, as shown in Fig. 15, 16, 17, 18, and Fig. 19. The effect of the independ- ent variable can be seen by the variation of the line, with a large deviation from the horizontal considered a significant effect. In this experiment, the independent variables affect each of the dependent variables, the manufactured section properties, to differing degrees. The effective diameter, Deff , is the dominant factor for the cross-sectional area, ACT , the second moment of area, ICT , and the radius of gyration, Rg,CT . By contrast, the main effects of build angle, shape, and material on those section properties are relatively small. The main effects of the shape on the second moment of area shows ICT improving as the polygon order decreases from circle to octagon, square, and finally triangle. However, the magnitude in variation is similar to that caused by the incli- nation angle, αin , and material. There is an expectation that the stiffness shape factor, ∅e B , and the failure shape factor, ∅f B , are independent of scale (i.e., the effective diameter, Deff ), but dependent on the cross-sectional shape. The main effects plots for both the elastic, ∅e B , and failure shape fac- tors, ∅f B , for manufactured struts, show that the shape varia- ble is the most dominant, with the triangles giving the lowest values. However, the effective diameter, Deff , also produces a significant effect. The material shows a small effect on all the section properties, with the titanium having slightly higher values than the aluminium. The International Journal of Advanced Manufacturing Technology (2023) 126:3555–3577 The International Journal of Advanced Manufacturing Technology (2023) 126:3555–3577 3569 Fig. 12 Box plots of R퐂퐓 com- pared with R퐢퐝퐞퐚퐥 (green hori- zontal lines), where(a) to (d) are aluminium strut elements, with an effective diameter of 0.5 mm, 1.0 mm, 2.0 mm, and 3.0 mm, and (e) to (h) are titanium strut elements, with an effective diameter of 0.5 mm, 1.0 mm, 2.0 mm, and 3.0 mm (outliers hidden) 1 3 1 3 3570 The International Journal of Advanced Manufacturing Technology (2023) 126:3555–3577 The International Journal of Advanced Manufacturing Technology (2023) 1 3 The International Journal of Advanced Manufacturing Technology (2023) 126:3555–3577 3571 failure shape factor highlights an opportunity for improved strength based on geometric orientation. An important observation from these graphs is that the median result typi- cally sits below unity, meaning that most orientations result in reduced strength rather than improved strength. This indi- cates that care should be taken with cross-section orientation relative to the load direction. Fig. 13 Box plots of CT stiffness shape factor, ∅e B,퐂퐓 , compared with ideal stiffness shape factor, ∅e B,퐢퐝퐞퐚퐥 (green horizontal lines), where (a) to (d) are aluminium strut elements, with an effective diameter of 0.5 mm, 1.0 mm, 2.0 mm, and 3.0 mm, and (e) to (h) are tita- nium strut elements, with an effective diameter of 0.5 mm, 1.0 mm, 2.0 mm, and 3.0 mm (outliers hidden) ◂ and titanium (Fig. 13e–h), generally, there is less variability within individual titanium strut elements than within indi- vidual aluminium strut elements, as can be seen by the size of the IQR for individual box plots. This is most notable in the 35° and 45° cases. The exception appears to be in strut elements with smaller Deff of 0.5 mm, where the alu- minium and titanium both show relatively large variation in ∅e B . Also, at lower effective diameters, the elastic shape factors of the as-manufactured triangular cross-sections are producing results more in line with a circular cross-section. While at higher effective diameters, the elastic shape factor of the as-manufactured strut elements better matches the ide- alised trend for each shape. The transition for this behaviour occurs at Deff of 1.0 mm for the titanium, and between an Deff of 2.0 to 3.0 mm for the aluminium. 5 Discussion Structural mechanics theory suggests that of the regular polygons, square and triangular cross-sections provide the greatest structural efficiency over the circular cross- section, with elastic shape factors of 1.05 and 1.21, respec- tively, when aligned to the load direction. This suggests an opportunity for improved stiffness simply through the choice of more efficient cross-section shapes and align- ing them to the load direction. For polygons with the equivalent cross-sectional area, I is the key parameter when considering stiffness and buckling resistance. For irregular cross-sections, both I and ∅f B have orientation dependencies. This orientation dependence is well known 1 3 1 The International Journal of Advanced Manufacturing Technology (2023) 126:3555–3577 The International Journal of Advanced Manufacturing Technology (2023) 126:3555–3577 3572 ge h m, t The International Journal of Advanced Manufacturing Technology (2023) 126:3555–3577 Fig. 14 Box plots of CT strength shape factor, ∅f B,퐂퐓,of aluminium and titanium strut elements compared with a range of ideal strength shape fac- tor,∅f B,퐢퐝퐞퐚퐥 , values represented as green (lower value) and orange (upper value) horizon- tal lines, where (a) to (d) are aluminium strut elements, with an effective diameter of 0.5 mm, 1.0 mm, 2.0 mm, and 3.0 mm, and (e) to (h) are titanium strut elements, with an effective diameter of 0.5 mm, 1.0 mm, 2.0 mm, and 3.0 mm (outliers hidden) 1 3 Fig. 15 Main effect plot for the CT area, A퐂퐓 , among independent variables Fig. 16 Main effect plot for the second moment of area, I퐂퐓 , among independent variables 3573 The International Journal of Advanced Manufacturing Technology (2023) 126:3555–3577 The International Journal of Advanced Manufacturing Technology (2023) 126:3555–3577 The International Journal of Advanced Manufacturing Technology (2023) 126:3555–3577 3573 Fig. 15 Main effect plot for the CT area, A퐂퐓 , among independent variables Fig. 15 Main effect plot for the CT area, A퐂퐓 , among independent variables Fig. 15 Main effect plot for the CT area, A퐂퐓 , among independent variables Fig. 16 Main effect plot for the second moment of area, I퐂퐓 , among independent variables Fig. 16 Main effect plot for the second moment of area, I퐂퐓 , among independent variables Fig. 16 Main effect plot for the second moment of area, I퐂퐓 , among independent variables 1 3 The International Journal of Advanced Manufacturing Technology (2023) 126:3555–3577 The International Journal of Advanced Manufacturing Technology (2023) 126:3555–3577 3574 Fig. 17 Main effect plot for the radius of gyration, Rg,퐂퐓 , among independent variables Fig. 17 Main effect plot for the radius of gyration, Rg,퐂퐓 , among independent variables Fig. 18 Main effect plot for the elastic shape factor, ∅e B,퐂퐓 , among independent variables Fig. 18 Main effect plot for the elastic shape factor, ∅e B,퐂퐓 , among independent variables Fig. 18 Main effect plot for the elastic shape factor, ∅e B,퐂퐓 , among independent variables 3 The International Journal of Advanced Manufacturing Technology (2023) 126:3555–3577 3575 Fig. 19 Main effect plot for the minimum failure shape factor, ∅f B,퐂퐓 , among independent variables Fig. The International Journal of Advanced Manufacturing Technology (2023) 126:3555–3577 19 Main effect plot for the minimum failure shape factor, ∅f B,퐂퐓 , among independent variables and exploited in the fields of civil and structural engineer- ing through the use of shaped sections such as I-beams, although to date has yet to be applied to lattice structures. Given the failure shape factor’s dependence on orientation, it has been shown that strut elements are particularly sensi- tive to shape defects introduced during the AM process. To determine alternative cross-section shape suitability, the desired performance and the number of manufactur- ing defects that can be tolerated must be considered. For example, while a traditional I-beam section would provide exceptional stiffness, due to the thin sections of the web and flanges, they would be particularly susceptible to defects at small scales and it is only appropriate for two diametrically opposed loading conditions; in complex loading conditions, this section is less than optimal. The second moment of area of regular polygons is independent of the loading direc- tion (unlike irregular polygons). Thus, resistance to elastic bending could be improved by switching to the triangular or square cross-section, regardless of load direction. tive to shape defects introduced during the AM process. Titanium strut elements possessed more consistent geo- metric characteristics along their length of build for all sizes, although they exhibited an unexpected increase in cross- sectional area with decreasing polygon order. For square and triangular cross-sections, ∅e B,CT and ∅f B,CT approached the predicted values of ∅e B,ideal and ∅f B,ideal with increasing Deff. Aluminium strut elements were subject to significant variations in geometric characteristics along their length of build, with the shape being indistinguishable for results with an Deff under 2 mm. A strong dependency on inclina- tion angle, 훼in , was observed, with increasing cross-sectional area and variation in said area, a trend carried into all other characteristics. For the square and triangular cross-sections, ∅e B,CT and ∅f B,CT approached the predicted values of ∅e B,ideal and ∅f B,ideal with increasing Deff , although results were lower than ideal when the Deff was below 2 mm. A manufactur- ability limit of 2 mm is observed for all cross-sections in aluminium, with results below this being indistinguishable from the circular case. Titanium strut elements possessed more consistent geo- metric characteristics along their length of build for all sizes, although they exhibited an unexpected increase in cross- sectional area with decreasing polygon order. 7 Future work The methodology was applied to two sets of additively manufactured strut elements, one in aluminium alloy AlSi10Mg and another in titanium alloy Ti6Al4V, with varying effective diameters, polygon orders, and inclination angles. The following key observations were made. Future work will be dedicated to exploring the production of parts using multi jet fusion (MJF), which can fabricate parts significantly faster than LB-PBF, using the devel- oped methodology to determine the manufacturability and mechanical responses of parts fabricated using the MJF process. The comparison of this work and producing parts using MJF opens up the possibility of better under- standing the advantage and disadvantages of each process relative to the required application. Porosity also will be considered as one of the section properties that affect the manufacturable and mechanical performance. • Iideal for the square and triangular cross-sections have a 5% and 21% improvement, respectively, compared to the idealised circular cross-section. This indicates an increased elastic buckling and bending resistance of fabricated strut elements.f • Iideal of circular and octagonal cross-sections differ by only 0.2%, displaying that polygon orders greater than eight do not significantly affect I . This is most beneficial as representing struts with larger order polygons typically require larger file sizes. These file size requirements may then be reduced without losing as-manufactured shape accuracy. Fewer faces also benefit FEA, with lower com- putational costs. Acknowledgements The authors acknowledge the use of facilities within RMIT Advanced Manufacturing Precinct and the RMIT Micros- copy and Microanalysis Facility. (https://www.rmit.edu.au/about/our- locations-and-acilities/facilities/researchfacilities/advanced-manufactur ing-precinct). Funding Open Access funding enabled and organized by CAUL and its Member Institutions • It was found that the variation between ICT and Iideal in aluminium is generally greater than that in titanium, indicating titanium produces less manufacturing defects. These variations are increased with lower inclination angles, 훼in.f Data availability The data is available and only can be provided by request of the journal. Code availability The code is available and only can be provided by request of the journal. • The effective diameter has a major impact on the manu- facturability of the strut elements, particularly on alu- minium at Deff ≤2.0 mm. At these sizes, the variation in the dependent variables is significantly larger than the expected ranges and the effect of shape is not dis- cernible over the inclination angle. The International Journal of Advanced Manufacturing Technology (2023) 126:3555–3577 For square and triangular cross-sections, ∅e B,CT and ∅f B,CT approached the predicted values of ∅e B,ideal and ∅f B,ideal with increasing Deff.i p B,ideal B,ideal g eff Aluminium strut elements were subject to significant variations in geometric characteristics along their length of build, with the shape being indistinguishable for results with an Deff under 2 mm. A strong dependency on inclina- tion angle, 훼in , was observed, with increasing cross-sectional area and variation in said area, a trend carried into all other characteristics. For the square and triangular cross-sections, ∅e B,CT and ∅f B,CT approached the predicted values of ∅e B,ideal and ∅f B,ideal with increasing Deff , although results were lower than ideal when the Deff was below 2 mm. A manufactur- ability limit of 2 mm is observed for all cross-sections in aluminium, with results below this being indistinguishable from the circular case. There are future opportunities to extend the methodology and analysis to other loading cases, cross-sections, and mate- rials. Both ∅e B , characterising elastic bending and buckling resistance, and ∅f B , characterising bending failure, have been considered as these are often the dominant behaviours in lattice structures under compression. Other shape factors, such as torsion, must be considered for more complex load- ing cases. This could lead to an investigation of alternative cross-sections that provide structural efficiencies, such as channels and hollow sections [32]. Furthermore, this meth- odology can be applied to investigate the manufacturability and as-manufactured structural efficiency of other AM sys- tems [24, 25] and materials. 1 3 3 The International Journal of Advanced Manufacturing Technology (2023) 126:3555–3577 3576 6 Summary elements show this improvement in both ∅e B,CT and ∅f B,CT , for each incremental increase in Deff . Aluminium strut elements show poor replication of both shape factors at all but the largest effective diameter.ff Laser-based powder bed fusion processes observe a high frequency of geometrical variation between the idealised models and the as-manufactured specimens. This research has developed a design tool to algorithmically quantify the structural impact of as-manufactured geometrical defects. • The main effects analysis showed that the effective diam- eter is the dominant factor for strut cross-sectional area, second moment of area, and radius of gyration. The shape was the dominant factor for the elastic shape fac- tor and failure shape factor; however, effective diameter and build angle also had a strong influence. • The main effects analysis showed that the effective diam- eter is the dominant factor for strut cross-sectional area, second moment of area, and radius of gyration. The shape was the dominant factor for the elastic shape fac- tor and failure shape factor; however, effective diameter and build angle also had a strong influence. The as-manufactured strut elements were imaged using μCT and then reconstructed into a stack of as-manufactured strut element cross-section images. The geometric param- eters including, ACT , ICT , ∅e B,CT , and ∅f B,CT have been quanti- fied using the proposed method and verified against idealised cases. 7 Future work A transition is seen at Deff = 3.0 mm, where the aluminium struts show dependence on shape and inclination angle. For titanium, this transition is more apparent between 0.5 and 1.0 mm f Declarations Ethics approval Not applicable. References 19. Zhang XZ et al (2018) Toward manufacturing quality Ti-6Al-4V lattice struts by selective electron beam melting (SEBM) for lat- tice design. Jom 70(9):1870–1876 1. Gibson I et al (2021) Additive manufacturing technologies. Springer International Publishing 1. Gibson I et al (2021) Additive manufacturing technologies. Springer International Publishing 2. Leary M, Mazur M, Williams H, Yang E, Alghamdi A, Lozanovski B, Shidid D, Farahbod-Sternahl L, Witt G, Kelbassa I, Choong P, Qian M, Brandt M (2018) Inconel 625 lattice structures manu- factured by selective laser melting (SLM): mechanical properties, deformation and failure modes. Mater Des 157:179–199 20. Shidid D et al (2016) Just-in-time design and additive manufacture of patient-specific medical implants. Phys Procedia 83:4–14 21. Fleck NA (2002) New approaches to structural mechanics, shells and biological structures kluwer. New Approaches to Structural Mechanics, Shells and Biological Structures. Springer Nether- lands : Imprint: Springer.f 3. Mun J, Ju J, and Thurman J (2014) Indirect additive manufactur- ing based casting (I AM casting) of a lattice structure. in ASME International Mechanical Engineering Congress and Exposition, Proceedings (IMECE). 22. Deshpande VS, Fleck NA, Ashby MF (2001) Effective prop- erties of the octet-truss lattice material. J Mech Phys Solids 49(8):1747–1769f 4. Frazier WE (2014) Metal additive manufacturing: a review. J Mater Eng Perform 23(6):1917–1928f 4. Frazier WE (2014) Metal additive manufacturing: a review. J Mater Eng Perform 23(6):1917–1928f 23. Maconachie T et al. (2021) The effect of topology on the quasi- static and dynamic behaviour of SLM AlSi10Mg lattice structures. Int J Adv Manuf Technol. 5. Alghamdi A et al (2019) Effect of polygon order on additively manufactured lattice structures: a method for defining the thresh- old resolution for lattice geometry. Int J Adv Manuf Technol 105:2501–2511 24. Kang D et al (2019) Multi-lattice inner structures for high-strength and light-weight in metal selective laser melting process. Mater Des 175:107786f 6. Leary M et al (2018) Inconel 625 lattice structures manufactured by selective laser melting (SLM): mechanical properties, deforma- tion and failure modes. Mater Des 157:179–199 25 Clifford M, Simmons K, Shipway P (2009) An introduction to mechanical engineering: Part 1. CRC Press 26. Gentle R, Edwards P, Bolton W (2001) Mechanical engineering systems. Elsevier, pp 261–265 7. Lozanovski B et al (2019) Computational modelling of strut defects in SLM manufactured lattice structures. Mater Des 171:107671 27. Hally D (1987) Calculation of the moments of polygons. Defence Research Establishment Suffield Ralston (Alberta) 8. Ethics approval Not applicable. Consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors declare no competing interests. Competing interests The authors declare no competing interests. • ∅e B and ∅f B are useful to determine the manufacturabil- ity of different regular polygon cross-sections. As the effective diameter increased, ∅e B,CT and ∅f B,CT distribu- tions were more closely aligned with the range of their idealised values, ∅e B,ideal and ∅f B,ideal , indicating greater accuracy in achieving idealised geometry. Titanium strut Open Access This article is licensed under a Creative Commons Attri- bution 4.0 International License, which permits use, sharing, adapta- tion, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated 1 3 3577 The International Journal of Advanced Manufacturing Technology (2023) 126:3555–3577 16. Alomar Z, Concli F (2020) A review of the selective laser melt- ing lattice structures and their numerical models. Adv Eng Mater 12(12):2000611 otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. 17. Noronha J et al (2021) Manufacturability of Ti-Al-4V hol- low-walled lattice struts by laser powder bed fusion. JOM 73(12):4199–4208l 18. Bächle M, Kohal RJ (2004) A systematic review of the influ- ence of different titanium surfaces on proliferation, differentiation and protein synthesis of osteoblast-like MG63 cells. Clin Oral Implants Res 15(6):683–692 Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. References Zhai X, Jin L, Jiang J (2022) A survey of additive manufacturing reviews. MSAM 1(4):21 28. Ochshorn J (2009) Structural elements for architects and builders. Elsevier 9. Downing D et al (2020) Heat transfer in lattice structures during metal additive manufacturing: numerical exploration of tempera- ture field evolution. Rapid Prototyp J 25(5):911–928 29 Alghamdi A et al (2021) Buckling phenomena in AM lattice strut elements: a design tool applied to Ti-6Al-4V LB-PBF. Mater Des 208:109 10. Zhang B, Li Y, Bai Q (2017) Defect formation mechanisms in selective laser melting: a review. Chin J Mech Eng 30(3):515–527 30. Ashby MF, Cebon D (1993) Materials selection in mechanical design. Le J de Phys IV 3(C7):C7–C1 11. Echeta I et al. (2019) Review of defects in lattice structures manu- factured by powder bed fusion. Int J Adv Manuf Technol, 1–20. 31. Almalki A et al (2022) A digital-twin methodology for the non-destructive certification of lattice structures. JOM 74(4):1784–1797 12. Leary M et al. (2021) Surface roughness, in Fundamentals of Laser Powder Bed Fusion of Metals, I. Yadroitsev, et al., Editors. 32 Jywe W-Y, Liu C-H, Chen CO-K (1999) The min–max prob- lem for evaluating the form error of a circle. Measurement 26(4):273–282 13. Li R et al (2012) Balling behavior of stainless steel and nickel powder during selective laser melting process. Int J Adv Manuf Technol 59(9):1025–1035f 14. Alghamdi A et al. (2020) Effect of additive manufactured lattice defects on mechanical properties: an automated method for the enhancement of lattice geometry. Int J Adv Manuf Technol, 1–15. Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 15. Ali H, Ghadbeigi H, Mumtaz K (2018) Processing parameter effects on residual stress and mechanical properties of selective laser melted Ti6Al4V. J Mater Eng Perform 27(8):4059–4068 1 3 1 3 1 3 3
W3095208426.txt	https://link.springer.com/content/pdf/10.1007/s12592-020-00360-3.pdf	de		Der Lizenzierungsweg Fernstudium in der Sozialen Arbeit: Der Boom des großen Unbekannten	Soziale Passagen	2,020	cc-by	4,814	Soz Passagen (2020) 12:345–357 https://doi.org/10.1007/s12592-020-00360-3 PRAXIS HOCHSCHULE Der Lizenzierungsweg Fernstudium in der Sozialen Arbeit: Der Boom des großen Unbekannten Nikolaus Meyer · Christina Buschle Eingegangen: 9. März 2020 / Angenommen: 7. September 2020 / Online publiziert: 3. November 2020 © Der/die Autor(en) 2020 Zusammenfassung Eine Möglichkeit in das Berufsfeld der Sozialen Arbeit einzumünden besteht im Besuch eines Fernstudiengangs. Der vorliegende Beitrag beleuchtet die Entwicklung der Studierendenzahlen bei Fernstudiengängen aus dem Bereich der Sozialen Arbeit. Dafür werden sowohl Ergebnisse des Statistischen Bundesamtes als auch aus einer Sekundäranalyse zu Einstellungen von Fernstudierenden der Sozialen Arbeit abgebildet. Denn trotz steigender Bedeutung für die Professionalisierung des Berufsfeldes, gemessen an den Studierendenzahlen, ist das Fernstudium Sozialer Arbeit bisher kaum Gegenstand empirischer Analysen. Deutlich wird, dass das Fernstudium für den Lizenzierungsprozess eine hohe Bedeutung hat und ebenso als Möglichkeit der individuellen Professionalisierung für die Beschäftigten. Schlüsselwörter Professionalisierung · Soziale Arbeit · Hochschulforschung · Fernstudium · Lizenzierung Prof. Dr. N. Meyer () Fachbereich Sozialwesen, Hochschule Fulda, Leipziger Straße 123, 36037 Fulda, Deutschland E-Mail: nikolaus.meyer@sw.hs-fulda.de Prof. Dr. C. Buschle Arbeitsbereich Erwachsenenpädagogik, IUBH Internationale Hochschule, Kaiserplatz 1, 83435 Bad Reichenhall, Deutschland E-Mail: c.buschle@iubh-fernstudium.de K 346 N. Meyer, C. Buschle The licensing path distance learning in social work: the boom of the great unknown Abstract Attending a distance learning course is one way of entering the professional field of social work. This article examines the development of student numbers in such distance learning courses. We discuss data from the Federal Statistical Office as well as findings from a secondary analysis of attitudes of distance learning students in social work. So far, and despite its increasing importance for the professionalisation of the field, this alternative path has hardly been subject of empirical analyses. Our findings emphasise the significance of distance learning for the process of licensing as well as for individual professionalisation of employees. Keywords Professionalization · Social work · University research · Distance learning · Licensing 1 Einleitung Die berufssoziologischen Arbeiten von Everett Hughes (1984) markieren mit den Begriffen „Lizenz“ und „Mandat“ zwei relevante Kategorien zum Verständnis von Berufsgruppen (vgl. Nittel 2000; Schütze 1992). Während nun das Mandat den gesellschaftlichen Auftrag ausdrückt und für die spätere Berufspraxis zentral ist, werden die Novizinnen in jeder Berufsgruppe an einem zentralen Ort in die konkreten Praktiken der spezifischen Berufsgruppe „eingeweiht“. Hier erwerben die angehenden Fachkräfte die formale Berechtigung (Lizenz) zum Vollzug auch riskanter Handlungen an ihren Adressatinnen (Nittel 2000; Schütze 1992). Zentrale Orte dieses Lizenzierungsprozesses sind in der Sozialen Arbeit1 in besonderer Weise Hochschulen für angewandte Wissenschaft und Universitäten. Hier finden allerdings aktuell erhebliche Wandlungsprozesse statt (vgl. Meyer 2020, 2019a; Meyer und Schoneville 2020; Autorengruppe Fachkräftebarometer 2019). Zunächst auf institutioneller Ebene: Hier engagieren sich zunehmend private Hochschulen und dabei nehmen gerade die Zahlen dualer Studierender erheblich zu2. Gerade letztere Entwicklung wird in Profession und Disziplin Sozialer Arbeit sehr kritisch begleitet (Deutsche Gesellschaft für Soziale Arbeit (DGSA) 2019), kontrovers diskutiert (Otto 2018) und ist zunehmend Gegenstand eigenständiger Forschung (DGSA 2018; Meyer 2018). Trotz der eher geringen Zahl von rund 5 % der Studierenden im Verhältnis zu den Gesamtstudierenden, wird dem dualen Studium sozialer Berufe also eine erhebliche Aufmerksamkeit zu teil. Dabei geraten quantitativ zentrale 1 Der vorliegende Beitrag folgt zwei Vorgaben: 1.) Unter dem Begriff „Soziale Arbeit“ werden alle Studiengänge subsumiert, die von Destatis als Sozialwesen (Soziale Arbeit, Sozialpädagogik und Sozialwesen) sowie Pädagogik der frühen Kindheit ausgewiesen werden (vgl. Thole 2014). 2.) Der Beitrag orientiert sich an der Fächerklassifikation des Statistischen Bundesamtes (2018a, b). Zur Kritik an dieser mindestens intransparenten Differenzierung durch das Statistische Bundesamt: Meyer 2019b. 2 Im Wintersemester 2018/2019 wurden 4828 dual Studierende in den untersuchten Studiengängen von Destatis erfasst (Meyer 2020). Im Verhältnis zur Gesamtstudierendenzahl sind dies 5 % der immatrikulierten Personen. K Der Lizenzierungsweg Fernstudium in der Sozialen Arbeit: Der Boom des großen Unbekannten 347 Veränderungen bisher nur geringfügig in den Blick von Profession und Disziplin: Fernstudiengänge expandieren in nicht gekannter Weise. Bereits heute gibt es mehr Fernstudierende als Personen, die ihr Studium dual organisiert haben. Das Fernstudium als Organisationsmodus des Studiums und die Folgen für Professionalisierung sowie Professionalität werden dabei empirisch in der Sozialen Arbeit kaum untersucht. Er wird stattdessen vielmehr als didaktisches Problem reflektiert (vgl. Arnold et al. 2018). Vor diesem Hintergrund beschreibt der vorliegende Beitrag auf Basis einer Sonderauswertung von Daten des Statistischen Bundesamtes (Destatis) die Entwicklung der Studierendenzahlen in den verschiedenen Fernstudiengängen der Sozialen Arbeit. Darüber hinaus werden die Studierenden in Fernstudiengängen Sozialer Arbeit im Hinblick auf soziodemografische Faktoren sowie deren Erwartungen und Wünschen an das Studium auf Basis der Trendstudie Fernstudium der IUBH (Sommerfeld 2019) in den Blick genommen. Letztere ist dabei aktuell die einzige Quelle zur Studierendenstruktur sowie deren Erwartungen in Fernstudiengängen der Sozialen Arbeit, was angesichts der zunehmenden Bedeutung der Lizenzierungsphase überrascht. Gerade auch, weil die verschiedenen Hochschulen ein Fernstudium höchst unterschiedlich organisieren: Hier existieren Blended Learning-Szenarien neben reinen Onlinestudiengängen oder eher berufsbegleitenden Formen der Qualifizierung für die Soziale Arbeit. 2 Das Fernstudium in der Fächergruppe Soziale Arbeit Insgesamt haben im zuletzt durch Destatis3 ausgewiesenen Wintersemester 2018/2019 96.473 Personen einen Studiengang Soziale Arbeit (59.132), Sozialpädagogik (9052), Sozialwesen (24.875) oder Pädagogik der frühen Kindheit (3414) studiert (Meyer 2020). Im Verhältnis zu der Gesamtstudierendenzahl in den untersuchten Studiengängen studieren 10,6 % (10.261) der immatrikulierten Personen im Fernstudium4. Dies unterscheidet sich je nach Studiengang: So studierten 7459 Personen (davon 78 % Frauen) im Wintersemester 2018/2019 einen Fernstudiengang der Sozialen Arbeit, 1101 (davon 81,7 % Frauen) ein Fernstudium im Bereich Sozialpädagogik, 1009 (davon 71 % Frauen) einen entsprechenden Studiengang Sozialwesen sowie 692 Personen (davon 95 % Frauen) einen Fernstudiengang der Pädagogik der frühen Kindheit. In Abb. 1 zeigt sich der enorme Zuwachs in absoluten Zahlen innerhalb von rund zehn Jahren: Diese entsprechen im Fernstudiengang Soziale Arbeit einer prozentualen Zunahme von über 1047 % bei den Gesamtfernstudierendenzahlen zwischen 2007/2008 und 2018/2019, im Fernstudiengang Sozialpädagogik sogar um 100.800 % (sic!), im Fernstudiengang Sozialwesen um über 128 % sowie im Fernstudium der Pädagogik der frühen Kindheit um über 69.100 %. 3 Genauere Informationen zum methodischen Vorgehen liefert der Qualitätsbericht des Statistischen Bundesamtes „Studierende an Hochschulen“ (2019). 4 Eine Differenzierung nach den Abschlussarten der Fernstudierenden (Bachelor und Master) kann nicht auf der Grundlage der vorliegenden Destatis-Datensätze nicht getroffen werden. K 348 N. Meyer, C. Buschle 8000 7000 6000 5000 4000 3000 2000 1000 0 2007/2008 2017/2018 Soziale Arbeit 650 5141 7459 Sozialpädagogik 1 655 1101 Sozialwesen 441 992 1009 Pädagogik der frühen Kindheit 0 549 692 Soziale Arbeit Sozialpädagogik Sozialwesen 2018/2019 Pädagogik der frühen Kindheit Abb. 1 Entwicklung der Gesamtstudierendenzahlen in den Fernstudiengängen Soziale Arbeit, Sozialpädagogik, Sozialwesen und Pädagogik der frühen Kindheit in der Bundesrepublik Deutschland zwischen 2007/2008 und 2018/2019 (eigene Darstellung, in absoluten Zahlen) Auch die Bedeutungszunahme der privaten Hochschulen für angewandte Wissenschaften (HAW) im Fernstudium wird deutlich. Grundsätzlich ergänzen Hochschulen in privater oder auch kirchlicher Trägerschaft den öffentlichen Hochschulbereich. Diese Lage hat sich zwischenzeitlich völlig verändert (Abb. 2). Private Hochschulen für angewandte Wissenschaft sind im Segment des Fernstudiums keineswegs mehr eine Ergänzung der Hochschullandschaft, vielmehr dominieren sie diesen Bereich quantitativ deutlich: An staatlichen Hochschulen angewandter Wissenschaft studieren insgesamt 2908 Personen einen der genannten Studiengänge. An kirchlichen Hochschulen studieren 405 Personen einen entsprechenden Fernstudiengang. An privaten HAWs studieren dagegen insgesamt 6948 Personen einen Fernstudiengang. Ein Vergleich der prozentualen Anteile der verschiedenen Träger (Abb. 2) an den unterschiedlichen Studiengängen offenbart das deutliche Gewicht privater Hochschulen: Im Fernstudiengang Soziale Arbeit studieren 21 % der Studierenden an einer staatlichen, 5,2 % an einer kirchlichen sowie 74 % an einer privaten HAW. Im Fernstudiengang Sozialpädagogik studieren 7 % der Studierenden an einer staatlichen sowie 93 % an einer privaten HAW (vgl. Meyer 2019b). Im Fernstudiengang Sozialwesen studieren 98 % der Studierenden an einer staatlichen sowie 2 % an einer kirchlichen HAW. K Der Lizenzierungsweg Fernstudium in der Sozialen Arbeit: Der Boom des großen Unbekannten 349 5523 1548 1027 992 388 294 74 Soziale Arbeit Sozialpädagogik Staatliche HAWs 398 17 Sozialwesen Kirchliche HAWs Pädagogik der frühen Kindheit Private HAWs Abb. 2 Entwicklung der Studierendenzahl in den Studiengängen Soziale Arbeit, Sozialpädagogik, Sozialwesen sowie Pädagogik der frühen Kindheit nach Trägerart im Wintersemester 2018/2019 (eigene Darstellung, in absoluten Zahlen) Im Fernstudiengang Pädagogik der frühen Kindheit studieren 43 % der Studierenden an einer staatlichen sowie 58 % an einer privaten HAW. Die Studierendenzahlen entsprechen dabei weitgehend der institutionellen Verteilung: Zehn staatliche, eine kirchliche sowie sieben private HAWs und keine Universitäten sind im Wintersemester 2018/2019 in den genannten Fernstudiengängen aktiv. Eine beispielhafte Betrachtung der bei der Stiftung Akkreditierungsrat veröffentlichten Modulhandbücher aus dem Bereich Soziale Arbeit, für die anderen durch Destatis ausgewiesenen Studiengänge bedürfte es einer ähnlichen Untersuchung, zeigt dabei folgende Strukturen: An privaten HAWs sind die Fern-Bachelorstudiengänge in der Mehrheit mit einem Workload von 180 ECTS ausgewiesen, wobei die Praxisphase für die staatliche Anerkennung mit unterschiedlichen Zeitansätzen (800–960 h) ausnahmslos in den Studienverlauf integriert ist. Die Regelstudiendauer schwankt in diesen Studiengängen an privaten HAWs zwischen sechs und acht Semestern, wobei staatliche Hochschulen Studiengänge ausschließlich mit einer Regelstudienzeit von acht Semestern anbieten. Es werden an privaten HAWs keine Vorgaben – im Gegensatz zu der Mehrzahl der staatlichen HAWs – hinsichtlich relevanter Praxiserfahrung und -dauer vor der Aufnahme des Studiums gemacht. K 350 N. Meyer, C. Buschle Gleichzeitig differieren die Umrechnungszahlen von einem ECTS zwischen 25 und 30 h. In der Folge ergibt sich eine Differenz zwischen 4500 oder 5400 Studienstunden bei 180 ECTS. Unberücksichtigt blieben in der Auseinandersetzung mit den Akkreditierungsgutachten, hier bedürfte im Rahmen weiterer Forschung der Einsichtnahme in die Studien- und Prüfungsordnungen aller in der Differenzierung von Destatis genannten Hochschulen, die Anerkennungsmöglichkeiten der einzelnen Hochschulen bspw. mit Blick auf staatlich anerkannte Erzieherinnen oder andere Personengruppen5. 3 Die Ergebnisse der Trendstudie Fernstudium der IUBH Die vorliegenden Destatis-Daten zeigen die Bedeutungszunahme des Fernstudiums im Bereich der Sozialen Arbeit. Forschungsergebnisse zu Studierenden und deren Beweggründen sowie Einstellungen zum Fernstudium liegen dabei für den Bereich der Sozialen Arbeit aktuell nicht vor. Aus diesem Grund wird nachfolgend auf die Ergebnisse der Trendstudie Fernstudium der IUBH (Sommerfeld 2019) zurückgegriffen, die seit 2011 alle zwei bis drei Jahre durchgeführt wird. An der Umfrage haben sowohl Personen, die zum Befragungszeitpunkt bereits ein Fernstudium abgeschlossen haben, teilgenommen, als auch Personen, die in ein Fernstudium eingeschrieben waren, einen Fernstudiengang abgebrochen hatten oder Interesse an einem Fernstudium haben. Personen, die angaben sich kein Fernstudium vorstellen zu können, wurden von der Befragung ausgeschlossen. Die Erhebung der Trendstudie Fernstudium wurde im Oktober und November 2018 durchgeführt. Es beteiligten sich rund 5000 Personen, 3675 Personen haben den Fragebogen beendet (Sommerfeld 2019). Bisher wurde die Trendstudie lediglich kumuliert im Hinblick auf alle befragten Studiengänge veröffentlicht. Für den vorliegenden Beitrag wurden die Befragungsergebnisse einer Sekundäranalyse unterzogen: Ausgewertet wurden nur aktive Studierende, also keine Studieninteressierten oder bereits graduierte Studierende, die aktuell in einem Fernstudiengang einer deutschen Hochschule in der Fächergruppe Soziale Arbeit immatrikuliert sind. Entsprechend ergab sich ein Rücklauf von 304 Fragebögen, was einer Quote von knapp 6 % der erhaltenen Fragebogen entspricht. Aufgrund des hohen Non-response sind die Ergebnisse im Hinblick auf deren Verallgemeinerbarkeit mit Vorsicht zu bewerten. Vor dem professionstheoretischen Hintergrund und der damit verbundenen veränderten Bedeutung der Lizenzierungsphase, erscheint eine Auseinandersetzung mit den Erkenntnissen der Trendstudie, trotz deren Einschränkung in der Repräsentativität, als Möglichkeit einen Eindruck vom aktuellen Stand zu bekommen gleichwohl von hoher Bedeutung. Die 304 Studierenden der Sozialen Arbeit sind an neun HAWs in der Bundesrepublik immatrikuliert, wovon zwei in staatlicher und sieben in privater Trägerschaft sind. Bei den soziodemografischen Daten der Befragten zeigt sich, dass mit 74 % 5 Gleichzeitig wird bei der Durchsicht der Unterlagen der Stiftung Akkreditierungsrat deutlich, dass dort bei Destatis nicht als Fernstudienorte vermerkte weitere Hochschulstandorte bestanden haben oder bestehen müssen. Zur Problematik der Intransparenz bei der statistischen Zählung: Spanu et al. (2020). K Der Lizenzierungsweg Fernstudium in der Sozialen Arbeit: Der Boom des großen Unbekannten 351 die Zahl der Studentinnen in diesem Bereich überwiegt. Damit ist die Anzahl der weiblichen Studierenden im Bereich Soziale Arbeit tendenziell etwas höher als im Vergleich zur Geschlechterverteilung in der Trendstudie allgemein (68 % weiblich). Der Blick in die Anbieterstatistik „Strukturdaten Distance Learning/Distance Education 2019“ für das Wintersemester 2018/2019 weist einen Frauenanteil von 45 % bei den Studierenden im Bereich Distance Education aus (Fogolin 2019, S. 44)6, was den hohen Anteil an weiblichen Studierenden im Bereich der Sozialen Arbeit unterstreicht. Dieser Umstand ist im gesamten Qualifizierungsbereich der Sozialen Arbeit allerdings nicht unüblich: So sind 77,25 % aller Studierenden hier weiblich (Meyer 2020). Die befragten Fernstudierenden der Sozialen Arbeit sind in der Trendstudie zudem mehrheitlich in einem Alter zwischen 25 sowie 35 Jahren (41,1 %)7, was sich so ähnlich auch in den „Strukturdaten Distance Learning/Distance Education 2019“ zeigt (Fogolin 2019, S. 44) und auch den Erkenntnissen aus der Trendstudie der IUBH – ohne studiengangsspezifische Unterscheidung – tendenziell entspricht (Sommerfeld 2019, S. 16). Der höchste Bildungsabschluss der Fernstudierenden im Bereich Soziale Arbeit variiert zwischen (Fach-)Hochschulreife (60 %), einem bereits abgeschlossenen Hochschulstudium (Bachelor/Master: 11,5 %) und Hauptbzw. Realschulabschluss (17,1 %). Hier überwiegt also – ähnlich bei den Studierenden im Bereich Distance Education (Fogolin 2019, S. 44) – die allgemeine Hochschulzugangsberechtigung. Bei 49,7 % der befragten Studierenden haben die im Haushalt lebenden Personen und/oder die Eltern keinen Hochschulabschluss. Darüber hinaus wurden im Rahmen der Trendstudie weitere Erkenntnisse zur Lebenssituation der Studierenden in der Sozialen Arbeit generiert. Diese leben in einer festen Partnerschaft (28,6 %) oder sind verheiratet (32,9 %). Tendenziell verfügen die befragten Studierenden mit 56,3 % über keine Kinder unter 14 Jahren im eigenen Haushalt, wobei ein knappes Drittel der Studierenden (31,3 %) mit Kindern unter 14 Jahren im Haushalt lebt8. Ist dies der Fall, so sind es zumeist eines (10,9 %) oder zwei (14,8 %) Kinder. Die befragten Studierenden der Sozialen Arbeit kommen besonders häufig aus den westlichen Bundesländern, wobei sie hier relativ gleichverteilt in größeren (mehr als 50.000 Einwohnerinnen), mittleren (zwischen 10.000–50.000) sowie kleineren Kommunen (unter 10.000) leben. Gerade daraus wird deutlich, dass nicht die Erreichbarkeit einer Hochschule entscheidend zu sein scheint, sondern die bewusste Wahl eines Fernstudiengangs im Vordergrund steht. Dies unterstreicht auch die Angabe von 54,6 % der Studierenden, dass persönliche/ private Gründe die Aufnahme eines Präsenzstudiums verhindern. Einmal aufmerksam auf den jeweiligen Studiengang im Modus des Fernstudiums geworden, sind die Flexibilität des Studienmodells (75,3 %), die inhaltliche Ausrichtung des Studiengangs (63,6 %) sowie der geringe Anteil an Präsenzveranstaltungen (59,5 %) 6 Die Daten beziehen sich nicht speziell auf Studierende der Sozialen Arbeit, schließen Studierende aus den Bereichen Soziale Arbeit, Sozialpädagogik und Sozialwesen aber im Studienbereich Sozialwesen mit ein (Statistisches Bundesamt 2018b, S. 241). 7 Lediglich 26 % der befragten Studierenden sind älter als 36 Jahre und 21,4 % jünger als 25 Jahre. 8 Insgesamt planen etwa 10 % in Elternzeit die Aufnahme von Maßnahmen zur individuellen Professionalisierung (Statista 2019), wobei dies Frauen eher planen als Männer. K 352 N. Meyer, C. Buschle relevant, wobei insbesondere die Flexibilität den klassischen Vorteil eines Fernstudiengangs darstellt (Martin 2019). Darüber hinaus investieren die Studierenden der Sozialen Arbeit viel Zeit in ihr Studium: knapp 50 % studieren in einem Studienmodell mit über 30 h oder zwischen 15 und 30 h (36,8 %) pro Woche. Nach der realen Stundenzahl pro Woche befragt, geben 22,4 % der Studierenden 16–20 h pro Woche sowie 27 % 11–15 h pro Woche an. 17,4 % arbeiten mehr als 21 h pro Woche für das Fernstudium und 22,7 % bis zu zehn Stunden. Besonders stark werden dabei der Morgen (35,5 %), der frühe (45,4 %) sowie der späte Abend (44,1 %) zum Studium genutzt. Ebenso das Wochenende (61,2 %) oder Urlaube (40,5 %). In der Arbeitszeit setzen sich 6,3 % der Studierenden der Sozialen Arbeit mit Inhalten des Fernstudiums auseinander sowie gut 12 % in der Mittagspause. Damit liegen Studierende der Sozialen Arbeit in diesen beiden Tagesstrukturbereichen etwas hinter den Nutzungszahlen aller Studierender (9 % in der Arbeitszeit bzw. 14,2 % in der Mittagspause). Die geringere Integration des Studiums in die Arbeitszeit mag mit darin begründet liegen, dass die Arbeitgeberinnen der befragten Fernstudierenden der Sozialen Arbeit nur in Teilen die individuelle Professionalisierung (vgl. Nittel und Seltrecht 2008) unterstützen: Rückhalt durch den Arbeitgeber erfahren hier nur 24 % der Befragten, 48 % bekommen keine Unterstützung und gut 16 % arbeiten aktuell nicht. Mit welchem Ziel wird nun ein Fernstudium der Sozialen Arbeit in Angriff genommen? Die Studierenden streben in Summe zumeist (96,4 %) einen grundständigen Abschluss (Bachelor etc.) an. Insgesamt versprechen sich die Fernstudierenden der Sozialen Arbeit von der Aufnahme des Studiums eine Vertiefung ihres bereits vorhandenen Fachwissens (30,6 %) oder zu 35,2 % eine fachliche Veränderung. Über die Hälfte erwarten die Ermöglichung des eigenen beruflichen Aufstiegs, 39,5 % hoffen auf die Verbesserung von Arbeitsmarktchancen und 35,5 % auf eine Verbesserung der finanziellen Entlohnung. Der höchste Wert mit 53,5 % stellt der Wunsch nach persönlicher Weiterentwicklung dar. Auch wenn der Wunsch nach letzterer besonders hoch erscheint, so ist doch auffällig, dass mit der Wahl eines Fernstudiums eine Verbesserung der beruflichen Situation einhergeht. Bei 53,6 % baut der Fernstudiengang aus dem Bereich Soziale Arbeit entsprechend auch auf der bisherigen beruflichen Laufbahn auf. Vor diesem Hintergrund könnte die Wahl eines Fernstudiums in der Sozialen Arbeit als Instrument der individuellen Professionalisierung (wissenschaftliche berufliche Weiterbildung) für beruflich Tätige in dem Bereich verstanden werden9. Dennoch studieren 25 % einen solchen Fernstudiengang als Fachfremde, allerdings mit einer vorhandenen beruflichen Praxis und 10,5 % waren vor Aufnahme des Fernstudiums noch nicht berufstätig. Gerade bei Letzteren stellt 9 Robert Pelz und Markus Herklotz (2019) analysieren die Teilnahme von sächsischen Hochschulabsolventinnen. Sie stellen fest: „Die Mehrheit der [sächsischen, NM und CB] Absolventinnen und Absolventen (66 %) haben vor der wissenschaftlichen Weiterbildung ein Studium an einer Universität absolviert; etwas mehr als ein Drittel (34 %) an einer Hochschule für angewandte Wissenschaften“ (Pelz und Herklotz 2019, S. 90), wobei „[k]umuliert 26 % (...) nach einem bzw. 42 % nach zwei Jahren [nach einem ersten Studienabschluss, NM und CB] in eine Weiterbildung über[gehen]“ (Pelz und Herklotz 2019, S. 91). Weder Bildungsherkunft, Alter, Familienstand oder das Vorhandensein von mindestens einem Kind hat signifikanten Einfluss auf die Teilnahmehäufigkeit an wissenschaftlichen Weiterbildungen (Pelz und Herklotz 2019, S. 92). K Der Lizenzierungsweg Fernstudium in der Sozialen Arbeit: Der Boom des großen Unbekannten 353 sich die Frage warum sich diese Studierenden direkt für ein Fernstudium entschieden haben. Die Art der didaktischen Aufbereitung stellt für die Studierenden dabei ein wesentliches Entscheidungskriterium für die Aufnahme eines Fernstudiums dar: Eine geringe (11,5 %) oder sogar überhaupt keine Präsenzphase in den Lehrveranstaltungen (83,2 %) sind die am stärksten befürworteten Antwortoptionen. Vor diesem Hintergrund überrascht es nicht, dass mehr sozialer Austausch während des Studiums nur von 16,1 % der Studierenden gewünscht wird. Sind sich die Studierenden im Hinblick auf die besonderen didaktischen Optionen des Fernstudiums und deren Nutzen in der Gestaltung des eigenen Lebens bewusst, so wird der Ruf des Fernstudiums bei möglichen Arbeitgeber*innen indifferent eingeschätzt. Während 21,4 % der befragten Studierenden der Sozialen Arbeit glauben, dass ein Fernstudium bei einem Arbeitgeber weniger Wert als ein Präsenzstudium sei – sehen es gut 57 % als gleichwertig an. Dies sehen knapp 52 % aller befragten Fernstudierenden ebenso. 4 Diskussion und Ausblick Die Daten heben die Bedeutung des Fernstudiums für den Lizenzierungsprozess im Bereich der Sozialen Arbeit sowie als Möglichkeit der individuellen Professionalisierung für die Beschäftigten deutlich hervor. Dies hängt jedoch eng mit Fragen der kollektiven Professionalisierung zusammen, denn im Rahmen eines Fernstudiengangs scheint in erster Linie nicht der Erwerb einer rein formalen Berechtigung im Vordergrund zu stehen – der Studiengang baut ja bereits mehrheitlich auf bestehenden beruflichen Kenntnissen auf. Vielmehr scheint es der Wunsch nach persönlicher Weiterentwicklung sowie der Verbesserung der eigenen beruflichen Situation zu sein, welcher die Studierenden antreibt. Insofern stellt das Fernstudium Sozialer Arbeit für eine Mehrheit der befragten Personen gleichsam eine zweite Qualifizierung nach Abschluss einer beruflichen Erstausbildung in diesem Berufssegment und damit im eigentlichen Sinn eine wissenschaftliche Weiterbildung10 dar (vgl. Schmid et al. 2019; Wolter und Schäfer 2019). Diese führt gleichzeitig zur formalen Akademisierung bisheriger beruflicher Praxis, womit sich ein wichtiges Kriterium der Professionalisierung Sozialer Arbeit erfüllt (vgl. Meyer 2019a). Dieser Umstand ist aus zwei professionstheoretischen Gründen relevant: Professionalisierung beschreibt einen zeitlichen Prozess (vgl. Nittel 2000), der auf zwei verschiedenen Ebenen stattfindet. Sowohl auf der individuellen Ebene, hier verweist Professionalisierung auf den berufsbiografischen Prozess der Qualifizierung und die Erlangung von Professionalität durch eine Fachkraft, als auch auf die kollektive Ebene (vgl. Nittel und Seltrecht 2008; Nittel 2000). Auf dieser stellt Professionalisierung die fachliche Entwicklung und Profilierung sowie die Akademisierung von ganzen Berufsgruppen dar (vgl. Nittel und Seltrecht 2008) In diesem Sinne lässt sich in den Daten eine „Akademisierung auf Umwegen“ er- 10 „Insgesamt ist die unzureichende Datenbasis angesichts der steigenden Bedeutung der wWB [wissenschaftlichen Weiterbildung, NM und CB] erstaunlich“ (Shajek und Winterhager 2019, S. 53). K 354 N. Meyer, C. Buschle kennen, die für den Prozess der Professionalisierung Sozialer Arbeit langfristig positive Konnotationen aufweisen könnte. Gleichzeitig wird klar, dass diese „Akademisierung auf Umwegen“ eine besondere Hürde, zeitliche Überschneidungen und durch die fehlende Abstimmung zwischen den Qualifizierungsebenen sowie deren spezifischen Inhalten zu Doppelungen in der Auseinandersetzung mit den jeweiligen Wissensbeständen der Berufsgruppe führen wird. Auch die fehlende Anerkennungspraxis wird hier schlaglichtartig deutlich: An der Tatsache, dass es eine Praxis des Umwegs gibt, wird die Ungleichzeitigkeit der formal im Europäische Qualifikationsrahmen (EQR) gleichgestellten Abschlüsse Fachschulausbildung und Bachelor deutlich. Für die Befragten gibt es „handfeste“ Erwartungen – beruflicher Aufstieg, bessere Entlohnung oder Entfristung (vgl. Meyer und Wahl 2018; Nittel, Schütz und Tippelt 2014) –, die sie zur Akademisierung in ihrem bisherigen Arbeitsfeld antreibt. Gleichwohl wären die Abschlüsse schon längst gleichgestellt und trotzdem scheint es so etwas wie eine unterschiedliche Wertigkeit zu geben. Diese Perspektive lässt wiederrum keineswegs eine positive Entwicklung der Professionalisierung Sozialer Arbeit erkennen. Insgesamt ist das Geschlechterverhältnis eine wichtige Facette im Fernstudium: Hier sind tendenziell mehr Frauen als im Gesamtverhältnis aller Studierenden eingeschrieben, wobei dies besonders die Fernstudiengänge Sozialwesen und Pädagogik der frühen Kindheit betrifft. Dabei wird ein Fernstudiengang im Bereich der Sozialen Arbeit von Frauen wie Männer besonders in der Altersgruppe der 25 bis 35Jährigen nachgefragt. Gleichsam sind die Beweggründe für die Aufnahme eines als persönlich herausfordernd wahrgenommenen Fernstudiums (Sommerfeld 2019) bei den befragten Studierenden der Sozialen Arbeit von Bedeutung. Erkenntnisse aus der wissenschaftlichen Weiterbildung zeigen, dass in Gruppen nicht-traditioneller Studierender, also ohne familialen Erfahrungen mit einem Studium, der Wunsch zur Erhöhung des eigenen Wissensstandes offenbar besonders hoch ist (vgl. Gegenfurtner et al. 2019, S. 75). Dies gilt auch für die Befragten der IUBH-Studie (Sommerfeld 2019). Darüber hinaus fällt auf, dass Sozialpädagogik – im Gegensatz zur Gesamtstudierendenentwicklung (Meyer 2020) – im Fernstudium ähnlich stark wie der Studiengang Sozialwesen nachgefragt wird. Aus der Sonderauswertung der Daten des Bundesamtes für Statistik lässt sich für den Bereich des Fernstudiums außerdem zeigen, dass der Studiengang Pädagogik der frühen Kindheit sein starkes Wachstum eher hinter sich hat und sich nun auf hohem Niveau konsolidiert und fest in der Hochschullandschaft etabliert hat (Fachkräftebarometer Frühe Bildung 2019). Mit Blick auf die Trägerstruktur wird außerdem ein klarer Unterschied zwischen Präsenzstudium wie dualem Studium deutlich (Meyer 2020): Im prozentualen Verhältnis sind private Träger die Motoren der Entwicklung im Fernstudienbereich, der mehrheitlich an Hochschulen angewandter Wissenschaften vollzogen wird. Einzig der Studiengang Sozialwesen bildet eine Ausnahme. Hier sind staatliche HAWs – im Gegensatz zu privaten Hochschulen – besonders stark engagiert. Dies gilt für das im Präsenzstudium ebenso wie für das Fernstudium. K Der Lizenzierungsweg Fernstudium in der Sozialen Arbeit: Der Boom des großen Unbekannten 355 Abschließend bleibt ein ambivalentes Bild: Einerseits steigen die Zahlen von Fernstudierenden in der Sozialen Arbeit massiv an und gleichzeitig kennen wir die Fernstudierenden in der Sozialen Arbeit bisher so gut wie nicht. Da die Studienmodelle der verschiedenen Hochschulen sehr stark variieren, ist dies allerdings von elementarer Bedeutung. In einer späteren beruflichen Praxis, deren Hauptwerkzeug aber kommunikatives Handeln steht (vgl. von Spiegel 2013), muss diesem Aspekt in der Lizenzierungsphase besonderes Augenmerk geschenkt werden. Bisher haben Profession und Disziplin sich eher Fragen der Entwicklung des dualen Studiums gewidmet (vgl. DGSA 2019; Otto 2018) als die quantitativ bedeutsamere Entwicklung im Bereich des Fernstudiums wahrzunehmen. Die IUBH-Studie zeigt nun durchaus wichtige Einschätzungen der befragten Studierenden: Aus privaten, wie beruflichen Gründen kam ein Präsenzstudium für sie überhaupt nicht in Frage. Insofern entscheiden sich Studierende keineswegs „aus der Not heraus“ für ein Fernstudium, sondern im Gegenteil bewusst und alternativlos. Aktuell wissen wir über die Studierenden Sozialer Arbeit viel zu wenig, um damit die Präferenzen für die Wahl von Studienmodi (Präsenz, dual, Fern) erklären zu können und entsprechende Förderlinien zur Hochschulforschung fehlen. Vor diesem Hintergrund gilt es zunächst differenziert zu fragen, wer warum welches Studienmodell wählt und wie dieses inhaltlich adäquat auf die spätere berufliche Praxis vorbereiten kann. Funding Open Access funding enabled and organized by Projekt DEAL. 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Weitere Details zur Lizenz entnehmen Sie bitte der Lizenzinformation auf http://creativecommons.org/ licenses/by/4.0/deed.de. Literatur Arnold, P., Griesehop, H. R., & Füssenhäuser, C. (Hrsg.). (2018). Profilierung Sozialer Arbeit online. Innovative Studienformate und Qualifizierungswege. Wiesbaden: Springer VS. Autorengruppe Fachkräftebarometer (Hrsg.). (2019). Fachkräftebarometer Frühe Bildung 2019. München: DJI. Deutsche Gesellschaft für Soziale Arbeit (DGSA) (Hrsg.). (2018). Newsletter 1/2018. https://www.dgsa. de/fileadmin/Dokumente/Newsletter/Newsletter_DGSA_1_18-final.pdf. Zugegriffen: 8. Nov. 2018. Deutsche Gesellschaft für Soziale Arbeit (Hrsg.). (2019). Duale, trägernahe und reguläre Studiengänge Sozialer Arbeit – Qualitätsstandards für eine sich verändernde Hochschullandschaft. Ein Positionspapier des Vorstands der Deutschen Gesellschaft für Soziale Arbeit (DGSA). https://www.dgsa.de/ fileadmin/Dokumente/Aktuelles/DGSA_Stellungnahme_Qualit%C3%A4tskriterien_duale_Studieng %C3%A4nge_Soziale_Arbeit.pdf. Zugegriffen: 19. Apr. 2019. K 356 N. Meyer, C. Buschle Fogolin, A. (2019). Strukturdaten Distance Learning/Distance Education 2019. Bonn: Bundesinstitut für Berufsbildung. Gegenfurtner, A., Fisch, K., & Ebner, C. (2019). Teilnahmemotivation nicht-traditioneller Studierender an wissenschaftlicher Weiterbildung. Beiträge zur Hochschulforschung, 41(4), 58–83. Hughes, E. C. (1984). The sociological eye. New Brunswick: Transaction Books. Martin, A. (2019). Das Bedürfnis nach Flexibilität von Fernstudierenden. die hochschullehre, 5(2019), 839–854. Meyer, N. (2018). Trendstudiengang Soziale Arbeit?! Statistische Daten und ihre professionstheoretische Relevanz. Soziale Passagen. Journal Für Empirie und Theorie Sozialer Arbeit, 10(2), 299–308. https://doi.org/10.1007/s12592-018-0301-x. Meyer, N. (2019a). Zwischen Aufbruch und Gefährdung. Sozial Extra, 43(5), 335–340. https://doi.org/10. 1007/s12054-019-00219-9. Meyer, N. (2019b). Neue Unterschiede statt neuer Einigkeit. Sozial Extra, 43(4), 281–286. https://doi.org/ 10.1007/s12054-019-00194-1. Meyer, N. (2020). Spaltungen im Projekt „Professionalisierung Sozialer Arbeit“: Eine professionstheoretische Deutung am Beispiel der Gesamtstudierendenzahlen. neue praxis, 50(2), 122–140. Meyer, N., & Schoneville, H. (2020). Qualifizierung für die Soziale Arbeit. In W. Thole (Hrsg.), Grundriss Soziale Arbeit. Ein einführendes Handbuch. Wiesbaden: Springer VS. Meyer, N., & Wahl, J. (2018). Zwischen beruflichem Aufstieg und persönlicher Entwicklung. Sozial Extra, 42(1), 48–51. https://doi.org/10.1007/s12054-017-0113-9. Nittel, D. (2000). Von der Mission zur Profession? Stand und Perspektiven der Verberuflichung in der Erwachsenenbildung. Bielefeld: Bertelsmann. Nittel, D., & Seltrecht, A. (2008). Der Pfad der „individuellen Professionalisierung“. Ein beitrag zur kritisch-konstruktiven erziehungswissenschaftlichen Berufsgruppenforschung. Zeitschrift für Biographieforschung, Oral History und Lebensverlaufsanalyse, 21(1), 124–145. Nittel, D., Schütz, J., & Tippelt, R. (2014). Pädagogische Arbeit im System des lebenslangen Lernens. Ergebnisse komparativer Berufsgruppenforschung. Weinheim: Beltz Juventa. Otto, H.-U. (2018). Dual – Ende oder Wende des Studiums einer modernen Sozialen Arbeit. neue praxis, 48(3), 297–299. Pelz, R., & Herklotz, M. (2019). Wer bildet sich wissenschaftlich weiter? Beiträge zur Hochschulforschung, 41(4), 84–100. Schmid, C., Maschwitz, A., Wilkesmann, U., & Nickel, S. (2019). Wissenschaftliche Weiterbildung in Deutschland. Beiträge zur Hochschulforschung, 41(4), 10–35. Schütze, F. (1992). Sozialarbeit als „bescheidene“ Profession. In B. Dewe, W. Ferchhoff & F. Olaf-Radtke (Hrsg.), Erziehen als Profession. Zur Logik professionellen Handelns in pädagogischen Feldern (S. 132–170). Opladen: Leske + Budrich. Shajek, A., & Winterhager, N. (2019). Nutzen und Kosten wissenschaftlicher Weiterbildung. Beiträge zur Hochschulforschung, 41(4), 36–56. Sommerfeld, H. (Hrsg.). (2019). Trendstudie Fernstudium der IUBH. https://www.iubh-fernstudium.de/ wp-content/uploads/IUBH-Trendstudie_Fernstudium-2019_White-Paper_web.pdf. Zugegriffen: 22. Dez. 2019. Spanu, S., Meyer, N., & Karsten, M.-E. (2020, i. E.). Qualifizierung für die Qualifizierung zukünftiger Fachkräfte. Beschreibung und Diskussion empirischer Entwicklungen zur Ausbildung von Lehrkräften an Berufsbildenden Schulen mit der Fachrichtung Sozialpädagogik. neue praxis. v. Spiegel, H. (2013). Methodisches Handeln in der Sozialen Arbeit. Grundlagen und Arbeitshilfen für die Praxis. München: Reinhardt Verlag; UTB. Statista (Hrsg.) (2019). Was planen Sie während der Elternzeit zu tun?. https://de.statista.com/statistik/ daten/studie/727929/umfrage/geplante-aktivitaeten-in-der-elternzeit-in-deutschland/. Zugegriffen: 22. Dez. 2019. Statistisches Bundesamt (Destatis) (Hrsg.). (2018a). Bildung und Kultur. Studierende an Hochschulen – Fächersystematik. https://www.destatis.de/DE/Methoden/Klassifikationen/BildungKultur/ StudentenPruefungsstatistik.pdf;jsessionid=C85E62F78FF2F118DB34A36A783C3309. InternetLive1?__blob=publicationFile. Zugegriffen: 7. Jan. 2020. Statistisches Bundesamt (Destatis) (Hrsg.). (2018b). Bildung und Kultur. Prüfungen an Hochschulen. https://www.destatis.de/DE/Themen/Gesellschaft-Umwelt/Bildung-Forschung-Kultur/Hochschulen/ Publikationen/Downloads-Hochschulen/pruefungen-hochschulen-2110420177004.pdf?__ blob=publicationFileundv=4. Zugegriffen: 1. Febr. 2020. K Der Lizenzierungsweg Fernstudium in der Sozialen Arbeit: Der Boom des großen Unbekannten 357 Statistisches Bundesamt (Destatis) (Hrsg.). (2019). Studierende an Hochschulen. Qualitätsbericht. https:// www.destatis.de/DE/Methoden/Qualitaet/Qualitaetsberichte/Bildung/studenten.pdf?__ blob=publicationFile. Zugegriffen: 15. Okt. 2020. Thole, W. (2014). Die Soziale Arbeit. Praxis, Theorie, Forschung und Ausbildung. In W. Thole (Hrsg.), Grundriss Soziale Arbeit. Ein einführendes Handbuch (4. Aufl. S. 19–70). Wiesbaden: VS. Wolter, A., & Schäfer, E. (2019). Geschichte der wissenschaftlichen Weiterbildung. In W. Jütte & M. Rohs (Hrsg.), Handbuch Wissenschaftliche Weiterbildung (S. 1–28). Wiesbaden: Springer VS. K
https://openalex.org/W2128651676	https://europepmc.org/articles/pmc3610660?pdf=render	English	null	The Tolerogenic Peptide, hCDR1, Down-Regulates the Expression of Interferon-α in Murine and Human Systemic Lupus Erythematosus	PloS one	2,013	cc-by	7,943	Abstract doi:10.1371/journal.pone.0060394 Editor: Ari Waisman, Johannes Gutenberg University of Mainz, Germany Editor: Ari Waisman, Johannes Gutenberg University of Mainz, Germany Editor: Ari Waisman, Johannes Gutenberg University of Mainz, German Received September 21, 2012; Accepted February 27, 2013; Published March 28, 2013 Copyright: 2013 Sthoeger et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The authors have no support or funding to report. Funding: The authors have no support or funding to report. Competing Interests: The authors have declared that no competing interests exist. Competing Interests: The authors have declared that no competing interests exist. * E-mail: sthoeger@gmail.com (ZS); edna.mozes@weizmann.ac.il (EM) Abstract Background: The tolerogenic peptide, hCDR1, ameliorated manifestations of systemic lupus erythematosus (SLE) via the immunomodulation of pro-inflammatory and immunosuppressive cytokines and the induction of regulatory T cells. Because type I interferon (IFN-a) has been implicated to play a role in SLE pathogenesis, we investigated the effects of hCDR1 on IFN- a in a murine model of SLE and in human lupus. Methodology/Principal Findings: (NZBxNZW)F1 mice with established SLE were treated with hCDR1 (10 weekly injections). Splenocytes were obtained for gene expression studies by real-time RT-PCR. hCDR1 down-regulated significantly IFN-a gene expression (73% inhibition compared to vehicle treated mice, p = 0.002) in association with diminished clinical manifestations. Further, hCDR1 reduced, in vitro, IFN-a gene expression in peripheral blood mononuclear cells (PBMC) of 10 lupus patients (74% inhibition compared to medium, p = 0.002) but had no significant effects on the expression levels of IFN-a in PBMC of primary anti-phospholipid syndrome patients or of healthy controls. Lupus patients were treated for 24 weeks with hCDR1 (5) or placebo (4) by weekly subcutaneous injections. Blood samples collected, before and after treatment, were frozen until mRNA isolation. A significant reduction in IFN-a was determined in hCDR1 treated patients (64.4% inhibition compared to pretreatment expression levels, p = 0.015). No inhibition was observed in the placebo treated patients. In agreement, treatment with hCDR1 resulted in a significant decrease of disease activity. IFN-a appears to play a role in the mechanism of action of hCDR1 since recombinant IFN-a diminished the immunomodulating effects of hCDR1 on IL-1b, TGFb and FoxP3 gene expression. Conclusions/Significance: We reported previously that hCDR1 affected various cell types and immune pathways in correlation to disease amelioration. The present studies demonstrate that hCDR1 is also capable of down-regulating significantly (and specifically to lupus) IFN-a gene expression. Thus, hCDR1 has a potential role as a novel, disease specific treatment for lupus. Citation: Sthoeger Z, Zinger H, Sharabi A, Asher I, Mozes E (2013) The Tolerogenic Peptide, hCDR1, Down-Regulates the Expression of Interferon-a in Murine and Human Systemic Lupus Erythematosus. PLoS ONE 8(3): e60394. doi:10.1371/journal.pone.0060394 Citation: Sthoeger Z, Zinger H, Sharabi A, Asher I, Mozes E (2013) The Tolerogenic Peptide, hCDR1, D Human Systemic Lupus Erythematosus. PLoS ONE 8(3): e60394. Introduction matory cytokines (e.g. IL-1b, IFN-c, TNF-a,) [11,12] and up regulation of the immunosuppressive cytokine TGFb [11,13]. hCDR1 was shown to inhibit T cell receptor signaling following its binding to class II major histocompatibility complex (MHC) [14]. The induction of CD4 and CD8 regulatory T cells play a key role in the mechanism of action of hCDR1 [15–17]. Further, hCDR1 was shown to diminish T cell apoptosis [18,19]. Treatment with hCDR1 affected the B cell compartment as well. Thus, it down regulated the rate of maturation, differentiation and survival of B cells by reducing the levels of B cell activating factor (BAFF, BLyS) [20] as well as of molecules of the CD74/MIF pathway on B cells [21]. In addition, hCDR1 was shown to induce dendritic cells with immature phenotype and suppressed function that down regulate autoreactive T cells [22]. Systemic lupus erythematosus (SLE) is an autoimmune disorder characterized by the production of autoantibodies and impaired function of B and T cells accompanied by systemic clinical manifestations [1]. Various cytokines [2,3], factors affecting B cell activation and survival [4], apoptosis [5,6] and dysfunction of regulatory T-cells [7,8] were shown to be involved in the pathogenesis of murine and human SLE. Our laboratory designed a peptide, designated hCDR1 [9], that is based on the sequence of the complementarity determining region 1 (CDR1) of a human anti-DNA monoclonal antibody that bears a major idiotype (Id), namely the 16/6 Id [10]. hCDR1 was shown to ameliorate the serological and clinical manifestations of experimental SLE in mice with either induced (BALB/c) or spontaneous (NZBxNZW)F1 lupus [11]. The beneficial effects of hCDR1 were associated with a reduced production and expression of inflam- We have further demonstrated similar immunomodulatory effects of hCDR1 on peripheral blood mononuclear cells (PBMC) March 2013 \| Volume 8 \| Issue 3 \| e60394 March 2013 \| Volume 8 \| Issue 3 \| e60394 PLOS ONE \| www.plosone.org 1 hCDR1 Diminishes Interferon-a Expression in Lupus Figure 1. Treatment of SLE afflicted (NZBxNZW)F1 mice with hCDR1 results in the down regulation of IFN-a. (A) Mean percentage (6SE) results of 4 independent experiments in which the mRNA expression of IFN-a was determined in pools of spleen derived cells of SLE afflicted mice (10–12 mice per group) treated with vehicle, hCDR1, or the control, scrambled, peptide. Introduction The levels of gene expression were determined by real- time RT-PCR and calculated relatively to levels in cells from vehicle-treated mice (considered as 100%). (B) Mean concentrations (6SE) of IFN-a determined by ELISA in sera of the same groups of mice. doi:10.1371/journal.pone.0060394.g001 Figure 1. Treatment of SLE afflicted (NZBxNZW)F1 mice with hCDR1 results in the down regulation of IFN-a. (A) Mean percentage (6SE) results of 4 independent experiments in which the mRNA expression of IFN-a was determined in pools of spleen derived cells of SLE afflicted mice (10–12 mice per group) treated with vehicle, hCDR1, or the control, scrambled, peptide. The levels of gene expression were determined by real- time RT-PCR and calculated relatively to levels in cells from vehicle-treated mice (considered as 100%). (B) Mean concentrations (6SE) of IFN-a determined by ELISA in sera of the same groups of mice. doi:10.1371/journal.pone.0060394.g001 a has been considered recently as a therapeutic target for the treatment of human SLE. obtained from lupus patients. Thus, incubation of PBMC of lupus patients (but not of healthy volunteers) with hCDR1 resulted in a significant down regulation of gene expression of pro-inflamma- tory cytokines, apoptotic factors, and BLyS and up regulation of gene expression of immunosuppressive factors (Foxj1, Foxo3a, TGFb, Foxp3) [23]. In addition, hCDR1 increased the number as well as the function of CD4+CD25+Foxp3+regulatory T-cells in PBMC of lupus patients [23]. Further, we reported the beneficial effects of in vivo treatment with hCDR1 in five lupus patients with mild to moderate disease [24]. In agreement with its clinical beneficial effects, hCDR1 was shown to immunomodulate in vivo the expression of genes that play a role in SLE thus restoring the global immune dysregulation in those patients [24]. Since we have demonstrated that hCDR1 was capable of restoring the cytokine dysregulation observed in SLE and of down regulating the maturation and function of dendritic cells (that are activated by IFN-a and are a source for IFN-a production as well) we investigated, in the present studies, the effects of hCDR1 on IFN-a in a murine model of SLE and in human lupus. Effects of hCDR1 on IFN-a gene expression of PBMC of lupus patients lymphocytes of SLE-afflicted mice treated with vehicle, hCDR1 or control peptide. The gene expression of IFN-a was determined by real-time RT-PCR. The results are expressed as percentage gene expression relatively to that observed for vehicle treated mice, considered as 100%. As can be seen in the Figure, treatment with hCDR1 down-regulated significantly (p = 0.0005) the gene expression of IFN-a as compared to levels of the vehicle treated mice. No such effects were observed in splenocytes of mice treated with the control peptide. In spite of the limited sensitivity of the ELISA assay, the levels of IFN-a (56.8361.4 pg/ml) detected in sera of vehicle treated mice were similar to those reported for the sera of SLE patients [32]. As can been seen in Figure 1B, treatment with hCDR1 but not with the control peptide decreased significantly (p = 0.028) the sera levels of IFN-a. Thus, our studies demonstrated that hCDR1 treatment decreased IFN-a gene expression (Figure 1A) and its sera levels (Figure 1B). p p We further studied the in vitro effects of hCDR1 on IFN-a in lupus patients. To this end, PBMC were obtained from 10 lupus patients (8 females, 2 males) aged 32–65 years that were diagnosed with SLE according to four or more ACR diagnostic criteria [33]. Their main current clinical manifestations were arthritis (60%), mucocutaneous (50%), renal (20%) and pleuritis/pericarditis (20%). Eight patients were treated with Plaquonil (400 mg/d) and five with corticosteroids (2.5–10 mg/d of Prednisone) at the time of the study. The patients’ PBMC were cultured for 48 hours in the presence of hCDR1 (25 mg/ml) or medium alone. Thereafter, mRNA was isolated from the cells and IFN-a gene expression was determined by real-time RT-PCR. For control we cultured concomitantly PBMC that were obtained from 5 healthy volunteers and PBMC of 5 patients with primary anti-phospho- lipid syndrome (APS) that did not have lupus (either clinically or serologically). Figure 3 shows the results of these experiments. It can be seen that in vitro incubation of PBMC of lupus patients with hCDR1 diminished significantly (p = 0.004) the IFN-a gene expression as compared with PBMC of the same patients cultured with medium (considered as 100%). It should be noted that the baseline levels of IFN-a gene expression in PBMC of SLE patients were 3 folds higher than those determined for the healthy controls (p = 0.028). Treatment of SLE afflicted (NZBxNZW)F1 mice with hCDR1 down regulated gene expression of IFN-a Type I interferons, mainly interferon (IFN)-a, were suggested to play a major role in the pathogenesis of murine and human SLE [25]. Thus, elevated levels of IFN-a were demonstrated in sera of SLE afflicted mice as well as in sera of lupus patients [26,27], and IFN-a levels were reported to correlate with disease activity [28]. Similarly, high levels of IFN-a inducible gene expression (‘‘IFN- signature’’) were shown in blood cells of lupus patients [29]. Moreover, type I IFN receptor deficiency was reported to reduce significantly lupus like disease in a mouse model [30] and IFN-a neutralizing antibodies were shown to prevent the clinical manifestations in a lupus flare murine model [31]. Hence, IFN- Eight month old (NZBxNZW)F1 female mice were treated with 10 subcutaneously weekly injections of hCDR1 (50 mg/mouse), control (scrambled) peptide (50 mg/mouse) or the vehicle alone (10–12 mice per group in 4 independent experiments). Mice were bled periodically for the determination of sera anti-dsDNA autoantibody and IFN-a levels and were tested for proteinuria levels. All mice were sacrificed at the end of treatment, their kidneys were evaluated for immune complex deposits and spleen derived lymphocytes were used for mRNA preparation. Figure 1A shows the effect of treatment with hCDR1 on IFN-a gene expression. The results are of 4 independent experiments in which mRNA was prepared from pools of spleen derived PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org March 2013 \| Volume 8 \| Issue 3 \| e60394 March 2013 \| Volume 8 \| Issue 3 \| e60394 2 hCDR1 Diminishes Interferon-a Expression in Lupus Table 1. Effects of treatment with hCDR1 on SLE manifestations in mice. Treatment a dsDNA Ab b(OD) p c Proteinuria d (g/L) p c ICD e p c Vehicle 0.5060.08 — 8.3661.86 — 2.4360.2 — hCDR1 0.3060.05 0.04 1.9460.45 0.03 1.5760.2 0.002 Controlf 0.5260.08 NS 8.9461.86 NS 2.7860.2 NS aSLE-afflicted (NZB6NZW)F1 mice (10–12 mice per group in 4 independent experiments) were treated with weekly subcutaneous injections of the vehicle, hCDR1, or a control (scrambled peptide) for 10 weeks. bResults are of sera from mice that were bled after the end of treatment. Dilution of sera 1:1250. cStatistical evaluation was based on the Mann-Whitney U test to compare post –treatment effects between the vehicle –treated groups and the remaining treatment groups. dProteinuria was always measured at about the same time of day and all mice in an experimental cohort were tested together. Treatment of SLE afflicted (NZBxNZW)F1 mice with hCDR1 down regulated gene expression of IFN-a eImmune complex deposits (ICD) were assessed at sacrifice. fp = 0.04, 0.02 and 0.0001 between the control peptide and hCDR1 treated mice for dsDNA specific antibodies, proteinuria and ICD, respectively. doi:10.1371/journal.pone.0060394.t001 p p ( ) .04, 0.02 and 0.0001 between the control peptide and hCDR1 treated mice for dsDNA specific antibodies, proteinuria and ICD, respectively. 0 1371/journal pone 0060394 t001 Effects of hCDR1 on IFN-a gene expression of PBMC of lupus patients Figure 3 also shows that in vitro incubation of hCDR1 with PBMC of healthy donors or of patients with primary APS did not down regulate the levels of IFN-a gene expression in the treated cells. Thus, these results suggest that the hCDR1-induced reduction of IFN-a gene expression is specific to lupus patients. In agreement, treatment with hCDR1 ameliorated disease manifestations in the experimental mice. As can be seen in Table 1, that summarizes the beneficial effects of hCDR1 treatment, a significant reduction was determined in the levels of dsDNA autoantibodies, in proteinuria levels as well as in glomerular immune complex deposits (ICD) as compared to mice that were treated with the vehicle alone. It is also seen in the Table that no such effects were observed in mice that were treated with the control peptide. Immunohistology of kidney sections of representative mice of each group are shown in Figure 2. As can been seen in the Figure, intense ICD are demonstrated in the kidney sections of the vehicle and the control peptide treated mice but not in the hCDR1 treated group of mice. We also tested the supernatants of the lupus PBMC cultured with hCDR1 (or medium alone) for the presence of IFN-a by ELISA but apparently the assay was not sensitive enough and IFN-a could not be detected in the cultures. Figure 2. hCDR1 down regulates immune complex deposits in kidney sections. Immunohistology of kidney sections of representa- tive mice of each experimental group (vehicle, hCDR1 and control peptide treated mice). Magnification X400. doi:10.1371/journal.pone.0060394.g002 hCDR1 Diminishes Interferon-a Expression in Lupus hCDR1 Diminishes Interferon-a Expression in Lupus Figure 3. hCDR1 down regulates IFN-a gene expression in PBMC of SLE patients. PBMC of 10 SLE, 5 primary APS patients and 5 healthy controls were cultured (56106 cells/well) for 48 hours in the presence of medium or hCDR1 (25 mg/ml). Gene expression was determined by real-time RT-PCR. Results are presented as the mean6SE percentage of gene expression compared with cultures with medium (considered as 100%). * p = 0.004. doi:10.1371/journal.pone.0060394.g003 SLEDAI scores of the treated patients. In agreement, a significant decrease in the SLEDAI-2K score (45% reduction, p = 0.02) was observed in the hCDR1 treated patients but not in the placebo treated patients [24]. The role of IFN-a in the hCDR1-induced immunomodulation in SLE We further studied the possible mechanistic role of IFN-a in the hCDR1-induced immunomodulation in SLE. To this end, PBMC obtained from 3 SLE patients were cultured in triplicates for 48 hours in the presence of medium alone, hCDR1 (25 mg/ml) or hCDR1 (25 mg/ml) with human recombinant IFN-a at concen- trations of 100–10,000 U/ml. Thereafter, mRNA was isolated from the cells and IL-1b, TGFb and FoxP3 gene expression was determined by real-time RT-PCR. Figure 5 shows the results of these experiments using human recombinant IFN-a at a concentration of 5,000 U/ml (shown to have the optimal effect). It can been seen that, as was previously shown [23], hCDR1 significantly down-regulated IL-1b and up-regulated TGFb and FoxP3 gene expression in lupus PBMC (p = 0.05, 0.03 and 0.03 for IL-1b, TGFb and FoxP3, respectively). The addition of IFN-a to the cultures abolished completely those effects (p = 0.05, 0.016 and 0.028 between cultures of PBMC with hCDR1 and those with hCDR1+recombinant IFN-a for IL-1b, TGFb and FoxP3, respectively) suggesting that IFN-a plays a role in the immuno- modulating effects of hCDR1 in SLE. Figure 3. hCDR1 down regulates IFN-a gene expression in PBMC of SLE patients. PBMC of 10 SLE, 5 primary APS patients and 5 healthy controls were cultured (56106 cells/well) for 48 hours in the presence of medium or hCDR1 (25 mg/ml). Gene expression was determined by real-time RT-PCR. Results are presented as the mean6SE percentage of gene expression compared with cultures with medium (considered as 100%). * p = 0.004. doi:10.1371/journal.pone.0060394.g003 the SLE patients on IFN-a gene expression was then determined. Table 2 shows the expression of the IFN-a gene for the individual patients and Figure 4 presents the mean percent of gene expression at week 24 compared to the level prior to hCDR1 or vehicle treatment, at week 0 (defined as 100%; dotted line). As can be seen in Table 2 and Figure 4, treatment for 24 weeks with hCDR1 diminished significantly (p = 0.0005) the gene expression of IFN-a in the 5 treated patients. No significant effects were observed in the 4 lupus patients that were treated with the vehicle alone (placebo group; Table 2, Figure 4). the SLE patients on IFN-a gene expression was then determined. The role of IFN-a in the hCDR1-induced immunomodulation in SLE Table 2 shows the expression of the IFN-a gene for the individual patients and Figure 4 presents the mean percent of gene expression at week 24 compared to the level prior to hCDR1 or vehicle treatment, at week 0 (defined as 100%; dotted line). As can be seen in Table 2 and Figure 4, treatment for 24 weeks with hCDR1 diminished significantly (p = 0.0005) the gene expression of IFN-a in the 5 treated patients. No significant effects were observed in the 4 lupus patients that were treated with the vehicle alone (placebo group; Table 2, Figure 4). The effect of in vivo treatment of lupus patients with hCDR1 on IFN-a gene expression We have further evaluated the effect of hCDR1 treatment on IFN-a gene expression in 9 lupus patients with mild to moderate disease. The patients were treated with weekly subcutaneous injections of either hCDR1 (5 patients) or vehicle alone (4 patients). The inclusion and exclusion criteria, patients’ charac- terization and clinical outcome were described previously [24]. Blood samples were collected from the patients in PAXgene tubes prior and following 24 weeks of treatment for the preparation of mRNA. The in vivo effect of weekly administration of hCDR1 to Figure 2. hCDR1 down regulates immune complex deposits in kidney sections. Immunohistology of kidney sections of representa- tive mice of each experimental group (vehicle, hCDR1 and control peptide treated mice). Magnification X400. doi:10.1371/journal.pone.0060394.g002 March 2013 \| Volume 8 \| Issue 3 \| e60394 PLOS ONE \| www.plosone.org 3 Discussion The main finding of the present study is that the tolerogenic peptide hCDR1 is capable of suppressing IFN-a gene expression in a murine SLE model and in lupus patients. This suppressive effect was specific since it was not observed following treatment of (NZBxNZW) F1 lupus prone mice with the control scramble peptide, or in PBMC obtained from healthy volunteers or APS patients. Moreover, the down regulation of IFN-a gene expression correlates to the therapeutic beneficial effects of hCDR1 in murine and in human lupus. Figure 4 also shows that the significant inhibition of IFN-a gene expression following treatment with hCDR1 was in agreement with the observed clinical effects. Thus, as shown in the Figure, lupus disease activity as determined by the BILAG score decreased significantly (p = 0.03) in the hCDR1 treated but not in the placebo treated group of patients. Not shown in the Figure are the In previous studies, we were able to demonstrate that the tolerogenic peptide, hCDR1, ameliorates manifestations of SLE in murine models [9] and in a small cohort of lupus patients [24]. Figure 4. hCDR1 down regulates in vivo IFN-a gene expression in SLE patients. SLE patients were treated (subcutaneously, once a week) with either hCDR1 (0.5, 1, or 2.5 mg) or placebo. Gene expression in blood samples obtained from the patients was determined by real-time RT-PCR. Results are presented as mean percentage of gene expression (6SE) at week 24 compared to the levels at week 0 (defined as 100%; dotted line). Also shown in the Figure is the mean percent reduction in the BILAG score following 24 weeks of treatment with either hCDR1 or placebo as compared to the baseline score (week 0) considered as 100% (dotted line). doi:10.1371/journal.pone.0060394.g004 Figure 4. hCDR1 down regulates in vivo IFN-a gene expression in SLE patients. SLE patients were treated (subcutaneously, once a week) with either hCDR1 (0.5, 1, or 2.5 mg) or placebo. Gene expression in blood samples obtained from the patients was determined by real-time RT-PCR. Results are presented as mean percentage of gene expression (6SE) at week 24 compared to the levels at week 0 (defined as 100%; dotted line). Also shown in the Figure is the mean percent reduction in the BILAG score following 24 weeks of treatment with either hCDR1 or placebo as compared to the baseline score (week 0) considered as 100% (dotted line). Discussion doi:10.1371/journal.pone.0060394.g004 March 2013 \| Volume 8 \| Issue 3 \| e60394 March 2013 \| Volume 8 \| Issue 3 \| e60394 PLOS ONE \| www.plosone.org 4 hCDR1 Diminishes Interferon-a Expression in Lupus Table 2. The effect of in vivo treatment with hCDR1 on IFN-a gene expression in PBMC of SLE patient Patient No. Treatment Dose (mg) IFN-a (% Expression relative to baseline) 70103 hCDR1 0.5 20 70106 hCDR1 0.5 20 70101 hCDR1 1.0 17 70403 hCDR1 1.0 44 70104 hCDR1 2.5 82 70404 Placebo — 135 70405 Placebo — 141 70102 Placebo — 228 70107 Placebo — 96 SLE patients with mild and moderate disease manifestations were treated (subcutaneously) with either hCDR1 or placebo. IFN-a gene expression in blood samples was determined by real-time RT-PCR. Results are presented as the percentage of gene expression at week 24 compared to that at week 0 (before the study was initiated), defined as100%. doi:10.1371/journal.pone.0060394.t002 SLE patients with mild and moderate disease manifestations were treated (subcutaneously) with either hCDR1 or placebo. IFN-a gene expression in blood samples was determined by real-time RT-PCR. Results are presented as the percentage of gene expression at week 24 compared to that at week 0 (before the study was initiated), defined as100%. doi:10.1371/journal.pone.0060394.t002 SLE patients with mild and moderate disease manifestations were treated (subcutaneously) with either hCDR1 or placebo. IFN-a gene expression in blood samples was determined by real-time RT-PCR. Results are presented as the percentage of gene expression at week 24 compared to that at week 0 (before the study was initiated), defined as100%. doi:10 1371/journal pone 0060394 t002 Those beneficial effects resulted from the effects of hCDR1 on different immune cell types (including dendritic [22], T [9,17,34] and B cells [20,21,35]) and on cytokines [9,11]. Thus, hCDR1 down regulated (in vivo and in vitro, in murine SLE models and in human lupus) pro-inflammatory cytokines [11,23,24], apoptotic factors [18,19,23,24,36], B-cell stimulatory factors (BAFF/BLyS) [20,24] and up regulated immunosuppressive cytokines [9,11,23,24,37] with the induction of CD4+CD25+FoxP3+regu- latory T-cells [15,23]. Second, hCDR1 significantly decreased, in vitro, IFN-a gene expression in PBMC of lupus patients but not in PBMC obtained from healthy volunteers or primary APS patients (Figure 3). Thus, as was previously shown for other cytokines and immunosuppres- sive factors [9,23], the effect of hCDR1 on IFN-a is specific to lupus. hCDR1 Diminishes Interferon-a Expression in Lupus types that are involved in the pathogenicity of lupus. In the present study we demonstrated down regulating effect of hCDR1 on one of the important cytokines that is involved in lupus etiology and pathogenesis, namely IFN-a. Recent reports showing that IFN-a has the potential to influence the development and progression of SLE suggest this cytokine as a therapeutic target. A number of mechanisms were suggested to account for the pathogenic effects of IFN-a. It has been reported that dendritic cells mature and become more prone to activate T cells in the presence of IFN-a [38]. Further, activity of regulatory T cells (Tregs) was shown to be suppressed by the in vitro treatment of dendritic cells with IFN-a [39] and the increased levels of IFN-a in lupus patients was reported to contribute, at least in part, to the diminished Tregs activity observed in patients with SLE [40]. Type I interferons were shown to directly improve B-cell survival in vitro [41] and to reduce the sensitivity of B cells to FasL-mediated apoptosis [42]. In addition, IFN-a may affect B cell survival, maturation and differentiation, indirectly by inducing dendritic cells and macro- phages to produce BLyS [43]. Moreover, the present study suggests that IFN-a plays a mechanistic role in the immunomod- ulating effects of hCDR1 in SLE since the addition of recombinant IFN-a diminished the effects of hCDR1 on cytokine expression in PBMC of SLE patients (Figure 5). In agreement, the addition of anti-IFN-a-antibodies to PBMC of SLE patients (in the absence of hCDR1) down regulated IL-1b and up regulated TGFb and FoxP3 gene expression, similarly to the immunomodulation of these genes by hCDR1 (Mozes et al., unpublished results) further supporting the role of IFN-a. IL-2 production only in cases of lupus associated responses [14,34,37,45] and did not affect responses to unrelated antigens. Similarly, we demonstrated that treatment of SLE–like disease in SCID mice, transplanted with PBMC of SLE patients, led to the suppressed production of the human anti-dsDNA autoantibodies as well as to the amelioration of SLE manifestations. Nevertheless, no significant effects could be observed on the levels of human anti-tetanus toxoid antibodies [47] in the treated mice. Moreover, only Tregs induced by hCDR1 ameliorated disease manifestations when transferred into SLE afflicted (NZBxNZW)F1 mice [15]. Tregs originating from a control peptide or the vehicle treated mice did not have a significant clinical effect on mice with established lupus [15]. Evaluation of murine lupus disease activity Anti-dsDNA autoantibody levels were measured using l phage dsDNA, as previously described [11]. Proteinuria was measured by a standard semi-quantitative test, using an Albustix kit (Bayer Diagnostic, Newbury, UK). Detection of glomerular immune complex deposits was performed as described earlier [11]. The intensity of immune complex deposits (immunohistology) was graded as follows: 0, no immune complex deposits; 1, low intensity; 2, moderate intensity; and 3, high intensity of immune complexes. The analysis was performed by two people blinded to whether the mice belonged to control or experimental groups. An important aim in the treatment of lupus, as well as any other diseases, is to suppress the SLE related autoimmune responses and, at the same time, to spare the normal function of the immune system. One of the challenges using therapeutic means that neutralize IFN-a is to inhibit the SLE-related over production of IFN-a and to leave intact the anti-viral activity of IFN-a. A similar problem arises when certain cell types (e.g., B cells) are depleted in order to suppress autoimmune responses. We have shown, in the present study that the inhibitory effect of hCDR1 on the expression of the IFN-a gene is specific to lupus and it does not affect healthy controls and patients with APS (Figure 3). In agreement, we have previously shown that hCDR1 inhibited in vitro murine and human T cell proliferation as well as IFN-c and hCDR1 Diminishes Interferon-a Expression in Lupus The specific effect of hCDR1 was further confirmed by the fact that hCDR1 induced functional Tregs were not capable of inhibiting myasthenia gravis associated responses (Mozes E. et al. unpublished results). Thus, the efficient and specific beneficial effects of hCDR1 at the different checkpoints and on the various factors involved in lupus, as exemplified in the present study by its effect on one of the central cytokine, IFN-a, suggest a potential role for this tolerogenic peptide in the treatment of lupus patients. Treatment of (NZBxNZW)F1 mice Eight-month old female mice (10–12 mice per group) with established lupus manifestations were treated in 4 independent experiments with 10 weekly subcutaneous injections of hCDR1 (50 mg/mouse), control peptide (50 mg/mouse) or vehicle alone (phosphate buffered saline). Mice Female (NZBxNZW)F1 mice were purchased from The Jackson Laboratory (Bar harbor, ME, USA). Murine experiments were approved by the Animal Care and Use Committee of the Weizmann Institute of Science. pp g Thus, the diminished expression of IFN-a following treatment with hCDR1 demonstrated in our study may affect SLE manifestations via any or all the above suggested mechanisms. Indeed, we have previously reported that treatment with hCDR1 down regulated the maturation and activation of dendritic cells [22] resulting in the induction of functional Tregs and suppressed autoreactive T cell activity in SLE models and in lupus patients [9]. Furthermore, hCDR1 was shown to reduce BAFF/BLyS production and to up-regulate B cell apoptosis via the up regulation of pro-apoptotic molecules (e.g. Caspase 8) and the down regulation of anti-apoptotic molecules (Bcl-2, Bcl-xL) [20,24]. Nevertheless, it should be kept in mind that even though the effects of IFN-a can explain many SLE features, only a fraction of SLE patients displays elevated levels of IFN-a. Furthermore, other pathogenic cytokines that function together with IFN-a or independently, and cell marker molecules [2,44] were shown to be involved in lupus and therefore, reducing IFN-a probably affects only partially this complex, multifactorial disease. Thus, blocking a single cytokine might not be the full answer for controlling SLE. Indeed, we have previously shown that treatment with hCDR1 leads to a cascade of events that affect activated dendritic cells, T and B cells and their products as well as important pathways involved in the pathogenesis of lupus [9,45,46]. Synthetic peptides A peptide, GYYWSWIRQPPGKGEEWIG, (hCDR1) [37] that is based on the complementarity determining region (CDR) 1 of the human anti-DNA monoclonal antibody, bearing a major idiotype (16/6 Id) [10] was synthesized by Polypeptide Labora- tories (CA, USA). A peptide, SKGIPQYGGWEGWRYEI, containing the same amino acids as hCDR1 in a scrambled order was used as a control. Discussion Third, treatment of five lupus patients with hCDR1 for twenty-four consecutive weeks resulted in significant inhibition of IFN-a gene expression (Table 2, Figure 4). Concomitantly, disease activity (defined by both, BILAG and SLEDAI) in the hCDR1 treated patients decreased significantly (Figure 4 and [24]). No such effects were observed in the four other lupus patients who were treated with the vehicle alone [Table 2, Figure 4]. Taken together, the present studies clearly demonstrated lupus-specific inhibitory effects of our tolerogenic peptide on IFN-a in SLE. We demonstrate here a significant down regulation of IFN-a gene expression by hCDR1 in three different lupus related experimental systems. First, treatment of (NZBxNZW) F1 lupus prone mice with hCDR1, but not with the vehicle or the control (scrambled) peptide, resulted in significant down regulation of IFN-a gene expression (Figure 1A) and IFN-a sera levels (Figure 1B) which correlated to the serological and clinical beneficial effects of hCDR1 in this model (Table 1, Figure 2). We have previously demonstrated the ability of the tolerogenic peptide, hCDR1, to ameliorate SLE manifestations by immuno- modulating specifically a variety of cytokines, molecules and cell Figure 5. IFN-a diminishes hCDR1 immunomodulatory effects on PBMC of SLE patients. PBMC of 3 SLE patients were cultured (56106 cells/well) for 48 hours in the presence of medium, hCDR1 (25 mg/ml) or hCDR1 (25 mg/ml) and human recombinant IFN-a (rIFN-a) at a concentration of 5,000 U/ml. Gene expression (for IL-1b, TGFb and FoxP3) were determined by real-time RT-PCR. Results are presented as the mean6SEpercentage of gene expression compared with cultures of PBMC incubated with medium alone (considered as 100%). p = 0.05, p = 0.03, *p = 0.015 and 0 = not significant. doi:10.1371/journal.pone.0060394.g005 Figure 5. IFN-a diminishes hCDR1 immunomodulatory effects on PBMC of SLE patients. PBMC of 3 SLE patients were cultured (56106 cells/well) for 48 hours in the presence of medium, hCDR1 (25 mg/ml) or hCDR1 (25 mg/ml) and human recombinant IFN-a (rIFN-a) at a concentration of 5,000 U/ml. Gene expression (for IL-1b, TGFb and FoxP3) were determined by real-time RT-PCR. Results are presented as the mean6SEpercentage of gene expression compared with cultures of PBMC incubated with medium alone (considered as 100%). p = 0.05, p = 0.03, *p = 0.015 and 0 = not significant. doi:10.1371/journal.pone.0060394.g005 g doi:10.1371/journal.pone.0060394.g005 March 2013 \| Volume 8 \| Issue 3 \| e60394 PLOS ONE \| www.plosone.org 5 Statistical analysis Results are presented as Mean6standard error (SE). The nonparametric Mann-Whitney and unpaired Student’s T tests were used for statistical analysis. p values of 0.05 or less were considered statistically significant. References ameliorates spontaneous and induced lupus manifestations in correlation with cytokine immunomodulation. J Clin Immunol 24: 579–590. ameliorates spontaneous and induced lupus manifestations in correlation with cytokine immunomodulation. J Clin Immunol 24: 579–590. 1. Tsokos GC (2011) Systemic lupus erythematosus. N Engl J Med 365: 2110– 2121. ameliorates spontaneous and induced lupus manifestations in correlation with cytokine immunomodulation. J Clin Immunol 24: 579–590. 1. Tsokos GC (2011) Systemic lupus erythematosus. N Engl J Med 365: 2110– 2121. 2. Horwitz DA, Jacob CO (1994) The cytokine network in the pathogenesis of systemic lupus erythematosus and possible therapeutic implications. Springer Semin Immunopathol 16: 181–200. 2. Horwitz DA, Jacob CO (1994) The cytokine network in the pathogenesis of systemic lupus erythematosus and possible therapeutic implications. Springer Semin Immunopathol 16: 181–200. 12. Sharabi A, Haviv A, Zinger H, Dayan M, Mozes E (2006) Amelioration of murine lupus by a peptide, based on the complementarity determining region-1 of an autoantibody as compared to dexamethasone: different effects on cytokines and apoptosis. Clin Immunol 119: 146–155. 3. Segal R, Bermas BL, Dayan M, Kalush F, Shearer GM, et al. (1997) Kinetics of cytokine production in experimental systemic lupus erythematosus: involvement of T helper cell 1/T helper cell 2-type cytokines in disease. J Immunol 158: 3009–3016. 13. Sela U, Hershkoviz R, Cahalon L, Lider O, Mozes E (2005) Down-regulation of stromal cell-derived factor-1alpha-induced T cell chemotaxis by a peptide based on the complementarity-determining region 1 of an anti-DNA autoantibody via up-regulation of TGF-beta secretion. J Immunol 174: 302–309. 4. Cheema GS, Roschke V, Hilbert DM, Stohl W (2011) Elevated serum B lymphocyte stimulator levels in patients with systemic immune-based rheumatic diseases. Arthritis Rheum 44: 1313–1319. 14. Sela U, Dayan M, Hershkoviz R, Cahalon L, Lider O, et al. (2006) The negative regulators Foxj1 and Foxo3a are up-regulated by a peptide that inhibits systemic lupus erythematosus-associated T cell responses. Eur J Immunol 36: 2971–2980. 5. Tre´be´den-Ne`gre H, Weill B, Fournier C, Batteux F (2003) B cell apoptosis accelerates the onset of murine lupus. Eur J Immunol 33: 1603–1612. 15. Sharabi A, Zinger H, Zborowsky M, Sthoeger ZM, Mozes E (2006) A peptide based on the complementarity-determining region 1 of an autoantibody ameliorates lupus by up-regulating CD4+CD25+cells and TGF-beta. Proc Natl Acad Sci U S A 103: 8810–8815. 6. Emlen W, Niebur J, Kadera R (1994) Accelerated in vitro apoptosis of lymphocytes from patients with systemic lupus erythematosus. In vivo studies IFN-a levels in murine sera and in human PBMC supernatants were determined by Platinum ELISA sets (eBioscience, San Diego, Ca.) according to the manufacturer’s instructions. The 9 lupus patients that participated in the clinical study had a mild to moderate disease with SLE-disease activity index 2000 (SLEDAI-2K) [48] of 6–12 (inclusive) and stable lupus-related medications [24]. hCDR1 dissolved in Captisol (Sulfobutyl ether cyclodextrin sodium, CyDex, Inc., KS, USA) was injected subcutaneously weekly for 24 consecutive weeks at doses of 0.5 mg (2 patients), 1 mg (2 patients) or 2.5 mg (1 patient). Four patients were treated with Captisol alone. Patients were evaluated clinically by the SLEDAI-2K and the British Islets Lupus Assessment Group (BILAG) [49] scores. Venous blood samples prior (week 0) and following treatment (week 24) were collected in PAXgene (PreanalytiX, Switzerland) tubes and frozen at -70uC until mRNA isolation. Author Contributions Conceived and designed the experiments: ZS EM. Performed the experiments: ZS HZ IA. Analyzed the data: ZS HZ EM. Contributed reagents/materials/analysis tools: ZS EM. Wrote the paper: ZS AS EM. In vitro experiments Peripheral blood mononuclear cells (PBMC) were isolated from heparinized venous blood using UNI-SEP maxi for density gradient separation (NOVAmed Ltd., Jerusalem, Israel). PBMC (56106/ml) were cultured in triplicates in enriched RPMI-1640 medium containing 10% fetal calf serum [23] for 48 hours in the presence of hCDR1 (25 mg/ml) or medium alone as control. In some experiments, PBMC were also cultured with hCDR1 (25 mg/ml) and various concentrations (100–10,000 U/ml) of human recombinant IFN-a (Millipore, Temacula, Ca, USA). PBMC were then washed (x3 in RPMI-1640) and mRNA was extracted for gene expression as described below. GATTTCGTTGTG-3’), human FoxP3 (5’-CCACAACATG- GACTACTT-3’, 5’-CGTTTCTTGCGGAACT-3’), and human GAPDH (59-CTGCCAACGTGTCAGT-39, 59- GTTGAGGG- CAATGCCA-39). The levels of b-actin (murine studies) and GAPDA (human studies) were used to normalize the gene expression levels of the other genes. GATTTCGTTGTG-3’), human FoxP3 (5’-CCACAACATG- GACTACTT-3’, 5’-CGTTTCTTGCGGAACT-3’), and human GAPDH (59-CTGCCAACGTGTCAGT-39, 59- GTTGAGGG- CAATGCCA-39). The levels of b-actin (murine studies) and GAPDA (human studies) were used to normalize the gene expression levels of the other genes. Real-time RT-PCR Total RNA was isolated from spleen derived murine lympho- cytes, human PBMC or blood samples collected in PAXgene tubes. The RNA was reversed transcribed to prepare cDNA using Moloney murine leukemia virus reverse transcribtase (Promega, Madison, WI, USA). The resulting cDNA was subjected to real- time RT-PCR using Light Cycler ((Roche Mannheim, Germany) according to the manufecturer’s instructions. Primer sequences (forward and reversed, respectively) were: mouse IFN-a1 (59- CTGCAAGGCTGTCTGA-39, 59-GCACATTGGCAGAGGA- 39), mouse b-actin, (59-GTGACGTTGACATCCG-39, 59-CAG- TAACAGTCCGCCT-39), human IFNa1 (59- TGTGATCTCCCTGAGACC-39, 59-AGATGGAGTCCG- CATT-39), human IL-1b (5’-CAGAAAACATGCCCGT-3’, 5’- GCACTACCCTAAGGCAG-3’), human TGF-b (5’-GCAA- GACTATCGACATGG-3’, 5’-ACTTGTCATA- GATTTCGTTGTG-3’), human FoxP3 (5’-CCACAACATG- GACTACTT-3’, 5’-CGTTTCTTGCGGAACT-3’), and human GAPDH (59-CTGCCAACGTGTCAGT-39, 59- GTTGAGGG- CAATGCCA-39). The levels of b-actin (murine studies) and GAPDA (human studies) were used to normalize the gene expression levels of the other genes. Total RNA was isolated from spleen derived murine lympho- cytes, human PBMC or blood samples collected in PAXgene tubes. The RNA was reversed transcribed to prepare cDNA using Moloney murine leukemia virus reverse transcribtase (Promega, Madison, WI, USA). The resulting cDNA was subjected to real- time RT-PCR using Light Cycler ((Roche Mannheim, Germany) according to the manufecturer’s instructions. Primer sequences (forward and reversed, respectively) were: mouse IFN-a1 (59- CTGCAAGGCTGTCTGA-39, 59-GCACATTGGCAGAGGA- 39), mouse b-actin, (59-GTGACGTTGACATCCG-39, 59-CAG- TAACAGTCCGCCT-39), human IFNa1 (59- TGTGATCTCCCTGAGACC-39, 59-AGATGGAGTCCG- CATT-39), human IL-1b (5’-CAGAAAACATGCCCGT-3’, 5’- GCACTACCCTAAGGCAG-3’), human TGF-b (5’-GCAA- GACTATCGACATGG-3’, 5’-ACTTGTCATA- GATTTCGTTGTG-3’), human FoxP3 (5’-CCACAACATG- GACTACTT-3’, 5’-CGTTTCTTGCGGAACT-3’), and human GAPDH (59-CTGCCAACGTGTCAGT-39, 59- GTTGAGGG- CAATGCCA-39). The levels of b-actin (murine studies) and GAPDA (human studies) were used to normalize the gene expression levels of the other genes. hCDR1 Diminishes Interferon-a Expression in Lupus pated in a large clinical trial with hCDR1 (Edratide) [24]. Included are all patients from the two Medical Centers who completed the study and from whom blood samples were taken at least twice (before treatment initiation and at week 24) for mRNA preparation. All lupus patients were diagnosed according to the American College of Rheumatology (ACR) diagnostic criteria [33]. All participants signed an informed consent form prior to the initiation of the studies. The studies were approved by the Ethic Committees of the Medical Centers and were conducted according to all good clinical practice (GCP) rules. Patients Ten lupus patients (8 females and 2 males), 5 patients (all females) with primary APS and 5 (4 females and 1 male) age matched healthy controls participated in the in vitro experiments. We also present here data of 9 lupus patients (8 females and 1 male) from two Israeli Medical Centers. These patients partici- March 2013 \| Volume 8 \| Issue 3 \| e60394 March 2013 \| Volume 8 \| Issue 3 \| e60394 PLOS ONE \| www.plosone.org 6 hCDR1 Diminishes Interferon-a Expression in Lupus J Autoimmun 33: 77–82. 40. Yan B, Ye S, Chen G, Kuang M, Shen N, et al. (2008) Dysfunctional CD4+,CD25+regulatory T cells in untreated active systemic lupus erythema- tosus secondary to interferon-alpha-producing antigen-presenting cells. Arthritis Rheum 58: 801–812. g p 25. Lee PY, Reeves WH (2006) Type I interferon as a target of treatment in SLE. Endocr Metab Immune Disord Drug Targets 6: 323–330. 41. Braun D, Caramalho I, Demengeot J (2002) IFN-alpha/beta enhances BCR- dependent B cell responses. Int Immunol 14: 411–419. g g 26. Crow MK, Kirou KA (2004) Interferon-alpha in systemic lupus erythematosus. Curr Opin Rheumatol 16: 541–547. 42. Badr G, Saad H, Waly H, Hassan K, Abdel-Tawab H, et al. (2010) Type I interferon (IFN-alpha/beta) rescues B-lymphocytes from apoptosis via PI3K- delta/Akt, Rho-A, NFkappaB and Bcl-2/Bcl(XL). Cell Immunol 263: 31–40. p 27. Ytterberg SR, Schnitzer TJ (1982) Serum interferon levels in patients with systemic lupus erythematosus. Arthritis Rheum 25: 401–406. 28. Kirou KA, Lee C, George S, Louca K, Peterson MG, et al. (2005) Activation of the interferon-alpha pathway identifies a subgroup of systemic lupus erythema- tosus patients with distinct serologic features and active disease. Arthritis Rheum 52: 1491–1503. pp ( ) 43. Litinskiy MB, Nardelli B, Hilbert DM, He B, Schaffer A, et al. (2002) DCs induce CD40-independent immunoglobulin class switching through BLyS and APRIL. Nat Immunol 3: 822–829. 44. Mitani Y, Takaoka A, Kim SH, Kato Y, Yokochi T, et al. (2001) Cross talk of the interferon-alpha/beta signalling complex with gp130 for effective interleu- kin-6 signalling. Genes Cells 6: 631–640. 29. Baechler EC, Batliwalla FM, Karypis G, Gaffney PM, Ortmann WA, et al. (2003) Interferon-inducible gene expression signature in peripheral blood cells of patients with severe lupus. Proc Natl Acad Sci U S A 100: 2610–2615. 45. Sela U, Dayan M, Hershkoviz R, Lider O, Mozes E (2008) A peptide that ameliorates lupus up-regulates the diminished expression of early growth response factors 2 and 3. J Immunol 180: 1584–1591. 30. Santiago-Raber ML, Baccala R, Haraldsson KM, Choubey D, Stewart TA, et al. (2003) Type-I interferon receptor deficiency reduces lupus-like disease in NZB mice. J Exp Med 197: 777–788. J p 31. Zagury D, Le Buanec H, Mathian A, Larcier P, Burnett R, et al. (2009) IFNalpha kinoid vaccine-induced neutralizing antibodies prevent clinical manifestations in a lupus flare murine model. Proc Natl Acad Sci U S A 106: 5294–5299. 46. hCDR1 Diminishes Interferon-a Expression in Lupus 19. Sharabi A, Luger D, Ben-David H, Dayan M, Zinger H, et al. (2007) The role of apoptosis in the ameliorating effects of a CDR1-based peptide on lupus manifestations in a mouse model. J Immunol 179: 4979–4987. 35. Ben-David H, Sharabi A, Parameswaran R, Zinger H, Mozes E (2009) A tolerogenic peptide down-regulates mature B cells in bone marrow of lupus- afflicted mice by inhibition of interleukin-7, leading to apoptosis. Immunology 128: 245–252. J 20. Parameswaran R, Ben David H, Sharabi A, Zinger H, Mozes E (2009) B-cell activating factor (BAFF) plays a role in the mechanism of action of a tolerogenic peptide that ameliorates lupus. Clin Immunol 131: 223–232. 36. Sharabi A, Lapter S, Mozes E (2010) Bcl-xL is required for the development of functional regulatory CD4 cells in lupus-afflicted mice following treatment with a tolerogenic peptide. J Autoimmun 34: 87–95. 21. Lapter S, Ben-David H, Sharabi A, Zinger H, Telerman A, et al. (2011) A role for the B-cell CD74/macrophage migration inhibitory factor pathway in the immunomodulation of systemic lupus erythematosus by a therapeutic tolero- genic peptide. Immunology 132: 87–95. 37. Sthoeger ZM, Dayan M, Tcherniack A, Green L, Toledo S, et al. (2003) Modulation of autoreactive responses of peripheral blood lymphocytes of patients with systemic lupus erythematosus by peptides based on human and murine anti-DNA autoantibodies. Clin Exp Immunol 131: 385–392. 22. Sela U, Sharabi A, Dayan M, Hershkoviz R, Mozes E (2009) The role of dendritic cells in the mechanism of action of a peptide that ameliorates lupus in murine models. Immunology 128: e395–405. murine anti-DNA autoantibodies. Clin Exp Immunol 131: 385–392. 38. Farkas A, Tonel G, Nestle FO (2008) Interferon-alpha and viral triggers promote functional maturation of human monocyte-derived dendritic cells. Br J Dermatol 158: 921–929. 23. Sthoeger ZM, Sharabi A, Dayan M, Zinger H, Asher I, et al. (2009) The tolerogenic peptide hCDR1 downregulates pathogenic cytokines and apoptosis and upregulates immunosuppressive molecules and regulatory T cells in peripheral blood mononuclear cells of lupus patients. Hum Immunol 70: 139– 145. 39. Gigante M, Mandic M, Wesa AK, Cavalcanti E, Dambrosio M, et al. (2008) Interferon-alpha (IFN-alpha)-conditioned DC preferentially stimulate type-1 and limit Treg-type in vitro T-cell responses from RCC patients. J Immunother 31: 254–262. 24. Sthoeger ZM, Sharabi A, Molad Y, Asher I, Zinger H, et al. (2009) Treatment of lupus patients with a tolerogenic peptide, hCDR1 (Edratide): immunomodula- tion of gene expression. References J Immunol 152: 3685–3692. 7. Valencia X, Yarboro C, Illei G, Lipsky PE (2007) Deficient CD4+CD25high T regulatory cell function in patients with active systemic lupus erythematosus. J Immunol 178: 2579–2588. 16. Sharabi A, Mozes E (2008) The suppression of murine lupus by a tolerogenic peptide involves foxp3-expressing CD8 cells that are required for the optimal induction and function of foxp3-expressing CD4 cells. J Immunol 181: 3243– 3251. 8. Paust S, Cantor H (2005) Regulatory T cells and autoimmune disease. Immunol Rev 204: 195–207. 17. Arazi A, Sharabi A, Zinger H, Mozes E, Neumann AU (2009) In vivo dynamical interactions between CD4 Tregs, CD8 Tregs and CD4+CD25- cells in mice. PLoS One 4: e8447. 9. Mozes E, Sharabi A (2010) A novel tolerogenic peptide, hCDR1, for the specific treatment of systemic lupus erythematosus. Autoimmun Rev 10: 22–26. 10. Waisman A, Shoenfeld Y, Blank M, Ruiz PJ, Mozes E (1995) The pathogenic human monoclonal anti-DNA that induces experimental systemic lupus erythematosus in mice is encoded by a VH4 gene segment. Int Immunol 7: 689–696. 18. Rapoport MJ, Sharabi A, Aharoni D, Bloch O, Zinger H, et al. (2005) Amelioration of SLE-like manifestations in (NZBxNZW)F1 mice following treatment with a peptide based on the complementarity determining region 1 of an autoantibody is associated with a down-regulation of apoptosis and of the pro-apoptotic factor JNK kinase. Clin Immunol 117: 262–270. 11. Luger D, Dayan M, Zinger H, Liu JP, Mozes E (2004) A peptide based on the complementarity determining region 1 of a human monoclonal autoantibody March 2013 \| Volume 8 \| Issue 3 \| e60394 March 2013 \| Volume 8 \| Issue 3 \| e60394 7 PLOS ONE \| www.plosone.org hCDR1 Diminishes Interferon-a Expression in Lupus Sharabi A, Sthoeger ZM, Mahlab K, Lapter S, Zinger H, et al. (2009) A tolerogenic peptide that induces suppressor of cytokine signaling (SOCS)-1 restores the aberrant control of IFN-gamma signaling in lupus-affected (NZB6NZW)F1 mice. Clin Immunol 133: 61–68. 32. Shahin D, El-Refaey AM, El-Hawary AK, Salam AA, Machaly S, et al. (2011) Serum Interferon-alpha level in first degree relatives of systemic lupus erythematosus patients: correlation with autoantibodies titers. The Egyptian J Med Hum Genetics12:139–146. 47. Mauermann N, Sthoeger Z, Zinger H, Mozes E (2004) Amelioration of lupus manifestations by a peptide based on the complementarity determining region 1 of an autoantibody in severe combined immunodeficient (SCID) mice engrafted with peripheral blood lymphocytes of systemic lupus erythematosus (SLE) patients. Clin Exp Immunol 137: 513–520. 33. Tan EM, Cohen AS, Fries JF, Masi AT, McShane DJ, et al. (1982) The 1982 revised criteria for the classification of systemic lupus erythematosus. Arthritis Rheum 25: 1271–1277. 48. Gladman DD, Iban˜ez D, Urowitz MB (2002) Systemic lupus erythematosus disease activity index 2000. J Rheumatol 29: 288–291. 34. Sela U, Mauermann N, Hershkoviz R, Zinger H, Dayan M, et al. (2005) The inhibition of autoreactive T cell functions by a peptide based on the CDR1 of an anti-DNA autoantibody is via TGF-beta-mediated suppression of LFA-1 and CD44 expression and function. J Immunol 175: 7255–7263. 49. Yee CS, Isenberg DA, Prabu A, Sokoll K, Teh LS, et al. (2008) BILAG-2004 index captures systemic lupus erythematosus disease activity better than SLEDAI-2000. Ann Rheum Dis 67: 873–876. March 2013 \| Volume 8 \| Issue 3 \| e60394 PLOS ONE \| www.plosone.org 8
https://openalex.org/W2657450723	https://bmchealthservres.biomedcentral.com/track/pdf/10.1186/s12913-017-2363-4	English	null	Supporting nurse practitioners’ practice in primary healthcare settings: a three-level qualitative model	BMC health services research	2,017	cc-by	7,643	Chouinard et al. BMC Health Services Research (2017) 17:437 DOI 10.1186/s12913-017-2363-4 Chouinard et al. BMC Health Services Research (2017) 17:437 DOI 10.1186/s12913-017-2363-4 Open Access * Correspondence: catherine.larouche.4@umontreal.ca 2University of Montreal, Faculty of Nursing, University of Montreal Public Health Research Institute (IRSPUM), Pavillon Marguerite-d’Youville, 2375, chemin de la Côte-Ste-Catherine, Quebec H3T 1A8, Canada Full list of author information is available at the end of the article © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Supporting nurse practitioners’ practice in primary healthcare settings: a three-level qualitative model Véronique Chouinard1, Damien Contandriopoulos2, Mélanie Perroux3 and Catherine Larouche2 ique Chouinard1, Damien Contandriopoulos2, Mélanie Perroux3 and Catherine Larouche2* Background medical clinics where public hospitals cover the salary and benefits of nursing staff in exchange for clinics’ providing extended opening hours and increased care continuity) [10]. Regardless of their practice environ- ment, it is mandatory for every PHCNP in Quebec to sign a “partnership contract” with one or more family physicians stipulating rules of collaboration as well as each one’s professional roles and responsibilities. Several studies have shown that greater reliance on nurse practitioners can alleviate pressures on primary healthcare systems, such as those caused by public expenditure cuts and demographic changes. This cost-effective measure has the potential to improve primary care services accessibility without lowering quality of care or patient satisfaction levels [1–8]. According to Quebec’s regulatory frameworks, PHCNPs technically come under the responsibility of a nursing department. These nursing departments are present in every Health and Social Services Centre (CSSS),1 and are generally responsible for setting and pursuing the vision for high-quality nursing services and ensuring the condi- tions are in place to achieve this quality in the various or- ganizations making up the CSSS. However, on the ground, the responsibility for PHCNPs’ integration often involves many other actors apart from the nursing departments. Their integration into primary care is, however, not devoid of challenges, notably because the role attributed to primary healthcare nurse practitioners (PHCNPs) expands nursing practice and consequently implies re- thinking the nature of each professional’s role in primary healthcare teams [9]. Developing adequate team-focused support practices is thus essential for PHCNPs’ integra- tion and ultimately for improving those teams’ capacity to contribute to primary healthcare quality accessibility and efficiency [10]. We define support practices as all ac- tivities intended to respond to the needs of the PHNCP or of other professionals collaborating with the PHNCP, and analyze them from a multi-level perspective – clinical, team, and systemic. The scientific literature and various reports have shown that, at the local level, a number of obstacles can hinder successful insertion of PHCNPs into primary healthcare settings, such as feelings of isolation in mainly medical-centred environments, limited opportun- ities to communicate with other nurses from similar settings, lack of team preparation prior to PHCNPs’ inte- gration, confusion in role definitions, and misuse of the nurses’ capacities [12–14]. Poghosyan et al.’s quantitative study, in which nearly 600 PHCNPs were surveyed, indi- cated that they were often dissatisfied with their rela- tionship with their immediate supervisor [15]. Abstract Background: While greater reliance on nurse practitioners in primary healthcare settings can improve service efficiency and accessibility, their integration is not straightforward, challenging existing role definitions of both registered nurses and physicians. Developing adequate support practices is therefore essential in primary healthcare nurse practitioners’ integration. This study’s main objective is to examine different structures and mechanisms put in place to support the development of primary healthcare nurse practitioner’s practice in different healthcare settings, and develop a practical model for identifying and planning adequate support practices. Methods: This study is part of a larger multicentre study on primary healthcare nurse practitioners in the province of Quebec, Canada. It focuses on three healthcare settings into which one or more primary healthcare nurse practitioners have been integrated. Case studies have been selected to cover a maximum of variations in terms of location, organizational setting, and stages of primary healthcare nurse practitioner integration. Findings are based on the analysis of available documentation in each primary healthcare setting and on semi-structured interviews with key actors in each clinical team. Data were analyzed following thematic and cross-sectional analysis approaches Results: This article identifies three types of support practices: clinical, team, and systemic. This three-level analysis demonstrates that, on the ground, primary healthcare nurse practitioner integration is essentially a team-based, multilevel endeavour. Despite the existence of a provincial implementation plan, the three settings adopted very different implementation structures and practices, and different actors were involved at each of the three levels. The results also indicated that nursing departments played a decisive role at all three levels. Conclusions: Based on these findings, we suggest that support practices should be adapted to each organization’s environment and experience and be modified as needed throughout the integration process. We also stress the importance of combining this approach with a strong coordination mechanism involving managers who have in- depth understanding of nursing professional roles and scopes of practice. Making primary healthcare nurse practitioner integration frameworks more flexible and clarifying and strengthening the role of senior nursing managers could be the key to successful integration. Keywords: Primary healthcare nurse practitioners, Primary healthcare service organization, Scope of practice, Support practices Chouinard et al. BMC Health Services Research (2017) 17:437 Page 2 of 9 Page 2 of 9 A three-level approach to support practices The scientific literature frequently highlights the import- ance of support practices to overcome the main integra- tion challenges presented above [11, 16–22]. However, it rarely proposes specific practical models for planning and implementing adequate support practices [10, 11], most likely because support practices encompass a wide array of activities and the meaning of the concept re- mains vague. Background Certain features of many healthcare systems (i.e., wide variety of practice settings, physical distance between key players, differences in legal frameworks) also add to the difficulty of successfully developing this advanced practice. While there is relatively abundant literature on the advantages and challenges of integrating nurse practi- tioners into primary care settings, the optimal structures and practices to support their integration have remained largely unexplored [10, 11]. The present study focuses on three primary care settings in Quebec (Canada) to examine the types of managerial structures and mechanisms put in place to support the development of PHCNPs’ practice. We sug- gest dividing support practices into three types—clinical, team and systemic—for a thorough understanding of their impact on PHCNPs’ integration. Our analysis of these three types of support demonstrates that practices should be adapted to the environment and experience of the organizations and be modified as needed throughout the integration process. In a broader perspective, our results also indicate that nursing departments play a decisive role in PHCNPs’ integration at all three levels. We suggest that clarifying and strengthening their role could therefore be the key to successful integration. Systemic support clinical, team, and systemic. These three forms of support overlap in many ways, and clearly distinguishing among them is sometimes difficult when observing actual prac- tices on the ground. However, since healthcare organiza- tions are complex systems in which various structures and processes interact at different levels, disaggregating the concept of support into its multiple components is useful to identify which support practices are more effective. The next sections provide more detail on the definition and delimitation of the multi-level support practices model we developed and used for this study. ‘Systemic level’ support practices are those related to ad- aptations to and of the broader environment within which PHCNPs are integrated. Cameron and Masterson [30] argue that nurse managers can play a crucial role in nurse practitioners’ integration into primary care set- tings, but that their capacity to do so depends on their level of knowledge regarding this new role, on their capacity for strategic action within the organization, and on the level of responsibility attributed to them in this function. Other systemic support practices pertain to fi- nancial support or service reimbursement rules [11, 22, 31] and the constitution and organization of coordinating committees [11]. Notably, studies on the topic high- light the importance of including nurse managers in regional coordinating committees related to PHCNPs and of establishing good communication mechanisms between PHCNPs and nursing departments [11, 32]. Clinical support What we defined as ‘clinical level’ support consists of interventions aimed at facilitating PHCNPs’ clinical work. It includes the most immediate aspects of support in their work environment, such as access to clinical information and resources, capacity development oppor- tunities, and training, as well as measures to help them occupy the full scope of nursing practice. For example, relationships between PHCNPs and their partner family physicians [16, 20], and opportunities to exchange know- ledge and experience with other nurse practitioners or clinical nurses occupying similar roles in other settings, are clinical support elements that affect PHCNPs’ work and capacity development [26, 27]. MacPhee [26] argues, along these lines, that nurse managers are indispensable collaborators in transferring information and resources to PHCNPs. The challenges of integration We define support practices as all activities that are intended to respond to the needs of the PHNCP or of other professionals collaborating with the PHNCP. Support practices are designed to resolve problems, meet challenges, or improve certain processes [23]. They can take various forms (e.g. clinical, psychological, admin- istrative) and be implemented at different organizational levels [24, 25]. To capture these organizational levels of support practices—going from the local to a broader macro level—we organized practices into three categories: PHCNPs practising in the province of Quebec are regis- tered nurses who have successfully completed a university master’s-level nurse practitioner program. According to Quebec’s 2010–2015 strategic health plan, PHCNPs are expected to provide primary care in three practice environ- ments: local community health centres (CLSCs—public organizations providing primary care and social services); hospital-based family medicine units (FMUs—hospital affil- iated medical clinics which train medical residents in family medicine); and family medicine groups (FMGs—private Chouinard et al. BMC Health Services Research (2017) 17:437 Page 3 of 9 Methods This study was part of a larger multicentre study focused on formulating recommendations regarding PHCNPs’ integration and best practices in Quebec’s healthcare system [9, 10]. The project was based on six case studies of settings deemed by Quebec’s Ministry of Health and Social Services (MSSS) to be successful examples of PHCNPs integration. Each case is defined as a clinical team into which one or more PHCNPs have been inte- grated. Beyond sharing the characteristic of being per- ceived as successful examples, cases were selected according to a logic of maximum of variation sampling in terms of location, organizational setting, and stages of PHCNP integration. Results department, as in Case 1, seemed to translate into higher levels of support. In the two other cases, PHCNPs had to deal with two different managers and a complex hierarchical structure even though, in practice, they often requested the help of nursing department managers before consulting their immediate supervisor. More than direct clinical support however, the vertical support of- fered by the nursing departments was mostly focused on facilitating exchanges among PHCNPs. Coordinators from the nursing department would, for example, organize exchanges among PHCNPs working in different settings on a regular basis. The coordinators used these occasions to stay in regular contact with PHCNPs despite geograph- ical distance, identify their training needs, and become informed of potential difficulties arising in each setting. All participants in the study referred to this as an invalu- able form of support uniting managers and clinicians. The three cases selected for in-depth analysis were a small CLSC in a rural area, a small FMG in a rural area, and a large FMU in an urban setting. Stage of NP inte- gration ranged from two to 7 years, and the three set- tings had significantly different organizational structures. In the first case, PHCNPs were under the direct respon- sibility of the CSSS Director of Nursing, while in the two other cases, supervision was shared between at least two players. PHCNPs in the second setting reported struc- turally to the Assistant Director of the General Services Department and functionally to the Director of Nursing, whereas PHCNPs in the third setting reported to an ad- ministrative assistant while receiving clinical advice from the CSSS Director of Nursing and a nursing practice clinical manager (see Table 1). Lastly, patient manage- ment in the settings under study followed either a ‘joint’ or a ‘consultative’ model. A model is considered joint when the NP and the physician partner follow the same panel of patients. In such a model, both professionals may see the same patients at different points in their treatment. Conversely, a model is considered consulta- tive when the NP and the physician partner each follow a different panel of patients and the physician is consulted as needed. [10, 36, 37]. Within the healthcare setting, horizontal support between PHCNPs emerged as another important aspect of clinical support. Such exchanges generally consisted of sharing clinical experiences, documentation, and advice on day-to-day work arrangements in the clinic. Team support A PHCNP’s arrival in a new practice environment requires mutual adaptation, as the PHCNP adjusts to the team’s established patterns of functioning, while the team adjusts its functioning to include the PHCNP. ‘Team level’ support consists of measures taken to re- organize roles, redesign task distribution, and manage interpersonal relations in a team before and after the PHCNP’s integration. Among the few studies on this type of support, those that have focused on challenges related to managing relationships and redesigning roles within the team suggest that fostering role complemen- tarity, increasing communication channels to reduce hierarchical differences, and building common goals while leaving space for team members to organize them- selves and manage their own difficulties are keys to successfully meeting those challenges [19, 28]. Reay et al. suggest adopting a ‘balcony’ perspective, from which the manager focuses not only on the nurse practitioners, but on the team as a whole, and formulates overall goals to guide members in their actions and adaptation to changes [19]. In all cases, integrating a new role into a team can be seen as an opportunity to rethink the team’s organizational structure [9, 29]. Findings in the present article focus on three of the above six cases. They are based on the analysis of available documentation in each setting and on 18 semi-structured interviews with key actors in each clinical team: PHCNPs, physician partners of the PHCNPs, directors of nursing in the CSSSs, administrative staff, and other nurses. Inter- views were conducted in the healthcare settings and lasted about an hour each. Interviews were audio-recorded, transcribed, and read for accuracy. Interviews were conducted in French (cita- tions in this article have been translated into English). All data were compiled and coded following a thematic analysis approach (systematic identification of recurring themes) [33, 34]. We employed two methods to interpret the data. First, we used pattern matching to highlight patterns linking data from the literature with empirical data and patterns appearing within the interviews [35]. The primary structure used to organize and code data was the typology of support levels presented above. We then performed cross-sectional analysis by comparing similarities and differences between the case studies. Page 4 of 9 Chouinard et al. BMC Health Services Research (2017) 17:437 Page 4 of 9 Results Even in cases where no formal measures were taken to support horizontal exchanges (Case 2), the PHCNP maintained contacts with other PHCNPs in more or less informal ways, such as meeting outside work hours. The existence of this parallel network shows the need for this type of support. Another successful horizontal support practice was the dyadic integration of nurses in the health- care settings. Although the relationship between the two nurses varied according to their years of experience (senior–junior in Case 1 and junior–junior in Case 3), the I. Clinical support They were more invested in deal- ing with day-to-day relationships among team members, while the nursing department managers adopted more of a counselling role. In general, the head physicians had, to a certain extent, developed their own mecha- nisms for supervising and integrating the PHCNPs into their clinics. “It was a very important source of support. It was so stressful in the beginning. It’s like another way to work, everything is new. So, with two of us, I was able to compare what I was going through with someone…that, for me, I felt like this, like that…” (PHCNP, Case 3). Being at least two in the same clinic also presented advantages in terms of work efficiency and coverage during absences. g Our analysis also showed that patient management was a challenge when more than three partner physicians were involved, especially in settings based on a joint patient man- agement model. The consultative model (Cases 1 and 2) was easier to manage and fostered collaborative relation- ships between MDs and PHCNPs, whereas the joint model (Case 3) tended to lead more toward supervision-based relationships. Apart from the question of the number of physicians, most of the coordination mechanisms between physicians and PHCNPs seemed to be based on mutual adjustment and were modified over time. In all cases, the need for physician support was much higher in the first 6 months and diminished as the PHCNP developed greater confidence and expertise. As the PHCNP developed complementary services (Case 2), relationships with the physician became increasingly consultative and less inter- dependent. Certain formal measures, such as narrowing the type of caseload selected (Cases 2–3) and having a set con- sultation plan between PHCNP and physician (Case 2) helped PHCNPs develop independence and fostered satisfy- ing interprofessional consultations. The need for formal mechanisms depended mostly on setting size and quality of communication between members. Thus, although nursing department managers, phys- ician managers, and the PHCNPs’ partner physicians shared many responsibilities for PHCNPs’ integration, they seemed nevertheless to operate in relatively parallel networks, with few structures facilitating communication between them. I. Clinical support Analysis of the three cases showed, first, that the nursing department always took part in clinical support, regardless of which department was officially supervising the PHCNPs. However, direct supervision by the nursing Table 1 Case description and management structures Case 1: Rural CLSC Case 2: Rural FMG Case 3: Urban FMU Size: Small (2 PHCNPs, 4 nurses, 2 physicians) Stage of NP integration: 2 and 7 years Patient management: Consultative model Size: Small (1 PHCNP, 4 part-time doctors, 1 NP, 1 secretary) Stage of NP integration: 2 years Patient management: Consultative model Size: Large (2 PHCNPs) Stage of NP integration: 2 years Patient management: Joint model Legend: : hierarchical link; --- : non-hierarchical link Table 1 Case description and management structures Table 1 Case description and management structures Size: Small (1 PHCNP, 4 part-time doctors, 1 NP, 1 secretary) Stage of NP integration: 2 years Patient management: Consultative model Size: Small (2 PHCNPs, 4 nurses, 2 physicians) Stage of NP integration: 2 and 7 years Patient management: Consultative model Size: Small (2 PHCNPs, 4 nurses, 2 physicians) Stage of NP integration: 2 and 7 years Patient management: Consultative model Page 5 of 9 Page 5 of 9 Chouinard et al. BMC Health Services Research (2017) 17:437 Chouinard et al. BMC Health Services Research (2017) 17:437 fact of there being two PHCNPs in one setting gave them the opportunity to create alliances, develop and share a vision of their role, validate ideas related to their clinical practice or their integration into the setting, and suggest, when needed, changes to make the most of advanced nursing practices. a broader vision of the whole process. The general services managers (immediate supervisors in Cases 2 and 3), on the other hand, did not have extensive know- ledge of the PHCNPs’ role and so their support was limited to basic financial and administrative interven- tions (schedule planning, space assignments, etc.). The physicians in charge of the clinics also appeared to play an important role in team support. They were in- vited to participate in evaluating the work of PHCNPs coordinated by the nurse managers. They also organized meetings to facilitate communication between all profes- sionals in the clinic and served as a reference point for questions and comments from other physician partners on the PHCNP’s role. II. Team support There was no uniform process common to all three cases to support the teams after the introduction of the PHCNPs. Team support was provided by different insti- tutions and people inside and outside the team itself. In the smaller settings (Cases 1 and 2), integration pro- cesses seemed to work more smoothly, as logistical adjustments were simpler than in the larger setting and communication between members of the smaller teams was generally more fluid. I. Clinical support In some settings, notably in Case 2, where the physicians did not come under the financial and administrative responsibility of the CSSS, the nurs- ing department managers felt they did not have the necessary legitimacy to intervene in the healthcare setting, even though they were officially in charge of the PHCNPs’ integration and of paying their salary. They had to rely essentially on the personal collaboration links they had established with the clinic’s head physician to support, indirectly, the PHNCPs. “We can encourage the physician in charge of the clinic to think about this. We can do that, but we can’t necessarily force him to say… ‘look, us, we’d like our nurse practitioner to do such and such.’ It’s always a matter of collaborating with our physician in charge.” (Assistant to the General Services Department Director, Case 2). III. Systemic support With regard to systemic support, the nursing depart- ment was again the key player. However, while responsi- bilities for clinical and team support were generally delegated to a nursing department manager, it was the Directors of Nursing (a position often labeled as Chief Nursing Officer in other jurisdictions) themselves who took on most of the systemic, political, and macro-level tasks pertaining to the recognition of PHCNPs’ Nursing managers seemed to be the most useful source of support for the PHCNPs, as they could help in defining and developing the PHCNPs’ role and provided Chouinard et al. BMC Health Services Research (2017) 17:437 Page 6 of 9 practices. One example mentioned a few times was the fact that the nursing directors represented the PHCNPs’ interests at regional and provincial levels regarding recognition of their right to prescribe diagnostic tests or drugs. These types of interventions occurred at all stages of PHCNPs’ integration in primary care delivery struc- tures. The nursing department was also involved in ad- vocacy and education to promote the administrative integration of additional PHCNPs in other institutions within the CSSS. Despite their involvement in such sup- port activities, the Directors of Nursing had few formal opportunities to meet with colleagues from other CSSSs in their region, and some had built their own informal networks with other nursing departments. large number of team members. The absence of stan- dardized management structures and practices was less of a challenge in smaller contexts, where there was greater reliance on individual and informal coordination mechanisms. It should also be mentioned that, in all cases, despite the existence of formal coordination struc- tures, many of the communication processes between PHCNPs, nursing departments, and the settings were, in practice, based on informal and personal relationships. p p p The contingency theory approach developed in the field of organization studies can be helpful in formalizing those observations. More specifically, from a contingency theory perspective, size matters. Structural arrangements that make sense in a large organization might make no sense at all in a small one [38, 39]. The same logic applies to other parameters, such as environment complexity. Imple- mentation processes whose evolution is difficult to predict and that are carried out in complex environments (diffuse responsibilities and participation) are ill-suited for standardized structures. III. Systemic support For example, regarding PHCNPs’ implementation in primary care, it might make more sense for the MSSS to simply list best practices and set targets and schedules rather than to establish committees and appoint participants. Moreover, any implementation process is, by its very nature, evolving. Thus implementa- tion structures and practice will have to adapt and evolve as well. The gradual transformation of the main physician partner/PHCNP clinical relationship from strict supervi- sion to counselling and collaboration is one example of adaptation to changing needs [27]. Ultimately, the goal should be to achieve a balance between a shared and broad vision of the PHCNP’s role, on one hand, and the constraints and needs of the clinical environment, on the other [40]. According to the integration plan developed by the MSSS, systemic integration was to be supported by local and regional implementation committees. However, while our respondents saw the role of the nursing department as unequivocally central in systemic support practices, their opinions about the role and usefulness of these committees were generally mixed. Only one pri- mary healthcare setting had established an official local implementation committee, and the three settings had variable experiences with the government-mandated regional committee. In general, the need for such struc- tures did not seem very pressing after the first stages of PHCNPs’ integration, since these committees were intended to plan broad general directions rather than to discuss emerging problems. As the actors were already in regular interaction with each other in the CSSS, and most decisions were taken outside of formal meetings, this additional committee was seen as not really necessary. Discussion and recommendations for practice pp p Our study makes two original contributions to the field. The first is in suggesting that supporting PHCNPs’ integration is a multilevel endeavour. As we have argued, there are distinct needs and responsibil- ities at the clinical, team, and systemic levels. Some actors may be involved at all three levels, but it is likely that different actors will be better positioned for different levels of support, making PHCNPs’ integra- tion an intrinsically team-based process. This brings us to the second contribution to the field, which is the observation that, as support practices for PHCNP in- tegration involve a variety of actors from different backgrounds and structural positions, there is a need for strong but adaptive coordination structures. A balance must be struck between relying on highly formalized statutory committees—which will likely be less adaptable—and counting exclusively on informal communication and personal involvement, whose effectiveness is unpredictable and unreliable. This is where contingency theory arguments—that there are no best organizational structures to produce strong but adaptive coordination—provide a useful conceptual framework. The best structure will be the one best fitted to the contingencies of a given organizational system. When designing the structure for PHCNPs’ integration support, it is essential to leave enough room for local adaptation while making sure there are people account- able for the outcomes. This goes back to the importance of having a senior manager—someone with in-depth un- derstanding of professional roles and scopes of practice—- coordinating the functioning of implementation structures from a ‘balcony’ or ‘whole-picture’ vantage point [19]. The second recommendation also relates to the role of nursing actors in support practices but, in this case, at the horizontal level. For example, the PHCNPs all stressed the positive impact of there being more than one PHCNP in the same clinic, as it provided opportunities for exchange and communica- tion. Fostering horizontal support would also make it possible to take advantage of senior PHCNPs’ experi- ence to train junior PHCNPs, thereby reducing reli- ance on physicians for such training. Moreover, in contexts where the number of nurse practitioners being integrated into primary care settings is meant to increase, being able to share successes and chal- lenges with those who have already experienced the process becomes even more vital. At a higher level, nursing administrators and managers also benefit from horizontal exchanges with other nursing depart- ments in the region. Discussion and recommendations for practice The three levels of support approach, however, constitutes an additional tool to clar- ify the role of nursing departments and understand how it can be most productively enacted at each support level. Notably, our findings showed that the nursing department’s role was not limited to the clinical level, but also contributed significantly at the team and systemic support levels. This strongly advocates, on one hand, for assigning nursing departments the responsibil- ity for PHCNPs’ direct supervision [41] and, on the other, for giving nursing departments the means to be informed and included in team and macro level support structures and practices right from the beginning of the integration process, even if they do not have direct authority in certain primary care settings. It appears there is still room for improvement in that area, given the lack of legitimacy felt by the nursing department managers when intervening at the team level, for example. department in integrating PHCNPs in primary care insti- tutions are consistent with those of other studies on the topic [11, 26, 30, 32]. The three levels of support approach, however, constitutes an additional tool to clar- ify the role of nursing departments and understand how it can be most productively enacted at each support level. Notably, our findings showed that the nursing department’s role was not limited to the clinical level, but also contributed significantly at the team and systemic support levels. This strongly advocates, on one hand, for assigning nursing departments the responsibil- ity for PHCNPs’ direct supervision [41] and, on the other, for giving nursing departments the means to be informed and included in team and macro level support structures and practices right from the beginning of the integration process, even if they do not have direct authority in certain primary care settings. It appears there is still room for improvement in that area, given the lack of legitimacy felt by the nursing department managers when intervening at the team level, for example. integration process in terms of scope of practice and role redefinition, and this appears to be more product- ive than working from a narrower task-based perspec- tive. However, this general observation needs to be considered in the light of specific contexts. For ex- ample, in our study, PHCNPs’ partner physicians also played a central role, as they were the ones best posi- tioned to support PHCNPs’ clinical role development. Discussion and recommendations for practice Formalizing the time commit- ment and creating meeting opportunities could be useful to support this need for horizontal support. Discussion and recommendations for practice Discussion and recommendations for practice A first observation to be made is that there does not seem to be a unique and straightforward process to inte- grate PHCNPs and support development of their prac- tice in primary care settings. The three settings studied were examples of relatively successful integration, but all relied on very different implementation structures and practices. Our interpretation is that each setting had to invent its own support model from resources available in its organization, past experience, and managers’ per- sonal interests, strengths, and weaknesses. Although the government’s implementation plan included structural elements, those were poorly suited to local practices and lacked support from participants. Even though one set of support structures and prac- tices cannot benefit and be relevant to all settings equally, two general recommendations can still be formed in light of the successful support practices we observed in this study. The first relates to the role of the nursing department. Despite the different responsibilities attributed to the nursing departments in each case study, in all cases, and at all three levels of support, the Director of Nursing or a representative had a central and positive effect on the integration process; hence the importance of clarifying and supporting the nursing department’s responsibilities. In contrast to the General Services Department, which usually offered only basic administrative support, the nursing department’s inter- ventions also had the objective of developing a broader vision of what PHCNPs’ role in primary care should be. Supporting and clarifying that department’s responsibil- ities would also be helpful to reduce the number of actors involved and the overlapping of tasks. Our find- ings on the centrality of the role played by the nursing The major drawback of this high variability in local practices was that most issues were addressed in an ad hoc way, leading to overlaps and the involvement of many actors from different organizational levels. This was especially true in larger settings, as it was more diffi- cult to coordinate changes and adjustments on an infor- mal basis, given the complexity of the structure and the Chouinard et al. BMC Health Services Research (2017) 17:437 Page 7 of 9 Page 7 of 9 department in integrating PHCNPs in primary care insti- tutions are consistent with those of other studies on the topic [11, 26, 30, 32]. Ethics approval and consent to participate This study was approved by the research ethics committees of the Montreal Health and Social Services Agency and the University of Montreal. All participants interviewed in this project provided their written consent. This study was approved by the research ethics committees of the Montreal Health and Social Services Agency and the University of Montreal. All participants interviewed in this project provided their written consent. 14. MSSS. Évaluation de l’implantation et des effets des premiers groupes de médecine de famille au Québec. Québec: Ministère de la Santé et des Services sociaux, Direction de l’évaluation; 2008. http://publications.msss. gouv.qc.ca/msss/fichiers/2008/08-920-02.pdf Additional file 5. Laurant M, Reeves D, Hermens R, Braspenning J, Grol R, Sibbald B. Substitution of doctors by nurses in primary care. Cochrane Database Syst Rev. 2005;2:CD001271. 5. Laurant M, Reeves D, Hermens R, Braspenning J, Grol R, Sibbald B. Substitution of doctors by nurses in primary care. Cochrane Database Syst Rev. 2005;2:CD001271. Additional file 1: Interview Guide (Combined). (DOCX 34 kb) 6. Laurant MG, Hermens RP, Braspenning JC, Akkermans RP, Sibbald B, Grol RP. An overview of patients preference for, and satisfaction with, care provided by general practitioners and nurse practitioners. J Clin Nurs. 2008;17:2690–8. 6. Laurant MG, Hermens RP, Braspenning JC, Akkermans RP, Sibbald B, Grol RP. An overview of patients preference for, and satisfaction with, care provided by general practitioners and nurse practitioners. J Clin Nurs. 2008;17:2690–8. Publisher’s Note 15. Poghosyan L, Shang J, Liu J, Poghosyan H, Liu N, Berkowitz B. Nurse practitioners as primary care providers: creating favorable practice environments in New York state and Massachusetts. Health Care Manag Rev. 2015;40:46–55. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 16. Read SM. Nurse-led care: the importance of management support. Nurs Times Res. 1999;4:408–21. Endnote 1At the time of the study, Health and Social Services Centres (Centres de santé et de services sociaux) were mandated to oversee the activities of public health institutions on a given territory, including CLSCs, re- habilitation centres, long-term care facilities, and often hospitals. The system has since been modified (in 2015), resulting in further centralization and administrative in- tegration of institutions into what are now called CISSS: Centres intégrés de santé et de services sociaux, and CIUSSS: Centres intégrés universitaires de santé et de services sociaux. Our analysis suggests that PHCNPs’ integration and continuing development in primary care settings are more than a local matter. This tallies with other results in the field suggesting that PHCNPs’ integration is not a straightforward process. Because PHCNPs’ scope of practice and roles are at the intersection of medicine and nursing, their integration challenges existing role definitions of both registered nurses and physicians. As other studies have found [11, 17, 21, 31, 36, 41], man- agers trained in nursing seem better equipped than their non-nursing counterparts to conceptualize the Page 8 of 9 Page 8 of 9 Chouinard et al. BMC Health Services Research (2017) 17:437 Funding g This study was supported by the Quebec Ministry of Health and Social Services/Canadian Institute of Health Research– Partnerships for Health System Improvement program (PHE 114115); the Quebec Nursing Intervention Research Network; the Faculty of Nursing, University of Montreal; and the Ordre régional des infirmières et infirmiers de Montreal/Laval. 8. Russell GM, Dahrouge S, Hogg W, Geneau R, Muldoon L, Tuna M. Managing chronic disease in Ontario primary care: the impact of organizational factors. Ann Fam Med. 2009;7:309–18. 8. Russell GM, Dahrouge S, Hogg W, Geneau R, Muldoon L, Tuna M. Managing chronic disease in Ontario primary care: the impact of organizational factors. Ann Fam Med. 2009;7:309–18. 9. Contandriopoulos D, Brousselle A, Breton M, Sangster-Gormley E, Kilpatrick K, Dubois CA, Brault I, Perroux M. Nurse practitioners, canaries in the mine of primary care reform. Health Policy. 2016;120(6):682–9. 9. Contandriopoulos D, Brousselle A, Breton M, Sangster-Gormley E, Kilpatrick K, Dubois CA, Brault I, Perroux M. Nurse practitioners, canaries in the mine of primary care reform. Health Policy. 2016;120(6):682–9. Received: 17 November 2016 Accepted: 7 June 2017 20. Almost J, Laschinger HK. Workplace empowerment, collaborative work relationships, and job strain in nurse practitioners. J Am Acad Nurse Pract. 2002;14:408–20. Availability of data and materials The written material analyzed in the current study is available from the corresponding author on reasonable request. However, the interview transcriptions cannot be communicated as per the agreements in the written consent form approved by the research ethics committees. An English version of the Interview Guide is available as a Additional file 1. 10. Contandriopoulos D, Brousselle A, Dubois CA, Perroux M, Beaulieu MD, Brault I, Kilpatrick K, D’Amour D, Sansgter-Gormley E. A process-based framework to guide nurse practitioners integration into primary healthcare teams: results from a logic analysis. BMC Health Serv. research. 2015;15(1):78. 11. DiCenso A, Martin-Misener R, Bryant-Lukosius D, Bourgeault I, Kilpatrick K, Donald F, Kaasalainen S, Harbman P, Carter N, Kioke S, Abelson J. Clinical nurse specialists and nurse practitioners in Canada: a decision support synthesis. Ottawa, ON: Canadian Health Services Research Foundation, 2010. Acknowledgements Not applicable. 7. Wong ST, Farrally V. The utilization of nurse practitioners and physician assistants: a research synthesis. Prepared for the Michael Smith Foundation for Health Research. 2013. http://www.msfhr.org/sites/default/files/ Utilization_of_Nurse_Practitioners_and_Physician_Assistants.pdf. Accessed 23 June 2016. 7. Wong ST, Farrally V. The utilization of nurse practitioners and physician assistants: a research synthesis. Prepared for the Michael Smith Foundation for Health Research. 2013. http://www.msfhr.org/sites/default/files/ Utilization_of_Nurse_Practitioners_and_Physician_Assistants.pdf. Accessed 23 June 2016. Authors’ contributions VC, DC, MP, and CL analyzed and interpreted the data. CL and MP were major contributors in writing the manuscript. All authors commented, read and approved the final manuscript. 12. Beaulieu M-D, Denis J-L, D’Amour D, Goudreau J, Haggerty J, Hudon É, et al. L’implantation des Groupes de médecine de famille: le défi de la réorganisation de la pratique et de la collaboration interprofessionnelle - Étude de cas dans cinq GMF de la première vague au Québec. Montréal: Chaire Docteur Sadok Besrour en médecine familiale; 2006. Consent for publication 13. ASSSM. Le modèle montréalais de prise en charge. Évaluation de l’implantation des groupes de médecine de famille (GMF) et des cliniques- réseau (CR). Montreal: Agence de la Santé et des Services sociaux; 2010. http://pro.santemontreal.qc.ca/fileadmin/asssm/Medecins/1_gerer_ma_ pratique/3_pratique_en_GMF/isbn978-2-89510-764-4.pdf Competing interests The authors declare that they have no competing interests. Author details 1 16. Read SM. Nurse-led care: the importance of management support. Nurs Times Res. 1999;4:408–21. 1University of Montreal Hospital Centre (CHUM), University of Montreal Public Health Research Institute (IRSPUM), C.P. 6128 succ. Centre-Ville, Montreal, Quebec H3C 3J7, Canada. 2University of Montreal, Faculty of Nursing, University of Montreal Public Health Research Institute (IRSPUM), Pavillon Marguerite-d’Youville, 2375, chemin de la Côte-Ste-Catherine, Quebec H3T 1A8, Canada. 3University of Montreal Public Health Research Institute (IRSPUM), C.P. 6128 succ. Centre-Ville, Montreal, Quebec H3C 3J7, Canada. 17. Sangster-Gormley E, Martin-Misener R, Burge F. A case study of nurse practitioner role implementation in primary care: what happens when new roles are introduced? BMC Nurs. 2013;12:1. 17. Sangster-Gormley E, Martin-Misener R, Burge F. A case study of nurse practitioner role implementation in primary care: what happens when new roles are introduced? BMC Nurs. 2013;12:1. 18. de Guzman A, Ciliska D, DiCenso A. Nurse practitioner role implementation in Ontario public health units. Can J Public Health. 2010;101:309–13. 18. de Guzman A, Ciliska D, DiCenso A. Nurse practitioner role implementation in Ontario public health units. Can J Public Health. 2010;101:309–13. 19. Reay T, Golden-Biddle K, Germann K. Challenges and leadership strategies for managers of nurse practitioners. J Nurs Manag. 2003;11:396–403. Received: 17 November 2016 Accepted: 7 June 2017 References 21. Sangster-Gormley E, Martin-Misener R, Downe-Wamboldt B, Dicenso A. Factors affecting nurse practitioner role implementation in Canadian practice settings: an integrative review. J Adv Nurs. 2011;67:1178–90. 1. Martin-Misener R, Downe-Wamboldt B, Cain E, Girouard M. Cost effectiveness and outcomes of a nurse practitioner–paramedic–family physician model of care: the long and Brier Islands study. Prim Health Care Res Dev. 2009;10:14–25. 22. Sullivan-Bentz M, Humbert J, Cragg B, Legault F, Laflamme C, Bailey PH, et al. Supporting primary health care nurse practitioners’ transition to practice. Can Fam Physician. 2010;56:1176–82. 2. DiCenso A, Bryant-Lukosius D. The long and winding road: integration of nurse practitioners and clinical nurse specialists into the Canadian health- care system. Can J Nurs Res. 2010;42:3–8. 23. Poghosyan L, Nannini A, Clarke S. Organizational climate in primary care settings: implications for nurse practitioner practice. J Am Acad Nurse Pract. 2013;25:134–40. 3. DiCenso A, Bryant-Lukosius D, Bourgeault I, Martin-Misene R, Donald F, Abelson J, et al. CHSRF decision support synthesis: clinical nurse specialist and nurse practitioner roles – summary report : roundtable with decision makers and recommendations for practice and policy. Ottawa: Canadian Health Services Research Foundation; 2009. 24. Yegdich T. Clinical supervision and managerial supervision: some historical and conceptual considerations. J Adv Nurs. 1999;30:1195–204. 24. Yegdich T. Clinical supervision and managerial supervision: some historical and conceptual considerations. J Adv Nurs. 1999;30:1195–204. 25. American Medical Directors Association. Collaborative and supervisory relationships between attending physicians and advanced practice nurses in long-term care facilities. Geriatr Nurs. 2011;32:7–17. 4. Clarin OA. Strategies to overcome barriers to effective nurse practitioner and physician collaboration. J Nurs Pract. 2007;3:538–48. Page 9 of 9 26. MacPhee M. Strategies and tools for managing change. J Nurs Adm. 2007;37:405–13. 27. Kilminster S, Jolly B, van der Vleuten CP. A framework for effective training for supervisors. Med Teach. 2002;24:385–9. 28. Liu N, Finkelstein SR, Poghosyan L. A new model for nurse practitioner utilization in primary care: increased efficiency and implications. Health Care Manag Rev. 2013;1:10–20. 29. Carter N, Martin-Misener R, Kilpatrick K, Kaasalainen S, Donald F, Bryant- Lukosius D, et al. The role of nursing leadership in integrating clinical nurse specialists and nurse practitioners in healthcare delivery in Canada. Nurs Leadersh. 2010;23:167–85. 30. Cameron A, Masterson A. Managing the unmanageable? Nurse Executive Directors and new role developments in nursing. J Adv Nurs. 2000;31(5):1081–8. 31. Tarrant F, Associates. Literature review of Nurse Practitioner Legislation & Regulation. Ottawa: Canadian Nurses Association; 2005. Chouinard et al. BMC Health Services Research (2017) 17:437 References www.ibrarian.net/ navon/paper/Literature_Review_of_Nurse_Practitioner_Legislati.pdf. 32. Ducharme J, Buckley J, Alder R, Pelletier C. The application of change management principles to facilitate the introduction of nurse practitioners and physician assistants into six Ontario emergency departments. Healthc Q. 2009;12:70–7. 33. Paillé P. De l’analyse qualitative en général et de l’analyse thématique en particulier. Recherches Qualitatives. 1996;15:179–94. 34. Denzin NK, Lincoln YS. The SAGE handbook of qualitative research (4th Ed.) Thousand Oaks, CA: SAGE Publications; 2011. 34. Denzin NK, Lincoln YS. The SAGE handbook of qualitative research (4th Ed.) Thousand Oaks, CA: SAGE Publications; 2011. 35. Yin RK. Case Study Research: Design and Methods (4th Ed). Thousand Oaks: SAGE Publications; 2009. 35. Yin RK. Case Study Research: Design and Methods (4th Ed). Thousand Oaks: SAGE Publications; 2009. 36. DiCenso A, Matthews S. Report of the Nurse Practitioner Integration Task Team submitted to the Ontario Minister of Health and Long-Term Care. Toronto; 2007. http://ipspl.info/docs/DiCenso%20&%20Matthews%20- %202007.pdf. Accessed 23 June 2016. 37. Bush NJ, Watters T. The emerging role of the oncology nurse practitioner: a collaborative model within the private practice setting. Oncol Nurs Forum. 2001:1425–31. 37. Bush NJ, Watters T. The emerging role of the oncology nurse practitioner: a collaborative model within the private practice setting. Oncol Nurs Forum. 2001:1425–31. 38. Mintzberg H. The structuring of organisations. Upper Saddle River, NJ: Pearson Education; 1979. 38. Mintzberg H. The structuring of organisations. Upper Saddle River, NJ: Pearson Education; 1979. 39. Donaldson L. The contingency theory of organizations. Thousand Oaks, CA: SAGE Publications; 2001. 39. Donaldson L. The contingency theory of organizations. Thousand Oaks, CA: SAGE Publications; 2001. 40. Woods LP. The contingent nature of advanced nursing practice. J Adv Nurs. 1999;30:121–8. 40. Woods LP. The contingent nature of advanced nursing practice. J Adv Nurs. 1999;30:121–8. 41. Bryant-Lukosius D, DiCenso A, Browne G, Pinelli J. Advanced practice nursing roles: development, implementation and evaluation. J Adv Nurs. 2004;48:519–29. 41. Bryant-Lukosius D, DiCenso A, Browne G, Pinelli J. Advanced practice nursing roles: development, implementation and evaluation. J Adv Nurs. 2004;48:519–29. 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https://openalex.org/W3092343623	https://zenodo.org/record/4089675/files/PK_article_52174.pdf	English	null	Two new species of Oreocharis (Gesneriaceae) from karst regions in Yunnan and notes on O. tetraptera and O. brachypoda from China	PhytoKeys	2,020	cc-by	4,882	China 1 Launched to accelerate biodiversity research A peer-reviewed open-access journal China 1 Launched to accelerate biodiversity research A peer-reviewed open-access journal Two ne PhytoKeys 162: 1–12 (2020) doi: 10.3897/phytokeys.162.52174 http://phytokeys.pensoft.net Two ne PhytoKeys 162: 1–12 (2020) doi: 10.3897/phytokeys.162.52174 http://phytokeys.pensoft.net f Oreocharis (Gesneria RESEARCH ARTICLE Keywordsl flora of Yunnan, limestone area, morphology, new taxon, Oreocharis cademic editor: Eric Roalson \| Received 18 March 2020 \| Accepted 24 July 2020 \| Published 7 October Citation: Cai L, Huang Z-J, Wen F, Dao Z-L (2020) Two new species of Oreocharis (Gesneriaceae) from karst regions in Yunnan and notes on O. tetraptera and O. brachypoda from China. PhytoKeys 162: 1–12. https://doi.org/10.3897/ phytokeys.162.52174 Abstract Two new species of Gesneriaceae, Oreocharis aimodisca and O. longipedicellata, from the limestone area of Yunnan Province, China, are described and illustrated. Their morphological relationship with similar species is discussed and colour photographs, detailed descriptions, distribution and habitat, as well as the IUCN endangered status are provided. We also discuss the accuracy of the scientific names of the described species O. tetrapterus from Guangxi, China in 2019 and O. brachypodus from Guizhou, China, in 2015, and put forward corrections related to name form. Two new species of Oreocharis (Gesneriaceae) from karst regions in Yunnan and notes on O. tetraptera and O. brachypoda from China Lei Cai1, Zhang-Jie Huang2,3, Fang Wen2,3, Zhi-Ling Dao1 1 Yunnan Key Laboratory for Integrative Conservation of Plant Species with Extremely Small Populations, and Key Laboratory for Plant Diversity and Biogeography of East Asia, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, Yunnan, China 2 Guangxi Key Laboratory of Plant Conservation and Restoration Ecology in Karst Terrain, Guangxi Institute of Botany, Guangxi Zhuang Autonomous Region and Chinese Academy of Sciences, Guilin 541006, Guangxi, China 3 Gesneriad Conservation Center of China, Guilin Botanical Garden, Chinese Academy of Sciences, Guilin 541006, Guangxi, China Corresponding author: Fang Wen (wenfang760608@139.com), Zhi-Ling Dao (daozhl@mail.kib.ac.cn) Academic editor: Eric Roalson \| Received 18 March 2020 \| Accepted 24 July 2020 \| Published 7 Octo Copyright Lei Cai et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Introduction At least 30 new taxa of Oreocharis Benth. (Gesneriaceae) have been described and of ficially published (e.g., Cai et al. 2017, 2019; Do et al. 2017; Chen et al. 2018; Guo et al. 2018; Möller et al. 2018; Pan et al. 2019; Yang et al. 2019) after the generic Copyright Lei Cai et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. At least 30 new taxa of Oreocharis Benth. (Gesneriaceae) have been described and of ficially published (e.g., Cai et al. 2017, 2019; Do et al. 2017; Chen et al. 2018; Guo et al. 2018; Möller et al. 2018; Pan et al. 2019; Yang et al. 2019) after the generic Copyright Lei Cai et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Lei Cai et al. / PhytoKeys 162: 1–12 (2020) 2 redefinition based on new evidence following the development of molecular phyloge netics in 2011 (Möller et al. 2011) and several later adjustments of the species (Mid dleton et al. 2013; Chen et al. 2014; Möller et al. 2014; Möller 2015). Oreocharis s.l. hitherto comprises more than 140 taxa, mainly distributed in South and Southwest China (Wen et al. 2014, 2019) and a few species extending into North Vietnam (nine species), Myanmar (two species), Bhutan (one species), India (one species), Japan (one species) and Thailand (one species) (Xuyen et al. 2016; Do et al. 2017; Möller et al. 2017, 2018; Chen et al. 2018). Li and Li (2015) and Pan et al. (2019) each described new species of Oreocharis. One of the taxa with four corolla lobes is from Guangxi, China, which they named O. tetrapterus F.Wen, B.Pan & T.V.Do (Pan et al. 2019). The other has sessile or shorter petiole leaves and four stamens with anthers coherent in pairs from Guizhou, China, which was named as O. brachypodus J.M. Li & Z.M. Li (Li and Li 2015). These scientific names are improperly formed because the Latin forms ‘tetrapterous’ and ‘brachypodus’ are masculine and the Latin word of this genus, ‘Oreocharis’, is feminine. Introduction We revise the Latin name to ‘tetraptera’ and ‘brachypoda’ here and provide appropriate notes.i In 2018, during field investigations in the limestone area in Southeast Yunnan, China, an unknown species of Gesneriaceae without flowers was collected, then was in troduced to, and cultivated in, Guilin Botanical Garden (GBG). We first observed flow ering plants which were cultivated in GBG in August 2019. Thereafter, in September 2019, another unknown species of Gesneriaceae with flowers was collected from Shi zong County, eastern Yunnan. We confirmed that they are both members of Oreocharis, based on the characteristics of leaves in a basal rosette, four separated fertile stamens and capsules dehiscing predominantly on one side. After a careful examination of the related specimens and taxonomic publications of Oreocharis from the adjacent regions (Wang et al. 1990, 1998; Li and Wang 2005), we concluded that these two species are both new to science. Here, Oreocharis aimodisca and O. longipedicellata are described and illustrated and their morphological characters are compared to closely-related species. Oreocharis tetraptera F.Wen, B.Pan & T.V.Do Orthographic variant. Oreocharis tetrapterus F.Wen, B.Pan & T.V.Do in Pan et al. 2019: 83. Orthographic variant. Oreocharis tetrapterus F.Wen, B.Pan & T.V.Do in Pan et al. 2019: 83. Type. China. Guangxi: Hezhou City, Lisong Town, Gupo Mountain, 24°39'N, 111°36'E, elev. ca. 950 m, on moist surface of granite rocks, in flower, 25 August 2018, Wen Fang WF160825-01 (holotype: IBK!, isotype: IBK!). Type. China. Guangxi: Hezhou City, Lisong Town, Gupo Mountain, 24°39'N, 111°36'E, elev. ca. 950 m, on moist surface of granite rocks, in flower, 25 August 2018, Wen Fang WF160825-01 (holotype: IBK!, isotype: IBK!). Material and methods Extensive fieldwork has been undertaken in the east and southeast of Yunnan, Chi na, in recent years. Samples of the two new species were respectively collected from the fields of Shizong County and living plants cultivated in Guilin Botanical Garden (GBG) which initially introduced from Malipo County, Yunnan, China. All available specimens of Oreocharis s.l., stored in herbaria (E, HITBC, IBK, HN, K, KUN, P, PE and VMN), Chinese Virtual Herbarium (http://www.cvh.ac.cn/) in China and Global Plants on JSTOR (https://plants.jstor.org/) were examined. We studied all morpholog ical characters with dissecting microscopes and described the morphological characters by using the terminology presented by Wang et al. (1990, 1998). The photographs and the specimens were taken in the field and GBG by the first and correspondence authors. All specimens seen are indicated by ‘!’. Two new species of Oreocharis (Gesneriaceae) from China 3 Oreocharis brachypoda J.M. Li & Z.M. Li Orthographic variant. Oreocharis brachypodus J.M. Li & Z.M. Li in Li and Li 2015: 296. Orthographic variant. Oreocharis brachypodus J.M. Li & Z.M. Li in Li and Li 2015: 296. Type. China. Guizhou: in the vicinity of Tongren city, on rather cool rocks and very steep banks of cool, clammy soil that grows a fine film of moss, elev. 1300 m, 9 April Type. China. Guizhou: in the vicinity of Tongren city, on rather cool rocks and very steep banks of cool, clammy soil that grows a fine film of moss, elev. 1300 m, 9 April 2014, Jia-Mei Li 2304 (holotype: HEAC!); ibid. Jia-Mei Li 2305 (paratype: HEAC!).hfi Notes. The gender of the genus name, Oreocharis, is feminine, but the suffix of the scientific name, “-us,” is typically masculine. For Oreocharis tetrapterus (Pan et al. 2019), the correct orthography of the name of the new species is O. tetraptera, is writ ten by using an inaccurate gender, namely “tetrapterus”, in the citation of the type of the new species (p. 85), in the discussion of the Etymology (p. 86) and in the notes of the illustration (pp. 86, 87 and 88). In the other new taxon, Oreocharis brachypodus (Li and Li 2015), the correct orthography of the epithet “brachypoda” should be used to replace “brachypodus”. The inaccurately-used name gender appeared in the cita tion of the type of the new species (p. 296) and in the notes of the illustration (pp. 297 and 298). Thus, here we correct and revise two new species’ names as Oreocharis tetraptera and O. brachypoda. Oreocharis aimodisca Lei Cai, Z.L.Dao & F.Wen, sp. nov. urn:lsid:ipni.org:names:77211926-1 Figures 1–3 Diagnosis. Oreocharis aimodisca is morphologically similar to O. longifolia (Craib) Mich.Möller & A.Weber and O. muscicola (Diels) Mich.Möller & A.Weber in the appearance and colour of its flowers, but differs from the latter two species in its leaf blade oval to ovate, base cordate or auriculate, margin crenate, peduncle densely brown villous and pubescent, corolla outside densely pubescent and four separated fertile sta mens, pistil densely pubescent and disc blood red. Type. China. Yunnan: Shizong County, Wulong Town, Dachang Village, Xiaofakuai, 24°39'N, 104°10'E, elev. ca. 2122 m, on the surface of wet rocks, in flower, 10 Septem ber 2019, Lei Cai & Pin Zhang CL275 (holotype: KUN!, isotypes: KUN!, IBK!). Lei Cai et al. / PhytoKeys 162: 1–12 (2020) 4 Lei Cai et al. Oreocharis brachypoda J.M. Li & Z.M. Li / PhytoKeys 162: 1–12 (2020) 4 Figure 1. Oreocharis aimodisca Lei Cai, Z.L.Dao & F.Wen, sp. nov. A habit B pistil with disc and calyx C old capsule D front view of flower E opened corolla showing stamens and staminode F side view of a flower G adaxial view of the anther. Drawn by Xuan-Lin Zhu. Figure 1. Oreocharis aimodisca Lei Cai, Z.L.Dao & F.Wen, sp. nov. A habit B pistil with disc and calyx C old capsule D front view of flower E opened corolla showing stamens and staminode F side view of a flower G adaxial view of the anther. Drawn by Xuan-Lin Zhu. Description. Perennial herb, rhizome 5–18 mm long, 3–5 mm in diameter. Leaves 6–18, basal, petiole 2.5–10.5 cm long, brown villous and pubescent, leaf blade oval to ovate, 2.5–7.0 × 1.8–5.5 cm, adaxially densely appressed pubescent, abaxially puberulent, densely brown pubescent along veins, lateral veins 3–6 on each side of midrib, adaxially inconspicuous, abaxially conspicuous, apex acute, base cordate or auriculate, slightly oblique sometimes, margin crenate. Cymes ax illary 2–5, inflorescence 1–5-flowered; peduncle 5.5–16 cm long, brown villous and pubescent; bracts 2, lanceolate to narrowly triangle, 5–8 × 1.5–2.8 mm, out side brown pubescent, inside glabrous, margin nearly entire to denticulate; pedicel 1.2–3.5 cm long, densely pubescent. Calyx 8–12 mm long, 5-lobed to the base, lobes unequal, linear-lanceolate or narrowly triangular, 8–12 × 1.5–2.2 mm, both sides densely pubescent, margin denticulate. Corolla yellow, 2.8–3.6 cm long, out Two new species of Oreocharis (Gesneriaceae) from China 5 Figure 2. Oreocharis aimodisca Lei Cai, Z.L.Dao & F.Wen, sp. nov. in natural habitat A, B plants with flowers in the wild C habitat. Photographed by Lei Cai. Figure 2. Oreocharis aimodisca Lei Cai, Z.L.Dao & F.Wen, sp. nov. in natural habitat A, B plants with flowers in the wild C habitat. Photographed by Lei Cai. side densely pubescent, inside puberulent in the throat and on adaxial lobes, the lower part forms red stripes on the throat and lobes, tube coarsely tubular, gradually expanded from base to the throat, 2.0–2.6 cm long, 6–10 mm in diameter; limb 2-lipped; adaxial lip 2-lobed to middle, semicircular, lobes 4–5 × 4–5 mm, abaxial lip 3-lobed to middle, semicircular, 5–6 × 5–7 mm. Oreocharis brachypoda J.M. Li & Z.M. Li Stamens 4, 1.5–1.8 cm long, adnate to corolla 4–7 mm from the base; filaments linear, sparsely pubescent; an thers oblong, 2-loculed, dehiscing longitudinally, connective glabrous; staminode 1, 0.6–0.8 mm long, inserted ca. 3 mm from the base. Disc ca. 1.2 mm high, blood red, margin undulate. Pistil 1.6–2.4 cm long; ovary long cylindrical, densely pu bescent, 1.0–1.4 cm long; style 6–10 mm long, densely pubescent; stigma bilobed, flabellate. Capsule linear, 3.5–4.8 cm long. l Phenology. Flowering from August to September; fruiting from September to December. Distribution and habitat. Oreocharis aimodisca is currently known from two ad jacent populations at the type locality, Shizong County, East Yunnan, China. The new species commonly growing with other plants in shady and wet places on the middle part of mountain slopes under primary evergreen broad-leaf forest and shrubbery on karstic limestone at an elevation of over 2000 m. Lei Cai et al. / PhytoKeys 162: 1–12 (2020) 6 Figure 3. Oreocharis aimodisca Lei Cai, Z.L.Dao & F.Wen, sp. nov. A habit B adaxial (lower half) and abaxial leaf surface (upper half) C side view of a flower D front view of flowers E pistil with disc and calyx F opened corolla showing stamens and staminode G opened corolla with pistil and calyx H fresh fruits I old capsules. Photographed by Lei Cai. Figure 3. Oreocharis aimodisca Lei Cai, Z.L.Dao & F.Wen, sp. nov. A habit B adaxial (lower half) and abaxial leaf surface (upper half) C side view of a flower D front view of flowers E pistil with disc and calyx F opened corolla showing stamens and staminode G opened corolla with pistil and calyx H fresh fruits I old capsules. Photographed by Lei Cai. Figure 3. Oreocharis aimodisca Lei Cai, Z.L.Dao & F.Wen, sp. nov. A habit B adaxial (lower half) and abaxial leaf surface (upper half) C side view of a flower D front view of flowers E pistil with disc and calyx F opened corolla showing stamens and staminode G opened corolla with pistil and calyx H fresh fruits I old capsules. Photographed by Lei Cai. Figure 3. Oreocharis aimodisca Lei Cai, Z.L.Dao & F.Wen, sp. nov. Etymology. The original specific epithet ‘aimodisca’ derives from the Greek ‘αίμα’ meaning blood red and ‘δίσκος’ meaning discus. Vernacular name. The Chinese name of the new species is “Diān Dōng Mǎ Líng Jù Tái” (滇东马铃苣苔). The first two words, “Diān Dōng,” mean east of Yunnan, the next four words, “Mǎ Líng Jù Tái,” mean Oreocharis in Mandarin. Conservation status. Based on our field investigations, the new species is currently only known from the type locality with two contiguous subpopulations, in total ca. one thousand mature individuals were present within 5000 m2 (AOO). Since no special surveys were carried out for delimiting its distribution and information about threats is not very clear, this species was provisionally considered to be Critically Endangered [CR B2(a)] in terms of the IUCN Red List Categories and Criteria (IUCN 2019). Oreocharis brachypoda J.M. Li & Z.M. Li A habit B adaxial (lower half) and abaxial leaf surface (upper half) C side view of a flower D front view of flowers E pistil with disc and calyx F opened corolla showing stamens and staminode G opened corolla with pistil and calyx H fresh fruits I old capsules. Photographed by Lei Cai. Etymology. The original specific epithet ‘aimodisca’ derives from the Greek ‘αίμα’ meaning blood red and ‘δίσκος’ meaning discus.h Etymology. The original specific epithet ‘aimodisca’ derives from the Greek ‘αίμα’ meaning blood red and ‘δίσκος’ meaning discus.h Vernacular name. The Chinese name of the new species is “Diān Dōng Mǎ Líng Jù Tái” (滇东马铃苣苔). The first two words, “Diān Dōng,” mean east of Yunnan, the next four words, “Mǎ Líng Jù Tái,” mean Oreocharis in Mandarin.i Conservation status. Based on our field investigations, the new species is currently only known from the type locality with two contiguous subpopulations, in total ca. one thousand mature individuals were present within 5000 m2 (AOO). Since no special surveys were carried out for delimiting its distribution and information about threats is not very clear, this species was provisionally considered to be Critically Endangered [CR B2(a)] in terms of the IUCN Red List Categories and Criteria (IUCN 2019). Two new species of Oreocharis (Gesneriaceae) from China 7 Table 1. Morphological comparison of Oreocharis aimodisca sp. nov., O. longifolia and O. muscicola. Characters O. aimodisca O. longifolia O. Oreocharis brachypoda J.M. Li & Z.M. Li muscicola Leaf-blade oval to ovate, base cordate or auriculate, margin crenate narrowly elliptic to oblanceolate, base attenuate, margin serrulate narrowly elliptic to lanceolate, base often slightly oblique, narrowly to broadly cuneate, margin serrate to serrate-crenate, sometimes doubly so Petiole brown villous and pubescent grey to brownish pubescent densely rust-brown villous Peduncle densely brown villous and pubescent sparsely brownish villous to pubescent rust-brown villous and glandular- pubescent Bract lanceolate to narrowly triangle, outside brown pubescent margin nearly entire to denticulate oblanceolate, outside pubescent, margin entire lanceolate, outside rust-brown villous, margin entire Calyx lobes linear-lanceolate or narrowly triangular, both sides densely pubescent, margin denticulate narrowly ovate, outside sparsely brownish pubescent, inside glabrous, margin entire lanceolate, outside sparsely white pubescent and rust-brown villous, margin entire Corolla outside densely pubescent, inside puberulent in the throat and on adaxial lobes outside sparsely glandular puberulent, inside sparsely glandular puberulent outside sparsely puberulent, inside glandular pubescent Filaments sparsely pubescent glabrous sparsely puberulent Anthers oblong, separated reniform, connected in pairs reniform, connected in pairs Pistil densely pubescent glabrous glabrous Disc blood red yellow yellow-green . Morphological comparison of Oreocharis aimodisca sp. nov., O. longifolia and O. muscicola. Taxonomic affinities. Oreocharis aimodisca is morphologically similar to O. longi folia and O. muscicola in the corolla yellow and coarsely tubular; however, it is different from the latter two species by the shape of the leaf blade, indumentum characters of the peduncle, pedicel, calyx, corolla and pistil and separated stamens. The comparison of morphological characters on related species is provided in Table 1. Oreocharis longipedicellata Lei Cai & F.Wen, sp. nov. urn:lsid:ipni.org:names:77211927-1 Figures 4, 5 Diagnosis. Oreocharis longipedicellata morphologically resembles O. panzhouensis Lei Cai, Y.Guo & F.Wen in its ovate leaf blade, yellow flower, four separated fertile sta mens, oblong anthers and bilobed, flabellate stigma, but can be easily distinguished from this species in the peduncle 20–28 cm long, bract lanceolate to elliptic, margin denticulate, the calyx 5-lobed to the base, stamens adnate to corolla 3–4 mm from base and the pistil 1.5–2 cm long. Type. China. Yunnan: Malipo County, Mengdong, on the surface of moist rocks (Cultivated in GCCC nursery, Guilin Botanical Garden, Chinese Academy of Sciences) in flower, 24 August 2019, Fang Wen WF190824-01 (holotype: KUN!, isotype: IBK!). Type. China. Oreocharis brachypoda J.M. Li & Z.M. Li Yunnan: Malipo County, Mengdong, on the surface of moist rocks (Cultivated in GCCC nursery, Guilin Botanical Garden, Chinese Academy of Sciences) in flower, 24 August 2019, Fang Wen WF190824-01 (holotype: KUN!, isotype: IBK!). Description. Perennial herb, rhizome 0.8–2 cm long, 3–5 mm in diameter. Leaves 8–15, basal, petiole 3.5–8.0 cm long, densely brown villous, leaf blade elliptic to ovate, 3.0–5.5 × 2.4–4.5 cm, adaxially densely pubescence, abaxially pubescent, densely brown villous along veins, lateral veins 3–6 on each side of midrib, apex rounded, base slightly oblique sometimes, cordate to auricula-cordate, margin cre 8 Lei Cai et al. / PhytoKeys 162: 1–12 (2020) 8 Figure 4. Oreocharis longipedicellata Lei Cai & F.Wen, sp. nov. A habit B front view of flower C open corolla showing stamens and staminode D pistil with disc E calyx lobes. Drawn by Xuan-Lin Zhu. Figure 4. Oreocharis longipedicellata Lei Cai & F.Wen, sp. nov. A habit B front view of flower C opened corolla showing stamens and staminode D pistil with disc E calyx lobes. Drawn by Xuan-Lin Zhu. Figure 4. Oreocharis longipedicellata Lei Cai & F.Wen, sp. nov. A habit B front view of flower C opened corolla showing stamens and staminode D pistil with disc E calyx lobes. Drawn by Xuan-Lin Zhu. Two new species of Oreocharis (Gesneriaceae) from China 9 Two new species of Oreocharis (Gesneriaceae) from China 9 Figure 5. Oreocharis longipedicellata Lei Cai & F.Wen, sp. nov. A plants cultivated in GBG B adaxial and abaxial leaf surface C front view of flowers D side view of a flower E petiole F pistil with disc and calyx G, H opened corolla showing stamens and staminode. Photographed by Fang Wen. Figure 5. Oreocharis longipedicellata Lei Cai & F.Wen, sp. nov. A plants cultivated in GBG B adaxial and abaxial leaf surface C front view of flowers D side view of a flower E petiole F pistil with disc and calyx G, H opened corolla showing stamens and staminode. Photographed by Fang Wen. Figure 5. Oreocharis longipedicellata Lei Cai & F.Wen, sp. nov. A plants cultivated in GBG B adaxial and abaxial leaf surface C front view of flowers D side view of a flower E petiole F pistil with disc and calyx G, H opened corolla showing stamens and staminode. Photographed by Fang Wen. nate, with brown villous. Oreocharis brachypoda J.M. Li & Z.M. Li Cymes axillary 2–5, inflorescence 4–8-flowered; peduncle 20–28 cm long, brown villous; bracts 2, lanceolate to elliptic, 10–12 × 2.5–5.0 mm, adaxially densely villous, abaxially glabrous, sometimes upper part pubescent, margin denticulate; pedicel 2.0–3.5 cm long, densely villous. Calyx 6–9 mm long, 5-lobed to the base, lobes triangular lanceolate to narrowly triangular, 6–9 mm long, 1.5–2 mm wide, outside brown villous, inside glabrous, margin denticulate. Corolla sigmoid, yellow, 2.2–2.8 cm long, outside pubescent and glandular-pubescent, inside glan dular-pubescent in the throat and on adaxial lobes, tube cylindrical, slightly bent near the mouth, 1.8–2.2 cm long, 5–7 mm in diameter; limb 2-lipped; adaxial lip 2-lobed to near base, semicircular, lobes 4–5 × 3.8–4.2 mm, abaxial lip 3-lobed to nate, with brown villous. Cymes axillary 2–5, inflorescence 4–8-flowered; peduncle 20–28 cm long, brown villous; bracts 2, lanceolate to elliptic, 10–12 × 2.5–5.0 mm, adaxially densely villous, abaxially glabrous, sometimes upper part pubescent, margin denticulate; pedicel 2.0–3.5 cm long, densely villous. Calyx 6–9 mm long, 5-lobed to the base, lobes triangular lanceolate to narrowly triangular, 6–9 mm long, 1.5–2 mm wide, outside brown villous, inside glabrous, margin denticulate. Corolla sigmoid, yellow, 2.2–2.8 cm long, outside pubescent and glandular-pubescent, inside glan dular-pubescent in the throat and on adaxial lobes, tube cylindrical, slightly bent near the mouth, 1.8–2.2 cm long, 5–7 mm in diameter; limb 2-lipped; adaxial lip 2-lobed to near base, semicircular, lobes 4–5 × 3.8–4.2 mm, abaxial lip 3-lobed to Lei Cai et al. / PhytoKeys 162: 1–12 (2020) 10 Table 2. Morphological comparison between Oreocharis longipedicellata sp. nov. and O. panzhouensis. Characters O. longipedicellata O. panzhouensis Peduncle 20–28 cm long 4.5–8 cm long Bract lanceolate to elliptic, margin denticulate linear to subulate, margin entire Calyx 5-lobed to the base, lobes lanceolate to narrowly trian gular, outside brown villous 5-lobed to the middle, lobes equal, broadly triangular, outside pubescent and sparsely brown villous Corolla sigmoid, tube cylindrical, lobes reflexed outwards slightly not sigmoid, tube campanulate, lobes not reflexed outwards Stamens 10–13 mm long, adnate to corolla 3–4 mm from base 5–10 mm long, adnate to corolla 5–6 mm from base Pistil 15–20 mm long, ovary long cylindrical, 10–12 mm long; style 4–6 mm long 8–14 mm long, ovary cylindrical, 5–8 mm long; style 2–4 mm long base, semicircular to oval, 6–8 × 5–7 mm. Oreocharis brachypoda J.M. Li & Z.M. Li Stamens 4, 1.0–1.3 cm long, adnate to corolla 3–4 mm from the base; filaments linear, glabrous; anthers oblong, 2-loculed, dehiscing longitudinally, connective glabrous; staminode 1, 0.6–1.0 mm long, in serted ca. 1 mm from the base. Disc ca. 1.5 mm high, yellow, margin undulate. Pistil 1.5–2.0 cm long, glabrous; ovary long cylindrical, 10–12 mm long; style 4–6 mm long; stigma bilobed, flabellate. Fruit unknown. g gl Phenology. Flowering from August to October; fruiting unknown. Distribution and habitat. Oreocharis longipedicellata is currently known by only one population at the type locality, Mengdong, Malipo County, southeastern Yunnan, in the China and Vietnam border area. The species was observed to grow on the surface of moist rocks in the karst region. Etymology. The specific epithet ‘longipedicellata’ refers to the relatively-long peduncle of the new species. This species has almost the longest pedicels in the genus Oreocharis.h h Vernacular name. The Chinese name of the new species is “Cháng Gěng Mǎ Líng Jù Tái” (长梗马铃苣苔). The first two words, “Cháng Gěng,” mean the long pedun cle. The next four words mean Oreocharis in mandarin. h Conservation status. The new species could be endangered, but more data is needed to evaluate as the field distribution information is not sufficiently detailed.fi Taxonomic affinities. Oreocharis longipedicellata most resembles recently pub lished O. panzhouensis in the yellow flower, four separated stamens, calyx 5-lobed to the middle and stigma bilobed, flabellate. Nevertheless, it differs from the latter species in several other characteristics (see Table 2). Acknowledgements We are grateful to Ms. Xuan-Lin Zhu for the beautiful illustrations and processing the figures. Thanks also to Mr. Pin Zhang, Mr. Zheng-Yun Lu and Mr. Yu-Yang Lei for their help during the fieldwork. Special thanks to Stephen Maciejewski, The Gesneriad Society, Michael LoFurno, Adjunct Professor, Temple University, Philadelphia, USA and Two new species of Oreocharis (Gesneriaceae) from China 11 Zhi-Qian Zou for their editorial assistance. This study was financially jointly supported by the Science & Technology Basic Resources Investigation Program of China (Grant no. 2017FY100100), Yunnan Science and Technology Innovation Team Program for PSESP (Plant Species with Extremely Small Populations) Conservation and Utilization (Grant No. 2019HC015), the National Natural Science Foundation of China (31860047), the Natural Science Foundation of Guangxi (2017GXNSFAA198006) and the Fund of Guangxi Key Laboratory of Plant Conservation and Restoration Ecology in Karst Terrain (19-050-6). References Cai L, Guo Y, Zhang RM, Dao ZL, Wen F (2019) Oreocharis panzhouensis (Gesneriaceae), a new species from karst regions in Guizhou, China. Phytotaxa 393(3): 287–291. https:// doi.org/10.11646/phytotaxa.393.3.5 Cai L, Huang H, Dao ZL, Wu ZK (2017) Oreocharis parviflora, a new species of Gesneriaceae from northwestern Yunnan, China. Phytotaxa 329(2): 167–172. https://doi.org/10.11646/ phytotaxa.329.2.7 Chen WH, Nguyen QH, Chen RZ, Nguyen TH, Nguyen SK, Nguyen VT, Möller M, Middleton DJ, Shui YM (2018) Two new species of Oreocharis (Gesneriaceae) from Fan Si Pan, the highest mountain in Vietnam. PhytoKeys 94: 95–106. https://doi.org/10.3897/phytokeys.94.21329 Chen WH, Shui YM, Möller M (2014) Two new combinations in Oreocharis Benth. (Gesne riaceae) from China. Candollea 69(2): 179–182. https://doi.org/10.15553/c2014v692a10 Do VT, Wei YG, Wen F (2017) Oreocharis caobangensis (Gesneriaceae), a new species from Cao Bang Province, northern Vietnam. Phytotaxa 302(1): 65–70. https://doi.org/10.11646/ phytotaxa.302.1.6 Guo ZY, Li ZY, Xiang XG (2018) Oreocharis duyunensis (Gesneriaceae), a new species from Guizhou, China. Nordic Journal of Botany 36(9): e01514. https://doi.org/10.1111/njb.01514 IUCN (2019) Guidelines for Using the IUCN Red List Categories and Criteria. Ver. 14. Pre pared by the Standards and Petitions Subcommittee of the IUCN Species Survival Com mission. http://cmsdocs.s3.amazonaws.com/RedListGuidelines.pdf Li JM, Li ZM (2015) Oreocharis brachypodus (Gesneriaceae), a new taxon from Guizhou, Chi na. Phytotaxa 204(4): 296–299. https://doi.org/10.11646/phytotaxa.204.4.6 Li ZY, Wang YZ (2005) Plants of Gesneriaceae in China. Henan Science & Technology Pub lishing House, Zhengzhou, Henan, 14–47. Middleton DJ, Weber A, Yao TL, Sontag S, Möller M (2013) The current status of the spe cies hitherto assigned to Henckelia (Gesneriaceae). Edinburgh Journal of Botany 70(3): 385–404. https://doi.org/10.1017/S0960428613000127 Möller M (2015) Transfer of Tremacron hongheense to Oreocharis (Gesneriaceae). Phytotaxa 239(3): 295–296. https://doi.org/10.11646/phytotaxa.239.3.12 Möller M, Atkins HJ, Bramley GL, Middleton DJ, Baines R, Nguyen VD, Bui HQ, Barber S (2018) Two new species of Oreocharis (Gesneriaceae) from northern Vietnam. Edinburgh Journal of Botany 75(3): 309–319. https://doi.org/10.1017/S0960428618000148 Lei Cai et al. / PhytoKeys 162: 1–12 (2020) 12 Möller M, Chen WH, Shui YM, Atkins H, Middleton DJ (2014) A new genus of Gesneriaceae in China and the transfer of Briggsia species to other genera. Gardens’ Bulletin (Singapore) 66: 195–205. Möller M, Middleton DJ, Nishii K, Wei YG, Sontag S, Weber A (2011) A new delineation for Oreocharis incorporating an additional ten genera of Chinese Gesneriaceae. Phytotaxa 23(1): 1–36. https://doi.org/10.11646/phytotaxa.23.1.1 Möller M, Nampy S, Janeesha AP, Weber A (2017) The Gesneriaceae of India: Consequences of updated generic concepts and new family classification. References Rheedea 27(1): 23–41. https:// doi.org/10.22244/rheedea.2017.27.1.5 Pan B, Tang GD, Do TV, Maciejewski S, Deng CL, Wen F (2019) Oreocharis tetrapterus (Gesneriaceae), a new species from East Guangxi, China. PhytoKeys 131: 83–89. https:// doi.org/10.3897/phytokeys.131.35434 Wang WT, Pan KY, Li ZY (1990) Gesneriaceae. In: Wang WT (Ed.) Flora Reipublicae Popu laris Sinicae (Vol. 69). Science Press, Beijing, 141–271. Wang WT, Pan KY, Li ZY, Weitzman AL, Skog LE (1998) Gesneriaceae. In: Wu ZY, Raven PH (Eds) Flora of China (Vol. 18). Science Press, Beijing & Missouri Botanical Garden Press, St. Louis, 254–401. Wen F, Li S, Xin ZB, Fu LF, Hong X, Cai L, Qin JQ, Pan B, Pan FZ, Wei YG (2019) The updated plant list of Gesneriaceae in China under the new Chinese naming rules. Guangxi Sciences 26(1): 37–63. Wen F, Wei YG, Fu LF, Xin ZB, Li S, Huang ZJ, Meng DC (2014 onward) The Checklist of Gesneriaceae in China. [Free download from] http://gccc.gxib.cn/about-46.aspx Xuyen DT, Phuong VX, Hoan HV, Duc NA (2016) Genus Opithandra B.L. Burtt and Species Opithandra dinghushanensis W.T. Wang as new records for the flora of Vietnam from Bac Huong Hoa Nature Reserve, Quang Tri Province. VNU Journal of Science: Natural Sciences and Technology 32(1S): 142–146. Yang LE, Cen HF, Sun H, LoFurno M, Maciejewski S, Goretsky WJ, Wen F (2019) Oreocharis rubrostriata (Gesneriaceae), a new species from Guangxi, China. Kew Bulletin 74(23): 1–5. https://doi.org/10.1007/s12225-019-9810-9
https://openalex.org/W2076766031	https://zenodo.org/records/2495649/files/article.pdf	English	null	STUDIES IN ASEPTIC TECHNIC	Journal of the American Medical Association	1,915	public-domain	6,932	2. Brewer, George E.: Operative Surgery at the City Hospital with a Completed Report on the Study of Wound Infection, Med. Rec., New York, March 13, 1897. 3. Brewer, George E.: Med. Rec., New York, March 26, 1898. 4. Brewer, George E.: Studies in Surgical Technic with a Report on Operative Surgery at the City Hospital for 1898 and 1899, Med. News, Sept. 22, 1900. STUDIES IN ASEPTIC TECHNIC WITH A REPORT OF SOME RECENT OBSERVATIONS AT THE ROOSEVELT HOSPITAL GEORGE EMERSON BREWER, M.D. NEW YORK That the conditions were unfavorable was evidenced by the fact that there was not in the hospital during that period a single modern sterilizing autoclave, that the ligature and suture material was prepared in a wholly indifferent and unscientific manner and that neither the operating-room nurse nor any member of the house staff had had any training in modern aseptic methods. One of the most important duties of an attending surgeon to a hospital is to devise and carry out in his operative work, a system of aseptic technic which will insure the minimum of infection in his operative wounds. Every infection occurring in a clean wound and every infected wound which is not rendered sterile by the primary operation, when this is possible, results in increased suffering and delay in convalescence to the patient and an increased expenditure of money, time, and effort on the part of the hospital and attending staff. A second report2 was published one year later, after the hospital had been provided with two new modern operating-rooms and a complete modern sterilizing apparatus ; and after an exhaustive bactériologie exam¬ ination of our methods and material had been made by the late Prof. Philip Hanson Hiss of Columbia University. During this year, the report stated, 9 per cent, of our clean wounds had become infected. Take for instance the operation for the removal of a cancerous breast. Although this is often one of the most extensive operations in surgery, the wound should heal firmly in ten days and the patient should leave the hospital in a fortnight. If, however, through any error in technic the wound becomes infected, heal- ing may be delayed for weeks or months, and the return to health and earning capacity of the patient may be indefinitely postponed. Each additional day that such a patient remains in the hospital as a result of infection, and every dollar that the hospital expends for his or her additional care should be recorded as a waste product. Moreover, the prolonged occupancy of a ward bed for such a reason often deprives other needy patients of the possibility of hospital care. p During the following year many changes in technic were made to bring about further improvement in our results. These were all described at length in a paper3 read before the Suffolk Medical Society in Boston. 1. Brewer, George E.: Operative Surgery at the City Hospital with a Preliminary Report on the Study of Wound Infection, New York Med. Jour., May 2, 1896. report "never should have been published as it was a disgrace to the profession and would bring it into dis¬ repute." I replied that to my mind the report possessed at least one merit and that was, that it was an abso¬ lutely truthful statement of the facts which had occurred during an honest effort to do clean surgery under most unfavorable conditions. report "never should have been published as it was a disgrace to the profession and would bring it into dis¬ repute." I replied that to my mind the report possessed at least one merit and that was, that it was an abso¬ lutely truthful statement of the facts which had occurred during an honest effort to do clean surgery under most unfavorable conditions. STUDIES IN ASEPTIC TECHNIC WITH A REPORT OF SOME RECENT OBSERVATIONS AT THE ROOSEVELT HOSPITAL GEORGE EMERSON BREWER, M.D. NEW YORK Downloaded From: http://jama.jamanetwork.com/ by a Simon Fraser University User on 06/02/2015 STUDIES IN ASEPTIC TECHNIC WITH A REPORT OF SOME RECENT OBSERVATIONS AT THE ROOSEVELT HOSPITAL GEORGE EMERSON BREWER, M.D. NEW YORK During the progress of these studies I visited many of the larger and more important hospitals in this city and also in Boston, Baltimore, and Philadelphia, with a view to ascertaining the methods of hand and skin disinfection, sterilization of instruments, dress¬ ings and suture material, in use by the leading sur¬ geons ; also the operating-room technic and in general the results obtained. When obtaining permission of the attending surgeon to observe the preparation of the patients, the surgeons, and assistants, and for the making of ward visits when dressings were changed, the question generally was asked, "How much infec¬ tion do you expect in clean cases?" The answer almost invariably was "practically none." Later con¬ versations with members of the house staff and obser¬ vations during ward visits led to the belief that while the "practically none" represented the honest opinion of the visiting surgeon, such an opinion was not based on accurate records. Indeed I am thoroughly con¬ vinced, that if I had been asked the same question at any time during the past ten years during which no effort was made to keep accurate records of infections occurring in my service, my off-hand estimate of the percentage of infection would fall far below the actual figures. In fact Tarn strongly of the opinion that the only way to obtain the best technical results is to keep an accurate record of infection in every patient sub¬ mitted to operation, for it is only by this means that one can be kept aware of his technical transgressions. ld b it i ibl f th d t d t patients. During the first six years of service at the Roosevelt Hospital, while I acted as first assistant to Professor Weir, the number of clean wounds which became infected was exceedingly small, although no exact record was kept. kept. Shortly after I was appointed senior attending sur¬ geon, a veritable storm of infection occurred, five clean wounds became infected as a result of operations (by both surgical divisions) in a single day, and the patients operated on in the succeeding few days also showed a high percentage of infections. One of these cases proved fatal. Two or three days after this out¬ break of infection the hospital pathologist came to the operating-room just as we were about to begin our afternoon work. STUDIES IN ASEPTIC TECHNIC WITH A REPORT OF SOME RECENT OBSERVATIONS AT THE ROOSEVELT HOSPITAL GEORGE EMERSON BREWER, M.D. NEW YORK Cultures were taken from the gloves of all who were to participate in the operation, also from the gowns, caps, towels, sheets, sponges, instru¬ ments, ligature and suture material. Of twelve flasks of catgut and silk, eleven showed growth. All the other cultures remained sterile. It was thus clearly demonstrated that this wave of infection was directly due to some grave error in the preparation of our catgut. The person responsible for this was dismissed, the entire stock of contaminated gut was destroyed and the epidemic of infection was at an end. ept g It would be quite impossible for the modern student of technic to appreciate the. difficulties which were encountered on every side, in the five years of constant effort to improve the technical results in an institu¬ tion where the conditions were so unfavorable. Dur¬ ing this period I was a constant observer and student of the methods being employed in the Syms operating- room by the late Dr. Charles McBurney, who estab¬ lished a standard of surgical technic in the Roosevelt Hospital which was equal if not superior to that of any other American or European clinic. Many of the changes made in our procedures at the City Hospital were the direct result of helpful consultations with Dr. McBurney who was always ready and willing to discuss the various problems presented and to give friendly and sound advice. p A year or two later two cases of infection by the Bacillus aerogenes capsulatus occurred within a period of two or three days, one on the first and one on the second surgical division. Both cases proved fatal. A thorough investigation of our methods and material was made, including an elaborate series of tests of our steam sterilizers, which were of an old model, and had been in constant use for over fifteen years. While the source of the gas bacillus was never discovered, the investigation demonstrated a definite defect in our autoclaves. These were practically rebuilt and ren¬ dered in every way as efficient as any of the most modern design. Repeated bactériologie tests since that time have demonstrated that our material is not only free from living bacteria, but also spore-free. For this valuable work, and also for many months of patient investigation on the preparation of our catgut, the hospital is indebted to Dr. Karl Connell of the attend¬ ing surgical staff. STUDIES IN ASEPTIC TECHNIC WITH A REPORT OF SOME RECENT OBSERVATIONS AT THE ROOSEVELT HOSPITAL GEORGE EMERSON BREWER, M.D. NEW YORK Shortly after I was appointe geon, a veritable storm of infec wounds became infected as a both surgical divisions) in patients operated on in the su showed a high percentage of in cases proved fatal. Two or th break of infection the hospital operating-room just as we w afternoon work. Cultures wer of all who were to participate from the gowns, caps, towels, ments, ligature and suture mat of catgut and silk, eleven sh other cultures remained sterile demonstrated that this wave o due to some grave error in catgut. The person responsible the entire stock of contamina and the epidemic of infection w A year or two later two ca Bacillus aerogenes capsulatus o of two or three days, one on t second surgical division. Bo A thorough investigation of ou was made, including an elabora steam sterilizers, which were had been in constant use for ov the source of the gas bacillus the investigation demonstrated autoclaves. These were prac dered in every way as efficie modern design. Repeated bact time have demonstrated that o free from living bacteria, but a valuable work, and also for m investigation on the preparat hospital is indebted to Dr. Kar ing surgical staff. For some years, both before gation, our results seemed s accidental infections so far apa was made to ascertain the exac During the early part of 191 unexplained infections occurre gurate on the first surgical di of every patient operated on wound area before operatio whether clean or infected at t dressings. This report was rea held every Friday morning af any case of unexplained infec committee to investigate, and conference conditions of that institution were unnecessary, and that even better results could be obtained by the some¬ what simpler but equally effective technic employed by Dr. McBurney and my new chief, Dr. Robert F. Weir. While this was in part due to more perfect operating- room equipment and to a more thoroughly trained staff of assistants and nurses, the most important factor seemed to be the better physical condition of the patients. STUDIES IN ASEPTIC TECHNIC WITH A REPORT OF SOME RECENT OBSERVATIONS AT THE ROOSEVELT HOSPITAL GEORGE EMERSON BREWER, M.D. NEW YORK In this report the use of dilute solutions of a 40 per cent, solution of formaldehyd for wound disinfection and dressings was advocated, and also the employment of sterile rubber gloves by all participating in the operation. During the period covered by this third report, the percentage of wound infections was reduced to 7. Two years later a fourth and final report4 was made of our studies in technic at this institution. This included a number of other changes in our methods with a careful investigation of several cases in which asepsis was lost as a result of our operative proce¬ dures. From a careful analysis of our results we were able to report that during our last year the percentage of infection in clean cases had been reduced to 3.2 per cent., which, at that time, compared favorably with the results in the other New York hospitals where the patients were recruited from those in the more fortunate walks of life, and were not the victims of starvation, alcoholism, and chronic disease which made up such a large percentage of those admitted to the wards of the City Hospital. y p p y p While accidental infections will occasionally occur even in the best regulated institutions, and while it is frequently impossible to render a given septic wound sterile by the most approved and skilful operative treatment, a conscientious surgeon by perfect technic and an ever vigilant watchfulness will reduce these unfortunate accidents to the minimum. i h i My interest in operative technic with a view to avoiding wound infection, dates back to the year 1895, when I began my first service as attending surgeon at the City Hospital. In a report1 published the follow¬ ing May, I called attention to the fact that during an active service of six months, 39 per cent, of all clean operative wounds occurring in my service became infected. STUDIES IN ASEPTIC TECHNIC WITH A REPORT OF SOME RECENT OBSERVATIONS AT THE ROOSEVELT HOSPITAL GEORGE EMERSON BREWER, M.D. NEW YORK This report resulted in considerable unfavor¬ able comment from many of my professional col¬ leagues, one even going so far as to state that the During the progress of these studies I visited many of the larger and more important hospitals in this city and also in Boston, Baltimore, and Philadelphia, with a view to ascertaining the methods of hand and skin disinfection, sterilization of instruments, dress¬ ings and suture material, in use by the leading sur¬ geons ; also the operating-room technic and in general the results obtained. When obtaining permission of the attending surgeon to observe the preparation of the patients, the surgeons, and assistants, and for the making of ward visits when dressings were changed, the question generally was asked, "How much infec¬ tion do you expect in clean cases?" The answer almost invariably was "practically none." Later con¬ versations with members of the house staff and obser¬ vations during ward visits led to the belief that while the "practically none" represented the honest opinion of the visiting surgeon, such an opinion was not based on accurate records. Indeed I am thoroughly con¬ vinced, that if I had been asked the same question at any time during the past ten years during which no effort was made to keep accurate records of infections occurring in my service, my off-hand estimate of the percentage of infection would fall far below the actual figures. In fact Tarn strongly of the opinion that the only way to obtain the best technical results is to keep an accurate record of infection in every patient sub¬ mitted to operation, for it is only by this means that one can be kept aware of his technical transgressions. It would be quite impossible for the modern student of technic to appreciate the. difficulties which were encountered on every side, in the five years of constant effort to improve the technical results in an institu¬ tion where the conditions were so unfavorable. Dur¬ ing this period I was a constant observer and student of the methods being employed in the Syms operating- room by the late Dr. Charles McBurney, who estab¬ lished a standard of surgical technic in the Roosevelt Hospital which was equal if not superior to that of any other American or European clinic. STUDIES IN ASEPTIC TECHNIC WITH A REPORT OF SOME RECENT OBSERVATIONS AT THE ROOSEVELT HOSPITAL GEORGE EMERSON BREWER, M.D. NEW YORK Many of the changes made in our procedures at the City Hospital were the direct result of helpful consultations with Dr. McBurney who was always ready and willing to discuss the various problems presented and to give friendly and sound advice. I have referred to these experiences in another hospital for the reason that I wish to call attention to the fact that in an institution where the patients as a rule were seriously handicapped by a low vital resistance due to malnutrition, dissipation, and dis¬ ease, a somewhat exaggerated technic was necessary to obtain results comparable with those in other hospi¬ tals. Another important factor was that of air infec¬ tion. At the City Hospital the air of the operating- rooms was constantly contaminated by floating germs, largely of the pus-producing varieties. This was fre¬ quently demonstrated by exposure of gelatin or agar plates in the operating-room during our surgical work. To overcome this source of infection, we found it nec¬ essary to cover all instrument trays, wash basins, Irri¬ gators, and pitchers with tents or covers of sterile mus¬ lin, and to employ irrigation of the wound during our operative procedures. It was only by these methods that our best results were obtained. When, in 1899, I received an appointment as junior surgeon to the Roosevelt Hospital, I was surprised and gratified to see that many of the unusual technical procedures used in the City Hospital to meet the local conditions of that institution were unnecessary, and that even better results could be obtained by the some¬ what simpler but equally effective technic employed by Dr. McBurney and my new chief, Dr. Robert F. Weir. While this was in part due to more perfect operating- room equipment and to a more thoroughly trained staff of assistants and nurses, the most important factor seemed to be the better physical condition of the patients. During the first six years of service at the Roosevelt Hospital, while I acted as first assistant to Professor Weir, the number of clean wounds which became infected was exceedingly small, although no exact record was kept. STUDIES IN ASEPTIC TECHNIC WITH A REPORT OF SOME RECENT OBSERVATIONS AT THE ROOSEVELT HOSPITAL GEORGE EMERSON BREWER, M.D. NEW YORK Shortly after I was appointed senior attending sur¬ geon, a veritable storm of infection occurred, five clean wounds became infected as a result of operations (by both surgical divisions) in a single day, and the patients operated on in the succeeding few days also showed a high percentage of infections. One of these cases proved fatal. Two or three days after this out¬ break of infection the hospital pathologist came to the operating-room just as we were about to begin our afternoon work. Cultures were taken from the gloves of all who were to participate in the operation, also from the gowns, caps, towels, sheets, sponges, instru¬ ments, ligature and suture material. Of twelve flasks of catgut and silk, eleven showed growth. All the other cultures remained sterile. It was thus clearly demonstrated that this wave of infection was directly due to some grave error in the preparation of our catgut. The person responsible for this was dismissed, the entire stock of contaminated gut was destroyed and the epidemic of infection was at an end. A year or two later two cases of infection by the Bacillus aerogenes capsulatus occurred within a period of two or three days, one on the first and one on the second surgical division. Both cases proved fatal. A thorough investigation of our methods and material was made, including an elaborate series of tests of our steam sterilizers, which were of an old model, and had been in constant use for over fifteen years. While the source of the gas bacillus was never discovered, the investigation demonstrated a definite defect in our autoclaves. These were practically rebuilt and ren¬ dered in every way as efficient as any of the most modern design. Repeated bactériologie tests since that time have demonstrated that our material is not only free from living bacteria, but also spore-free. For this valuable work, and also for many months of patient investigation on the preparation of our catgut, the hospital is indebted to Dr. Karl Connell of the attend¬ ing surgical staff. For some years, both before and after this investi¬ gation, our results seemed so satisfactory and the accidental infections so far apart that no special inquiry was made to ascertain the exact number or percentage. STUDIES IN ASEPTIC TECHNIC WITH A REPORT OF SOME RECENT OBSERVATIONS AT THE ROOSEVELT HOSPITAL GEORGE EMERSON BREWER, M.D. NEW YORK During the early part of 1911, however, one or two unexplained infections occurred which led me to inau¬ gurate on the first surgical division, a weekly report of every patient operated on, the condition of the wound area before operation, and the condition whether clean or infected at the first and subsequent dressings. This report was read at a staff conference, held every Friday morning after the ward visit; and any case of unexplained infection was referred to a committee to investigate, and to report at the next conference During the progress of these studies I visited many of the larger and more important hospitals in this city and also in Boston, Baltimore, and Philadelphia, with a view to ascertaining the methods of hand and skin disinfection, sterilization of instruments, dress¬ ings and suture material, in use by the leading sur¬ geons ; also the operating-room technic and in general the results obtained. When obtaining permission of the attending surgeon to observe the preparation of the patients, the surgeons, and assistants, and for the making of ward visits when dressings were changed, the question generally was asked, "How much infec¬ tion do you expect in clean cases?" The answer almost invariably was "practically none." Later con¬ versations with members of the house staff and obser¬ vations during ward visits led to the belief that while the "practically none" represented the honest opinion of the visiting surgeon, such an opinion was not based on accurate records. Indeed I am thoroughly con¬ vinced, that if I had been asked the same question at any time during the past ten years during which no effort was made to keep accurate records of infections occurring in my service, my off-hand estimate of the percentage of infection would fall far below the actual figures. In fact Tarn strongly of the opinion that the only way to obtain the best technical results is to keep an accurate record of infection in every patient sub¬ mitted to operation, for it is only by this means that one can be kept aware of his technical transgressions. It would be quite impossible for the modern student of technic to appreciate the. difficulties which were encountered on every side, in the five years of constant effort to improve the technical results in an institu¬ tion where the conditions were so unfavorable. STUDIES IN ASEPTIC TECHNIC WITH A REPORT OF SOME RECENT OBSERVATIONS AT THE ROOSEVELT HOSPITAL GEORGE EMERSON BREWER, M.D. NEW YORK Dur¬ ing this period I was a constant observer and student of the methods being employed in the Syms operating- room by the late Dr. Charles McBurney, who estab¬ lished a standard of surgical technic in the Roosevelt Hospital which was equal if not superior to that of any other American or European clinic. Many of the changes made in our procedures at the City Hospital were the direct result of helpful consultations with Dr. McBurney who was always ready and willing to discuss the various problems presented and to give friendly and sound advice. I have referred to these experiences in another hospital for the reason that I wish to call attention to the fact that in an institution where the patients as a rule were seriously handicapped by a low vital resistance due to malnutrition, dissipation, and dis¬ ease, a somewhat exaggerated technic was necessary to obtain results comparable with those in other hospi¬ tals. Another important factor was that of air infec¬ tion. At the City Hospital the air of the operating- rooms was constantly contaminated by floating germs, largely of the pus-producing varieties. This was fre¬ quently demonstrated by exposure of gelatin or agar plates in the operating-room during our surgical work. To overcome this source of infection, we found it nec¬ essary to cover all instrument trays, wash basins, Irri¬ gators, and pitchers with tents or covers of sterile mus¬ lin, and to employ irrigation of the wound during our operative procedures. It was only by these methods that our best results were obtained. When, in 1899, I received an appointment as junior surgeon to the Roosevelt Hospital, I was surprised and gratified to see that many of the unusual technical procedures used in the City Hospital to meet the local conditions of that institution that even better results could b what simpler but equally effect Dr. McBurney and my new ch While this was in part due to room equipment and to a more of assistants and nurses, the seemed to be the better phy patients. During the first six years of Hospital, while I acted as firs Weir, the number of clean infected was exceedingly sm record was kept. possible, and to make changes only when we felt that by such changes our results could be improved. While the earliest reports of our investigations were not kept in permanent form, we have a complete list of these reports since Jan. 1, 1912. by changes improved. In the following tables we shall give the results of our method in clean cases, in which infection could only be introduced during our operative procedures by some error in technic ; also we will state the num¬ ber of infected cases rendered sterile by our operative treatment. reports Jan. , The form in which these weekly reports was read was: First, the name of each patient with the clinical diagnosis; the anesthetic used; the operator; the con¬ dition of the wound area, clean, infected, or border¬ line ; the condition of asepsis at the first or subsequent dressings, "maintained" or "lost" in clean cases, "estab¬ lished" or "unestablished" in the infected. At the end of the report a summary was given of which the following is an example. I regret that we have not accurate records of the results in our borderline cases. While these often were read at our weekly conferences, they do not appear with any regularity in the records which have been preserved. This, while regrettable, does not in any way lessen the value of our report, for infections occurring in this group could never be attributed to technical errors, as the wound was always contami¬ nated at some time during the operation. By border¬ line cases we refer to those in which an otherwise clean operative wound was at some time in contact with an infected area or material, but in which it was possible generally to avoid gross infection by extra care. In this group we would include such cases as a gastro-enterostomy, an appendectomy in which it was impossible to cauterize the stump before inverting it, and a cholecystostomy with infected bile. Total number of operations for the week ending: July 11, 1912. 34 Wound area clean. 16 Wound area infected. 13 Borderline cases. S Asepsis maintained in. 16 Asepsis lost in. 0 Asepsis established (in infected cases) .... 4 Asepsis unestablished (in infected cases).. 9 Deaths during the week. 1 After the weekly report was read, there also was added a summary of all cases since January 1 or July 1 in a given year, for six months or the period of ser¬ vice of each unit of the house staff. Without going more into detail I will submit the total records for each six months since Jan. 1, 1912, together with a few brief comments which will give a fair idea of the infec¬ tion occurring in our service for the past two and a half years. SUMMARY OF RESULTS Total cases from Jan. 1 to July 1, 1912. .457 Clean .250 Infected .139 Borderline . 69 Asepsis maintained in.244 Asepsis lost in. 6 Percentage of infection in clean cases.. 2.4 years. During this period our technic may be summarized as follows : In discussing this report, our entire staff expressed surprise and disappointment. We all had the idea that our infections would not amount to more than 1 per cent, of our clean cases. During the next six months, we adopted the plan of watching each other operate, Dr. Darrach, Dr. Russell and myself each acting as censor at different times, and critically observing every step of the operation. As the result of these observa*- tions we found that the suture and reserve instrument tables were too near the operating table and that the towels covering these were not infrequently contami¬ nated by orderlies and unstërile nurses while bringing in the patient, handling hand-lights or the cautery apparatus. While these contaminations were not directly to the instruments or suture material, it was possible indirectly to destroy their sterility by first handling the contaminated towels and later the instru¬ ments, sponges, or other material. A number of other possible errors in technic were noted, and after consul¬ tation with Dr. Peck we rearranged the tables and fixtures in the operating-room, adopted new stoppers for our ligature flasks and water bottles, had the patient transferred to the operating table, prepared and draped in an adjoining room, and in a number of other ways altered our routine technic. These changes resulted in a decided improvement in our results, as will be seen in the following tabulation: Whenever possible a tub bath was given to the patient the night before operation, followed by shav¬ ing the wound area, and the application of a soap poul¬ tice. STUDIES IN ASEPTIC TECHNIC WITH A REPORT OF SOME RECENT OBSERVATIONS AT THE ROOSEVELT HOSPITAL GEORGE EMERSON BREWER, M.D. NEW YORK e d y I have referred to these experiences in another hospital for the reason that I wish to call attention to the fact that in an institution where the patients as a rule were seriously handicapped by a low vital resistance due to malnutrition, dissipation, and dis¬ ease, a somewhat exaggerated technic was necessary to obtain results comparable with those in other hospi¬ tals. Another important factor was that of air infec¬ tion. At the City Hospital the air of the operating- rooms was constantly contaminated by floating germs, largely of the pus-producing varieties. This was fre¬ quently demonstrated by exposure of gelatin or agar plates in the operating-room during our surgical work. To overcome this source of infection, we found it nec¬ essary to cover all instrument trays, wash basins, Irri¬ gators, and pitchers with tents or covers of sterile mus¬ lin, and to employ irrigation of the wound during our operative procedures. It was only by these methods that our best results were obtained. Wh i 1899 I i d i j i g g For some years, both before and after this investi¬ gation, our results seemed so satisfactory and the accidental infections so far apart that no special inquiry was made to ascertain the exact number or percentage. D i th l f 1911 h g g For some years, both before and after this investi¬ gation, our results seemed so satisfactory and the accidental infections so far apart that no special inquiry was made to ascertain the exact number or percentage. During the early part of 1911, however, one or two unexplained infections occurred which led me to inau¬ gurate on the first surgical division, a weekly report of every patient operated on, the condition of the wound area before operation, and the condition whether clean or infected at the first and subsequent dressings. This report was read at a staff conference, held every Friday morning after the ward visit; and any case of unexplained infection was referred to a committee to investigate, and to report at the next conference. When, in 1899, I received an appointment as junior surgeon to the Roosevelt Hospital, I was surprised and gratified to see that many of the unusual technical procedures used in the City Hospital to meet the local Downloaded From: http://jama.jamanetwork.com/ by a Simon Fraser University User on 06/02/2015 Downloaded From: http://jama.jamanetwork.com/ by a Simon Fraser University User on 06/02/2015 4 Asepsis established in.102 Percentage of infection in clean cases. 1.8 Total cases from Jan. 1 to July 1, 1913.474 Clean .213 Infected .148 Borderline .113 Asepsis maintained in.209 Asepsis lost in. 4 Asepsis established in.102 Percentage of infection in clean cases. 1.8 Montgomery, Ala., the late Dr. W. W. Miller of Wash¬ ington, and Dr. Armitage Whitman of this city, whose service terminated July 1, 1914. Montgomery, Ala., the late Dr. W. W. Miller of Wash¬ ington, and Dr. Armitage Whitman of this city, whose service terminated July 1, 1914. Montgomery, Ala., the late Dr. W. W. Miller of Wash¬ ington, and Dr. Armitage Whitman of this city, whose service terminated July 1, 1914. July 1, During the progress of these studies in technic after accurate records were kept, we determined to widen the field of our observations somewhat and to include in our weekly reports the record of all catheteriza- tions which occurred on our service. The reason for this will be patent to every hospital attending, for we are all familiar with the increased suffering and pro¬ longed convalescence which so often follows a cys¬ titis resulting from perhaps a single postoperative catheterization. A weekly record therefore was kept of each catheterization occurring on the service, with the name of the house officer or nurse who performed it, together with a report of the urinary analysis both before and after. While this plan was only recently adopted and the number of observations is too small to be of much value and is therefore not included in our lists, it is gratifying to report that in no instance since records have been kept has catheterization resulted in infection. As the result of a staff conference at this time, we decided to adopt the long-sleeved muslin operating- gown, changed between each operation. While I have always been opposed to this procedure for the reason that accidental contamination on the skin of the bare arm was more easily felt than on even a light muslin covering, I yielded to the wish of the majority of the staff, and the results since that change' seem to indi¬ cate its merit: Total cases from July 1, 1913, to Jan. 1, 1914. .439 Clean .243 Infected .125 Borderline. 71 Asepsis maintained in.239 Asepsis lost in. 4 Asepsis established in. 70 Percentage of infection in clean cases. 1.6 During a part of the time covered by these observa¬ tions, a record of all cystoscopies were included in our weekly conferences. These were not introduced into this report for the reason that the records are imper¬ fect during a part of the time, possibly because the results were invariably the same. It is, however, only fair to Dr. Edward F. Kilbane, our cystoscopist, to state that during a period of eight years, he has made over 1,050 cystoscopies in the Syms Operating Build¬ ing on both private and ward patients, without infec¬ tion of a sterile urinary tract occurring in a single instance, and without infection being transferred to a sterile ureter or kidney from an infected ureter or bladder. In our investigation of the infections occurring in clean wounds prior to July 1, 1913, no facts were elicited which would enable us to ascribe them to fac¬ tors other than technical errors occurring during oper¬ ation. Just what these errors were was not always determined, but there was no evidence to indicate that the infection was due to conditions beyond our control. beyond In investigating the four infections occurring dur¬ ing the period from July 1 to Jan. 1, 1914, it was found that three of the four cases occurred quite close together and all had been operated on under local anesthesia. Bactériologie examination proved that the novocain solutions were contaminated, as a result of faulty preparation. During this period the solutions for local infiltration anesthesia were prepared in the drug department of the hospital, and they were deliv¬ ered to the operating-room nurse ready for use. It will thus be seen that these three infections could not with reason be ascribed to errors in operating-room technic, as none of the staff or operating-room nurses had anything to do with the preparation of the infected solutions. . Although not strictly germane to the subject, it may also be of interest to state that during the fourteen years which have elapsed since my appointment to the attending staff of the Roosevelt Hospital, not a single infection has occurred in a rather large series of arthrotomies of the knee joint, undertaken for the repair of a fractured patella, ruptured quadriceps or its tendon, or for the removal of joint mice, injured or dislocated semilunar cartilages. Downloaded From: http://jama.jamanetwork.com/ by a Simon Fraser University User on 06/02/2015 After from four to eight hours, this was removed, the part scrubbed with soap and hot water for five minutes, and a wet mercuric chlorid dressing applied. On the operating-table this dressing was removed by a sterile assistant, the parts rescrubbed for one minute, douched with ether, alcohol, and a 1: 5,000 solution of mercuric chlorid. The wound area was surrounded by sterile towels, and the entire body cov¬ ered by sterile sheets. Sterile caps, gowns, and gloves were worn by all participating in the operation. The towels around the wound area were changed as often as soiled, and always before the insertion of the cutaneous sutures. The method of sterilizing our material will not be described, as it did not differ in any material way from that employed in any modern surgical clinic. Suffice it to say that frequent bactériologie examinations dem¬ onstrated that our material was sterile. We did not at that time use the long-sleeved gowns, completely covering our arms, and did not wear face masks in ordinary cases. The reason for the latter omission was, not that we disapproved of the plan, but by a system of hand signals, we were able to avoid talking during most routine operations. during operations. Our technic was changed slightly in two classes of cases, operative treatment of fractures, and open oper¬ ations on the knee joint. In these cases face masks or helmets were worn, and nothing entered the wound which had been touched even with the gloved hand, all manipulations being carried out by instruments. b th t Total cases from July 1, 1912, to Jan. 1, 1913. .445 Clean .234 Infected .131 Borderline . 80 Asepsis maintained in.231 Asepsis lost in. 3 Asepsis established in. 60 Percentage of infection in clean cases. 1.2 a pu at o s g y It will thus be seen that our technic was by no means an elaborate one, our aim being to keep it as simple as Downloaded From: http://jama.jamanetwork.com/ by a Simon Fraser University User on 06/02/2015 Total cases from Jan. 1 to July 1, 1913.474 Clean .213 Infected .148 Borderline .113 Asepsis maintained in.209 Asepsis lost in. 4 Asepsis established in.102 Percentage of infection in clean cases. 1.8 Total cases from Jan. 1 to July 1, 1913.474 Clean .213 Infected .148 Borderline .113 Asepsis maintained in.209 Asepsis lost in. This statement includes all cases, in both surgical divisions, in which an incision has been made into the sterile knee joint by any member of the staff. If therefore we exclude these three infections, the number of infections in clean cases during that par¬ ticular six months would be reduced to 1, or 0.4 per cent. yTo recapitulate briefly the facts bearing on the main object of this communication, namely, to give the per¬ centage of infections occurring in our clean cases dur¬ ing each of the five periods of six months since Jan. 1, 1912, it will be seen that for the first six months in 1912, the percentage of infection occurring in clean operative wounds was 2.4 per cent., for the second six months 1.2 per cent. ; for the first six months of 1913, 1.8 per cent., for the last half of 1913 1.6 per cent., if we include the three cases in which infection occurred as a result of using an unsterile solution of novocain for local anesthesia, for which none of the operating-room staff was in any way responsible, or 0.4 per cent, if these are excluded. For the six months ending July 1, 1914, no infection occurred in the 273 clean cases. f i Total cases from Jan. 1 to July 1, 1914..SS9 Clean .273 Infected .220 Borderline . 66 Asepsis maintained in.273 Asepsis lost in. 0 Asepsis established in. 64 During the last two months of this period the ser¬ vice was in charge of Dr. Charles N. Dowd, who suc¬ ceeded me as senior surgeon to the first division on May 1, but as the routine technic remained practically unchanged, the entire period of six months is included in this report. i i If, as seems fair, we exclude the three cases referred to above, during the year from July 1, 1913, to July 1, 1914, only one infection for which the operating staff could be held responsible occurred in 516 cases, or a little less than 0.2 per cent. report. Many years ago I established the custom of giving a prize to the house surgeon on whose service of six months, no clean case became infected. This prize has been won by three men, Dr. John H. Blue, now of Downloaded From: http://jama.jamanetwork.com/ by a Simon Fraser University User on 06/02/2015
https://openalex.org/W2946859074	https://europepmc.org/articles/pmc6498670?pdf=render	English	null	Type D personality is a predictor of prolonged acute brain dysfunction (delirium/coma) after cardiovascular surgery	BMC psychology	2,019	cc-by	7,741	Abstract 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. * Correspondence: yinoue@md.tsukuba.ac.jp 1Department of Emergency and Critical Care Medicine, Faculty of Medicine, University of Tsukuba, Tsukuba, Ibaraki, Japan Full list of author information is available at the end of the article Type D personality is a predictor of prolonged acute brain dysfunction (delirium/coma) after cardiovascular surgery Yujiro Matsuishi1, Nobutake Shimojo1, Takeshi Unoki2, Hideaki Sakuramoto3, Chiho Tokunaga4, Yasuyo Yoshino5, Haruhiko Hoshino1, Akira Ouchi1, Satoru Kawano1, Hiroaki Sakamoto4, Yuji Hiramatsu4 and Yoshiaki Inoue1* Matsuishi et al. BMC Psychology (2019) 7:27 https://doi.org/10.1186/s40359-019-0303-2 Matsuishi et al. BMC Psychology (2019) 7:27 https://doi.org/10.1186/s40359-019-0303-2 * Correspondence: yinoue@md.tsukuba.ac.jp Correspondence: yinoue@md.tsukuba.ac.jp 1Department of Emergency and Critical Care Medicine, Faculty of Medicine, University of Tsukuba, Tsukuba, Ibaraki, Japan Full list of author information is available at the end of the article p y jp 1Department of Emergency and Critical Care Medicine, Faculty of Medicine, University of Tsukuba, Tsukuba, Ibaraki, Japan Full list of author information is available at the end of the article p y jp 1Department of Emergency and Critical Care Medicine, Faculty of Medicine, University of Tsukuba, Tsukuba, Ibaraki, Japan Full list of author information is available at the end of the article Abstract Background: Previous studies have shown a relationship between delirium and depressive symptoms after cardiac surgery with distress personalities linking to negative surgical outcomes. The aim of the present study is to further investigate the association between patients with Type D (distressed) personality with regards to delirium after cardiac surgery. Methods: We conducted a consecutive-sample observational cohort pilot study with an estimated 142 patients needed. Enrollment criteria included patients aged ≥18 years who were undergoing planned cardiovascular, thoracic and abdominal artery surgery between October 2015 to August 2016 at the University of Tsukuba Hospital, Japan. All patients were screened by Type-D Personality Scale-14 (DS14) as well as the Hospital Anxiety and Depression Scale (HADS) the day before surgery. Following surgery, daily data was collected during recovery and included severity of organ dysfunction, sedative/analgesic exposure and other relevant information. We then evaluated the association between Type D personality and delirium/coma days (DCDs) during the 7-day study period. We applied regression and mediation modeling for this study. Results: A total of 142 patients were enrolled in the present study and the total prevalence of delirium was found to be 34% and 26% of the patients were Type D. Non-Type D personality patients experienced an average of 1.3 DCDs during the week after surgery while Type D patients experienced 2.1 days over the week after surgery. Multivariate analysis showed that Type D personality was significantly associated with increased DCDs (OR:2.8, 95%CI:1.3–6.1) after adjustment for depressive symptoms and clinical variables. Additionally, there was a significant Type D x depression interaction effect (OR:1.7, 95% CI:1.2–2.2), and depressive symptoms were associated with DCDs in Type D patients, but not in non-Type D patients. Mediation modeling showed that depressive symptoms partially mediated the association of Type D personality with DCDs (Aroian test =0.04). Conclusions: Type D personality is a prognostic predictor for prolonged acute brain dysfunction (delirium/coma) in cardiovascular patients independent from depressive symptoms and Type D personality-associated depressive symptoms increase the magnitude of acute brain dysfunction. Keywords: Delirium, Delirium/coma days, Type D personality, Depression, Thoracic surgery, Intensive care units, Critical care © The Author(s). © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. * Correspondence: yinoue@md.tsukuba.ac.jp 1Department of Emergency and Critical Care Medicine, Faculty of Medicine, University of Tsukuba, Tsukuba, Ibaraki, Japan Full list of author information is available at the end of the article Background and delirium [26]. There is still a lack of associative evidence for Type D personality, delirium and the mediating effects of depressive symptoms for this re- lationship. Some points of improvement were noted in this previous study allowing for closer examination into important factors such as the severity and dur- ation of delirium/coma take account for better patient outcomes. Delirium is a common post-surgical neuropsycho- logical complication among cardiac patients and onset occurs rapidly due to the development of physio- logical abnormalities characterized by fluctuating course, attention deficits, disorganized thinking, and an altered level of consciousness [1].The prevalence of delirium within this post-surgical, cardiac patient population is reported to be between 26 to 52% [2]. This figure is in line with previous studies which re- port that preoperative cognitive impairment and de- pression in cardiac surgical patients are associated with greater risk of developing delirium [3, 4]. In addition, risk of delirium increases cumulatively with intraoperative and postoperative factors, such as lon- ger cardiopulmonary bypass times [5] and/or use of benzodiazepine [6]. Importantly, delirium was inde- pendently associated with negative outcomes, such as higher mortality [7], decline in cognitive ability [8], increased length of stay and hospital readmissions [8]. However, outside of the prevalence, duration of delir- ium dose affect mortality [9]. Additionally, reports have measured delirium associated with terminal con- ditions [10] and from this insight, the concept of measuring both delirium and coma days was born [11–13]. The main concept is that psychiatric disor- ders can often manifest alongside physical ailments and even if the physical condition causes the initial psychiatric insult, ongoing depressive symptoms can enact a positive feedback loop to worsen the physical condition. To this end, previous studies reported that depressive symptoms are associated with delirium in cardiac patients [14]. However, a recent study re- ported that heart disease outcomes are not based on psychiatric condition alone but also patient personal- ities [15–19]. The distress personality (also known as Type D) is based on personality type and is defined by complex and highly negative emotions plus social inhibition [20] This total personality is associated with increase depressive symptoms [21]. Surprisingly, about 30% of cardiac surgery patients that carry this personality [22] suffer adverse consequences [23] and previous research showed a significant association be- tween Type D personality and hard endpoint-adjusted hazard ratios (HR:2.24, 95% CI [1.37–3.66]) in meta-analysis of 12 studies on 5341 patients [24]. Background Despite this initial evidence linking Type D personal- ity with hazard ratios, a full explanation of the correl- ation between personality and postoperative delirium which lead to high mortality is still lacking. While previous research has reported that personality traits of neuroticism and conscientiousness are associated with delirium in hip fracture patients [25] another re- port found no association between Type D personality We hypothesize that a Type D personality affects post- operative delirium/coma days and by using regression and mediation modeling, the present study was able to revisit the association between Type D character and the development of postoperative delirium/coma days after cardiac surgery. Matsuishi et al. BMC Psychology (2019) 7:27 Matsuishi et al. BMC Psychology (2019) 7:27 Page 2 of 10 Page 2 of 10 Material and methods Patient selection A list of enrolled and approved patients was obtained by operation room staff a week before surgery and enroll- ment criteria included patients aged ≥18 years that were undergoing scheduled cardiovascular, thoracic and ab- dominal artery operations between October 2015 and August 2016. Patients were excluded if they had stroke, were deaf or otherwise unable to speak, or had current or previous major depression. This information was ob- tained from medical records. The Institutional Review Board (IRB) of the University of Tsukuba Affiliated Hos- pital approved the present study (H27–085) and written informed consent was obtained from patients prior to surgery. Data collection prior to surgery Data collection prior to surgery We recorded baseline preoperative factors, including age, sex, baseline medical history, and cardiac func- tion, and calculated the European System score for Cardiac Operative Risk EvaluationII (EuroSCOREII) from these data [27] . EuroSCOREII is a cardiac risk score for predicting mortality after cardiac surgery that takes into account patient-related factors, cardiac-related factors, previous cardiac surgery, and operation-related factors. The validation of the Euro- SCOREII with Japanese patients has been previously reported [27]. All patients underwent the following evaluations the day before the surgery: (a) the Type-D personality Scale-14 (DS14) [28]; (b) the Hospital Anxiety and Depression Scale (HADS) [29] and (c) the Mini-Mental State Examination (MMSE) [30]. The DS14 was specifically developed to assess Negative Affectivity (NA) and Social Inhibition (SI). This scale contains fourteen items and these subscales consist of seven items, and each item is rated from false (0) to true (4) on a 5-point Likert scale. Scores equal to or above 10 on both NA and SI were used to determine a Type D personality. HADS is a self-administered Matsuishi et al. BMC Psychology (2019) 7:27 Matsuishi et al. BMC Psychology (2019) 7:27 Matsuishi et al. BMC Psychology (2019) 7:27 Page 3 of 10 Page 3 of 10 The sample size was calculated with the software G * Power 3.1. Using Wilcoxon-Mann-Whitney testing, and effect size was d = 0.49 based on the pilot study. We deter- mined that a sample size of 142 patients would be re- quired for a significance level (α) of 0.05 and test power (1-β) of 0.80. The sample size was calculated with the software G * Power 3.1. Using Wilcoxon-Mann-Whitney testing, and effect size was d = 0.49 based on the pilot study. We deter- mined that a sample size of 142 patients would be re- quired for a significance level (α) of 0.05 and test power (1-β) of 0.80. scale for the evaluation of anxiety and depression in non-psychiatric patients. Each item is rated on a 4-point Likert scale and increases measure degree of severity. In the present study, only the depressive HADS scale was used. The MMSE was used to assess presence and severity of cognitive impairment. The validation of the Japanese versions of DS14, HADS and MMSE has been previously reported [31–34]. Delirium assessment Delirium and coma were assessed using the Richmond Agitation - Sedation Scale (RASS) [36] and Confusion Assessment Method for the ICU (CAM-ICU) [37] twice daily for the 7-day study period. The assessments were all performed by IRB-approved researchers. Patients with RASS −4 and −5 were determined to be comatose and if delirium/coma was observed even once for a given day, it was noted that delirium/coma was prevalent for that particular day. Delirium/coma days (DCDs) As Type D personality and depressive symptoms are generally considered co-morbid, and previous studies reported that having these two factors suspected to inflate bad outcomes for cardiac patients [40, 41]. Therefore, we attempted to construct an interaction model. Interaction modeling can analyze the relation- ship of the inflation between two factors (covariates) for outcome of the interest. Although the basic as- sumption of regression modeling is the independence of each factor, we suspected a significant interaction and therefore used a two-step process where we first constructed an isolated main effect model (model 1) then iteratively included interaction modeling (model 2). In model 2, the odds ratio of the main effect (Type D personality and depressive symptoms) was not significant, possibly due to the ability to capture only a segment of the main effect. DCDs are defined as days acute brain dysfunction (delir- ium and coma) within the study period. Delirium obser- vation, however, took into account the comatose days to avoid lead time bias. Care was taken when recording both delirium and coma to avoid focusing on one of the DCDs conditions at the exclusion of the other (as seen in previous reports) which could have skewed or biased the data [11, 12]. Intra- and post-operative data collection Intraoperative data, including aortic clamping time, was recorded. Post-operative daily data, including severity of organ dysfunction calculated by modified Sequential Organ Failure Assessment (mSOFA) and Benzodiazepine, Propofol, Dexmedetomidine dosage, were collected during ICU and general ward stays. Modified Sequential Organ Failure Assessment (mSOFA) is an assessment score calculated with SpO2/ FiO2, liver function, cardiovascular, hypotension, central nervous system function, and renal creatinine levels. This system has been validated as a good predictor of post-operative mortality [35]. Data collection prior to surgery DS14 and HADS were provided by paper and scoring was done after the experimental period, blinding the researchers to patient Type D status during testing. Regression modeling The outcomes of interest were DCDs within the 7-day study period. DCDs are defined as days with acute brain dysfunction (delirium and coma) within the study period. Because previous studies have noted a heavily skewed distribution of DCDs, we instead de- cided to use Proportional Odds Logistic Regression (POLR), which does not require the normal distribu- tion, in examining the relationship between Type D personality and DCDs. Furthermore, we also adjusted for the following additional covariates chosen a priori in our model: EuroSCOREII, mSOFA without a cen- tral nervous system component, use of sedative medi- cine, and MMSE. EuroSCOREII for adjusting patient baseline characteristics including sex, age, history of complications, and intraoperative factors including ur- gency and intervention procedures. We used mSOFA for adjusting for daily severity of the patient. As cen- tral nervous system (CNS) components would be cor- related with the outcome of interest we excluded this component to protect the integrity of our analysis. Additionally, The variance inflation factor (VIF) were observed to assess multicollinearity among the vari- ables. As previous studies reported [38, 39], we tested continuous values of SI and NA (which are compo- nents of Type D personality) independently as a sub-analysis. Sample size calculations Before this study, we conducted a month-long pilot study where a total of 22 patients, were enrolled and we observed a mean of 0.7 (SD ± 1.4) delirium/coma days (DCDs) in the Type D personality group and a mean of 0.2 (SD ± 0.3) DCDs in the control group. Matsuishi et al. BMC Psychology (2019) 7:27 Page 4 of 10 Matsuishi et al. BMC Psychology (2019) 7:27 Moderator model Model 2 for DCDs included interaction between Type D personality and depressive symptoms, and this interaction was found to be significant (Type D personality×depressive symptoms: OR = 1.7, 95% CI = 1.2–2.2). (Table 2). This interaction effect indicates that Type D per- sonality moderated the association of depressive symptoms with DCDs; i.e., depressive symptoms had a deleterious effect in terms of prolonged brain dys- function among Type D patients, but depressive symptoms were not associated with DCDs in non-Type D patients (Fig. 3). Fig. 1 Participant flow chart. This figure shows participant flow chart including exclusion criteria, and final enrollment patients for the investigation Patient characteristics From October 2015 to August 2016, we enrolled a total of 142 patients (see Fig. 1 illustrating participant flow). Of the 174 patients, the following two groups were ex- cluded from the study: A) 16 patients: 2 deaf or unable to speak, 2 could not speak Japanese and 12 had stroke B) 16 patients that freely exercised their legal right to re- fuse participation. Table 1 presents baseline patient study characteristics. y 45% of the patient takes valve surgery and the me- dian age at enrollment was 67 (± 14) and 63% of the patients were male. The average EuroSCOREII was 2.0 (± 2.0) and the average of 7-days modified Se- quential Organ Failure Assessment was 3.5 (± 2.1). Non-Type D personality patients experienced coma days average of 0.8 ± 1.1 during the week after surgery while Type D patients experienced 0.9 ± 1.0, and Non-Type D personality patients experienced a delir- ium average of 0.4 ± 0.8 during the week after surgery while Type D patients experienced 1.1 ± 1.5, thus Non-Type D personality patients experienced 1.3 ± 1.6 DCDs during the week after surgery while Type D patients experienced 2.1 ± 1.9 DCDs over the week after surgery (Fig. 2). All patients survived during the study period. Out of the 49 patients (34%) with Regression modeling Regression modeling VIF was less than 3. Therefore, multicollinearity was con- sidered not to be problematic. Type D personality factors [odds ratio (OR) = 2.8, 95% confidence interval (CI) = 1.3–6.1], HADS-Depression (OR = 1.1, 95% CI = 1.0– 1.3), mSOFA (OR = 1.7, 95% CI = 1.3–2.2), Benzodi- azepine (OR = 9.8, 95% CI = 2.4–40.3) and Propofol (OR = 1.1, 95% CI = 1.0–1.2) were associated with sig- nificantly increased DCDs (Table 2). This indicates that these factors were independently associated with pro- longed acute brain dysfunction in the 7-day post-operative period. We also tested continuous values of SI and NA (which are components of Type D personality) independently as a sub-analysis NA (OR = 1.09, 95% CI = 1.03–1.15) and SI (OR = 1.05, 95% CI = 1.0–1.1) themselves were also associated with significantly decreased DCDs (Table 3) and NA and SI interaction was not significant. (OR = 0.9, 95% CI = 0.9–1.0) (Table 4). Mediation modeling delirium in total population and 32 patients (30%) in Non-Type D personality 17 patients (45%) in Type D personality patients experienced delirium, 37 patients (26%) were found to have a Type D personality. To determine the mediating effect of depressive symp- toms on the relationship between Type D personality and DCDs, mediation analyses were conducted using the Baron and Kenny approach [42] (bootstrapping method and Aroian testing) [43] and adjusted for the same co- variate factors in regression modeling. All statistical ana- lyses were performed using SPSS version 25 (SPSS, Inc., Chicago, IL). First, Type D personality (X) significantly predicts DCDs (Y) (β = 0.93; p < 0.01). Second, Type D personality (X) significantly predicts depressive symptoms (M) (β = 1.35; p < 0.01). Third, in regression analysis, both Type D personality (X) and depressive symptoms (M) are predictors for DCDs (Y) (β = 0.78; p < 0.01), (β = 0.109; p = 0.02). First, Type D personality (X) significantly predicts DCDs (Y) (β = 0.93; p < 0.01). Second, Type D personality (X) significantly predicts depressive symptoms (M) (β = 1.35; p < 0.01). Mediation model The mediation analyses involved Type D personality (X; independent variable), depressive symptoms (M; medi- ator), and DCDs (Y; dependent variable) and were ad- justed for the same covariate factors in regression modeling (Fig. 4). The analysis was performed according to Baron and Kenny’s method [42] as follows: First, Type D personality (X) significantly predicts DCDs (Y) (β = 0.93; p < 0.01). Second, Type D personality (X) significantly predicts depressive symptoms (M) (β = 1.35; p < 0.01). Fig. 1 Participant flow chart. This figure shows participant flow chart including exclusion criteria, and final enrollment patients for the investigation Third, in regression analysis, both Type D personality (X) and depressive symptoms (M) are predictors for DCDs (Y) (β = 0.78; p < 0.01), (β = 0.109; p = 0.02). Page 5 of 10 Matsuishi et al. Mediation model BMC Psychology (2019) 7:27 Table 1 Baseline characteristics of study patients variable Total population N = 142 Type D personality N = 37 Non-Type D personality N = 105 Age ± SD 67 ± 14 64 ± 13 67 ± 14 Male n (%) 90 (63) 24(64) 66(62) Surgical procedure n (%) CABG 26 (18) 7 (18) 19 (18) CABG+Valve surgery 10 (7) 2 (5) 8 (7) Valve surgery 65 (45) 14 (37) 51 (48) Thoracic blood vessel replacement 7 (4) 3 (8) 4 (3) Thoracic blood vessel replacement+VALVE surgery 6 (4) 1 (2) 5 (4) Abdominal blood vessel replacement 5 (3) 1 (2) 4 (3) Endovascular aortic repair 16 (11) 5 (13) 11 (10) ASD/VSD closer 2 (1) 1 (2) 1 (1) Heart tumor resection 3 (2) 2 (5) 1 (1) Ventricular aneurysm resection 2 (1) 1 (2) 1 (1) MMSE ± SD 28 ± 1.52 28 ± 1.50 28 ± 1.53 Depressive symptom a ± SD 1.9 ± 2.7 3.0 ± 2.9 1.6 ± 2.5 DS 14 Negative Affectivity (NA) 6.6 ± 6.0 14.8 ± 4.2 3.7 ± 3.2 Social Inhibition (SI) 8.7 ± 6.6 16.0 ± 4.7 6.2 ± 5.1 EuroSCOREII ± SD 2.0 ± 2.0 1.7 ± 1.5 2.1 ± 2.1 Aortic clamping times, min (IQR) 135 (0, 206) 136 (34, 214) 135 (0, 208) mSOFA b ± SD 3.5 ± 2.1 3.4 ± 1.9 3.5 ± 2.2 Benzodiazepine (mg/kg/day) b ± SD 0.06 ± 0.5 0.06 ± 0.41 0.06 ± 0.53 Propofol (mg/kg/day) b ± SD 2.8 ± 6.6 3.0 ± 7.3 2.3 ± 4.1 Dexmedetomidine (μg/kg/day) b ± SD 0.8 ± 3.0 0.6 ± 1.2 0.9 ± 3.4 Prevalence of delirium n (%) 49 (34) 17 (45) 32 (30) Delirium/coma days ± SD 1.5 ± 1.7 2.1 ± 1.9 1.3 ± 1.6 Coma days ± SD 0.9 ± 1.1 0.9 ± 1.0 0.8 ± 1.1 Delirium days ± SD 0.6 ± 1.0 1.1 ± 1.5 0.4 ± 0.8 a: measured by Hospital Anxiety and Depression Scale (HADS) b: used average of 7 days IQR interquartile range, SD standard deviation, MMSE mini-mental state examination, EuroSCOREII European System for Cardiac Operative Risk Evaluation II, mSOFA modified Sequential Organ Failure Assessment ndard deviation, MMSE mini-mental state examination, EuroSCOREII European System for Cardiac Operative Risk Evaluation II, mSOFA lure Assessment The subsequent Aroian test, which tests the statistically significant difference in results between univariate and regression analyses with respect to Type D personality (X) for DCFDs (Y), was significantly different (p = 0.04). Mediation model dysfunction (measured as delirium/coma days) during 7 days after operation, after adjusting for severity and vari- ous predicting factors. Although a previous study had shown that the prevalence of Type D personality is rela- tively high (46%) in Japan among healthy subjects [44], the present study is the first to show that the Japanese preva- lence rates are comparable to European cardiac surgery patients [22]. One possible reason for the difference be- tween the current findings and the previous Japanese study could be that the earlier study was conducted in the rural areas of Japan, which have a higher population of the elderly, thus inflating the prevalence of Type D personality. Based on the above analysis, our present findings show that Type D personality is an independent predictor of DCDs and that depressive symptoms had a partial medi- ating effect on the relationship between Type D person- ality and DCDs after adjustment. Discussion Several previous studies showed that Type D per- sonality was associated with depressive symptoms [21, The present study is the first to demonstrate that Type D personality patients experience longer acute brain Matsuishi et al. BMC Psychology (2019) 7:27 Page 6 of 10 Matsuishi et al. BMC Psychology Fig. 2 Distribution of normal, delirium, and coma days, stratified by Type D personality. This is the distribution of normal, coma, delirium days for normal and Type D personality Fig. 2 Distribution of normal, delirium, and coma days, stratified by Type D personality. This is the distribution of normal, coma, delirium days for normal and Type D personality have a partial mediating effect between Type D per- sonality and acute brain dysfunction during the 7-day period after surgery. 45] and these were in turn were associated with delir- ium [3, 46]. Our present results are in line with these earlier results but we differed in our methods by employing regression (including interaction) models and mediation modeling to analyze statistical signifi- cance within our findings that depressive symptoms Based on these analyses, we found a theoretical re- lationship between distressed personality and depres- sive symptoms [47]. Depressive symptoms can be said Table 2 Regression model for prolonged delirium/coma days Multivariate model 1 OR (95% CI) a VIF Multivariate model 2 OR (95% CI) a VIF EuroSCOREII 1.1 (0.9–1.3) 1.3 1.1 (0.9–1.3) 1.3 MMSE 0.9 (0.7–1.1) 1.1 0.9 (0.7–1.1) 1.1 Type D personality (Present or not) 2.8 (1.3–6.1)* 1.0 2.4 (5.4–1.0)* 1.1 Depressive symptoms b 1.1 (1.0–1.3)* 1.1 0.9 (0.8–1.1) 1.7 Aortic clamping time 0.9 (0.9–1.0) 1.4 0.9 (0.9–1.0) 1.4 mSOFA c 1.7 (1.3–2.2)* 1.7 1.7 (1.3–2.7)* 1.7 Benzodiazepine d 9.8 (2.4–40.3)* 1.1 16.1 (3.7–69.8)* 1.1 Propofol d 1.1(1.0–1.2) * 1.9 1.1(1.1–1.3) * 1.9 Dexmedetomidine e 1.1(0.9–1.2) 1.7 1.1(0.9–1.2) 1.7 Type D personality × Depressive symptom f 1.7 (1.2–2.2)* 1.6 a: P values obtained from Ordered Logistic Regression P value<0.05 b: measured by Hospital Anxiety and Depression Scale (HADS) c: Exclude GCS, used average of 7 days d: Used average of 7 days. mg/day/kg e: Used average of 7 days. μg/day/kg f: Centering was performed MMSE mini-mental state examination, EuroSCOREII European System for Cardiac Operative Risk Evaluation II, mSOFA modified Sequential Organ Failure Assessment Table 2 Regression model for prolonged delirium/coma days Matsuishi et al. Discussion BMC Psychology (2019) 7:27 Page 7 of 10 Table 3 Sub-analysis of each tendency of regression model for prolonged delirium/coma days Multivariate model 3 OR (95% CI) a VIF Multivariate model 4 OR (95% CI)a VIF EuroSCOREII 1.1 (0.9–1.3) 1.3 1.1 (0.9–1.3) 1.2 MMSE 0.9 (0.7–1.1) 1.0 0.9 (0.7–1.3) 1.1 Negative Affectivity (NA) 1.09 (1.03–1.15) 1.0 Social Inhibition (SI) 1.05 (1.0–1.1) * 1.0 Depressive symptomsb 1.1 (1.0–1.3)* 1.1 1.1 (1.0–1.3) * 1.1 Aortic clamping time 0.9 (0.9–1.0) 1.0 0.9 (0.9–1.0) 1.0 mSOFAc 1.7 (1.3–2.2)* 1.6 1.6 (1.2–2.1)* 1.6 Benzodiazepined 9.9 (2.4–40.2)* 1.0 9.8 (2.3–40.9)* 1.0 Propofold 1.1(1.0–1.2)* 1.8 1.1(1.0–1.2) * 1.8 Dexmedetomidinee 1.1(0.9–1.2) 1.7 1.1(0.9–1.3) 1.7 a: P values obtained from Ordered Logistic Regression P value<0.05 b: measured by Hospital Anxiety and Depression Scale (HADS) c: Exclude GCS, used average of 7 days d: Used average of 7 days. mg/day/kg e: Used average of 7 days. μg/day/kg MMSE mini-mental state examination, EuroSCOREII European System for Cardiac Operative Risk Evaluation II, mSOFA modified Sequential Organ Failure Assessment c: Exclude GCS, used average of 7 days d: Used average of 7 days. mg/day/kg e: Used average of 7 days. μg/day/kg MMSE mini-mental state examination, EuroSCOREII European System for Cardiac Operative Risk Evaluation II, mSOFA modified Sequential Organ Failure Assessment g y g y g e: Used average of 7 days. μg/day/kg MMSE mini-mental state examination, EuroSCOREII European System for Cardiac Operative Risk Evaluation II, mSOFA modified Sequential Organ Failure Assessment solid predictive factor for delirium [48]; however, there is no knowledge of the association between Type D personality and depressive symptoms for pro- longed acute brain dysfunction. We assume that Type D personality patients might underreport their symp- toms even if they are in such an at-risk population for depression. Therefore, this propensity to underre- port depressive symptoms underscores the need for solid evaluative tools to screen out Type D personal- ities from patient pools for more intensive monitoring to assist in their recoveries. We suggest further re- searches should focus on this interaction and medi- ation when studies for acute brain dysfunction include Type D personality or depressive symptoms as a factor. We also observed a NA and SI-independent effect for DCDs. From this result, we assumed that each component of the Type D person- ality worsens acute brain dysfunction after cardiovas- cular surgery. Limitation large, population-based study [45]. However, not only is Type D personality associated with inflammation, it is also linked to endothelial dysfunction. Interestingly, a previous study has reported that Type D personality is associated with decreased endothelial progenitor cells in patients with heart failure [53] and a recent study in patients with coronary artery disease showed that the association of Type D personality with endothelial dysfunction was ro- bust across time [54]. It was already shown that inflamma- tion biomarkers and these receptors associated with onset of delirium [55] and endothelial dysfunction associated with acute brain dysfunction during critical illness [56]. Further research is needed to explore whether the under- lying mechanism of the observed relationship between Type D personality and delirium could be neural inflam- mation and/or endothelial factors. large, population-based study [45]. However, not only is Type D personality associated with inflammation, it is also linked to endothelial dysfunction. Interestingly, a previous study has reported that Type D personality is associated with decreased endothelial progenitor cells in patients with heart failure [53] and a recent study in patients with coronary artery disease showed that the association of Type D personality with endothelial dysfunction was ro- bust across time [54]. It was already shown that inflamma- tion biomarkers and these receptors associated with onset of delirium [55] and endothelial dysfunction associated with acute brain dysfunction during critical illness [56]. Further research is needed to explore whether the under- lying mechanism of the observed relationship between Type D personality and delirium could be neural inflam- mation and/or endothelial factors. There are several limitations in the present study. First, since this study is a cross-sectional design, the direction of the mediation between Type D personality and de- pressive symptoms cannot be confirmed. Second, the Type D personality scale (DS14) and depressive symp- tom scale (HADS) might have some overlapping ques- tions. Additionally, the stress and dysphoria that naturally results from impending surgery might have skewed testing that was done the day before surgery. However, a previous study showed that Type D person- ality and depression are distinct manifestations of psy- chological distress [57]. Hence, we think that our current finding that shows a cross between independent variable and mediating effect might be valid. Third, des- pite the good response rate (90%), the non-consenting Fig. 4 Mediation model for delirium/coma days. Discussion Previous research showed that SI modulates the effect of NA on cardiac prognosis fol- lowing percutaneous coronary intervention [49]. Fur- ther research with a proper sample size is needed to check for any modulating effect for acute brain dysfunction. to have an additive deleterious effect on DCDs when combined with Type D personality. Thus, we should be aware that patients with Type D personalities may experience delirium and brain dysfunction after car- diac surgery and should be monitored carefully for depressive symptoms. Depressive symptoms are a Table 4 Sub-analysis of each tendency’s regression modeling interaction for prolonged delirium/coma days Multivariate model 5 OR (95% CI)a VIF EuroSCOREII 1.1 (0.9–1.3) 1.3 MMSE 0.9 (0.9–1.1) 1.1 Negative Affectivity (NA)b 1.0 (1.0–1.1) 2.5 Social Inhibition (SI)b 1.0(0.9–1.0) 1.8 Negative Affectivity (NA) × Social Inhibition (SI) 0.9(0.9–1.0) 1.7 Depressive symptomsc 1.1 (1.0–1.3)* 1.1 Aortic clamping time 0.9 (0.9–1.0) 1.0 mSOFAd 1.7 (1.3–2.2)* 1.6 Benzodiazepinee 11 (2.6–46.2)* 1.0 Propofole 1.1(1.0–1.2)* 1.8 Dexmedetomidinef 1.1(0.9–1.3) 1.7 a: P values obtained from Ordered Logistic Regression *P value<0.05 b: Centering was performed c: measured by Hospital Anxiety and Depression Scale (HADS) d: Exclude GCS, used average of 7 days e: Used average of 7 days. mg/day/kg f: Used average of 7 days. μg/day/kg MMSE mini-mental state examination, EuroSCOREII European System for Cardiac Operative Risk Evaluation II, mSOFA modified Sequential Organ Failure Assessment Table 4 Sub-analysis of each tendency’s regression modeling interaction for prolonged delirium/coma days y Another potential mechanism through which Type D personality might have a negative influence on acute brain dysfunction may include inflammation and endo- thelial dysfunction. Previous observational studies showed that Type D personality was significantly asso- ciated with increased levels of IL-6 and TNF-α [50, 51]. In addition, another study showed that Type D person- ality is significantly associated with elevation of another pro-inflammatory marker, C-reactive protein [52], in a Matsuishi et al. BMC Psychology (2019) 7:27 Page 8 of 10 Matsuishi et al. BMC Psychology Fig. 3 Association of depressive symptoms with prolonged brain dysfunction, stratified by Type D personality. The interactive effect of Type D personality and depressive symptoms on DCDs. Adjusted for the covariate factors used in regression modeling Fig. 3 Association of depressive symptoms with prolonged brain dysfunction, stratified by Type D personality. The interactive effect of Type D personality and depressive symptoms on DCDs. Adjusted for the covariate factors used in regression modeling Limitation The mediation effect of depressive symptoms regarding the association of Type D personality with DCDs, adjusted for the same covariates used in regression modeling Fig. 4 Mediation model for delirium/coma days. The mediation effect of depressive symptoms regarding the association of Type D personality with DCDs, adjusted for the same covariates used in regression modeling Fig. 4 Mediation model for delirium/coma days. The mediation effect of depressive symptoms regarding the association of Type D personality with DCDs, adjusted for the same covariates used in regression modeling Page 9 of 10 Page 9 of 10 Matsuishi et al. BMC Psychology (2019) 7:27 Page 9 of 10 Matsuishi et al. BMC Psychology (2019) 7:27 patients (who were not assessed) may have refused con- sent because of a higher level of depressive symptoms, leading to some bias in the results. Received: 25 August 2018 Accepted: 16 April 2019 References 1. American Psychiatric Association. Diagnostic and Statistical Manual of Mental Disorders : DSM-5. Washington: American Psychiatric Publishing; 2014. 1. American Psychiatric Association. Diagnostic and Statistical Manual of Mental Disorders : DSM-5. Washington: American Psychiatric Publishing; 2014. Competing interests Competing interests Competing interests The authors declare that they have no competing interests. 19. Williams L, O’Carroll RE, O’Connor RC. Type D personality and cardiac output in response to stress. Psychol Health. 2009;24(5):489–500. Competing interests The authors declare that they have no competing interests. 20. Denollet J, Van Heck GL. Psychological risk factors in heart disease: what Type D personality is (not) about. J Psychosom Res. 2001;51(3):465–8. Publisher’s Note 21. Spindler H, Kruse C, Zwisler AD, Pedersen SS. Increased anxiety and depression in danish cardiac patients with a type D personality: cross- validation of the type D scale (DS14). Int J Behav Med. 2009;16(2):98–107. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 22. Dannemann S, Matschke K, Einsle F, et al. Is type-D a stable construct? An examination of type-D personality in patients before and after cardiac surgery. J Psychosom Res. 2010;69(2):101–9. Authors’ contributions d d h d 14. Rudolph JL, Jones RN, Levkoff SE, et al. Derivation and validation of a preoperative prediction rule for delirium after cardiac surgery. Circulation. 2009;119(2):229–36. YM designed the study and carried out sample collection, data analysis, and wrote the manuscript. NS, UT, HS1, SK and YI participated in designing study. YY, HH and AO participated in sample collection. CT, HS2 and YH support clinical aspects including informed consent. All authors read and approved the final manuscript. 15. Denollet J, Pedersen SS, Vrints CJ, Conraads VM. Usefulness of type D personality in predicting five-year cardiac events above and beyond concurrent symptoms of stress in patients with coronary heart disease. Am J Cardiol. 2006;97(7):970–3. Abbreviations CAM-ICU: Confusion Assessment Method for the ICU; CNS: Central nerve system; DCDs: Delirium/coma days; DS14: Type-D personality Scale-14; Euro- SCORE II: European System score for Cardiac Operative Risk Evaluation II; HADS: Hospital Anxiety and Depression Scale; IL-6: Interleukin-6; MMSE: Mini- Mental State Examination; mSOFA: Modified Sequential Organ Failure Assessment; NA: Negative Affectivity; POLR: Proportional Odds Logistic Regression; RASS: Richmond Agitation - Sedation Scale; SD: Standard deviation; SI: Social Inhibition; TNF-α: Tumor Necrosis Factor α; VIF: The variance inflation factor 5. Andrejaitiene J, Sirvinskas E. Early post-cardiac surgery delirium risk factors. Perfusion. 2012;27(2):105–12. 6. McPherson JA, Wagner CE, Boehm LM, et al. Delirium in the cardiovascular ICU: exploring modifiable risk factors. Crit Care Med. 2013;41(2):405–13. 6. McPherson JA, Wagner CE, Boehm LM, et al. Delirium in the cardiovascular ICU: exploring modifiable risk factors. Crit Care Med. 2013;41(2):405–13. 7. Martin B-J, Buth KJ, Arora RC, Baskett RJF. Delirium: a cause for concern beyond the immediate postoperative period. Ann Thorac Surg. 2012;93(4): 1114–20. 8. Koster S, Hensens AG, Schuurmans MJ, Van Der Palen J. Consequences of delirium after cardiac operations. Ann Thorac Surg. 2012;93(3):705–11. 8. Koster S, Hensens AG, Schuurmans MJ, Van Der Palen J. Consequences of delirium after cardiac operations. Ann Thorac Surg. 2012;93(3):705–11. 9. Bellelli G, Mazzola P, Morandi A, et al. Duration of postoperative delirium is an independent predictor of 6-month mortality in older adults after hip fracture. J Am Geriatr Soc. 2014;62(7):1335–40. Conclusion Type D personality is a prognostic predictor for prolonged acute brain dysfunction (delirium/coma) in cardiovascular patients independent from depressive symptoms. Further- more, Type D personality-associated depressive symptoms increase the magnitude of acute brain dysfunction. 2. Brown CH. Delirium in the cardiac surgical ICU. Curr Opin Anaesthesiol. 2014;27(2):117–22. 3. Kazmierski J, Kowman M, Banach M, et al. Incidence and predictors of delirium after cardiac surgery: results from the IPDACS study. J Psychosom Res. 2010;69(2):179–85. 4. Stransky M, Schmidt C, Ganslmeier P, et al. Hypoactive delirium after cardiac surgery as an independent risk factor for prolonged mechanical ventilation. J Cardiothorac Vasc Anesth. 2011;25(6):968–74. 4. Stransky M, Schmidt C, Ganslmeier P, et al. Hypoactive delirium after cardiac surgery as an independent risk factor for prolonged mechanical ventilation. J Cardiothorac Vasc Anesth. 2011;25(6):968–74. Availability of data and materials 13. Pandharipande PP, Sanders RD, Girard TD, et al. Effect of dexmedetomidine versus lorazepam on outcome in patients with sepsis: an apriori-designed analysis of the MENDS randomized controlled trial. Crit Care (London, England). 2010;14(2):R38. The datasets used and/or analyzed during the present study are available from the corresponding author on reasonable request. Acknowledgments ld l k h We would like to thank all of the patients for participating in this study. We would also like to thank Dr. Bryan J. Mathis of the University of Tsukuba Medical English Communication Center for critical reading of this manuscript. 10. Pandharipande P, Jackson J, Ely EW. Delirium: acute cognitive dysfunction in the critically ill. Curr Opin Crit Care. 2005;11(4):360–8. 11. Inoue S, Vasilevskis EE, Pandharipande PP, et al. The impact of Lymphopenia on delirium in ICU patients. PLoS One. 2015;10(5):1–11. Consent for publication Not applicable. Consent for publication Not applicable. 18. Williams L, O’Connor RC, Grubb N, O’Carroll R. Type D personality predicts poor medication adherence in myocardial infarction patients. Psychol Health. 2011;26(6):703–12. Competing interests The authors declare that they have no competing interests. 23. Lin I-M, Wang S-Y, Chu I-H, et al. The Association of Type D personality with heart rate variability and lipid profiles among patients with coronary artery disease. Int J Behav Med. 2017;24(1):101–9. Ethics approval and consent to participate 16. Denollet J, Sys SU, Stroobant N, Rombouts H, Gillebert TC, Brutsaert DL. Personality as independent predictor of long-term mortality in patients with coronary heart disease. Lancet (London, England). 1996;347(8999):417–21. The Institutional Review Board (IRB) of the University of Tsukuba Affiliated Hospital approved the present study (H27-085) and written informed consent was obtained from patients prior to surgery. 17. Kupper N, Denollet J, Type D. Personality as a risk factor in coronary heart disease: a review of current evidence. Curr Cardiol Rep. 2018;20(11):104. Funding 12. Page VJ, Ely EW, Gates S, et al. Eff ect of intravenous haloperidol on the duration of delirium and coma in critically ill patients ( Hope-ICU ): a randomised , double-blind , placebo-controlled trial. Lancet Oncol. 2013;1(7): 515–23. No funding received. Author details 1 1Department of Emergency and Critical Care Medicine, Faculty of Medicine, University of Tsukuba, Tsukuba, Ibaraki, Japan. 2Department of Adult Health Nursing, School of Nursing, Sapporo City University, Sapporo, Japan. 3Adult Health Nursing, College of Nursing, Ibaraki Christian University, Hitachi, Ibaraki, Japan. 4Department of Cardiovascular Surgery, Faculty of Medicine, University of Tsukuba, Tsukuba, Ibaraki, Japan. 5Department of Nursing, Kanto Gakuin University College of Nursing, Yokohama, Kanagawa, Japan. 1Department of Emergency and Critical Care Medicine, Faculty of Medicine, University of Tsukuba, Tsukuba, Ibaraki, Japan. 2Department of Adult Health Nursing, School of Nursing, Sapporo City University, Sapporo, Japan. 3Adult Health Nursing, College of Nursing, Ibaraki Christian University, Hitachi, 4 23. Lin I-M, Wang S-Y, Chu I-H, et al. The Association of Type D personality with heart rate variability and lipid profiles among patients with coronary artery disease. Int J Behav Med. 2017;24(1):101–9. 24. Grande G, Romppel M, Barth J. Association between type D personality and prognosis in patients with cardiovascular diseases: a systematic review and meta-analysis. Ann Behav Med. 2012;43(3):299–310. Page 10 of 10 Page 10 of 10 Matsuishi et al. BMC Psychology (2019) 7:27 Matsuishi et al. BMC Psychology (2019) 7:27 25. Shin JE, Kyeong S, Lee JS, et al. A personality trait contributes to the occurrence of postoperative delirium: a prospective study. BMC Psychiatry. 2016;16(1):1–13. 50. Hur S, Han G-S, Cho B-J. Changes in glucose, TNF-α and IL-6 blood levels in middle-aged women associated with aerobic exercise and meditation training. J Phys Ther Sci. 2014;26(12):1933–6. 51. Denollet J, Conraads VM, Brutsaert DL, De Clerck LS, Stevens WJ, Vrints CJ. Cytokines and immune activation in systolic heart failure: the role of Type D personality. Brain Behav Immun. 2003;17(4):304–9. 26. Tully PJ, Baker RA, Winefi HR, Turnbull DA. Depression, anxiety disorders and Type D personality as risk factors for delirium after cardiac surgery. Aust N Z J Psychiatry. 2010;44(May):1005–11. 52. Einvik G, Dammen T, Hrubos-Strøm H, et al. Prevalence of cardiovascular risk factors and concentration of C-reactive protein in Type D personality persons without cardiovascular disease. Eur J Cardiovasc Prev Rehabil. 2011; 18(3):504–9. 27. Nashef SAM, Roques F, Sharples LD, et al. Euroscore II. Eur J Cardio-thoracic Surg. 2012;41(4):734–45. 28. Denollet J. DS14: standard assessment of negative affectivity, social inhibition, and Type D personality. Psychosom Med. 2005;67(1):89–97. 53. Van Craenenbroeck EM, Denollet J, Paelinck BP, et al. Author details 1 Circulating CD34 +/KDR+ endothelial progenitor cells are reduced in chronic heart failure patients as a function of Type D personality. Clin Sci. 2009;117(4):165–72. 29. Zigmond AS, Snaith RP. The hospital anxiety and depression scale. Acta Psychiatr Scand. 1983;67(6):361–70. 30. Folstein MF, Folstein SE, McHugh PR. “Mini-mental state”. A practical method for grading the cognitive state of patients for the clinician. J Psychiatr Res. 1975;12(3):189–98. 54. Denollet J, van Felius RA, Lodder P, et al. Predictive value of Type D personality for impaired endothelial function in patients with coronary artery disease. Int J Cardiol. 2018;259(2017):205–10. y 31. Ideno Y, Takayama M, Hayashi K, Takagi H, Sugai Y. Evaluation of a Japanese version of the mini-mental state examination in elderly persons. Geriatr Gerontol Int. 2012;12(2):310–6. 55. Ritter C, Tomasi CD, Dal-Pizzol F, et al. Inflammation biomarkers and delirium in critically ill patients. Crit Care. 2014;18(3):1–6. 56. Hughes CG, Morandi A, Girard TD, et al. Association between endothelial dysfunction and acute brain dysfunction during critical illness. Anesthesiology. 2013;118(3):631–9. 32. Shigemori K, Ohgi S, Okuyama E, Shimura T, Schneider E. The factorial structure of the mini mental state examination (MMSE) in Japanese dementia patients. BMC Geriatr. 2010;10:36. 57. Denollet J, Schiffer AA, Spek V. A general propensity to psychological distress affects cardiovascular outcomes: evidence from research on the type D (distressed) personality profile. Circ Cardiovasc Qual Outcomes. 2010; 3(5):546–57. 33. Ishihara S, Uchibori T, Imai A, Makita S. Development of the Japanese version of Type D scale for patients with coronary heart disease. Japanese J Heal Psychol. 2015;27:177–84. 34. Higashi A, Yashiro H, Kiyota K, et al. Validation of the hospital anxiety and depression scale in a gastro-intestinal clinic. Nihon Shokakibyo Gakkai Zasshi. 1996;93(12):884–92. 35. Grissom CK, Brown SM, Kuttler KG, et al. A modified sequential organ failure assessment score for critical care triage. Disaster Med Public Health Prep. 2010;4(4):277–84. 36. Sessler CN, Gosnell MS, Grap MJ, et al. The Richmond agitation-sedation scale: validity and reliability in adult intensive care unit patients. Am J Respir Crit Care Med. 2002;166(10):1338–44. 37. Ely EW, Margolin R, Francis J, et al. Evaluation of delirium in critically ill patients: validation of the confusion assessment method for the intensive care unit (CAM-ICU). Crit Care Med. 2001;29(7):1370–9. 38. Stevenson C, Williams L. Type D personality, quality of life and physical symptoms in the general population: a dimensional analysis. Psychol Health. 2014;29(3):365–73. 39. Horwood S, Anglim J. Author details 1 A critical analysis of the assumptions of Type D personality : comparing prediction of health-related variables with the five factor model. Personal Individ Differ. 2017;117:172–6. 40. Widdershoven J, Kessing D. How are depression and Type D personality associated with outcomes in chronic heart failure patients? Curr Hear Fail Rep. 2013;10:244–53. 41. Denollet J, Pedersen S. Prognostic value of Type D personality compared with depressive symptoms. Arch Intern Med. 2008;168(4):431–2. 41. Denollet J, Pedersen S. Prognostic value of Type D personality compared with depressive symptoms. Arch Intern Med. 2008;168(4):431–2. 42. Baron RM, DA K. The moderator-mediator variable distinction in social the moderator-mediator variable distinction in social psychological research: conceptual, strategic, and statistical considerations. J Pers Soc Psychol. 1986 51(6):1173–82. 43. Aroian LA. The probability function of the product of two normally distributed variables. Ann Math Stat. 1947;18:265–71. 43. Aroian LA. The probability function of the product of two normally distributed variables. Ann Math Stat. 1947;18:265–71. 44. Kasai Y, Suzuki E, Iwase T, Doi H, Takao S. Type D personality is associated with psychological distress and poor self-rated health among the elderly: a population-based study in Japan. PLoS One. 2013;8(10):e77918. 44. Kasai Y, Suzuki E, Iwase T, Doi H, Takao S. Type D personality is associated with psychological distress and poor self-rated health among the elderly: a population-based study in Japan. PLoS One. 2013;8(10):e77918. 45. Van Dooren FEP, Verhey FRJ, Pouwer F, et al. Association of Type D personality with increased vulnerability to depression : is there a role for in fl ammation or endothelial dysfunction ? – the Maastricht study. J Affect Disord. 2016;189:118–25. 46. Phillip JT, Robert AB. Depression, anxiety, and cardiac morbidity outcomes after coronary artery bypass surgery: a contemporary and practical review. J Geriatr Cardiol. 2012;9(2):197–208. 47. Abramson LY, Metalsky GI, Alloy LB. Hopelessness depression: a theory- based subtype of depression. Psychol Rev. 1989;96(2):358–72. 48. Smith PJ, Attix DK, Weldon BC, Monk TG. Depressive symptoms and risk of postoperative delirium. Am J Geriatr Psychiatry. 2016;24(3):232–8. 48. Smith PJ, Attix DK, Weldon BC, Monk TG. Depressive symptoms and risk of postoperative delirium. Am J Geriatr Psychiatry. 2016;24(3):232–8. 49. Denollet J, Pedersen SS, Ong ATL, Erdman RAM, Serruys PW, Van Domburg RT. Social inhibition modulates the effect of negative emotions on cardiac prognosis following percutaneous coronary intervention in the drug-eluting stent era. Eur Heart J. 2006;27(2):171–7. 49. Denollet J, Pedersen SS, Ong ATL, Erdman RAM, Serruys PW, Van Domburg RT. Author details 1 Social inhibition modulates the effect of negative emotions on cardiac prognosis following percutaneous coronary intervention in the drug-eluting stent era. Eur Heart J. 2006;27(2):171–7.
https://openalex.org/W2150868856	https://air.unimi.it/bitstream/2434/159879/1/1476-511X-10-73.pdf	English	null	Effects of n-3 PUFAs on breast cancer cells through their incorporation in plasma membrane	Lipids in health and disease	2,011	cc-by	13,853	© 2011 Corsetto et al. ; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Lipids in Health and Disease This Provisional PDF corresponds to the article as it appeared upon acceptance. Fully formatted PDF and full text (HTML) versions will be made available soon. Effects of n-3 PUFAs on breast cancer cells through their incorporation in plasma membrane This peer-reviewed article was published immediately upon acceptance. 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Background PUFAs are important molecules for membrane order and function; they can modify inflammation-inducible cytokines production, eicosanoid production, plasma triacylglycerol synthesis and gene expression. Recent studies suggest that n-3 PUFAs can be cancer chemopreventive, chemosuppressive and auxiliary agents for cancer therapy. Ν−3 PUFAs could alter cancer growth influencing cell replication, cell cycle, and cell death. The question that remains to be answered is how n-3 PUFAs can affect so many physiological processes. We hypothesize that n-3 PUFAs alter membrane stability, modifying cellular signalling in breast cancer cells. Methods Two lines of human breast cancer cells characterized by different expression of ER and EGFR receptors were treated with AA, EPA or DHA. We have used the MTT viability test and expression of apoptotic markers to evaluate the effect of PUFAs on cancer growth. Phospholipids were analysed by HPLC/GC, to assess n-3 incorporation into the cell membrane. Paola A. Corsetto, Gigliola Montorfano, Stefania Zava, Ilaria E. Jovenitti, Andrea Cremona, Dipartimento di Scienze Molecolari Applicate ai Biosistemi, Università degli Studi di Milano, Italy. 1 Passed away in April 2010 § Corresponding author Angela Maria Rizzo, Dipartimento di Scienze Molecolari Applicate ai Biosistemi, Università degli Studi di Milano, Via D. Trentacoste 2, 20134 Milan, Italy. Phone: +39 02 503 15777.Fax: +39 02 503 15775 E-mail: angelamaria.rizzo@unimi.it paola.corsetto@unimi.it gigliola.montorfano@unimi.it stefania.zava@unimi.it ilaria jovenitti@unimi it Results We have observed that EPA and DHA induce cell apoptosis, a reduction of cell viability and the expression of Bcl2 and procaspase-8. Moreover, DHA slightly reduces the concentration of EGFR but EPA has no effect. Both EPA and DHA reduce the activation of EGFR. N-3 fatty acids are partially metabolized in both cell lines; AA is integrated without being further metabolized. We have analysed the fatty acid pattern in membrane phospholipids where they are incorporated with different degrees of specificity. N-3 PUFAs influence the n-6 content and vice versa. Conclusions Our results indicate that n-3 PUFA feeding might induce modifications of breast cancer membrane structure that increases the degree of fatty acid unsaturation. This paper underlines the importance of nutritional factors on health maintenance and on disease prevention. Background Breast cancer is the most common cancer among women worldwide, with an estimated 1.4 million new breast cancer cases only in 2008. Epidemiologic and experimental studies suggest that dietary fatty acids influence the development and subsequent progression of breast cancer [1-3]. The role that long-chain n-3 polyunsaturated fatty acids (PUFAs), eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3), play in the aetiology of cancer has been highlighted by animal experiments and in vitro studies [4, 5]. A number of mechanisms have been proposed for the anticancer actions of n-3 PUFAs. The most prominent mechanism for the chemopreventive action of n-3 PUFAs is their suppressive effect on the production of arachidonic acid (AA)-derived prostanoids, particularly prostaglandin E2 (PGE2), which has been implicated in the immune response to inflammation, cell proliferation, differentiation, apoptosis, angiogenesis and metastasis [6]. The n-3 PUFAs might alter the growth of tumour cells by influencing cell replication, by interfering with components of the cell cycle or by increasing cell death either by way of necrosis or apoptosis [7, 8]. For example, these fatty acids are involved in regulating the tumour p53 proapoptotic signal and superoxide dismutase (SOD) levels, telomere shorting and tumour angiogenesis [9]. In vitro treatment with DHA arrested cell-cycle progression in human-derived breast cancer and malignant melanoma cells [10, 11]. Similarly, in vitro treatment with EPA is reported to arrest the growth of K-562 human leukemic and many other cancer cells accompanied by down-regulation of cyclin expression in some instances [12, 13, 14]. In addition, recent studies of human breast cancer have shown that n-3 PUFAs up- regulate syndecan 1 (SDC-1), which has been shown to play a role in cell adhesion [15, 16], inhibit matrix metalloproteinases [17] and decrease invasion of tumour cells. SDC-1 induces apoptosis in myeloma cells and some studies suggest a similar property in breast cancer cells [18, 19]. The transcriptional pathway for the n-3 PUFA regulation of SDC-1 expression involves the nuclear hormone receptor peroxisome proliferator-activated receptor gamma (PPARγ) [20]. Moreover n-3 PUFAs down- regulate the expression of HER2/neu, a well characterized oncogene that plays a key role in aetiology, progression and chemosensitivity of various types of human cancer in which this oncogene is over-expressed. HER2/neu encodes transmembrane tyrosine kinase orphan receptor p185Her2/neu, which regulates biological functions including cellular proliferation, differentiation, motility and apoptosis [21]. PUFAs EPA (cis-5,8,11,14,17-eicosapentaenoic acid sodium salt), DHA (cis-4,7,10,13,16,19- docosahexaenoic acid sodium salt) and AA (arachidonic acid sodium salt) were purchased from Sigma-Aldrich, USA. The PUFAs were dissolved in ethanol and stored at –80°C under nitrogen gas. Background Nevertheless the mechanism by which n-3 PUFAs inhibit the growth of breast cancer cells is not well understood, but it has been suggested that these fatty acids might change the fluidity and structure of the cell membrane. In fact, changes in the structural characteristics of the plasma membrane in mammalian cells can modify the activity of proteins that function as ion channels, transporters, receptors, signal transducers or enzymes [21-25]. In this study, we have investigated the impact of EPA, DHA and AA on breast cancer cell growth, on cell signalling in apoptosis and on epidermal growth factor receptor (EGFR) activity. We hypothesize that the alteration of cellular cycle, of gene expression and the induction of apoptosis determined from n-3 PUFAs are also a consequence of membrane architecture modifications. For these reasons we have analyzed PUFA incorporation in breast cancer membrane and their PL-specific enrichment. Cell lines and culture conditions Human breast cancer cell lines MDA-MB-231 (ER-negative) and MCF-7 (ER- positive) were kindly provided by Dr P. Degan from the IST (Italian National Cancer Research Institute, Genoa Italy, Laboratory of Molecular Mutagenesis and DNA Repair). Both cell lines are derived from human mammary adenocarcinoma; the MCF7 line retains several characteristics of differentiated mammary epithelium, including the ability to process estradiol via cytoplasmic estrogen receptors. The MDA-MB-231 cells over-express EGFR. These cell lines were maintained in DMEM (Gibco-BRL, Life Tecnologies Italia srl, Italy) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, 100 mg/ml streptomycin and 2 mM glutamine. Medium for treatments (MFT) was DMEM supplemented with 10% FBS. Cells were grown at 37°C in a 5% CO2 atmosphere with 98% relative humidity. Cell viability assay The numbers of viable cells exposed to fatty acids were evaluated by the MTT (3- (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) colorimetric assay [26]. Initially, cells were seeded and cultured in 96-well plates for 48 h to allow adhesion to the plate and to reach 50–60% confluence. After this period, the culture medium was changed to the experimental medium supplemented with EPA or DHA or AA then cultured for 72 h. We studied the effects of different concentrations of PUFAs (50–300 µM). The final concentration of ethanol (<1%) in the culture medium had no antiproliferative effect on any cell line tested; therefore, 10 µl of MTT stock solution (5 mg/ml in PBS, pH 7.5) was added to each well and incubated for 4 h as a control. Then 100 µl of solubilizing solution (10% SDS in 0.01M HCl) was added and incubated overnight. Plates were read at 540 nm in a plate reader. All reagents were purchased from Sigma-Aldrich, USA. Data points represent the mean of eight wells and the results are expressed as relative growth rate (RGR) in comparison to controls that were exposed to a concentration of ethanol equal to that in the samples exposed to fatty acids. Antibodies The mouse monoclonal anti-Bcl2 antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) and the C20 goat polyclonal anti-procaspase-8 p18 antibody were used to study the n-3 PUFA induction of the apoptosis process. The 1005 rabbit polyclonal anti-EGFR antibody and the 11C2 mouse monoclonal anti-pEGFR antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) were used to investigate the alterations of EGFR receptors after treatment with PUFAs. The monoclonal anti-actin (AC-40) antibody (Sigma-Aldrich, USA) was used to normalize gel loading. Bound primary antibodies were visualized by secondary horseradish peroxidase (HRP)-linked antibodies (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) and immunoreactivity was assessed by chemiluminescence (ECL, Amersham). Cell treatment Cell culture experiments were done with the MDA-MB-231 and MCF-7 cell lines to determine the concentrations of EPA (230 µM), DHA (200 µM), required to inhibit growth by 20–30%, and AA (200 µM). Cells were seeded at 1.5 × 104 cells/cm2 for MDA-MB-231 and at 3 × 104 cells/cm2 for MCF-7 in 18 ml of medium containing 10% FBS and allowed to adhere for 48 h, then the medium was replaced with 18 ml of fresh medium (DMEM, 10% FBS) containing the experimental fatty acids and incubated for 72 h without changing the medium. Experiments included untreated cells that were not exposed to any exogenous fatty acids but to an equal content of ethanol during the incubation to serve as controls. After 72 h, cells were harvested using trypsin-EDTA and centrifuged at 900 rpm for 10 min. The supernatant was removed and the pellets were subjected to lipid analysis. For western blot analysis, cells were harvested by scraping in phosphate-buffered saline containing 0.4 mM Na3VO4. Cells were centrifuged and then suspended in 1.4 ml of lysis buffer (1% Triton X-100, 10 mM Tris buffer, pH 7.5, 150 mM NaCl, 5 mM EDTA, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, 75 mU/ml aprotinin), kept on ice for 20 min and then disrupted by 10 strokes in a tight-fitting Dounce homogenizer. The cell lysate was centrifuged (5 min at 1300g) and the supernatant was transferred to an eppendorf tube. Total protein was determined by the Lowry assay [27]. Lipid composition analysis Cell lipids were extracted with three different chloroform/methanol mixtures (1:1, 1:2 and 2:1, v/v) and partitioned with a theoretical upper phase (chloroform/methanol/water, 47:48:1, by vol.) and then with water. The organic phase was dried and then suspended in chloroform/methanol (2:1, v/v) for the analysis of total and PL fatty acids. Purification of single PL moieties was achieved with an HPLC-ELSD system (Jasco, Japan) equipped with one pump, a SCL-10 Advp, a degasser module and a Rheodyne manual injector with 20 µl sample loop and a column (length 250 mm, I.D 4.6 mm and film thickness 5 µm) packed with silica normal-phase LiChrospher Si 60 (LiChroCART 250-4, Merck, Darmstadt, Germany). The chromatographic separation was achieved with a linear binary gradient of 0% B to 100% B in 14 min and then 100% B for 9 min. The total chromatographic run time was 40 min/sample; 23 min analysis, 12 min to restore initial conditions and 5 min for re-equilibration. Eluent A was chloroform/methanol/water (80:19.5:0.5, by vol.) and eluent B was chloroform/methanol/water (60:34:6, by vol.) and the flow rate of the eluent was 1.0 ml/min. An evaporative light-scattering detector (ELSD) was used to detect and quantify the separated PL species. re-equilibration. Eluent A was chloroform/methanol/water (80:19.5:0.5, by vol.) and eluent B was chloroform/methanol/water (60:34:6, by vol.) and the flow rate of the eluent was 1.0 ml/min. An evaporative light-scattering detector (ELSD) was used to detect and quantify the separated PL species. After elution, the eluate was split with one part going to the detector and nine parts to a Gilson fraction collector model 201 to collect the different PL classes for GC analysis. Total fatty acids and PL fatty acids were determined as methylesters by gas chromatography (GC). The methyl esters were obtained by reaction with 3.33% (w/v) sodium methoxide in methanol and injected into an Agilent Technologies (6850 series II) gas chromatograph equipped with a flame ionization detector (FID) and a capillary column (AT Silar) (length 30 m, film thickness 0.25 µm). The carrier gas was helium, the injector temperature was 250°C, the detector temperature was 275°C, the oven temperature was set at 50°C for 20 min and then increased to 200°C at 10°C min–1 for 20 min. Western blot analysis for Bcl2, caspase-8, EGFR and pEGFR Control and treated cell lysates (100 µg protein/lane) were separated by SDS-PAGE (10% polyacrylamide gel), transferred to a polyvinylidene difluoride (PVDF) membrane and analysed by western blot with anti-Bcl2 (1:100), anti-caspase-8 (1:500) and β-actin (1:1800) antibodies. The PVDF membrane was blocked for 1 h in blocking buffer 5% (w/v) dried non-fat milk in Tris-buffered saline (T-TBS: 10 mM Tris–HCl, pH 7.5, 150 mM NaCl, 0.1% (v/v) Tween®20) followed by incubation with an appropriate primary antibody in blocking buffer at room temperature for 2 h. The blots were washed with T-TBS and then incubated with the proper secondary antibody in blocking buffer at room temperature for 1 h. The protein bands were visualized using ECL western blot detection reagents (PerkinElmer, USA). For the analysis of EGFR and p-EGFR, cells treated or not with DHA and EPA were cultured in MFTs supplemented with 10 nM EGF (Sigma-Aldrich, St. Louis, MO, USA) and incubated at 37°C for 15 min of stimulation. Cells were washed twice with ice-cold phosphate-buffered saline (PBS) and lysed as described above. Equal amounts of protein (100 µg/lane) from each treatment were separated by SDS-PAGE (10% polyacrylamide gel) and transferred onto a PVDF membrane then blocked in blocking buffer at room temperature for 1 h. Primary antibodies to EGFR and p- EGFR were diluted 1:200 in blocking buffer at room temperature for 2 h and then with an appropriate secondary antibody at room temperature for 1 h. Parallel blots were probed under the same conditions with primary antibody β-actin diluted 1:1800 to confirm equal protein loading. For the analysis of EGFR and p-EGFR, cells treated or not with DHA and EPA were cultured in MFTs supplemented with 10 nM EGF (Sigma-Aldrich, St. Louis, MO, USA) and incubated at 37°C for 15 min of stimulation. Cells were washed twice with ice-cold phosphate-buffered saline (PBS) and lysed as described above. Equal amounts of protein (100 µg/lane) from each treatment were separated by SDS-PAGE (10% polyacrylamide gel) and transferred onto a PVDF membrane then blocked in blocking buffer at room temperature for 1 h. Primary antibodies to EGFR and p- EGFR were diluted 1:200 in blocking buffer at room temperature for 2 h and then with an appropriate secondary antibody at room temperature for 1 h. Parallel blots were probed under the same conditions with primary antibody β-actin diluted 1:1800 to confirm equal protein loading. Western blot analysis for Bcl2, caspase-8, EGFR and pEGFR The relative intensities of band signals were determined by digital scanning densitometry and β-Actin was used to normalize the results to protein content. Statistical analysis The data are presented as mean ± SD. Student’s unpaired t-test was used for comparisons between treated and control cells and the level of statistical significance was set at P <0.05 and P <0.01. The effects of treatment with PUFAs on breast cancer cell growth To evaluate the effects of PUFAs on breast cancer proliferation, cells were incubated for 3 days in medium supplemented with n-3 and n-6 PUFAs (EPA, DHA and AA). The effect on cell viability of MDA-MB-231 and MCF7 cells was assessed and quantified by the MTT assay. As shown in Fig. 1, cells were treated with various concentrations of n-3 and n-6 PUFAs in the range 50–300 µM. DHA and EPA induce a dose-dependent reduction of cell viability at concentrations > 200 µM (Fig. 1A and 1B). In contrast, AA (Fig. 1C), the major n-6 PUFA, had no significant effect on MCF7 cell viability. The MCF7 cell line was more resistant than the MDA-MB-231 cell line to all treatments with PUFAs. From these experiments, we extrapolated the dose to be used in successive experiments to assess n-3 PUFA incorporation into cell membrane PLs: 230 µM for EPA, 200 µM for DHA, which correspond to 70~80% viability for both cell lines, and 200 µM for AA. DHA and EPA induce apoptosis in breast cancer cells In order to delineate the possible mechanism(s) by which EPA and DHA induce apoptosis we examined the cytoplasmic levels of the Bcl2 protein. Figure 2A and 2B indicate that there was a slight reduction of Bcl2 level in MCF7 cells after treatment with 200 µM DHA, whereas treatment with 230 µM EPA determined the loss of signal; the expression of Bcl2 is also decreased when MDA-MB-231 cells are treated with 230 µM EPA and the protein is not detectable after incubation with 200 µM DHA. µM DHA. Furthermore, apoptosis involves the activation of procaspase-8 (55 kDa) by its cleavage to caspase-8 (18 kDa); this smaller protein together with caspase-3 mediates the rapid dismantling of cellular organelles and architecture [28]. The expression of procaspase-8 was determined by western blot analysis. In Fig. 3A and 3B it is possible to observe a reduction of the proform of caspase-8 for both cell lines treated with EPA and DHA; the reduction was statistically significant after DHA treatment in both cell lines, and also after EPA treatment of MCF7 cells. 4A). As shown in Fig. 4B, DHA significantly reduces the EGFR level (70%) compared to control cells and completely inhibit EGFR activation in cells treated with DHA or DHA/EGF. EPA and DHA alter the EGFR and pEGFR levels in MDA-MB-231 cells EGFR is usually activated in response to extracellular ligands (EGF) by its phosphorylation; ligand binding leads to homo- or heterodimerization with another ligand-bound ErbB receptor, and transmits extracellular mitogenic signals to downstream target signalling cascades that involve cell survival and proliferation, such as phosphatidylinositol 3-kinase (PI3K)/Akt, mitogen-activated protein kinase (MAPK) and signal transducer and activator of transcription 3 (STAT3) [29]. In human breast cancer cell lines, such as MDA-MB-231, the EGFR level is elevated compared with that in other breast cancer cells, such as MCF7 [30]; for this reason, we have studied the effects of DHA and EPA on EGFR activity mainly in MDA- MB-231 cells. Figure 4 reports the effects of EPA and DHA treatments on expression and activation of EGFR in presence of EGF. EPA did not modify EGFR expression in breast cancer cells; while EGF stimulation significantly increase EGFR phosphorylation to about 140%; co-treatment with EPA/EGF significantly inhibit EGFR activation down to about 40% compared to control non stimulated cells (Fig. 4A). Total fatty acid profile after treatment with PUFA Treatment with AA, EPA or DHA alters the FA profile in MDA-MB-231 and MCF7 cells compared with control cells (Table 1). Treatment of both cell lines with AA resulted in a significant increase of AA content in total cell lipids, from 14.40% to 46.85% in MDA-MB-231 and from 12.73% to 44.26% in MCF7. Furthermore, the data for MDA-MB-231 show a significant decrease of EPA, docosapentaenoic acid (DPA) and DHA, whereas the data for MCF7 cells show a significant reduction of only EPA and DHA. When both cell lines were treated with EPA, the content of this FA in total cell lipids was increased significantly and we observed a significant reduction of AA. Unexpectedly, we found an increase of DPA content, indicating that EPA is incorporated into cells and is further metabolized by elongation. The treatment with DHA determines a significant increase of DHA content in both cell lines and an increase of EPA content in MCF7, probably due to a retro conversion; a significant reduction of AA content was also measured. Effects of treatment with PUFAs on PL composition in breast cancer cells Effects of treatment with PUFAs on PL composition in breast cancer cells Table 2 and 3 give the fatty acid composition of specific PLs in MDA-MB-231 and MCF7 cells treated with n-3 or n-6 PUFAs; to simplify the tables SD, are reported as plain numbers above the bold mean value. After treatment with AA, the content of this n-6 PUFA was increased significantly in PE and PC in MDA-MB-231 (Fig. 5, Table 2). The treatment induced a reduction of omega-3 PUFAs, particularly EPA, that was significantly decreased in all phospholipids but SM; while DHA content was decreased after AA treatment only in PE and PC, the other two omega- 3 fatty acids namely DPA and ALA (C18:3) were decreased in all phospholipids but SM. Incubation with EPA caused an increase of EPA content in all PLs in MDA-MB-231 cells. In particular, the incorporation of EPA was different in relation to the PL moiety with highest levels of incorporation in PI and PC. There was a decrease of monounsaturated FA in PE and PC, and a significant increase of polyunsaturated FA in PI and PC. Furthermore, an increase of DPA content was found in all PLs, especially PC. The content of AA was significantly decreased in all phospholipids but SM. After treatment of MDA-MB-231 cells with DHA, the content of this fatty acid was significantly increased in all cell membrane PLs, but not in SM. We measured a significant decrease of the content of EPA in PE, PS, PI and PC. The concentration of AA was significantly reduced in PE and PI and SM as the result of treatment with DHA. In MCF7 cells (Fig. 6, Table 3), the treatment with AA induced a significant increase of this fatty acid in all PLs, except in SM; a significant reduction of EPA and of DHA in PE, PI, PS and PC was also measured. After treatment with EPA, the EPA and DPA content was significantly increased, especially in PE, PI, PS and PC. The concentration of DHA was significantly decreased in PE and PI, whereas the AA content was significantly reduced in PE, and PI. The exposure of MCF7 cells to DHA determined a significant increase of DHA in all PLs, but not in SM, and of EPA content in PE, PI, and PC, whereas the content of AA was significantly reduced only in PE, PI and PS. Effects of treatment with PUFAs on PL composition in breast cancer cells Also in these cells a significant decrease of monounsaturated fatty acids is always present when the cells are treated with n-6 and n-3 PUFAs; while saturated fatty acids are in most cases constant. Moreover also 18:2, 18:3 (n-3) and 20:3 (n-6) are significantly decrease after PUFA treatment. As far as phospholipids content concerns, it is worth noting the significant decrease of SM content (from 11.32% to 9.02%, data not shown) in MCF7 after treatment with DHA even if, sphingomyelin is the phospholipid less influenced in its fatty acid composition by PUFA treatment. The other treatments did not modify the distribution of PL in both cell lines. DISCUSSION Breast cancer is the leading cause of the death among women in the world. The principal effective endocrine therapy for treatment on this type of cancer is anti- estrogens, but therapeutic choices are limited for estrogen receptor (ER) negative tumor, which are more aggressive. Moreover the development of ER positive cancer cells that are resistant to chemotherapeutic agents is a major factor responsible to the successful treatment of breast cancer. This is a strong input to discover new approaches in vitro. Several epidemiologic and clinical studies have shown that n-3 PUFAs are able to provide beneficial effects in a wide variety of pathologies ranging from autoimmune and inflammatory diseases to neurological and psychiatric disorders and, in particular, to several types of malignancy, including ovarian, pancreatic, prostate, renal, colorectal and breast cancer [31-33]. This study was prompted by the observation that MDA-MB-231 and MCF7 breast cancer cell lines showed a significant reduction in cell number following treatment with n-3 PUFAs. The same conclusion is not possible for the AA incubation. We hypothesize that this reduction in cell number results from both proliferation reduction and induction of apoptosis. Apoptosis is a genetically controlled form of cell death that is conserved from worms to humans. Deregulation of apoptosis is a hallmark of all cancer cells and the agents that activate apoptosis in cancer cells could be considered as anti-cancer therapeutics [34]. In some mammalian cells, apoptosis can be triggered by members of the Fas/TNF receptor family. When activated by receptor aggregation, Fas and TNFR1 induce the activation of a set of cysteine proteases called caspases. Studies designed to elucidate the mechanism(s) by which Fas and TNFR1 stimulation lead to caspase activation are underway. In the case of Fas, receptor aggregation by the Fas ligand induces the formation of a death-inducing signalling complex (DISC) of proteins comprising Fas itself, the adaptor protein FADD and the inactive zymogen form of caspase-8. Shortly after formation of the DISC, procaspase-8 is cleaved and the active protease is released. Once activated, caspase-8 is thought to activate other downstream caspases by proteolytic cleavage of their zymogen forms, thus amplifying the caspase signal [35]. Our results demonstrate the activation of caspase-8 in response to incubation with n-3 PUFAs by a reduction of the levels of its zymogen form in both cell lines. DISCUSSION In many cells, over- expression of either Bcl2 or Bcl-xl inhibits apoptosis, affecting the release of cyt-c This study was prompted by the observation that MDA-MB-231 and MCF7 breast cancer cell lines showed a significant reduction in cell number following treatment with n-3 PUFAs. The same conclusion is not possible for the AA incubation. We hypothesize that this reduction in cell number results from both proliferation reduction and induction of apoptosis. Apoptosis is a genetically controlled form of cell death that is conserved from worms to humans. Deregulation of apoptosis is a hallmark of all cancer cells and the agents that activate apoptosis in cancer cells could be considered as anti-cancer therapeutics [34]. In some mammalian cells, apoptosis can be triggered by members of the Fas/TNF receptor family. When activated by receptor aggregation, Fas and TNFR1 induce the activation of a set of cysteine proteases called caspases. Studies designed to elucidate the mechanism(s) by which Fas and TNFR1 stimulation lead to caspase activation are underway. In the case of Fas, receptor aggregation by the Fas ligand induces the formation of a death-inducing signalling complex (DISC) of proteins comprising Fas itself, the adaptor protein FADD and the inactive zymogen form of caspase-8. Shortly after formation of the DISC, procaspase-8 is cleaved and the active protease is released. Once activated, caspase-8 is thought to activate other downstream caspases by proteolytic cleavage of their zymogen forms, thus amplifying the caspase signal [35]. Our results demonstrate the activation of caspase-8 in response to incubation with n-3 PUFAs by a reduction of the levels of its zymogen form in both cell lines. In many cells, over- expression of either Bcl2 or Bcl-xl inhibits apoptosis, affecting the release of cyt-c and apoptosis-inducing factor (AIF) from the mitochondrial intramembrane space to the cytosol. Once released, AIF is translocated to the nucleus where it is capable of inducing nuclear chromatin condensation and large-scale DNA fragmentation that mediates a caspase-independent mitochondrial apoptotic pathway [36]. Cyt-c, together with dATP, binds to apoptotic proteinase activating factor-1 (Apaf-1) and this complex promotes procaspase-9 autoactivation. The active forms of caspase-8 and caspase-9 might activate the downstream effectors caspase-3, -6 and -7, resulting in the cleavage of crucial cellular proteins and apoptosis. We have observed a significant difference in the amount of Bcl2 present in the DHA-treated MDA-MB- 231 cells and EPA-treated MCF7 cells compared to the control group. DISCUSSION The absence of Bcl2 when compared to the control is suggestive that the cell might be more likely to proceed to apoptosis. EGFR is an interesting target for tumour therapy, because it is over-expressed in many human tumours such as lung and breast cancers [37]. MDA-MB-231 cells express high levels of EGFR and are a good model to study EGFR modulation by n-3 PUFAs. This receptor is a member of the ErbB receptor tyrosine kinase family, which consists of EGFR (or HER1 or ErbB1), HER2/ErbB2, HER3/ErbB3 and HER4/ErbB. Ligand binding to EGFR induces its dimerization with another EGFR or with other members of the ErbB family, and activates tyrosine kinase residues on the intracellular domains of the protein through autophosphorylation. EGFR recruits downstream signalling proteins, triggering signal cascades along a number of pathways that eventually lead to cell growth, migration and apoptosis resistance [38, 39]. We found that the phosphorylated EGFR levels are reduced after treatment with n-3 PUFAs (EPA or DHA) in MDA-MB-231 cells, whereas the EGFR level was decreased only after incubation with DHA. The entire mechanism by which n-3 PUFAs exert their beneficial effects is not fully understood. We have hypothesized that the induction of apoptosis, the reduction of cell proliferation and the inhibition of EGFR activity by these fatty acids might be the consequences of cell membrane alterations induced by FA. Our data indicate that EPA and DHA are incorporated in breast cancer membrane. In particular the EPA treatment determines an increase of EPA and DPA content, and a reduction of SFA, MUFA and n-6 PUFA concentration in both cell lines. This suggests an incorporation of EPA which is further metabolised. In fact, EPA is converted to 22:5 n-3 (DPA) by elongase (Elovl)-5 and then by Elovl-2 to 24:5, n-3. The next step requires desaturation of 24:5 by ∆6 desaturase to produce 24:6, n-3. This product is translocated from the endoplasmic reticulum to the peroxisome, where the β oxidation pathway involves acyl chain shortening of C2 to produce DHA [40]. Also DHA incubation determines an increase of EPA, of DHA, and in general of the unsaturation degree in both cell lines. We have also observed that PUFAs are incorporated into the breast cancer membrane with different specificity for each PL moiety. The enrichment is significant, especially in PE, PI and PC. The transbilayer distribution of lipids across biological membranes is asymmetric. DISCUSSION The choline- containing lipids PC and SM are enriched primarily on the external leaflet of the plasma membrane or the topologically equivalent luminal leaflet of internal organelles. In contrast, the amine-containing glycerophospholipids PE and PS are located preferentially on the cytoplasmic leaflet. Other minor PLs, such as phosphatidic acid (PA), PI and phosphatidylinositol-4,5-bisphosphate (PIP2) are also enriched on the cytofacial side of the membrane. Specific alterations of the molecular composition of the plasma membrane occur during apoptosis. Hence, cells undergoing apoptosis express signals, including lipids, proteins and modified sugar moieties that facilitate recognition and ingestion by macrophages. Loss of transmembrane PL asymmetry, with consequent exposure of PS in the external monolayer, occurs in both normal and pathologic conditions. PS externalization is induced early in the process of apoptosis. On the basis of our findings, the data suggest that the incorporation of n-3 PUFAs is mainly into cytofacial leaflet PLs, altering the membrane environment to impact on the activation of cell signalling. Moreover, a significant decrease of SM was evident in cells treated with DHA. Moreover, a significant decrease of SM was evident in cells treated with DHA. Once lipid asymmetry has been established, it is maintained by a combination of slow transbilayer diffusion, protein–lipid interactions and protein-mediated transport. The most significant contributors to the maintenance and dissipation of transbilayer lipid asymmetry are proteins that catalyse the movement of lipids across the membrane. Two classes of transport activities have been described that are responsible for the ATP-dependent transport of lipids. The best characterized activity is flippase, which transports PS from the outer monolayer to the cytoplasmic surface of the plasma membrane and requires ATP and Mg2+ but its activity is inhibited by Ca2+. A second ATP-dependent activity, catalysed by flippases, transports lipids in the opposite direction. The third class of lipid transporter consists of the Ca2+- activated scramblases that catalyse the PS externalization [41, 42]. Growing evidence indicates that excessive concentrations of FA affect cell functions by altering the activity of various ion transporters and channels, including Ca2+. Zhang et al. have found that PUFAs, but not monounsaturated or saturated FAs, cause [Ca2+]i mobilization in NT2 human tetracarcinoma cells by causing release of this proton from mitochondria [43]. Furthermore, Djemli-Shipkolye et al. showed that FA modifications in membranes could be correlated with the variations observed in the activity of ATPase, for instance of Mg-ATPase [44]. DISCUSSION This effect could influence the flippase and scramblase activities, and thus the transbilayer lipid asymmetry. Once lipid asymmetry has been established, it is maintained by a combination of slow transbilayer diffusion, protein–lipid interactions and protein-mediated transport. The most significant contributors to the maintenance and dissipation of transbilayer lipid asymmetry are proteins that catalyse the movement of lipids across the membrane. Two classes of transport activities have been described that are responsible for the ATP-dependent transport of lipids. The best characterized activity is flippase, which transports PS from the outer monolayer to the cytoplasmic surface of the plasma membrane and requires ATP and Mg2+ but its activity is inhibited by Ca2+. A second ATP-dependent activity, catalysed by flippases, transports lipids in the opposite direction. The third class of lipid transporter consists of the Ca2+- activated scramblases that catalyse the PS externalization [41, 42]. Growing evidence indicates that excessive concentrations of FA affect cell functions by altering the activity of various ion transporters and channels, including Ca2+. Zhang et al. have found that PUFAs, but not monounsaturated or saturated FAs, cause [Ca2+]i mobilization in NT2 human tetracarcinoma cells by causing release of this proton from mitochondria [43]. Furthermore, Djemli-Shipkolye et al. showed that FA modifications in membranes could be correlated with the variations observed in the activity of ATPase, for instance of Mg-ATPase [44]. This effect could influence the flippase and scramblase activities, and thus the transbilayer lipid asymmetry. Moreover PUFA incorporation induces an alteration of SFA, MUFA and PUFA content in membrane phospholipids; these data suggest a metabolic rearrangement in Moreover PUFA incorporation induces an alteration of SFA, MUFA and PUFA content in membrane phospholipids; these data suggest a metabolic rearrangement in cells in order to try to balance the ratio between saturated and unsaturated fatty acids. In addition membranes constitute a meeting point for lipids and proteins. Thousands of cellular proteins interact with membranes in different ways, for example integral (transmembrane, as EGFR) proteins are embedded in the lipid bilayer and their activity is sensitive to changes in the lipid environment. Recently, multiple studies Moreover PUFA incorporation induces an alteration of SFA, MUFA and PUFA content in membrane phospholipids; these data suggest a metabolic rearrangement in cells in order to try to balance the ratio between saturated and unsaturated fatty acids. In addition membranes constitute a meeting point for lipids and proteins. DISCUSSION Thousands cells in order to try to balance the ratio between saturated and unsaturated fatty acids. In addition membranes constitute a meeting point for lipids and proteins. Thousands of cellular proteins interact with membranes in different ways, for example integral (transmembrane, as EGFR) proteins are embedded in the lipid bilayer and their activity is sensitive to changes in the lipid environment. Recently, multiple studies demonstrated very rapid ERα actions at level of the plasma membrane [45]. O’Malley and collaborators have demonstrated that ERα on the membrane initially activates cytoplasmic kinases, which in turn phosphorylate and activate coactivators proteins in the cytoplasm. These coactivators then travel to the nucleus and modulate ERα-mediated transcriptional events [46]. Then n-3 PUFAs, modifying the unsaturated degree, the permeability, the flip-flop process and the fluidity of the plasma membrane, might alter the activity of these proteins. This hypothesis will be investigated in our laboratory. Abbreviations PL (phospholipid), FA (fatty acid), PUFA (polyunsaturated fatty acid), MUFA (monounsaturated fatty acid), SFA (saturated fatty acid), DHA (docosahexaenoic acid), EPA (eicosapentaenoic acid), AA (Arachidonic acid), PE (phosphatidylethanolamine), PI (phosphatidylinositol), PC (phosphatidylcholine), PS (phosphatidylserine), SM (sphingomyelin). 5. CONCLUSIONS We suggest that n-3 PUFAs induce modifications of membrane structure and function of breast cancer cells, thereby increasing the degree of unsaturation. These changes of plasma membrane might modify the membrane architecture and signal transduction causing a reduction of cell proliferation and apoptosis induction. Competing interests The authors declare that they have no competing interests Authors’ contributions PAC carried out cell treatments, MTT tests, WB assays and drafted the manuscript, GM performed lipid analysis, SZ was responsible for cell cultures, IEJ performed lipid analysis, AC performed lipid analysis, BB Coordinated the study, AMR conceived and designed the study, performed analysis and interpretation of data and drafted the manuscript. All authors have read and approved the final manuscript. Acknowledgements This paper is dedicated to the memory of Prof. Bruno Berra, who dedicated his life to the biochemistry of lipids. Financial support to Dr Angela M. Rizzo came from VI PQ: Nutra Snack Project and from the Italian Space Agency (ASI). Financial support to Dr Angela M. Rizzo came from VI PQ: Nutra Snack Project and from the Italian Space Agency (ASI). References [1] H Bartsch, J Nair, RW Owen: Dietary polyunsaturated fatty acids and cancers of the breast and colorectum: emerging evidence for their role as risk modifiers. Carcinogenesis 1999, 20: 2209-18 of the breast and colorectum: emerging evidence for their role as risk modifiers. Carcinogenesis 1999, 20: 2209-18 [2] DP Rose, JM Connolly: Omega-3 fatty acids as cancer chemopreventive agents. Pharmacol Ther 1999, 83: 217-44 [3] WE Hardman: Omega-3 fatty acids to augment cancer therapy. J Nutr 2002, 132: 3508S-12S [4] CY Lee, WH Sit, ST Fan, K Man, IW Jor, LL Wong, ML Wan, KC Tan-Un, JM Wan: The cell cycle effects of docosahexaenoic acid on human metastatic hepatocellular carcinoma proliferation. Int J Oncol 2010, 36(4): 991-8 [5] N Habermann, A Schön, EK Lund, M Glei: Fish fatty acids alter markers of apoptosis in colorectal adenoma and adenocarcinoma cell lines but fish consumption has no impact on apoptosis-induction ex vivo. Apoptosis 2010 15: 621-630 [6] SC Larsson, M Kumlin, M Ingelman-Sundberg, A Wolk: Dietary long-chain n-3 fatty acids for the prevention of cancer: a review of potential mechanisms. Am. J Clin Nutr 2004, 79: 935-945 [7] S Serini, E Piccioni, N Merendino, G Calviello: Dietary polyunsaturated fatty acids as inducers of apoptosis: implications for cancer. Apoptosis 2009, 14(2): 135-52. [8] CJ Field, PD Schley: Evidence for potential mechanisms for the effect of conjugated linoleic acid on tumor metabolism and immune function: lessons from n-3 fatty acids. Am J Clin Nutr 2004, 79: 1190S-8S [9] UN Das: A radical approach to cancer. Med Sci Monit 2002, 8:RA79–92 [2] DP Rose, JM Connolly: Omega-3 fatty acids as cancer chemopreventive agents. Pharmacol Ther 1999, 83: 217-44 [2] DP Rose, JM Connolly: Omega-3 fatty acids as cancer chemopreventive agents. Pharmacol Ther 1999, 83: 217-44 [3] WE Hardman: Omega-3 fatty acids to augment cancer therapy. J Nutr 2002, 132: 3508S-12S [3] WE Hardman: Omega-3 fatty acids to augment cancer therapy. J Nutr 2002, 132: 3508S-12S [4] CY Lee, WH Sit, ST Fan, K Man, IW Jor, LL Wong, ML Wan, KC Tan-Un, JM Wan: The cell cycle effects of docosahexaenoic acid on human metastatic hepatocellular carcinoma proliferation. Int J Oncol 2010, 36(4): 991-8 [5] N Habermann, A Schön, EK Lund, M Glei: Fish fatty acids alter markers of apoptosis in colorectal adenoma and adenocarcinoma cell lines but fish consumption has no impact on apoptosis-induction ex vivo. Apoptosis 2010 15: 621-630 [6] SC Larsson, M Kumlin, M Ingelman-Sundberg, A Wolk: Dietary long-chain n-3 fatty acids for the prevention of cancer: a review of potential mechanisms. Am. J Clin Nutr 2004, 79: 935-945 [7] S Serini, E Piccioni, N Merendino, G Calviello: Dietary polyunsaturated fatty acids as inducers of apoptosis: implications for cancer. Apoptosis 2009, 14(2): 135-52. [10] SK Kachhap, PP Dange, RH Santani, SS Sawant, SN Ghosh: Effect of n-3 fatty acid (docosahexaenoic acid ) on BRCA1 gene expression and growth in MCF-7 cell line. Cancer Biother Radiochem 2001, 16: 257-63 [11] AP Albino, G Juan, F Traganos, L Reinhart, J Connolly, DP Rose, Z Darzynkiewicz: Cell cycle arrest apoptosis of melanoma cells by docohexaenoic acid: association with decreased PRb phosphorylation. Cancer Res 2000, 60: 4139-45 [10] SK Kachhap, PP Dange, RH Santani, SS Sawant, SN Ghosh: Effect of n-3 fatty acid (docosahexaenoic acid ) on BRCA1 gene expression and growth in MCF-7 cell line. Cancer Biother Radiochem 2001, 16: 257-63 [11] AP Albino, G Juan, F Traganos, L Reinhart, J Connolly, DP Rose, Z Darzynkiewicz: Cell cycle arrest apoptosis of melanoma cells by docohexaenoic acid: association with decreased PRb phosphorylation. Cancer Res 2000, 60: 4139-45 [12] LCM Chiu, VEC Ooi, JMF Wan: Eicosapentaenoic acid modulates cyclin expression and arrests cell cycle progression in human leukemic K-562 cells. Int J Oncol 2001, 19: 845-9 [13] PB Lai, JA Ross, KC Fearon, JD Anderson, DC Carter: Cell cycle arrest and induction of apoptosis in pancreatic cancer cells exposed eicosapentaenoic acid in vitro. Br J Cancer 1996, 74: 1375-83 [14] RG Clarke, EK Lund, P Latham, AC Pinder, IT Johnson: Effect of eicosapentaenoic acid on the proliferation and incidence of apoptosis in the colorectal cell line HT29. Lipids 1999, 34: 1287-95 [15] W Liu, ED Litwack, MJ Stanley, JK Langford, AD Lander, RD Sanderson: Heparan sulfate proteoglycans as adhesive and anti-invasive molecules. Syndecans and glypican have distinct functions. [3] WE Hardman: Omega-3 fatty acids to augment cancer therapy. J Nutr 2002, 132: 3508S-12S J Biol Chem 1998, 273: 22825- 22832 [16] A Woods, ES Oh, JR Couchman: Syndecan proteoglycans and cell adhesion. Matrix Biol 1998, 17: 477-483 [17] GP Kaushal, X Xiong, AB Athota, TL Rozypal, RD Sanderson, T Kelly: Syndecan-1 expression suppresses the level of myeloma matrix metalloproteinase-9. Br J Haematol 1999, 104: 365-373 acid: association with decreased PRb phosphorylation. Cancer Res 2000, 60: 4139-45 [12] LCM Chiu, VEC Ooi, JMF Wan: Eicosapentaenoic acid modulates cyclin expression and arrests cell cycle progression in human leukemic K-562 cells. Int J Oncol 2001, 19: 845-9 [13] PB Lai JA Ross KC Fearon JD Anderson DC Carter: Cell cycle arrest and [15] W Liu, ED Litwack, MJ Stanley, JK Langford, AD Lander, RD Sanderson: Heparan sulfate proteoglycans as adhesive and anti-invasive molecules. Syndecans and glypican have distinct functions. J Biol Chem 1998, 273: 22825- 22832 [16] A Woods, ES Oh, JR Couchman: Syndecan proteoglycans and cell adhesion. Matrix Biol 1998, 17: 477-483 [17] GP Kaushal, X Xiong, AB Athota, TL Rozypal, RD Sanderson, T Kelly: Syndecan-1 expression suppresses the level of myeloma matrix metalloproteinase-9. Br J Haematol 1999, 104: 365-373 [18] BF Lieberbach, RD Sanderson: Expression of syndecan-1 inhibits cell invasion into type I collagen. J Biol Chem 1994, 269: 20013-20019 [16] A Woods, ES Oh, JR Couchman: Syndecan proteoglycans and cell adhesion. Matrix Biol 1998, 17: 477-483 [19] MV Dhodapkar, E Abe, A Theus, M Lacy, JK Laqngford, B Barlogie, RD Sanderson: Syndecan-1 is a multifunctional regulator of myeloma pathobiology: control of tumor cell survival, growth, and bone cell differentiation. Blood 1998, 91: 2679-2688 [20] IJ Edwards, H Sun, Y Hu, IM Berquin, JT O’Flaherty, JM Cline, LL Rudel, YQ Chen: In vivo and in vitro regulation of syndecan 1 in prostate cells by n- 3polyunsaturated fatty acids. J Biol Chem 2008, 283: 18441-18449 [21] JA Menendez, A Vazquez-Martin, S Ropero, R Colomer, R Lupu: HER2 (erbB- 2)-targeted effects of the omega-3 polyunsaturated fatty acid, alpha-linolenic acid (ALA; 18:3n-3), in breast cancer cells: the "fat features" of the "Mediterranean diet" as an "anti-HER2 cocktail". Clin Transl Oncol 2006, 8: 812-820 [22] CD Stubbs, AD Smith: The modification of mammalian membrane polyunsaturated fatty acid composition in relation to membrane fluidity and function. Biochim Biophys Acta 1984, 779: 89-137 [23] RF Grimble, PS Tappia: Modulatory influence of unsaturated fatty acids on the biology of tumour necrosis factor-α. [3] WE Hardman: Omega-3 fatty acids to augment cancer therapy. J Nutr 2002, 132: 3508S-12S Immunol Cell Biol 2000, 78: 31-39 [26] T Mosmann: Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 1983, 65: 55-63 [22] CD Stubbs, AD Smith: The modification of mammalian membrane [3] WE Hardman: Omega-3 fatty acids to augment cancer therapy. J Nutr 2002, 132: 3508S-12S Biochem Soc Trans 1995, 23: 282-287 [24] CA Jolly, YH Jiang, RS Chapkin, DN McMurray: Dietary (n-3) polyunsaturated fatty acids suppress murine lymphoproliferation, interleukin-2 secretion, and the formation of diacylglycerol and ceramide. J Nutr 1997, 127: 37- 43 [25] MA De Pablo, DC Alvarez: Modulatory effects dietary lipids on immune system functions. Immunol Cell Biol 2000, 78: 31-39 [26] T Mosmann: Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 1983, 65: 55-63 [27] OH Lowry, NJ Rosebrough, AL Farr, RJ Randall: Protein measurement with [19] MV Dhodapkar, E Abe, A Theus, M Lacy, JK Laqngford, B Barlogie, RD [19] MV Dhodapkar, E Abe, A Theus, M Lacy, JK Laqngford, B Barlogie, RD Sanderson: Syndecan-1 is a multifunctional regulator of myeloma pathobiology: control of tumor cell survival, growth, and bone cell differentiation. Blood 1998, 91: 2679-2688 [20] IJ Edwards, H Sun, Y Hu, IM Berquin, JT O’Flaherty, JM Cline, LL Rudel, YQ Chen: In vivo and in vitro regulation of syndecan 1 in prostate cells by n- 3polyunsaturated fatty acids. J Biol Chem 2008, 283: 18441-18449 [21] JA Menendez, A Vazquez-Martin, S Ropero, R Colomer, R Lupu: HER2 (erbB- 2)-targeted effects of the omega-3 polyunsaturated fatty acid, alpha-linolenic acid (ALA; 18:3n-3), in breast cancer cells: the "fat features" of the "Mediterranean diet" as an "anti-HER2 cocktail". Clin Transl Oncol 2006, 8: 812-820 3polyunsaturated fatty acids. J Biol Chem 2008, 283: 18441 18449 [21] JA Menendez, A Vazquez-Martin, S Ropero, R Colomer, R Lupu: HER2 (erbB- 2)-targeted effects of the omega-3 polyunsaturated fatty acid, alpha-linolenic acid (ALA; 18:3n-3), in breast cancer cells: the "fat features" of the "Mediterranean diet" as an "anti-HER2 cocktail". Clin Transl Oncol 2006, 8: 812-820 [22] CD Stubbs, AD Smith: The modification of mammalian membrane polyunsaturated fatty acid composition in relation to membrane fluidity and function. Biochim Biophys Acta 1984, 779: 89-137 [23] RF Grimble, PS Tappia: Modulatory influence of unsaturated fatty acids on the biology of tumour necrosis factor-α. Biochem Soc Trans 1995, 23: 282-287 [24] CA Jolly, YH Jiang, RS Chapkin, DN McMurray: Dietary (n-3) polyunsaturated fatty acids suppress murine lymphoproliferation, interleukin-2 secretion, and the formation of diacylglycerol and ceramide. J Nutr 1997, 127: 37- 43 [25] MA De Pablo, DC Alvarez: Modulatory effects dietary lipids on immune system functions. [22] CD Stubbs, AD Smith: The modification of mammalian membrane 2010, 82(4-6):179-87 [32] M Rondanelli, A Giacosa, A Opizzi, C Pelucchi, C La Vecchia, G Montorfano, M Negroni, B Berra, P Politi, AM Rizzo: Effect of omega-3 fatty acids supplementation on depressive symptoms and on health-related quality of life in the treatment of elderly women with depression: a double-blind, placebo- controlled, randomized clinical trial. J Am Coll Nutr 2010, 29(1):55-64 [33] RE Patterson, SW Flatt, Na Newman, L Natarajan, CL Rock, Thomson CA, BJ Caan, BA Parker, JP Pierce: Marine Fatty Acid Intake Is Associated with Breast Cancer Prognosis. J Nutr 2011, 14(2): 201-6 [34] B Fadeel: Plasma membrane alterations during apoptosis: role in corpse clearance. Antioxid Redox Signal 2004, 6: 269-275 [35] MR Wilson: Apoptotic signal transduction: emerging pathways. Biochem Cell Biol 1998, 76: 573-582 [36] E Daugas, D Nochy, L Ravagnan, M Loeffler, SA Susin, N Zamzami, G Kroemer. Apoptosis-inducing factor (AIF): a ubiquitous mitochondrial oxidoreductase involved in apoptosis. FEBS Lett 2000, 476: 118-123 [31] KN Akhtar: Polyunsaturated fatty acids in the modulation of T-cell [31] KN Akhtar: Polyunsaturated fatty acids in the modulation of T-cell signalling. Prostaglandins Leukot Essent Fatty Acids. 2010, 82(4-6):179-87 [32] M Rondanelli, A Giacosa, A Opizzi, C Pelucchi, C La Vecchia, G Montorfano, M Negroni, B Berra, P Politi, AM Rizzo: Effect of omega-3 fatty acids supplementation on depressive symptoms and on health-related quality of life in the treatment of elderly women with depression: a double-blind, placebo- controlled, randomized clinical trial. J Am Coll Nutr 2010, 29(1):55-64 [33] RE Patterson, SW Flatt, Na Newman, L Natarajan, CL Rock, Thomson CA, BJ Caan, BA Parker, JP Pierce: Marine Fatty Acid Intake Is Associated with Breast Cancer Prognosis. J Nutr 2011, 14(2): 201-6 [34] B Fadeel: Plasma membrane alterations during apoptosis: role in corpse clearance. Antioxid Redox Signal 2004, 6: 269-275 [22] CD Stubbs, AD Smith: The modification of mammalian membrane polyunsaturated fatty acid composition in relation to membrane fluidity and function. Biochim Biophys Acta 1984, 779: 89-137 [23] RF Grimble, PS Tappia: Modulatory influence of unsaturated fatty acids on polyunsaturated fatty acid composition in relation to membrane fluidity and function. Biochim Biophys Acta 1984, 779: 89-137 [23] RF Grimble, PS Tappia: Modulatory influence of unsaturated fatty acids on the biology of tumour necrosis factor-α. Biochem Soc Trans 1995, 23: 282-287 polyunsaturated fatty acid composition in relation to membrane fluidity and function. Biochim Biophys Acta 1984, 779: 89-137 43 [25] MA De Pablo, DC Alvarez: Modulatory effects dietary lipids on immune system functions. Immunol Cell Biol 2000, 78: 31-39 [26] T Mosmann: Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 1983, 65: 55-63 [27] OH Lowry, NJ Rosebrough, AL Farr, RJ Randall: Protein measurement with the Folin phenol reagent. J Biol Chem 1951, 193: 265-275 [28] CS Mitsiades, V Poulaki, G Fanourakis, E Sozopoulos, D McMillin, Z Wen, G Voutsinas, S Tseleni-Balafouta, N Mitsiades: Fas signalling in thyroid carcinomas is diverted from apoptosis to proliferation. Clin Cancer Res 2006, 12: 3705-3712 [29] R Zandi, AB Larsen, P Andersen, MT Stockhausen, HS Poulse: Mechanisms for oncogenic activation of the epidermal growth factor receptor. Cell Signal 2007, 19: 2013-2023 [28] CS Mitsiades, V Poulaki, G Fanourakis, E Sozopoulos, D McMillin, Z Wen, G Voutsinas, S Tseleni-Balafouta, N Mitsiades: Fas signalling in thyroid carcinomas is diverted from apoptosis to proliferation. Clin Cancer Res 2006, 12: 3705-3712 [29] R Zandi, AB Larsen, P Andersen, MT Stockhausen, HS Poulse: Mechanisms for oncogenic activation of the epidermal growth factor receptor. Cell Signal 2007, 19: 2013-2023 [30] JS Biscardi, AP Belsches, SJ Parsons. Characterization of human epidermal growth factor receptor and Src interactions in human breast tumor cells. Mol Carcinog 1998, 21: 261-272 [30] JS Biscardi, AP Belsches, SJ Parsons. Characterization of human epidermal growth factor receptor and Src interactions in human breast tumor cells. Mol Carcinog 1998, 21: 261-272 [31] KN Akhtar: Polyunsaturated fatty acids in the modulation of T-cell signalling. Prostaglandins Leukot Essent Fatty Acids. [35] MR Wilson: Apoptotic signal transduction: emerging pathways. Biochem Cell Biol 1998, 76: 573-582 [37] DS Salomon, R Brandt, F Ciardiello, N Normanno: Epidermal growth factor- related peptides and their receptors in human malignancies. Crit Rev Oncol Hematol 1995, 19: 183-232 [38] Y Yarden, MX Sliwkowski: Untangling the ErbB signalling network. Nat Rev Mol Cell Biol 2001, 2: 127-137 [39] M Scaltriti, J Baselga: The epidermal growth factor receptor pathway: a model for targeted therapy. Clin Cancer Res 2006, 12: 5268-5272 [40] H Sprecher: The roles of anabolic and catabolic reactions in the synthesis and recycling of polyunsaturated fatty acids. Prostaglandins Leukot Essent fatty Acids 2002, 67: 79-83 [41] DL Daleke: Regulation of phospholipid asymmetry in the erytrocyte membrane. Curr Opin Hematol 2008, 15: 191-195 [42] B Fadeel, D Xue: The ins and outs of phospholipid asymmetry in the plasma membrane: roles in health and disease. Crit Rev Biochem Mol Biol 2009, 44: 264- 277 [43] BX Zhang, X Ma, W Zhang, CK Yeh, A Lin, J Luo, EA Sprague, RH Swerdlow, MS Katz: Polyunsaturated fatty acids mobilize intracellular Ca2+ in NT2 human teratocarcinoma cells by causing release of Ca2+ from mitochondria. Am. J Physiol Cell Physiol 2006, 290: C1321-1333 [44] A Djemli-Shipkolye, D Raccah, G Pieroni, P Vague, TC Coste, A Gerbi: Differential effect of omega3 PUFA supplementations on Na,K-ATPase and Mg- ATPase activities: possible role of the membrane omega6/omega3 ratio. Membr Biol 2003, 191: 37-47 [45] I Nemere, RJ Pietras, PF Blackmore: Membrane receptors for steroid hormones: signal transduction and physiological significance. J Cell Biochem 2003 88 438 445 related peptides and their receptors in human malignancies. Crit Rev Oncol Hematol 1995, 19: 183-232 [38] Y Yarden, MX Sliwkowski: Untangling the ErbB signalling network. Nat Rev Mol Cell Biol 2001, 2: 127-137 [39] M Scaltriti, J Baselga: The epidermal growth factor receptor pathway: a model for targeted therapy. Clin Cancer Res 2006, 12: 5268-5272 [40] H Sprecher: The roles of anabolic and catabolic reactions in the synthesis and recycling of polyunsaturated fatty acids. Prostaglandins Leukot Essent fatty Acids 2002, 67: 79-83 [40] H Sprecher: The roles of anabolic and catabolic reactions in the synthesis and recycling of polyunsaturated fatty acids. Prostaglandins Leukot Essent fatty Acids 2002, 67: 79-83 [41] DL Daleke: Regulation of phospholipid asymmetry in the erytrocyte membrane. Curr Opin Hematol 2008, 15: 191-195 membrane: roles in health and disease. Crit Rev Biochem Mol Biol 2009, 44: 264- 277 [43] BX Zhang, X Ma, W Zhang, CK Yeh, A Lin, J Luo, EA Sprague, RH Swerdlow, MS Katz: Polyunsaturated fatty acids mobilize intracellular Ca2+ in NT2 human teratocarcinoma cells by causing release of Ca2+ from mitochondria. Am. J Physiol Cell Physiol 2006 290: C1321-1333 Physiol Cell Physiol 2006, 290: C1321-1333 [44] A Djemli-Shipkolye, D Raccah, G Pieroni, P Vague, TC Coste, A Gerbi: Differential effect of omega3 PUFA supplementations on Na,K-ATPase and Mg- ATPase activities: possible role of the membrane omega6/omega3 ratio. Membr Biol 2003, 191: 37-47 [45] I Nemere, RJ Pietras, PF Blackmore: Membrane receptors for steroid hormones: signal transduction and physiological significance. J Cell Biochem 2003, 88: 438-445 [46] BW O’Malley: A life-long search for the molecular pathways of steroid hormone action. Mol Endocrinol 2005, 19: 1402-141. related peptides and their receptors in human malignancies. Crit Rev Oncol Hematol 1995, 19: 183-232 able 1: Total fatty acid composition of PUFA treated breast cancer cells MDA-MB-231 MCF-7 CTR AA EPA DHA CTR AA EPA DHA C:16:0 13.91±2.19 13.48±2.91 6.57±0.91 8.66±1.25 16.03±4.06 15.52±1.56 14.7±1.76 11.14±3.59 C 16:1 1.61±0.85 1.19±0.87 0.87±0.58 0.77±0.42* 8.25±3.27 2.97±0.86 3.95±0.54 2.94±1.53** C 18:0 17.57±1.97 10.46±2.05* 5.15±1.03 6.39±1.30 14.83±1.58 15.73±1.76 10.41±2.49 9.46±0.89 C 18:1 26.14±4.74 15.91±4.21* 8.15±0.95 9.65±1.29 30.14±2.91 12.12±1.19 12.21±1.94 9.77±1.12 C 18:2 6.05±2.38 3.07±0.65 2.38±0.17 2.46±0.32 4.23±0.73 2.48±1.27 2.26±0.42 1.66±0.21 C 18:3 1.54±4.10 0.68±0.37 0.22±0.16 0.32±0.10 1.32±0.87 0.96±0.53* 0.59±0.36* 0.63±0.61* C 20:3 1.64±0.21 1.60±0.10 0.72±0.37** 1.18±0.70* 2.41±0.67 1.28±0.11 0.67±0.09 0.78±0.16** C 20:4 (AA) 14.40±2.92 46.85±10.48* 2.56±0.87 3.91±0.50 12.73±2.90 44.26±3.80 3.13±0.57 3.20±0.55 C 20:5 (EPA) 2.42±0.70 0.70±0.23 38.75±3.52 1.98±0.43 3.22±0.98 0.74±0.75 34.16±3.89 4.90±0.51 C 22:5 (DPA) 7.04±1.10 3.61±0.44 33.30±1.83 2.94±0.62 1.10±0.71 1.29±0.45 15.80±3.32 0.96±0.14 C 22:6 (DHA) 7.68±1.55 2.45±0.60 1.29±0.35 61.76±3.93 5.73±2.36 2.65±0.68 2.12±0.31 54.55±6.02 SFA 33.35±5.50 23.94±4.88* 11.76±1.90 15.05±2.52 30.86±3.21 31.24±2.89 25.11±3.81 20.60±4.18 MUFA 27.74±4.69 17.10±4.61* 9.01±0.82 10.41±1.06 38.39±4.36 15.09±1.62 16.16±2.02 12.71±2.27 n-3 PUFA 18.68±3.93 7.44±0.56 73.56±3.16 67.00±3.45 11.37±3.71 5.63±1.58 52.67±6.24 61.05±5.96 n-6 PUFA 22.09±3.15 51.52±9.81 5.67±1.37 7.55±0.70 19.38±3.58 48.03±4.00 6.06±0.76 5.64±0.59** Breast cancer cells were treated with solvent (ethanol) as control, AA (20:4, n-6), EPA (20:5, n-3), and DHA(22:6, n-3) . Fatty acid composition was analyzed from n = 10 experiments and expressed in % means ± SD. Asterisks indicate the significant differences between treated- and control cells (, P<0.05; , P<0.01). Table 2: Phospholipids fatty acid composition of PUFA-treated MDA-MB-231. C16:0 C16:1 C18:0 C18:1 C18:2 C18:3 C20:3 C20:4 C20:5 C22:5 C22:6 SFA MUFA PUFA n-6 PUFA n-3 PUFA Omega-6/ Omega-3 AA/EPA AA/DHA PE CTR 5.13 0.80 19.58 18.11 3.12 0.62 1.58 30.38 3.65 6.14 7.85 24.71 18.90 53.34 35.08 18.26 1.95 8.96 3.93 s.d. 1.87 0.59 2.40 2.59 0.97 0.19 0.37 4.39 0.90 1.01 1.06 3.16 2.80 4.03 3.42 2.01 0.30 2.99 0.79 PE AA 6.16 1.29 22.60 15.64* 1.52* 0.30* 1.15* 40.72* 0.66* 3.75* 3.09* 28.76* 16.93* 51.08* 43.39* 7.70* 5.69* 81.49* 13.38* s.d. 0.86 1.12 0.88 0.84 0.14 0.03 0.35 1.29 0.49 0.74 0.45 1.73 0.52 0.89 1.16 0.80 0.61 37.02 1.79 PE EPA 5.64 0.55 20.44 13.68* 1.66* 0.33* 0.84* 8.13* 22.52* 18.86* 1.62* 26.09 14.23* 53.96 10.63* 43.34* 0.25* 0.36* 5.10 s.d. 1.35 0.24 5.47 0.92 0.35 0.07 0.28 1.21 1.33 2.80 0.26 5.28 1.07 3.47 1.13 3.78 0.04 0.07 1.14 PE DHA 6.44 1.14 19.87 8.80* 1.34* 0.27* 0.84* 12.15* 2.10* 2.26* 42.31* 26.31 10.57* 56.55 13.46* 47.24* 0.26* 6.41 0.25* s.d. related peptides and their receptors in human malignancies. Crit Rev Oncol Hematol 1995, 19: 183-232 1.86 0.64 3.99 0.62 0.46 0.09 0.45 2.73 0.71 0.45 3.04 5.38 1.95 7.81 3.21 2.96 0.01 3.60 0.02 PI CTR 4.32 1.31 34.32 14.48 2.50 0.50 2.88 25.54 1.44 4.65 3.98 40.05 14.35 41.67 30.34 11.33 3.23 22.84 8.98 s.d. 1.73 1.31 5.38 4.35 1.74 0.35 0.66 3.93 0.65 1.12 1.74 5.37 5.32 5.09 6.33 3.34 1.68 11.79 9.39 PI AA 8.23* 2.40 33.71 13.31 1.44* 0.29* 0.65* 30.03* 0.40* 2.16* 2.94 41.95 15.26 37.99 32.11 5.87* 5.51* 75.95* 10.23 s.d. 2.67 1.24 2.05 1.54 0.40 0.08 0.30 2.86 0.04 0.15 0.36 3.09 2.39 2.53 2.70 0.40 0.69 15.180 1.75 PI EPA 6.16* 1.12 24.38 14.81 2.34 0.47 1.15* 11.53* 14.30* 16.40* 1.32* 30.55* 15.93 48.87* 15.02* 33.85* 0.45* 0.84* 10.35 s.d. 1.23 0.50 2.72 6.18 1.19 0.24 0.48 3.47 5.28 4.92 0.74 2.27 6.31 6.89 2.60 5.10 0.07 0.15 6.91 PI DHA 10.09* 2.99 32.88 10.90 1.96 0.39 1.81* 10.22* 0.96* 2.55* 20.43* 42.96 13.89 39.71 16.48* 22.57* 0.81* 10.77* 0.53* s.d. 4.52 1.72 4.57 3.28 0.51 0.10 0.91 3.93 0.24 0.36 2.58 8.07 4.59 6.02 6.13 4.12 0.48 3.14 0.15 PS CTR 6.22 2.16 26.07 23.76 3.69 1.10 2.27 13.12 2.50 5.74 7.52 32.40 24.94 38.00 20.12 16.73 1.25 6.03 1.90 s.d. 1.88 1.96 9.75 6.53 1.62 1.07 0.99 7.67 1.20 1.69 3.71 9.19 7.87 11.64 10.49 4.72 0.68 3.59 1.64 PS AA 13.48* 4.69* 21.00 25.00 2.00* 0.48* 0.29* 14.87 0.41* 1.82* 6.07 34.38 29.80 26.37* 17.81 8.56* 2.12* 47.07* 2.86 s.d. 1.77 1.71 4.77 4.62 0.61 0.21 0.16 4.00 0.23 0.63 1.75 3.20 3.57 4.01 4.27 0.98 0.70 29.02 1.92 PS EPA 6.17 1.38 14.67* 18.78 1.76* 0.35* 1.34 6.96* 16.53* 23.17* 2.48* 27.92 19.97 48.44 10.06* 38.38* 0.29* 0.48* 2.18 s.d. 1.30 0.65 4.30 3.79 0.55 0.11 1.12 4.17 0.34 7.45 0.59 13.86 4.06 13.24 4.27 12.21 0.14 0.27 0.83 PS DHA 11.48* 2.44 28.32 10.43* 1.60* 0.32* 0.42* 9.99 1.23* 2.41* 30.84* 36.77 12.26* 45.48 10.63 34.49* 0.32* 9.65 0.33* s.d. 3.46 1.48 13.14 1.56 0.29 0.06 0.13 2.22 0.40 0.83 8.78 17.15 3.15 11.23 3.27 8.40 0.09 3.81 0.05 PC CTR 23.82 3.13 13.90 35.51 6.45 1.29 1.43 8.17 1.20 1.88 2.29 37.73 38.64 22.62 16.04 6.58 2.50 7.50 3.86 s.d. related peptides and their receptors in human malignancies. Crit Rev Oncol Hematol 1995, 19: 183-232 3.82 1.19 2.11 3.70 0.84 0.17 0.37 2.43 0.44 0.64 0.72 3.25 3.55 2.82 2.26 1.15 0.47 3.56 1.43 PC AA 27.29* 3.84 11.20* 21.03* 2.66* 0.53* 1.02* 27.61* 0.10* 1.45* 1.33* 38.50 24.87* 34.80* 31.29* 3.51* 8.89* 277.85* 18.21* s.d. 1.51 0.42 0.69 1.98 0.18 0.04 0.13 3.19 0.01 0.23 0.28 1.43 2.03 3.27 3.13 0.15 0.56 34.12 2.17 PC EPA 25.10 1.63* 9.05* 20.73* 4.33* 0.87* 0.83* 3.62* 15.71* 14.26* 1.36 34.14 22.36* 40.96* 8.77* 32.17* 0.28* 0.24* 3.42 s.d. 3.84 0.25 1.94 1.79 0.66 0.13 0.42 0.53 2.09 5.43 0.93 4.74 1.92 6.92 0.36 6.75 0.05 0.04 1.29 PC DHA 23.54 2.89 13.68 16.85* 3.07* 0.61* 0.96* 7.16 1.78* 1.80 23.90* 37.41 20.77* 39.77* 11.80* 27.96* 0.43* 4.12* 0.33* s.d. 2.72 0.88 1.65 3.35 0.37 0.08 0.25 0.98 0.45 0.66 4.15 4.53 1.62 3.56 0.42 3.75 0.08 0.54 0.09 SM CTR 20.03 2.74 14.34 21.96 3.21 0.80 1.11 8.09 1.17 3.16 6.05 33.78 22.98 24.11 12.93 11.18 1.34 11.07 1.73 s.d. 9.10 1.62 8.11 7.61 1.60 0.73 1.09 3.47 1.17 2.87 3.57 16.17 9.73 6.62 4.26 4.31 0.45 7.33 0.83 SM AA 18.75 2.94 10.40 20.80 2.24 0.45 0.53* 12.30* 0.75 0.63* 6.68 33.32 23.22 22.58 14.80 8.45* 1.76* 30.33* 1.86 s.d. 5.27 1.67 1.00 4.33 0.93 0.19 0.15 0.96 0.54 0.34 0.94 14.58 4.99 1.84 1.45 0.29 0.23 10.50 0.26 SM EPA 12.67* 1.99 6.43* 24.50 2.07* 0.41 0.38* 7.85 4.38* 6.28* 2.93* 19.11* 24.70 26.51 13.17 16.24 0.90 1.78* 3.47 s.d. 3.19 0.78 0.67 2.85 0.71 0.14 0.06 2.91 1.67 2.13 1.48 3.51 7.82 6.22 5.26 7.02 0.45 0.83 2.00 SM DHA 19.07 5.01* 14.14 13.62* 2.89 0.58 1.72 5.60* 1.20 1.24* 8.35 33.21 19.47 25.69 10.21* 15.48 0.79* 5.76* 0.70* s.d. 6.89 0.79 3.32 1.01 0.42 0.08 0.73 1.88 0.53 0.23 2.52 9.19 2.32 7.32 1.37 7.31 0.37 3.21 0.37 Value are expressed as % means bold numbers. SD plain numbers, n= CTR 15, Treated 6; * P<0.01 Table 2: Phospholipids fatty acid composition of PUFA-treated MDA-MB-231. C16:0 C16:1 C18:0 C18:1 C18:2 C18:3 C20:3 C20:4 C20:5 C22:5 C22:6 SFA MUFA PUFA n-6 PUFA n-3 PUFA Omega-6/ Omega-3 AA/EPA AA/DHA PE CTR 5.13 0.80 19.58 18.11 3.12 0.62 1.58 30.38 3.65 6.14 7.85 24.71 18.90 53.34 35.08 18.26 1.95 8.96 3.93 s.d. related peptides and their receptors in human malignancies. Crit Rev Oncol Hematol 1995, 19: 183-232 1.87 0.59 2.40 2.59 0.97 0.19 0.37 4.39 0.90 1.01 1.06 3.16 2.80 4.03 3.42 2.01 0.30 2.99 0.79 PE AA 6.16 1.29 22.60* 15.64* 1.52* 0.30* 1.15* 40.72* 0.66* 3.75* 3.09* 28.76* 16.93* 51.08* 43.39* 7.70* 5.69* 81.49* 13.38* s.d. 0.86 1.12 0.88 0.84 0.14 0.03 0.35 1.29 0.49 0.74 0.45 1.73 0.52 0.89 1.16 0.80 0.61 37.02 1.79 PE EPA 5.64 0.55 20.44 13.68* 1.66* 0.33* 0.84* 8.13* 22.52* 18.86* 1.62* 26.09 14.23* 53.96 10.63* 43.34* 0.25* 0.36* 5.10 s.d. 1.35 0.24 5.47 0.92 0.35 0.07 0.28 1.21 1.33 2.80 0.26 5.28 1.07 3.47 1.13 3.78 0.04 0.07 1.14 PE DHA 6.44 1.14 19.87 8.80* 1.34* 0.27* 0.84* 12.15* 2.10* 2.26* 42.31* 26.31 10.57* 56.55 13.46* 47.24* 0.26* 6.41 0.25* s.d. 1.86 0.64 3.99 0.62 0.46 0.09 0.45 2.73 0.71 0.45 3.04 5.38 1.95 7.81 3.21 2.96 0.01 3.60 0.02 PI CTR 4.32 1.31 34.32 14.48 2.50 0.50 2.88 25.54 1.44 4.65 3.98 40.05 14.35 41.67 30.34 11.33 3.23 22.84 8.98 s.d. 1.73 1.31 5.38 4.35 1.74 0.35 0.66 3.93 0.65 1.12 1.74 5.37 5.32 5.09 6.33 3.34 1.68 11.79 9.39 PI AA 8.23* 2.40 33.71 13.31 1.44* 0.29* 0.65* 30.03* 0.40* 2.16* 2.94 41.95 15.26 37.99 32.11 5.87* 5.51* 75.95* 10.23 s.d. 2.67 1.24 2.05 1.54 0.40 0.08 0.30 2.86 0.04 0.15 0.36 3.09 2.39 2.53 2.70 0.40 0.69 15.180 1.75 PI EPA 6.16* 1.12 24.38 14.81 2.34 0.47 1.15* 11.53* 14.30* 16.40* 1.32* 30.55* 15.93 48.87* 15.02* 33.85* 0.45* 0.84* 10.35 s.d. 1.23 0.50 2.72 6.18 1.19 0.24 0.48 3.47 5.28 4.92 0.74 2.27 6.31 6.89 2.60 5.10 0.07 0.15 6.91 PI DHA 10.09* 2.99 32.88 10.90 1.96 0.39 1.81* 10.22* 0.96* 2.55* 20.43* 42.96 13.89 39.71 16.48* 22.57* 0.81* 10.77* 0.53* s.d. 4.52 1.72 4.57 3.28 0.51 0.10 0.91 3.93 0.24 0.36 2.58 8.07 4.59 6.02 6.13 4.12 0.48 3.14 0.15 C16:0 C16:1 C18:0 C18:1 C18:2 C18:3 C20:3 C20:4 C20:5 C22:5 C22:6 SFA MUFA PUFA n-6 PUFA n-3 PUFA Omega-6/ Omega-3 AA/EPA AA/DHA PE CTR 7.76 4.23 22.01 23.50 3.46 0.69 1.70 24.93 5.28 0.72 5.71 29.77 27.73 42.50 30.09 12.41 2.47 4.75 4.58 s.d. 1.18 1.91 1.92 3.14 0.69 0.14 0.28 3.13 0.69 0.56 1.37 1.86 3.83 3.88 2.66 1.96 0.37 0.50 1.04 PE AA 8.24 1.52* 33.44* 8.05* 1.38* 0.28* 0.49* 42.32* 0.70* 1.03 2.56* 41.68* 9.57* 48.75* 44.19* 4.57* 9.97* 76.64* 17.85* s.d. related peptides and their receptors in human malignancies. Crit Rev Oncol Hematol 1995, 19: 183-232 1.62 0.95 1.22 1.04 0.20 0.04 0.25 2.15 0.47 0.39 0.81 1.44 1.57 2.19 1.94 0.86 1.80 30.68 5.10 PE EPA 9.52 2.19* 27.35* 8.58* 1.50* 0.30* 0.53* 7.37* 33.91* 7.68* 1.07* 36.87* 10.77* 52.36* 9.40* 42.96* 0.23* 0.22* 5.42 s.d. 2.55 1.22 2.29 2.80 0.52 0.10 0.26 1.10 4.88 2.26 0.50 1.58 4.00 5.13 1.76 6.77 0.08 0.06 0.45 PE DHA 9.26 1.36* 25.39 6.97* 2.08* 0.42* 1.03 6.62* 9.45* 0.52 36.92* 34.65* 8.33* 57.02* 9.73* 47.30* 0.21* 0.71* 0.19* s.d. 2.80 1.10 4.61 2.50 1.24 0.25 1.00 1.43 2.19 0.33 7.43 4.38 2.66 6.35 1.95 6.27 0.05 0.13 0.08 PI CTR 10.84 2.51 31.95 18.13 3.43 0.69 5.50 14.10 2.22 1.64 8.99 42.79 20.65 36.56 23.02 13.54 1.80 8.04 1.84 s.d. 5.40 2.32 5.42 6.55 1.29 0.26 1.53 3.89 1.65 1.00 3.07 5.35 7.91 7.50 4.66 3.77 0.47 4.32 1.20 PI AA 8.22 1.04* 35.22* 6.79* 1.45* 0.29* 1.01* 40.65* 0.96* 1.52 2.83* 43.44 7.83* 48.73* 43.12* 5.61* 8.34* 69.09* 23.36* s.d. 1.78 0.73 2.36 3.69 0.34 0.07 0.13 3.84 0.80 1.41 1.77 2.35 4.03 2.81 3.70 1.65 2.63 49.53 17.92 PI EPA 8.81 1.66 33.04 6.85* 2.18* 0.44* 1.48* 10.41* 24.41* 8.60* 2.13* 41.84 8.51* 49.65* 14.07* 35.57* 0.40* 0.44* 4.76 s.d. 1.58 1.04 1.55 2.31 0.79 0.16 0.61 0.92 4.29 1.69 2.18 1.73 2.04 2.96 1.61 3.59 0.08 0.10 3.02 PI DHA 9.06 1.23* 24.60* 13.54 4.52 0.91 3.11 7.63* 7.71* 0.87* 26.82* 33.66* 14.77* 51.57* 15.26* 36.31* 0.43* 1.16* 0.31* s.d. 3.81 0.91 5.96 5.74 2.54 0.51 3.09 1.55 3.67 0.76 6.04 6.91 5.63 7.46 5.64 5.29 0.19 0.48 0.17 PS CTR 10.79 3.53 24.95 27.92 4.30 0.86 4.21 11.93 3.18 2.19 6.14 35.75 31.45 32.81 20.43 12.37 1.83 5.17 2.49 s.d. 3.01 2.22 8.41 7.49 1.77 0.35 1.57 5.37 1.91 2.00 3.36 8.35 8.32 8.96 7.21 4.27 0.84 4.46 1.57 PS AA 11.97 1.92 29.81 12.97* 2.24* 0.45* 1.07* 33.50* 1.21* 1.69 3.18* 41.78* 14.89* 43.33* 36.80* 6.53* 6.49* 38.97* 12.22* s.d. 4.28 2.83 7.69 4.92 1.33 0.27 0.43 11.48 0.63 2.12 1.47 5.84 7.35 9.32 10.79 2.15 3.45 31.62 6.48 PS EPA 16.78* 1.07* 20.16 7.31* 1.27* 0.25* 1.36* 7.62 33.04* 8.11* 3.05 36.93 8.37* 54.70* 10.25* 44.45* 0.22* 0.22* 4.62 s.d. related peptides and their receptors in human malignancies. Crit Rev Oncol Hematol 1995, 19: 183-232 3.53 0.69 11.14 3.65 0.53 0.11 0.66 3.42 12.94 3.14 3.04 11.06 3.52 14.23 3.84 10.79 0.06 0.06 4.12 PS DHA 9.83 1.78* 24.73 13.57* 3.40 0.68 1.75* 6.67* 5.00 1.55 31.06* 34.56 15.35* 50.10* 11.82* 38.28* 0.32* 1.85* 0.22* s.d. 3.89 1.47 6.84 3.83 1.70 0.34 1.30 3.26 2.94 0.59 2.17 6.22 3.88 4.33 5.70 4.53 0.18 1.50 0.12 PC CTR 28.42 13.13 6.51 36.22 4.19 0.84 1.52 5.18 1.18 0.59 2.23 34.93 49.35 15.73 10.89 4.83 2.61 5.70 3.91 s.d. 4.83 5.72 1.57 4.15 0.85 0.17 0.66 1.79 0.62 0.92 2.01 3.83 4.82 5.01 2.61 2.95 0.85 3.65 3.51 PC AA 28.28 4.97* 10.89* 16.67* 1.92* 0.38* 0.58* 34.61* 0.19* 0.29 1.21* 39.16* 21.65* 39.19* 37.11* 2.08* 18.12* 222.12* 30.38* s.d. 2.59 1.87 2.23 0.69 0.05 0.01 0.05 1.68 0.10 0.07 0.33 0.63 1.61 1.91 1.61 0.33 2.44 100.56 8.64 PC EPA 36.02 8.05 5.93 19.61* 2.64* 0.53* 0.56* 3.72 20.12* 1.75 1.07 41.95* 27.66* 35.69* 6.92* 23.48* 0.34* 0.21* 4.37 s.d. 4.52 3.18 0.62 4.19 0.29 0.06 0.34 1.29 8.27 1.39 0.63 0.91 6.34 0.53 1.68 9.30 0.17 0.10 2.54 PC DHA 29.09 3.68* 9.94* 14.94* 3.37* 0.67* 1.42 5.14 10.01* 0.39 21.34* 39.03* 18.62* 42.35* 9.93 32.42* 0.32* 0.51* 0.26* s.d. 5.46 2.60 1.71 3.86 0.46 0.09 0.39 0.99 1.36 0.20 5.63 4.11 5.19 6.44 1.47 6.01 0.07 0.06 0.08 SM CTR 29.95 3.73 15.64 18.03 3.22 0.64 1.86 13.35 4.32 3.66 5.59 45.60 21.76 32.64 18.43 14.22 1.60 4.45 6.27 s.d. 10.86 3.23 5.22 12.01 2.59 0.52 1.75 7.22 2.64 4.77 6.24 14.67 13.34 11.00 7.83 7.61 1.01 4.12 7.52 SM AA 17.58* 2.89 11.48* 20.36 2.56 0.51 2.16 17.74 4.16 6.86 5.97 29.06* 23.25 47.70* 22.46 15.86 1.50 6.56 3.41 s.d. 3.70 1.81 2.48 14.89 1.24 0.25 1.20 4.82 2.46 2.41 2.77 5.41 15.15 13.59 5.90 5.92 0.48 5.17 1.33 SM EPA 22.22* 13.52* 10.43* 5.06* 1.62* 0.32* 5.71 10.28 7.24 7.54* 16.06 32.65* 18.57 48.78* 17.62 31.16* 0.59* 1.49* 0.74* s.d. 2.85 2.24 1.59 0.20 0.65 0.13 3.16 3.56 2.52 1.84 8.51 3.98 2.43 5.54 4.86 6.18 0.22 0.52 0.37 SM DHA 27.05 5.19 11.33* 23.02 3.99 0.80 1.30 9.78 2.65* 4.38 10.53 38.38 28.21 33.41 15.06* 18.35 0.94 4.00 1.05* s.d. related peptides and their receptors in human malignancies. Crit Rev Oncol Hematol 1995, 19: 183-232 10.74 4.69 2.93 20.63 1.93 0.39 0.89 5.93 0.60 3.81 8.19 12.47 18.30 15.33 6.22 11.75 0.41 2.60 0.47 d % b ld b SD l i b CTR 15 T d 6 * P 0 01 Figure 2 - Effects of n-3 PUFA on Bcl2 expression Both cell lines were treated with DHA (200 µM) or EPA (230 µM) for 72 h. Control and treated cell lysates are separated on 10% SDS-PAGE and transferred to PVDF membrane. A: The expression of the anti-apoptotic protein Bcl2 was assessed by western blot. B: Semi-quantitative analysis performed by plate scanning. β actin was used to normalize results of protein content. B: Semi-quantitative analysis performed by plate scanning. β actin was used to normalize results of protein content. Figure 1 - Effects of PUFA on viability of breast cancer cells The effect on cell viability of PUFA in MDA-MB-231 and MCF7 cells is assessed and quantified by MTT assay. Cells are treated with various concentrations of EPA (A), DHA (B), and AA (C). Cells are seeded and cultured for 48 h in a 96-well plate, after this period, the medium is replaced with fresh medium for treatments with AA, EPA, or DHA and incubated for further 72 h. The numbers of viable cell exposed to fatty acids are evaluated by a colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay. Data represent the mean of eight values and results are expressed as Relative Growth Rate (RGR) in comparison with controls (100%). * p<0.05; p<0.01 compared to control cells. Figure 3 - Effects of n-3 PUFAs on caspase-8 expression . The determination of the integrity of the procaspase-8 after treatment with DHA (200 µM) and EPA (230 µM) for 72 h was assessed in both cell lines. The determination of the integrity of the procaspase-8 after treatment with DHA (200 µM) and EPA (230 µM) for 72 h was assessed in both cell lines. A: MCF7 cells treated with DHA (200 µM) or EPA (230 µM) for 72 h . A: MCF7 cells treated with DHA (200 µM) or EPA (230 µM) for 72 h . B: MDA-MB-231 cells treated with DHA (200 µM) or EPA (230 µM) for 72 h B: MDA-MB-231 cells treated with DHA (200 µM) or EPA (230 µM) for 72 h B: MDA-MB-231 cells treated with DHA (200 µM) or EPA (230 µM) for 72 h Semi-quantitative analysis performed by plate scanning. β-actin was used to normalize results of protein content. p<0.01 compared to control cells; n=3 Figure 5 - Content of AA (C20:4), EPA (C20:5), DPA (20:6), DHA (C22:6), SFA and MUFA in PLs of MDA-MB-231 cells treated with LCPUFAs Figure 5 - Content of AA (C20:4), EPA (C20:5), DPA (20:6), DHA (C22:6), SFA and MUFA in PLs of MDA-MB-231 cells treated with LCPUFAs MDA-MB-231 cells were treated with AA, EPA, and DHA. Purification of single PL moieties was achieved with an HPLC-ELSD system. PL fatty acids were determined as methyl esters by gas chromatography (GC). Data are reported as percentage of total fatty acids. Controls are not exposed to any exogenous fatty acids. * p<0.01; n=3. Figure 4 - EGFR expression and EGF stimulation (p-EGFR) in MDA-MB-231 breast cancer cells after n-3 PUFAs treatment A: MDA-MB-231 treated with 230 µM EPA for 72 h, lane 1 control, lane 2 230 µM EPA, lane 3 EGF, lane 4 EGF+EPA. B: MDA-MB-231 treated with 200 µM DHA for 72 h, lane 1 control, lane 2 200 µM DHA, lane 3 EGF, lane 4 EGF+DHA. The relative intensities of band signals, reported in graphics, are determined by digital scanning densitometry. β-actin was used to normalize results of protein content. * p<0.05; p<0.01 compared to control cells; n=3. A MCF7 CTR EPA CTR DHA Procaspase-8 MCF7 0 20 40 60 80 100 120 EPA DHA CTR TREATMENT CTR EPA CTR DHA Procaspase-8 MDA-MB-231 0 20 40 60 80 100 120 CTR TREATMENT B MDA-MB-231 -actin ProCASPASE 8 ProCASPASE 8 A MCF7 -actin CTR EPA CTR DHA CTR EPA CTR DHA ProCASPASE 8 A MCF7 -actin Figure 6 - Content of AA (C20:4), EPA (C20:5), DPA (20:6), DHA (C22:6), SFA and MUFA in PLs of MCF7 cells treated with LCPUFAs MCF7 cells were treated with AA, EPA, and DHA. Purification of single PL moieties was achieved with an HPLC-ELSD system. PL fatty acids were determined as methyl esters by gas chromatography (GC). Data are reported as percentage of total fatty acids. Controls are not exposed to any exogenous fatty acids. * p<0.01; n=3. 0 50 100 150 50 100 150 200 220 240 260 280 300 EPA ( M) RGR (%) MDA MCF7 ** * 0 50 100 150 50 100 150 200 220 240 260 280 300 DHA ( M) RGR (%) * ** 100 150 R (%) A B C MDA MCF7 MDA MCF7 0 50 100 150 50 100 150 200 220 240 260 280 300 EPA ( M) RGR (%) MDA MCF * A 50 100 150 200 220 240 260 280 300 EPA ( M) 0 50 100 150 50 100 150 200 220 240 260 280 300 DHA ( M) RGR (%) * ** 0 50 100 150 50 100 150 200 250 300 AA ( M) RGR (%) ** B C Figure 1 0 50 100 150 50 100 150 200 220 240 260 280 300 DHA ( M) RGR (%) * ** B B 0 50 100 150 50 100 150 200 250 300 AA ( M) RGR (%) C Figure 1 Bcl2 MCF7 0 20 40 60 80 100 120 EPA DHA CTR TREATMENT Bcl2 MDA-MB-231 0 20 40 60 80 100 120 CTR TREATMENT A MCF7 Bcl2 く-actin B MDA-MB-231 CTR EPA CTR DHA Bcl2 く-actin CTR EPA CTR DHA Bcl2 MCF7 0 20 40 60 80 100 120 EPA DHA CTR TREATMENT A MCF7 Bcl2 く-actin CTR EPA CTR DHA Bcl2 く-actin B MDA-MB-231 CTR EPA CTR DHA Bcl2 く-actin B Bcl2 く-actin Bcl2 MDA-MB-231 0 20 40 60 80 100 120 EPA DHA CTR TREATMENT CTR EPA CTR DHA Procaspase-8 MCF7 0 20 40 60 80 100 120 EPA DHA CTR TREATMENT CTR EPA CTR DHA DA-MB-231 actin ProCASPASE 8 ProCASPASE 8 MCF7 -actin ProCASPASE 8 -actin Procaspase-8 MCF7 0 20 40 60 80 100 120 EPA DHA CTR TREATMENT 0 20 40 60 80 100 120 EPA DHA CTR TREATMENT CTR EPA CTR DHA Procaspase-8 MDA-MB-231 0 20 40 60 80 100 120 EPA DHA CTR TREATMENT B MDA-MB-231 -actin ProCASPASE 8 Figure 3 CTR EPA CTR DHA B MDA-MB-231 -actin ProCASPASE 8 Procaspase-8 MDA-MB-231 0 20 40 60 80 100 120 EPA DHA CTR TREATMENT Procaspase-8 MDA-MB-231 Figure 3 EGFR CTR EPA EGF EGF/EPA EGFR 0 50 100 CTR DHA EGF EGF/DHA p-EGFR CTR EPA EGF EGF/EPA * ** p-EGFR 0 50 100 150 CTR DHA EGF EGF/DHA * EGF CTR DHA DHA/EGF B pEGFR EGFR -actin CTR EGF EPA/EGF EPA pEGFR EGFR -actin EGF CTR DHA DHA/EGF B pEGFR EGFR -actin CTR EGF EPA/EGF EPA pEGFR EGFR -actin EGF CTR DHA DHA/EGF EGF CTR DHA DHA/EGF EGF CTR DHA DHA/EGF B EGF CTR DHA DHA/EGF pEGFR EGFR -actin CTR EGF EPA/EGF EPA pEGFR EGFR -actin A A CTR EGF EPA/EGF EPA CTR EGF EPA/EGF EPA EGFR R EPA EGF EGF/EPA EGFR 0 50 100 CTR DHA EGF EGF/DHA EGFR 0 50 100 CTR EPA EGF EGF/EPA EGFR 0 50 100 CTR DHA EGF EGF/DHA EGF/DHA p-EGFR 0 50 100 150 CTR DHA EGF EGF/DHA * p-EGFR 0 50 100 150 CTR EPA EGF EGF/EPA * ** PE MDA 0 10 20 30 40 50 C20:4 C20:5 C 22:5 C 22:6 CTR AA EPA DHA * * * * * * * * * * * * PE MDA 0 10 20 30 SFA MUFA CTR AA EPA DHA * * * * PI MDA 0 10 20 30 C20:4 C20:5 C 22:5 C 22:6 CTR AA EPA DHA * * * * * * * * * * * PI MDA 0 10 20 30 40 50 60 SFA MUFA CTR AA EPA DHA * PS MDA 0 10 20 30 40 C20:4 C20:5 C 22:5 C 22:6 CTR AA EPA DHA * * * * * * * * * PS MDA 0 10 20 30 40 50 60 SFA MUFA CTR AA EPA DHA * PC MDA 0 10 20 30 C20:4 C20:5 C 22:5 C 22:6 CTR AA EPA DHA * * * * * * * * * PC MDA 0 10 20 30 40 50 SFA MUFA CTR AA EPA DHA * * * SM MDA 12 14 CTR AA EPA DHA * SM MDA 50 60 CTR AA EPA DHA PE MDA 0 10 20 30 40 50 C20:4 C20:5 C 22:5 C 22:6 CTR AA EPA DHA * * * * * * * * * * * * PE MDA 0 10 20 30 SFA MUFA CTR AA EPA DHA * * * * PI MDA 0 10 20 30 C20:4 C20:5 C 22:5 C 22:6 CTR AA EPA DHA * * * * * * * * * * * PI MDA 0 10 20 30 40 50 60 SFA MUFA CTR AA EPA DHA * PS MDA 0 10 20 30 40 C20:4 C20:5 C 22:5 C 22:6 CTR AA EPA DHA * * * * * * * * * PS MDA 0 10 20 30 40 50 60 SFA MUFA CTR AA EPA DHA * PC MDA 0 10 20 30 C20:4 C20:5 C 22:5 C 22:6 CTR AA EPA DHA * * * * * * * * * PC MDA 0 10 20 30 40 50 SFA MUFA CTR AA EPA DHA * * * SM MDA 0 10 20 30 40 50 60 SFA MUFA CTR AA EPA DHA * PE MCF7 0 10 20 30 40 50 C20:4 C20:5 C 22:5 C 22:6 CTR AA EPA DHA * * * * * * * * * * PE MCF7 0 10 20 30 40 50 SFA MUFA CTR AA EPA DHA * * * * * * PI MCF7 0 10 20 30 40 50 C20:4 C20:5 C 22:5 C 22:6 CTR AA EPA DHA * * * * * * * * * * * PI MCF7 0 10 20 30 40 50 SFA MUFA CTR AA EPA DHA * * * * PS MCF7 0 10 20 30 40 50 C20:4 C20:5 C 22:5 C 22:6 CTR AA EPA DHA * * * * * * * PS MCF7 0 10 20 30 40 50 SFA MUFA CTR AA EPA DHA * * * * PC MCF7 0 10 20 30 40 C20:4 C20:5 C 22:5 C 22:6 CTR AA EPA DHA * * * * * * PC MCF7 0 10 20 30 40 50 SFA MUFA CTR AA EPA DHA * * * * * * SM MCF7 30 CTR AA EPA DHA SM MCF7 50 60 CTR AA EPA DHA * PE MCF7 0 10 20 30 40 50 C20:4 C20:5 C 22:5 C 22:6 CTR AA EPA DHA * * * * * * * * * * PE MCF7 0 10 20 30 40 50 SFA MUFA CTR AA EPA DHA * * * * * * PI MCF7 0 10 20 30 40 50 C20:4 C20:5 C 22:5 C 22:6 CTR AA EPA DHA * * * * * * * * * * * PI MCF7 0 10 20 30 40 50 SFA MUFA CTR AA EPA DHA * * * * PS MCF7 0 10 20 30 40 50 C20:4 C20:5 C 22:5 C 22:6 CTR AA EPA DHA * * * * * * * PS MCF7 0 10 20 30 40 50 SFA MUFA CTR AA EPA DHA * * * * PC MCF7 0 10 20 30 40 C20:4 C20:5 C 22:5 C 22:6 CTR AA EPA DHA * * * * * * PC MCF7 0 10 20 30 40 50 SFA MUFA CTR AA EPA DHA * * * * * * SM MCF7 30 CTR AA EPA DHA SM MCF7 50 60 CTR AA EPA DHA * PE MCF7 0 10 20 30 40 50 C20:4 C20:5 C 22:5 C 22:6 CTR AA EPA DHA * * * * * * * * * * PE MCF7 0 10 20 30 40 50 SFA MUFA CTR AA EPA DHA * * * * * * 0 10 C20:4 C20:5 C 22:5 C 22:6 * * * 0 10 SFA MUFA PI MCF7 0 10 20 30 40 50 C20:4 C20:5 C 22:5 C 22:6 CTR AA EPA DHA * * * * * * * * * * * PI MCF7 0 10 20 30 40 50 SFA MUFA CTR AA EPA DHA * * * * PS MCF7 0 10 20 30 40 50 C20:4 C20:5 C 22:5 C 22:6 CTR AA EPA DHA * * * * * * * PS MCF7 0 10 20 30 40 50 SFA MUFA CTR AA EPA DHA * * * * PC MCF7 0 10 20 30 40 C20:4 C20:5 C 22:5 C 22:6 CTR AA EPA DHA * * * * * * PC MCF7 0 10 20 30 40 50 SFA MUFA CTR AA EPA DHA * * * * * * SM MCF7 0 10 20 30 C20:4 C20:5 C 22:5 C 22:6 CTR AA EPA DHA * * SM MCF7 0 10 20 30 40 50 60 SFA MUFA CTR AA EPA DHA * * Figure 6 PI MCF7 0 10 20 30 40 50 C20:4 C20:5 C 22:5 C 22:6 CTR AA EPA DHA * * * * * * * * * * * PI MCF7 0 10 20 30 40 50 SFA MUFA CTR AA EPA DHA * * * * PS MCF7 0 10 20 30 40 50 SFA MUFA CTR AA EPA DHA * * * * PC MCF7 0 10 20 30 40 C20:4 C20:5 C 22:5 C 22:6 CTR AA EPA DHA * * * * * * PC MCF7 0 10 20 30 40 50 SFA MUFA CTR AA EPA DHA * * * * * * SM MCF7 0 10 20 30 C20:4 C20:5 C 22:5 C 22:6 CTR AA EPA DHA * * Figure 6 SM MCF7 0 10 20 30 40 50 60 SFA MUFA CTR AA EPA DHA * *
https://openalex.org/W2017608206	https://bmcprimcare.biomedcentral.com/counter/pdf/10.1186/1471-2296-15-100	English	null	Extending the authority for sickness certification beyond the medical profession: the importance of ‘boundary work’	BMC family practice	2,014	cc-by	9,701	© 2014 Welsh et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Abstract Background: The study aimed to explore the views of general practitioners (GPs), nurses and physiotherapists towards extending the role of sickness certification beyond the medical profession in primary care. g y y Methods: Fifteen GPs, seven nurses and six physiotherapists were selected to achieve varied respondent characteristics including sex, geographical location, service duration and post-graduate specialist training. Constant-comparative qualitative analysis of data from 28 semi-structured telephone interviews was undertaken. Results: The majority of respondents supported the extended role concept; however members of each professional group also rejected the notion. Respondents employed four different legitimacy claims to justify their views and define their occupational boundaries in relation to sickness certification practice. Condition-specific legitimacy, the ability to adopt a holistic approach to sickness certification, system efficiency and control-related arguments were used to different degrees by each occupation. Practical suggestions for the extension of the sickness certification role beyond the medical profession are underpinned by the sociological theory of professional identity. Conclusions: Extending the authority to certify sickness absence beyond the medical profession is not simply a matter of addressing practical and organisational obstacles. There is also a need to consider the impact on, and preferences of, the specific occupations and their respective boundary claims. This paper explores the implications of extending the sick certification role beyond general practice. We conclude that the main policy challenge of such a move is to a) persuade GPs to relinquish this role (or to share it with other professions), and b) to understand the ‘boundary work’ involved. Keywords: Professional boundaries, Sick certification, Qualitative methods, Sociology of professions, Primary care Extending the authority for sickness certification beyond the medical profession: the importance of ‘boundary work’ h, Tom Sanders, Jane C Richardson, Gwenllian Wynne-Jones, Clare Jinks and Christian D Mallen Victoria K Welsh, Tom Sanders, Jane C Richardson, Gwenllian Wynne-Jones, Clare Jinks and C Victoria K Welsh, Tom Sanders, Jane C Richardson, Gwenllian Wynne-Jones, Clare Jinks and Christian D Mallen RESEARCH ARTICLE Open Access Welsh et al. BMC Family Practice 2014, 15:100 http://www.biomedcentral.com/1471-2296/15/100 RESEARCH ARTICLE Open Access Welsh et al. BMC Family Practice 2014, 15:100 http://www.biomedcentral.com/1471-2296/15/100 Welsh et al. BMC Family Practice 2014, 15:100 http://www.biomedcentral.com/1471-2296/15/100 Open Access Correspondence: t.sanders@keele.ac.uk Arthritis Research UK Primary Care Centre, Primary Care Sciences, Keele University, Keele, Staffordshire ST5 5BG, UK Background Thus, rather than emphasising the relative merits afforded by alternative therapy within the hospital setting (such as its holistic character) practitioners in Mizrachi et al’s [11] study sought acceptance from medicine by indi- cating a need for scientific research to validate the effects of their therapies, and often referred to patients as ‘cases’, indicating that they were adopting a medical discourse. The rhetorical reduction of the patient to a medical case was therefore indicative of their desire to emulate. A ra- ther different example is the broadening of the notion of ‘science’ to encompass wider rationalities such as the ef- fectiveness and efficiency of treatments, or adherence to research-based clinical protocols [10,20]. Of course, it is not necessarily the case that professions are consistent in their legitimation discourses; Foley and Fairclough [21] found that midwives used discourses of both ‘medicine’ and ‘collaboration’, which they deployed in different ways depending on the context of their work. They reported that use of the language of medicine by midwives was an attempt to establish themselves as equal to doctors, because they too used ‘science’ in their work. However, at other times they placed themselves in a cooperative relationship with physicians as a means of validating their location in the professional status hierarchy. nurses may be offset by the lower productivity of nurses and potential increase in doctor-workload due to nurses meeting previously unmet needs or generating demand for care where previously none existed [6]. Boundaries between professions are fundamental to professional identity and as such, occupations often undertake ‘boundary work’ to maintain such identity. By ‘boundary work’ we refer here to the process by which professions attempt to maintain ownership over a sphere of work. In previous studies such practices included the process by which occupations made claims to specialist knowledge or through direct negotiation between occupa- tions to demarcate work boundaries [7-9]. In the context of changing healthcare policies and organisational struc- tures, boundary work is particularly important to maintain control over a sphere of work. Although there is much interest in evaluating extended roles in primary care, there is a paucity of literature exploring the impact of boundary work on role extension in primary care. The research presented here sought to explore the views of GPs, practice nurses and physiotherapists towards the extension of sickness certification beyond the medical pro- fession, to identify areas of consensus and disagreement. Background for non-medical health professionals including nurses and physiotherapists. Indeed, primary care nurses are increasingly becoming the first point of contact for healthcare and are managing chronic disease [3] and physiotherapists often manage patients with minimal input from the general practitioner [4]. In the UK, demand on primary health care continues to rise as the population ages, health reforms focus on shifting secondary care services into the community and the service delivery targets continue to be devel- oped, for example the linking of physician pay to out- comes as with the Quality Outcomes Framework [1]. Role extension, defined as the ‘substitution of doctors’ traditional role’ can be a useful means of achieving in- creasing healthcare system efficiency [2]. In the current setting, this is particularly relevant to primary health care teams, who have evolved to include extended roles Role extension in primary care has generally met posi- tive reviews with advanced roles of primary care nurses deemed successful [5]. Physiotherapist role extension to act as first point-of-contact practitioners has received high levels of support from GPs and physiotherapists [6]. However, concerns over the negative impact of role extension in primary care have been raised. For example, previous research highlights that cost savings achieved through the substitution of doctors for primary care * Correspondence: t.sanders@keele.ac.uk Arthritis Research UK Primary Care Centre, Primary Care Sciences, Keele University, Keele, Staffordshire ST5 5BG, UK Welsh et al. BMC Family Practice 2014, 15:100 http://www.biomedcentral.com/1471-2296/15/100 Page 2 of 11 Page 2 of 11 One possibility is that such discourses exhibit a moral content, the bases of which might range from judgments about different patient groups as ‘good or rubbish’ [17] to ‘atrocity stories’ in which health visitors distinguished themselves from doctors with reference to the negative at- titudes of the latter [18]. A second possibility is that the notion of ‘science’ might be reconceptualised or reused in various ways. For instance, the notion of ‘clinical iso- morphism’ [19] has been used to signify the readiness of one health profession to adopt the scientific norms of an- other. Background The notion of ‘boundary work’, a key component of which is the idea of ‘legitimacy claims’, was used as a supporting theory to explain attitudes towards the role extension con- cept by three groups of health care professionals. Professional boundaries The literature on professional boundaries shows that juris- dictions must be legitimated in the context of where pro- fessionals carry out their work and interact with other professions [10,11]. The manner of these legitimization practices has taken many forms, indicating the often sub- tle ways that occupational groups attempt to assert au- thority over the content and scope of their work. For instance, groups may attempt to protect their claims to a specific jurisdiction through the delegation of ‘unwanted’ tasks to others; thus general medical practitioners (GPs) may pass on routine work to nurses whilst seeking to re- tain their overarching status as ‘expert’ [12,13]. Abbott [10] argued that by the late 20th Century many professions had come to rely heavily on science as a means of legitim- ation. In this context, ‘science’ includes both its narrow definition and a broader understanding as rationality and efficiency, a point to which we return below. Several stud- ies have highlighted how distinctions are made when prac- titioners appeal to the scientific basis of their work [14,15]. Science narrowly conceived is, however, not static. In- creasing sub-specialisation in medicine is perhaps one manifestation of this [16]. In any event, such practices always have ‘discursive’ characteristics. For all of the above reasons professional work boundar- ies need to be considered in any analysis of occupational behaviour change, to acknowledge the wider context of the NHS multidisciplinary workforce. The extension of sickness certification to professional groups other than medicine is likely to result in considerable inter- professional boundary negotiation. Sickness certification: the international context Research on the role of health professionals and their attitudes towards sickness certification is scarce, and international comparisons indicate wide variations in beliefs and corresponding behaviours among patients and primary care practitioners [32-38]. Research from Scandinavia has made significant progress in under- standing GPs’ attitudes towards sickness certification and work absence. One study found that the strongest indicator of sickness certification is the extent of concord- ance between patients’ and GPs’ evaluations of reduced work capacity [39], whilst a diagnosis of musculoskeletal disease or mental illness increased the likelihood of work absence; perhaps reflecting societal pressure and ex- pectation to exempt people with certain health condi- tions from participation in work [40]. Other studies from Scandinavia highlight the difficult challenge of fit- ness for work assessments in the presence of clinical uncertainty about the patients’ presenting complaint, particularly in the absence of objective signs [41]. In such circumstances, GPs may be inclined to accept the patient’s complaint and issue a sick certificate [39]. Re- search from Scandinavia and the UK also shows that GPs consider work related issues to be less relevant to their primary role and may be ill equipped to assess people’s capacity to work [42,43]. In a recent survey, al- most two thirds of employers claimed that occupational health specialists, not GPs, were best placed to assess people’s fitness to work [44]. It is clear from the inter- national literature that significant variation exists in GPs’ assessments of work capacity and decisions to issue a sick certificate. Given the cost implications for global economies, an improved understanding of how the delivery of sickness certification could be improved is needed, and this includes more in-depth research on the possibility of extending authority to other healthcare practitioners [45-48]. Evidence demonstrates that work is generally good for health, yet the predominant national philosophy that ill- ness is incompatible with work remains [29,30]. In light of this evidence, the Government introduced a strategy on health, work and wellbeing to encourage and assist individuals with ill-health to return to work [29,30]. Part of this strategy, the ‘Fit for Work Service’, emphasises a multidisciplinary approach in encouraging an early re- turn to work which includes increasing responsibilities placed upon nurses and physiotherapists. Government policy on managing health and work is becoming more proactive, as opposed to passive in providing disability benefits. Sickness certification in the UK context In 2011, 131 million days were lost due to sickness ab- sences in the UK [22], costing the UK economy £17 billion [23]. Furthermore, 2.07 million adults of work- ing age were out of work due to long-term sickness ab- sence in 2010 (ONS [22]). For those unable to work due to ill-health, sickness certificates provide support- ing evidence for health-related benefits claims [24]. In the UK, only medical doctors are legally able to certify sickness absence [25] despite evolving extended roles within the primary health care team. GPs are contrac- tually obliged to certify short- and medium-term sickness For all of these reasons, we might expect such dis- courses in the workplace to be dynamic and opportunistic. Welsh et al. BMC Family Practice 2014, 15:100 http://www.biomedcentral.com/1471-2296/15/100 Page 3 of 11 focus specifically on the relevance of occupational bound- aries in role extension policy introduced in the NHS. absence [26]. Estimates suggest GPs spend six consulta- tions every half-day session discussing work and health and a full-time GP expects to sign approximately ten sick- ness certificates every week [27]. In 2001, the Government estimated that extending the authority to certify sickness absence to primary care nurse practitioners would save 2.4 million GP appointments and 51,000 hours of GP time per year. As demands are increasingly placed upon GPs, role extension in sickness certification is an important proposition to consider, particularly as GPs hold mixed views towards their sickness certification role. Some GPs value their participation and feel they are best placed to fulfil this role, others prefer to have the role removed [28]. Recently, a majority of surveyed GPs thought primary care nurses and physiotherapists should have the authority to certify at least some sickness absence [27]. Sickness certification: the international context There has been the introduction of the Fit for Work Service [24] and a new White Paper Fitness for Work: The Government response to “health at work” has set out the Government’s policy in relation to the development of a health and work advisory service provid- ing access to state funded occupational health, improving sickness absence management in the workplace alongside support for healthcare professionals and a reform of the benefits system [31]. However, a shift towards proactive health and work management in the UK means that di- verse skills are required to help people stay in work and manage their work related difficulties. One potential barrier to this could be the possible reluctance of GPs to relinquish their responsibilities for managing work and health to other occupational groups (eg. primary care nurses) or to share this role. More recently, research in the UK and abroad has begun to examine the impact of illness on work absence, placing greater emphasis on the broader role of em- ployers and organisational policies in facilitating people’s return to work [49-51]. In light of re-focused UK Government priorities towards work and health, the current role extension in primary care and renewed support for role extension in sickness certification to nurses and physiotherapists, this paper seeks to further explore views towards primary care sick- ness certification role extension, and the potential prac- tical benefits and barriers such a move would involve. We report the views of practice nurses, physiotherapists and GPs to establish whether there is support for role exten- sion, and if so, the key challenges to its introduction. We Methods The decision to end fieldwork was based on ‘thematic saturation’, where new insights in relation to the research question were no longer emerging from the interviews. qualifications, contract basis (partner, salaried, locum, full- time, or part-time) and sex. Nurses and physiotherapists were recruited through snowball sampling. Nurses were recruited through their GP colleagues to explore the in- fluence of working relationships and common working environments upon views. Five nurses were matched to their GP colleagues and two were unmatched. The rea- son for this was that we could not recruit all nurses in the same practices as their GP colleagues, though five matched pairs offered a useful insight into how the views towards role extension among GPs and nurses working together compared. As with any ‘snowball’ sampling approach, it is possible that participants shar- ing a common working space may hold similar or ‘com- patible’ attitudes towards a particular working pattern or behaviour. At the same time such views may help to better understand the underlying reasons and whether and to what extent these are influenced by local organ- isational factors, or whether attitudes towards role ex- tension are largely formed on the basis of professional differences or training. Six physiotherapists were randomly recruited through local research networks. The topic guide used broad prompts to explore views towards GP roles, views on role extension and practical requirements of role extension. One researcher (VKW) undertook semi- structured telephone interviews lasting 30–60 minutes. Written informed consent was obtained for study par- ticipation, interview recording and quotation use. Inter- views were transcribed verbatim. A total of 28 respondents were interviewed. Nine GPs were male, the median time in practice was 21 years (range: 5–32 years), ten worked full-time, eleven were practice partners (three were salaried GPs, one worked as a locum) and three had advanced occupational health training. All the nurses were female, the median time in practice was 14 years (range: 2-33 years) and four had advanced training, although not in occupational health. The GPs and nurses worked in a range of large, medium and small practices in cities and towns. Four of the physio- therapists interviewed were male, which is not representa- tive of the average proportion of male physiotherapists in UK clinical practice. Methods A random sample of 125 GPs were selected from a list of 397 GPs who consented to receiving further study invita- tions as part of a previous research study [27]. Purposive sampling was subsequently employed to select 15 GPs from the 26 who consented to participate. Participants were selected on the basis of practice location, practice list size, service duration, postgraduate occupational health Page 4 of 11 Welsh et al. BMC Family Practice 2014, 15:100 http://www.biomedcentral.com/1471-2296/15/100 characteristics such as age, sex and training in occupa- tional health did not appear to influence opinions to- wards a particular viewpoint; either in favour or against role extension. Despite preference towards support for role extension, notable exceptions from each profes- sional group existed. Support for role extension did not always mean a rejection of the GP sickness certification role. As with any qualitative study, the ultimate aim is to explore participants’ views in depth rather than to achieve generalizable findings. We over recruited GPs into the interview study because they are primarily re- sponsible for issuing sickness certificates in the UK, and therefore we sought to utilise their views as a starting point for our analysis, and using the nurse and physio- therapist interviews to compare and contrast the views of GPs. Moreover, limited in-depth qualitative evidence exists of GPs’ perceptions of sickness certification and attitudes to service re-design such as role extension. Al- though a smaller number of nurses and physiotherapists were recruited into the study, the data had reached ‘sat- uration’ point in relation to the role extension concept (if not in relation to other less directly relevant issues) and thus we decided that further interviews were not needed. A topic guide was used (the same for each partici- pant group) which included questions on how participants approached sickness certification decisions during clinical practice, their views towards extending or sharing respon- sibility for such decisions, and in which circumstances role extension would be appropriate. The topic guide was amended to some degree during fieldwork as new insights and themes emerged from the interviews. These new themes were subsequently included in the topic guide and explored in the remaining interviews. One example of a new theme to emerge from the initial interviews was the idea of ‘boundary work’ and participants’ claims to the jur- isdiction of ‘sickness certification’ practice. Methods The data cannot strictly speaking be viewed as a direct reflection of clinical prac- tice because we have relied entirely on the perceptions of three occupations rather than observed behaviour. Each occupational group may therefore have their own motives and agendas to support their particular views about sick- ness certification, and may not necessarily represent ‘how things are done’ in daily clinical practice. However, the organise the data. The first transcript was independently coded by two researchers (VKW, JR). The initial codes were discussed and revised so that agreement about their appropriateness was reached by the analysis team. These codes were applied to several transcripts, followed by dis- cussion and comparison. Any differences in coding were discussed until a consensus was reached. The emerging coding frame was applied to the remaining transcripts by a single researcher (VKW). Themes were compared across participants and within individual accounts. The four key themes arose directly from the data analysis. The research team reached consensus about the interpretation of these themes and how they might best be ‘labelled’ and defined. The concept of ‘legitimacy claims’ which is a key compo- nent of ‘boundary work’ carried out by healthcare profes- sionals seemed to provide an appropriate theoretical ‘lens’ for interpreting our findings. The data cannot strictly speaking be viewed as a direct reflection of clinical prac- tice because we have relied entirely on the perceptions of three occupations rather than observed behaviour. Each occupational group may therefore have their own motives and agendas to support their particular views about sick- ness certification, and may not necessarily represent ‘how things are done’ in daily clinical practice. However, the data lend itself to an exploration of professionals’ diverse agendas and motives towards extending the sick certifica- tion role, and which revealed a number of barriers and possibilities for introducing service redesign in primary care. Ethics approval was obtained from a local NHS eth- ics committee. Methods Although male physiotherapists may differ in their views to some extent, it is unlikely that their perceptions about role extension in relation to sickness certification would dramatically deviate from female phys- iotherapists. The median time in practice was seven years (range: 1-40 years), five worked full-time and all worked in multiple locations. Two physiotherapists possessed advanced training in occupational health and four had advanced training in musculoskeletal health. Fifteen re- spondents were interviewed at work and 13 were inter- viewed at home. New themes ceased to emerge after 13 GP and six nurse interviews, but continued to emerge during the final physiotherapist interview. Respondent The main author’s background as a general practitioner with a special interest in social science influenced the the- oretical focus taken to the research question and analysis of the data. However, the concept of ‘boundary work’ emerged from the analysis and we reviewed the literature on professional boundaries during the course of the field- work in order to interpret the findings. The initial aims of the research were not to investigate ‘boundary work’ but to explore different views towards role extension in rela- tion to the sickness certification role by three groups of healthcare professionals. Data analysis was continuous and iterative throughout data collection to enable exploration of emerging themes. Thematic analysis was undertaken using constant com- parative methodology, facilitated by NVivo9 to code and Page 5 of 11 Welsh et al. BMC Family Practice 2014, 15:100 http://www.biomedcentral.com/1471-2296/15/100 organise the data. The first transcript was independently coded by two researchers (VKW, JR). The initial codes were discussed and revised so that agreement about their appropriateness was reached by the analysis team. These codes were applied to several transcripts, followed by dis- cussion and comparison. Any differences in coding were discussed until a consensus was reached. The emerging coding frame was applied to the remaining transcripts by a single researcher (VKW). Themes were compared across participants and within individual accounts. The four key themes arose directly from the data analysis. The research team reached consensus about the interpretation of these themes and how they might best be ‘labelled’ and defined. The concept of ‘legitimacy claims’ which is a key compo- nent of ‘boundary work’ carried out by healthcare profes- sionals seemed to provide an appropriate theoretical ‘lens’ for interpreting our findings. Results Respondents employed four key arguments, or ‘profes- sional legitimacy claims’, to maintain professional bound- aries and ultimately, to maintain professional identity. Practical suggestions for implementation (Figure 1) are explained by professional legitimacy claims. A unique numerical identifier is included at the end of each quotation to identify interview participants. A holistic approach All professions recognised the need to adopt a holistic approach towards sickness certification. Each group ap- peared to equate “the full picture” with the ability to practice holistically. Information access seemed a central requirement of holistic practice. “…if you’ve got more complicated cases, I just don’t think that would be suitable for a nurse… it’s very complicated, it’s not easy to get the patient back to work. You have to be careful that you’re happy that the patient is fit and sometimes there are lots of other psychological reasons why they don’t want to return to work.” GP 304 GPs legitimised their certification role and defined their occupational boundaries through highlighting their monopoly over the holistic approach: “Of course you have the advantage that you usually know the patient very well so you know a lot more of the background…that does help…we see everything that goes on with the patient and that puts us in a unique position, we get a complete overview of the patient. Whereas, a physiotherapist is, understandably, more specialist in that area so they’re not going to see the whole patient the same way that we are.” GP304 “I suppose it depends on what the condition was. If someone came in with chronic back pain, obviously we couldn't assess that person. But if it was for…fairly self-limiting illnesses…one of our patients had a really nasty insect bite, worked in a lab. We'd been seeing him regularly. So in that situation I guess we could have done a sick note…” Nurse 321 Additionally, GPs referred to their gatekeeper role to patient information through raising concerns over the appropriateness of fit note completion without access to full records and the ethical challenges of information sharing: Physiotherapists, however, employed their specialist knowledge to define their role and alluded to GPs’ knowledge gap to legitimise role extension claims: “When you get referred to a physio, generally they're focusing on one element. It may be straightforward if they're seeing someone rehabilitating from a total knee replacement or something but it's not always that straightforward and they're not likely to know the background issues at all…they'll have the sketchiest of referral information but they may never have been referred from primary care. So they're not forced to know anything about the background. And our notes might be writing ‘patient clearly fit enough to work. Needs to return. Condition-specific legitimacy Respondents based their claims on perceptions of their own and other professions’ specialist expertise to justify their part in the certification process and the role exten- sion concept. Narratives of each professional group were based around the distinction between “straight-forward” and “woolly” medical problems. Figure 1 Suggested requirements for extended role sickness certification implementation and their underlying professional legitimacy claims. Figure 1 Suggested requirements for extended role sickness certification implementation and their underlying profession Page 6 of 11 Welsh et al. BMC Family Practice 2014, 15:100 http://www.biomedcentral.com/1471-2296/15/100 Welsh et al. BMC Family Practice 2014, 15:100 http://www.biomedcentral.com/1471-2296/15/100 GPs highlighted their skill in managing complex multi- morbidity, indicating that sickness certification by nurses or physiotherapists for such cases would be less accept- able whilst nurses reinforced GP views of nurses certify- ing straight-forward cases. System efficiency All professional groups recognised the potential of role extension to increase system efficiency including GP workload reduction, improved healthcare access for sick- ness certification and removal of work duplication. “…if the way that nurses deal with everything else was to do with it, then I don’t think you would get a – it would just be blank cheques being written, or else they’d always refer them to a doctor…” GP528 “…if the way that nurses deal with everything else was to do with it, then I don’t think you would get a – it would just be blank cheques being written, or else they’d always refer them to a doctor…” GP528 Despite their contrasting views towards role extension, GPs employed this claim to support “streamlining the fairly obvious” cases to others to manage to save GPs time and enable them to focus on more “urgent” patient problems. Examples of scenarios in which role extension would improve organisational efficiency included patients requiring post-operative monitoring by nurses (for ex- ample, wound dressings or stitch excision); patients re- ceiving a diagnosis and management plan from other frontline healthcare providers (for example, the emer- gency department and walk-in-centres) who only have to attend the GP specifically for a sickness certificate; patients receiving a diagnosis, management plan and follow-up from a nurse practitioner but have to see the GP for a sickness certificate (for example, chronic disease monitoring); minor illnesses and injuries (for example, gastroenteritis and tonsillitis) and patients undergoing treatment with the physiotherapist who only needed to see the GP specifically for a sickness certificate. Every matched pair gave similar narratives about improving efficiency, indicating that shared values may arise from a shared organisational culture. The use of protocols and guidelines to assist certification decisions by non-medical staff was suggested to mitigate increased GP workload. Each professional group raised concerns over the potential for patients to manipulate a new extended role system: “…when you introduce multiple people doing the same thing there’s always a potential for inter-observer vari- ation. I think the guy’s fine and fine to go to work. The physio doesn’t. And there may be a little bit of playing one side off [against] the other. The guy doesn’t want to go back to work and: ‘Well, ***** you. System efficiency If you're not going to give me a line I'm going to go to the physio.” GP757 Respondents linked manipulation with increasing de- mand for physiotherapist and nursing services and pos- sible inappropriate certification practice. GPs seemed to use system efficiency arguments as an indirect means of exercising influence, defining other oc- cupations’ ‘acceptable’ roles. GPs may support role exten- sion if it saves them time, not because physiotherapists or nurses are the better professions to do it. A holistic approach Told I will not issue any sick notes anymore’ and they won't have any access to that record.” GP760b “…because physiotherapists are at the heart of the rehabilitation process, the person needs to have time off work to help their recovery, we could identify those patients quite well… I think your average GP can sometimes struggle with just assessing a hip, if it needs to be replaced or not and to then identify if that person’s got any emotional overlay on top of that hip pain…I just don’t think it’s in their remit….” Physiotherapist 11 A strong sense of certification within usual clinical remit emerged, reinforced by matched pairs presenting similar arguments of condition-specific legitimacy. The Table 1 below presents situations that respondents deemed “appropriate” for extended role certification. “…it’s only ever been GPs who are at the centre of all the networks of information about a patient and there would be patient confidentiality problems if you were relaying it. It would be unnecessary to relay it as you’ve got it all.” GP145 Table 1 Medical problems deemed ‘appropriate’ for extended role sickness certification by GPs, nurses and physiotherapists Primary care nurses Wound care including chronic ulceration, animal bites Self-limiting medical conditions including chest infections, urinary tract infections, Chronic disease management including diabetes, chronic obstructive pulmonary disease, cardiovascular disease Physiotherapists Musculoskeletal conditions including low back pain, tennis elbow, shoulder pain, knee pain, acute injuries, chronic pain Table 1 Medical problems deemed ‘appropriate’ for extended role sickness certification by GPs, nurses and physiotherapists Primary care nurses Wound care including chronic ulceration, animal bites Self-limiting medical conditions including chest infections, urinary tract infections, Chronic disease management including diabetes, chronic obstructive pulmonary disease, cardiovascular disease Physiotherapists Musculoskeletal conditions including low back pain, tennis elbow, shoulder pain, knee pain, acute injuries, chronic pain Physiotherapists similarly acknowledged their inability to access primary care records due to geographical loca- tion and the mobile nature of their work. They attributed the lack of information access as a limitation in holistic practice and thus ability to make informed decisions over sickness certification. One physiotherapist described this obstacle to holistic practice as ‘insurmountable’. Thus, physiotherapists equated access to patient information held by GPs with holistic care. Page 7 of 11 Page 7 of 11 Welsh et al. BMC Family Practice 2014, 15:100 http://www.biomedcentral.com/1471-2296/15/100 Welsh et al. BMC Family Practice 2014, 15:100 http://www.biomedcentral.com/1471-2296/15/100 prescribed and printed out the drugs. A holistic approach And then they say ‘oh you know, I don’t feel I can go back to work’, I agree, and I have to say,‘oh well you will have to come back and see the Doctor’. Or I’ll have to go off and find a Doctor, which is really time-wasting.” Nurse304 Nurses and physiotherapists legitimised their role ex- tension by highlighting their ability to spend more time with patients than GPs, thus achieving a “more in depth” approach to “get to the bottom of what is going on”. In contrast to his colleagues, one GP stated how reduced consultation times and increasing target-driven medicine precluded his ability to practice holistically and therefore undertake sickness certification. Conversely, the physiotherapist who did not agree with natural role progression claimed an extended role would result in increased workload. Some GPs raised concerns over a ‘just-checking’ ideation, whereby nurses and physio- therapists would create more work for GPs by requesting case discussions prior to certification decisions: Control and responsibility One physiotherapist was particularly wary of this dele- gation for little reward. Both GPs and nurses referred to the necessity of protocols and guidelines to inform nurse- led certification practice, although each profession's views had different underpinning reasons. GPs advocated guide- line use to ensure ‘appropriate’ certification and to exercise control over their nursing colleagues’ practice: GPs sought to maintain control over the sickness certification process with claims to be best placed to manage ‘complex’ health problems in patients with knowledge of their specific healthcare needs, and by using a more holistic approach than the other profes- sional groups. GPs advocated role extension to nurses only if conducted under supervision and with the aid of clear protocols, or to save GP time by streamlining the more ‘simple cases’ which did not require a GP’s expertise. Physiotherapists however voiced support for role extension, claiming to possess specialist knowledge of musculoskeletal problems, a common cause for work ab- sence. Although they did not claim to practice holistically, they perceived role extension would improve system ef- ficiency as patients would not need to visit a GP as fre- quently, helping to reduce their workload. In addition, physiotherapists could spend more time with patients to address work absence difficulties and the role exten- sion concept offered physiotherapists the opportunity to extend their skills and perhaps professional status (see Sanders et al.) [52]. Nurses also claimed to have more time to spend with patients and perceived role exten- sion as a natural role progression. They claimed to offer greater accessibility to healthcare than could be offered by GPs, though some would only discharge such a role in the presence of clear protocols to guide their decisions. GPs’ overall control over patient records deprived nurses and physiotherapists of information about patients’ back- ground and medical history; a critical requirement for making sickness certification decisions. “…they’re [practice nurses] very good actually because they don’t go anywhere beyond their competence, which you would expect anyway…they’re entirely willing to take on the policies and the principles that we’ve set them.” GP528 “…they’re [practice nurses] very good actually because they don’t go anywhere beyond their competence, which you would expect anyway…they’re entirely willing to take on the policies and the principles that we’ve set them.” GP528 The less experienced nurses appeared more risk-averse, reluctant to take on added responsibility and subsequent accountability. Control and responsibility Each group used the perceived professional hierarchy to express their professional legitimacy claims differently. The ability to control the form and content of clinical work is a central tenet of the medical profession gener- ally and a significant guiding principal for GPs. GP nar- ratives reflected this through using words and phrases including “oversee”, “like to know what is going on” and “being the central coordinator”. Some GPs refrained from talking about control, preferring instead to use complicit status claims through phrases including “theoretically” and “some people”. Physiotherapists supported their legitimacy claims to role extension through suggesting it “would save an extra trip to the doctors”. Nurses employed accessibility to healthcare to support their role extension claims by highlighting the relative ease of obtaining nurse- appointments. All nurse and five physiotherapist respondents viewed sickness certification as a natural role progression: Nurses used the professional hierarchy and existing GP authority to protect their responsibility for patient care and therefore justify role extension, referring to GPs as “back up”. Physiotherapists saw role extension as an opportunity to strengthen their professional status as “…for me, it would seem like a natural progression of my role…I’ve done everything else: they’ve come in, I’ve assessed them, we’ve discussed what their treatment options could be, we’ve decided on a plan, I’ve “…for me, it would seem like a natural progression of my role…I’ve done everything else: they’ve come in, I’ve assessed them, we’ve discussed what their treatment options could be, we’ve decided on a plan, I’ve Page 8 of 11 Welsh et al. BMC Family Practice 2014, 15:100 http://www.biomedcentral.com/1471-2296/15/100 own credibility in the eyes of their colleagues or as a means of presenting their own skills as superior [14]. Our professionals identified four overlapping legitimacy claims which were used in a variety of ways to support or reject role extension; condition specific legitimacy, holistic care, system efficiency, and, control and responsibility. These discourses were used interchangeably by each of our professions giving rise to a mixed picture which re- vealed diverse opinions about the appropriateness of service redesign in relation to sickness certification practice. autonomous respected practitioners using words including “empower” and “advantage”. All GPs highlighted conflicts of duty to the patient and to the State during work-and- health related consultations and the impact on the doctor-patient relationship. GPs were aware of the pos- sible perception of “dumping” their least desired tasks. Control and responsibility Therefore, protocols were referred to as a means of deferring responsibility to the GP and offering “protection”. Physiotherapists did not refer to the use of protocols or guidelines during their interviews and GPs did not relate the use of guidelines to physiotherapists’ extended role, perhaps indicating their superior status in the eyes of GPs. Discussion d h Extending the authority to certify sickness absence beyond the medical profession in primary care is not a simple matter of addressing organisational obstacles. Role exten- sion is underpinned by the sociological theories of profes- sional identity and boundary work. Respondents employed legitimacy claims to support their views on extension of the sickness certification role beyond the medical profes- sion. Respondents generally supported the concept of role extension for sickness certification, although this support came with conditions including recognition (physiothera- pists), use of guidelines (nurses) and maintenance of over- all patient care control (GP). Rejections of the extended role concept were based upon the perceived challenges to GPs’ dominance, the apparent inability of non-medical professionals to practice holistically and their lack of access to full medical records, the potential for system manipulation and increased GP workload. To summarise the findings, two overarching trends are evident in the data. First, the claim to specialist skill or knowledge by GPs and physiotherapists to deal with sickness certification decisions was a key factor in deter- mining role extension; this was most prevalent in the claims made by GPs and physiotherapists, with the former largely resisting role extension, and the latter supporting it. The second trend related to widespread use of organisa- tional and system efficiency discourses. All three profes- sions utilised the system efficiency discourse, though most strikingly it was used by nurses and physiotherapists, each claiming that role extension would enhance system effi- ciency and therefore patient care. In relation to the field of The sociological literature on professional boundaries has been dominated by analyses of the way professions use ‘science’ to differentiate their unique contribution to patient care as superior to the contribution of other groups or professionals. As noted previously, clinicians may adopt a discourse of ‘science’ as means of strengthening their Page 9 of 11 Welsh et al. BMC Family Practice 2014, 15:100 http://www.biomedcentral.com/1471-2296/15/100 heart failure, Sanders and Harrison [53] also found that occupations lower down the ‘status hierarchy’ in a hospital setting (eg. specialist nurses) predominantly used a system efficiency claim to differentiate their specific contribution to patient care from cardiologists and geriatricians, who did not have time to address patients’ information needs and provide preventative care. undertaking. Although the majority of respondents sup- ported the concept, the views of the ‘sceptics’ are equally important. Study limitations R d i h Respondents with a range of characteristics were inter- viewed to ensure a spread of opinions was captured. The relatively small sample risks overlooking alternative views, particularly in the physiotherapist group where some add- itional insights continued to emerge during the final inter- view. However, these were not related to the ‘core’ themes of legitimacy claims and boundary work explored in the current analysis. Thus we are reasonably confident that the main subject of analysis presented in this paper has been explored in depth without key issues having been missed or overlooked. Physiotherapist recruitment through associations with the host Research Centre raises the pos- sibility that responses were tailored to avoid impact upon future relationships. However, this is unlikely given the range of physiotherapist views elicited. Most physiothera- pists had received specialist musculoskeletal training, a sampling strength since the second most common reason for sickness certification is musculoskeletal ill-health [55]. The interviewer’s occupation, a GP-trainee, was disclosed prior to interview commencement. This may have influ- enced interviews, for example one physiotherapist used de- tailed clinical language when referring to musculoskeletal pain and the impact on sickness certification, perhaps to present themselves as technically competent to the GP trainee researcher. In addition a nurse interviewee expressed some ‘negative’ views towards GPs. Telephone interviews may restrict rapport development and recognition of non- verbal cues. In this study, the degree of anonymity afforded through telephone use noticeably encouraged participation in a potentially sensitive topic area [56]. We are also aware that the relatively low response rate from GPs may lead to the exploration of views from a select group of respon- dents, although the recruitment of GPs has always pre- sented this dilemma in other qualitative (and quantitative) research. We do not seek to claim that our findings are ‘representative’ or generalizable to the entire population of GPs, nurses and physiotherapists working in the UK, but that the findings identify important insights some (if not most) are likely to be held by a wider group of clinicians. Further in-depth qualitative research on this topic is there- fore required to build on the themes presented here. The equation of a holistic approach with access to GP held information is not widely reported in the literature. Study limitations R d i h It could be argued that physiotherapists should not be precluded from delivering holistic care, including sick- ness certification, on the basis of information access since the amount of information known is a matter be- tween healthcare professionals and patients. Discussion d h For instance, implementing such a change is not simply a case of overcoming practical and organisa- tional barriers. Our study demonstrates that in relation to role extension, professions hold deeply-entrenched values that are underpinned by professional identities. Further exploration of these values is required to understand spe- cific professional responses to organisational change and aid planning and implementation of future primary care role extension, including the task of sickness certification. Neither physiotherapists nor nurses explicitly used the discourse of ‘medicine’ or ‘science’ to differentiate them- selves from GPs, as in Foley and Fairclough’s [21] study which reported that midwives used a discourse of ‘medi- cine’ to attempt to establish themselves as equal to doc- tors, because they too used ‘science’ in their work. Both occupations referred to the practical benefits they could bring to primary care through enhancing system efficiency and thus their global contribution to patient care. The close proximity of nurses to GPs in clinical practice may also have had an influence on their claims, with nurses uti- lising only a managerial discourse of system efficiency without claiming to possess the same technical expertise as GPs. Perhaps seeking only to show how they could add value to an existing set of practices rather than replacing or upstaging the current GPs’ role. They claimed to have more time to dedicate to patients, but stopped short of espousing superior knowledge claims. Physiotherapists however used their technical expertise in the manage- ment of musculoskeletal problems to distinguish their contribution from GPs as potentially superior. They adopted both the language of technical skill and system efficiency to support role extension. On the whole phys- iotherapists and nurses utilised a largely ‘proactive’ stance emphasising their positive contribution to sickness certifi- cation. GP’s claims were largely ‘defensive’; attempting to exclude nurses and physiotherapists from the jurisdiction of sickness certification practice, whilst allowing minor ad- justments to the current system such as through ‘delega- tion of dirty work’, a strategy used to reinforce the medical model of dominance by doctors through determination of nursing and physiotherapy boundaries [54]. Acknowledgements 21. Foley L, Faircloth CA: Medicine as discursive resource: legitimation in the work narratives of midwives. Sociol Health Illn 2003, 25:165–184. Study approval was granted from the North Staffordshire Research Ethics Committee (10/H1204/10). Study approval was granted from the North Staffordshire Research Ethics Committee (10/H1204/10). 22. ONS: 2012. http://www.ons.gov.uk/ons/dcp171776_265016.pdf. 22. ONS: 2012. http://www.ons.gov.uk/ons/dcp171776_265016.pdf. This paper summarises independent research funded by the National Institute for Health Research (NIHR) under its Academic Clinical Fellowship and its Doctoral Research Fellowship schemes. The views expressed in this paper are those of the author(s) and not necessarily those of the NHS, the NIHR or the Department of Health. 23. CBI absence and workplace survey May 2011. http://www.cbi.org.uk/media/ 955604/2011.05-healthy_returns_-_absence_and_workplace_health_survey_2011. pdf. 24. Health, Work and Well-being Directorate: Reforming the medical statement. Consultation on draft regulations, 2009. London: Department of Work and Pensions; 2009. http://www.dsdni.gov.uk/reforming-the-medical- statement-consultation-28may2009.pdf [accessed 6 Mar 2012]. VKW is funded through the National Institute of Health Research Academic Clinical Fellowship. CDM is funded by an Arthritis Research UK Clinical VKW is funded through the National Institute of Health Research Academic Clinical Fellowship. CDM is funded by an Arthritis Research UK Clinical Scientist Award. Gwenllian Wynne-Jones is funded by a National Institute for Health Research Post-Doctoral Fellowship (PDF-2009-02-54). VKW is funded through the National Institute of Health Research Academic Clinical Fellowship. CDM is funded by an Arthritis Research UK Clinical statement-consultation-28may2009.pdf [accessed 6 Mar 2012]. y Scientist Award. Gwenllian Wynne-Jones is funded by a National Institute for Health Research Post-Doctoral Fellowship (PDF-2009-02-54). Scientist Award. Gwenllian Wynne-Jones is funded by a National Institute for Health Research Post-Doctoral Fellowship (PDF-2009-02-54). 25. Department for Work and Pensions Annual Report & Accounts 2012-13. London: The Stationary Office. https://www.gov.uk/government/uploads/ system/uploads/attachment_data/file/264555/dwp-annual-report-accounts- 2012-2013.pdf. We gratefully acknowledge the GPs, nurses and physiotherapists who took part in the study. We gratefully acknowledge the GPs, nurses and physiotherapists who took part in the study. 26. Department of Health: General Medical Services: standard contract, 2009. London: GMC; 2009. Received: 14 January 2014 Accepted: 13 May 2014 Published: 17 May 2014 27. Wynne-Jones G, Mallen CD, Main CJ, Dunn KM: Sickness certification and the general practitioner: what really happens in practice? Fam Pract 2010, 27(3):344–350. Competing interests 15. Welsh S, Kelner M, Wellman B, Boon H: Moving forward? Complementary and alternative practitioners seeking self-regulation. Sociol Health Illn 2004, 26:216–241. There are no competing interests to declare. 16. Fryer GE: The United States medical profession: an abnormal form of the division of labour. Sociol Health Illn 1991, 13:213–230. 16. Fryer GE: The United States medical profession: an abn Authors’ contributions division of labour. Sociol Health Illn 1991, 13:213–230. 17. Jeffery R: Normal rubbish: deviant patients in casualty departments. VW carried out the qualitative fieldwork, took a lead in the analysis and drafted the manuscript. TS contributed to the analysis, conception of the manuscript and writing up. JR contributed to the analysis, conception of the manuscript and writing up. GWJ contributed to the analysis and writing up. CJ contributed to the analysis and helped to draft the manuscript. CM contributed to the analysis, conception of the manuscript and writing up. All authors read and approved the final manuscript. VW carried out the qualitative fieldwork, took a lead in the analysis and drafted the manuscript. TS contributed to the analysis, conception of the manuscript and writing up. JR contributed to the analysis, conception of the manuscript and writing up. GWJ contributed to the analysis and writing up. 17. Jeffery R: Normal rubbish: deviant patients in casualty d 17. Jeffery R: Normal rubbish: deviant patients in casualty departments. Sociol Health Illn 1979, 1(1):91–98. Sociol Health Illn 1979, 1(1):91–98. 18. Dingwall R: The Social Organisation of Health Visitor Training. London: Croom Helm; 1977. 19. Scott WR: Organisations: Rational, Natural and Open Systems. 3rd edition. New Jersey: Prentice Hall; 1992. CJ contributed to the analysis and helped to draft the manuscript. CM contributed to the analysis, conception of the manuscript and writing up. All authors read and approved the final manuscript. 20. Harrison S, Dowswell G, Wright J: Practice nurses and clinical guidelines in a changing primary care context: an empirical study. J Adv Nurs 2002, 39(3):1–10. References 28. Hiscock J, Ritchie J: The role of GPs in sickness certification. (Research report No 148). London: Department of Work and Pensions; 2001. 1. Roland M: Linking physician pay to quality of care: a major experiment in the UK. N Engl J Med 2004, 351:1448–1454. 1. Roland M: Linking physician pay to quality of care: a major experiment in the UK. N Engl J Med 2004, 351:1448–1454. 29. Health, Work and Well-Being Directorate: Reforming the Medical Statement: Gov- ernment response to the consultation on draft regulations: The Social Security (Medical Evidence) and the Statutory Sick Pay (Medical Evidence) (Amendment) Regulations, 2010. London: Department of Work and Pensions; 2010. 2. Department of Health, NHS Service Delivery and Organisation R&D Programme: Evaluating models of service delivery. Briefing paper: Extending the practice of allied health professionals in the NHS. London: National Coordinating Centre for the Service Delivery and Organisation; 2006. 2. Department of Health, NHS Service Delivery and Organisation R&D Programme: Evaluating models of service delivery. Briefing paper: Extending the practice of allied health professionals in the NHS. London: National Coordinating Centre for the Service Delivery and Organisation; 2006. 30. Black C: Review of the health of Britain's working age population: Working for a healthier tomorrow. London: The Stationary Office; 2008. 3. Royal College of General Practitioners: Primary care practice and its team: RCGP information sheet. London: Royal College of General Practitioners; 2007. 31. Department for Work and Pensions: Fitness for work: the Government response to ‘Health at work – an independent review of sickness absence’; 2013. 4. Holdsworth LK, Webster VS, McFadyen AK: Physiotherapists’ and general practitioners’ views of self-referral and physiotherapy scope of practice: results from a national trial. Physiotherapy 2008, 94(3):236–244. 32. Aronsson G, Gustafsson K: Sickness presenteeism: prevalence, attendance-pressure factors, and an outline of a model for research. J Occup Env Med 2005, 47(9):958–966. 33. Barnes MC, Buck R, Williams G, Webb K, Aylward M: Beliefs about common health problems and work: a qualitative study. Soc Sci Med 2008, 67(4):657–665. 5. Kelly A, Neale J, Rollings R: Barriers to extended nurse prescribing among practice nurses. Community Pract 2010, 83(1):21–24. 5. Kelly A, Neale J, Rollings R: Barriers to extended nurse prescribing among practice nurses. Community Pract 2010, 83(1):21–24. p y 6. Laurant M, Reeves D, Hermens R, Braspenning J, Grol R, Sibbald B: Substitution of doctors by nurses in primary care. Cochrane Database Syst Rev 2004, 4:CD001271. doi:10.1002/ 14651858.CD001271. Conclusions The use of protocols to guide tasks appropriate for nurse- delegation in primary care is known [7]; the present study suggests that GPs also used protocols and guidelines to exert control through defining occupational boundaries. Extending the authority to certify sickness absence to nurses and physiotherapists in primary care is a complex Page 10 of 11 Welsh et al. BMC Family Practice 2014, 15:100 http://www.biomedcentral.com/1471-2296/15/100 References 6. Laurant M, Reeves D, Hermens R, Braspenning J, Grol R, Sibbald B: Substitution of doctors by nurses in primary care. Cochrane Database Syst Rev 2004, 4:CD001271. doi:10.1002/ 14651858.CD001271. 34. Blaxter M: Health. California: Cambridge: Policy Press; 2004. 35. Johansson G, Lundberg I: Adjustment latitude and attendance requirements as determinants of sickness absence or attendance. Empirical tests of the illness flexibility model. Soc Sci Med 2004, 58(10):1857–1868. 7. Allen D: The nursing-medical boundary: a negotiated order? Sociol Health Illn 1997, 19:498–520. 7. Allen D: The nursing-medical boundary: a negotiated order? Sociol Health Illn 1997, 19:498–520. 36. Nettleton S: ‘I just want permission to be ill’: towards a sociology of medically unexplained symptoms. Soc Sci Med 2006, 62(5):1167–1178. 8. Svensson R: The interplay between doctors and nurses: a negotiated order perspective. Sociol Health Illn 1996, 18:379–398. 37. Pinder R: Bringing back the body without the blame? The experience of ill and disabled people at work. Sociol Health Illn 1995, 17(5):605–631. 9. Gieryn TF: Boundary work and the demarcation of science from non-science: strains and interests in professional ideologies of scientists. Am Sociol Rev 1983, 48:781–795. 38. Williams GH: Chronic illness and the pursuits of virtue in everyday life. In Worlds of Illness: Biographical and Cultural Perspectives of Health and Disease. Edited by Radley A. London: Routledge; 1993. 10. Abbott A: The System of Professions: An Essay on the Division of Expert Labour. Chicago. Chicago: University of Chicago Press; 1988. 39. Norrmén G, Svärdsudd K, Andersson DKG: How primary health care physicians make sick listing decisions: The impact of medical factors and functioning. BMC Fam Pract 2008, 9:3. 11. Mizrachi N, Shuval JT, Gross S: Boundary at work: alternative medicine in biomedical settings. Sociol Health Illn 2005, 27:20–43. 12. Charles-Jones H, Latimer J, May C: Transforming general practice: the redistribution of medical work in primary care. Sociol Health Illn 2003, 25:71–92. 40. Krohne K, Brage S: How GPs in Norway conceptualise functional ability: a focus group study. Br J Gen Pract 2008, 58(557):850–855. 13. Tolliday H: Clinical autonomy. In Health Services: Their nature and organization and the role of patients, doctors, nurses and the complementary professions. Edited by Jacques E. London: Heinemann; 1978. 41. Hussey S, Hoddinott P, Wilson P, Dowell J, Barbour R: Sickness certification system in the United Kingdom: qualitative study of views of general practitioners in Scotland. Br Med J 2004, 328:88. 10 January. 42. References Von Knorring M, Sundberg L, Lofgren A, Alexanderson K: Problems in sickness certification of patients: a qualitative study on views of 26 physicians in Sweden. Scan J Prim Health 2008, 26:22–28. 14. Timmons S, Tannner J: A disputed occupational boundary: operating theatre nurses and operating department practitioners. Sociol Health Illn 2004, 26:645–666. Page 11 of 11 Page 11 of 11 Welsh et al. BMC Family Practice 2014, 15:100 http://www.biomedcentral.com/1471-2296/15/100 Welsh et al. BMC Family Practice 2014, 15:100 http://www.biomedcentral.com/1471-2296/15/100 43. Cohen D, Marfell N, Webb K, Robling M, Mansel A: Managing long-term worklessness in primary care: a focus group study. Occup Med 2010, 60:121–126. 44. Vallance-Owen A: Health Insurance. ; 2009. 45. Gulbrandsen P, Hofoss D, Nylenna M, Saltyte-Benth J, Aasland OG: General practitioners’ relationship to sickness certification. Scand J Prim Health 2007, 25(1):20–26. 46. Harvey S, Henderson M, Lelliott P, Hotopf M: Mental health and employment: much work still to be done. Br J Psychiat 2009, 194:201–203. 47. Vaez M, Rylander G, Nygren A, Asberg M, Alexanderson K: Sickness absence and disability pension in a cohort of employees initially on long term sick leave due to psychiatric disorders in Sweden. Soc Psych Psych Epid 2007, 42:381–388. 48. Stansfield S, Feeney A, Head J, Canner R, North F, Marmot M: Sickness absence for psychiatric illness: the Whitehall II study. Soc Sci Med 1995, 40(2):189–197. 49. Munir F, Leka S, Griffiths A: Dealing with self management of chronic illness at work: predictors for self disclosure. Psychol Health 2005, 13(4):717–733. 50. Munir F, Yarker J, Haslam C, Long H, Leka S, Griffiths A, Cox S: Work factors related to psychological and health-related distress among employees with chronic illnesses. J Occup Rehabil 2007, 17(2):259–277. 51. Munir F, Yarket J, Haslam C: Sickness absence management: encouraging attendance or ‘risk-taking’ presenteeism in employees with chronic illness? Disabil Rehabil 2008, 30(19):1461–1472. 52. Sanders T, Ong BN, Sowden G, Foster N: Implementing change in physiotherapy: professions, contexts and interventions. J Health Organisat Manag 2014, 28(1):96–114. 53. Sanders T, Harrison S: Professional legitimacy claims in the multidisciplinary workplace: the case of heart failure care. Sociol Health Illn 2008, 30(2):289–308. 54. Hughes EC EC: Men and their Work. Toronto: Free Press of Glencoe, 1958. Quoted by Nancarrow SA, Borthwich AM. Dynamic professional boundaries in the healthcare workforce. Sociol Health Illn 2005, 27(7):897–919. 55. References Wynne-Jones G, Mallen CD, Mottram S, Main CJ, Dunn KM: The identification of UK sickness certification rates, standardised for age and sex. Br J Gen Pract 2009, 59(564):510–515. 56. Welsh VK, Mallen CD, Wynne-Jones G, Jinks C: Exploration of GPs’ views and use of the fit note: a qualitative study in primary care. Br J Gen Pract 2012, 62(598):e363–e370. 56. Welsh VK, Mallen CD, Wynne-Jones G, Jinks C: Exploration of GPs’ views and use of the fit note: a qualitative study in primary care. Br J Gen Pract 2012, 62(598):e363–e370. doi:10.1186/1471-2296-15-100 Cite this article as: Welsh et al.: Extending the authority for sickness certification beyond the medical profession: the importance of ‘boundary work’. BMC Family Practice 2014 15:100. 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https://openalex.org/W3006042865	https://europepmc.org/articles/pmc6961302?pdf=render	English	null	Early administration of fibrinogen concentrate is associated with improved survival among severe trauma patients: a single-centre propensity score-matched analysis	World journal of emergency surgery	2,020	cc-by	7,004	RESEARCH ARTICLE Open Access Itagaki et al. World Journal of Emergency Surgery (2020) 15:7 https://doi.org/10.1186/s13017-020-0291-9 Itagaki et al. World Journal of Emergency Surgery (2020) 15:7 https://doi.org/10.1186/s13017-020-0291-9 Itagaki et al. World Journal of Emergency Surgery https://doi.org/10.1186/s13017-020-0291-9 Early administration of fibrinogen concentrate is associated with improved survival among severe trauma patients: a single-centre propensity score-matched analysis Yuki Itagaki1* , Mineji Hayakawa2, Kunihiko Maekawa2, Tomoyo Saito2, Akira Kodate1, Yoshinori Honma2, Asumi Mizugaki2, Tomonao Yoshida2, Takayoshi Ohyasu2, Kenichi Katabami2 and Takeshi Wada2 Abstract Background: Fibrinogen plays an important role in haemostasis during the early phase of trauma, and low fibrinogen levels after severe trauma are associated with haemostatic impairment, massive bleeding, and poor outcomes. Aggressive fibrinogen supplementation may improve haemostatic function, as fibrinogen levels deteriorate before other routine coagulation parameters in this setting. Therefore, we evaluated whether early administration of fibrinogen concentrate (FC) was associated with improved survival in severe trauma patients. Methods: This single-centre retrospective study evaluated patients with severe trauma (injury severity score ≥16) who were admitted to our emergency department between January 2010 and July 2018. The exclusion criteria included age < 18 years, cardiac arrest before emergency department arrival, cervical spinal cord injury not caused by a high-energy accident, and severe burn injuries. The FC and control groups included trauma patients who received and did not receive FC within 1 h after emergency department arrival, respectively. Propensity scores were used to balance the two groups based on the trauma and injury severity score (TRISS), heart rate at emergency department admission, and age. The primary outcome was the in-hospital survival rate. Results: The propensity scoring model had a c-statistic of 0.734, the Hosmer-Lemeshow chi-squared value was 7.036 (degrees of freedom = 8), and the non-significant p value of 0.533 indicated a good model fit. The propensity score matching created 31 matched pairs of patients, who had appropriately balanced characteristics. The FC group had a significantly higher in-hospital survival rate than the control group (log-rank p = 0.013). The FC group also used significantly higher amounts of red blood cells and fresh frozen plasma within 6 h after emergency department admission. However, the two groups had similar transfusion amounts between 6 and 24 h after emergency department admission. Conclusions: The present study revealed that early FC administration was associated with a favourable survival rate among severe trauma patients. Therefore, FC may be useful for the early management of trauma-induced coagulopathy and may improve outcomes in this setting. Keywords: Cryoprecipitate, Fibrinogen, Fibrinogen concentrate, Fresh frozen plasma, Trauma-induced coagulopathy * Correspondence: koaraninaritaizo@gmail.com * Correspondence: koaraninaritaizo@gmail.com 1Emergency and Critical Care Center, Sapporo City General Hospital, 1-1 Nishi13, Kita 11, Kita-ku, Sapporo, Hokkaido 060-8604, Japan Full list of author information is available at the end of the article Background factor XIII, and von Willebrand factor (unlike FC), cryopre- cipitate also requires thawing before administration [26] and carries a risk of viral infection, similar to FFP [35, 36]. Therefore, although aggressive fibrinogen replacement therapy using FFP or cryoprecipitate provides favourable outcomes [17, 37], this benefit must be balanced with the immediate availability and rapid administration of FC, which does not require thawing or confirmation of ABO compatibility [38]. Furthermore, FC administration may in- crease plasma fibrinogen levels more easily than FFP [31] and may produce a greater increase in fibrinogen levels more rapidly than both FFP and cryoprecipitate [39]. g Trauma remains a major cause of death [1, 2], which is primarily related to uncontrolled bleeding during the early phase of trauma [3]. Traumatic haemorrhage may be exacerbated by coagulopathy (i.e. trauma-induced coagulopathy). Although the pathophysiology of trauma- induced coagulopathy remains incompletely understood [4–8], we speculate that it is generated by the following mechanisms: (1) coagulation activation, (2) hyperfibri- no(geno)lysis, and (3) consumption coagulopathy [5, 6]. Coagulation activation caused by massive tissue injuries cause excessive thrombin generation, which leads to the fibrinogen consumption. Hyperfibrino(geno)lysis is caused by the acute release of tissue-plasminogen acti- vator, which is induced by tissue hypoperfusion and massive tissue injuries-induced coagulation activation. Various coagulation factors and platelets are consumed by coagulation activation and hyperfibrino(geno)lysis. Nevertheless, trauma-induced coagulopathy is often clearly present on emergency department (ED) arrival and is asso- ciated with massive haemorrhage, increased transfusion needs, and a high mortality rate [9–14]. Unfortunately, in patients with severe trauma, haemostatic impairment is worsened by haemodilution, hypothermia, and acidosis during the early phases of treatment [5, 7, 15, 16]. There- fore, better management of trauma-induced coagulopathy is needed to improve the outcomes of these patients. p y y p p Several reports have indicated that FC administration is effective for patients with severe trauma [18, 38, 40, 41]. For instance, Wafaisade et al. retrospectively examined the effects of FC administration and reported that it helped improve the short- and not long-term mortality rate [18]. Nevertheless, their FC group included patients who were treated in the ED and intensive care unit; this partially obscured the effects of early FC administration [18]. A single-centre randomised controlled trial (RCT) using real- time thromboelastometry also revealed that relative to FFP, coagulation factor concentrates (including FC, prothrombin complex concentrate, and factor XIII concentrate) helped improve outcomes in patients with severe trauma [41]. © The Author(s). 2020 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Page 2 of 10 Page 2 of 10 Itagaki et al. World Journal of Emergency Surgery (2020) 15:7 Background However, that trial failed to clarify how the patients’ out- comes varied according to the use of FC, prothrombin com- plex concentrate, and factor XIII concentrate [41]. Akbari et al. also performed a single-centre RCT and reported that patients with severe trauma who received FC had a signifi- cantly lower mortality rate and shorter duration of hospital- isation than those who received FFP and the control group [38]. However, that report failed to clearly describe the timing of FC administration [38]. Therefore, to the best of our knowledge, no studies have specifically examined the early administration of FC in patients with severe trauma. The present study aimed to determine whether this strategy improved survival, based on a propensity score-matched analysis. p p Fibrinogen plays an important role in haemostasis during the early phase of trauma [16–22], as low fibrinogen levels impair the firmness of the fibrin clots that help to control haemostasis. Fibrinogen also accelerates platelet aggrega- tion [6, 23, 24], and many studies have indicated that low fibrinogen levels at ED arrival are associated with haemo- static impairment, massive bleeding, and poor outcomes in patients with severe trauma [11–14, 16, 24–28]. Further- more, fibrinogen levels deteriorate faster than other haemo- static components during the early phase of severe trauma [6, 11, 27, 29]. Therefore, decreased fibrinogen levels are an important marker for trauma-induced coagulopathy, and fi- brinogen supplementation is needed to help maintain haemostatic function [6, 16]. Recent European guidelines have suggested that fibrinogen concentrations should be maintained at > 1.5–2.0 g/L in patients with severe trauma [30], and there is increasing awareness that fibrinogen concentrate (FC) can be effective for managing massive haemorrhage in these patients. Fibrinogen supplementation can be achieved by using fresh frozen plasma (FFP) and cryoprecipitate [31]. However, FFP must be thawed via a time-consuming process [32], and ABO compatibility must be confirmed before administering FFP [33]. Certain trauma centres have recently begun early coagulation factor supplementation using pre-thawed FFP; however, thawed plasma has a short shelf-life and must be discarded if it is not used [34]. While cryoprecipitate contains factor VIII, Definitions The patients were divided into an FC group (received FC within 1 h after ED arrival) and a control group (no FC or FC received at 1–24 h after ED arrival). The deci- sion to administer FC, its timing, and the FC amount were fully at the discretion of the attending physicians. The administered FC was commercially available freeze- dried human fibrinogen (Fibrinogen HT i.v. 1 g “JB”, Japan Blood Products Organization, Tokyo, Japan). On sub-group analysis, severe brain injury was defined as injury with a head abbreviated injury scale (AIS) of ≥3. In the two sub-groups, namely, patients with blunt trauma and with severe brain injury, additional analyses were performed using the same methods. All analyses were performed using SPSS software (ver- sion 25; IBM Japan, Tokyo, Japan). All reported p values were two-tailed, and differences were considered statisti- cally significant at p values of < 0.05. Patient selection and data collection This single-centre retrospective study evaluated elec- tronic medical records from a tertiary emergency and critical care centre (Hokkaido University Hospital). The study protocol was approved by our institutional review board, and the requirement for informed consent was waived owing to the retrospective design. Adult patients with severe trauma (injury severity score ≥16) who were directory admitted to our ED be- tween January 2010 and July 2018 were eligible for inclu- sion. Patients were excluded based on the following criteria: (a) age < 18 years, (b) cardiac arrest before ED arrival, (c) cervical spinal cord injury not caused by a Page 3 of 10 Page 3 of 10 Itagaki et al. World Journal of Emergency Surgery (2020) 15:7 Itagaki et al. World Journal of Emergency Surgery high-energy accident, and (d) severe burn injuries. The records of eligible patients were searched to collect data regarding trauma severity, laboratory test results from ED arrival, clinical characteristics, treatments, transfu- sion amounts, and patient outcomes. and a calliper width of 0.2 standardised deviations for the propensity score. We used the standardised difference to evaluate the covariate balances after the propensity score matching, with absolute standardised differences of > 0.1 considered indicative of a meaningful imbalance. The two groups were then compared using the Mann-Whitney U and chi-squared tests, as appropriate. In-hospital survival outcomes were compared using the Kaplan-Meier method and log-rank tests. Statistical analysis During the study period, 480 patients with severe trauma were directly transferred to our ED from the accident site. After the exclusion of ineligible patients, 148 eligible patients were divided into the FC group (38 patients) and the control group (110 patients) (Fig. 1). The pa- tients’ overall characteristics are shown in Table 1; it shows that the FC group had a significantly higher crit- ical status on ED admission. The FC group included trauma patients who received FC within 1 h after ED arrival (n = 38), and the control group included 110 Propensity score matching was used to balance the groups’ characteristics and clinical variables. The propen- sity scores for early FC administration were estimated using a logistic regression model, based on the trauma and injury severity score, heart rate at ED admission, and age, all of which are related to the early administration of FC. Patients with and without early FC administration were then matched 1:1 based on their propensity scores, using the nearest neighbour method without replacement, Fig. 1 Study flowchart. The fibrinogen concentrate (FC) group included trauma patients who received FC within 1 h after their emergency department admission. The control group included patients who did not receive FC within 1 h after emergency department admission. ISS, injury severity score Fig. 1 Study flowchart. The fibrinogen concentrate (FC) group included trauma patients who received FC within 1 h after their emergency department admission. The control group included patients who did not receive FC within 1 h after emergency department admission. ISS, injury severity score Itagaki et al. FC fibrinogen concentrate, ED emergency department, AIS abbreviated injury scale, PT-INR prothrombin time-international normalised ratio, FDP fibrin/fibrinogen degradation products , ED emergency department, AIS abbreviated injury scale, PT-INR prothrombin time-international normalised ratio, FDP fibrin/fibrinogen Statistical analysis The FC group had a significantly higher in- hospital survival rate (log-rank p = 0.013) and a significantly lower 28-day in-hospital mortality rate (6/31 patients [19.3%] vs. 14/31 patients [45%], p = 0.03). During the first 28 days, 16% of patients in the FC group (5/31 patients) had died owing to a brain injury, which was not signifi- cantly lower than the 32% rate in the control group (10/31 patients). The rates of haemorrhage-related deaths in the FC and control groups were 0% (0/31 patients) and 6% (2/ 31 patients), respectively. Table 3 shows the haemostatic treatments and transfu- sion requirements in the matched groups, which revealed that the two groups had similar frequencies of haemostatic interventions. The two groups had similar amounts of total FC during the first 24 h after ED admission (p = 0.96). The FC group had higher transfusion amounts dur- ing the first 6 h after ED admission; however, no signifi- cant inter-group differences were observed between 6 and 24 h after ED admission. In patients with blunt trauma (n = 142), the propensity score matching process ultimately selected 29 patients from each group (Additional file 1: Table S1), and the FC group had a significantly higher survival rate than the control group (p = 0.034) (Additional file 2: Figure S1). We additionally analysed patients with severe brain injury (head AIS ≥3, n = 97). The propensity score matching process ultimately selected 20 patients from each group (Additional file 1: Table S2); the FC group tended to have a higher survival rate than the control group; however, the difference lacked statistical significance (p = 0.174) (Additional file 2: Figure S2.). Several previous reports have indicated that FC admin- istration provides various advantages in patients with severe trauma [18, 38, 40, 41]; however, those studies did not specifically examine the time point(s) for FC ad- ministration. In our centre, FFP is mainly used for the supplementation of coagulation factors in patients with severe trauma. In addition, FC can be used before starting the FFP administration and/or to boost the fibrinogen levels during FFP administration. Therefore, we evaluated the effects of early FC administration in this setting, which revealed fairly clear advantages for this early treatment strategy. Although the difference was not significant, we observed that the FC group had approximately one-half the number of brain injury-related deaths in the control group. Statistical analysis World Journal of Emergency Surgery (2020) 15:7 Page 4 of 10 Table 1 Characteristics of the patients Table 1 Characteristics of the patients Control group n = 110 FC group n = 38 p value Age (year) 54 (39–71) 53 (32–72) 0.565 Gender, male 86 (78.2%) 20 (52.6%) 0.005 Blunt trauma 106 (96.4%) 36 (94.7%) 0.647 Mechanism of the injury Traffic accident (in the car) 29 (26.4%) 13 (4.2%) 0.313 Traffic accident (pedestrian) 29 (26.4%) 13 (34.2%) Fall 39 (35.5%) 10 (26.3%) Invert 6 (5.5%) 0 (0.0%) Stab wound/cutting 3 (2.7%) 2 (5.3%) Others 4 (3.6%) 0 (0.0%) Injury to the admission to ED 0–30 (min) 12 (10.9%) 9 (23.7%) 0.138 30–60 (min) 64 (58.2%) 21 (55.3%) 60–90 (min) 21 (19.1%) 3 (7.9%) 90– (min) 13 (11.8%) 5 (13.2%) Revised trauma score 6.72 (5.03–7.84) 5.68 (4.09–6.08) < 0.001 Probability of survival 0.842 (0.630–0.943) 0.567 (0.230–0.832) < 0.001 Vital signs on the admission to ED Heart rate (per min) 86 (72–105) 110 (86–120) 0.010 Glasgow Coma Scale 11 (6–14) 6 (3–13) 0.035 Systolic Blood Pressure (mmHg) 120 (99–151) 93 (68–137) 0.014 Respiratory rate (per min) 22 (16–25) 21 (17–30) 0.489 Injury severity score 25 (20–32) 34 (25–41) < 0.001 Abbreviated injury scale Head/neck 4 (1–5) 4 (0–5) 0.264 AIS face 0 (0–0) 0 (0–1) 0.482 AIS chest 3 (0–4) 3 (0–4) 0.104 AIS abdomen 0 (0–2) 0 (0–3) 0.065 AIS extremity 1 (0–3) 2 (0–3) 0.047 AIS external 1 (0–1) 1 (0–1) 0.082 Blood gas analysis pH 7.35 (7.30–7.38) 7.24 (7.11–7.35) < 0.001 Base deficit (mmol/L) 2.5 (0.3–5.4) 6.95 (2.7–14.63) < 0.001 Lactate (mmol/L) 3.2 (2.3–4.7) 5.8 (3.6–10.0) < 0.001 Laboratory tests Platelet (× 103/μL) 200 (147–235) 193 (146–234) 0.632 PT-INR 1.06 (0.98–1.158) 1.22 (1.12–1.40) < 0.001 Fibrinogen (mg/dL) 193 (159–235) 156 (137–219) 0.034 FDP (μg/mL) 76.4 (30.0–165.0) 99.5 (45.0–188.0) 0.349 D-dimer (μg/mL) 47.8 (19.5–104.2) 58.3 (28.3–104.9) 0.633 FC fibrinogen concentrate, ED emergency department, AIS abbreviated injury scale, PT-INR prothrombin time-international normalised ratio, FDP fibrin/fibrinogen degradation products Itagaki et al. World Journal of Emergency Surgery (2020) 15:7 Page 5 of 10 Page 5 of 10 patients who did not receive FC within 1 h after ED arrival (39/110 received FC within 1–24 h after admis- sion and 71/110 did not receive FC). low fibrinogen levels at ED arrival are associated with haemostatic impairment, massive bleeding, and poor out- comes in patients with severe trauma [11–14, 16, 24–28]. Statistical analysis Therefore, early fibrinogen supplementation will help manage trauma-induced coagulopathy [16–22]. The propensity score model had a c-statistic of 0.734, which indicated good discrimination between the patients assigned to the FC and control groups. The Hosmer- Lemeshow chi-squared value was 7.036 (degrees of freedom = 8), and the non-significant p value of 0.533 indicated a good model fit. The propensity score matching process ultimately selected 31 patients from each group, and the characteristics of the matched patients are shown in Table 2. The two groups had generally well-balanced characteristics including the probability of survival, which provides a comprehensive assessment of trauma severity. Most imbalanced variables were more severe in the FC group than in the control group. Fibrinogen levels deteriorate faster than other haemo- static components during the early phase of severe trauma [6, 11, 27, 29], and early fibrinogen supplementation is crucial for maintaining haemostatic function [16]. In this context, two RCTs have examined the feasibility of early FC administration [39, 42]. Nascimento et al. performed a single-centre RCT that examined FC administration within 50 min after ED admission of patients with severe trauma, and concluded that this approach helped increase plasma fibrinogen levels; however, they acknowledged the need for larger RCTs [42]. However, a second multi- centre RCT examined FC administration within 45 min after ED admission in patients with severe trauma, and found that this approach was not feasible as only 69% of the patients received the intervention within 45 min (ver- sus an intended proportion of 90% of patients receiving the early intervention) [39]. Interestingly, both trials were granted a waiver for obtaining informed consent by the relevant ethics committees [39, 42]. However, if an RCT is planned to evaluate the effects of early FC administration for patients with severe trauma, the same non-consent process may not be approved in other regions including Japan. The present study revealed that early FC adminis- tration can easily be performed within 1 h, and was associ- ated with a favourable survival rate after severe trauma in a real clinical setting. Moreover, the FC and control groups had used similar total amounts of FC during the first 24 h after ED admission; however, delayed FC administration (i.e. at 1–24 h after ED admission) was not associated with the same improvement in se- vere trauma outcomes. Figure 2 shows the matched groups’ Kaplan-Meier survival curves. Statistical analysis Further- more, on sub-group analysis of patients with severe brain injury, although there were no significant differences, we observed that early FC administration tended to improve Discussion The present study is the first to indicate that early FC administration (< 1 h after ED admission) may be useful for patients with severe trauma, based on a propensity score-matched analysis. Many studies have indicated that Itagaki et al. World Journal of Emergency Surgery (2020) 15:7 Page 6 of 10 the survival rate of the patients. Discussion In patients with severe brain injury hyperfibrinolysis is frequently observed at ad- of intracranial haematomas, trauma-induced coagulopathy, and poor outcomes [43 45–47] In this context FC may Table 2 Characteristics of the propensity score-matched patients Control group n = 31 FC group n = 31 Matched standardised difference p value Age (years) 55 (36–72) 53 (32–74) −0.006 0.978 Gender, male 25 (80.6) 19 (61.3) −0.435 0.093 Blunt trauma 29 (93.5%) 29 (93.5%) < 0.001 1.000 Mechanism of the injury Traffic accident (in the car) 12 (38.7%) 12 (38.7%) < 0.001 1.000 Traffic accident (pedestrian) 11 (35.5%) 11 (35.5%) < 0.001 Fall 6 19.4%) 6 19.4%) < 0.001 Invert 0 (0.0%) 0 (0.0%) < 0.001 Stab wound/cutting 2 (6.5%) 2 (6.5%) < 0.001 Others 0 (0.0%) 0 (0.0%) < 0.001 Injury to the admission to the ED 0–30 (min) 6 (19.3%) 6 (19.3%) < 0.001 0.963 30–60 (min) 18 (58.0%) 18 (58.0%) < 0.001 60–90 (min) 4 (12.9%) 3 (9.67%) −0.102 90– (min) 3 (9.67%) 4 (12.9%) 0.102 Revised trauma score 5.03 (4.09–6.90) 5.68 (4.45–6.38) 0.086 0.750 Probability of survival 0.684 (0.276–0.878) 0.724 (0.275–0.886) 0.043 0.894 Vital signs on the admission to ED Heart rate (per min) 94 (68–120) 105 (80–120) 0.119 0.578 Glasgow coma scale 6 (3–13) 7 (4–14) 0.271 0.228 Systolic Blood Pressure (mmHg) 112 (91–150) 100 (70–140) −0.135 0.383 Respiratory rate (per min) 19 (16–24) 24 (16–30) 0.459 0.105 Injury severity score 30 (21–36) 34 (25–41) 0.353 0.182 AIS head and neck 4 (0–5) 4 (0–5) 0.016 0.895 AIS face 0 (0–0) 0 (0–1) −0.403 0.414 AIS chest 3 (0–4) 3 (0–4) −0.164 0.699 AIS abdomen 0 (0–2) 0 (0–3) 0.660 0.087 AIS extremity 1 (0–3) 2 (0–3) −0.295 0.668 AIS external 1 (0–1) 1 (0–1) 0.419 0.678 Blood gas analysis pH 7.33 (7.24−7.37) 7.25 (7.15−7.35) −0.283 0.095 Base deficit (mmol/L) 3.7 (2.1−6.7 ) 6.3 (2.4–12.5) −0.343 0.130 Lactate (mmol/L) 4.1 (2.9–5.5) 4.9 (3.0–7.9) 0.219 0.260 Laboratory data Platelet (× 103/μL) 208 (163–240) 193 (114–235) −0.158 0.559 PT-INR 1.08 (1.03–1.27) 1.20 (1.10–1.40) −0.158 0.078 Fibrinogen (mg/dL) 169 (124–200) 164 (138–219) 0.113 0.647 FDP (μg/mL) 120.0 (66.6–267.0) 93.5 (43.0–188.0) 0.082 0.391 D-dimer (μg/mL) 67.5 (46.5–207.7) 56.9 (20.4–125.0) −0.019 0.240 FC fibrinogen concentrate, ED emergency department, AIS Abbreviated Injury Scale, PT-INR prothrombin time-international normalised ratio, FDP fibrin/fibrinogen degradation products Table 2 Characteristics of the propensity score-matched patients of intracranial haematomas, trauma-induced coagulopathy, and poor outcomes [43, 45–47]. A patients who has performed burr hole surgery was included FC fibrinogen concentrate, ED emergency department, RBC red blood cell, FFP fresh frozen plasma, PC platelet concentrate Discussion Therefore, a multi-centre RCT is needed to address these limitations; a planned RCT aiming to identify the optimal timing and dose of fibrinogen supplemen- tation during trauma resuscitation will be of particu- lar value [33]. fibrinogen, which deteriorates owing to hyperfibrinolysis in patients with severe brain injury. Therefore, FC supplemen- tation may suppress intracranial haematoma enlargement and reduce the risk of death owing to severe brain injury. and reduce the risk of death owing to severe brain injury. In situations with severe bleeding, Geeraedts et al. have suggested that “blind” coagulation management (without point-of-care guidance, such as thromboelasto- metry) underestimates the real demand for coagulation factors [48]. However, based on the severe decrease in plasma fibrinogen levels during the early phase of severe trauma, we empirically administer FC based on trauma severity alone, before confirming the laboratory test results. Thus, we intentionally “overestimate” the de- mand for fibrinogen; this is in contrast with the finding reported by Geeraedts et al., who stated that this ap- proach naturally underestimates demand. Although Schöchl et al. have reported the utility of point-of-care guidance [32], the use of FC and its timing in the present study were totally dependent on the attending physicians’ discretion. Therefore, although our “blind” coagulation management using FC without point-of-care guidance may “overestimate” the demand for FC in severe trauma cases, we were still able to effectively administer FC earlier than if we had relied on point-of-care guidance. In the present study, the FC group had significantly higher amounts of transfusions during the first 6 h after ED arrival, although the FC and control groups had similar total amounts of transfusions (RBC, FFP, and PC) between 6 h and 24 h after ED arrival. Furthermore, the patients in FC group were more recently treated than those in the control groups (this data has not been presented). Therefore, the higher transfusion amounts during the first 6 h and recent advanced treatments may have affected the survival rate in the FC group. Neverthe- less, we speculate that the FC may have helped prevent early trauma-related deaths, which may have increased the total need for transfusions in the FC group, thereby intro- ducing the so-called “survival bias”. Th d h l li i i Th g j y In situations with severe bleeding, Geeraedts et al. Discussion In this context, FC may help restore haemostasis by complementing plasma the survival rate of the patients. In patients with severe brain injury, hyperfibrinolysis is frequently observed at ad- mission to the ED [43, 44]; this contributes to enlargement of intracranial haematomas, trauma-induced coagulopathy, and poor outcomes [43, 45–47]. In this context, FC may help restore haemostasis by complementing plasma Itagaki et al. World Journal of Emergency Surgery (2020) 15:7 Page 7 of 10 Itagaki et al. World Journal of Emergency Surgery (2020) 15:7 Page 7 of 10 Itagaki et al. World Journal of Emergency Surgery Fig. 2 Kaplan-Meier curves for the fibrinogen concentrate (FC) and control groups Fig. 2 Kaplan-Meier curves for the fibrinogen concentrate (FC) and control groups Table 3 Hemostatic treatments and transfusion amounts in the propensity-matched groups Table 3 Hemostatic treatments and transfusion amounts in the propensity-matched groups Control group n = 31 FC group n = 31 p value Interventions to emergency haemostasis 5 (16.1%) 10 (32.2%) 0.138 Thoracotomy and/or laparotomy 3 (9.7%) 8 (25.8%) 0.096 Transarterial embolization 3 (9.7%) 2 (6.5%) 0.641 Other emergency interventions 10 (32.2%) 14 (45.1%) 0.297 Craniotomy 8 (25.8%) 9 (29.0%) 0.753 Orthopaedic surgery 2 (6.5%) 4 (9.7%) 0.390 Other 0 (0.0%) 2 (9.7%) 0.151 Transfusion During the first 6 h after the admission to ED RBC (unit) 2 (0–10) 8 (2–22) 0.016 FFP (unit) 4 (0–10) 14 (4–23) 0.009 PC (unit) 0 (0–0) 0 (0–20) 0.059 From 6 to 24 h after the admission to ED RBC (unit) 0 (0–4) 2 (0–5) 0.387 FFP (unit) 0 (0–7) 4 (0–9) 0.133 PC (unit) 0 (0–15) 0 (0–20) 0.771 FC administration during 24 h after the admission to ED 19 (61.2%) 31 (100%) < 0.001 First FC administration after the admission to ED 0–1 h 0 31 (100%) < 0.001 1–3 h 10 (32.2%) 0 (0.0%) 3–24 h 9 (29.0%) 0 (0.0%) Total amounts during 24 h after the admission to ED 3 (3–3) 3 (3–3) 0.96 *A patients who has performed burr hole surgery was included FC fibrinogen concentrate, ED emergency department, RBC red blood cell, FFP fresh frozen plasma, PC platelet concentrate Itagaki et al. World Journal of Emergency Surgery (2020) 15:7 Page 8 of 10 Page 8 of 10 Itagaki et al. World Journal of Emergency Surgery (2020) 15:7 Itagaki et al. World Journal of Emergency Surgery care. This limitation has been mentioned previously. Acknowledgements We thank Editage (www.editage.com) for English language editing. Supplementary information l f y Supplementary information accompanies this paper at https://doi.org/10. 1186/s13017-020-0291-9. Additional file 1: Table S1. Characteristics of the propensity score matched patients with blunt trauma. Table S2. Characteristics of the propensity score matched patients with severe traumatic Additional file 2: Figure S1. Kaplan-Meier curves for the fibrinogen concentrate (FC) and control groups in the pair-matched blunt trauma patients. Figure S2. Kaplan-Meier curves for the fibrinogen concentrate (FC) and control groups in the pair-matched severe head trauma patients In the present study, the FC group had significantly higher amounts of transfusions during the first 6 h after ED arrival, although the FC and control groups had similar total amounts of transfusions (RBC, FFP, and PC) between 6 h and 24 h after ED arrival. Furthermore, the patients in FC group were more recently treated than those in the control groups (this data has not been presented). Therefore, the higher transfusion amounts during the first 6 h and recent advanced treatments may have affected the survival rate in the FC group. Neverthe- less, we speculate that the FC may have helped prevent early trauma-related deaths, which may have increased the total need for transfusions in the FC group, thereby intro- ducing the so-called “survival bias”. Discussion have suggested that “blind” coagulation management (without point-of-care guidance, such as thromboelasto- metry) underestimates the real demand for coagulation factors [48]. However, based on the severe decrease in plasma fibrinogen levels during the early phase of severe trauma, we empirically administer FC based on trauma severity alone, before confirming the laboratory test results. Thus, we intentionally “overestimate” the de- mand for fibrinogen; this is in contrast with the finding reported by Geeraedts et al., who stated that this ap- proach naturally underestimates demand. Although Schöchl et al. have reported the utility of point-of-care guidance [32], the use of FC and its timing in the present study were totally dependent on the attending physicians’ discretion. Therefore, although our “blind” coagulation management using FC without point-of-care guidance may “overestimate” the demand for FC in severe trauma cases, we were still able to effectively administer FC earlier than if we had relied on point-of-care guidance. Abbreviations S bb d AIS: Abbreviated Injury Scale; FC: Fibrinogen concentrate; ED: Emergency department; RBC: Red blood cell; FFP: Fresh frozen plasma; PC: Platelet concentrate Conclusions The present study revealed a favourable survival rate after early FC administration in patients with severe trauma. In this setting, FC may be an ideal early treat- ment for managing trauma-induced coagulopathy and may help improve patient outcomes. Consent for publication Not applicable Consent for publication Not applicable Consent for publication Not applicable Funding None g The present study has several limitations. The most important limitation is the small number of patients enrolled. The second is the single-centre retrospective study design; however, all eligible patients had available data regarding all variables from ED admission and be- fore FC administration. Third, although we used propen- sity scores to balance the groups’ characteristics, some variables remained imbalanced. However, it is important to note that most imbalanced variables were more severe in the FC group (vs. the control group); this suggests that FC may have improved the survival ratio even in relatively severe cases. Fourth, the demand for transfu- sions during the first 6 h of admission to the ED was increased in the FC group. We considered that this result may have been affected by two possible causes, namely, survival bias and recent advances in trauma Availability of data and materials Availability of data and materials All relevant data are presented in the published manuscript. Authors’ contributions YI collected and interpreted the data and drafted the manuscript. MH conceived the study, analysed and interpreted the data, and drafted the manuscript. KM, TS, AK, YH, AM, TY, TO, KK, and TW read the manuscript and revised it for important intellectual content. All authors read and approved the final manuscript. YI collected and interpreted the data and drafted the manuscript. MH conceived the study, analysed and interpreted the data, and drafted the manuscript. KM, TS, AK, YH, AM, TY, TO, KK, and TW read the manuscript and revised it for important intellectual content. All authors read and approved the final manuscript. Ethics approval and consent to participate The retrospective protocol of this study was approved by our institutional review board, and the requirement for informed consent was waived. References Dynamics of fibrinogen in acute phases of trauma. J Intensive Care. 2017;5(1):3. 7. Gando S, Hayakawa M. Pathophysiology of trauma-induced coagulopathy and management of critical bleeding requiring massive transfusion. Semin Thromb Hemost. 2016;42(2):155–65. 7. Gando S, Hayakawa M. Pathophysiology of trauma-induced coagulopathy and management of critical bleeding requiring massive transfusion. Semin Thromb Hemost. 2016;42(2):155–65. 27. Floccard B, Rugeri L, Faure A, Saint Denis M, Boyle EM, Peguet O, Levrat A, Guillaume C, Marcotte G, Vulliez A, et al. Early coagulopathy in trauma patients: an on-scene and hospital admission study. Injury. 2012;43(1):26–32. 8. Gando S, Wada H, Thachil J. Scientific Standardization Committee on DIC of the International Society on Thrombosis and Haemostasis: differentiating disseminated intravascular coagulation (DIC) with the fibrinolytic phenotype from coagulopathy of trauma and acute coagulopathy of trauma-shock (COT/ACOTS). J Thromb Haemost. 2013;11(5):826–35. 28. Chambers LA, Chow SJ, Shaffer LE. Frequency and characteristics of coagulopathy in trauma patients treated with a low- or high-plasma- content massive transfusion protocol. Am J Clin Pathol. 2011;136(3):364–70. 29. Hiippala S. Replacement of massive blood loss. Vox Sang. 1998;74(Suppl 2): 399–407. 9. Brohi K, Cohen MJ, Davenport RA. Acute coagulopathy of trauma: mechanism, identification and effect. Curr Opin Crit Care. 2007;13(6):680–5. 30. Spahn DR, Bouillon B, Cerny V, Duranteau J, Filipescu D, Hunt BJ, Komadina R, Maegele M, Nardi G, Riddez L, et al. The European guideline on management of major bleeding and coagulopathy following trauma: fifth edition. Crit Care. 2019;23(1):98. 10. Brohi K, Cohen MJ, Ganter MT, Schultz MJ, Levi M, Mackersie RC, Pittet JF. Acute coagulopathy of trauma: hypoperfusion induces systemic anticoagulation and hyperfibrinolysis. J Trauma. 2008;64(5):1211–7 discussion 1217. 31. Collins PW, Solomon C, Sutor K, Crispin D, Hochleitner G, Rizoli S, Schochl H, Schreiber M, Ranucci M. Theoretical modelling of fibrinogen supplementation with therapeutic plasma, cryoprecipitate, or fibrinogen concentrate. Br J Anaesth. 2014;113(4):585–95. 11. Hayakawa M, Gando S, Ono Y, Wada T, Yanagida Y, Sawamura A. Fibrinogen level deteriorates before other routine coagulation parameters and massive transfusion in the early phase of severe trauma: a retrospective observational study. Semin Thromb Hemost. 2015;41(1):35–42. 32. Schöchl H. Goal-directed coagulation management of major trauma patients using thromboelastometry (ROTEM®)-guided administration of fibrinogen concentrate and prothrombin complex concentrate. Crit Care. 2010;14:R55. 12. Sawamura A, Hayakawa M, Gando S, Kubota N, Sugano M, Wada T, Katabami K. Disseminated intravascular coagulation with a fibrinolytic phenotype at an early phase of trauma predicts mortality. Thromb Res. 2009;124(5):608–13. 33. Author details 1 d Author details 1Emergency and Critical Care Center, Sapporo City General Hospital, 1-1 Nishi13, Kita 11, Kita-ku, Sapporo, Hokkaido 060-8604, Japan. 2Department of Emergency Medicine, Hokkaido University Hospital, Sapporo, Japan. Page 9 of 10 Page 9 of 10 Itagaki et al. World Journal of Emergency Surgery (2020) 15:7 Received: 22 October 2019 Accepted: 6 January 2020 References Winearls J, Wullschleger M, Wake E, Hurn C, Furyk J, Ryan G, Trout M, Walsham J, Holley A, Cohen J, et al. Fibrinogen Early In Severe Trauma studY (FEISTY): study protocol for a randomised controlled trial. Trials. 2017; 18(1):241. 13. Nakamura Y, Ishikura H, Kushimoto S, Kiyomi F, Kato H, Sasaki J, Ogura H, Matsuoka T, Uejima T, Morimura N, et al. Fibrinogen level on admission is a predictor for massive transfusion in patients with severe blunt trauma: analyses of a retrospective multicentre observational study. Injury. 2017; 48(3):674–9. 34. Schöchl H, Nienaber U, Maegele M, et al. Transfusion in trauma- thromboelastometry-guided coagulation factor concentrate-based therapy versus standard fresh frozen plasma-based therapy. Critical Care. 2011;15:R83. 14. Hayakawa M, Maekawa K, Kushimoto S, Kato H, Sasaki J, Ogura H, Matauoka T, Uejima T, Morimura N, Ishikura H, et al. High D-dimer levels predict a poor outcome in patients with severe trauma, even with high fibrinogen levels on arrival: a multicenter retrospective study. Shock. 2016;45(3):308–14. 35. Nascimento B, Goodnough LT, Levy JH. Cryoprecipitate therapy. Br J Anaesth. 2014;113(6):922–34. 36. Schlimp CJ: Impact of fibrinogen concentrate alone or with prothrombin complex concentrate (+/−fresh frozen plasma) on plasma fibrinogen level and fibrin-based clot strength (FIBTEM) in major trauma: a retrospective study. Scandinavian Journal of Trauma, Resuscitation and Emergency Medicine 2013. 15. Engstrom M, Schott U, Romner B, Reinstrup P. Acidosis impairs the coagulation: a thromboelastographic study. J Trauma. 2006;61(3):624–8. 16. Fries D, Martini WZ. Role of fibrinogen in trauma-induced coagulopathy. Br J Anaesth. 2010;105(2):116–21. 37. Holcomb JB, Tilley BC, Baraniuk S, Fox EE, Wade CE, Podbielski JM, del Junco DJ, Brasel KJ, Bulger EM, Callcut RA, et al. Transfusion of plasma, platelets, and red blood cells in a 1:1:1 vs a 1:1:2 ratio and mortality in patients with severe trauma: the PROPPR randomized clinical trial. JAMA. 2015;313(5):471–82. 17. Stinger HK, Spinella PC, Perkins JG, Grathwohl KW, Salinas J, Martini WZ, Hess JR, Dubick MA, Simon CD, Beekley AC, et al. The ratio of fibrinogen to red cells transfused affects survival in casualties receiving massive transfusions at an army combat support hospital. J Trauma. 2008;64(2 Suppl):S79–85 discussion S85. 38. Akbari E, Safari S, Hatamabadi H. The effect of fibrinogen concentrate and fresh frozen plasma on the outcome of patients with acute traumatic coagulopathy: a quasi-experimental study. Am J Emerg Med. 2018;36(11):1947–50. 18. Received: 22 October 2019 Accepted: 6 January 2020 19. Ranucci M, Solomon C. Supplementation of fibrinogen in acquired bleeding disorders: experience, evidence, guidelines, and licences. Br J Anaesth. 2012; 109(2):135–7. 20. Levy JH, Szlam F, Tanaka KA, Sniecienski RM. Fibrinogen and hemostasis: a primary hemostatic target for the management of acquired bleeding. Anesth Analg. 2012;114(2):261–74. References 1. Naghavi M, Abajobir AA, Abbafati C, Abbas KM, Abd-Allah F, Abera SF, Aboyans V, Adetokunboh O, Afshin A, Agrawal A, et al. Global, regional, and national age-sex specific mortality for 264 causes of death, 1980–2016: a systematic analysis for the Global Burden of Disease Study 2016. Lancet. 2017;390(10100):1151–210. 1. Naghavi M, Abajobir AA, Abbafati C, Abbas KM, Abd-Allah F, Abera SF, Aboyans V, Adetokunboh O, Afshin A, Agrawal A, et al. Global, regional, and national age-sex specific mortality for 264 causes of death, 1980–2016: a systematic analysis for the Global Burden of Disease Study 2016. Lancet. 2017;390(10100):1151–210. 21. Nienaber U, Innerhofer P, Westermann I, Schochl H, Attal R, Breitkopf R, Maegele M. The impact of fresh frozen plasma vs coagulation factor concentrates on morbidity and mortality in trauma-associated haemorrhage and massive transfusion. Injury. 2011;42(7):697–701. 22. Meyer MA, Ostrowski SR, Windelov NA, Johansson PI. Fibrinogen concentrates for bleeding trauma patients: what is the evidence? Vox Sang. 2011;101(3):185–90. 2. Yeboah D, Mock C, Karikari P, Agyei-Baffour P, Donkor P, Ebel B. Minimizing preventable trauma deaths in a limited-resource setting: a test-case of a multidisciplinary panel review approach at the Komfo Anokye Teaching Hospital in Ghana. World J Surg. 2014;38(7):1707–12. 23. Levy JH, Welsby I, Goodnough LT. Fibrinogen as a therapeutic target for bleeding: a review of critical levels and replacement therapy. Transfusion. 2014;54(5):1389–405 quiz 1388. 3. Kauvar DS, Wade CE. The epidemiology and modern management of traumatic hemorrhage: US and international perspectives. Crit Care. 2005; 9(Suppl 5):S1–9. 24. Schöchl H, Cotton B, Inaba K, Nienaber U, Fischer H, Voelckel W, Solomon C. FIBTEM provides early prediction of massive transfusion in trauma. Crit Care. 2011;15(6):R265. 4. Gando S. Acute coagulopathy of trauma shock and coagulopathy of trauma: a rebuttal. You are now going down the wrong path. J Trauma. 2009;67(2):381–3. 25. Inaba K, Karamanos E, Lustenberger T, Schochl H, Shulman I, Nelson J, Rhee P, Talving P, Lam L, Demetriades D. Impact of fibrinogen levels on outcomes after acute injury in patients requiring a massive transfusion. J Am Coll Surg. 2013;216(2):290–7. 5. Hayakawa M. Pathophysiology of trauma-induced coagulopathy: disseminated intravascular coagulation with the fibrinolytic phenotype. J Intensive Care. 2017;5(1):14. 26. Rourke C, Curry N, Khan S, Taylor R, Raza I, Davenport R, Stanworth S, Brohi K. Fibrinogen levels during trauma hemorrhage, response to replacement therapy, and association with patient outcomes. J Thromb Haemost. 2012; 10(7):1342–51. 6. Hayakawa M. References Kushimoto S, Shibata Y, Yamamoto Y. Implications of fibrinogenolysis in patients with closed head injury. J Neurotrauma. 2003;20(4):357–63. 47. Kushimoto S, Shibata Y, Yamamoto Y. Implications of fibrinogenolysis in patients with closed head injury. J Neurotrauma. 2003;20(4):357–63. 48. Geeraedts LM Jr, Demiral H, Schaap NP, Kamphuisen PW, Pompe JC, Frolke JP. ‘Blind’ transfusion of blood products in exsanguinating trauma patients. Resuscitation. 2007;73(3):382–8. References Wafaisade A, Lefering R, Maegele M, Brockamp T, Mutschler M, Lendemans S, Banerjee M, Bouillon B, Probst C. Trauma Registry of DGU: Administration of fibrinogen concentrate in exsanguinating trauma patients is associated with improved survival at 6 hours but not at discharge. J Trauma Acute Care Surg. 2013;74(2):387–3 discussion 393-385. 39. Curry N, Foley C, Wong H, Mora A, Curnow E, Zarankaite A, Hodge R, Hopkins V, Deary A, Ray J, et al. Early fibrinogen concentrate therapy for Page 10 of 10 Itagaki et al. World Journal of Emergency Surgery (2020) 15:7 major haemorrhage in trauma (E-FIT 1): results from a UK multi-centre, randomised, double blind, placebo-controlled pilot trial. Crit Care. 2018; 22(1):164. 40. Schöchl H, Forster L, Woidke R, Solomon C, Voelckel W. Use of rotation thromboelastometry (ROTEM) to achieve successful treatment of polytrauma with fibrinogen concentrate and prothrombin complex concentrate. Anaesthesia. 2010;65(2):199–203. 41. Innerhofer P, Fries D, Mittermayr M, Innerhofer N, von Langen D, Hell T, Gruber G, Schmid S, Friesenecker B, Lorenz IH, et al. Reversal of trauma- induced coagulopathy using first-line coagulation factor concentrates or fresh frozen plasma (RETIC): a single-centre, parallel-group, open-label, randomised trial. Lancet Haematol. 2017. 42. Nascimento B, Callum J, Tien H, Peng H, Rizoli S, Karanicolas P, Alam A, Xiong W, Selby R, Garzon AM, et al. Fibrinogen in the initial resuscitation of severe trauma (FiiRST): a randomized feasibility trial. Br J Anaesth. 2016; 117(6):775–82. 43. Hayakawa M, Maekawa K, Kushimoto S, Kato H, Sasaki J, Ogura H, Matsuoka T, Uejima T, Morimura N, Ishikura H, et al. Hyperfibrinolysis in severe isolated traumatic brain injury may occur without tissue hypoperfusion: a retrospective observational multicentre study. Crit Care. 2017;21(1):222. 44. Gando S, Nanzaki S, Kemmotsu O. Coagulofibrinolytic changes after isolated head injury are not different from those in trauma patients without head injury. J trauma. 1999;46(6):1070–6. 44. Gando S, Nanzaki S, Kemmotsu O. Coagulofibrinolytic changes after isolated head injury are not different from those in trauma patients without head injury. J trauma. 1999;46(6):1070–6. 45. Kaufman HH, Moake JL, Olson JD, Miner ME, duCret RP, Pruessner JL, Gildenberg PL. Delayed and recurrent intracranial hematomas related to disseminated intravascular clotting and fibrinolysis in head injury. Neurosurgery. 1980;7(5):445–9. 46. Kaufman HH, Hui KS, Mattson JC, Borit A, Childs TL, Hoots WK, Bernstein DP, Makela ME, Wagner KA, Kahan BD, et al. Clinicopathological correlations of disseminated intravascular coagulation in patients with head injury. Neurosurgery. 1984;15(1):34–42. 47. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
https://openalex.org/W4321994768	https://zenodo.org/records/7656453/files/Exploring%20the%20Relationship%20between-20022023-2.pdf	English	null	Exploring the Relationship between Learner Characteristics and Language Learning: Implications for Language Instruction	Zenodo (CERN European Organization for Nuclear Research)	2,023	cc-by	8,853	LITERATURE REVIEW Previous research has shown that learner characteristics can influence language learning outcomes. For example, motivation, as one of the learner’s characteristics, has been found to be a significant predictor of language learning success (Dörnyei, 2001; Gardner, 1985). Age has also been found to be a significant factor, with younger learners often outperforming older learners in language learning (Piske, MacKay, & Flege, 2001). Gender has been studied less frequently, but some studies have suggested that there may be differences in the way males and females acquire language (McWhorter, 2012). Cognitive style, which refers to the preferred way of processing information, has also been found to be an important factor in language learning (Griffiths, 2013). Exploring the Relationship between Learner Characteristics and Language Learning: Implications for Language Instruction NAELA ABDULLAH TAMI The Higher Institute of Telecommunication and Navigation, Kuwait, 2023 DOI: https://doi.org/10.5281/zenodo.7656453 Published Date: 20-February-2023 NAELA ABDULLAH TAMI The Higher Institute of Telecommunication and Navigation, Kuwait, 2023 DOI: https://doi.org/10.5281/zenodo.7656453 Published Date: 20-February-2023 Abstract: Language learning is a complex process that is influenced by a range of learner characteristics. This paper aims to explore the various learner characteristics that impact language acquisition and the ways in which these characteristics can be leveraged by teachers to create a more effective and engaging learning environment. This paper also discusses learner characteristics such as cognitive style, prior knowledge, motivation, personality, and maturity and their impact on language learning. It also explores the ways in which teachers can identify and address individual learner characteristics to support their language learning. Keywords: Language learning, learner characteristics. Language Instruction. ISSN 2348-3156 (Print) International Journal of Social Science and Humanities Research ISSN 2348-3164 (online) Vol. 11, Issue 1, pp: (134-144), Month: January - March 2023, Available at: www.researchpublish.com ISSN 2348-3156 (Print) International Journal of Social Science and Humanities Research ISSN 2348-3164 (online) Vol. 11, Issue 1, pp: (134-144), Month: January - March 2023, Available at: www.researchpublish.com Research Publish Journals INTRODUCTION 1984) Personal and emotional factors, such as self-esteem, stress, and mental health, can also impact learning. These factors can affect an individual's ability to engage with new material and can impact their overall academic performance. Personal and emotional factors, such as self-esteem, stress, and mental health, can also impact learning. These factors can affect an individual's ability to engage with new material and can impact their overall academic performance. Finally, learning strategies are approaches or techniques used by learners to acquire, retain, and recall information effectively. These can include methods such as note-taking, summarizing, and active listening, among others. Understanding and implementing effective learning strategies can help individuals process and make sense of new information in ways that are meaningful and relevant to them. (Kornell, N., & Bjork, R. A. (2008) Finally, learning strategies are approaches or techniques used by learners to acquire, retain, and recall information effectively. These can include methods such as note-taking, summarizing, and active listening, among others. Understanding and implementing effective learning strategies can help individuals process and make sense of new information in ways that are meaningful and relevant to them. (Kornell, N., & Bjork, R. A. (2008) In order to effectively teach and support students, it is important to understand the various learner characteristics that can impact the learning process. Moreover, it can help students to better understand their own strengths and weaknesses as learners, allowing them to develop more effective study habits and learning strategies. Those learner characteristics include: I. learner characteristics is learning style. What is meant by Learning style? Learning style refers to an individual's preferred way of acquiring and processing information. It refers to the distinct ways in which learners approach and engage with new information and concepts. Fleming, N. D. (2011). Some common learning styles include: Learning style refers to an individual's preferred way of acquiring and processing information. It refers to the distinct ways in which learners approach and engage with new information and concepts. Fleming, N. D. (2011). Some common learning styles include: I.1. Visual: learners who prefer to learn through visual aids such as pictures, graphs, and charts. I.1. Visual: learners who prefer to learn through visual aids such as pictures, graphs, and charts. Visual learners are individuals who prefer to learn through visual aids, such as pictures, diagrams, graphs, and charts. Some common characteristics of visual learners. INTRODUCTION The acquisition of the four learning skills is essential for successful language learning. However, learners differ in their ability to develop these skills. Understanding the role of learner characteristics in the development of the four learning skills can help educators to design effective language teaching methods and strategies. This paper aims to highlight the relationship between learner characteristics and the development of language acquisition. (Krashen, S. D. 1982) Learning is a complex process that is influenced by a range of learning characteristics which refer to the unique qualities, traits, abilities, Past learning experiences that influence how individuals learn and process information. One such characteristic is learning style, which refers to an individual's preferred way of learning and processing information. This can include visual, auditory, and kinaesthetic learning styles, among others. Understanding one's own learning style can be beneficial in improving academic performance and maximizing learning potential. (Pashler, H., McDaniel, M., Rohrer, D., & Bjork, R. 2008) Cognitive development is another factor that can impact learning. This refers to the stage of mental development an individual is in and how this affects their ability to process and understand new information. Understanding cognitive development can help educators tailor teaching methods to better match the needs of individual learners. Page \| 134 Page \| 134 ( ) International Journal of Social Science and Humanities Research ISSN 2348-3164 (online) Vol. 11, Issue 1, pp: (134-144), Month: January - March 2023, Available at: www.researchpublish.com Motivation is an internal drive that fuels engagement in the learning process and the achievement of academic goals. It is an important factor in the success of the learning process and can be influenced by a variety of internal and external factors. (Pintrich, P. R. 2003). Attitude is another factor that can influence learning. An individual's overall disposition and approach to learning can impact their ability to engage with new information and their willingness to take risks and ask questions. Past learning experience is also an important factor in the learning process. It refers to the background knowledge and experiences that an individual brings to the learning process and can impact their ability to engage with new material. Cultural and linguistic background can also impact learning. Individuals from different cultural and linguistic backgrounds may approach learning differently and may require different teaching methods to be successful. (Kolb, D. A. INTRODUCTION First, they like to see information presented in a clear and organized way, using visual aids such as mind maps, flow charts, and diagrams. Second, they prefer to read and write in an organized and structured manner, using headings, bullet points, and tables to break information down into manageable chunks. Third, they learn best by observing and visualizing information, such as watching demonstrations or seeing diagrams of concepts. They may struggle with lectures that rely heavily on verbal information, as they need to see information to understand it. They may have a strong spatial awareness and be able to visualize objects in their mind's eye. Finally, they may have a strong ability to retain images and patterns and can often recall visual information more easily than verbal information. (Felder, R. M., & Silverman, L. K. 1988) Here are some activities that may be beneficial for visual learners in the classroom: Here are some activities that may be beneficial for visual learners in the classroom: a. Use of visual aids such as graphs, charts, and diagrams to help explain concepts and information. b. Use of mind maps or concept maps to show relationships between ideas and concepts. c. Use of visual organizers, such as Venn diagrams, to compare and contrast information. d. Use of visual aids in presentations, such as slideshows or videos, to supplement verbal explanations. e. Providing reading materials with clear headings, bullet points, and tables to help break down information into manageable chunks. e. Providing reading materials with clear headings, bullet points, and tables to help break down information into manageable chunks. f. Use of colour coding to highlight important information and make it easier to remember. g. Allowing students to create their own visual aids, such as diagrams or graphs, to help them understand and retain information. h. Use of images, such as photographs or illustrations, to provide context and understanding of concepts. Page \| 135 Research Publish Journals Kinaesthetic learners are learners who prefer to learn through physical movement and hands-on activities. They like to be physically active and hands-on, such as through experimentation, building, or moving around. They may struggle with traditional classroom activities, such as sitting still and listening to lectures, they may have good dexterity and physical coordination. they may learn best through practical experiences and may have a hard time retaining information that is only presented in a lecture format, they may have a strong ability to understand concepts through physical manipulation and exploration. Some examples of activities that may be beneficial for kinaesthetic learners in the classroom include: Some examples of activities that may be beneficial for kinaesthetic learners in the classroom include: a. Hands-on projects, such as building models or conducting experiments. a. Hands-on projects, such as building models or conducting experiments. b. Physical games and simulations that involve movement and activity. b. Physical games and simulations that involve movement and activity. c. Group activities and discussions that involve movement and physical interaction, such as role-playing ties and discussions that involve movement and physical interaction, such as role-playing or dramatization d. Field trips or outdoor activities that allow for hands-on exploration and learning. d. Field trips or outdoor activities that allow for hands-on exploration and learning. d. Field trips or outdoor activities that allow for hands-on exploration and learning. e. Active problem-solving tasks, such as physically manipulating objects to solve a problem. I.4. Reading/Writing learners are individuals who prefer to learn through reading and writing. Some common characteristics of Reading/Writing learners that they enjoy reading and writing, and may prefer to learn through written materials such as textbooks, articles, or notes. They may prefer to take notes by writing information down, rather than listening to it. They may have good writing skills, and enjoy writing essays, reports, or other written assignments. They may have strong attention to detail and be able to focus well when reading or writing. They may have a strong ability to recall and understand information that they have read and may prefer to study by reading and re-reading information. (Kavale, K. A., & Forness, S. R.2000) I.4. Reading/Writing learners are individuals who prefer to learn through reading and writing. Some common characteristics of Reading/Writing learners that they enjoy reading and writing, and may prefer to learn through written materials such as textbooks, articles, or notes. Research Publish Journals ISSN 2348-3156 (Print) International Journal of Social Science and Humanities Research ISSN 2348-3164 (online) Vol. 11, Issue 1, pp: (134-144), Month: January - March 2023, Available at: www.researchpublish.com I.2. Auditory learners are individuals who prefer to learn through hearing information, such as lectures, discussions, and audio recordings. Some common characteristics of auditory learners include: They like to listen to information and have it explained to them, such as through lectures, discussions, or audio recordings. They may prefer to take notes by listening and verbally summarizing information, rather than writing it down. They may learn best through verbal explanations and discussion and may struggle with visual aids that don't include a verbal component. They may have good listening skills and be able to focus well in a noisy environment. They may have a strong ability to remember information that they have heard, such as verbal instructions or spoken details. They may prefer to discuss and reflect on information, either alone or with others, to fully understand it. (Martino, W., & Monfils, M. H. 2017) Here are some activities that may be beneficial for auditory learners in the classroom: Here are some activities that may be beneficial for auditory learners in the classroom: a. Lectures and verbal explanations, such as teacher-led discussions or presentations. b. Audio recordings of lectures or discussions, such as podcasts or audio books. c. Classroom discussions and debates, where students can listen and respond to each other's ideas. d. Verbal storytelling, such as acting out scenes or historical events. e. Group work, where students can collaborate and discuss information and ideas. f. Verbal quizzes or oral exams, where students can listen to questions and answer verbally. g. Verbal summarization activities, such as having students verbally summarize key concepts or information. se of music or sound effects to help students understand and remember information. (Molloy, E. (2017) I.3. Kinaesthetic learners are learners who prefer to learn through physical movement and hands-on activities. They like to be physically active and hands-on, such as through experimentation, building, or moving around. They may struggle with traditional classroom activities, such as sitting still and listening to lectures, they may have good dexterity and physical coordination. they may learn best through practical experiences and may have a hard time retaining information that is only presented in a lecture format, they may have a strong ability to understand concepts through physical manipulation and exploration. I.3. Research Publish Journals They may prefer to take notes by writing information down, rather than listening to it. They may have good writing skills, and enjoy writing essays, reports, or other written assignments. They may have strong attention to detail and be able to focus well when reading or writing. They may have a strong ability to recall and understand information that they have read and may prefer to study by reading and re-reading information. (Kavale, K. A., & Forness, S. R.2000) Some examples of activities that may be beneficial for Reading/Writing learners in the classroom include: Some examples of activities that may be beneficial for Reading/Writing learners in the classroom include: a. Writing assignments, such as essays, reports, or journal entries, to help reinforce learning. b. Reading and discussion groups, where students can read and discuss written materials. c. Writing workshops or writing centers, where students can receive feedback on their writing. d. Independent reading and writing time, where students can read and write at their own pace. Page \| 136 Research Publish Journals ISSN 2348-3156 (Print) International Journal of Social Science and Humanities Research ISSN 2348-3164 (online) Vol. 11, Issue 1, pp: (134-144), Month: January - March 2023, Available at: www.researchpublish.com e. Writing summaries or reflection pieces, where students can reflect on what they have learned and summarize the key concepts and ideas. I.5. Logical/Mathematical learners are individuals who prefer to learn through logical reasoning and mathematical concepts. Some common characteristics of Logical/Mathematical learners include solving problems and finding patterns, and may have a strong aptitude for mathematics and science. They prefer to learn through hands-on, experiential learning, such as hands-on activities and experiments. They are good at breaking down complex information into smaller parts and understanding how they fit together. They also enjoy finding the logic behind things and have a strong ability to think abstractly. They usually prefer to work with numbers and data, and enjoy activities that involve mathematical calculations and problem-solving. Some examples of activities that may be beneficial for Logical/Mathematical learners in the classroom i a. Hands-on experiments and projects, such as building models or conducting science experiments. b. Mathematical and scientific problem-solving activities, such as puzzles, logic games, and mathematical simulations. c. Use of data and statistics to help understand concepts, such as creating graphs or analysing data sets. d. Group problem-solving activities, such as working on math or science projects as a team. e. Using mathematical and logical reasoning to analyze real-world problems and find solutions. 6. reflective learners features and best class activities Reflective learners are individuals who prefer to learn through reflection and contemplation. Some common characteristics of Reflective learners include: They enjoy thinking deeply and considering the implications of what they are learning. They may have a strong ability to understand complex ideas and theories, and to think critically about them. They may be introspective and introverted, and may prefer quiet, solitary activities. They may prefer to take their time to process information, and may need time to reflect before they are ready to engage with the material. Some examples of activities that may be beneficial for Reflective learners in the classroom include: a. Independent study time, where students can reflect on what they have learned and think about how it applies to their lives. b. Journaling or reflective writing activities, where students can reflect on what they have learned and consider its implications. c. I.8. autonomous learners features and best class activities: I.8. autonomous learners features and best class activities: Autonomous learners are individuals who prefer to learn independently and with a high degree of control over their own learning process. Some common characteristics of Autonomous learners include having control over their own learning process, and may prefer to learn at their own pace. They may have a strong ability to set and achieve learning goals and may be highly motivated and self-directed. They may prefer to work alone and may find group work or collaborative activities distracting. They may have a strong ability to self-evaluate and monitor their own progress and may enjoy learning through self-reflection and self-assessment. They may have a strong desire for independence and may prefer to learn through self-directed projects and activities. Some examples of activities that may be beneficial for Autonomous learners in the classroom include: a. Independent study time, where students can pursue their own interests and learning goals. b. Self-paced online courses, or online learning platforms, where students can learn at their own pace. c. Independent projects, where students can explore topics of their own interest and design their own learning experiences. c. Independent projects, where students can explore topics of their own interest and design their own learning experiences. d Self-reflection exercises and self-assessment activities where students can monitor their own progress and reflect on c. Independent projects, where students can explore topics of their own interest and design their own learning experiences. d. Self-reflection exercises and self-assessment activities, where students can monitor their own progress and reflect on their own learning. c. Independent projects, where students can explore topics of their own interest and design their own learning experiences. d. Self-reflection exercises and self-assessment activities, where students can monitor their own progress and reflect on their own learning. d. Self-reflection exercises and self-assessment activities, where students can monitor their own progress and reflect on their own learning. e. Opportunities for self-directed exploration and experimentation, such as hands-on labs, makerspaces, or design challenges. e. Opportunities for self-directed exploration and experimentation, such as hands-on labs, makerspaces, or design challenges. II. Another common learner characteristics Besides Learning style is The Cognitive Development Learning. Cognitive development refers to the changes and advancements in a person's mental processes, such as thinking, reasoning, and problem-solving, from childhood to adulthood. Research Publish Journals Research Publish Journals Solitary activities, such as silent reading or self-reflection exercises. c. Solitary activities, such as silent reading or self-reflection exercises. d. Quiet activities that allow for reflection, such as yoga, meditation, or mindfulness practices. Opportunities for independent, self-directed learning, such as online courses or independent study proje 7 impulsive learners e. Opportunities for independent, self-directed learning, such as online courses or independent study projects. I.7. impulsive learners Impulsive learners are individuals who prefer to learn through spontaneous and unplanned experiences. Some common characteristics of Impulsive learners may include have a tendency to act on impulse and may find it difficult to focus on tasks for extended periods of time. They may enjoy hands-on, experiential learning, and may prefer to learn through direct, concrete experiences. They may be impulsive and may struggle with self-control, which can lead to difficulties in the classroom setting. They may prefer to work in short, intense bursts, and may become easily bored with repetitive or routine activities. They may have a strong desire for instant gratification, and may be easily distracted by new and exciting stimuli. Some examples of activities that may be beneficial for Impulsive learners in the classroom include: of activities that may be beneficial for Impulsive learners in the classroom include: a. Hands-on, experiential activities, such as field trips, simulations, or projects that allow for exploration and discovery. Page \| 137 Page \| 137 ISSN 2348-3156 (Print) International Journal of Social Science and Humanities Research ISSN 2348-3164 (online) Vol. 11, Issue 1, pp: (134-144), Month: January - March 2023, Available at: www.researchpublish.com b. Games and interactive activities, such as puzzles, simulations, and interactive media, that provide instant feedback and keep the learner engaged. c. Active learning experiences, such as physical activities, sports, or other forms of movement that allow for exploration and discovery through the body. c. Active learning experiences, such as physical activities, sports, or other forms of movement that allow for exploration and discovery through the body. d. Opportunities for creative expression, such as art, music, or dance, that allow for spontaneous and unplanned exploration. d. Opportunities for creative expression, such as art, music, or dance, that allow for spontaneous and unplanned exploration. e. A flexible and dynamic learning environment that allows for spontaneous exploration, collaboration, and experimentation. e. A flexible and dynamic learning environment that allows for spontaneous exploration, collaboration, and experimentation. ISSN 2348-3156 (Print) International Journal of Social Science and Humanities Research ISSN 2348-3164 (online) Vol. 11, Issue 1, pp: (134-144), Month: January - March 2023, Available at: www.researchpublish.com ( ) International Journal of Social Science and Humanities Research ISSN 2348-3164 (online) Vol. 11, Issue 1, pp: (134-144), Month: January - March 2023, Available at: www.researchpublish.com III. The third common learner characteristics is Motivation and its affect over the leaners' achievement. Motivation is a crucial factor that influences the achievement of learners. It refers to an individual's internal drive or willingness to engage in a particular behaviour or activity. When learners are motivated, they are more likely to be focused, engaged, and persistent in their learning. (Pintrich, P. R.2003) There are several different types of motivation, including intrinsic motivation (the drive to engage in an activity for its own sake) and extrinsic motivation (the drive to engage in an activity in order to receive a reward or avoid punishment). Both types of motivation can have a significant impact on learners' achievement. Intrinsic motivation is often seen as the most powerful form of motivation, as it is driven by a genuine interest in the activity itself. When learners are intrinsically motivated, they are more likely to persist in their learning, even in the face of challenges or difficulties. Intrinsically motivated learners are also more likely to engage in self-directed learning and to seek out new learning opportunities. (Ryan, R. M., & Deci, E. L. 2017) Extrinsic motivation, on the other hand, can be less effective in promoting long-term learning and achievement. Although extrinsic motivators, such as grades or awards, may provide short-term incentives for learners, they can also reduce intrinsic motivation and lead to a focus on the reward rather than the learning itself. Extrinsic motivation, on the other hand, can be less effective in promoting long-term learning and achievement. Although extrinsic motivators, such as grades or awards, may provide short-term incentives for learners, they can also reduce intrinsic motivation and lead to a focus on the reward rather than the learning itself. Promoting motivation and enhancing achievement among learners, is important for teachers to create a supportive and engaging learning environment that provides opportunities for learners to explore their interests, engage in hands-on activities, and receive feedback and recognition for their efforts. Additionally, teachers can help to build intrinsic motivation by providing meaningful and relevant learning experiences that connect to learners' personal interests and goals. ISSN 2348-3156 (Print) International Journal of Social Science and Humanities Research ISSN 2348-3164 (online) Vol. 11, Issue 1, pp: (134-144), Month: January - March 2023, Available at: www.researchpublish.com Promoting motivation and enhancing achievement among learners, is important for teachers to create a supportive and engaging learning environment that provides opportunities for learners to explore their interests, engage in hands-on activities, and receive feedback and recognition for their efforts. Additionally, teachers can help to build intrinsic motivation by providing meaningful and relevant learning experiences that connect to learners' personal interests and goals. How can a teacher motivate the learners to increase their achievement? There are several strategies that teachers can use to motivate their learners and enhance their achievement: How can a teacher motivate the learners to increase their achievement? There are several strategies that teachers can use to motivate their learners and enhance their achievement: a. Create a supportive and engaging learning environment: This can be achieved by creating a positive and inclusive classroom culture, providing clear expectations and rules, and recognizing and rewarding positive behaviours and achievements. b. Provide meaningful and relevant learning experiences: By connecting the curriculum to the interests and needs of the learners, teachers can increase motivation and engagement. Teachers can also provide hands-on, experiential learning opportunities that allow learners to apply what they have learned in real-world contexts. c. Encourage learner autonomy and choice: Allowing learners to make choices about their learning experiences can increase motivation and engagement. For example, teachers can provide options for learners to choose topics for research projects, or allow them to work in small groups or individually on assignments. d. Provide regular and meaningful feedback: Feedback is an important part of the learning process, as it helps learners to understand their strengths and areas for improvement. Teachers can provide feedback in a variety of formats, including written comments, oral feedback, and grades. e. Encourage goal-setting and reflection: By setting learning goals and reflecting on their progress, learners can become more motivated and engaged in their learning. Teachers can encourage learners to set personal learning goals and to reflect on their progress towards achieving these goals. f. Foster a growth mindset: Encouraging learners to view challenges as opportunities for growth and learning can increase motivation and achievement. Teachers can model a growth mindset by encouraging learners to take risks and embrace challenges in their learning. (Dörnyei, Z. 2001) IV. The Fourth learner characteristics is attitude. Attitude is a key factor that can have a significant impact on English language learners. I.8. autonomous learners features and best class activities: Cognitive development is a key aspect of overall human development, and it is influenced by a combination of genetic and environmental factors. II. Another common learner characteristics Besides Learning style is The Cognitive Development Learning. Cognitive development refers to the changes and advancements in a person's mental processes, such as thinking, reasoning, and problem-solving, from childhood to adulthood. Cognitive development is a key aspect of overall human development, and it is influenced by a combination of genetic and environmental factors. Jean Piaget, a Swiss psychologist, is one of the most well-known theorists of cognitive development. According to Piaget, cognitive development occurs through a series of stages, with each stage building on the previous one. The four stages of Piaget's theory are: 1. The Sensory-Motor Stage (birth to 2 years): This stage is characterized by infants' use of their senses and movements to understand and interact with their environment. 2. The Preoperational Stage (2 to 7 years): This stage is characterized by the development of symbolic thought and the ability to use mental representations. Children at this stage begin to understand concepts such as quantity and classification. 3. The Concrete Operational Stage (7 to 12 years): This stage is characterized by the development of logical thinking and the ability to manipulate and transform mental representations. Children at this stage develop the ability to understand cause and effect relationships. 4. The Formal Operational Stage (12 years and older): This stage is characterized by the development of abstract and hypothetical reasoning. Individuals at this stage have the ability to think logically and systematically about complex problems. 4. The Formal Operational Stage (12 years and older): This stage is characterized by the development of abstract and hypothetical reasoning. Individuals at this stage have the ability to think logically and systematically about complex problems. Learning can be seen as a key component of cognitive development, as it provides opportunities for children and adults to practice and refine their mental processes. In the classroom, teachers can support cognitive development by providing opportunities for students to engage in hands-on, experiential learning activities, to reflect on their own learning, and to collaborate and engage in problem-solving tasks with their peers. (Fiorella, L., & Mayer, R. E. 2015) Page \| 138 Page \| 138 Research Publish Journals ISSN 2348-3156 (Print) International Journal of Social Science and Humanities Research ISSN 2348-3164 (online) Vol. 11, Issue 1, pp: (134-144), Month: January - March 2023, Available at: www.researchpublish.com M., & Spada, N. 2006) Learning strategies: Learners who have had prior language learning experience may have developed effective learning strategies that they can apply to their English language learning. For example, they may be more familiar with using self- directed learning resources or seeking feedback from others. (Lightbown, P. M., & Spada, N. 2006) Transfer of learning: Learners who have prior experience learning another language may be able to transfer some of their language learning skills and strategies to their English language learning. This can include knowledge of grammar and vocabulary, as well as strategies for pronunciation and listening comprehension. Transfer of learning: Learners who have prior experience learning another language may be able to transfer some of their language learning skills and strategies to their English language learning. This can include knowledge of grammar and vocabulary, as well as strategies for pronunciation and listening comprehension. Speed of language development: Learners who have had prior language learning experience may develop their English language skills more quickly, as they have already developed a foundation of language learning skills and strategies. Speed of language development: Learners who have had prior language learning experience may develop their English language skills more quickly, as they have already developed a foundation of language learning skills and strategies. It is important for teachers to consider the impact of learners' past language learning experiences when planning their lessons and activities, and to be mindful of how these experiences may affect learners' attitudes and motivation towards English language learning (Gardner & Lambert, 1972). It is important for teachers to consider the impact of learners' past language learning experiences when planning their lessons and activities, and to be mindful of how these experiences may affect learners' attitudes and motivation towards English language learning (Gardner & Lambert, 1972). Personal and emotional factors are very important, and they have their effect over learners during the learning process. Positive emotions such as motivation, engagement, and confidence can enhance learning, while negative emotions such as anxiety, stress, and frustration can interfere with it. Additionally, personal factors such as past experiences, self-esteem, and individual learning styles can also play a role in how a learner approaches and processes information. It's important for educators to be aware of these factors and create a supportive and inclusive learning environment that takes these into consideration. ISSN 2348-3156 (Print) International Journal of Social Science and Humanities Research ISSN 2348-3164 (online) Vol. 11, Issue 1, pp: (134-144), Month: January - March 2023, Available at: www.researchpublish.com Effective learning strategies are of great importance for language learners as they can greatly influence language acquisition and success. A study by Oxford (1990) found that strategy use was positively correlated with language achievement, and another study by Rigney (2002) found that language learners who used more strategies achieved higher levels of language proficiency. Moreover, research has shown that certain strategies are more effective for different learners and learning situations (Nunan, 1999). For example, using visualization techniques may be effective for visual learners, while using mnemonics may be effective for auditory learners. Some effective learning strategies that are of great importance for language learners who can benefit from include: P \| 140 a. Immersion: surrounding oneself with the target language through reading, listening, speaking, and watching media. Page \| 140 ISSN 2348-3156 (Print) International Journal of Social Science and Humanities Research ISSN 2348-3164 (online) Vol. 11, Issue 1, pp: (134-144), Month: January - March 2023, Available at: www.researchpublish.com ( ) International Journal of Social Science and Humanities Research ISSN 2348-3164 (online) Vol. 11, Issue 1, pp: (134-144), Month: January - March 2023, Available at: www.researchpublish.com Negative attitudes towards English language learning can be caused by a number of factors, including lack of confidence, difficulty with the language, and feeling overwhelmed by the challenges of learning a new language. To address these attitudes, teachers can provide targeted support, such as extra tutoring or language-specific instruction, and encourage learners to adopt a growth mindset, viewing challenges as opportunities for growth and learning. It is also important for teachers to be aware of cultural and linguistic factors that may influence learners' attitudes towards English language learning. For example, learners from cultures where English is not the dominant language may have negative attitudes towards the language, due to historical or cultural associations. Teachers can help to mitigate these attitudes by fostering a positive and inclusive learning environment, and by recognizing and celebrating the linguistic and cultural diversity of their learners. A positive attitude towards learning English is essential for success, and teachers can play a key role in fostering this attitude among their learners. By creating a supportive learning environment, providing meaningful and relevant learning experiences, and encouraging a growth mindset, teachers can help English language learners to develop a positive and motivated approach to their learning. V. The Fifth common learner characteristics is past language learning experience which can significantly affect the success and motivation of English language learners (Dornyei, 2001). Some of the ways past language learning experience can impact learners include: V. The Fifth common learner characteristics is past language learning experience which can significantly affect the success and motivation of English language learners (Dornyei, 2001). Some of the ways past language learning experience can impact learners include: Attitudes and motivation: Past language learning experiences, particularly positive ones, can shape learners' attitudes and motivation towards English language learning. Learners who have had successful language learning experiences in the past may be more confident and motivated to learn English, while those who have had negative experiences may be less motivated and more resistant to learning. Learning strategies: Learners who have had prior language learning experience may have developed effective learning strategies that they can apply to their English language learning. For example, they may be more familiar with using self- directed learning resources or seeking feedback from others. (Lightbown, P. ISSN 2348-3156 (Print) International Journal of Social Science and Humanities Research ISSN 2348-3164 (online) Vol. 11, Issue 1, pp: (134-144), Month: January - March 2023, Available at: www.researchpublish.com A positive attitude towards learning English can lead to increased motivation, engagement, and achievement, while a negative attitude can hinder progress and lead to frustration and demotivation. Positive attitudes towards learning English can be fostered by creating a supportive and inclusive learning environment, providing opportunities for learners to use English in real-world contexts, and by recognizing and celebrating the progress and achievements of individual learners. Page \| 139 Page \| 139 Research Publish Journals Research Publish Journals ISSN 2348-3156 (Print) International Journal of Social Science and Humanities Research ISSN 2348-3164 (online) Vol. 11, Issue 1, pp: (134-144), Month: January - March 2023, Available at: www.researchpublish.com ISSN 2348 3156 (Print) International Journal of Social Science and Humanities Research ISSN 2348-3164 (online) Vol. 11, Issue 1, pp: (134-144), Month: January - March 2023, Available at: www.researchpublish.com e. Listening practice: listening to native speakers and practicing comprehension through audio materials, such as podcasts or audio books. e. Listening practice: listening to native speakers and practicing comprehension through audio materials, such as podcasts or audio books. f. Cultural immersion: learning about the culture and customs of the target language to deepen understanding and appreciation. f. Cultural immersion: learning about the culture and customs of the target language to deepen understanding and appreciation. g. Personalized practice: customizing language learning to individual needs, interests, and learning styles. VI. Maturity is also considered as one of the most important learning characteristic that refers to the level of psychological and emotional development of a learner. Maturity can influence a learner's ability to handle stress, manage emotions, and make responsible decisions. Maturity can also impact a learner's motivation and self-discipline, as well as their ability to work independently and collaborate with others. Maturity can play a significant role in language learning, and there are several ways in which differences in maturity can influence the language acquisition process. (Dörnyei, Z. 2010). Some of these differences include: 1. Motivation: Maturity level can impact a learner's motivation to study a language. More mature learners may be self- motivated and focused on their long-term goals, while less mature learners may need more structure and external incentives to stay engaged. (Deci, E. L., & Ryan, R. M. 2000) 2. Attention span: Differences in maturity can affect a learner's ability to focus and retain information for longer periods of time. Mature learners may be able to concentrate for longer, while less mature learners may have a shorter attention span. 3. Emotional control: Maturity can impact a learner's ability to regulate emotions, particularly in stressful or challenging situations. More mature learners may be better able to manage their emotions and remain calm, while less mature learners may become frustrated or overwhelmed more easily. 4. Self-discipline: Differences in maturity can also impact a learner's self-discipline and ability to study and practice regularly on their own. Research Publish Journals Mature learners may have better time management skills and be more self-directed, while less mature learners may need more guidance and structure. It's important for language teachers to take into consideration the maturity level of their students and to provide appropriate support and guidance based on individual needs differences in Maturity among children, teenagers and adults that influence language learning Maturity level can play a significant role in language learning, and there are several differences in maturity among children, teenagers, and adults that can influence the language acquisition process. Some of these differences include: a. Children: Children are typically less mature than teenagers and adults, and may have shorter attention spans, lower levels of self-discipline, and more limited emotional control. Children may also need more structure and guidance in their language learning, as well as hands-on, interactive activities to keep them engaged. ( Gallahue, D. L., & Ozmun, J. C. 2012) b. Teenagers: Teenagers are often more mature than children, but may still struggle with self-discipline and attention span. Teenagers may be more motivated by peer pressure and social interaction, and may benefit from opportunities to use the language in real-life situations. (Gallahue, D. L., & Ozmun, J. C. 2012) c. Adults: Adults are typically more mature and self-directed than children and teenagers, and may have better attention span, self-discipline, and emotional control. Adults may also have more life experience and a clearer understanding of their learning goals, which can help them stay motivated and focused. It's important for language teachers to take into consideration the maturity level of their students and to provide appropriate support and guidance based on individual needs. Different teaching methods and materials may be more or less effective for different age groups, and teachers should adjust their approach accordingly. It's important for language teachers to take into consideration the maturity level of their students and to provide appropriate support and guidance based on individual needs. Different teaching methods and materials may be more or less effective for different age groups, and teachers should adjust their approach accordingly. In general, mature learners are more self-directed and have a clearer understanding of their learning goals and objectives. They are also more resilient and adaptable, able to overcome obstacles and setbacks in their learning journey. In general, mature learners are more self-directed and have a clearer understanding of their learning goals and objectives. Research Publish Journals VII. Teaching methods Staying current with research: Keeping up to date with research in the field of language teaching can help teachers stay informed about best practices and new developments in the field. (Harmer, J. 2007) By implementing these strategies, English teachers can continuously improve their teaching methods and support their students in achieving their language learning goals. By implementing these strategies, English teachers can continuously improve their teaching methods and support their students in achieving their language learning goals. There is one important question for teachers to ask, “How can English teachers know their students’ weaknesses and strength?” The answer to this question will enable them to build their teaching strategies and method, to get the best results from teaching. There is one important question for teachers to ask, “How can English teachers know their students’ weaknesses and strength?” The answer to this question will enable them to build their teaching strategies and method, to get the best results from teaching. The best way for any teacher to know their students’ weaknesses and strength is the profile of their students which plays a significant role in their learning as it provides insight into their individual strengths, weaknesses, and learning preferences. Understanding the profile of the student allows the teacher to tailor their teaching methods to meet the needs of individual learners and create a more inclusive and effective learning environment. The best way for any teacher to know their students’ weaknesses and strength is the profile of their students which plays a significant role in their learning as it provides insight into their individual strengths, weaknesses, and learning preferences. Understanding the profile of the student allows the teacher to tailor their teaching methods to meet the needs of individual learners and create a more inclusive and effective learning environment. The student profile can include information on their learning style, prior knowledge, motivation, personality, and maturity. By considering these factors, the teacher can make informed decisions about the types of activities, materials, and assessments that are best suited to the individual learner. For example, if a student is a visual learner, the teacher can incorporate more visual aids into their lessons. If a student has a high level of prior knowledge, the teacher can challenge them with more advanced material. Additionally, by understanding the student profile, teachers can better support their students in reaching their full potential. Research Publish Journals They are also more resilient and adaptable, able to overcome obstacles and setbacks in their learning journey. Page \| 141 ISSN 2348-3156 (Print) International Journal of Social Science and Humanities Research ISSN 2348-3164 (online) Vol. 11, Issue 1, pp: (134-144), Month: January - March 2023, Available at: www.researchpublish.com It's important for educators to take into consideration the maturity level of their students when planning and delivering instruction. For example, a more mature learner may benefit from more complex and challenging tasks, while a less mature learner may need more structure and support. VII. Teaching methods English teachers can greatly impact their learners' achievement by continuously developing and refining their teaching methods. By staying up-to-date with the latest research and best practices in language teaching, English teachers can adopt innovative and effective instructional strategies that engage and motivate their students. This may involve incorporating technology and multimedia resources into the classroom, utilizing task-based and communicative approaches, and providing opportunities for students to practice their language skills in authentic and meaningful contexts. Additionally, English teachers can also assess their students' learning regularly and provide targeted feedback to help them identify areas for improvement and grow as language learners. (Willis, J., & Willis, D. 2007). By adopting these strategies and continuously refining their teaching methods, English teachers can create a supportive and effective learning environment that maximizes their learners' potential for achievement. They can develop their teaching methods to improve their learners' achievement in several ways like: a. Continuous Professional Development (CPD): Participating in professional development opportunities such as workshops, conferences, or online courses can help teachers stay up to date with current teaching methods and materials, as well as best practices for language teaching. (Richards, J. C. 2015) a. Continuous Professional Development (CPD): Participating in professional development opportunities such as workshops, conferences, or online courses can help teachers stay up to date with current teaching methods and materials, as well as best practices for language teaching. (Richards, J. C. 2015) b. Reflection and evaluation: Regularly reflecting on their teaching and seeking feedback from students, colleagues, and supervisors can help teachers identify areas for improvement and make changes to their teaching methods. c. Adapting teaching methods: English teachers can try different teaching methods to find what works best for their students. This can include incorporating active learning activities, using technology, or incorporating different types of assessment. d. Collaboration: Collaborating with other English teachers or language experts can provide opportunities for sharing ideas and resources, as well as learning from others' experiences. e. Student-centered teaching: Focusing on student needs and making teaching decisions based on individual learner characteristics can help teachers provide a more effective and engaging learning environment. f. Differentiation: Differentiating instruction to meet the needs of individual learners can help ensure that all students have equal opportunities for success. g. Research Publish Journals VII. Teaching methods For example, if a student lacks motivation, the teacher can work with them to identify their interests and incorporate them into their learning experience. Page \| 142 ISSN 2348-3156 (Print) International Journal of Social Science and Humanities Research ISSN 2348-3164 (online) Vol. 11, Issue 1, pp: (134-144), Month: January - March 2023, Available at: www.researchpublish.com In conclusion, considering the student profile is an important aspect of language teaching, as it allows the teacher to create a more effective and engaging learning environment that supports the individual needs of each learner. English teachers can identify their students' strengths and weaknesses in other several ways, like: English teachers can identify their students' strengths and weaknesses in other several ways, like: 1. Observations: Regularly observing students during class and taking note of their engagement, participation, and performance can provide insight into their strengths and weaknesses. 1. Observations: Regularly observing students during class and taking note of their engagement, participation, and performance can provide insight into their strengths and weaknesses. 2. Assessment: Administering assessments such as quizzes, tests, or written assignments can provide concrete data on students' proficiency in various language skills. 3. Feedback from students: Encouraging students to provide feedback on their own learning experiences can help teachers understand their individual needs and areas for improvement. 4. One-on-one meetings: Scheduling regular one-on-one meetings with students to discuss their progress and provide support can help teachers identify strengths and weaknesses. 5. Student portfolios: Encouraging students to maintain portfolios of their work can provide evidence of their growth and development over time. 6. Learning style assessments: Administering assessments to determine students' learning styles can help teachers understand how they process and retain information, and make adjustments to their teaching methods accordingly. By using a combination of these methods, English teachers can gain a comprehensive understanding of their students' strengths and weaknesses and make informed decisions about how best to support their learning. On the other hand, teachers can design lessons that incorporate activities that match their students' learning styles to make the learning experience more effective and engaging. For example, if a student is an auditory learner, the teacher can incorporate listening activities, such as dictation or role-plays, into the lesson. If a student is a visual learner, the teacher can include graphic organizers or visual aids in the lesson. VII. Teaching methods For kinaesthetic learners, hands-on activities, such as simulations or games, can be incorporated into the lesson. By considering their students' learning styles, teachers can create a more inclusive and effective learning environment that supports the needs of all learners. It's important to note, however, that students may have a combination of learning styles, and the teacher may need to adjust their approach accordingly. Additionally, it's important for teachers to provide a variety of activities and materials to cater to different learning styles, as well as to offer opportunities for students to practice different language skill. THE CONCLUSION In conclusion, there is a complex relationship between learner characteristics and language learning. While factors such as age, motivation, and learning style can impact language learning outcomes, it is important to note that language learning is a highly individualized process and the role of each factor can vary from learner to learner. For example, research suggests that younger learners may have an advantage in acquiring native-like pronunciation, while older learners may have greater knowledge of grammar and vocabulary. Similarly, learners with high levels of motivation and a positive attitude towards language learning tend to achieve better outcomes, but there is no one-size-fits-all solution to motivation. Furthermore, learning styles can influence how learners process and retain information, but again, individual differences mean that some learners may thrive in a more structured classroom environment, while others may prefer a more experiential, communicative approach. Therefore, language educators should be aware of the potential impact of learner characteristics on language learning, but should also take a flexible, adaptive approach to instruction that meets the needs of each individual learner. By doing so, language educators can create a supportive and effective learning environment that enables all learners to reach their full potential in language learning. Page \| 143 Research Publish Journals Page \| 143 Research Publish Journals Page \| 143 Page \| 143 ISSN 2348-3156 (Print) International Journal of Social Science and Humanities Research ISSN 2348-3164 (online) Vol. 11, Issue 1, pp: (134-144), Month: January - March 2023, Available at: www.researchpublish.com International Journal of Social Science and Humanities Research ISSN 2348-3164 (online) Vol. 11, Issue 1, pp: (134-144), Month: January - March 2023, Available at: www.researchpublish.com REFERENCES [1] Felder, R. M., & Silverman, L. K. (1988). Learning and teaching styles in engineering education. Engineering education, 78(7), 674-681. [2] Kolb, D. A. (1984). Experiential learning: Experience as the source of learning and development. Prentice-Hall. [3] Dunlosky, J., Rawson, K. A., Marsh, E. J., Nathan, M. J., & Willingham, D. T. (2013). Improving students' learning with effective learning techniques: Promising directions from cognitive and educational psychology. Psychological Science in the Public Interest, 14(1), 4-58. [4] Bjork, R. A., & Bjork, E. L. (2011). Making things hard on yourself, but in a good way: Creating desirable difficulties to enhance learning. In M. A. Gernsbacher, R. W. Pew, L. M. Hough, & J. R. Pomerantz (Eds.), Psychology and the real world: Essays illustrating fundamental contributions to society (pp. 56-64). New York: Worth Publishers. [5] Kornell, N., & Bjork, R. A. (2008). Learning concepts and categories: Is spacing the "enemy of induction"? Psychological Science, 19(3), 585-592. [6] Kavale, K. A., & Forness, S. R. (2000). What is a learning disability? Learning Disability Quarterly, 23(2), 93-104. [7] Pashler, H., McDaniel, M., Rohrer, D., & Bjork, R. (2008). Learning styles: Concepts and evidence. Psychological Science in the Public Interest, 9(3), 105-119. [8] Martino, W., & Monfils, M. H. (2017). The effectiveness of auditory learning for memory and cognitive development in early childhood education. International Journal of Child Care and Education Policy, 11(1), 1-11. [9] Molloy, E. (2017). Understanding the auditory learner: An exploration of the benefits and challenges. Journal of Education and Practice, 8(29), 22-30. [10] Fiorella, L., & Mayer, R. E. (2015). A cognitive theory of multimedia learning: Implications for design principles. The Cambridge Handbook of Multimedia Learning, 2nd Edition, 41-60 [11] Fleming, N. D. (2011). Teaching and learning styles: VARK strategies. Neil D. Fleming, [12] Gallahue, D. L., & Ozmun, J. C. (2012). Understanding motor development: Infants, children, adolescents, adults. McGraw-Hill Education. [13] Ryan, R. M., & Deci, E. L. (2017). Self-determination theory: Basic psychological needs in motivation, development, and wellness. Guilford Press. [14] Elliot, A. J., & Dweck, C. S. (2005). Handbook of competence and motivation. Guilford Press. [15] Deci, E. L., & Ryan, R. M. (2000). The "what" and "why" of goal pursuits: Human needs and the self-determination of behavior. Psychological Inquiry, 11(4), 227-268. [16] Ryan, R. M., & Deci, E. L. (2017). Self-determination theory: Basic psychological needs in motivation, development, and wellness. Guilford Press. [17] Elliot, A. REFERENCES J., & Dweck, C. S. (2005). Handbook of competence and motivation. Guilford Press. [18] Dörnyei, Z. (2001). Motivational strategies in the language classroom. Cambridge University Press. [19] Dörnyei, Z. (2010). The psychology of second language acquisition. Oxford University Press. [20] Krashen, S. D. (1982). Principles and practice in second language acquisition. Prentice Hall. [21] Willis, J., & Willis, D. (2007). Doing Task-based Teaching. Oxford University Press. 22] Harmer, J. (2007). The Practice of English Language Teaching (4th ed.). Longman. 23] Richards, J. C. (2015). Key issues in language teaching. Routledge. [23] Richards, J. C. (2015). Key issues in language teaching. Routledge. [24] Pintrich, P. R. (2003). A motivational science perspective on the role of student motivation in learning and teaching contexts. Journal of Educational Psychology, 95(4), 667-686. Page \| 144 Page \| 144 Research Publish Journals
https://openalex.org/W3159398135	https://repozitorij.uni-lj.si/Dokument.php?id=153806&dn=	English	null	Exploring Equity in Public Transportation Planning Using Smart Card Data	Sensors	2,021	cc-by	14,345	Citation: Ghasemlou, K.; Ergun, M.; Dadashzadeh, N. Exploring Equity in Public Transportation Planning Using Smart Card Data. Sensors 2021, 21, 3039. https://doi.org/10.3390/ s21093039 Academic Editors: Tamer Nadeem, Michail Makridis, Anastasios Kouvelas, Tomer Toledo and Rui Jiang Keywords: public transportation; smart card data; equity; cost beneﬁt analysis; travel behavior; mobility pattern; transport planning; human centric planning Received: 29 January 2021 Accepted: 12 April 2021 Published: 26 April 2021 Received: 29 January 2021 Accepted: 12 April 2021 Published: 26 April 2021 Article Exploring Equity in Public Transportation Planning Using Smart Card Data Kiarash Ghasemlou 1,, Murat Ergun 2 and Nima Dadashzadeh 3 Kiarash Ghasemlou 1,, Murat Ergun 2 and Nima Dadashzadeh 3 1 Graduate School of Science, Engineering and Technology, Istanbul Technical University, 34467 Istanbul, Turkey 1 Graduate School of Science, Engineering and Technology, Istanbul Technical University, 34467 Istanbul, Turkey 2 Civil Engineering Faculty, Istanbul Technical University, 34467 Istanbul, Turkey; ergunmur@itu.edu.tr 3 Faculty of Civil and Geodetic Engineering, University of Ljubljana, 1000 Ljubljana, Slovenia; nima.dadashzadeh@fgg.uni-lj.si * Correspondence: ghasemlou@itu.edu.tr Abstract: Existing public transport (PT) planning methods use a trip-based approach, rather than a user-based approach, leading to neglecting equity. In other words, the impacts of regular users—i.e., users with higher trip rates—are overrepresented during analysis and modelling because of higher trip rates. In contrast to the existing studies, this study aims to show the actual demand characteristic and users’ share are different in daily and monthly data. For this, 1-month of smart card data from the Kocaeli, Turkey, was evaluated by means of speciﬁc variables, such as boarding frequency, cardholder types, and the number of users, as well as a breakdown of the number of days traveled by each user set. Results show that the proportion of regular PT users to total users in 1 workday, is higher than the monthly proportion of regular PT users to total users. Accordingly, users who have 16–21 days boarding frequency are 16% of the total users, and yet they have been overrepresented by 39% in the 1-day analysis. Moreover, users who have 1–6 days boarding frequency, have a share of 66% in the 1-month dataset and are underrepresented with a share of 22% in the 1-day analysis. Results indicated that the daily travel data without information related to the day-to-day frequency of trips and PT use caused incorrect estimation of real PT demand. Moreover, user-based analyzing approach over a month prepares the more realistic basis for transportation planning, design, and prioritization of transport investments. sensors sensors sensors 1. Introduction The sustainable development of transportation has become a key point of interest on the part of scholars and policy-makers over recent decades. In this context, sustainability not only refers to environmentally friendly transportation systems but also to those that are economically and socially sustainable. In line with sustainable transport development, accessibility and mobility for all are two new paradigms altering conventional transport planning and policy-making. Sustainable mobility [1] and accessible transport [2] are not new concepts in transportation. For instance, bus priority methods are one of strategies to support sustainable and equitable transport planning by giving priority to bus on trafﬁc congestion [3,4]. Beyazit [5] noted that sustainable transport and mobility plays a crucial role in distributing socio-economic beneﬁts or losses, as well as social justice. Therefore, the efﬁciency of the exiting method regarding sustainable transport planning and projects analyses should be critically evaluated and improved considering issues such as social justice [5–7], equity [8,9], and social inclusion (exclusion) [8,9]. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). On the other hand, methods that are used in the evaluation and prioritization of pub- lic transport (PT) investment projects are more trip-based rather than user-based [10,11]. https://www.mdpi.com/journal/sensors Sensors 2021, 21, 3039. https://doi.org/10.3390/s21093039 Sensors 2021, 21, 3039 2 of 23 Using a trip-based approach, the needs of users with more boarding frequency are over- represented in transport planning and modelling. In addition, some routes are used by more people overall, even though the number of users is rare on a daily basis. This being the case, the best example comes in the form of medical-based PT routes (trips to medical centers). The shortening or lifting of these PT routes, even if the number of trips is small, affects more people than the number of those using the line each day. This is because, along such routes, different users take the routes every day. On contrary, some routes have high boarding ﬁgures by day but are taken by the same users daily, such as commuter routes at rush hour. 1. Introduction The use of anonymous big data, e.g., PT Smart Card Fare Collection (PT-SCFC) data, has begun to ﬁgure more predominantly in the analysis of travel behavior and mobility patterns [12–14]. However, the misrepresentation and mis-modelling that characterizes the above in the form of using such large data can be found in a number of recently published studies [15–19]. In contrast to existing studies and considering the number of days and number of trips, or boardings that PT users make over a month, this study shows how more appropriate modelling can result in socially equitable opportunity and services to PT users. In the following sections the statement of the art related to the equity in transporta- tion and PT-SCFC application in PT is given. Afterward, a case study together with the associated data, and data-cleaning process is detailed. Then, results of the analysis and the related discussion on equity in PT planning is given. Finally, the highlights of the study together with the ﬁndings and suggestion for potential future studies is provided. 2.2. Smart Card Data Application in PT Planning PT smart card data beneﬁts scholars and practitioners in understanding urban dynam- ics and human activities [12,33]. For instance, smart card data can be used to estimate the Origin-Destination (OD) of PT users, long-term network planning, demand forecasting, operational purposes like timetable and schedule adjustments as well as PT funding and investment decisions [13,33–40]. PT smart card data includes information on boarding numbers, their times, and locations. As tons of data are gathered by fare collection systems every day, data exploration and data-driven segmentation on users has been become an essential issue. Data-driven segmentation analyses consist of spatial and temporal probabil- ity. The ﬁrst refers to users’ tendency to board a given station for their trips, while the later refers to users’ tendency to travel at a particular hour of the day i.e., trip frequency [41]. Espinoza et al. [42] measured PT users’ behavior change in travel over time by splitting data into different time windows using three algorithms (Transition Probability Matrix (TPM), Spatiotemporal Edit Distance Method (EDM) and Regions of Interest and Feature Vector (RoIs-FV)). It was found that the results obtained for the same users can be different using different algorithms. However, the behavior of the variability through time is similar for the three algorithms evaluated. g Most existing studies on the smart card data evaluation are provided on a daily bases and there are very limited studies on the monthly trip frequency of PT users. Benenson and Ben-Elia [43] have illustrated the unexpected ﬂexibility of PT usage by considering their daily boarding numbers. This study has examined data on a weekly basis only and focuses on trip counts alone. The results ﬁnd that the rate of trips made once or twice in the total weekly data are higher than expected. Some studies considered the variability of demand on PT and temporal rhythms in travel and activity patterns [42,44–47]. However, the actual PT demand is difﬁcult to be obtained as it changes continuously over the time (period of the day, day of the week, season, or holiday) [48]. For instance, Morency et al. [47] showed that spatial and temporal variability of PT use can be measured using smart card data considering bus stops used for boarding and frequency of using of the bus stop. 2.1. Equity in PT Planning Equity and social inclusion (exclusion) as a transport planning issue has been deﬁned by Litman [20,21] as the fair distribution of beneﬁts and cost impacts of all users. In addition, social exclusion (the transport of disadvantaged people) referred to the barriers (households do not own personal car, low-income individuals, persons with disabilities or reduced mobility, such as the elderly) preventing people from using public services like transport, education, and jobs, etc. It was concluded that inadequate transport planning, policies, and decisions have direct equity impacts and cause social exclusion. Transport equity and social inclusion analyses, in particular, in PT projects, are very difﬁcult to conduct due to their complexity, spatiotemporal dimensions, impacts measurement, and users’ categorization [8,22–26]. There are also a number of psychosocial barriers to PT use when it comes to the elderly, who have speciﬁc need for a socially sustainable transportation system that should constitute a paramount consideration [27]. To address these challenges, three indicators proposed by Di Ciommo et al. [8], including: (i) indicators that make it possible to assess how much beneﬁts or costs are being received by different population groups, (ii) indicators to disaggregate population groups from each other, and (iii) indicators to determine the equity of an observed distribution of a particular beneﬁt or cost (e.g., transit subsidies or direct access to key activities). Equity in PT planning can be evaluated from different dimensions—namely, spatial and temporal dimensions considering socio- economic characteristics like age, gender, income, and health condition (e.g., disability). • Socio-spatial equity: equitable access to transport services in terms of spatial and land-use patterns [28,29], such as transport accessibility in urban area vs. sub-urbans or rural area, as well as in CBD (central business district) area vs. non-CBD area; • Temporal equity: equitable access to transport services considering the time-critical nature of accessibility needs [24], such as transport accessibility for users with low trip frequency or high trip frequency (regular users) during peak-hour or off-peak-hour. To evaluate PT transport projects, Cost Beneﬁt Analysis (CBA) is one of the most common techniques [10,11,30–32]. There are also other project appraisal methods such as the Multi Criteria Analysis (MCA), social-based analysis, decision-analysis, simulation, and mathematical modelling. 2.1. Equity in PT Planning CBA has some limitations, such as the complexity of the concept Sensors 2021, 21, 3039 3 of 23 3 of 23 in application and the weakness in assessing environmental, health, and social issues, such as equity and social justice—as these elements cannot be examined in monetary terms [5]. For instance, it has been found that the CBA framework and project appraisal considering only “travel time savings” has various equity effects [10]. The results also show that CBA appraisal are not accurate enough to measure distributional impacts of equity. 3.1. Data and Study Area of Trips % Walking 135,085 21% 539,252 70% 223,257 27% 76,573 39% 974,168 40% Private car 172,874 21% 29,605 70% 296,496 27% 66,062 39% 565,037 23% Shuttle service 201,878 31% 108,173 14% 13,135 2% 7397 4% 330,583 14% Public transport 149,105 23% 89,976 12% 285,815 35% 48,230 24% 573,125 23% Total 658,943 100% 767,005 100% 818,703 100% 198,262 100% 2,442,913 100% There are 361 ofﬁcial bus routes in KMM, but 293 of these currently are active and operational. KMM’s bus transport network includes a total of 7400 bus stops, but the maximum number of active bus stops comes to 4869. Figure 1 shows the study area and PT assignment results, which is modelled using VISUM macroscopic transport modelling software [52]. In this study, the smart card data from the KMM were used. The dataset included 1-month smart card data of November 2018. The PT smart card data of KMM included unique card ID, card types such as normal, student, elderly, and people with disabilities cards, boarding station ID, card boarding (validation) time, cost boarding, type of boarding, route, direction, and vehicle ID. When the user boards the buses, the smart card’s fare was validated. The card validation process consisted of the following steps: the Global Positioning System (GPS) reader on the bus identiﬁes the stop where the boarding is made. The system validates the run (correct route) at this location as the bus system contains planned runs for a single day (a run refers to a sequence of stops to be deserved; it usually represents one direction of a route) Card numbers dates times validation status and Table 1. Modal share and trip purposed distribution [51]. Table 1. Modal share and trip purposed distribution [51]. Transportation Modes Home-Based Work Trips Home-Based School Trips Home-Based Other Trips Non-Home-Based Trips Total Trips No. of Trips % No. of Trips % No. of Trips % No. of Trips % No. of Trips % Walking 135,085 21% 539,252 70% 223,257 27% 76,573 39% 974,168 40% Private car 172,874 21% 29,605 70% 296,496 27% 66,062 39% 565,037 23% Shuttle service 201,878 31% 108,173 14% 13,135 2% 7397 4% 330,583 14% Public transport 149,105 23% 89,976 12% 285,815 35% 48,230 24% 573,125 23% Total 658,943 100% 767,005 100% 818,703 100% 198,262 100% 2,442,913 100% There are 361 ofﬁcial bus routes in KMM, but 293 of these currently are active and operational. 2.2. Smart Card Data Application in PT Planning K-mean algorithm was used to cluster transit use cycles and homogenous days and weeks of travel among card segments and at various times of the year. Likewise, Raux et al. [45] have used the sequential alignment method in measuring the variability in day-to-day travel-activity behavior according to interpersonal and intrapersonal differences of attributes. They concluded that intrapersonal variability is greater than the interpersonal one considering the random part of behavior, while intrapersonal variability is lower than the interpersonal one due to habitual part of behavior. p Liu et al. [49] underlined the relationship between user demographic characteristics (e.g., the role of gender and age) and the variability of travel behavior. Results showed that female users exhibit higher intrapersonal variability than their male counterparts. Weekly patterns are the most diverse for users aged 70+, followed by the users aged 65–69. More recently, Egu and Bonnel [44] combined clustering algorithm with day-to-day intrapersonal similarity metric to explore day-to-day intrapersonal variability of PT usage. It was found that there is no one-size-ﬁts-all approach to the problem of day-to-day variability of transit usage. According to their studies, low-frequency users represent only 1% of the total journey, although it is 14% of the total data. This study is not related to determination of real demand of PT users and equity issues. Clustering method used in the existing studies caused losing some of information related to day-to-day individual behavior, and they focused more on most common day-to-day usage pattern results. Sensors 2021, 21, 3039 4 of 23 The literature review shows that in studies based on smart card data, there was no comparison in terms of overrepresented and underrepresented users and trips between whole data and 1 day data. Therefore, this study aims to show the importance of deﬁning temporal frequency to better determine the actual share of users and demand in transport planning. We deﬁne a new approach in equity analysis of transport planning that reveals the actual demand characteristic, and users’ share are different in daily data (which has been usually used by the existing transport planning methods) compared to the real demand and users’ share. Thus, our study has purposefully focused on the trip and PT usage frequency of users to be extracted from a 1-month set of PT-SCFC data. These are the main contribution of our study with respect to the related work. 3.1. Data and Study Area Kocaeli is one of the most populated provinces in Turkey, with a population of around 1.9 million (2019) across 12 districts, 13 municipalities, including one metropolitan and 12 district municipalities, and a population density of 575 people per km2. Brieﬂy, 93.7% of the population live in cities, while the rest live in villages. Kocaeli Metropolitan Mu- nicipality (KMM) is located at the easternmost end of the Marmara Sea around the Gulf of Izmit [50]. Public transport is one the most frequent transportation modes, with a daily average ridership of around 408,000 passengers. Among motorized transportation, PT share repre- sents 37% of the total modal share [51]. Information about the data on the mode share of PT according to trip purpose is given in Table 1. Table 1. Modal share and trip purposed distribution [51]. Transportation Modes Home-Based Work Trips Home-Based School Trips Home-Based Other Trips Non-Home-Based Trips Total Trips No. of Trips % No. of Trips % No. of Trips % No. of Trips % No. of Trips % Walking 135,085 21% 539,252 70% 223,257 27% 76,573 39% 974,168 40% Private car 172,874 21% 29,605 70% 296,496 27% 66,062 39% 565,037 23% Shuttle service 201,878 31% 108,173 14% 13,135 2% 7397 4% 330,583 14% Public transport 149,105 23% 89,976 12% 285,815 35% 48,230 24% 573,125 23% Total 658,943 100% 767,005 100% 818,703 100% 198,262 100% 2,442,913 100% There are 361 ofﬁcial bus routes in KMM, but 293 of these currently are active and operational. KMM’s bus transport network includes a total of 7400 bus stops, but the maximum number of active bus stops comes to 4869. Figure 1 shows the study area and PT assignment results, which is modelled using VISUM macroscopic transport modelling software [52]. In this study, the smart card data from the KMM were used. The dataset included Table 1. Modal share and trip purposed distribution [51]. Transportation Modes Home-Based Work Trips Home-Based School Trips Home-Based Other Trips Non-Home-Based Trips Total Trips No. of Trips % No. of Trips % No. of Trips % No. of Trips % No. 3.1. Data and Study Area KMM’s bus transport network includes a total of 7400 bus stops, but the maximum number of active bus stops comes to 4869. Figure 1 shows the study area and PT assignment results, which is modelled using VISUM macroscopic transport modelling software [52]. In this study, the smart card data from the KMM were used. The dataset included 1-month smart card data of November 2018. The PT smart card data of KMM included unique card ID, card types such as normal, student, elderly, and people with disabilities cards, boarding station ID, card boarding (validation) time, cost boarding, type of boarding, route, direction, and vehicle ID. When the user boards the buses, the smart card’s fare was validated. The card validation process consisted of the following steps: the Global Positioning System (GPS) reader on the bus identiﬁes the stop where the boarding is made. The system validates the run (correct route) at this location as the bus system contains planned runs for a single day (a run refers to a sequence of stops to be deserved; it usually represents one direction of a route). Card numbers, dates, times, validation status, and stop numbers are stored at each boarding. This information is downloaded to the central server at of the end of each day. Smart card boarding data was collected over a period of 30 days for each card. Each record was divided into nine binary variables. This day Sensors 2021, 21, 3039 5 of 23 division was common to transport planning studies in Turkey, though other countries may have different time intervals. division was common to transport planning studies in Turkey, though other countries may have different time intervals. division was common to transport planning studies in Turkey, though other countrie have different time intervals. Figure 1. Public transport (PT) network assignment in our study area; Kocaeli metropolitan municipality. igure 1. Public transport (PT) network assignment in our study area; Kocaeli metropolitan municipality. T) network assignment in our study area; Kocaeli metropolitan municipality. The data set was a compilation of 12,250,983 boarding validations made by 808,834 card holders on the Kocaeli Public Transport network between 1 November and 30 November 2018. The average daily number of users came to 201,212 people with a standard devia- tion of 35,155. 3.1. Data and Study Area Sunday November 14th had the minimum number of users with 121,711 (240,461 boarding), while Thursday November 18th had the maximum number of users by 227,347 (466,280 boarding). As the boarding numbers vary on weekdays and weekends, it would be more accurate to analyze them separately. Step 1. Data Extraction 1.1. Extracting data from SQL server of KMM; g 1.2. Converting extracted data to daily basis Excel ﬁle; 3.2. Data Analysis Method A monthly smart card dataset with information on around 12 million raw boardings (validation) extracted from a SQL Server in KMM was obtained. Then, Python and Excel were used for tracking each unique ID of card holders in the ensemble of the data set. The analysis was conducted as an aggregate view of 808,834 card holders. Then, various data mining techniques and tools (see [53], for more information) were used to clear and prepare the dataset align with the objectives of this study. The steps of data analysis in our study were as follows: data extraction from SQL server, data cleaning, dataset development, data ﬁltering, and the categorization of both Card ID-based and PT route-based categorizations, as seen in Figure 2. Looking at the ﬂowchart, data cleaning and categorization algorithm procedures succeed in the following: Step 2. Data Cleaning 2.1. Identifying the data without coordination information; 2.2. Identifying the data with false coordination information; 2.3. Modifying data coordination information according to General Transit Feed Speciﬁca- tion (GTFS) data coordination (PT schedules and associated geographic information provided by Google); 2.3. Modifying data coordination information according to General Transit Feed Speciﬁca- tion (GTFS) data coordination (PT schedules and associated geographic information provided by Google); Sensors 2021, 21, 3039 6 of 23 2.4. Categorizing card holders into six user groups with similar characteristics to come across more meaningful analysis. 2.4. Categorizing card holders into six user groups with similar characteristics to come across more meaningful analysis. 2.4. Categorizing card holders into six user groups with similar characteristics to come across more meaningful analysis. Step 3. Data set development and clustering Step 4. Data analysis and ﬁltering 4.1. CARD ID-based data analysis and ﬁltering 4.1.1. Identifying both workday (weekday) trips and weekend trips in ID-based dataset, and assign a dummy variable value (0 and 1) to them; 4.1.2. Identifying the number of days commuted by every card holder ID in entire dataset, determine the average daily trips, average monthly trips, and their standard deviation (STD); 4.1.3. Identifying the workday trips by every card holder ID, in entire dataset, deter- mine the average daily trips, average monthly trips, and their STD; 4.1.4. Identifying the weekend trips by every card holder ID, in entire dataset, deter- mine the average daily trips, average monthly trips, and their STD; 4.1.5. Identifying the number of days commuted by PT based on the day commuted (all days, workday, and weekend) and modiﬁed user groups (6 clustered user groups), determining the average daily trips, average monthly trips, and their STD. 4.1.1. Identifying both workday (weekday) trips and weekend trips in ID-based dataset, and assign a dummy variable value (0 and 1) to them; 4.1.2. Identifying the number of days commuted by every card holder ID in entire dataset, determine the average daily trips, average monthly trips, and their standard deviation (STD); 4.1.3. Identifying the workday trips by every card holder ID, in entire dataset, deter- mine the average daily trips, average monthly trips, and their STD; 4.1.4. Identifying the weekend trips by every card holder ID, in entire dataset, deter- mine the average daily trips, average monthly trips, and their STD; 4.1.5. Identifying the number of days commuted by PT based on the day commuted (all days, workday, and weekend) and modiﬁed user groups (6 clustered user groups), determining the average daily trips, average monthly trips, and their STD. 4.2. PT Route-based data analysis and ﬁltering 4.2.1. Filter the data based on PT routes; 4.2.2. For each PT route, identify the workday trips and weekend trips in entire ID dataset, and assign a dummy variable value (0 and 1) to them; 4.2.3. For each PT route, identify the number of days commuted by every card holder ID in entire dataset, determine the average daily trips, average monthly trips, and their STD; 4.2.3. For each PT route, identify the number of days commuted by every card holder ID in entire dataset, determine the average daily trips, average monthly trips, and their STD; 4.2.4. Step 3. Data set development and clustering 3.1. Filtering the dataset based on ID numbers; 3.1. Filtering the dataset based on ID numbers; 3.2. Extracting the following information for each ID (card holder) and add to ID dataset: daily number of boarding, trip-day information (which day and total number of days in 1 month), and card type. This refers to the calculation of group characteristics of each cardholder ID per day (frequency of PT use, boarding rate per workdays and weekends) and the calculation of its average values per user (including the average frequency of use and boardings per workday etc.); 3.3. Clustering natural cardholder groups according to their average value g g p g g 3.4. Clustering cardholder frequency groups (30 groups according to number of day that cardholder used PT); 3.5. Creating PT route-based dataset and extracting the boarding data of bus routes. Step 4. Data analysis and ﬁltering For each PT route, identify the workday trips by every card holder ID in entire data set, determine the average daily trips, average monthly trips, and their STD; 4.2.4. For each PT route, identify the workday trips by every card holder ID in entire data set, determine the average daily trips, average monthly trips, and their STD; 4.2.5. For each PT route, identify the weekend trips by every card holder ID in entire dataset, determine the average daily trips, average monthly trips, and their STD; 4.2.6. For each PT route, identify the number of days commuted based on the day commuted (all days, workday, and weekend) and modiﬁed user groups (i.e., six similar card holder groups), determine the average daily trips, average monthly trips, and their STD. 7 of 23 Sensors 2021, 21, 3039 Figure 2. Flow chart of the data mining and categorization in this study. Figure 2. Flow chart of the data mining and categorization in this study. To apply group characterization, ﬁrst the whole data set (1 month) is considered as an ensemble data set. Then, the raw datasets were subdivided into large, homogeneous clusters on the basis of trip patterns observed over a monthly basis. The goal was to split users in homogeneous groups according to their behavior and illustrate the similarity between each group. Afterward, card holders were categorized according to the number of days they used PT. For this, the following equations were deﬁned to examine the impact of user groups’ frequency and to detect this group of users in data set. g y • SCg,i,WD: number of card holder of group “g”, which have boarding in “i” days in weekday or weekend/holiday; Step 4. Data analysis and ﬁltering Percentage of card holder groups in 1-month dataset = SCg,i,WD/∑ n i=1 SCg,i,WD (1) Percentage of card holder groups in 1-day dataset = i ∗SCg,i,WD n /∑ n i=1 i ∗SCg,i,WD n (2) Percentage of card holder boardings in 1-day dataset = i ∗TCg,i,WD n /∑ n i=1 i ∗TCg,i,WD n , (3) where n i 1 n Percentage of card holder boardings in 1-day dataset = i ∗TCg,i,WD n /∑ n i=1 i ∗TCg,i,WD n , (3) where Percentage of card holder boardings in 1-day dataset = i ∗TCg,i,WD n /∑ n i=1 i ∗TCg,i,WD n , (3) where where where • WD: notation for weekday or weekend/holiday; whether the day of boarding (board- ing) is weekend or holiday; WD = 0, else WD = 1; • WD: notation for weekday or weekend/holiday; whether the day of boarding (board- ing) is weekend or holiday; WD = 0, else WD = 1; g y • SCg,i,WD: number of card holder of group “g”, which have boarding in “i” days in weekday or weekend/holiday; g y • SCg,i,WD: number of card holder of group “g”, which have boarding in “i” days in weekday or weekend/holiday; Sensors 2021, 21, 3039 8 of 23 • TCg,i,WD: number of boarding of card holder of group “g”, which have boarding in “i” days in weekday or weekend/holiday; • TCg,i,WD: number of boarding of card holder of group “g”, which have boarding in “i” days in weekday or weekend/holiday; y y y • n: number of days; for 1 month, n = 30; for weekday, n = 21; and for weekend, n = 9. • n: number of days; for 1 month, n = 30; for weekday, n = 21; and for weekend, n = 9. Later, we examined the variability of the group belongings to 1 month of observation. This provided us an initial idea about the regularity of the habits over time. It also identiﬁed unusual weeks in terms of travel behavior. In this stage, the objective was to determine more “natural” groupings of users in order to split them into homogeneous groups according to behaviors and to show the comportment of each group. Step 4. Data analysis and ﬁltering In the raw dataset extracted from the SQL database, there can be seen various types of PT smart cards, namely, 01: Normal, 10 Normal with Credit Card, 04: Student, 05: Reduced fare card, 06: Teachers, 07: Bus drivers, 16: new card for aged 65+, 65: aged 65+, 75: Disabled with Accompanying, 74: Disabled, 13: Limited-use Card (1–5 boarding limit), 66: Local Press Card, 67: National Press Card, 68: Staff Card, 69: Free of charge type 1, 70: Postman Card, 72: Allowance card for employees, 73: Trainee Card, 76: Temporary Staff, 77: TUIK (Turkish Statistics Ofﬁce) employee, 78: Veteran and Martyr Card, 79: Municipality parking staff, and 87: Free of charge type 2. According to the sample dataset, PT users were reclassiﬁed and clustered through a data mining method according to their card types’ characteristics, namely, 1: Normal, 2: Student, 3: Elderly, 4: People with Disabilities (PwD) and their accompany people/relatives, 5: limited-used, and 6: others. The number of groups is ﬁxed according to the usage frequency of PT during workdays within the month. Lastly, we carried out a route-based analysis on users in terms of card type, number of boarding per day, number of unique card holder per day, and monthly frequency of usage (in workdays) over the course of 1 month. 4. Results and Discussions 4.1. Group Categorization Analysis 4.1. Group Categorization Analysis Table 2 presents the original card types and revised card types, and their boarding (boarding) information. Table 2 presents the original card types and revised card types, and their boarding (boarding) information. Table 2. Original and revised card types information based on users travel behavior Table 2. Original and revised card types information based on users travel behavior. Original and revised card types information based on users travel behavior. Org. Card Type Rev. Card Type Unique Card # of Boarding Org. Card Type Rev. Card Type Unique Card # of Boarding 1 1 382,442 2,271,551 73 6 564 6750 10 1 16,030 79,413 78 6 521 4307 4 2 216,333 2,641,065 76 6 306 3475 65 3 57,788 425,234 79 6 225 1687 16 3 605 3804 67 6 168 2798 13 5 73,333 73,896 70 6 128 1750 5 6 22,883 148,718 72 6 87 1124 6 6 9950 104,110 77 6 46 264 69 6 3664 35,078 66 6 2 13 68 6 2203 25,918 74 4 16,254 170,436 87 6 1855 14,785 75 4 3447 20,185 It was expected that the card holders of type 6 (Teachers) and type 4 (Students) would be categorized in the same group, but according to their average frequency of PT usage over the course of 1 month (workdays and weekends), it was found that the travel behavior was different. For example, students had an average monthly 22.3 boarding per person, while teachers had 18.1. In addition, the frequency of days in which students used PT came to 12.2 per month, while this value for teachers came to 10.4 per month. In this regard, Figure 3 illustrates the share of user groups based on their smart card boarding per day, while Table 3 presents the share of user groups based on their smart card boarding on weekly basis. 9 of 23 Sensors 2021, 21, 3039 Figure 3 Distribution of boarding by different user groups Figure 3. Distribution of boarding by different user groups. Figure 3. Distribution of boarding by different user groups. Table 3. The distribution of card holders in weekdays and weekend. 4. Results and Discussions Clustered User Groups Boarding Unique Card ID Boarding Rate Workday STD Weekend STD Workday STD Weekend STD Workday STD Weekend STD 1: Normal 36.6% 0.6% 42.3% 0.8% 38.0% 0.5% 42.2% 0.4% 1.96 0.01 2.01 0.04 2: Student 45.9% 0.8% 40.3% 1.0% 44.9% 0.7% 39.7% 0.8% 2.08 0.02 2.04 0.03 3: Elderly 7.7% 0.3% 7.4% 0.4% 7.0% 0.3% 7.3% 0.3% 2.21 0.03 2.04 0.05 4: PwD 3.5% 0.1% 3.8% 0.2% 3.1% 0.1% 3.5% 0.2% 2.31 0.02 2.19 0.02 5: Limited Use 0.5% 0.1% 1.0% 0.2% 1.0% 0.1% 1.9% 0.3% 1.04 0.01 1.03 0.01 6: Others 5.8% 0.1% 5.1% 0.3% 5.9% 0.1% 5.3% 0.3% 1.97 0.01 1.93 0.02 It is evident that the total number of smart card boardings according to a particular user group differs in each day of the week based on user group. Table 3 also summarize the distribution of card holders in weekdays and weekends. In one hand, it is clear that holders of card type 1 (Normal or Adult card) and type 5 (1–5 boardings) take a higher share of the weekend boarding data (type 1: 42.3%, type 5: 1.0%) compared to their share in weekdays boarding data (type 1: 36.6%, type 5: 0.5%). On the other hand, it can be seen that student card holders have a larger share during the week. In terms of average number of boardings, card type 3 (Elderly card holders) and card type 4 (PwD card holders) have signiﬁcantly higher boarding rates on weekdays and weekends. This shows the possibility that these users demand more travel. It is also possible that the transportation system is not planned well enough for this group of the users and they have to travel considering more transfer between bus routes. In terms of the use of PT boardings data to estimate the OD, the question of whether this boarding rate is due to the journey or the transfer between bus lines can be gleaned. However, this is beyond the scope of our study and further studies to this end are suggested in Section 5.2. 4.2. Monthly’s Trip-Frequency Analysis on User Groups 4.2. Monthly’s Trip-Frequency Analysis on User Groups The data of smart card users according to three parameters was analyzed—namely, trip frequency over the month, the boarding rate of users per day, and the type of card. For this, users are categorized according to the number of days, which they boarded over the course of 1 month. Figure 4a illustrates the distribution of user groups and their boardings over the month, according to their trip frequency, in which the 1-day trip-frequency group denotes ors 2021, 21, 3039 10 those who traveled on only 1 day in the month, while the 30-days trip-frequency gr represents those who traveled every day in a month. Figure 4b depicts average num of boarding per day by each user in different trip-frequency groups (See Appendi Table A1 and Figure A1 for more information). Figure 4. (a) Number and share of card holders and boarding and (b) average boarding per card holder of each group. As can be seen from the distributions in Figure 4a, users with less frequency Sensors 2021, 21, 3039 10 of 23 10 of 23 those who traveled on only 1 day in the month, while the 30-days trip-frequency group represents those who traveled every day in a month. Figure 4b depicts average number of boarding per day by each user in different trip-frequency groups (See Appendix A; Table A1 and Figure A1 for more information). those who traveled on only 1 day in the month, while the 30-days trip-frequency group represents those who traveled every day in a month. Figure 4b depicts average number of boarding per day by each user in different trip-frequency groups (See Appendix A; Table A1 and Figure A1 for more information). Figure 4. (a) Number and share of card holders and boarding and (b) average boarding per card holder of each group. Figure 4. (a) Number and share of card holders and boarding and (b) average boarding per card holder of each group. As can be seen from the distributions in Figure 4a, users with less frequency are seen less in the total data, users with 1–6 days frequency and 63% have 21% ratio in the 1-day average data. On the other hand, Figure 4b shows that users that have minimum 1 boarding over workdays have 1.57 boarding per day on average. 4.3. Workdays’ Trip-Frequency Analysis on User Groups Weekend travel behavior varies more than that of weekdays due to increased vari- ations of the purpose of trips (i.e., since there are no work or school-based trips at the weekend), as well as a reduced number of active buses and the number of active bus lines. In our case, the rate of users who did not travel on weekends came to 36% (64% traveled the weekend). The share of weekend trips came to 22.48% of the total. The share of weekday trips, meanwhile, corresponded to 88.72%. Users who traveled both on weekdays and weekends (64%) composed of 9.37% of total users and 8.14% of total boarding data. Users who traveled only on the weekend came to 11.3% of total users, which corresponds to 8.1% of total weekend boardings and 1.8% of total boarding data. g g Thus, user behavior analysis should be conducted for workdays and weekends sepa- rately. Yet this study focuses on workday trips in particular. Table 4 presents the share of boarding and number of users for each trip-frequency group. Table 4. Workday analysis: number of boarding and users. Trip Freq. Group Boarding Person Workday SDT Workday SDT 1-day 3.42% 0.67% 4.27% 0.74% 2-days 3.68% 0.37% 4.02% 0.38% 3-days 3.87% 0.29% 4.18% 0.30% 4-days 3.84% 0.23% 4.11% 0.23% 5-days 3.74% 0.21% 3.96% 0.19% 6-days 3.49% 0.13% 3.68% 0.13% 7-days 3.32% 0.09% 3.49% 0.08% 8-days 3.24% 0.09% 3.36% 0.09% 9-days 3.13% 0.07% 3.26% 0.07% 10-days 3.10% 0.08% 3.18% 0.09% 11-days 2.98% 0.12% 3.08% 0.12% 12-days 2.98% 0.14% 3.06% 0.15% 13-days 3.14% 0.16% 3.18% 0.17% 14-days 3.26% 0.20% 3.29% 0.19% 15-days 3.69% 0.23% 3.67% 0.22% 16-days 4.19% 0.25% 4.12% 0.23% 17-days 4.90% 0.24% 4.78% 0.23% 18-days 5.95% 0.22% 5.74% 0.22% 19-days 7.36% 0.20% 7.07% 0.20% 20-days 10.18% 0.20% 9.54% 0.19% 21-days 16.56% 0.21% 14.95% 0.18% Table 4. Workday analysis: number of boarding and users. Table 4. Workday analysis: number of boarding and users. Based on the results of Table 4, it is evident that user groups with low frequency have lower boarding rates compared to other groups, and this rate increases as frequency in- creases. In addition, users who travel 16–21 days on average during workdays corresponds to 42.6% of users who traveled every day and 49.14% of boarding data. It is also seen that users with low fecundity (traveling for 1–6 days) represent 22.14% of average daily users and 22.04% of boarding data. 4.2. Monthly’s Trip-Frequency Analysis on User Groups The maximum boarding number comes to 2.57 on average per day, which is related to users who have taken the PT vehicles every day of a month. This is due to the fact that users with low trip frequency have a lower boarding rate in only 1 day of data. In terms of trip frequency, they will be Sensors 2021, 21, 3039 11 of 23 11 of 23 seen much less frequently as they have lower rates. In addition, it can be concluded that it would be more accurate to divide the total data in terms of weekdays and weekends. 4.3. Workdays’ Trip-Frequency Analysis on User Groups g Considering user distribution over the month, it is clear that the majority of active users are in 1–6 days trip-frequency groups (those who travel 1–6 days per month). On the other hand, it is clear that high trip-frequency users carry a higher share in the total data. The reason for this is that these group of users travel more days of the week and have a higher number of daily rides. The distribution of user groups and their boardings per day is given in the graph below, obtained from the workdays trip-frequency analysis (see detailed results in Appendix A; Tables A2 and A3). Sensors 2021, 21, 3039 12 of 23 12 of 23 The proportion of regular PT users out of the total in 1 workday is higher than that of total users in our 1-month dataset. For instance, the card holders which have 1–6 days boarding frequency have a share of 66% in the whole dataset, while they would be underrepresented with a share of 22% according to a 1-day analysis. Moreover, card holders with a 16–21 days boarding frequency came to 16% of all cardholders and would be thus overrepresented as 39% in 1-day analysis. p y y As shown in Figure 5, the share of users with low frequency has low boarding rate, while users with high frequency have a high rate of the total workdays boarding. Moreover, it is clear that users’ frequency increases with a rise in the average number of boardings. When cardholders are evaluated on the basis of deﬁned groups, the relevant weekday trips and user shares are given in the graphs below. In this study, the coefﬁcient of variation (CV) as a statistical measure was used to evaluate the variation of data distribution around the mean of data and calculated by dividing the STD by the mean. Depending on the ﬁeld of research, the acceptance values of CV vary between <5% (medicine) and <20% (engineering) [54,55]. When CV values are assessed in our case, the values came to over 20% only for card holders of type 2 (students) with a trip-frequency of 1- and 2-day out of all workdays over the month. These trip-frequency groups represent a small share (0.85%) among all boarding data. Therefore, it is assumed that the mean represents this group. Please see Appendix A Table A3 for more detailed information. Figure 5. 4.3. Workdays’ Trip-Frequency Analysis on User Groups Share of boarding per workdays (blue) vs. real share of persons (red) in entire dataset. Figure 5. Share of boarding per workdays (blue) vs. real share of persons (red) in entire dataset. To highlight how frequency-based user group boardings and cardholder share can be over/under representing (over-weighting and under-weighting) more clearly, the division of the real share of each group in single-month data for workdays is shown in Figure 6. As seen in Figure 6, groups of users with a higher frequency of use are more over- represented, while groups with a lower frequency of use are more underrepresented. In addition, it can also be observed that higher user groups have a higher number of boardings per day (causing a higher more share than in 1-day data) and are signiﬁcantly more overrepresented. p User groups with lower frequency are under-represented because they are less likely to appear in a day’s data, and on the contrary, user groups with high frequency are more likely to appear in a daily data. For example, the probability of a user group traveling only one day to be seen in one day is 0.48 (1 day/21 workdays). However, the user group traveling every day (the group traveling for 21 days) will deﬁnitely be seen on any selected working Sensors 2021, 21, 3039 13 of 23 13 of 23 day and the probability will be 1. Consequently, user groups with a high probability of being seen when looking at the selected daily data are represented much more than their shares in the total monthly data. day and the probability will be 1. Consequently, user groups with a high probability of being seen when looking at the selected daily data are represented much more than their shares in the total monthly data. Figure 6. Raito of workday share to 1 month share of each frequency group. Figure 6. Raito of workday share to 1 month share of each frequency group. The representation shares of different frequency groups in 1-day data are directly related to their shares in 1-month data. In this context, although the shares of the data used in this study and the public transport data belonging to the other center are different depending on the user behavior, the representation tendencies in the 1-day data will be similar to the trends in this study. 4.3. Workdays’ Trip-Frequency Analysis on User Groups By considering Figure 4b, higher frequency groups have higher probability of seeing in a daily trip data when a comparison is made between daily travel and real user shares of these groups, because of having higher trip rates in each day’s addition to higher probability related to their frequency of using PT. On the contrary, due to the fact that low-frequency groups also have lower number of trips, their share in 1-day data are much less represented than their user share in 1-day data. As shown in Figure 7, different card holder groups have varying trip frequency behavior. Card holders from type 1 have the highest share among low frequency types, while card holders of type 2, behave exactly in the opposite way and higher frequency group of users have a higher share in terms of boardings and users. Card holders from type 3, the group of 7-day frequency, showed the highest share among other frequencies of this type of card holder. Card holders type 5 (1–5 limited boarding), are mostly user groups with a frequency of 1 day on workdays. Card holders type 4 and 6 have signiﬁcantly similar distribution rate among trip frequency groups. User groups which are in higher frequency groups are users who travel on regularly bases. This means that as share of high frequency group of card holders increases, the probability of being seen in a day’s data will also increase. In this context, the share of student card holders is increasing steadily with more frequent user groups. They have 68% share of higher frequency groups, which are traveling every day. It means that more regular use of public transportation are students and educational purposed trips that more regularly occur by public transport when compared to the purposed trips. It is also possible to say that the students are represented more in one day PT data than their actual shares in PT users. Yet, the opposite is true for Normal card users, and the share of student card holders is decreasing steadily in more frequent user groups. Accordingly, the share of these user groups decreases up to 20% among the users who travel every day. It is concluded Sensors 2021, 21, 3039 14 of 23 14 of 23 that the regular users’ purposed trips (other than educational trips) are underrepresented in a day’s public transport data. 4.4. Bus Line-Based Analysis on Workday Trips Moreover, we have also carried out a bus line-based analysis using 1-month data to understand and show more concrete evidence regarding the (in)efﬁciency and impact on PT services planned. Hence, in each PT line such as bus, rail, and ferry lines, the types of card holders and their monthly boarding frequency (how many boarding/day) have been examined. All of the 293 KMM’s PT lines, two bus lines—namely, lines no. 118 and no. 23, with share of 1.45% (rank 14) and 1.41% (rank 15) of total PT ridership, respectively—have been selected for further analysis and comparison. Both of which are serving users in KMM districts that also have Tramway lines (a light rail PT system). Users and boarding information of the selected bus lines is as follows: • Bus lines no. 118 and 23 have on average 6348 and 6802 boarding per workday, respectively. • Average daily card holder numbers of these lines are 5014 and 5531 per workday, respectively. • The total number of card holders per workdays are 38,628 and 45,921 for bus lines no. 118 and no. 23, respectively. • Bus lines no. 118 and 23 have on average 6348 and 6802 boarding per workday, respectively. • Bus lines no. 118 and 23 have on average 6348 and 6802 boarding per workday, respectively. • Average daily card holder numbers of these lines are 5014 and 5531 per workday, respectively. • The total number of card holders per workdays are 38,628 and 45,921 for bus lines no. g g p y p y • Average daily card holder numbers of these lines are 5014 and 5531 per workday, respectively. • The total number of card holders per workdays are 38,628 and 45,921 for bus lines no. 118 and no. 23, respectively. • The total number of card holders per workdays are 38,628 and 45,921 for bus lines no. 118 and no. 23, respectively. The main reason to select these bus lines is to show that although both have similar numbers of users and boardings on a daily basis, they have totally different characteristics. For instance, bus line no. 23 is more frequently used by students, while bus line no. 118 is more frequently used by normal cardholders. Furthermore, line no. 118 is more frequently used by the Elderly and PwDs card holders than line 23 and the frequency of use by card holders varies between them. 4.3. Workdays’ Trip-Frequency Analysis on User Groups that the regular users’ purposed trips (other than educational trips) are underrepresented in a day’s public transport data. y p p Figure 7. Share of different user groups on workdays in a month according to: (a) number of p and (b) number of boardings in each user group. It is seen that different card users and their travel shares change within the Freq groups. It means that especially users with higher frequency are users who travel reg The probability of a cardholder type to be seen in a day’s data increases as the tenden Figure 7. Share of different user groups on workdays in a month according to: (a) number of person and (b) number of boardings in each user group. It is seen that different card users and their travel shares change within the Frequency groups. It means that especially users with higher frequency are users who travel regularly The probability of a cardholder type to be seen in a day’s data increases as the tendency to use regular public transport increases. Figure 7. Share of different user groups on workdays in a month according to: (a) number of persons and (b) number of boardings in each user group. It is seen that different card users and their travel shares change within the Frequency groups. It means that especially users with higher frequency are users who travel regularly. The probability of a cardholder type to be seen in a day’s data increases as the tendency to use regular public transport increases. Sensors 2021, 21, 3039 15 of 23 15 of 23 In general, and as one of the main hypotheses suggested by this study, it is shown that an evaluation of users’ daily behavior is not enough and that different user groups (with different trip-frequency) show different travel (boarding) patterns. 4.4. Bus Line-Based Analysis on Workday Trips As a result, the number of daily passengers (cardholders) and daily boardings will not be enough to determine the real number of users of each line. Line 23 has more daily boardings and users (cardholders); however, line 118 is used more frequently over the course of the month. In other words, the number of boardings and users on bus line no. 118 comes to less than the number of boardings and users on bus line no. 23. Meanwhile, considering the monthly number of users, bus line no. 23 serves more PT users (45,921) compared to bus line no. 118 (38,628). The distribution of users and their boarding frequency over workdays over a month are given at Figure 8a (bus line no. 118) and Figure 8b (bus line no. 23). In Figure 8a,b, it is seen that the user groups with lower frequency in Line 118 are using this line more than line 23. In this context, the share of passengers using line 118 less than 4 days in a month are 85%, while the share of these users using line 23 is 75%. Consequently, although the number of real users in this line are more than line 23 in the monthly data, line 23’s number of users and travel in daily data are higher. Table 5 presents the distribution of daily users and daily boarding among different card type holders, and the real number of users are using bus lines no. 23 and no. 118 in a month. Table 5. Distribution of users and boarding frequency considering card types: daily vs. real (in a month). Table 5. Distribution of users and boarding frequency considering card types: daily vs. real (in a month). Card Type Bus Line No. 23 Bus Line No. 118 Person Day TRIP DAY Real # of Users Person Day Trip Day Real # of Users 1 Normal 23.3% 24.4% 34.0% 50.1% 52.5% 55.7% 2 Students 70.7% 70.1% 56.8% 32.5% 31.1% 25.4% 3 Elderly 2.3% 2.1% 4.0% 9.1% 8.6% 10.5% 4 PwD 1.2% 1.1% 1.6% 3.4% 3.3% 3.1% 5 Limited Use 0.1% 0.1% 0.4% 0.1% 0.1% 0.3% 6 Others 2.3% 2.1% 3.4% 4.8% 4.4% 5.1% 16 of 23 16 of 23 Sensors 2021, 21, 3039 Figure 8. Comparing 1-day data and 1-month data in terms of number of users and bo (a) Bus Line No. 118 and (b) Bus Line No. 23. Figure 8. 5. Conclusions Generally, PT systems’ evaluation and planning are more trip-based rather than user- based, e.g., in conventional methods such as cost beneﬁt analysis, multi criteria analysis, and social-based analysis, which causes a miscalculation (overestimation). In other words, the needs of users with more frequency outweigh and are overrepresented in conventional transport projects analyses. To address this mis-estimation, this study proposes an equity- based analysis of PT users’ travel data to estimate the real percentage of each cardholder group in sufﬁcient detail. g p Around 12 million boardings’ worth of data collected from PT-SCFC systems by KMM was used to analyze the travel behavior of users over the course of 1 month. It was found that travel and mobility pattern analysis on a large time period PT-SCFC data i.e., 1 month instead of 1-day result in more accurate determination of the real share of PT users. To have a socially sustainable PT system, a particular and separate evaluation of the needs of users with lower frequency has to be done. PT planning according to the needs of these group may cause an increase in demand for PT. On the other hand, comparative to non-regular users, users with a higher frequency are considered, regardless of travel purposes, it seems that the current transportation service is well planned. The reason for this is that this group is already overrepresented in daily travel analyses. Frequency- based analyses not only give us the chance to evaluate transport, particularly PT, but can also assist with evaluating the impact of transport investment in terms of changing user behaviors and shifting to more sustainable transport modes. This study shows the vast extent of inequity in the number of trips used by exiting models (conventional transport planning methods). In fact, this deﬁciency will continue in terms of equity as long as 1-day user analysis/modeling continues to be used. This deﬁciency can only be eliminated if a long-time data analysis is considered in transport modeling and planning. To clarify, the limitations of existing public transport modeling and planning discussed in this study can not only be resolved by using long-time data. It is difﬁcult to determine the real number of users without an understanding of the frequency of transport use. To address this deﬁcit at the strategy and planning level, user categorizations ought to be constructed based on usage frequency. 4.4. Bus Line-Based Analysis on Workday Trips Comparing 1-day data and 1-month data in terms of number of users and boarding for (a) Bus Line No. 118 and (b) Bus Line No. 23. As given in Table 5, card holder type no. 1 is underrepresented in daily data for both bus lines. For instance, in bus line no. 23, normal card holders have a share of 23.3% of all daily users, while the real share of card holders type no. 1 represents 34% of monthly users. On the other hand, all other card types, in particular students (type no. 2), are overrepresented in daily data In bus line no 23 student card holders have a share of As given in Table 5, card holder type no. 1 is underrepresented in daily data for both bus lines. For instance, in bus line no. 23, normal card holders have a share of 23.3% of all daily users, while the real share of card holders type no. 1 represents 34% of monthly users. As given in Table 5, card holder type no. 1 is underrepresented in daily data for both bus lines. For instance, in bus line no. 23, normal card holders have a share of 23.3% of all daily users, while the real share of card holders type no. 1 represents 34% of monthly users. On the other hand, all other card types, in particular students (type no. 2), are overrepresented in daily data. In bus line no. 23, student card holders have a share of 70.7% among daily users, while the real share of these users among monthly users comes to 56.8%. On the other hand, all other card types, in particular students (type no. 2), are overrepresented in daily data. In bus line no. 23, student card holders have a share of 70.7% among daily users, while the real share of these users among monthly users comes to 56.8%. This situation approves the hypothesis of the study in terms of line-based evaluation as well. Therefore, taking only the daily number of users and boarding as a performance measure/criterion causes misleading evaluations of real performance due to the variation (distribution) of users and boarding frequency over a month. 17 of 23 Sensors 2021, 21, 3039 17 of 23 5. Conclusions It can be concluded that this information should either be contained within the dataset itself or be obtained from other data (e.g., survey data). Questions over frequency of use should also consider households and other surveys conducted in SUMP [56], while Transportation Master Plan studies and user groups should be created accordingly. This solution allows for a better determination of the actual number of users. In addition, it can give important contributions at planning stage of SUMP, mainly during scenario development, measure appraisal and selection, and monitoring. The signiﬁcance of this study is not speciﬁc to Kocaeli (Turkey), but rather reveals a weakness in terms of public transportation planning in general. Further studies ought to devote more attention to examining other, more advanced models/analyses to derive more comparative groups and users’ behaviors. Our research ﬁndings, limitation, and some recommendation for future studies are given below: 5.1. Research Highlights • Card holders who have a 1–day boarding frequency represent 66% of the whole dataset, while they represent 22% in a single workday. • Card holders with a 16–21 days boarding frequency represent 16% of the whole dataset, while they represent 39% in 1 workday. y p y • Regular users also have a higher boarding rate per day and will be much more overrepresented in single-day data. • The elderly, those with disabilities, the elderly and disabled: in terms of the average number of boardings, elderly card holders (group 3), and PwD card holders (group 4) have higher (c. 10–15%) boarding rates on weekdays and weekends compared to other card holder groups. This shows that these users need more travel access. Sensors 2021, 21, 3039 18 of 23 18 of 23 Another ﬁnding shows that PT routes and lines are not planned based on their travel needs (medical centers, elderly house, organizations for PwD, etc.), consequently, this increases their number of transfers between lines. • Some trip routes are used by more people, even though the number of users is rarer on a daily basis, such as PT routes to medical centers (medical trips). • Some trip routes are used by more people, even though the number of users is rarer on a daily basis, such as PT routes to medical centers (medical trips). • Since the smart card data are boarding (transaction) based, more boardings may not really mean more trips. 5.2. Limitations and Directions for Further Studies • We proposed a novel approach using 1-month data, which can be paved the way for further studies using long-period data like yearly dataset. • Some users use different cards or a card belong to somebody else (e.g., family members, relatives, or friends) as there is no card control (veriﬁcation) or enforcement system in KMM’s PT network. Moreover, some PT users do not have a smart card or enough charge while boarding, consequently, they have to use the driver’s card or other passengers’ cards. Thus, one of the limitations of our study is the possibility of the mis-grouping of those users having low boarding frequency. This issue needs further development in the proposed method. • It can be seen that the elderly and PwD have signiﬁcantly higher boarding rates on weekdays and weekends. Using the PT boarding data for OD estimation, one may study whether this boarding rate is due to their high travel tendency or the transfer between bus lines. If it was due to their high transfer rate between the bus lines, it means transport infrastructure is not designed/planned based on vulnerable users’ group. • In this study, 1-month PT-SCFC data has been used to analyze the travel behavior of users. The study can be extended using more than 1-month data or 1-year data to include the effects of holidays, summer seasons, etc. on the travel behavior of PT users. • The study did not consider gender variables in clustering user groups. The gender gap and gender-responsive public transportation have been featured in many studies [57,58] and this could represent an additional point of exploration for future research. • A simple and effective statistical analysis was conducted to come to the research hy- pothesis; however, advanced data analysis techniques could be employed to improve the proposed methodology. More advanced analyses may reveal more details about the nature of the phenomenon. • A simple and effective statistical analysis was conducted to come to the research hy- pothesis; however, advanced data analysis techniques could be employed to improve the proposed methodology. More advanced analyses may reveal more details about the nature of the phenomenon. Author Contributions: Conceptualization: K.G., N.D., and M.E.; investigation: K.G.; methodology and data analysis: K.G. and M.E.; supervising: N.D. and M.E.; writing—original draft, K.G. and N.D.; writing—review and editing, N.D. and M.E. All authors have read and agreed to the published version of the manuscript. reported. 5. Conclusions In other words, more boardings are likely to result from more transfer due to PT network limitations. • Monthly users and boarding frequencies, instead of daily data, can be examined in public transportation planning, investments, improvements, and evaluated as a performance criterion. p • Travel behavior and mobility pattern of PT users varies on weekends compared to workdays due to reduced PT vehicles’ frequency (headway) and reduced number of active PT lines. Appendix A Table A1. Distribution of boarding frequency of public transport (PT) users over 1-month data. Trip Freq. Group # of Days Which Users Have a Min. 1 Boarding Share of Card Holder Share of Boarding Number of Card Holders Share of Card holders in Entir Dataset Avg. Boarding Per Card Holder 1-day 212,901 3.5% 2.7% 212,901 26.3% 1.57 2-days 192,544 3.2% 2.9% 96,272 11.9% 1.87 3-days 210,165 3.5% 3.2% 70,055 8.7% 1.88 4-days 214,556 3.6% 3.3% 53,639 6.6% 1.90 5-days 211,370 3.5% 3.3% 42,274 5.2% 1.91 6-days 205,074 3.4% 3.2% 34,179 4.2% 1.92 7-days 197,015 3.3% 3.1% 28,145 3.5% 1.92 8-days 188,800 3.1% 3.0% 23,600 2.9% 1.94 9-days 176,418 2.9% 2.8% 19,602 2.4% 1.95 10-days 172,460 2.9% 2.7% 17,246 2.1% 1.95 11-days 164,780 2.7% 2.6% 14,980 1.9% 1.96 12-days 162,276 2.7% 2.6% 13,523 1.7% 1.98 13-days 158,288 2.6% 2.6% 12,176 1.5% 1.98 14-days 158,466 2.6% 2.6% 11,319 1.4% 1.98 15-days 159,615 2.6% 2.6% 10,641 1.3% 2.00 16-days 168,112 2.8% 2.7% 10,507 1.3% 2.00 17-days 177,548 2.9% 2.9% 10,444 1.3% 2.01 18-days 196,038 3.2% 3.2% 10,891 1.3% 2.01 19-days 216,201 3.6% 3.6% 11,379 1.4% 2.02 20-days 251,860 4.2% 4.2% 12,593 1.6% 2.04 21-days 317,583 5.3% 5.3% 15,123 1.9% 2.05 22-days 304,788 5.0% 5.2% 13,854 1.7% 2.09 23-days 295,688 4.9% 5.1% 12,856 1.6% 2.12 24-days 273,936 4.5% 4.8% 11,414 1.4% 2.16 25-days 268,800 4.5% 4.8% 10,752 1.3% 2.18 26-days 242,554 4.0% 4.4% 9329 1.2% 2.24 27-days 189,945 3.1% 3.5% 7035 0.9% 2.26 28-days 149,324 2.5% 2.8% 5333 0.7% 2.33 29-days 114,057 1.9% 2.2% 3933 0.5% 2.39 30-days 85,200 1.4% 1.8% 2840 0.4% 2.57 Table A1. Distribution of boarding frequency of public transport (PT) users over 1-month data. As seen in Tables A3 and A4, card holders in type 1 with a 2-day trip frequency have a share of 6.79% of total boarding in workdays over the 1-month dataset. For this frequency group, which has boarding only 2 days out of 21 workdays, we do not know in which days of November they will take PT. To do so, we ﬁrst calculated the percentage of the possibility of their boarding on 21 workdays of November 2018 (see table below). Then, we calculated the mean, STD, and CV for this group. 5.2. Limitations and Directions for Further Studies Funding: This research received no external funding. Data Availability Statement: Restrictions apply to the availability of these data. Data were ob- tained from the Kocaeli Metropolitan Municipality and are available from the corresponding author (ghasemlou@itu.edu.tr) with the permission of the Kocaeli Metropolitan Municipality. Sensors 2021, 21, 3039 19 of 23 19 of 23 Acknowledgments: This article is a part of the Ph.D. thesis of the corresponding author at Istanbul Technical University, Turkey. We would like to thank Kocaeli Metropolitan Municipality, public transport division, and Bo˘gaziçi Proje Engineering Inc. for providing us with smart card data and transport master plan reports. Conﬂicts of Interest: The authors declare no conﬂict of interest. Acknowledgments: This article is a part of the Ph.D. thesis of the corresponding author at Istanbul Technical University, Turkey. We would like to thank Kocaeli Metropolitan Municipality, public transport division, and Bo˘gaziçi Proje Engineering Inc. for providing us with smart card data and transport master plan reports. Conﬂicts of Interest: The authors declare no conﬂict of interest. Conﬂicts of Interest: The authors declare no conﬂict of interest. Appendix A The mean came to 6.79% (STD = 0.49%) with a CV value of 7.27% (which is <20%), which shows this mean (6.79%) can be properly represented for the sample group of users with 2-day boarding frequency. As seen in Tables A3 and A4, card holders in type 1 with a 2-day trip frequency have a share of 6.79% of total boarding in workdays over the 1-month dataset. For this frequency group, which has boarding only 2 days out of 21 workdays, we do not know in which days of November they will take PT. To do so, we ﬁrst calculated the percentage of the possibility of their boarding on 21 workdays of November 2018 (see table below). Then, we calculated the mean, STD, and CV for this group. The mean came to 6.79% (STD = 0.49%) with a CV value of 7.27% (which is <20%), which shows this mean (6.79%) can be properly represented for the sample group of users with 2-day boarding frequency. As seen in Tables A3 and A4, card holders in type 1 with a 2-day trip frequency have a share of 6.79% of total boarding in workdays over the 1-month dataset. For this frequency group, which has boarding only 2 days out of 21 workdays, we do not know in which days of November they will take PT. To do so, we ﬁrst calculated the percentage of the possibility of their boarding on 21 workdays of November 2018 (see table below). Then, we calculated the mean, STD, and CV for this group. The mean came to 6.79% (STD = 0.49%) with a CV value of 7.27% (which is <20%), which shows this mean (6.79%) can be properly represented for the sample group of users with 2-day boarding frequency. 20 of 23 20 of 23 Sensors 2021, 21, 3039 Figure A1. Share of card holders vs. share of boarding over a month. Figure A1. Share of card holders vs. share of boarding over a month. Figure A1. Share of card holders vs. share of boarding over a month. Table A2. Distribution of users and boarding over workdays considering 1-month data vs. one-data according to their trip frequency. Appendix A Monthly Dataset Considered 1-Day Data Considered Trip-Frequency Group # of Boarding # of Persons Share of Boarding Per Workday Real Share of Persons in Entire Dataset Average Daily Boarding Average Daily Persons 1-day 324,499 199,101 3.4% 27.7% 3.4% 4.3% 2-days 349,716 93,754 3.7% 13.1% 3.7% 4.0% 3-days 367,098 65,030 3.9% 9.1% 3.9% 4.2% 4-days 364,439 47,898 3.8% 6.7% 3.8% 4.1% 5-days 355,237 36,995 3.7% 5.2% 3.7% 4.0% 6-days 331,553 28,614 3.5% 4.0% 3.5% 3.7% 7-days 315,060 23,289 3.3% 3.2% 3.3% 3.5% 8-days 307,429 19,625 3.2% 2.7% 3.2% 3.4% 9-days 297,678 16,887 3.1% 2.4% 3.1% 3.3% 10-days 294,019 14,819 3.1% 2.1% 3.1% 3.2% 11-days 283,139 13,075 3.0% 1.8% 3.0% 3.1% 12-days 283,032 11,881 3.0% 1.7% 3.0% 3.1% 13-days 298,710 11,421 3.1% 1.6% 3.1% 3.2% 14-days 309,643 10,970 3.3% 1.5% 3.3% 3.3% 15-days 350,036 11,405 3.7% 1.6% 3.7% 3.7% 16-days 398,111 12,002 4.2% 1.7% 4.2% 4.1% 17-days 464,997 13,113 4.9% 1.8% 4.9% 4.8% 18-days 564,845 14,880 5.9% 2.1% 5.9% 5.7% 19-days 699,227 17,351 7.4% 2.4% 7.4% 7.1% 20-days 966,314 22,257 10.2% 3.1% 10.2% 9.5% 21-days 1,572,397 33,214 16.6% 4.6% 16.6% 15.0% rs and boarding over workdays considering 1-month data vs. one-data according to their trip frequency. Table A2. Distribution of users and boarding over workdays considering 1-month data vs. one-data acco Sensors 2021, 21, 3039 21 of 23 Table A3. Workday trip-frequency distribution among the categorized user groups. Trip Freq. Group 1 Normal 2 Student 3 Elderly 4 PwD 5 Limited-Use 6 Others Board. Person Board. Person Board. Person Board. Person Board. Person Board. Appendix A Person 1-day 5.44% 5.77% 1.08% 1.28% 2.73% 3.37% 1.65% 2.12% 98.38% 98.70% 2.42% 2.89% 2-days 6.79% 6.98% 1.32% 1.57% 4.01% 4.78% 2.48% 3.08% 1.30% 1.08% 3.26% 3.72% 3-days 6.87% 7.06% 1.44% 1.67% 4.79% 5.54% 3.00% 3.57% 0.22% 0.17% 3.68% 4.13% 4-days 6.56% 6.69% 1.50% 1.71% 5.34% 5.98% 3.26% 3.82% 0.02% 0.02% 3.94% 4.33% 5-days 6.12% 6.21% 1.57% 1.77% 5.50% 6.08% 3.44% 3.90% 0.00% 0.00% 4.11% 4.45% 6-days 5.44% 5.51% 1.62% 1.81% 5.32% 5.77% 3.44% 3.82% 0.00% 0.00% 3.94% 4.23% 7-days 4.84% 4.89% 1.67% 1.88% 5.59% 6.00% 3.76% 4.14% 0.00% 0.00% 3.77% 4.04% 8-days 4.36% 4.36% 1.93% 2.14% 5.20% 5.42% 3.64% 3.95% 0.00% 0.00% 3.93% 4.14% 9-days 3.98% 4.02% 2.08% 2.27% 5.01% 5.13% 3.62% 3.86% 0.00% 0.00% 3.65% 3.85% 10-days 3.71% 3.74% 2.22% 2.37% 4.91% 4.91% 3.72% 3.88% 0.00% 0.00% 3.64% 3.81% 11-days 3.43% 3.50% 2.28% 2.45% 4.61% 4.63% 3.67% 3.83% 0.00% 0.00% 3.41% 3.55% 12-days 3.16% 3.23% 2.53% 2.67% 4.33% 4.30% 3.50% 3.56% 0.00% 0.00% 3.54% 3.68% 13-days 3.13% 3.17% 2.90% 3.01% 4.23% 4.12% 3.93% 3.94% 0.00% 0.00% 3.52% 3.62% 14-days 3.07% 3.13% 3.32% 3.40% 3.67% 3.47% 3.81% 3.91% 0.00% 0.00% 3.40% 3.53% 15-days 3.23% 3.27% 4.02% 4.04% 3.86% 3.67% 4.17% 4.05% 0.00% 0.00% 3.70% 3.79% 16-days 3.42% 3.43% 4.87% 4.84% 3.92% 3.60% 4.22% 4.10% 0.08% 0.04% 4.41% 4.39% 17-days 3.60% 3.62% 6.11% 5.99% 3.80% 3.50% 4.70% 4.77% 0.00% 0.00% 5.50% 5.40% 18-days 4.05% 3.93% 7.92% 7.79% 4.03% 3.66% 5.34% 5.10% 0.00% 0.00% 5.73% 5.63% 19-days 4.63% 4.48% 10.40% 10.19% 4.12% 3.63% 6.24% 5.97% 0.00% 0.00% 6.17% 5.87% 20-days 5.62% 5.30% 14.95% 14.39% 5.68% 4.89% 9.05% 8.38% 0.00% 0.00% 8.69% 7.82% 21-days 8.55% 7.69% 24.25% 22.76% 9.35% 7.52% 19.36% 16.27% 0.00% 0.00% 15.60% 13.13% Table A3. Workday trip-frequency distribution among the categorized user groups. Table A4. Distribution of share of boarding over a month. Table A4. Distribution of share of boarding over a month. Date of November 2018 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Share of boarding (trip) 7% 5% 5% 5% 6% 9% 9% 5% 4% 4% 5% 5% 8% 9% 5% Date of November 2018 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 Share of boarding (trip) 4% 5% 5% 6% 8% 9% 5% 5% 5% 5% 7% 9% 10% 10% 9% References 2. OECD Improving Transport Accessibility for All: Guide to Good Practice; European Conference of Ministers of Transport (ECMT) Publications: Paris, France, 2006; ISBN 9282101398. 3. Dadashzadeh, N.; Ergun, M. Spatial bus priority schemes, implementation challenges and needs: An overview and directions for future studies. Public Transp. 2018, 10, 545–570. [CrossRef] 4. Dadashzadeh, N. Effects of Bus PRIORITY Methods on Adjacent Mixed Trafﬁc; Istanbul Technical University: Istanbul, Turkey, 2019. 5. Beyazit, E. Evaluating Social Justice in Transport: Lessons to be Learned from the Capability Approach. Transp. Rev. 2011, 31, 117–134. [CrossRef] 4. Dadashzadeh, N. Effects of Bus PRIORITY Methods on Adjacent Mixed Trafﬁc; Istanbul Technical University: Istanbul, Turkey, 2019. 5. Beyazit, E. Evaluating Social Justice in Transport: Lessons to be Learned from the Capability Approach. Transp. Rev. 2011, 31, 117–134. [CrossRef] ff f j fﬁ y y 5. Beyazit, E. Evaluating Social Justice in Transport: Lessons to be Learned from the Capability Approach. Transp. Rev. 2011, 31, 117–134. [CrossRef] 6. Vanoutrive, T.; Cooper, E. How just is transportation justice theory? The issues of paternalism and production. Transp. Res. Part A Policy Pract. 2019, 122, 112–119. [CrossRef] 6. Vanoutrive, T.; Cooper, E. How just is transportation justice theory? The issues of paternalism and production. Transp. Res. Part A Policy Pract. 2019, 122, 112–119. [CrossRef] y 7. Martens, K. Transport Justice: Designing Fair Transportation Systems; Routledge Taylor & Francis Group: New York, NY, USA, 2017; ISBN 9780415638319. 7. Martens, K. Transport Justice: Designing Fair Transportation Systems; Routledge Taylor & Francis Group: New York, NY, USA, 2017; ISBN 9780415638319. 8. Di Ciommo, F.; Shiftan, Y. Transport equity analysis. Transp. Rev. 2017, 37, 139–151. [CrossRef] , ; , p q y y p , , [ ] 9. Kelobonye, K.; Zhou, H.; McCarney, G.; Xia, J. Measuring the accessibility and spatial equity of urban services under competition using the cumulative opportunities measure. J. Transp. Geogr. 2020, 85, 102706. [CrossRef] p q y y p 9. Kelobonye, K.; Zhou, H.; McCarney, G.; Xia, J. Measuring the accessibility and spatial equity of urban services under competition using the cumulative opportunities measure. J. Transp. Geogr. 2020, 85, 102706. [CrossRef] 10. Martens, K.; Di Ciommo, F. Travel time savings, accessibility gains and equity effects in cost–beneﬁt analysis. Transp. Rev. 2017, 37, 152–169. [CrossRef] 10. Martens, K.; Di Ciommo, F. Travel time savings, accessibility gains and equity effects in cost–beneﬁt analysis. Transp. Rev. 2017, 37, 152–169. [CrossRef] 11. Martens, K. References Socio-spatial and temporal dimensions of transport equity for London’s night time economy. Transp. Res. Part A Policy Pract. 2019, 121, 433–443. [CrossRef] 25. Boisjoly, G.; Serra, B.; Oliveira, G.; El-Geneidy, A. Inequity in transit: Evaluating public transport distribution through accessibility measurements in São Paulo, Rio de Janeiro, Curitiba and Recife, Brazil. J. Transp. Geogr. 2020, 82, 1–11. [CrossRef] 26. Palm, M.; Farber, S.; Shalaby, A.; Young, M. Equity Analysis and New Mobility Technologies: Toward Meaningful Interventions. J. Plan. Lit. 2020, 36, 31–45. [CrossRef] 27. Al-Rashid, M.A.; Goh, H.C.; Harumain, Y.A.S.; Ali, Z.; Campisi, T.; Mahmood, T. Psychosocial barriers of public transport use and social exclusion among older adults: Empirical evidence from Lahore, Pakistan. Int. J. Environ. Res. Public Health 2021, 18, 185. [CrossRef] 28. Martens, K. Accessibility and potential mobility as a guide for policy action. Transp. Res. Rec. 2015, 2499, 18–24. [CrossRef] 29. Currie, G. Quantifying spatial gaps in public transport supply based on social needs. J. Transp. Geogr. 2010, 18, 31–41. [CrossRef] 30 United Nations Cost Beneﬁt Analysis of Transport Infrastructure Projects; United Nations: Geneva Switzerland 2003 28. Martens, K. Accessibility and potential mobility as a guide for policy action. Transp. Res. Rec. 2015, 2499, 18–24. [CrossRef] 29. Currie, G. Quantifying spatial gaps in public transport supply based on social needs. J. Transp. Geogr. 2010, 18, 31–41. [CrossRef] 29. Currie, G. Quantifying spatial gaps in public transport supply based on social needs. J. Transp. Geogr. 2010, 18, 31–41. [CrossRef] Q y g p g p p p pp y J p g , , ations. Cost Beneﬁt Analysis of Transport Infrastructure Projects; United Nations: Geneva, Switzerland, 2003. 30. United Nations. Cost Beneﬁt Analysis of Transport Infrastructure Projects; United Nations: Geneva, Sw ﬁ y f p f j 31. Thomopoulos, N.; Grant-Muller, S.; Tight, M.R. Incorporating equity considerations in transport infrastructure evaluation: Current practice and a proposed methodology. Eval. Program Plann. 2009, 32, 351–359. [CrossRef] [PubMed] 32. Topuz Kiremitçi, S. A Model for Accessibility and Affordability in Urban Transportation: Istanbul Case; Istanbul Technical University: Istanbul, Turkey, 2017. 33. Pelletier, M.P.; Trépanier, M.; Morency, C. Smart card data use in public transit: A literature review. T Technol. 2011, 19, 557–568. [CrossRef] 34. Syarif, M.; Widyawan; Adji, T.B. Big data analytics: Estimation of destination for users of bus rapid transit (brt) public transportation in Jakarta. References Substance precedes methodology: On cost-beneﬁt analysis and equity. Transportation 2011, 38, 959–974. [CrossRef] 22 of 23 22 of 23 Sensors 2021, 21, 3039 12. Koutsopoulos, H.N.; Ma, Z.; Noursalehi, P.; Zhu, Y. Transit Data Analytics for Planning, Monitoring, Control, and Information. In Mobility Patterns, Big Data and Transport Analytics; Antoniou, C., Dimitriou, L., Pereira, F., Eds.; Elsevier B.V.: Amsterdam, The Netherlands, 2018; ISBN 9780128129708. 13. Yu, W.; Bai, H.; Chen, J.; Yan, X. Anomaly Detection of Passenger OD on Nanjing Metro Based on Access 2019, 7, 138624–138636. [CrossRef] n, J.; Yan, X. Anomaly Detection of Passenger OD on Nanjing Metro Based on Smart Card Big Data. IEEE 4–138636. [CrossRef] 14. Zhang, X.; Wang, Q. PeopleVis: A visual analysis system for mining travel behavior. In Proceedings of the 2017 IEEE 21st International Conference on Computer Supported Cooperative Work in Design, Wellington, New Zealand, 26–28 April 2017; pp. 463–468. 15. Lee, I. Estimating of Bus-Trip Destinations Using Temporal Travel patterns of Smart Card Data. Ph.D. Thesis, Seoul National University, Seoul, Korea, August 2019. g asson, J. Dynamic origin-destination estimation using smart card data: An entropy maximisation approach 909.02826. 16. Ali, A.A.; Eliasson, J. Dynamic origin-destination estimation using smart card data: An entropy m 2019, arXiv:1909.02826. 17. Alsger, A.; Tavassoli, A.; Mesbah, M.; Ferreira, L.; Hickman, M. Public transport trip purpose inference using smart card fare data. Transp. Res. Part C Emerg. Technol. 2018, 87, 123–137. [CrossRef] 18. Yu, C.; He, Z.C. Analysing the spatial-temporal characteristics of bus travel demand using the heat map. J. Transp. Geogr. 2017, 58, 247–255. [CrossRef] 19. Tavassoli, A.; Mesbah, M.; Hickman, M. Application of smart card data in validating a large-scale multi-modal transit assignment model. Public Transp. 2018, 10, 1–21. [CrossRef] 20. Litman, T. Social Inclusion as A Transport Planning Issue in Canada. In Proceedings of the Transport and Social Exclusion G7 Comparison Seminar; Transport Studies Group of the University of Westminster: London, UK, 2003; pp. 6–20. 21. Litman, T. Evaluating Transportation Equity: Guidance for Incorporating Distributional Impacts in Available online: https://www.vtpi.org/equity.pdf (accessed on 26 April 2021). 22. Pereira, R.H.M.; Schwanen, T.; Banister, D. Distributive justice and equity in transportation. Transp [CrossRef] 23. Camporeale, R.; Caggiani, L.; Fonzone, A.; Ottomanelli, M. Quantifying the impacts of horizontal and vertical equity in transit route planning. Transp. Plan. Technol. 2017, 40, 28–44. [CrossRef] 24. McArthur, J.; Robin, E.; Smeds, E. References Investigating day-to-day variability of transit usage on a multimonth scale with sma in Lyon. Travel Behav. Soc. 2020, 19, 112–123. [CrossRef] 45. Raux, C.; Ma, T.Y.; Cornelis, E. Variability in daily activity-travel patterns: The case of a one-week travel diary. Eur. Transp. Res. Rev. 2016, 26, 8. [CrossRef] 46. Liu, S.; Yamamoto, T.; Yao, E.; Nakamura, T. Exploring Travel Pattern Variability of Public Transport Users Through Smart Card Data: Role of Gender and Age. IEEE Trans. Intell. Transp. Syst. 2020, 1–10. [CrossRef] 47. Morency, C.; Trépanier, M.; Agard, B. Measuring transit use variability with smart-card data. Transp. Policy 2007, 14, 193–203. [CrossRef] 48. Agard, B. Mining Smart Card Data from an Urban Transit Network. In Encyclopedia of Data Warehousing and Mining, 2nd ed.; IGI Global: Hershey, PA, USA, 2011; pp. 1292–1302. pp , X.; Han, B. A Review of Big Data Applications in Urban Transit Systems. IEEE Trans. Intell. Transp. Syst ef] 49. Lu, K.; Liu, J.; Zhou, X.; Han, B. A Review of Big Data Applications in Urban Transit Systems. IEEE 2020, 1–18. [CrossRef] 2020, 1 18. [CrossRef] 50. KMM Kocaeli Province, Turkey. Available online: https://en.wikipedia.org/wiki/Kocaeli Province (accessed on 13 December 2020). , [ ] 50. KMM Kocaeli Province, Turkey. Available online: https://en.wikipedia.org/wiki/Kocaeli_Province (accessed on 13 December 2020). 50. KMM Kocaeli Province, Turkey. Available online: https://en.wikipedia.org/wiki/Kocaeli_Province (accessed on 13 December 2020). 51. Kocaeli Transportation Master Plan and Public Transportation Priority Systems: Updates for 2050; Bo˘gaziçi Proje Engineering Inc.: Kocaeli, Turkey, 2014. 50. KMM Kocaeli Province, Turkey. Available online: https://en.wikipedia.org/wiki/Kocaeli_Province (acces y p p g 51. Kocaeli Transportation Master Plan and Public Transportation Priority Systems: Updates for 2050; Bo˘gaziçi Proje Engineering Inc.: Kocaeli, Turkey, 2014. 52. PTV VISION VISUM: Travel Demand Modeling—User Manuals 2019. Available online: https://www.ptvgroup.com/en/ solutions/products/ptv-visum/ (accessed on 14 April 2021). ; Jing, P.; Yang, K. Smart card data mining of public transport destination: A literature review. Information 201 53. Li, T.; Sun, D.; Jing, P.; Yang, K. Smart card data mining of public transport destination: A literature revie [CrossRef] 54. Smith, G. European medicines agency guideline on bioanalytical method validation: What more is there to say? Bioanalysis 2012, 4, 865–868. [CrossRef] 55. Gervasi, O.; Murgante, B.; Misra, S.; Garau, C.; Blec, I.; Taniar, D.; Apduhan, B.O.; Maria, A.; Eufemia, A.C.R.; Goos, G. References In Proceedings of the 2019 International Conference of Artiﬁcial Intelligence and Information Technology (ICAIIT), Yogyakarta, Indonesia, 13–15 March 2019; pp. 209–214. 35. Ma, X.; Ji, Y.; Yuan, Y.; Van Oort, N.; Jin, Y.; Hoogendoorn, S. A comparison in travel patterns and determinants of user demand between docked and dockless bike-sharing systems using multi-sourced data. Transp. Res. Part A Policy Pract. 2020, 139, 148–173. [CrossRef] 36. Zhang, J.; Shen, D.; Tu, L.; Zhang, F.; Xu, C.; Wang, Y.; Tian, C.; Li, X.; Huang, B.; Li, Z. A real-time passenger ﬂow estimation and prediction method for urban bus transit systems. IEEE Trans. Intell. Transp. Syst. 2017, 18, 3168–3178. [CrossRef] 37. Tian, X.; Zheng, B. Study Group Travel Behaviour Patterns from Large-Scale Smart Card Data. In Proceedings of the 2019 IEEE International Conference on Big Data, Los Angeles, CA, USA, 9–12 December 2019; pp. 1232–1237. g g pp 38. Tian, X.; Zheng, B. Using Smart Card Data to Model Commuters’ Responses Upon Unexpected Tra the 2019 IEEE International Conference on Big Data, Seattle, WA, USA, 10–13 December 2018; pp. 8 39. Zhang, J.; Tu, L.; Zhang, F.; Yin, X.; Sun, J.; Chen, H.S.T. Flexible Express Bus Line Planning and Operating Based on Passenger Flow Analysis. In Proceedings of the 2018 21st International Conference on Intelligent Transportation Systems (ITSC), Maui, HI, USA, 4–7 November 2018; pp. 2511–2518. 23 of 23 23 of 23 Sensors 2021, 21, 3039 40. Dickens, M.; Hughes-Cromwick, M. Leveraging Big Data in the Public Transportation Industry. Available online: https: //www.apta.com/wp-content/uploads/Big-Data-Policy-Brief.pdf (accessed on 26 April 2021). p p p g y p p 41. Basu, A.; Zhao, J.; Koutsopoulos, H.; Mishalani, R. Data-driven customer segmentation and personalized information provision in public transit. In Proceedings of the TransitData 2019: 5th International Workshop and Symposium: Research and Applications on the Use of passive Data from Public Transport, Paris, France, 8–10 July 2019; pp. 2–18. p p , , , J y ; pp 42. Espinoza, C.; Munizaga, M.; Bustos, B.; Trépanier, M. Assessing the public transport travel behavior consistency from smart card data. Transp. Res. Procedia 2018, 32, 44–53. [CrossRef] 43. Benenson, I.; Marinov, M.; Ben-Elia, E. Unexpected ﬂexibility of public transit usage revealed from mining Israel’s smartcard data. In Proceedings of the TransitData 2019: 5th International Workshop and Symposium: Research and Applications on the use of Passive Data from Public Transport, Paris, France, 8–10 July 2019. 44. Egu, O.; Bonnel, P. p p 58. Al-Rashid, M.A.; Nahiduzzaman, K.M.; Ahmed, S.; Campisi, T.; Akgün, N. Gender-responsive public transportation in the Dammam metropolitan region, Saudi Arabia. Sustainability 2020, 12, 9068. [CrossRef] References Computational Science and Its Applications—ICCSA 2020; Gervasi, O., Murgante, B., Misra, S., Garau, C., Bleˇci´c, I., Taniar, D., Apduhan, B.O., Rocha, A.M.A.C., Tarantino, E., Torre, C.M., Karaca, Y., Eds.; Lecture Notes in Computer Science; Springer International Publishing: Cham, Switzerland, 2020; Volume 12255, ISBN 978-3-030-58819-9. g 56. Torrisi, V.; Garau, C.; Ignaccolo, M.; Inturri, G. “Sustainable Urban Mobility Plans”: Key Concepts and a Critical Revision on SUMPs Guidelines. In Proceedings of the Computational Science and Its Applications—ICCSA 2020; Gervasi, O., Murgante, B., Misra, S., Garau, C., Bleˇci´c, I., Taniar, D., Apduhan, B.O., Rocha, A.M.A.C., Tarantino, E., Torre, C.M., et al., Eds.; Springer: Cham, Switzerland, 2020; pp. 613–628. pp 57. Pirra, M.; Carboni, A.; Diana, M. Assessing gender gaps in educational provision, research and employment opportunities in the transport sector at the european level. Educ. Sci. 2020, 10, 123. [CrossRef] p p 58. Al-Rashid, M.A.; Nahiduzzaman, K.M.; Ahmed, S.; Campisi, T.; Akgün, N. Gender-responsive public transportation in the Dammam metropolitan region, Saudi Arabia. Sustainability 2020, 12, 9068. [CrossRef]
https://openalex.org/W2805184673	https://europepmc.org/articles/pmc6027556?pdf=render	English	null	Provision and continuation of antiretroviral therapy during acute conflict: the experience of MSF in Central African Republic and Yemen	Conflict and health	2,018	cc-by	6,054	CASE STUDY Open Access Provision and continuation of antiretroviral therapy during acute conflict: the experience of MSF in Central African Republic and Yemen Cecilia Ferreyra1* , Daniel O’Brien2, Beatriz Alonso1, Abdulbasset Al-Zomour3 and Nathan Ford Abstract Background: Unstable settings present challenges for the effective provision of antiretroviral treatment (ART). In this paper, we summarize the experience and results of providing ART and implementing contingency plans during acute instability in the Central African Republic (CAR) and Yemen. Case presentation: In CAR, MSF has provided HIV care in three conflict-affected rural regions; these were put on hold throughout the acute phase of violence. “Run-away bags” containing 3 or 4 months of ART were distributed to patients at MSF facilities. Among 1820 HIV patients enrolled into care, 1440 (79%) initiated ART. By December 2016, 782 (54%) patients were still under ART, 354 (25%) have been lost to follow up and 182 (13%) had died. In 2013, when violence disrupted services, 683 patients were receiving ART. Between September–December 2013, 594 (87%) patients received runaway bags and by February 2014, 313 (53%) of these patients returned to the clinic. In Yemen, when violence erupted, patients received a health card that included a helpline to call in case of drug shortages in admission to emergency stocks; this was not possible in CAR due to lack of a functioning telephone network. One thousand six hundred fifty-five PLWHA have been enrolled in care and 1470 (89%) initiated ART; 1056 (72%) are still followed on ART, 126 (9%) were lost to follow up, and 288 (20%) died. In January 2011 clashes began and by April 2011 MSF medical activities were interrupted. Of the 363 patients receiving ART, 363 (100%) received emergency bags to cover 9 months and by February 2012, 354 (98%) patients returned to care. In March 2015 a new wave of conflict affected Yemen, forcing HIV activities to revert to contingency planning. Conclusions: This experience provides further evidence that provision of HIV treatment and emergency drug stocks can be successfully provided to most patients in both conflict-affected settings. Keywords: HIV, Conflict, Contingency plan, Emergency response © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Background emergencies are particularly underserved. In 2014 it was estimated that 1.6 million people living with HIV were affected by humanitarian emergencies, of them 81% were in sub-Saharan Africa and almost two thirds (1 million) did not have access to ART [2]. Globally, an estimated 36.7 million people were living with HIV at the end of 2017 and 21 million of them were on antiretroviral therapy (ART). While this reflects substantial progress in scaling up access to care and treatment, still around half of the patients in need of ART have yet to be diagnosed or enrolled into care [1]. Among the many challenges faced in reaching more people with HIV, populations affected by humanitarian Conflict-affected settings present a particular challenge for the effective provision of HIV care for a multitude of reasons, including the impact of the conflict on the local and national health systems, population movement, and the enduring view among providers of care that the provision of ART in these settings is not feasible [3–5]. Furthermore, concerns have been raised that providing provision of ART in these settings is not feasible [3 Furthermore, concerns have been raised that provi * Correspondence: cecilia.ferreyra@barcelona.msf.org 1Médecins sans Frontières Spain, Barcelona, Spain Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated * Correspondence: cecilia.ferreyra@barcelona.msf.org 1Médecins sans Frontières Spain, Barcelona, Spain Full list of author information is available at the end of the article provision of ART in these settings is not feasible [3– Furthermore, concerns have been raised that provid eyra@barcelona.msf.org ain, Barcelona, Spain is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Ferreyra et al. Conflict and Health (2018) 12:30 https://doi.org/10.1186/s13031-018-0161-1 Ferreyra et al. Conflict and Health (2018) 12:30 https://doi.org/10.1186/s13031-018-0161-1 Program design and implementation HIV services began by addressing patients presenting at the inpatient department (IPD), malnourished children, patients with tuberculosis (TB) or any sign suggesting HIV infection, and prevention of mother-to-child trans- mission (PMTCT) at antenatal clinics (ANC). Later in 2010, a voluntary counselling and testing service (VCT) and health promotion activities were added. In this paper we share our experience of providing ART in two conflict affected settings in Africa and the Middle-east. The aim of this paper is summarize differ- ent operational approaches that have been successfully implemented in different contexts experiencing periods of acute instability, the Central African Republic (CAR) and Yemen. HIV protocols and ART eligibility criteria were in line with the National AIDS Program (NAP) until 2012 when, with the agreement from Ministry of Health (MOH), MSF introduced tenofovir (TDF) based fixed-dose combina- tions as first line therapy to replace existing stavudine (d4T) based regimens following 2010 WHO recommen- dations [20]. Option B+ for PMTCT was implemented in 2014. In November 2016 the NAP adopted the WHO recommendations to start ART to all individuals as soon as possible following an HIV diagnosis; however imple- mentation of such recommendations is still very slow. Page 2 of 7 Ferreyra et al. Conflict and Health (2018) 12:30 Page 2 of 7 mortality and the development of drug resistance. Possibly reflecting these risks, rates of treatment failure in CAR are high and estimated at 30% in adults and 50% in children [16, 17]. More than a fifth (3000) of patients experiencing treatment failure had developed drug resistance. The abil- ity of the health system to handle ART failure is insuffi- cient, with few qualified health workers, no second-line ART available, extremely limited access to virological monitoring, and frequent and long-term nationwide shortages of medical supplies [18]. Adding to these con- cerns, the breakdown of law and order led to widespread violations of human rights including sexual and gender based violence and rape [19]. ART could cause harm by putting patients at risk of drug resistance if ART was interrupted. Limited epidemio- logical data and programme experiences and lack of polit- ical will to prioritize the needs of displaced or conflict affected populations have all contributed to neglect HIV services in these settings [6]. Restricted movement can further deter clinic visits during periods of conflict, limit- ing access to care and treatment [7]. Data on the feasibility and impact of providing ART in conflict and emergency settings are limited. During the post-election conflict in Kenya during 2007–2008, treat- ment interruption was 71% higher compared to the period prior to the conflict, with Médecins sans Fron- tières (MSF) reporting around 30% of patients missing pills [8, 9]. A report from 22 MSF programs where ART was initiated in conflict or post-conflict settings, 64% of patients remained on ART with a rate of loss of follow up of 11% [10, 11]. MSF Spain has been present in CAR since 1996 in the north and central part of the country – Kabo, Batangafo and Ndele towns – 3 rural areas chronically affected by conflict (Fig. 1). In 2008 HIV care was integrated into the existing health services. MSF works in conflict-affected settings responding to the acute needs of these populations during the collapse of health services. In some of these settings HIV preva- lence is high, leading to considerable HIV-related health needs. In many of these settings without access to HIV services, HIV-associated mortality is very high among hospitalized patients [12, 13]. Central African Republic CAR has endured decades of instability, trapped in a cycle of constant and interminable conflict since the late 1990s. In some parts of the country as much as half the population are affected by conflict, with many displaced from their homes. The negative health effects of these con- flicts are considerable, in addition to impacting schooling for children, agricultural production, access to functioning markets and the degradation of essential infrastructure including roads. Fig. 1 MSF Project Kabo, CAR HIV prevalence in CAR is among the highest in Central and Western Africa, estimated at 4.9–10% amongst adults according to different UN sources [14, 15]. Before a surge in violence in 2013, around 15,000 people living with HIV (PLWH) were on ART. The biggest concern for the HIV programme was interruption of ART due to stock rup- tures and loss of HIV patients on treatment due to inse- curity and displacement. Both events may have affected thousands of PLWH and could have led to increased Ferreyra et al. Conflict and Health (2018) 12:30 Page 3 of 7 Page 3 of 7 Implementation of these activities was mostly organised by a HIV experienced international doctor providing training to national and international staff, implementing protocols, setting up data collection systems, and monitor- ing activities. HIV testing and counselling (HTC) was per- formed by trained community healthcare workers (CHW) using rapid test kits, while clinical consultations were run by nurses supported by a doctor in complicated cases. HIV and TB consultations were integrated into the OPD. Tracing of defaulters was done by CHWs and included tracing of patients under TB treatment, although these ac- tivities where not always done due to limitations on access linked with insecurity. information about what to do in case treatment interrup- tion occurred given the unpredictable situation. There were several patients living in more remote areas who could not reach the clinic and MSF staff could not move, leaving a number of patients’ without access to drugs (Fig 2). In January 2014 the international MSF team had to be evacuated and all HIV services where put on hold except for PMTCT that continued with the national staff that remained in the Hospital. HIV activities where reini- tiated in July 2014 when situation became more stable and the MSF team could return. The contingency plan was rapidly implemented to ensure continuation of treatment in case of population movement or MSF evacuation. Central African Republic Run away bags were distributed to 594 (87%) patients during the peak of vio- lence between September and December 2013. By Feb- ruary 2014, 313 (52%) of those patients had returned for consultation. By December 2016, since the beginning of the programme 782 (54%) patients started on ART were still under active follow up, 354 (25%) patients had been lost to follow up and 182 (13%) had died. Only 26 pa- tients (0.1%) had received a second line regimen. These outcomes, while not optimal, are in line with outcomes reported over similar time periods in stable settings: a recent report from 57 cohorts from 22 countries found that 52.1% of patients were retained on ART, 41.8% were lost to follow-up and 6.0% had died 5 years after ART initiation [21]. Clinical monitoring was done every 1–3 months de- pending on adherence and security conditions. No regular access to CD4 or viral load was available. Point of care devices to measure creatinine and haemoglobin were available to support ARV regimen choices. There was no NAP electronic database and MOH ART registers were used to report a list of indicators entered into the Health Information System (HIS) used by MSF Spain. Tracing of patients lost to care was not always possible due to secur- ity concerns, so the outcomes of many patients lost to care are not known. Overall, 1820 patients were diagnosed with HIV and enrolled into care between 2008 and 2016. 1440 (79%) patients initiated ART, including 90 (8%) children. Median age was 31.7 years old, 1231 (67%) patients were classified as WHO stage 3 or 4 at admission and 77% of the patients started on ART were women. 538 (37%) of the enrolled patients underwent CD4 cell count testing; 333 (61.8%) had CD4 cell count < 200 cell/mm3, 217 (12%) CD4 cell count 200–500 cells/mm3 and 50 (2.7%) patients had a CD4 cell count > 500 cells/mm3. By September 2013, when the context became increasingly unstable, 683 (60%) patients remained under ART and active follow-up. During 2016 viral load monitoring was implemented by sending dried blood spot samples to an external laboratory in South Africa; during this time 390 samples were sent and 212 (54%) results were available. 139 (66%) patients were virologicaly suppressed and 71 (33%) had a viral load higher than 1000 copies/ml. Central African Republic Those with a high VL were provided enhanced adherence support and switched to second line if needed. Context Yemen is frequently affected by intra-state conflicts. The HIV epidemic is described as a mature, low-level epidemic with prevalence estimated at 0.2% in the gen- eral population; however Yemen has a concentrated HIV epidemic among men who have sex with men (MSM) with an estimated prevalence of 5.9%. It is esti- mated that at the end of 2013 there were 35,000 people living with HIV in Yemen [22]. HIV activities were initially implemented by inter- national staff that performed mentoring activities with national doctors at the government HIV clinic in Sana’a. Clinical trainings were provided to medical and non- medical staff of the hospital as a way to increase aware- ness about HIV and decrease stigma. Since 2011 due to the unstable situation, the MSF HIV program has been entirely run by national staff. g MSF has been present in Yemen since 2007 in the Awhar region covering Abyan and Shabwa Governorates with medical programs responding to violence. In February 2009 an assessment of access to HIV services found high levels of stigma and discrimination; patients were being rejected from the hospitals because of their positive status, surgeries had been denied and HIV-positive pregnant women had to attend private clinics to deliver. In response, MSF decided to start HIV services supporting the National AIDS Programme in the capital of Sana’a with the main objective of increasing access to HIV care and reducing stigma and discrimination (Fig. 3). Patients were reviewed monthly for the first year after ART initiation, then every 3 months depending on adher- ence, per national guidelines. CD4 was done at baseline and every 6 months on treatment; targeted viral load was used to detect treatment failure but was not accessible for routine monitoring. Point of care devices to measure cre- atinine and haemoglobin were used to support ARV regi- men choice. In 2010 MOH implemented Tier.net as an M&E tool for the entire country, starting with Sana’a as pilot project [23]. Since the beginning of the project in 2010, 1655 pa- tients were diagnosed HIV positive, 1470 (89%) were started on ART, 126 (9%) were lost to follow up, 288 (20%) died and 1056 (72%) are still under care as of De- cember 2016. Thirty seven patients (4%) are receiving a second line regimen. When the crisis broke out in 2011, there were 363 patients under ART. Program design and implementation The main HIV activities were to provide support to the National AIDS Programme in Sana’s city at the ART clinic in Al-Ghomouri Hospital. MSF support included training of national staff, covering ARV stock ruptures and a major focus on health promotion and advocacy efforts towards reduction of stigma and discrimination aimed at the community, patients and hospital staff. Contingency planning A contingency plan to ensure continuation of ART in case of insecurity was implemented in 2010 covering different security scenarios, from low levels of insecurity with short-term break in services up to full evacuation of most of the staff and distribution of “run-away” packs contain- ing 2 extra months of ARVs plus a 1 week tail protection with AZT/3TC in cases of high insecurity [11]. Patients were informed about what to do in case of violence during routine counselling sessions and national staff were trained on how to prepare in case of evacuation. Fig. 2 MSF response during acute conflict in CAR 2013 In September 2013 the security situation deteriorated, with several armed attacks displacing the population in the MSF supported region with many people crossing the border to Chad. During the initial phase of the plan, patients came to the MSF-supported health clinics to receive extra ARVs (2 months’ supply) and were provided Fig. 2 MSF response during acute conflict in CAR 2013 Page 4 of 7 Ferreyra et al. Conflict and Health (2018) 12:30 Ferreyra et al. Conflict and Health (2018) 12:30 Yemen Context Since the war in 2015, a policy of “Treat all” was imple- mented by the NAP to facilitate treatment initiation of patient’s diagnosed HIV positive and avoid loss to follow up of patients prior to ART. Contingency planning In February 2011, government loyalists and opposition tribesmen clashed during protests in Yemen, especially in the capital Sana’a, against the regime of President Ali Abdullah Saleh. The situation subsequently devolved into open fighting between military forces loyal to the government, defecting military forces, and tribal militia in the capital Sana’a in May 2011. HIV testing was available at the clinic and patients could self-present or be referred. Community outreach activities including health promotion were carried out to increase HIV awareness and testing. HIV protocols and ART eligibility criteria were in line with the National AIDS Program (NAP) and in 2010 a TDF based fixed- dose combination as first line was implemented to re- place existing stavudine-based regimens following WHO recommendations [20]. Unlike in CAR, there was no contingency plan defined at the beginning of the program and the team was forced to develop a plan to ensure ART continuation. All HIV services were interrupted, including HIV testing and ART initiation for new patients. Fig. 3 MSF HIV Project Sanaa, Yemen All patients received a “health card” describing the ARV regimen they were receiving, what to do in case of inability to reach the health facility, and an emergency phone number to call in case they ran out of drugs. The nurse in charge of the pharmacy removed the patient’s registers and ARVs from the clinic to his house and started calling all patients to deliver run-away bags and managed the emergency phone line. Patients called the phone number if they had any questions prior to the next drug refill and arranged with the MSF team drug distribution in different areas of the city. An extra ART Fig. 3 MSF HIV Project Sanaa, Yemen Fig. 3 MSF HIV Project Sanaa, Yemen Ferreyra et al. Conflict and Health (2018) 12:30 Page 5 of 7 Page 5 of 7 compared to other projects where there have not been any extra person to start HIV care. site was allocated out of the area most affected by the clashes where patients could go to pick up drugs. This system lasted for 7 months and patients could call the MSF nurse if they were running out of drugs or needed psychosocial support. A contingency plan was developed building on these lessons to prepare for future episodes of instability and shared with the National AIDS Programme. Contingency planning Outcomes described in this case study are similar to other non-conflict affected African countries [25], although rates of loss to follow up were higher in CAR particularly after the 2013 peak of violence pos- sibly related to the high mobility of the population. Retention in care and viral suppression in CAR was similar to other reports in African countries without security issues. It is important to note that few patients are receiving second line regimens in our programs mainly due to the limited availability of virological monitoring to detect treatment failure. y g Between April and November 2011, 363 (100%) patients received run-away bags with an extra 2 months of drugs and by February 2012, 354 (98%) patients had returned for their follow up consultation. No patient reported having had to interrupt their treatment. After activities resumed in 2014 the war started again in April 2015, at which point the contingency plan was again enacted, with the support from peer patients and associations of PLWH, whose work helped ensure the continuation of treatment for patients. This time patients were given 3–4 months of drugs to try and minimise the risk of treatment disruptions. In Yemen the outcomes of the contingency plan were better than those observed in CAR despite not having a plan beforehand. This difference could be re- lated to the fact that Sana’a is an urban area where patients can still look for a safer place and it has more reliable access to telephone and communication networks, whereas the rural areas of CAR without ac- cess to phone networks and not many options to seek safer places, makes this setting more challenging to ensure access of and to patients. Conclusions Our experience adds to the growing evidence that provision of HIV services in conflict-affected settings is feasible with simplified models and strategies and should be included in the comprehensive medical response in conflict-affected populations. This includes contingency plans to respond in case of emergencies implemented from the beginning of the intervention and linked with proper counselling and patient information. Simplified protocols are consistent with a public health approach to ART delivery [24] and have been successfully applied in other African countries where basic laboratory com- modities and human resources are limited, without com- promising quality. These results have in part been achieved by helping to empower and educate patients since the beginning of the intervention and encouraging them to be responsible for their own treatment. Still today, very few actors and donors are willing to support HIV services in countries chronically affected by violence and HIV packages are rarely included in the initial response to emergencies, despite a consensus state- ment made in 2006 by WHO, UNHCR, UNAIDS, MSF and UNICEF that ARV delivery should be included as part of comprehensive HIV services in emergency settings [26]. Rates of loss to follow up are much higher in CAR despite improved counselling approaches and adequate defaulter tracing systems; this might be due to the fre- quent episodes of violence in the area and the continu- ous internal displacement. This experience demonstrates the value of implement- ing different strategies according to the challenges: in places with high HIV prevalence and low resources available, a step-wise approach initially targeting the sickest patients (e.g. those admitted to IPDs, malnutri- tion centres and TB wards) could be the preferred option until capacity is built to extend care to all HIV patients. In places where a lower number of HIV pa- tients are expected, strategies targeting all HIV infected populations in all medical departments could be feasible. In our program both strategies were tailored to the setting. CAR with a higher HIV prevalence started tar- geting the sicker patients while in Yemen a full package of HIV activities was implemented from the beginning of the intervention. In both settings the inclusion of a focal person for the implementation of HIV activities (e.g. Conclusions Differenti- ated models of care including simplification of protocols, task shifting, patient and community involvement [27], and contingency planning are additional elements that support ART delivery in such settings. Consent for publication Not applicable Competing interests The authors declare that they have no competing interests. Funding Not applicable 13. Ford N, Shubber Z, Meintjes G, Grinsztejn B, Eholie S, Mills C, Davies M-A, Vitoria M, Penazzato M, Nsanzimana S, Frigati L, et al. Causes of hospital admission among people living with HIV worldwide: a systematic review and meta-analysis. Lancet HIV. 2015;2(10):e438–44. https://doi.org/10.1016/ S2352-3018(15)00137-X. Epub 2015 Aug 11 13. Ford N, Shubber Z, Meintjes G, Grinsztejn B, Eholie S, Mills C, Davies M-A, Vitoria M, Penazzato M, Nsanzimana S, Frigati L, et al. Causes of hospital admission among people living with HIV worldwide: a systematic review and meta-analysis. Lancet HIV. 2015;2(10):e438–44. https://doi.org/10.1016/ S2352-3018(15)00137-X. Epub 2015 Aug 11 Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. There is a need to ensure access to HIV services, including ART in conflict settings which have some of the lowest rates of treatment coverage world-wide. Places like South Sudan, or most of the Western and Central African countries (WCA) are among the re- gions with ART coverage below 50% [28]; improved access to HIV services are urgently needed in these settings if we want to reach the UNAIDS 90–90-90 targets. Funding and support to implement these ac- tivities is a major concern when other “competing priorities” are prioritized and main donors maintain a focus of funding for specific fields of interest. Donor support could be crucial to close the gaps when talk- ing about ART services in acute emergencies [29]. Our case studies demonstrate the importance and feasibility of providing HIV care in conflict affected populations in both low and high prevalence HIV set- tings. Simplified protocols and strategies tailored to the context should be thought about when providing access to health care in these settings. Conclusions training of national staff, setting up of basic activities and supervision of the HIV program) was an added value and it allowed a more rapid start of activities This experience demonstrates the value of implement- ing different strategies according to the challenges: in places with high HIV prevalence and low resources available, a step-wise approach initially targeting the sickest patients (e.g. those admitted to IPDs, malnutri- tion centres and TB wards) could be the preferred option until capacity is built to extend care to all HIV patients. In places where a lower number of HIV pa- tients are expected, strategies targeting all HIV infected populations in all medical departments could be feasible. Preferably, contingency plans should be in place from the beginning of the programme, prior to the onset of the emergency. Nevertheless, our experience in Yemen shows that even in the absence of a prior contingency plan, sim- ple actions can be taken rapidly to ensure continuity of care. Pharmacy management should also be included with the contingency plan to ensure the availability of enough drugs for the times of acute instability when drugs for 4– 6 months are given to patients. This could jeopardize fur- ther supply if not planned beforehand. In our program both strategies were tailored to the setting. CAR with a higher HIV prevalence started tar- geting the sicker patients while in Yemen a full package of HIV activities was implemented from the beginning of the intervention. In both settings the inclusion of a focal person for the implementation of HIV activities (e.g. training of national staff, setting up of basic activities and supervision of the HIV program) was an added value and it allowed a more rapid start of activities The recommendation to treat all HIV positive patients regardless of clinical or immunological status provides an opportunity to improve access to ART in conflict set- tings where very often there is no access to CD4 or viral Page 6 of 7 Ferreyra et al. Conflict and Health (2018) 12:30 load monitoring and ART coverage lies far behind other countries in the Sub-Saharan Africa region. Differenti- ated models of care including simplification of protocols, task shifting, patient and community involvement [27], and contingency planning are additional elements that support ART delivery in such settings. load monitoring and ART coverage lies far behind other countries in the Sub-Saharan Africa region. Abbreviations C l ANC: Antenatal care; ART: Antiretroviral treatment; ARV: Antiretroviral; CAR: Central African Republic; CHW: Community health worker; D4T: Stavudine; HIS: Health information system; HIV: Human immunodeficiency virus; HTC: HIV testing and counselling; IPD: Inpatients department; M&E: Monitoring and evaluation; MOH: Ministry of Health; MSF: Médecins sans Frontières; MSM: Men who have sex with men; NAP: National AIDS Program; OPD: Outpatient department; PLWHA: People living with HIV AIDS; PMTCT: Prevention mother- to-child transmission; TB: Tuberculosis; TDF: Tenofovir; UN: United Nations; VCT: Voluntary counselling and testing; VL: Viral load; WCA: Western and Central African region; WHO: World health organization 8. Pyne-Mercier LD, John-Stewart GC, Richardson BA, et al. The consequences of post-election violence on antiretroviral HIV therapy in Kenya. AIDS Care. 2011;23(5):562–8. https://doi.org/10.1080/09540121.2010.525615. 9. Reid T, Van Engelgem I, Telfer B, Manzi M. Providing HIV care in the aftermath of Kenya's post-election violence MSF lessons learned January- march 2008. Confl Health. 2008; https://doi.org/10.1186/1752-1505-2-15. 9. Reid T, Van Engelgem I, Telfer B, Manzi M. Providing HIV care in the aftermath of Kenya's post-election violence MSF lessons learned January- march 2008. Confl Health. 2008; https://doi.org/10.1186/1752-1505-2-15. 10. Culbert H, Tu D, O'Brien D, Ellman T, Mills C, Ford N, Amisi T, Chan K, Venis S. HIV treatment in a conflict setting: outcomes and experiences from Bukavu, Democratic Republic of the Congo. PLoS. https://doi.org/10.1371/ journal.pmed.0040129 10. Culbert H, Tu D, O'Brien D, Ellman T, Mills C, Ford N, Amisi T, Chan K, Venis S. HIV treatment in a conflict setting: outcomes and experiences from Bukavu, Democratic Republic of the Congo. PLoS. https://doi.org/10.1371/ journal.pmed.0040129 11. O'Brien D, Venis S, Greig J, Shanks L, Ellman T, Sabapathy K, Frigati L, Mills C. Provision of antiretroviral treatment in conflict settings: the experience of Médecins sans Frontières. Confl Health. 2010;4:12. https://doi.org/10.1186/1752-1505-4-12. 11. O'Brien D, Venis S, Greig J, Shanks L, Ellman T, Sabapathy K, Frigati L, Mills C. Provision of antiretroviral treatment in conflict settings: the experience of Médecins sans Frontières. Confl Health. 2010;4:12. https://doi.org/10.1186/1752-1505-4-12. Received: 21 December 2017 Accepted: 2 April 2018 Received: 21 December 2017 Accepted: 2 April 2018 Authors’ contributions 14. UNAIDS, Report on the global AIDS epidemic 2012 15. UNFPA. Principaux resultats de la serologie vih prevalence du vih de la quatrieme enquete nationale a indicateurs multiples, 2010. BA supported with field compilation of data and revision of the manuscript. AA supported with data collection at field and revision of the manuscript. NF and DO supported with the writing of the manuscript and results interpretation. CF designed the study, analysed data and did the main manuscript writing. All authors read and approved the final manuscript. 15. UNFPA. Principaux resultats de la serologie vih prevalence du vih de la quatrieme enquete nationale a indicateurs multiples, 2010. 16. Charpentier C, Gody JC, Mbitikon O, Moussa S, Matta M, Péré H, Fournier J, Longo Jde D, Bélec L. Virological response and resistance profiles after 18 to 30 months of first- or second−/third-line antiretroviral treatment: a cross-sectional evaluation in HIV type 1-infected children living in the Central African Republic. AIDS Res Hum Retrovir. 2012;28(1):87–94. https://doi.org/10.1089/AID.2011.0035. Availability of data and materials The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Acknowledgements l f Aurora Revuelta for valuable contributions on the manuscript revision. Jean François Saint Sauveur; for supporting the publication of this field experience. 12. Ogoina D, Obiako R, Muktar H, Adeiza M, Babadoko A, Hassan A, Bansi I. Morbidity and mortality patterns of hospitalised adult HIV/AIDS patients in the era of highly active antiretroviral therapy: a 4-year retrospective review from Zaria, northern Nigeria. AIDS Res Treat. 2012;2012 https://doi.org/10. 1155/2012/940580. 12. Ogoina D, Obiako R, Muktar H, Adeiza M, Babadoko A, Hassan A, Bansi I. Morbidity and mortality patterns of hospitalised adult HIV/AIDS patients in the era of highly active antiretroviral therapy: a 4-year retrospective review from Zaria, northern Nigeria. AIDS Res Treat. 2012;2012 https://doi.org/10. 1155/2012/940580. References 1. UNAIDS Global report on the global AIDS epidemic 2016. 2. UNICEF, UNAIDS, UNHCR. HIV in humanitarian emergencies, information note. 2015. 1. UNAIDS Global report on the global AIDS epidemic 2016. 2. UNICEF, UNAIDS, UNHCR. HIV in humanitarian emergencies, information note. 2015. 3. Response, Steering Committee for Humanitarian. The sphere project: humanitarian chapter and minimum standards in disaster response. 2004. 4. IASC, Inter-Agency Standing Commitee. Guidelines for HIV/AIDS interventions in emergency settings. 2003. 5. Ellman T, Culbert H, Torres-Feced V. Treatment of AIDS in conflict-affected settings: a failure of imagination. Lancet. https://doi.org/10.1016/S0140- 6736(05)17802-7. 6. Mills EJ, Ford N, Singh S, Eyawo O. Providing antiretroviral Care in Conflict Settings. Curr HIV/AIDS Rep. 2009;6(4):201–9. 7. Vreeman R, Nyandiko W, Sang E, Musick B, Braitstein P, Wiehe S. Impact of the Kenya postelection crisis on clinic attendance and medication adherence for HIV-infected children in western Kenya. Confl Health. 2009;3: 5. https://doi.org/10.1186/1752-1505-3-5. Author details 1Méd i 1Médecins sans Frontières Spain, Barcelona, Spain. 2Médecins sans Frontières, Amsterdam, Netherlands. 3National AIDS Program, Sanaa, Yemen. 4Centre for Infectious Disease Epidemiology and Research, University of Cape Town, Cape town, South Africa. Received: 21 December 2017 Accepted: 2 April 2018 Ferreyra et al. Conflict and Health (2018) 12:30 Ethics approval and consent to participate 17. Péré H, Charpentier C, Mbelesso P, et al. Virological response and resistance profiles after 24 months of first-line antiretroviral treatment in adults living in Bangui, Central African Republic. AIDS Res Hum Retrovir. 2012;28(4):315–23. https://doi.org/10.1089/aid.2011.0127. This research fulfilled the exemption criteria set by the Médecins sans Frontières Ethics Review Board for a posteriori analysis of routinely collected clinical data and this did not require MSF ERB review. It was conducted with permission from the Medical Director of MSF Spain. 17. Péré H, Charpentier C, Mbelesso P, et al. Virological response and resistance profiles after 24 months of first-line antiretroviral treatment in adults living in Bangui, Central African Republic. AIDS Res Hum Retrovir. 2012;28(4):315–23. https://doi.org/10.1089/aid.2011.0127. This research fulfilled the exemption criteria set by the Médecins sans Frontières Ethics Review Board for a posteriori analysis of routinely collected clinical data and this did not require MSF ERB review. It was conducted with permission from the Medical Director of MSF Spain. Page 7 of 7 Ferreyra et al. Conflict and Health (2018) 12:30 Ferreyra et al. Conflict and Health (2018) 12:30 . Bélec L, Mbopi-Kéou FX. HIV epidemic out of control in Central Afr 18. Bélec L, Mbopi-Kéou FX. HIV epidemic out of control in Central African Republic. Lancet. https://doi.org/10.1016/S0140-6736(12)62156-4. 19. UNHCR. Situational analysis of HIV activities in CAR after the coup d'état. 2013. 20. WHO, Antiretroviral therapy for HIV infection in adults and children. Recommendations for a public health approach. 2010 revision. 2010. Recommendations for a public health approach. 2010 revision. 201 21. Haas A, Zaniewski E, Anderegg N, Ford N, Fox MP, Vinikoor M, Dabis F, Nash D, Sinayobye J, Niyongabo T, Tanon A, Poda A, Adedimeji A, Edmonds A, Davies M-A, Egger M. Retention and mortality on antiretroviral therapy in sub-Saharan Africa: collaborative analyses of HIV treatment programs. J Int AIDS Soc. 2018;21(2). https://doi.org/10.1002/ijia2.25084. 22. NAP. Yemen National Aids Program report. 2013. 23. Osler M, Hilderbrand K, Hennessey C, Arendse J, Goemaere E, Ford N, Boulle A. A three-tier framework for monitoring antiretroviral therapy in high HIV burden settings. J Int AIDS Soc. 2014;17:18908. 24. Gilks CF, Crowley S, Ekpini R, Gove S, Perriens J, Souteyrand D, Mitoria M, Guerma T, De Cock K. The WHO public-health approach to antiretroviral treatment against HIV in resource-limited settings. Lancet. 2006;368(9534):505–1. 25. Brinkhof M, Pujades-Rodriguez M, Egger M. Mortality of patients lost to follow-up in antiretroviral treatment Programmes in Rsource-limited settings: systematic review and Metanalysis. PLoS One. http://www.who.int/ hac/techguidance/pht/HIV_AIDS_101106_arvemergencies.pdf. 26. UNHCR, WHO, MSF and UNAIDS. Consensus statement: delivering antiretroviral drugs in emergencies: neglected but feasible. Geneva; 2006. http://www.who. int/hac/techguidance/pht/HIV_AIDS_101106_arvemergencies.pdf 27. Zukoski AP, Thorbun S. Experiences of stigma and discrimination among adults living with HIV in a low HIV-prevalence context: a qualitative analysis. AIDS Patient Care STDs. 2009;23(4):267–76. 28. UNAIDS HIV Progress Report 2015. 29. Hanson B, Wodak A, Fiamma A, Coates T. Refocusing and prioritizing HIV programmes in conflict and post-conflict settings: funding recommendations. AIDS. 2008;22(Suppl 2):S95–103. https://doi.org/10.1097/01.aids.0000327441. 66656.da.
https://openalex.org/W4392356772	https://www.researchsquare.com/article/rs-3997224/latest.pdf	Latin	null	Bioactivity of essential oils from three species of Mentha against Plutella xylostella (Lepidoptera: Plutellidae)	Research Square (Research Square)	2,024	cc-by	10,486	Antônio de Almeida Paz Neto Universidade Federal Rural de Pernambuco, Laboratório de Investigação Química de Inseticidas Naturais Page 1/25 Universidade Federal Rural de Pernambuco, Laboratório de Investigação Química de Inseticidas Naturais Vaneska Barbosa Monteiro Universidade Federal Rural de Pernambuco, Laboratório de Investigação Química de Inseticidas Naturais Marcilio Martins Moraes Universidade Federal Rural de Pernambuco, Laboratório de Produtos Naturais Bioativos, Brazil João Paulo Ramos Melo Universidade Federal Rural de Pernambuco, Laboratório de Investigação Química de Inseticidas Naturais Tamara Thays Barbosa Leal Universidade Federal Rural de Pernambuco, Laboratório de Investigação Química de Inseticidas Naturais Research Article Keywords: Toxicity, feeding deterrence, repellence, phytotoxicity Posted Date: March 1st, 2024 DOI: https://doi.org/10.21203/rs.3.rs-3997224/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Additional Declarations: No competing interests reported. Research Article Additional Declarations: No competing interests reported. Page 1/25 Abstract Botanical derivatives constitute an important option for the reduction in the use of synthetic insecticides for the management of agricultural pests. The larval form of the diamond back moth, Plutella xylostella, is one of the main pests of the family Brassica and is resistant to synthetic insecticides. The aim of the present study was to test the potential of commercially available essential oils (EOs) from the genus Mentha on P. xylostella. EOs from M. arvensis, M. spicata and M. piperita were investigated. Topical toxicity and residual contact toxicity tests were conducted, along with the assessment of biological parameters, feeding deterrence and repellency. Phytotoxicity to Brassica leaves was also investigated. The oil from M. piperita was the most toxic to P. xylostella by topical contact, whereas the oil from M. spicata was the most toxic by residual contact. Overall, the Mentha oils were more toxic by topical contact than residual contact. The EOs from M. arvensis and M. spicata significantly altered the weight of the pupae and larval survival. All Mentha oils tested caused feeding deterrence in P. xylostella. The EO from M. piperita exhibited persistent repellence over time compared to the other EOs. The oils administered at the maximum doses tested did not cause phytotoxicity to Brassica leaves. The present results demonstrate the potential of essential oils from plants of the genus Mentha, altering biological and behavioral aspects of P. xylostella. Introduction The diamondback moth or cabbage moth, Plutella xylostella (L.) (Lepidoptera: Plutellidae), is a cosmopolitan pest that destroys species of Brassicaceae, such as cabbage, cauliflower and Brussels sprouts (Sarfraz et al., 2006; Gautam et al., 2018). The costs of pest management together with the loss of production caused by P. xylostella reach billions of dollars per year throughout the world (Zalucki et al., 2012). High selective pressure due to the unbridled use of insecticides has made this pest resistant to practically all classes of insecticides. Indeed, P. xylostella is the insect with the most records of resistance, with 1022 cases registered for 101 compounds (Mota-Sanchez & Wise, 2024). To hinder the occurrence of resistance and facilitate the management of P. xylostella, the possibility of its control by botanical insecticides has been investigated (Reddy et al., 2015; Araujo et al., 2020; Song et al., 2022). High selective pressure due to the unbridled use of insecticides has made this pest resistant to practically all classes of insecticides. Indeed, P. xylostella is the insect with the most records of resistance, with 1022 cases registered for 101 compounds (Mota-Sanchez & Wise, 2024). To hinder the occurrence of resistance and facilitate the management of P. xylostella, the possibility of its control by botanical insecticides has been investigated (Reddy et al., 2015; Araujo et al., 2020; Song et al., 2022). Botanical derivatives have biodegradability, various modes of action as well as low impact on the environment, non-target organisms and human health (Campos et al., 2019; Ahmed et al., 2022). In recent decades, essential oils have garnered considerable attention due to their biological properties resulting from secondary metabolites belonging to the chemical classes of monoterpenes, sesquiterpenes and phenylpropanoids, with recognized effects on arthropods, such as insecticidal (Ma et al., 2020), repellent (Pang et al., 2020; Santana et al., 2022) and antifeedant (da Camara et al., 2022) activities. The use of plant derivatives comprised of a complex mixture of chemical substances with different modes of action could delay the development of resistant pests (Pavela & Benelli, 2016), as commercial products have only one active ingredient, increasing the likelihood of the development of resistance. Introduction Page 2/25 Page 2/25 Besides lethal effects, the exposure of insects to sublethal doses of plant-derived compounds can interfere with the activity of digestive enzymes, such as α-amylase, α and b-glycosidase and TAG-lipase, thus reducing performance and affecting the efficiency of food conversion into biomass, with adverse effects on insect development (El-Hefny, 2019; Mirhaghparast et al., 2020). Nutrient ingestion and absorption are important components of the performance of herbivores and are directly correlated with effects on biological aspects of these insects (Le Gall and Behmer 2014). Plant derivatives can have a gamut of sublethal effects, such as the prolongation or reduction in the larval, pupal and egg stages, deformations, the suppression of the emergence of adults as well as adverse effects on survival, fecundity and the hatching of eggs (Stepanycheva et al., 2019; Mantzoukas et al., 2020; Hategekimana & Erler, 2020; Pavela et al., 2021). The family Lamiaceae comprises approximately 7000 plant species distributed among 240 genera (da Silva et al., 2021), with traditional use in folk medicine and as food condiments, especially plants of the genus Mentha (Singh & Pandey, 2018). Plants of this genus are characterized by the production of essential oils basically comprised of monoterpenes (menthol, menthone, carvone and piperitone), which have a large variety of biological properties, including effects on arthropods (Yildrim et al., 2013; Bossou et al., 2015; Jankowska et al., 2019; Tahri et al., 2022). Due to the recognized insecticidal property of essential oils (EOs) from species of Mentha as well as their established chemical standardization and commercial availability, EOs from M. arvensis, M. piperita and M. spicata were selected for the determination of the effects on P. xylostella. Previous studies on the EOs from M. piperita and M. spicata were restricted to the investigation of toxicity and the deterrence effect to 3rd instar larvae of P. xylostella (Koundal et al., 2018) and no study was found addressing the effect of the EO from M. arvensis on this pest. The present work is part of a broad investigation and contribution to knowledge on the chemical composition and insecticidal potential of aromatic plants. Considering the gap in knowledge on the action of Mentha in terms of the antifeedant effect and toxicity to different stages of life of P. xylostella, this work also investigated the main effects of the EOs on biological aspects of this pest as well as phytotoxicity to the host plant Brassica oleracea. Identification of components Identification of the components was based on GC-MS retention indices with reference to a homologous series of C8-C40 n-alkanes calculated using the Van der Dool and Kratz equation (Van den Dool and Kratz 1963), computer matching against the mass spectral library of the GC-MS data system (NIST 11 and WILEY 11th), co-injection with authentic standards as well as matching against other published mass spectra (Adams, 2007). Area percentages were obtained from the GC-FID response without the use of an internal standard or correction factors. Obtainment of pest and essential oils Obtainment of pest and essential oils The experiments were conducted at the Natural Insecticide Chemical Investigation Lab of the Agronomy Department of Universidade Federal Rural de Pernambuco (UFRPE) using a population of Plutella xylostella created years earlier without selection with insecticides. Rearing of P. xylostella was performed following the method described by Torres et al. (2006), with the insects maintained at a temperature of 25 ± 1ºC, with relative humidity of 74 ± 5% and a 12-hour light/darkness photoperiod. Page 3/25 Essential oils from Mentha arvensis, Mentha piperita and Mentha spicata were purchased from the Ferquima company. The commercial insecticides Azamax® and Decis 25 EC were purchased from agricultural product stores in the city of Recife, PE, Brazil. Gas chromatography-mass spectrometry Gas chromatography-mass spectrometry Qualitative analysis involving gas chromatography-mass spectrometry (GC-MS) (220-MS IT GC, Varian, Walnut Creek, CA, USA) was performed using a system with a mass selective detector, mass spectrometer in EI 70 eV with a scanning interval of 0.5 s and fragments from 40 to 550 Da. fitted with the same column and temperature program as that for the GC-FID experiments, with the following parameters: carrier gas = helium; flow rate = 1 mL min− 1; split mode (1:30); injected volume = 1 µL of diluted solution (1/100) of oil in n-hexane. Topical bioassay The effect of topical contact was investigated using the method described by Wei et al. (2015). A micropipette was employed (Kumar et al., 2014) for the application of 0.5 µL of each dose of EO on the prothoracic region of 3rd instar larvae of P. xylostella. The ranges of the doses used were established in preliminary tests. Solutions were obtained by the dilution of the EOs in water containing DMSO (0.5%) and the commercial insecticides in water and Triton-X100® (0.01%). Nine doses were used for the EOs from M. arvensis and M. piperita, ranging from 2.58 µg larva− 1 to 19.78 µg larva− 1 and from 2.67 µg larva− 1 to 9.79 µg larva− 1, respectively. Eight doses were used for the EOs from M. spicata, ranging from 2.3 µg larva− 1 to 15.18 µg larva− 1. A control treatment was also established with only water and dimethyl sulfoxide (DMSO) (0.5%). The following doses were tested for the conventional insecticide Decis (Deltametrina) 25 EC: 0.039 µg larva− 1 to 5 µg larva− 1 of deltamethrin diluted in water and Triton-X100® (0.01%). A control treatment was also established with only water and Triton-X100® (0.01%). Larvae were placed in Petri dishes with disks of collard greens measuring 5 cm in diameter separated into three groups of 10 individuals (30 larvae for each dose tested). The same quantity of individuals was used a second time for the test on another day (repetition in time). In the analysis with the maximum likelihood ratio, the slopes were compared for the determination of the hypotheses of parallelism and equalness (Robertson et al. 2007). When no difference was found, the tests performed on different days were combined, giving a final total of 60 larvae per dose tested for each treatment. Page 4/25 Page 4/25 Mortality achieved with the EOs was determined by counts of the number of dead larvae 24 hours after application of the doses. As mortality is slower for the commercial insecticides, the count of the number of dead larvae was performed 48 hours after the application of Decis 25 EC (Jan et al., 2015) and 72 hours after the application of Prêmio® 200 CS (Gong et al. 2014). Larvae were considered dead when not exhibiting any response when prodded with a brush. Topical bioassay Dose-mortality data were analyzed using the Probit model (Finney 1971) with the aid of the POLO plus software program (LeOra-Software, 2005) for the determination of LD25, LD50 and LD95 and respective 95% confidence intervals. Residual contact bioassay The immersion method was used. Disks of collard greens (5 cm in diameter) were immersed in 20 ml of the different concentrations of the test solution (essential oils and positive controls) and negative control for 10 seconds. After drying for 10 minutes at room temperature, ten 3rd instar larvae of P. xylostella were placed on each leaf disk. Mortality was recorded after 48 hours of exposure. Six repetitions were performed per treatment and repeated in time, corresponding to 120 larvae. To determine the toxicity of the EO solutions, the results were compared to the positive controls (chemical insecticide containing deltamethrin and botanical insecticide containing azadirachtin) as well as the negative control (distilled water + DMSO and dodecylbenzene sulfonic acid). Preliminary tests had been conducted to define the upper and lower concentrations of 0 and 100%. Concentrations were created with serial dilutions and ranged from 0.5 to 60 µL mL− 1 for the EO from M. arvensis, 5 to 80 µL mL− 1 for the EO from M. spicata, 7 to 100 µL mL− 1 for the EO from M. piperita, 2.5 to 170 µL mL− 1 for Azamax® and 12 to 800 µL mL− 1 for Decis®. Concentration-mortality data were analyzed using the Probit model (Finney 1971) with the aid of the POLO plus software program (LeOra- Software, 2005) for the determination of the LC50 and LC90 with respective 95% confidence intervals. Phytotoxicity test After the residual contact test, phytotoxicity tests were conducted with the concentration that caused the 95% mortality using the method adapted from Torres et al. (2006). Collard leaf disks measuring 5 cm in diameter were immersed in the solutions prepared with the EOs diluted in aqueous solutions and the negative control (1.0% DMSO, 0.1% de dodecylbenzene sulfonic acid and distilled water) for 10 seconds and then set to dry at room temperature. After 48 h, the phytotoxicity index (PI) was determined for each leaf disk with the aid of the AFSoft program using the following formula: PI = TA% – SA%, in which TA is the total area of the leaf and SA is the sound (unaffected) area of the leaf. The images were analyzed using the criteria of the phytotoxicity scale proposed by Monchiero et al. (2015): 0.00 to 9.99% = no effect or evident symptoms; 10.00 to 19.99% = negligible effects and symptoms; 20.00 to 29.99% = mild discoloration with no leaf scorch; 30.00 to 39.99% = moderate discoloration with leaf scorch; 40.00 to 99.99% = severe leaf scorch; 100% = severe leaf scorch and dead plant. Biological parameters Biological parameters Page 5/25 The assessment of the sublethal effects of the EOs on biological variables was based on the method described by Han et al. (2012). The following parameters were determined: survival from larva to adult; larval viability; weight of pupa; emergence rate; fecundity; longevity; egg viability of first generation (F1); larval duration of F1; and larval viability of F1. The experiment was begun with 100 larvae of P. xylostella, which were weighed and distributed among 10 Petri dishes to ensure equal final weight of the treatments. Each larva was submitted to topical application of the sublethal dose (LD25) of the EOs from M. arvensis (5.1 µg larva− 1), M. piperita (4 µg larva− 1) and M. spicata (5.1 µg larva− 1) previously calculated in the toxicity test. A collard green leaf disk measuring 5 cm in diameter was placed in each Petry dish. The leaf disks were exchanged daily until all larvae reached the pupal phase. The same procedure was performed with the control containing only water and DMSO (0.5%). Dead larvae in the period of 24 hours after exposure were removed and not counted in the determination of larval survival and viability (as this mortality was expected), thus avoiding the overestimation of the effect. Phytotoxicity test Therefore, counts of dead larvae began 48 hours after exposure, with the assessment of mortality and development. Larval development was recorded daily until the formation of the pupae, which were transferred to duly identified ELISA plates until the emergence of the adults. For each treatment, 15 pupae 24 hours of age were randomly selected and weighed on an analytical scale. Thus, larval viability, weight of the pupae and the periods of the pulpal phase and emergence were determined. Data on larval viability and weight of the pupae were submitted to analysis of variance (ANOVA) and differences between means were determined using Tukey’s test, with a P-value < 0.05 considered indicative of statistical significance. Data on the periods of the pupal phase and emergence were submitted to the nonparametric Kruskal–Wallis test. Larval survival data were analyzed using the log- rank survival test. All analyses were performed with the aid of the SAS statistical package (SAS Institute 2008). Egg viability bioassay Male and female pairs of P. xylostella from the previous experiment (24–36 hours of age) were randomly separated in plastic cups, forming seven groups of three pairs (total of 21 pairs per treatment). The cups were perforated with a pin to enable the passage of air and duly identified. A moistened filter paper disk carefully cut to occupy the entire space of the bottom of the cup was placed and a collard green disk measuring 2.5 cm in diameter was placed on the filter paper for oviposition of the females. The top was covered with PVC film. An opening was made in the PVC film with the aid of a precision knife and a cotton ball soaked in a 10% honey solution was placed in the opening. The cotton ball was exchanged daily. Page 6/25 Page 6/25 Only the eggs of the first laying found on the leaf disk were used for the observation of hatching. When the egg laying had finished, the test proceeded with the exchange of the cotton balls soaked in the honey solution until the death of all adults, with daily counts of the number of eggs laid by the females. The leaf disks with the first oviposition were placed in Petri dishes and the number of larvae hatched was counted daily. The ratio between the initial quantity of eggs and the number of larvae hatched was used to calculate egg viability. Female fecundity, longevity and egg viability were assessed in this test. Fecundity data were submitted to analysis of variance (ANOVA) and differences between means were determined using Tukey’s test, with a P-value < 0.05 considered indicative of statistical significance. For the analysis of longevity and egg viability, the data were submitted to the nonparametric Kruskal–Wallis test. All analyses were performed with the aid of the SAS statistical package (SAS Institute 2008). Effect on F1 For this experiment, 150 newly hatched larvae from the previous bioassay were distributed among three Petri dishes, totaling 50 larvae per dish for each treatment. The larvae were treated with the LD25 of each oil. A 5-cm leaf disk was placed in each dish, followed by the determination of development and mortality. After growing, the larvae were transferred to larger plastic recipients containing sections of collard green leaves and covered with a piece of voile fabric. The test was concluded when the larvae treated with the oils were unable to form pupae four days after the pupae formed in the control group. Larval viability and duration of the F1 generation were assessed using the nonparametric Kruskal–Wallis test. The analysis was performed with the aid of the SAS statistical package (SAS Institute 2008). Feeding deterrence and repellent activity bioassays The feeding deterrence and repellent activity tests were adapted from Akhtar et al. (2012) and involved the determination of the preference for treated or untreated leaves. Collard leaf disk measuring 2.2 cm in diameter were immersed in 20 ml in concentrations corresponding to the LC30 of the solutions (5 µL mL− 1, 2 µL mL− 1 and 3.75 µL mL− 1 for the EOs from M. avensis, M. spicata and M. piperita, respectively) for ten seconds and then set to dry at room temperature. A treated disk was placed at a distance of 2.0 cm from an untreated disk (immersed in distilled water with 1.0% DMSO and 0.1% dodecylbenzene sulfonic acid for 10 s) in a Petri dish 9.0 cm in diameter. A larva deprived of food for 4 hours was placed equidistant (1.0 cm) between the treated and control disk of each Petri disk and left to feed for 24 h. Ten repetitions were performed for each concentration of each treatment, with one Petri dish used for each repetition. After 24 h of exposure, the larvae were removed and areas of consumed leaf on the control and treated disks were determined with the aid of a Licor-3100 leaf area meter, which has high precision and reproducibility, with the resolution ranging from 0.1 to 1 mm2. Effect on F1 After determining the consumption of each leaf disk (treated and control), the feeding deterrence index (FDI) was calculated as follows: FDI = 100{(C – T) / (C + T)}, in which C and T (Isman 1993) refer to the areas consumed on the control and treated Page 7/25 Page 7/25 disks, respectively. Based on the FDI, the EO solutions were classified as phagodeterrent (positive values) or phagostimulant (negative values) (Ferreira et al., 2022). The repellent effect was recorded after 1, 2, 4, 6, 12 and 24 h of exposure, with the quantification of the number of larvae on the treated and control leaves. The repellence index (RI) was calculated at follows: RI = 2G / (G + P), in which G is the percentage of larvae found on the disk treated with the oils and positive control and P is the percentage of larvae found on the disk treated only with distilled water. RI values range from zero to 2. RI = 1 indicates neutral action; RI > 1 indicates attraction and RI < 1 indicates repellent action (Mazzonetto & Vendramim, 2003). The repellence intensity scale based on the indices proposed by Mazzonetto & Vendramim (2003) was used to classify the degree of repellence of the EOs and positive control to the larvae of P. xylostella. For this experiment, an entirely randomized design was employed with 10 repetitions per treatment. GC-MS analysis identified 27 compounds. The chemical composition of the Mentha oils is displayed in Table 1. The major constituents of the M. arvensis and M. piperita oils were menthol, menthone and isomenthone, respectively accounting for 38.41% and 45.42%; 21.99% and 21.50%; and 13.86% and 8.57% of the M. arvensis and M. piperita oils. The major constituents of the essential oil from M. spicata were carvone (66.40%) and limonene (19.16%). Results Chemical profile of essential oils GC-MS analysis identified 27 compounds. The chemical composition of the Mentha oils is displayed in Table 1. The major constituents of the M. arvensis and M. piperita oils were menthol, menthone and isomenthone, respectively accounting for 38.41% and 45.42%; 21.99% and 21.50%; and 13.86% and 8.57% of the M. arvensis and M. piperita oils. The major constituents of the essential oil from M. spicata were carvone (66.40%) and limonene (19.16%). Page 8/25 Table 1 Chemical composition of essential oils from Mentha arvensis, Mentha spicata, Mentha piperita Compound1 RI2 M. arvensis %3 M. piperita %3 M. spicata %3 β-Pineno 974 0.90 0.66 1.02 trans-Menta-2,8-dieno 977 0.40 0.10 0.14 trans-iso-Limoneno 981 1.22 0.85 1.00 Mirceno 985 0.58 0.21 1.05 dihidro-1,8-Cineol 989 0.54 - - Limoneno 1024 - 2.65 19.16 β-Felandreno 1025 0.23 0.38 0.23 1,8-Cineol 1026 0.21 5.02 - (Z)-β-Ocimeno 1030 - - 0.13 trans-Sabinenohidrate 1097 - 0.35 - Cânfora 1142 0.48 - - Mentone 1145 21.99 21.50 1.22 iso-Mentone 1153 13.86 8.57 0.77 Mentol 1164 38.41 45.42 2.91 neo-iso-Pulegol 1166 0.95 0.44 0.76 (E)-Isocitral 1177 1.64 2.40 - cis-p-Menta-1(7),8-dien-2-ol 1183 1.63 0.70 - (2E,4E)-Nonadenal 1210 0.44 - - Dihidrocarveolacetato 1222 0.14 - 0.58 Carvona 1239 - - 66.40 Piperitona 1244 4.43 6.49 0.39 neo-Dihidrocarveolacetato 1301 0.13 - 0.51 β-Cariofileno 1413 1.46 0.85 1.62 β-Cedreno 1416 0.79 - - 1Compounds are listed according to the elution on non-polar DB-5 column; 2Retention Indices from Table 1 1Compounds are listed according to the elution on non-polar DB-5 column; 2Retention Indices from the Literature; 3 Percentage of the compound in the essential oil. Compound1 RI2 M. arvensis %3 M. piperita %3 M. spicata %3 Hidratocarvona 1423 0.36 0.71 - γ-Elemeno 1435 0.19 - 0.15 Germacreno B 1555 0.10 0.35 0.14 Total 98.82 97.65 98.18 1Compounds are listed according to the elution on non-polar DB-5 column; 2Retention Indices from the Literature; 3 Percentage of the compound in the essential oil. 1Compounds are listed according to the elution on non-polar DB-5 column; 2Retention Indices from the Literature; 3 Percentage of the compound in the essential oil. Topical bioassay Topical bioassay The EOs from the species of Mentha exhibited substantial topical toxicity to 3rd instar larvae of Plutella xylostella. The dose-mortality results fit the Probit model. The mean lethal doses that killed 50% of the larvae (LD50) are displayed in Table 2. The P. xylostella larvae were more sensitive to the M. piperita oil, which had the lowest LD50, differing significantly from that of the other oils tested. However, the synthetic insecticide containing deltamethrin used as the positive control was at least sixfold more toxic than the Mentha oils. Table 2 Toxicity of essentials oils of Mentha piperita, Mentha arvensis and Mentha spicata topically applied to 3rd instar Plutella xylostella larvae Essential oils Na LD50 (95% CI)b LD95 (95% CI) Slope ± SE χ² (df)c Mentha piperita 540 5.25 (4.90–5.50) 10.20 (9.30–11.50) 5.6 ± 0.43 7.8 (7) Mentha arvensis 540 7.60 (6.52–8.65) 20.00 (16.5–26.9) 3.9 ± 0.29 13.2 (7) Mentha spicata 480 7.90 (7.20–8.60) 23.00 (19.40–29.00) 3.5 ± 0.31 9.1 (6) Deltamethrin (Positive Control) 540 0.90 (0.68–1.20) 29.70 (15.33–70.14) 1.1 ± 0.10 2.7 (6) anumber of Plutellla xylostella; b Lethal dose µg larva-1 (95% confidence limits) c χ ,chi-square and df, degrees of freedom Page 10/25 The toxicity of the Mentha oils to P. xylostella larvae through residual contact was demonstrated. The estimated mean lethal concentrations that killed 50% of the population (LC50) are displayed in Table 3. The larvae were more sensitive to the M. spicata and M. avensis oils, which had significantly lower LC50 values than the M. piperita oil. In this bioassay, the Mentha oils exhibited greater toxicity than the synthetic product deltamethrin. Table 3 Residual toxicity of essentials oils of Mentha piperita, Mentha arvensis and Mentha spicata against 3rd instar Plutella xylostella larvae Essential oils N LC50 (95% CI)b LC95 (95% CI) Slope ± SE χ2 Mentha arvensis 580 13.24 (10.84–16.27) 119.06 (80.85-202.27) 1.34 ± 0.11 0.25 (7) Mentha spicata 620 10.42 (8.93–12.04) 69.24 (44.72–87.66) 1.70 ± 0.16 0.46 (5) Mentha piperita 700 31.09 (27.61–35.27) 120.79 (97.28-158.74) 2.17 ± 0.15 0.71 (5) Deltamethrin (Positive Control) 840 3.90 (3.37–4.45) 14.74 (12.00–18.00) 2.29 ± 0.13 6.26 (5) anumber of Plutellla xylostella; b Lethal concentration mg mL-1 (95% confidence limits) c χ ,chi-square and df, degrees of freedom Phytotoxicity test The analysis of phytotoxicity to collard green leaves (Brassica oleracea) revealed the absence of effects/evident symptoms even at the highest concentrations of the M. arvensis and M. spicata oils tested, whereas phytotoxicity of the highest concentration of the M. piperita oil was classified as “negligible effects/symptoms”. An absence of phytotoxicity was found for the leaves treated with the negative control (distilled water + 1.0% DMSO and 0.1% dodecylbenzene sulfonic acid), whereas phytotoxicity was found for the positive controls Azamax® (28.95 ± 3.05%) and Decis® (72.49 ± 1.19%), with classifications of mild discoloration and severe leaf scorch, respectively (Table 4). Page 11/25 Table 4 Percentage of injury (phytotoxicity) promoted by Mentha essential oils and positive control (deltamethrin) on collard greens leaf discs Essential oils Phytotoxicity (%) ± S.E Phytotoxicity scalea Mentha arvensis 4.44 ± 0.31 No evident effects/symptoms Mentha spicata 5.93 ± 0.31 No evident effects/symptoms Mentha piperita 14.61 ± 0.36 Negligible effects/symptoms Deltamethrin 72.49 ± 1.19 Burnt/lost. aphytotoxicity scale proposed by Monchiero et al. (2015) Table 4 Biological parameters Biological parameters The effects of the Mentha oils larval viability, weight of the pupae, survival, hatching rate of F1 generation and larval viability of F1 after the exposure of the 3rd instar larvae to the sublethal dose (LD25) of the oils are displayed in Table 5. Topical exposure to the LD25 of the M. arvensis oil reduced larval viability by 12.9%, which was significantly lower than the mean of the control (F3,36 = 4.27; P < 0.01), but with no significant difference compared to the other oils. Larvae exposed to the M. arvensis and M. spicata oils formed significantly lighter pupae compared to those formed in the group treated with the M. piperita oil and the control (F3,56 = 7,74; P < 0.0002). Moreover, the M. arvensis and M. spicata oils diminished the life expectancy of P. xylostella between the 24-hour period after placement on the larvae to the adult phase, reducing the average larval survival time by 40.26% in comparison to the negative control. In contrast, the M. piperita oil had no statistically significant effect on average survival time (log-rank = 10.34; P > 0.016). The M. spicata oil significantly reduced the hatching rate of the F1 generation (F3,24 = 9.81; P < 0.02), whereas the other Mentha oils did not differ significantly from the control with regards to this variable. Phytotoxicity test The analysis of larval viability of the offspring revealed that the Mentha significantly reduced the percentage of larvae that reached the pupal phase, differing significantly from the control (χ² = 8.08; P > 0.04). The M. spicata oil caused the greatest reduction in the percentage of larvae, differing significantly from the M. arvensis and M. piperita oils, which, in turn, had similar results for this variable. whereas the other Mentha oils did not differ significantly from the control with regards to this variable. The analysis of larval viability of the offspring revealed that the Mentha significantly reduced the percentage of larvae that reached the pupal phase, differing significantly from the control (χ² = 8.08; P > 0.04). The M. spicata oil caused the greatest reduction in the percentage of larvae, differing significantly from the M. arvensis and M. piperita oils, which, in turn, had similar results for this variable. Page 12/25 Page 12/25 Page 12/25 Table 5 Effect of sublethal dose (LD25) of Mentha essential oils on the biological parameter of Plutella xylostella F1 Table 5 Effect of sublethal dose (LD25) of Mentha essential oils on the biological parameter of Plutella xylostella F1 generation Treatment Larval Viability (%) Weight of Pupa (mg) Larva-Adult Survival (day) Egg hatching (%) Larval survival 3rd instar- adult (day ± SE) Larval duration (day ± SE) Larval Viability (%) Negative control 99 (± 0.99)a5 6.4 (± 0.11)a 7.7 (± 0.12)a 90 (± 1.31)a 7.7 ± 0.12a 11.9 ± 0.23a 0.7 (± 0.00)a M. arvensis 86.1 (± 3.79)b 6.1 (± 0.10)b 3.1 (± 0.07)b 90 (± 3.10)ab 3.1 ± 0.07b 12.5 ± 0.27a 0.6 (± 0.03)b M. piperita 97.7 (± 1.51)ab 6.5 (± 0.06)a 4.2 (± 0.11)ab 90 (± 2.42)a 4.2 ± 0.11ab 12.1 ± 0.23a 0.6 (± 0.06)bc M. spicata 88.7 (± 4.58)ab 6.1 (± 0.07)b 3.1 (± 0.08)b 80 (± 3.00)b 3.1 ± 0.08b 12.6 ± 0.4a 0.4(± 0.03)c Means (± standard erro) followed by the same lowercase letter within a column do not differ significantly (P < 0.05, ANOVA; P > 0.05, Kruskal-Wallis; P > 0.05, Log-Rank) Effect of oils on F1 The effects of sublethal doses of the Mentha oils on 3rd instar larvae of P. xylostella in terms of larval survival to the adult phase, larval duration and viability of the offspring are displayed in Table 5. The M. arvensis and M. spicata oils reduced the average survival time of the larvae in comparison to the control (log-rank = 10.34; P > 0.016). The analysis of larval viability of the offspring revealed that the Mentha oils significantly reduced the percentage of larvae that reached the pupal phase, differing significantly from the control (χ² = 8.08; P > 0.04). The M. spicata oil caused the greatest reduction in the percentage of larval viability, differing significantly from the M. arvensis and M. piperita oils, which, in turn, achieved similar results to each other. However, none of the oils exerted an effect on larval duration of the offspring (χ² = 2.46; P > 0.48). Egg viability The mean number of eggs per female, longevity and hatching rate measured after exposure of the 3rd instar larvae to the Mentha oils are displayed in Table 6. The oils did not exert an effect on these biological variables, with the exception of the M. spicata, which significantly reduced the hatching rate in comparison to the control (F3,24 = 9.81; P < 0.02). The results also indicate that the oils did not affect the longevity of the adults: χ² = 1.47 for males (P > 0.69) and χ² = 0.96 for females (P > 0.81). The mean number of eggs per female, longevity and hatching rate measured after exposure of the 3rd instar larvae to the Mentha oils are displayed in Table 6. The oils did not exert an effect on these biological variables, with the exception of the M. spicata, which significantly reduced the hatching rate in comparison to the control (F3,24 = 9.81; P < 0.02). The results also indicate that the oils did not affect the longevity of the adults: χ² = 1.47 for males (P > 0.69) and χ² = 0.96 for females (P > 0.81). Page 13/25 Page 13/25 Page 13/25 Table 6 Effect of sublethal dose (LD25) of Mentha essential oils on fecundity, hatching percentage and longevity of Plutella xylostella adults Treatment Fecundity (eggs/female ± SE) % Hatching (± SE) Adult longevity Female (days ± SE) Male (days ± SE) Negative control 188.1 ± 15.07a 90 ± 1.31a 10.4 ± 1.13a 13.3 ± 1.49a M. arvensis 171.2 ± 22.34a 90 ± 3.01ab 10.4 ± 0.79a 13.8 ± 1.25a M. piperita 183.3 ± 11.91a 90 ± 2.42a 11.0 ± 1.15a 12.6 ± 1.13a M. spicata 157.3 ± 27.34a 80 ± 3.00b 9.3 ± 0.62a 14.5 ± 1.43a Means (± standard erro) followed by the same lowercase letter within a column do not differ significantly (P < 0.05, ANOVA; P > 0.05, Kruskal-Wallis; P > 0.05, Log-Rank) Eff t f il F1 Effect of oils on F1 Feeding deterrence and repellent activity bioassays Feeding deterrence and repellent activity bioassays The activity of feeding deterrence of 3rd instar larvae determined during the preference test are displayed in Table 7. All Mentha oils were considered phagodeterrent for the 3rd instar larvae of P. xylostella. The intensity of repellent activity varied throughout the four hours of assessment. The repellency promoted by the M. arvensis oil at two hours was reduced to “weak” after four hours of exposure, whereas the M. piperita oil exhibited high repellency throughout the entire exposure time. In contrast, the M. spicata oil had no repellent effect on 3rd instar larvae of P. xylostella (Table 8). Page 14/25 Table 7 Activity feeding deterrent of essential oils from the Mentha on Plutella xylostella (Lepidoptera: Plutellidae) in laboratory, after 24 hours of exposure Essential Oils χ2 (df)a Slope ± SE DC50 (µL mL− 1) (IC 95%)c %FDI (LC30)d Classification Mentha arvensis 5.32 (6) 1.65 ± 0.13 3.02 (2.61–3.60) 63.75% Phagodeterrent Mentha spicata 2.83 (6) 1.19 ± 0.11 2.79 (2.03–4.20) 48% Phagodeterrent Mentha piperita 4.62 (5) 2.89 ± 0.18 1.08 (0.98–1.18) 95% Phagodeterrent aχ2 = Chi square (P > 0.05) and Degrees of freedom c DC50 = Concentrations causing 50% feeding deterrence of larvae population d FDI = feeding deterrence index subjected to LC30 of oils (Mentha arvensis LC30 = 5µL mL − 1; Mentha spicata LC30 = 2µL mL-1; Mentha piperita LC30 = 3.75µL mL-1) Table 7 Table 8 Repellent activity of essential oils from the Mentha on Plutella xylostella (L.) (Lepidoptera: Plutellidae) in laboratory Treatment Exposure time (hours) RI ± SE Category Mentha arvensis 1 0.40 ± 0.03 High repellency 2 0.80 ± 0.06 Weak repellency 4 0.80 ± 0.06 Weak repellency Mentha spicata 1 0.33 ± 0.03 High repellency 2 1.11 ± 0.09 No repellency 4 1.11 ± 0.09 No repellency Mentha piperita 1 0.40 ± 0.03 High repellency 2 0.40 ± 0.03 High repellency 4 0.20 ± 0.02 Very high repellency Table 8 Discussion Species of the genus Mentha have been widely investigated with regards to the chemical composition of the essential oils (EOs). The chemical profiles determined for M. spicata, M. arvensis and M. piperita are compatible with the results of previous investigations, which show that the EOs are rich in pulegone, menthone, menthol, carvone, 1,8-cineole, limonene and β-caryophyllene (Singh & Pandey, 2018). However, Page 15/25 Page 15/25 none of the EOs from the species of Mentha investigated here had 1,8-cineole or β-caryophyllene as major components. A sample of M. spicata collected in Tunisia had carvone (40.8%) and limonene (20.8%) as the major constituents of the EO (Snoussi et al., 2015). In the present study, a higher percentage of carvone (66.40%) and a similar concentration of limonene (19.16%) were found. However, another study involving of a sample collected in Brazil described carvone (0-60.1%) as well as two constituents reported as major constituents of the EO not found in the present investigation: pulegone (0–54.0%) and 1,8-cineole (2.04– 28.8%) (de Souza et al., 2015). The chemical profile for the EO of M. arvensis was compatible with that reported in the literature. In a sample collected in Austria, the authors found the same major constituents as those identified in the present investigation: menthol (33.5%), menthone (24.0%) and isomenthone (10.5%) (Koschiera et al., 2002). These same compounds were identified as the major constituents of the EO from two cultivars of M. arvensis in different stages of growth, with the exception of isomenthone, the proportion of which ranged from 0.11 to 0.47% (Verma et al., 2010). The chemical composition found for M. piperita is compatible with that described in most studies found in the literature, which report menthol and menthone as the major constituents of EOs from different sources: commercial, perfume industry as well as imported from India (Iscan et al., 2002), Algeria (Djenane et al., 2012) and Iran (Saharkhiz et al., 2012). However, Rezende et al. (2017) and Yadegarinia et al. (2006) found other chemotypes of M. piperita rich in carvone (84.34%)/limonene (10.97%) and α- terpinene (19.7%)/piperitinone oxide (19.3%) in samples from Brazil and Iran, respectively. The qualitative and quantitative differences between the Mentha oils demonstrated by the chemical analysis in the present study and results found in the literature for the same species from different places of origin may be attributed to genetic factors as well as pedoclimatic conditions (Figueiredo et al., 2008). Discussion The insecticidal properties of EOs from species of Mentha for lepidopterans have been widely investigated (Darabi & Khajehali, 2017; Boulamtat et al., 2020; Lo Pinto et al., 2020; Aissaoui et al., 2021). However, no previous studies have assessed the toxicity of Mentha oils through topical application on larvae of P. xylostella. With regards to toxicity by residual contact, Koundal et al. (2018) reported that the oils from M. piperita and M. spicata collected in India were more toxic to P. xylostella than the oils investigated in the present study, with an estimated LC50 of 1.37 and 1.86 mg mL− 1, respectively. Such results may be explained by qualitative and quantitative differences in the constituents of the oils from different places of origin. Different methods can be used to assess the toxicity of EOs. Ttoxicity of the Mentha oils to P. xylostella was investigated in the present study through different forms of contact, both of which are widely used for the assessment of the acute toxicity of a xenobiotic compound (Paramasivam & Selvi, 2017). Although the oils exhibited considerable toxicity with both forms of contact, toxicity was always greater Page 16/25 Page 16/25 Page 16/25 through topical contact than residual contact. With the topical contact test, the exact quantity of oil placed on the cuticle of the thoracic region of the larvae of P. xylostella was known (0.5 µL). The cuticle is the first barrier to the penetration of compounds. The outer layer – denominated the epicuticle – is composed mainly of hydrocarbons, proteins and lipids, most of which are higher fatty acids and fatty alcohols with a lipophilic nature (Balabanidou et al., 2018; Gullan, 2017). Due to their lipophilic nature, the Mentha oils dissolved the outermost components of the cuticle during the topical contact test, enabling greater contact with the body of the insect and, consequently, greater mortality compared to that achieved with the residual contact test. With residual contact, it is not possible to know the exact quantity of oil that comes into contact with the insect. In this test, the major form of contamination by the product is through the appendages (Balabanidou et al., 2018). Studies have shown that the susceptibility of insects is greater when exposure is oral or topical compared to exposure by residual contact (Lawson & McDermott, 2023). Among the oils tested, M. Discussion piperita oil exhibited the greatest toxicity by topical contact, followed by the M arvensis and M. spicata, for which toxicity was equal between the two oils. The opposite order was found for toxicity by residual contact. It is difficult to attribute these results of greater toxicity only to qualitative and/or quantitative differences in the constituents of the oils. It is necessary to consider possible synergic and antagonistic action resulting from the diverse interactions of the constituents that make up each oil (Moraes et al., 2012; Akhtar & Isman, 2012). The direct contact of EOs with the host plant can result in cellular dysfunction, promoting the formation of chlorosis, necrosis and leaf scorch (Werrie et al., 2020). It is therefore important to assess the degree of phytotoxicity of a plant derivative prior to its use in pest control. Few studies have investigated the phytotoxicity of EOs from species of Mentha (Cloyd et al., 2009). Moreover, the main focus of such studies is the ability of the oils to inhibit the germination of the plants and/or root development (Santana et al., 2014; Kimbaris et al., 2017). Vapors of the oil from Mentha pulegium caused dispersed necrotic spots and mild chlorosis on the leaves of cucumber plants at a concentration of 8 µ/L of air (Topuz et al., 2018). Although the study cited involved the EO from a plant of the same genus as the species used in the present investigation, differences in the chemical composition and type of experiment employed impede a direct comparison of the results of the two studies. Investigations of the phytotoxicity of other EOs to the same host plant as that used in the present study (B. olerareae) exhibited higher levels compared to the Mentha oils. Phytotoxicity of the Mentha oils was less than 14.61%, whereas EOs from Syzygium aromaticum, Melaleuca alternifolia and Eucalyptus globulus investigated by de Melo et al. (2023) had rates of 27.54, 27.49 and 36.80%, respectively. Thus, the EOs from M. piperita, M. spicata and M. arvensis can be used in the management of P. xylostella on B. oleraceae through spraying the leaves with the concentration used in the present study. Doses of EOs lower than the LD50 can have sublethal impacts on target insects, affecting development and longevity (Pavela et al., 2021). No previous studies have assessed the biological activities of the Page 17/25 Page 17/25 Mentha oils investigated here for P. xylostella. Discussion However, experiments addressing the effects of EOs on biological parameters of other lepidopterans have been conducted with Mentha oils. Doses of the EO from M. spicata reduced the number of eggs, larval period, weight of the pupae and longevity of adults of Alabama argillacea (Santos et al., 2021). These results differ from those found in the present study, in which the sublethal dose of the M. spicata oil only reduced the weight of the pupae and larval-adult survival in P. xylostella. This divergence may be explained by the difference in the doses uses, which were lower in the present study. Although the effect of sublethal doses of the EO from M. piperita on the reduction in fecundity was demonstrated in pests from the same order, such as Plodia interpunctella (Jesser et al., 2017), the Mentha oils in the present study did not alter this parameter. Among the oils investigated, those from M. arvensis and M. spicata are more promising, significantly reducing the larval survival of P. xylostella. This result is relevant to the control of P. xylostella, as the use of a formulation with Mentha oils at a sublethal dose can minimize the impact of the pest on the plant. The feeding deterrent properties of plant derivatives are of considerable importance, especially for chewing insects, such as lepidopterans. Despite various studies on the feeding deterrence of EOs from M. spicata, M. piperita and M. arvensis for stored-grain pests (Rajkumar et al., 2019) and agricultural pests (Valcárcel et al., 2021), studies assessing feeding deterrence for P. xylostella are rare. Investigating the EO from M. spicata collected in India, Koundal et al. (2018) used a concentration of 10 mg/mL to obtain an FDI of 52.29%. Comparing this result to that of the present investigation, the quantity of M. spicata oil used to obtain an FDI of 48% was five times lower in our experiment. The difference in deterrence between the commercial M. spicata oil used the present study and that collected from plants in India may be attributed to qualitative and quantitative differences in the major and minor constituents of the oils. Repellence is another important property to assess in EOs. Few studies have addressed the repellent action of the Mentha oils studied here on P. xylotella. Koundal et al. (2018) found that the M. piperita oil was more repellent than the M. spicata oil. Discussion The same order of repellence was found in the present study for these oils. The investigations of the biology and behavior of P. xylostella exposed to the Mentha oils demonstrated that these oils are capable of causing biological changes and influencing the occurrence of the pest in the environment. More in-depth investigations are needed on the use of these oils in the field for the management of P. xylostella. Declarations Funding: The authors are grateful to the Conselho Nacional de Desenvolvimento Científico e Tecnológico (PQ-2 306514/2022-2), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (Nº 88887.474239/2020-00) and Laboratório Multiusuário de Análises Químicas (LABMAQ - UFRPE). Author Contribution A.A.P.N. and T.T.B.L. acquired and analyzed the data under the guidance of C.A.G.C. and J.P.R.M.; M.M.M. analyzed and interpreted the chemistry of essential oils; C.A.G.C. and V.B.M. drafted and revised the manuscript critically for important intellectual content. All authors read and approved the final manuscript. A.A.P.N. and T.T.B.L. acquired and analyzed the data under the guidance of C.A.G.C. and J.P.R.M.; M.M.M. analyzed and interpreted the chemistry of essential oils; C.A.G.C. and V.B.M. drafted and revised the manuscript critically for important intellectual content. All authors read and approved the final manuscript. References 1. Adams, R.P. (2007). Identification of essential oil components by gas chromatography/ quadrupole mass spectroscopy. Allured Publishing Corporation. 1. Adams, R.P. (2007). Identification of essential oil components by gas chromatography/ quadrupole mass spectroscopy. Allured Publishing Corporation. 2. Ahmed, N., Alam, M., Saeed, M., Ullah, H., Iqbal, T., Al-Mutairi, K.A. & Salman, M. (2021). Botanical Insecticides Are a Non-Toxic Alternative to Conventional Pesticides in the Control of Insects and Pests. Global Decline of Insects. IntechOpen. 3. Aissaoui, F., Hedjal-Chebheb, M., Soltani, A., Haouel‑Hamdi, S. Talhi, O. Ziani, B.E.C., J.M.B. Jemâa. (2021). Variations of chemical composition of two Algerian essential oils collected for different seasons and assessment of their insecticidal toxicity against three moth pests. Journal of Plant Diseases and Protection, 128, 1167–1176. https://doi.org/10.1007/s41348-021-00491-6 4. Akhtar, Y. & Isman, M.B. (2013). Plant natural products for pest management: the magic of mixtures. In Ishaaha, I. et al., (Eds), Advanced Technologies for Managing Insect Pests, (pp. 231–247). Springer. 5. Akhtar, Y., Pages, E., Stevens, A., Bradbury, R., Camara, C.A.G. & Isman, M.B. (2012). Effect of chemical complexity of essential oils on feeding deterrence in larvae of the cabbage looper. Physiological Entomology, 37(1), 81–91. https://doi.org/10.1111/j.1365-3032.2011.00824.x 6. Araújo, M.J.C., da Camara, C.A.G., Moraes, M.M. & Born, F.S. (2020). Insecticidal properties and chemical composition of Piper aduncum L., Lippia sidoides Cham. and Schinus terebinthifolius Raddi essential oils against Plutella xylostella L. Anais da Academia Brasileira de Ciências, 92 (suppl 1), e20180895. https://doi.org/10.1590/0001-3765202020180895 7. Balabanidou, V., Grigoraki, L. & Vontas, J. (2018). Insect cuticle: a critical determinant of insecticide resistance. Current Opinion in Insect Science, 27, 68-74. https://doi.org/10.1016/j.cois.2018.03.001 7. Balabanidou, V., Grigoraki, L. & Vontas, J. (2018). Insect cuticle: a critical determinant of insecticide resistance. Current Opinion in Insect Science, 27, 68-74. https://doi.org/10.1016/j.cois.2018.03.001 8. Bossou, A. D., Ahoussi, E., Ruysbergh, E., Adams, A., Smagghe, G., De Kimpe, N., Avlessi, F., Sohounhloue, D.C.K. & Mangelinckx, S. (2015). Characterization of volatile compounds from three Cymbopogon species and Eucalyptus citriodora from Benin and their insecticidal activities against Tribolium castaneum. Industrial Crops and Products, 76, 306-317. https://doi.org/10.1016/j.indcrop.2015.06.031 9. Boulamtat, R., Lhaloui, S., Sabraoui, A., El-Fakhouri, K., Oubayoucef, A., Mesfioui, A. & El-Bouhssini, M. (2020). Antifeedant and larvicidal activities of Mentha pulegium on chickpea pod borer Helicoverpa armigera (Lepidoptera: Noctuidae). International Journal Tropical Insect Science, 40, 151–156. https://doi.org/10.1007/s42690-019-00064-z Page 19/25 10. Campos, E.V.R., Proença, P.L.F., Oliveira, J.L., Bakshi, M., Abhilash, P.C. & Fraceto, L.F. (2019). References Use of botanical insecticides for sustainable agriculture: Future perspectives. Ecological Indicators, 105, Page 19/25 483-495. https://doi.org/10.1016/j.ecolind.2018.04.038 483-495. https://doi.org/10.1016/j.ecolind.2018.04.038 11. Cloyd, R.A., Galle, C.L., Keith, S.R., Kalscheur, N.A. & Kemp, K.E. (2009). Effect of commercially available plant-derived essential oil products on arthropod pests. Journal Econnomic Entomology, 102(4), 1567-1579. https://doi.org/10.1603/029.102.0422 12. da Camara, C.A.G., do Nascimento A.F., Monteiro, V.B. & Moraes, M.M. (2022). Larvicidal, ovicidal and antifeedant activities of essential oils and constituents against Spodoptera frugiperda. Archives of Phytopathology and Plant Protection, 55 (7), 851-873. https://10.1080/03235408.2022.2048557 13. da Silva, L.R.R., Ferreira, O.O., Cruz, J.N., de Jesus P.F.C, dos Anjos, T.O., Cascaes, M.M., da Costa W.A., Andrade, E.H.A. & de Oliveira, M.S. (2021). Lamiaceae essential oils, phytochemical profile, antioxidant, and biological activities. Evidence-Based Complementary and Alternative Medicine, 14, 6748052. https://10.1155/2021/6748052 14. Darabi, K. & Khajehali, J. (2017). Bioactivity of Essential Oils of Mentha Species and Cuminum cyminum L. on Anarta trifolii (Hufnagel) (Lepidoptera: Noctuidae). Journal of Essential Oil Bearing Plants, 20 (4), 1097 - 1106, https://doi.org/10.1080/0972060X.2017.1358674 15. de Melo, J.P.R., da Câmara, C.A.G. & de Moraes, M.M. (2023). Bioactivity of formulas containing essential oils from the family Myrtaceae for the management of deltamethrin-resistant Plutella xylostella (L.) (Lepidoptera: Plutellidae). Phytoparasitica, 51, 305–321. https://doi.org/10.1007/s12600-022-01043-w 16. de Sousa Barros A, de Morais SM, Ferreira PAT, Vieira, I.G.P., Craveiro, A.A., Fontenelle, R.O.S, de Menezes, J.E.S.A., da Silva, F.W.F. & de Sousa, H.A. (2015). Chemical composition and functional properties of essential oils from Mentha species. Industrial Crops and Products, 76, 55 - 7564. https://doi.org/10.1016/j.indcrop.2015.07.004 17. Djenane, D., Aïder, M., Yangüela, J., Idir, L., Gomez, D. & Roncales, P. (2012). Antioxidant and antibacterial effects of Lavandula and Mentha essential oils in minced beef inoculated with E. coli O157:H7 and S. aureus during storage at abuse refrigeration temperature. Meat Science, 92 (4), 667– 674. https://doi.org/10.1016/j.meatsci.2012.06.019 18. El-Hefny, A.A. (2019). Sublethal effects of the milky latex of Sodom Apple, Calotropis procera (Alton) on the growth, development, and some physiological aspects of the greater wax moth, Galleria mellonella (L.) (Lepidoptera: Pyralidae). Journal of Plant Protection and Pathology, 10 (10), 491-496. https://10.21608/jppp.2019.63674 19. Ferreira, E.A., Faca, E.C., de Souza, S.A., Fioratti, C.A.G., Mauad, J.R.C., Cardoso, C.A.L., Mauad, M. & Mussury, R.M. (2022). Antifeeding and oviposition deterrent effect of Ludwigia spp. (Onagraceae) against Plutella xylostella (Lepidoptera: Plutellidae). Plants, 11(19), 2656. https://doi.org/10.3390/plants11192656. 20. Figueiredo, A.C., Barroso, J.G., Pedro, L.G., & Scheffer, J.J.C. (2008). Factors affecting secondary metabolite production in plants: Volatile components and essential oils. Flavour and Fragrance Journal, 23(4), 213–226. https://doi.org/10.1002/ffj.1875 Page 20/25 21. Finney, D.J. (1971). 483-495. https://doi.org/10.1016/j.ecolind.2018.04.038 Probit Analysis, A statistical Treatment of the Sigmoid Response Curve. University Press. 22. Gautam, M.P., Singh, H., Kumar, S., Kumar, V., Singh, G. & Singh, S.N. (2018). Diamondback moth, Plutella xylostella (Linnaeus) (Insecta: Lepidoptera: Plutellidae) a major insect of cabbage in India: A review. Journal of Entomology and Zoology Studies, 6 (4), 1394-1399. 23. Gullan, P.J. & Cranston, P.S. (2017). Insetos: Fundamentos da Entomologia. Roca. 24. Han, W., Zhang, S., Shen, F., Liu, M., Ren, C. & Gao, X. (2012). Residual toxicity and sublethal effects of chlorantraniliprole on Plutella xylostella (Lepidoptera: Plutellidae). Pest Management Science, 68(8), 1184–90. https://doi.org/10.1002/ps.3282 25. Hategekimana, A. & Erler, F. (2020). Fecundity and fertility inhibition effects of some plant essential oils and their major components against Acanthoscelides obtectus Say (Coleoptera: Bruchidae). Journal of Plant Diseases and Protection 127, 615–623. https://doi.org/10.1007/s41348-020-00311- 3 26. Iscan, G., Kirimer, N., Kurkcuoglu, M., Baser, K. H., & Demirci, F. (2002). Antimicrobial screening of Mentha piperita essential oils. Journal of Agricultural and Food Chemistry, 50 (14), 3943–3946. https://doi.org/10.1021/jf011476k 27. Jan, M.T., Abbas, N., Shad, S.A. & Saleem, M.A. (2015). Resistance to organophosphate, pyrethroid and biorational insecticides in populations of spotted bollworm, Earias vittella (Fabricius) (Lepidoptera: Noctuidae), in Pakistan. Crop Protection 78: 247–252. https://doi.org/10.1016/j.cropro.2015.09.020 28. Jankowska, M., Lapied, B., Jankowski, W. & Stankiewicz, M. (2019). The unusual action of essential oil component, menthol, in potentiating the effect of the carbamate insecticide, bendiocarb. Pesticide Biochemistry and Physiology, 158: 101-111. https://doi.org/10.1016/j.pestbp.2019.04.013 29. Jesser, E.N., Werdin-González, J.O., Murray, A.P. & Ferrero, A.A. (2017). Efficacy of essential oils to control the Indian meal moth, Plodia interpunctella (Hübner) (Lepidoptera: Pyralidae). Journal of Asia-Pacific Entomology,20 (4), 1122-1129. https://doi.org/10.1016/j.aspen.2017.08.004 30. Kimbaris, A.C., González-Coloma, A., Andrés, M.F., Vidali, V.P., Polissiou, M.G. & Santana-Méridas, O. (2017). Biocidal compounds from Mentha sp. essential oils and their structure-activity relationships. Chemistry & biodiversity, 14(3). https://doi.org/10.1002/cbdv.201600270 31. Koschiera, E. H., Sedya, K. A., & Novak, J. (2002). Influence of plant volatiles on feeding damage caused by the onion thrips Thrips tabaci. Crop Protection, 21 (5), 419–425. https://doi.org/10.1016/S0261-2194(01)00124-7 32. Koundal, R., Dolma, S.K., Chand, G., Agnihotri, V.K. & Reddy, S.G.E. (2018). Chemical composition and insecticidal properties of essential oils against diamondback moth (Plutella xylostella L.). Toxin Reviews, 39 (4), 1556-9551. https://doi.org/10.1080/15569543.2018.1536668 33. Kumar, P., Mishra, S., Malik, A. & Satya, S. (2014). Biocontrol potential of essential oil monoterpenes against housefly, Musca domestica (Diptera: Muscidae). Ecotoxicology Environmental Safety, 100, 1- 6. https://doi.org/10.1016/j.ecoenv.2013.11.013 Page 21/25 34. Lawson, B.E. 483-495. https://doi.org/10.1016/j.ecolind.2018.04.038 & McDermott, E.G. (2023). Topical, contact, and oral susceptibility of adult Culicoides biting midges (Diptera: Ceratopogonidae) to fluralaner. Parasites & Vectors, 16(1), 281. https://doi.org/10.1186/s13071-023-05899-7 35. Le Gall, M. & Behmer, S.T. (2014). Effects of protein and carbohydrate on an insect herbivore: the vista from a fitness landscape. Integrative and Comparative Biology, 54, (5), 942–954. https://doi.org/10.1093/icb/icu102 36. LeOra-Software. (2005). POLO-Plus, POLO for Windows computer program, version 2.0. LeOra- Software, Petaluma, CA. 37. Lo Pinto, M., Vella, L. & Agrò, A. (2020). Adulticidal activity of essential oils of Mentha piperita L., Cupressus sempervirens L., and Eucalyptus globulus Labill. against the tomato leafminer Tuta absoluta Meyrick (Lepidoptera: Gelechiidae). Journal of Entomology and Zoology Studies, 8 (6), 1721 – 1928. 38. Ma, S., Jia, R., Guo, M., Qin, K. & Zhang, L. (2020). Insecticidal activity of essential oil from Cephalotaxus sinensis and its main components against various agricultural pests. Industrial Crops and Products, 150, 112403, https://doi.org/10.1016/j.indcrop.2020.112403. 39. Mantzoukas, S., Ntoukas, A., Lagogiannis, I., Kalyvas, N., Eliopoulos, P. & Poulas, K. (2020). Larvicidal action of cannabidiol oil and neem oil against three stored product insect pests: effect on survival time and in progeny. Biology (Basel), 9(10): 321. https://10.3390/biology9100321 40. Mazzonetto, F. & Vendramim, J.D. (2003). Efeito de pós de origem vegetal sobre Acanthoscelides obtectus (Say) (Coleoptera: Bruchidae) em feijão armazenado. Neotropical Entomology, 32 (1), 145 – 149. 41. Mirhaghparast, S.K., Zibaee, A., Hajizadeh, J. & Ramzi, S. (2020). Toxicity and physiological effects of the tea seed saponin on Helicoverpa armigera. Biocatalysis and Agricultural Biotechnology, 25, 101597, https://doi.org/10.1016/j.bcab.2020.101597 42. Monchiero, M., Gullino, M. L., Pugliese, M., Spadaro, D., & Garibaldi, A. (2015). Efcacy of diferent chemical and biological products in the control of Pseudomonas syringae pv. Actinidiae on kiwifruit. Australasian Plant Pathology, 44(1), 13–23. https://doi.org/10.1007/s13313-014-0328-1 43. Moraes, M.M., da Camara, C.A.G., dos Santos, M.L. & Fagg, C.W. (2012). Essential oil composition of Eugenia langsdorffii O. Berg.: relationships between some terpenoids and toxicity against Tetranychus urticae. Journal of the Brazilian Chemical Society, 23 (9), 1647-1656. https://doi.org/10.1590/S0103-50532012005000029 44. Mota-Sanchez, D. & Wise, J.C. (2024). The Arthropod Pesticide Resistance Database. Michigan State University. Retrieved February, 22, 2024 from http://www.pesticideresistance.org 44. Mota-Sanchez, D. & Wise, J.C. (2024). The Arthropod Pesticide Resistance Database. Michigan State University. Retrieved February, 22, 2024 from http://www.pesticideresistance.org 45. Pang, X., Feng, Y.X., Qi, X.J., Wang, Y., Almaz, B., Xi, C., & Du, S.S. (2020). Toxicity and repellent activity of essential oil from Mentha piperita Linn. 483-495. https://doi.org/10.1016/j.ecolind.2018.04.038 leaves and its major monoterpenoids against three stored product insects. Environmental Science and Pollution Research, 27 (7), 7618-7627. https://doi.org/10.1007/s11356-019-07081-y 45. Pang, X., Feng, Y.X., Qi, X.J., Wang, Y., Almaz, B., Xi, C., & Du, S.S. (2020). Toxicity and repellent activity of essential oil from Mentha piperita Linn. leaves and its major monoterpenoids against three stored product insects. Environmental Science and Pollution Research, 27 (7), 7618-7627. https://doi.org/10.1007/s11356-019-07081-y Page 22/25 46. Paramasivam, M. & Selvi, C. (2017). Laboratory bioassay methods to assess theinsecticide toxicity against insect pests-A review. Journal of Entomology and Zoology Studies, 5, 1441-1445. 47. Pavela, R. & Benelli G. (2016). Essential Oils as Ecofriendly Biopesticides? Challenges and Constraints. Trends in Plant Science, 21 (12), 1000–1007. https://doi.org/10.1016/j.tplants.2016.10.005 48. Pavela, R., Maggi, F., Mazzara, E., Torresi, J., Cianfaglione, K., Benelli, G. & Canale, A. (2021). Prolonged sublethal effects of essential oils from non-wood parts of nine conifers on key insect pests and vectors. Industrial Crops and Products, 168, 113590, https://doi.org/10.1016/j.indcrop.2021.113590 49. Rajkumar, V., Gunasekaran, C., Christy, I.K., Dharmaraj, J., Chinnaraj, P. & Paul, C.A. (2019). Toxicity, antifeedant and biochemical efficacy of Mentha piperita L. essential oil and their major constituents against stored grain pest. Pesticide Biochemistry Physiology. 156, 138-144. https://doi.org/10.1016/j.pestbp.2019.02.016 50. Reddy, S.G.E., Dolma, S.K., Koundal, R. & Singh, B. (2015). Chemical composition and insecticidal activities of essential oils against diamondback moth, Plutella xylostella (Lepidoptera: Yponomeutidae). Natural Product Research, 30 (16), 1834 - 1838. 51. Rezende, D. A. C. S., Souza, R. V., Magalhães, M. L., Caetano, A. R. S., Carvalho,M. S. S., de Souza, E. C., Guimarães, L.G.L., Nelson, D.L., Batista, L.R. & Cardoso, M.G. (2017). Characterization of the biological potential of the essential oils from five species of medicinal plants. American Journal of Plant Sciences. 8 (2), 154–170. https://doi.org/10.4236/ajps.2017.82012 52. Robertson, J.L., Russell, R.M., Preisler, H.K. & Savin, N.E. (2007). Bioassays with Arthropods. CRC Press. 53. Saharkhiz, M. J., Motamedi, M., Zomorodian, K., Pakshir, K., Miri, R., & Hemyari, K. (2012). Chemical composition, antifungal and antibiofilm activities of the essential oil of Mentha piperita L. ISRN Pharmaceutics. 2012, 718645. https://doi.org/10.5402/2012/718645 54. Santana, M.L.G., Melo, J.P.R., da Camara, C.A.G., Moraes, M.M., Araujo, C.A., Vasconcelos, G.J.N., Pereira, M.R.S. & Zartman, C.E. (2022). Lethal and sublethal effects of essential oils from Piper capitarianum Yunck and Piper krukoffii Yunck on Plutella xylostella. Anais da Academia Brasileira de Ciências, 94 (2), e20200072. https://doi.org/10.1590/0001-3765202220200072 55. 483-495. https://doi.org/10.1016/j.ecolind.2018.04.038 Santana, O., Fe Andrés, M., Sanz, J., Errahmani, N., Abdeslam, L. & González-Coloma A. (2014). Valorization of essential oils from Moroccan aromatic plants. Natural Product Communication, 9(8), 1109-1114. 56. Santos, A.A., Wanderley-Teixeira, V., Cruz, G.S., Dutra K.A., Navarro, D.M.A.F., de Oliveira, J.V., Lapa- Neto, C.J.C., Barbosa, D.R.S. & Teixeira, Á.A.C. (2021). Essential oil toxicity on biological and reproductive parameters of Alabama argillacea (Hübner) (Lepidoptera: Erebidae). Acta Histochemica, 123(4), 151714. https://doi.org/10.1016/j.acthis.2021.151714 57. Sarfraz, M., Dosdall, L.M. & Keddie, B.A. (2006). Diamondback moth–host plant interactions: implications for pest management. Crop Protection, 25 (7), 625– 639. Page 23/25 Page 23/25 58. SAS Institute. (2008). SAS/STAT User’s guide, version 8.02, TS level 2 MO. SAS Institute Inc., Cary, North Carolina. 59. Singh, P. & Pandey, A.K. (2018). Prospective of essential oils of the genus Mentha as biopesticides: a review. Frontiers in Plant Science, 9: 1295. https://10.3389/fpls.2018.01295 60. Snoussi, M., Noumi, E., Trabelsi, N., Flamini, G., Papetti, A. & De Feo, V. (2015). Mentha spicata essential oil: chemical composition, antioxidant and antibacterial activities against planktonic and biofilm cultures of Vibrio spp. strains. Molecules, 2 (8), 14402–14424. https:// doi.org/10.3390/molecules200814402 61. Song, C., Zhao, J., Zheng, R., Chi Hao & Yan, X. (2022). Chemical composition and bioactivities of thirteen non-host plant essential oils against Plutella xylostella L. (Lepidoptera: Plutellidae). Journal of Asia-Pacific Entomology, 25 (2), 101881. https://doi.org/10.1016/j.aspen.2022.101881 61. Song, C., Zhao, J., Zheng, R., Chi Hao & Yan, X. (2022). Chemical composition and bioactivities of thirteen non-host plant essential oils against Plutella xylostella L. (Lepidoptera: Plutellidae). Journal of Asia-Pacific Entomology, 25 (2), 101881. https://doi.org/10.1016/j.aspen.2022.101881 62. Stepanycheva, E., Petrova, M., Chermenskaya, T. & Pavela, R. (2019). Fumigant effect of essential oils on mortality and fertility of thrips Frankliniella occidentalis Perg. Environmental Science and Pollution Research, 26 (30): 30885-30892. https://10.1007/s11356-019-06239-y 62. Stepanycheva, E., Petrova, M., Chermenskaya, T. & Pavela, R. (2019). Fumigant effect of essential oils on mortality and fertility of thrips Frankliniella occidentalis Perg. Environmental Science and Pollution Research, 26 (30): 30885-30892. https://10.1007/s11356-019-06239-y 63. Tahri, D., Elhouiti, F., Chelghoum, M., Nebeg, H., Ouinten, M., & Yousfi, M. (2022). Biosynthesis and biological activities of carvone and carvotana cetone Derivatives. Revista Brasileira de Farmacognosia, 32(5), 708-723. https://doi.org/10.1007/s43450-022-00302-5 63. Tahri, D., Elhouiti, F., Chelghoum, M., Nebeg, H., Ouinten, M., & Yousfi, M. (2022). Biosynthesis and biological activities of carvone and carvotana cetone Derivatives. Revista Brasileira de Farmacognosia, 32(5), 708-723. https://doi.org/10.1007/s43450-022-00302-5 64. Topuz, E., Madanlar, N. & Erler, F. (2018). 483-495. https://doi.org/10.1016/j.ecolind.2018.04.038 Chemical composition, toxic and developmentand reproduction-inhibiting effects of some essential oils against Tetranychus urticae Koch (Acarina: Tetranychidae) as fumigants. Journal of Plant Diseases and Protection, 125, 377–387. https://doi.org/10.1007/s41348-018-0161-9 65. Torres, A.L., Boiça Júnior, A.L., Medeiros, C.A.M. & Barros, R. (2006). Efeito de extratos aquosos de Azadirachta indica, Melia azedarach e Aspidosperma pyrifolium no desenvolvimento e oviposição de Plutella xylostella. Bragantia, 65 (3): 447-457. https://doi.org/10.1590/S0006-87052006000300011 65. Torres, A.L., Boiça Júnior, A.L., Medeiros, C.A.M. & Barros, R. (2006). Efeito de extratos aquosos de Azadirachta indica, Melia azedarach e Aspidosperma pyrifolium no desenvolvimento e oviposição de Plutella xylostella. Bragantia, 65 (3): 447-457. https://doi.org/10.1590/S0006-87052006000300011 66. Valcárcel, F., Olmeda, A. S., González, M. G., Andrés, M. F., Rocha, J. N., & González-Coloma⊥, A. (2021). Acaricidal and insect antifeedant effects of essential oils from selected aromatic plants and their main components. Frontiers in Agronomy, 3, 1 - 12. https://doi.org/10.3389/fagro.2021.662802 67. Van den Dool, H. & Kratz, P.H. (1963). A Generalization of the retention index system including linear temperature programmed gas—liquid partition chromatography. Journal of Chromatography, 11, 463-471. 68. Verma, R. S., Rahman, L., Verma, R. K., Chauhan, A., Yadav, A. K., & Singh, A. (2010). Essential oil composition of menthol mint (Mentha arvensis) and peppermint (Mentha piperita) cultivars at different stages of plant growth from Kumaon region of Western Himalaya. Open acess Journal of Medicinal and Aromatic Plants, 1(1), 13-18. 69. Wei, H., Liu, J., Li, B., Zhan, Z., Chen, Y., Tian, H., Lin S. & Gu, X. (2015). The toxicity and physiological effect of essential oil from Chenopodium ambrosioides against the diamondback moth, Plutella xylostella (Lepidoptera: Plutellidae). Crop Protection, 76: 68-74. https://doi.org/10.1016/j.cropro.2015.06.013 Page 24/25 70. Werrie, P.Y., Durenne, B., Delaplace, P. & Fauconnier, M.L. (2020). Phytotoxicity of essential oils: opportunities and constraints for the development of biopesticides. A review. Foods, 9(9), 1291. https://doi.org/10.3390/foods9091291 71. Yadegarinia, D., Gachkar, L., Rezaei, M. B., Taghizadeh, M., Astaneh, S. A. & Rasooli, I. (2006). Biochemical activities of Iranian Mentha piperita L. and Myrtus communis L. essential oils. Phytochemistry, 67 (2), 1249–1255. https://doi.org/10.1016/j.phytochem.2006.04.025 72. Yıldırım, E., Emsen, B., & Kordalı, S. (2013). Insecticidal effects of monoterpenes on Sitophilus zeamais Motschulsky (Coleoptera: Curculionidae). Journal of Applied Botany and Food Quality, 86 (1), 198 – 204, https://10.5073/JABFQ.2013.086.027 73. Zalucki, M.P., Shabbir, A., Silva, R., Adamson, D., Shu-Sheng, L., & Furlong, M.J. (2012). 483-495. https://doi.org/10.1016/j.ecolind.2018.04.038 Estimating the economic cost of one of the world’s major insect pests, Plutella xylostella (Lepidoptera: Plutellidae): Just how long is a piece of string? Journal of Economic Entomology, 105 (4), 1115–1129. https://doi.org/10.1603/EC12107 Page 25/25
https://openalex.org/W2940311266	https://iconline.ipleiria.pt/bitstream/10400.8/8246/1/19%20-%20Towards%20a%20Conceptual%20Notion%20for%20a%20Universal%20Printing%20Machine.pdf	English	null	Towards a Conceptual Notion for a Universal Printing Machine	Applied mechanics and materials	2,019	cc-by	5,090	Introduction Our main goal is to establish a conceptual setting in which the theory of 3D printing can be developed. As far as we know, there has never been made any attempt to achieve such a goal. The results in [1] are somehow related but not in the same direction. The need for such a theory and frame work has been slowly developed in our minds during the last 15 years. In simple terms the idea of 3d printing is not difficult to pose, see for example [2]. The idea of a Universal Printing Machine on the other hand is not so easy to explain. For that reason we will use an analogy which was in fact our inpiration. The analogy is with the theoretical concept of a Turing Machine (see for example [3] and references therein). A Turing Machine can be thought of as consisting of a tape, with cells, in which there are written symbols, and a read-write head that moves along the tape. At each time the head can read the tape, overwrite it, and move to the left or to the right. It also has a collection of inner states which may change and determine different behaviours. This is an intuitive interpretation of the concept. Formally, from a purely mathematical point of view, it consists of five sets and a transition map, generalizing thus the notion of an automaton. This conceptualization is the base of all the theory of computation because everything that can be computed is computed by a Turing Machine (see for example the chapter on computability in [4], p. 262–265). We have identified the need for a similar conceptualization of 3D printing. We aim at achieving a theoretical model for a 3D printer. The model will be based on the notion of multilink, see [5]. A multilink is a mathematical structure which arose as a solution to encode many aspects of 3D printing. In particular, some specific algorithms that are fundamental for 3D printing, such as slicing, scanning, offsetting, segmentation, are suitably described using some special cases of multilinks (see [5] for some concrete examples). Other parts of the model shall include a collection of base materials that can be printed, and a collection of assembling strategies. These requirements are yet to be better understood and they will be useful in specifying the theoretical model. Keywords: Universal Printing Machine, Multi-link, Universal Tool-path Trajectory, Gradient of Mate- rials. Abstract. The idea of Universal Printing Machine is advanced. It is designed to serve as a theoretical model for 3D printing. Suitable mathematical structures are proposed as a framework for the Universal Printer. Applied Mechanics and Materials ISSN: 1662-7482, Vol. 890, pp 61-69 doi:10.4028/www.scientific.net/AMM.890.61 © 2019 The Author(s). Published by Trans Tech Publications Ltd, Switzerland. Applied Mechanics and Materials ISSN: 1662-7482, Vol. 890, pp 61-69 doi:10.4028/www.scientific.net/AMM.890.61 © 2019 The Author(s). Published by Trans Tech Publications Ltd, Switzerland. Applied Mechanics and Materials ISSN: 1662-7482, Vol. 890, pp 61-69 doi:10.4028/www.scientific.net/AMM.890.61 © 2019 The Author(s). Published by Trans Tech Publications Ltd, Switzerland. Submitted: 2017-11-13 Revised: 2018-06-18 Accepted: 2018-06-18 Online: 2019-04-09 Submitted: 2017-11-13 Revised: 2018-06-18 Accepted: 2018-06-18 Online: 2019-04-09 Applied Mechanics and Materials ISSN: 1662-7482, Vol. 890, pp 61-69 doi:10.4028/www.scientific.net/AMM.890.61 © 2019 The Author(s). Published by Trans Tech Publications Ltd, Switzerland. This article is an open access article under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0) State-of-the-Art and our Particular Motivation New printing processes and techniques are being developed almost everyday; there is a clear need for standardisation. Standardization is the focus of this article. First of all, the results of such an ambitious project, will only be fully understood and possibly measured several years from now when there has been widespread adoption of the standard. To achieve this standardisation we propose the development of a theoretical model for an ideal printing machine which will consist of a suitably refinement of the general abstract notion of a multilink (see Appendix). The concept of a universal printing machine is not intended to be a real printer. Rather it aims at a conceptualization of what it means to print a physical object, as well as to handle all the information that is necessary to achieve that goal. We have always looked for a conceptual and mathematical setting which would provide a clear, simple and organized way of thinking and reasoning about the practical problems that arise. As new methods, technologies, strategies, processing tools and materials evolve, our view has evolved and it is still evolving. So, whenever it was possible, we attempted to anticipate the future and to be ready for new challenges. We illustrate this evolution with the notion of triangulation. g g The idea of 3D printing assumes the existence of an oriented closed surface whose interior de- scribes a physically realisable object. This object is then printed layer by layer in an additive manner which in the first cases was by stereolithography (see e.g. [6]). For that reason, the file that is used to encode the information describing the closed and oriented surface is called an STL format file. An STL file a disjointed collection of arbitrary triangles in euclidean 3D-space (and as such some careful is needed to ensure that they describe a closed and oriented surface). The orientation of each individual triangle is specified by its normal vector. A first trivial observation is that this normal vector is not necessary. Indeed, the direction can be computed at the point of use with the so called right hand rule and the order in which the three vectors of a triangle are stored in the file. Introduction complexity of algorithms and debugging (with unification of concepts into a single theory, the complexity of algorithms can be split into simpler components thus facilitating the process of debugging) 6. complexity of algorithms and debugging (with unification of concepts into a single theory, the complexity of algorithms can be split into simpler components thus facilitating the process of debugging) These challenges have led us to identify the need for a standardization of the whole process of 3D printing which we have called the Universal Printing Machine. Introduction Indeed, the notion of a multilink (see Appendix) is simply a mathematical structure and it requires the identification of certain restrictions and specifications that allow a concrete interpretation. In proceeding in this way we have several advantages over the established knowledge on 3D print- ing, thus overpassing some of the main difficulties which occur when not having a good mathematical model, namely: 1. redundancy of data (STL files are still used as a standard, in spite of all the attempts that have been made during the last three decades to establish a new one) 1. redundancy of data (STL files are still used as a standard, in spite of all the attempts that have been made during the last three decades to establish a new one) 2. data not being efficiently encoded (the notion of multilink and an appropriate file format to encode it has the potential to solve both the problem of efficiently encoding information and defining a standards for its exchange) Direct Digital Manufacturing and Polymers 62 3. dependency from one machine to another (a theoretical notion of a Universal Printing Machine is by definition independent of any particular printing machine, and moreover it can be used to simulate any particular concrete printing machine) 3. dependency from one machine to another (a theoretical notion of a Universal Printing Machine is by definition independent of any particular printing machine, and moreover it can be used to simulate any particular concrete printing machine) 4. ad hoc algorithms (the notion of multilink, [5], see also the Appendix, can be used not only to encode information but it also has the potential to unify the algorithms that are needed in handling and transforming information) 4. ad hoc algorithms (the notion of multilink, [5], see also the Appendix, can be used not only to encode information but it also has the potential to unify the algorithms that are needed in handling and transforming information) 5. not a clear distinction between geometry, logic, topology and functional data (in the sense that when modelling a physical object to be printed, a designer should take into account that it is not merely a geometrical entity with some physical properties but rather something that will be produced in a printing machine) 6. What can be Printed? In this section we give some historical notes on the evolution of the notion of a printer. This will be useful in developing the concept of a Universal Printer Machine, since one of its goals is to be able to print everything which can be printed. Hence, the need to define what do we mean when we use the expression everything that can be printed. Let us then take a quick look at the process of printing, from Gutenberg Bible to the printing of a human heart [1, 2, 7]. The printing process has started with Gutenberg in 1450s where a bible was printed by the first time using mass-produced movable metal type in Europe. It marked the start of the ”Gutenberg Revolution” (). Before that, everything was made by craft, using human hands, in which each single piece was made individually. Nowadays the term 3D Printing has come into our daily life. Nevertheless, there are still some details that are stopping the technology from evolving into the direction of printing human organs such as a heart. This has mainly to do with the complexity of the organ. Let us suppose that there are no limits in technology and that we will, eventually, be able to print whatever we can imagine and model. We can only print something if we can design a specification for it. In practice there are always some limits to what a conventional printer can produce. This is encompassed with the way the printing process is designed and the way the information to be printed can be encoded. There will always be some limits to what we are able to print at each individual instance by a specific machine. Even if we assume that there are no technological limits. The truth is that we need to first make some specifications and only then state what the specific system is capable of doing. Different systems with different specifications will be able to do different things. We can use the concept of a universal machine, due to Alan Turing [11], base of what can be actually computed by a mathematical function. It turns out that even at that purely abstract level there are some limits to what can be computed and what cannot be computed, see for example [4], p. 264. State-of-the-Art and our Particular Motivation Another simple observation is that instead of considering a family of disjointed triangles it would be more efficient to consider a collection of unique vertices over which the collection of triangles would be incident to, thus describ- ing a triangle as a triple of pointers indicating the positions of the vertices rather than its geometrical coordinates. Thus avoiding the redundancy originated by the fact that a vertex is always incident to more than one triangle. Over the years, while trying to keep pace with the development of new technologies for 3D print- ing, we have also been adapting old systems and computing tools to build more robust and more effi- cient algorithms. The new methods and algorithms would arise by taking advantage from the progress Applied Mechanics and Materials Vol. 890 63 in scientific computing which was also taking place. For example, the need for the redundant nor- mal vectors in the earlier systems was not because researchers were not aware of the fact that the order would determine the direction. It was rather due to computing limitations, indeed it was a time consuming task to compute the normal vectors. As new systems and new technologies arise and are developed, we also need to develop clearer and simpler theoretical structures, which are more appro- priate models for the real world. Martins-Ferreira [5, 8] has described the passage from the concept of a triangulation into the more sophisticated one of double link. Martins-Ferreira et al [6, 10] has shown the evolution and the development of the methods and algorithms that we see as a key part of the standardisation for 3D printing. The Universal Printing Machine, will simulate any 3D printer in existence or to be invented in the future. It will be capable of printing everything that can be printed. It will have the advantage of defining standards and allowing developers not to be concerned with the current limitations of actual printers, but rather be imagining what will be printed in the future, as new printing strategies and technologies arise. We expect this to stimulate the development of new printers and printing processes, in order to address the reality of the theoretical possibilities of virtual printing in a universal way. State-of-the-Art and our Particular Motivation The notion of multilink [5], briefly recalled in the Appendix, seems to have the potential to become a generic mathematical structure to be used as a standardization for the whole process involved in 3D printing. Material systematization Determine which kind of materials can be printed, produce a catalogue with characteristics and properties. This should be combined with the structure of the Universal Printing Machine. Algorithms and computing Algorithms and more efficient and robust methods of storing the information need to be devel- oped. Algorithms and more efficient and robust methods of storing the information need to be devel- oped. What can be Printed? Similarly, in the printing domain, we may ask the same question: what is printable and what is not printable. We are interested in printing only the objects that can be defined. To do so we have to specify a collection of attributes (such as shape, material, assembly instructions) prior to the printing of the objects. Direct Digital Manufacturing and Polymers 64 Printing strategies and physical methods Explore all the possible ways for physically printing a 3D region from space using any methods which are suitable of being technically implemented. These results have to be combined with the structure of a Universal Printing Machine. The concept of an Universal Printing Machine is obtained by combining all three levels: materials; printing stratgies; storing of information and algorithms; that can be used as a future reference in the area of additive manufacturing and 3D-printing. If we can specify 3D objects following the new mathematical structures described in [5] this will have a direct impact on designers who will specify an object which is independent from context. This has clear advantages as it does not require new skills every time the technology evolves. Manifold Printing on Manifolds When we print a logo on a cup, we are printing on a surface but using 3D technology for moving the print head. This can be generalised to the printing design of any part on a given manifold into another manifold. Clearly we need a language in order to specify all the concepts that are suitable to be printed as well as all the technical issues that need to be addressed in order to achieve that goal. The problem can be addressed from a Mathematical perspective, in the same way as, in a certain sense, it has been done so far for 2D printing. Indeed, the kerning of some characters such as the letter f provides a simple an example of how complex can be the process of printing, see for example [7]. There are two main possible approaches that can be used as a model to encode the printing process in general. A continuous (or vectorized) approach which allows for arbitrary precision, and a discrete approach, allowing a fixed pre-assigned precision, which is directly related with the printer resolution, varying form one machine to another. A discrete theoretical frame work was develpped with the pur- pose of modelling data acquisition (e.g. MRI images), data manipulation (e.g. extracting iso-surfaces), data processing (e.g. generation of toolpath trajectories) and data realisation (e.g. physically manufac- turing). The model comprises a mathematical structure based on the notion of multi-link and uses a cubical-voxelised approach, see [5, 9]. For the moment, let us observe that there are three main levels where the concept of a Universal Printing Machine has to be developed. The level of computing and algorithms, the level of material systematization and the level of real printing strategies and technological processes. Conclusion The details of an Universal Printing Machine will be developed in future work. Here we have tried to identify a pathway forward. The mathematical structure of cubic link show that there is a great potential for that particular structure to play an important role on the future development of the concept of a Universal Printing Machine. Applied Mechanics and Materials Vol. 890 65 Appendix: Mathematical structures to be used in defining the notion of a Universal Printing Machine Appendix: Mathematical structures to be used in defining the notion of a Universal Printing Machine Here we collect some mathematical structures that have been developed with the purpose of achieving the desired concept of a Universal Printing Machine. The general structure of a multi-link Here we recall the definition of multi-link. This is a new mathematical structure which has many applications such as a geometric model of a surface, a slicing algorithm, a scanning trajectory path [5]. A multi-link is a set A, together with a collection of endomaps (generally isomorphisms) αi, a collection of projection maps (usually surjective) pj, and a geometrical realization map g into a vector space (usually a geometrical algebra, such as the Cayley algebras of the real number, complex numbers, quaternions or octonions) A pj αi 8 g / Rn Bj with i = 1, 2, . . . and k = 1, 2, . . . These maps are often subject to compatibility conditions, accordingly to the interpretation that is given to the elements in A. A concrete example of a multi-link of dimension 2 is what we called a double-link. It can be used to define any surface and in particular its relation to a triangulated surface is explained in [8]. A Double- link is a mathematical structure of sets and maps (forming a particular instance of a multi-link) as displayed A v A A A A A A A A f α,β 8 g / R4 F V such that αβαβ = 1A βαβα = 1A fα = f vβ = v such that αβαβ = 1A βαβα = 1A fα = f vβ = v αβαβ = 1A βαβα = 1A fα = f vβ = v vβ = v The simplest example is the case of the tetrahedron which is illustrated in the diagram below. Further details can be found in [12]. 1. A structural curve Appendix: Mathematical structures to be used in defining the notion of a Universal Printing Machine a1 β α a2 β // α a3 β ^>>>> α a4 β >>>> α O a5 β o α 'P P P P P P P P a6 β α 7n n n n n n n n a7 β @α wooooooo a8 β ~~~~~ α [88888888888888 a9 β / α 4 a10 β `@@@@ α / a11 β / α gOOOOOOO a12 β `@@@@ α g a1 β α a2 β // α a3 β ^>>>> α a4 β >>>> α O a5 β o α 'P P P P P P P P a6 β α 7n n n n n n n n a7 β @α wooooooo a8 β ~~~~~ α [88888888888888 a9 β / α 4 a10 β `@@@@ α / a11 β / α gOOOOOOO a12 β `@@@@ α g Direct Digital Manufacturing and Polymers 66 A Square-link is another instance of a multi-link. It is a simpler version of a surface in the sense that every face has to be a quadrilateral face and this forces the surface to be mapped into a patched square array organized by lines and columns. For example, in the pictures displayed below, the lines in the patch are determined by the direction of the indexing map β, while the columns in the patch are controlled by the indexing map α. A square link is a structure A g / α,β 8 H ≃R4 A g / α,β 8 H ≃R4 such that αβ = βα. such that αβ = βα. It is interpreted as a patch of oriented squares each square is indexed by and element x in A and identified with its origin vertex g(x), as illustrated β(x) / α(β(x)) x O / α(x) O g(β(x)) / g(α(β(x))) g(x) O / g(α(x)) O topology geometry topology geometry topology The notion of a square-link is easily generalized into the one of a cubic-link and it can further be considered into n-dimensions. A cubic link is a structure A g / α,β,γ 8 H ≃R4 such that αβ = βα, αγ = γα and γβ = βγ. such that αβ = βα, αγ = γα and γβ = βγ. β β γ γ γβ βγ It may be interpreted as illustrated below. γ(x) β / O γ α {wwwwwwwww βγ(x) O γ α zttttttttt αγ(x) β / O γ αβγ(x) O γ x β / α {wwwwwwwwww β(x) α zttttttttt α(x) β / αβ(x) We now give some more specific details on how to encode the modelling of an extrusion based universal printer. γ(x) β / O γ α {wwwwwwwww βγ(x) O γ α zttttttttt αγ(x) β / O γ αβγ(x) O γ x β / α {wwwwwwwwww β(x) α zttttttttt α(x) β / αβ(x) We now give some more specific details on how to encode the modelling of an extrusion based universal printer. We now give some more specific details on how to encode the modelling of an extrusion based universal printer. Brief description of a model for a Universal Printing Machine based on a multi-material variable extrusion head with several degrees of freedom Brief description of a model for a Universal Printing Machine based on a multi-material variable extrusion head with several degrees of freedom In this subsection we will give some details on how to encode the shape and position for a filament based extrusion on a Universal Printer Machine. The theoretical model needed to simulate the result of printing a multi-material, variable extrusion head filament is detailed in the list below: r, s: W →W; p: W →R3 r, s: W →W; p: W →R3 Applied Mechanics and Materials Vol. 890 Applied Mechanics and Materials Vol. 890 Applied Mechanics and Materials Vol. 890 67 where W is a set of linear indexes, r and s are the successor and reverse successor maps, while p is the geometric realization map. This means that if w ∈W is an index, then p(w) is a point in the curve while p(r(w)) is the previous point in the curve and p(s(w)) is the following point along the curve, as illustrated · · · / pr2(w) / pr(w) / p(w) / ps(w) / ps2(w) / · · · 2. A family of cross-sectioned curves with pre-established offsets, 2. A family of cross-sectioned curves with pre-established offsets, 2. A family of cross-sectioned curves with pre-established offsets, (βi, γi): Y × Z →Y × Z; gi : Y × Z →C, i ∈I interpreted in the following way: gi(y, z) is a point on the cross-sectioned curve i ∈I, with main index y ∈Y and offset index z ∈Z, as illustrated interpreted in the following way: gi(y, z) is a point on the cross-sectioned curve i ∈I, with main index y ∈Y and offset index z ∈Z, as illustrated gi(y, γi(y, z)) / gi(βi(y, z), γi(y, z)) gi(y, z) / O gi(βi(y, z), z) O This means that if, for a given i ∈I, we fix an offset index z ∈Z we obtain a contour curve which is interpreted as a cross-section of the filament path that is being extruded along the structural curve with direction given as in the following item. 3. A directional (or displacement) curve 3. A directional (or displacement) curve α: X →X; (d, m): X →(C × S2) × I with the following interpretation. The element d(x) ∈(C×S2) is a pair, consisting of a complex number and an element on the unit sphere. Brief description of a model for a Universal Printing Machine based on a multi-material variable extrusion head with several degrees of freedom The complex number defines a rotation angle and a scaling factor for each contour curve on the index patch of cross-sections, while the element in the unit sphere (for example given in spherical coordinates in the form of azimuth and elevation, see [13]) determines the direction orthogonal to the plane containing the indexed curve m(x) ∈ I. 4. An anchoring map 1. The set of linear indexes A is the linearisation of the cartesian product X × Y × Z; 3. The map f has the form 3. The map f has the form f(x, y, z) = P(x) + d(x) · gm(x)(y, z) + R(x, y, z) with with with the action d(x) · gm(x)(y, z) is the one described in [13] and it has the effect of rotating the 3D- space in such a way that the direction orthogonal to the planar curve with index m(x), which is the curve whose points are gm(x)(y,z), is parallel to the direction pointed by d(x). And finally, R(x, y, z) ∈[0, 1]3 is a perturbation value which places the point f(x, y, z) in the interior of the cube defined by the transition maps a, b, c. More specifically, if we let h(x, y, z) = P(x) + d(x) · gm(x)(y, z) and if R(x, y, z) = (R1(x, y, z), R2(x, y, z), R3(x, y, z)) ∈[0, 1]3 then, the desired geometric realization map for the cubic link, f(x, y, z), is obtained by putting f(x, y, z) = h(x, y, z) + 3 ∑ j=1 Rj(x, y, z) ∥hj(x, y, z)∥hj(x, y, z) with h1(x, y, z) = h(a(x, y, z)) −h(x, y, z) h2(x, y, z) = h(b(x, y, z)) −h(x, y, z) h3(x, y, z) = h(c(x, y, z)) −h(x, y, z) h1(x, y, z) = h(a(x, y, z)) −h(x, y, z) h2(x, y, z) = h(b(x, y, z)) −h(x, y, z) h3(x, y, z) = h(c(x, y, z)) −h(x, y, z) In addition, by simply choosing a subset of the indexes in the cubic-link, say S ⊆A, and restricting the maps a, b, c to it (in the sense that if (x, y, z) ∈S and a(x, y, z) ̸∈S, then we put a(x, y, z) = (x, y, z)) we obtain the equivalent to a discretization for the continuous process initially designed. 4. An anchoring map (l, n): X →W×] −1, 1[ which anchors each indexed curve m(x) into a point along the structural curve on the space, with the interpretation that if n(x) = 0 then it is placed exactly at position p(l(x)) ∈R4 with p the realization map of item 1. If n(x) > 0 then the curve indexed by m(x) ∈I is anchored at the point which is linearly interpolated with value n(x) ∈]0, 1[ between p(l(x)) and p(s(l(x))). In a similar way, if n(x) < 0, then the curve indexed by m(x) ∈I it is placed at the point which is linearly interpolated between with value −n(x) ∈]0, 1[ between p(l(x)) and p(r(l(x))). All said we are now in position to describe the dynamical process of running the x ∈X indexes as the positions where a cross-section is defined, with the shape of the curve (gi, βi, γi), with i = m(x), at position p(l(x)) ± n(x) with orthogonal direction d(x). This defines a cubic link, a, b, c: A →A; f: A →R3 1. The set of linear indexes A is the linearisation of the cartesian product X × Y × Z; 1. The set of linear indexes A is the linearisation of the cartesian product X × Y × Z; Direct Digital Manufacturing and Polymers 68 2. the maps a, b, c are determined as a(x, y, z) = (α(x), y, z) b(x, y, z) = (x, β(y, z), z) c(x, y, z) = (x, y, γ(y, z)) a(x, y, z) = (α(x), y, z) b(x, y, z) = (x, β(y, z), z) c(x, y, z) = (x, y, γ(y, z)) 3. The map f has the form Acknowledgement The Authors are grateful to Saba Abdulghani Oliveira da Silva for enlightening discussions on the subject. This work is supported by the Fundação para a Ciência e a Tecnologia (FCT) and Centro2020 through the Project references: UID/Multi/04044/2013 and PAMI - ROTEIRO/0328/2013 (Nº 022158) and also by CDRSP and ESTG from the Polytechnic Institute of Leiria. Applied Mechanics and Materials Vol. 890 Applied Mechanics and Materials Vol. 890 69 [13] N. Martins-Ferreira, Quaternion rotations and its applications, Scripta-Ingenia 8 (June) (2017) 20–24. References [1] A. Anastasiou et all, 3D printing: Basic concepts mathematics and technologies, 13th IEEE International Conference on BioInformatics and BioEngineering,Chania, Greece, 2013 (DOI: 10.1109/BIBE.2013.6701672) [2] Matthew B. Hoy, 3D Printing: Making Things at the Library, Medical Reference Services Quar- terly, 32:1 (2013) 93-99 (DOI: 10.1080/02763869.2013.749139) [3] R. Herken, (ed.), The Universal Turing Machine: A Half-Century Survey, New York: Oxford Uni- versity Press, 1988. [4] John D. Barrow, The World within the World, Oxford-New York: Oxford University Press, 1988. [5] N. Martins-Ferreira, The notion of multi-link, its applications and examples, Scripta-Ingenia 6 (December) (2016) 8–15. [6] M. Gaspar and N. Martins-Ferreira, Robust STL processing for extrusion-based manufacturing, Innovative Developments in Virtual and Physical Prototyping (2012) 273-278. [7] George A. Stevenson, Graphic Arts Encyclopedia Revised by William A. Pakan, Design Press, 3ed (1992) McGraw-Hill. [8] N. Martins-Ferreira, On the structure of a triangulation, Scripta-Ingenia 5 (December) (2015) 22–23. [9] N. Martins-Ferreira, An Image Segmentation Procedure based on the Superposition Principle of Quantum Mechanics, Procedure Manufacturing 12 (2017) 249-262. [10] N. Alves, P. Bartolo, N. Martins-Ferreira, M. B. Gaspar and A. Mateus, BioFab toolbox Soft- ware tools for biofabrication, High Value Manufacturing: Advanced Research in Virtual and Rapid Prototyping, 2013, 483-487. [11] A. M. Turing Computing Machinery and Intelligence, Mind 49 (1950) 433-460. [12] M. Gaspar, M. Leite, N. Martins-Ferreira and B. N. Panda, Computer construction of Platonic Solids, Scripta-Ingenia 4 (June) (2015) 6–11. [13] N. Martins-Ferreira, Quaternion rotations and its applications, Scripta-Ingenia 8 (June) (2017) 20–24.

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